Synthesis and biological activities of pseudopeptide analogues of LH-RH: Agonists and antagonists

Vol.

97, No.

3, 1980

December

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 1014-1023

16, 1980

SYNTHESISAND BIOLOGICALACTIVITIES OF PSEUDOPEPTIDE ANALOGUES OF LH-Ml: AGONISTSANDANTAGONISTS

F. Spatola: Nirankar S. Agarwal, Anthony L. Bettag, and John A. Yankeelov, Jr.?; Departments of Chemistry and Biochemistry, University of Louisville, Louisville, KY 40292 Arno

and

Cyril

Y. Bowers, Tulane University School of Medicine New Orleans, Louisiana 70112 and

Wylie W. Vale< The Salk Institute, Neuroendocrinology Laboratory, LaJolla, California Received

September

92037

22,198O

Sulrullary

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH2Slinkages as selected amide bond replacements. Agonists modified at the S-6, 6-7 and 9-10 of LH-RH, respectively. position had 2, < 0.1, and 10% of the in vitro activity Amongpotential antagonists, 6-7 posit%xogues showedonly minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LHand FSH inhibitory activity. In addition, a l-2 osition methylene thioether analogue of the parent [Ac-Prol, D-Phe2, D-Trp 3*i]LH-BH antagonist was completely inhibitory at 30 ng -in vitro and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither l-2 nor 9-10 methylene thioether position antagonists showed--in vivo antiovulatory activity at the 250 pg level. * To whomcorrespondence should be addressed. +

Deceased, April

** Abbreviations follicle

6, 1979.

used:

stimulating

LH-RH,

TFA,

sulfonyl;

hormone-releasing hormone; FSH,

hormone, LH, luteinizing

Cbz, benzyloxycarbonyl; amine;

luteinizing

DCC, dicyclohexylcarbodimide;

trifluoroacctic

Bzl, bcnzyl;

hormone, Boc, t-butyloxycarbonyl,

acid;

DMF, dimcthylformamidc;

pGlu, pyroglutamyl;

HPLC, high pressure liquid

DIEA, diisopropylethyl

thiomethylone

ether

p-tolucnc

t lc, thin layer chromatography;

chromatography; TEAP (Triethylammonium phosphate) ;

J, refers to the replacement of the normal amide linkage, the

Tos,

moiety,

OOOS-2glX/80/231014-10$01.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

[CH2S].

1014

[CONH]

by

Vol.

97, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

INTRODUCTION

Both

LH-RU

agents

superagonists

by virtue

bioactivity

of their

would

antiovulatory

that

prove

use of the link

resistant

ether

LH-Rlt as established position

potent

subjected

to this

pseudopeptide

selective

Boc-Protected followed Also,

chosen

the

bond

for

suitable

surrogate)

of each of these solid

described

Koch -L’ et al

(3)

JLH-RH

inhibitory

of but

other

to LH-RH based (4) have

also

been

of synthesizing activity. I

of the

three

methods

routes

elsewhere

for (7).

first

acids).

of the

amide

into

prepared prepared

psoudodipcptides

amino

analogues

the it

Boc-X J, Cly type

varieties

phase

contains

were

WC have prcparcd chiral

of these

to incorporate

(7)

has been first bond replacement

the pcptidc.

Tho

by Yankoelov

-ct --al

p (5)

by Spatola

ct al.,

(6).

of the

X$Y

typo

The procedure

pseudodipeptides

has been described

MATERIALS Synthetic

Our major

bonds

the objective

long-lasting

synthesis

GlyJIX,

X and Y represent

by standard

with

which

and then

by dipeptides then

the

dipeptide

dipeptides,

since

D-Trp3’6

amide

D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-GlyNH2

The strategy

(amide

have

(I).

Antagonists

the

the

6-7 and 9-10

and by

synthesized.

analogucs

for

LH-RH structure

(2)

D-Phe’,

peptide

we have examined

of the

modification that

to prepare

candidates

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2

LH-RH’ Antagonist

to prepare

been

[D-pGlu’,

analogues

LH- RH

lability

Prolonged

useful

as a replacement

the

and Stern

have also

inhibitor

[CH2S]

(1).

for

degradation,

within

known

by Marks

analogues

on the

moiety

positions by the

corequisite

In an effort

to enzymatic

thiomethylene

as antifertility

of LH and FSH release

activity.

was predicated

have potential

to be a necessary

[CUNH] in critical

focus

both

inhibition

appear

possessing would

and antagonists

into

for

(whcrc incorporation

the peptides

in the present

study.

AND METHODS

the pseudodipeptides The incorporation

1015

used of the

in this Boc-protected

study

have been derivatives

Vol.

97, No.

BIOCHEMICAL

3, 1980

Table

I

AND

Methodology

BIOPHYSICAL

For Solid

Time (Min)

1

CH2Cl*

L

TFA/anisole/CH2C12

3

CH2Cl *

4

DlEA/CH2c12

b

CH2C12

b

k&-amino

7

DCC/CH2C12 (2.5 equiv.)a

gob

s

CI12C12 (3x)

1

9

wx1

1

1

(3x1 (40:2:51)

(10: 90) (2x)

1

acid/CH2C12

(2.5 equiv.)”

of 2-12 hr. each for

into

peptides

laboratory.

Table

synthesis;

chiral

specified.

Side (Ser

amino chain

Vega Biochemicals,

acids

Bachem,

1% cross-linked

or

were

dipeptides,

although

catalyzed

deprotections,

Peptides

necessary

were

hydrogen

fluoride

30 min.

at O” C. G-15 using

are of the

in the

scavengers

from

containing The extracted

used

employed

for

used

were

amino

acids

polymers

extent

(0.4-0.5

products

25% or 30% HOAc.

1016

where

[Arg

and His)

purchased

lyophilized

pcptidc otherwise and

from resins

Boc-Glycine

or the No unusual

Boc-pseudo-

employed

and amino

and 10% ethyl

The major

phase

except

involving

by treatment

were

solid

mmol/g.

were

cleavage

resins

in our

meq. nitrogen/g)

with

antioxidants

10% anisole

were

reactions

fluoride the

tosyl

of 0.15-0.35

coupling

Boc-pscudodipcptidcs.

Benzhydrylamine

(DCC mediated)

and/or

hydrogen

cleaved

methods

Beckman Biochemicals.

to the

for

Boc-pseudodipcptides.

L-configuration

groups

condensed

pseudodipeptide

Gquivalents

methodology

polystyrene

Biochemicals,

3.0

standard

Common protected

precautions

Sephadex

the

protecting

and Tyr).

Beckman

follows

I describes

-

(3x)

by two couplings

LH-RH

2-10

(3x)

Substituted

to

25

1

b.

related

5 and

(5x1

of 1.5 thru

Boc-ProJIGly

Synthesis

IkilgL!nt

by two couplings

from

COMMUNICATIONS

9

Substituted

used wcrc

Psptide

Phase

il.

bcnzyl

RESEARCH

during

acid

hydrolyses.

with

anhydrous

methyl

sulfide

and desalted

peak was collected,

acid-

for over lyophilized

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 97, No. 3, 1980

and purified

by gel

reversed

phase

columns,

employing

liquid

and monitoring to remove tic

chromatography methanol/ammonium

HF’LC employing

and monitoring

separable integrating

purity

rats.

Zorbax

gradiont

acid

solvent

showed

the

analysis

all

resulting

including

(Table

weakly

products

amino

acid

of the

of the

rcpcatcdly

techniques

III),

absorbing free

via

Model

3837

conditions

pseudodipeptide

units

of the pseudodipeptide

partial

enzymatic

fragments

Details

impurities.

of HPLC-

hydrolysis

and position

and gradient

on a Hewlett-Packard

confirmed

systems

lyophilizcd

any possible

were

C-18

phosphatc/acetonitrile

were

The LH and FSH agonist

over

peptides

of nanograms

and NIAMDD rat The second dissociated

tests

via -

of the

rat

hemipituitaries

degradation,

chemical

latter

procedure

will

of the

Lh-LER-1240-2

standards (2.1

x NIH-FSH-Sl

--in vitro

system

anterior

pituitary

release

of luteinizing

of LH-RH was the basis

Values (0.60

cell

(91.

Multiple

tested rates

(8).

Sprague-

were

and adding

primary

cells

secretion

female

were

dctcrmincd

LH-RH and calculated

NIH-LH-Sl

in

(units/mg)

units/mg.).

utilized

were

old

systems.

activities

solution

elsewhere

hormone the

and antagonist

6 h as described

FSH RP-1

in two distinct

of 20-day

in Ringers

and an LH-RH standard

luteinizing

performed

the use of hemipituitaries

test

concentration

amino

analyses.

biological

the

to inhibit

using

by standard

The presence

and III

850)

elsewhere.

by incubating

the

II

and analysis

involved

peptides

II),

was by semi-prcparativc

was collected,

rearrangement)

mass spectrometric

The first

terms

peak

cases,

products.

The --in vitro

Dawley

buffer

procedure

apparent

compounds

be reported

acetate

and analyzed

In several

to ninhydrin-negative

ionization

Model

to > 95% as monitored

(through

derivatization,

(DuPont

a triethylammonium

by this

recorder. loss

purification

at 210 nm to detect

impurities

in

salt,

x, y, and z (Table

The analytical

units

The major

ammonium acetate

by analytical

caused

The final

at 254 nm.

on solvents

system

filtration.

for

their

by these hormone

of the

1017

cells.

stimulated antagonist

cultures dose abilities

of enzymatically levels

of pseudo-

to increase

The ability

of analogues

by a constant assay,

where

[D-pClu’,

[Tyr

(Pro$G1ySHZ

[DpGlu’,D-Phe*,

III

I\

v

D-Phe’.

HPLC

Tic

b.

System:

Solvent Systems: (7:3). :I EtOH:H,O

Solvent

LH-RH

[Ac-Pro$D-Phel-*,

x,

C.

A. B.

II.

Properties

HI’LC

of

C

C

B

B

System=Ret

Synthetic

14.7

12.0

10.7

12.9

l-7.0

11.9

12.7

‘II

HPLC

Lti-RH

9.05

(15:3:1?:10);

Triethylammonium Triethylammonium Triethylammonium

Solvent

n-bu011:H0,\c:t!20:I~)‘ridi~~c

Acetonitrile/O.ZSN Acetonitrile/O.ZSW Acetonitrile/O.2W

D-T~P~‘~]

D-Trp3’6

D-Trp3,

Pro $ G1ySH2’-“]LH-RH

[D-pGlu’,

D-Ser

D-Trp3,

$J Leu6-7]LH-RH

Gly $ Leu6-7]LH-RH

[D-pGlu’,D-Phe*,

a.

VIII

VII

VI

JI ClysV6]LH-RH

[Cly

II

‘-“]LH-RH

$J Leu6-‘]LH-RH

LH-RH

I

Peptide

Table

(MN)

y.

pH 3.5; pH 3.5; pH 3.5;

0.64

0.60

0.56

0.57

0.42

0.52

0.43

0.24

Rf*

r,-euotl:I!Or\c:EtO-\c:l120

phosphate; phosphate; phosphate;

Time

.Xnalogues.

12-25: 20-30cr 25-508

0.64

0.66

0.53

0.56

0.44

0.50

0.39

0.33

RfY

0.67

0.6s

0.60

0.61

Rf

(1:l:l:lj;

gradient gradient gradient

TLCb

min)

min) min)

(15

(15 (25

Vol.

97, No.

3, 1980

BIOCHEMICAL

'lablc

Structure

Clu

As&lo-acid

111.

Phc

AND

BIOPHYSICAL

Analysis Ratios Amino Acid Ratios

Trp

His

Ser

Tyr

ClY

0.97

1.04

RESEARCH

of Ui-IU

COMMUNICATIONS

AIldOgS

Lou

An?

h-0

0.87

1.13

1.02

a,b

Other

NU.

II

1.00

0.84

0.81

0.91

III

0.96:

0.81

0.91

0.78

IV

0.99

0.83

1.02

1.00

L

u.L)o

0.93

0.90

1.04

1.00

1.03

1.02

1.08

1.00

1.03

0.90

0.92

1.01

K.U. Tyr IJ(;I;/ N.D. Pro v tily 1.06

1.05

0.94

Cly0 VI

0.99

c

0.76

0.89

1.02

VI-1

1.05

1.10

1.64

0.92

1.03

Vlll

1.76

a.

Incorporated Pseudodipeptide

11.

N.D.

c.

U-&r

0.82

1.00

0.96 0.96

1.00

0.98

1.02

c N.D. Pro

0.90

1.10

0.94

0.99

Lcu

li, Lcu

+ Phc

due to low ninhydrin 1.89,

-

amino-acids

Cly

N.D. Pro

= not dotcrmincd

Q

0 D-NW

response

peaks are virtually

suporimposoblc.

D-Phe*, D-Trp 3'6]LH-RH was used as a standard and arbitrarily

assigned a

potency of 1. Antiovulatory

assays were carried

Worcester, Mass., using dissolved in corn oil the ova were counted

female

Sprague-Dawley

and injected the

next

out at the Mason Research Institute, rats.

The peptides were

subcutaneously on noon of proestrus

and

morning.

RESULTSAND DISCUSSION As

shown in Table IV, LH-RHanalogues whose only structural

involved biological III

amide

bond

activities.

variations

replacements at 5-6, 6-7 and 9-10 displayed widely divergent Though none were as active

as LH-RH, LH-RHpseudopcptides

and IV with amide bond replacements at 5-6 or 9-10 were l-2 orders of

magnitude more active containing

with respect to LH and FSHrelease than

a 6-7 modified linkage.

to be the most susceptible

Since the 6-7 linkage

the

has been

analogue shown

site of degradation of the LH-RHmoleculL1*.3!

1019

Vol.

97, No.

3, 1980

BIOCHEMICAL

TABLE IV.

AND

--IN VITRO AGONIST A"WITY

Pseudopeptide

[Gly$

Analogue

Ln-RH nglrnl of medium

P

A.ng/nl of medium

SEM 4)

P

3 450 5 187 849

5 23 3 44 198

"S .Ol ,001

470 7856 304 5562 9332

133 1462 87 648 981

ns .OOl ,001

29 1057 595 1256 .1607

15 148 74 105

. 001 ,001

179 5840 4487 8364 12846

33 607 702 829 1106

. 001 .OOl ,001

407 -6 25

49 11 14 8

.OOl ,001 ,001

5627 -155 -26 1966

281 112 240

.OOl ,001

282

,001

Leu6-711mH 0.6

0.6 30 100 1000

rlGlyWl2

g-'o]LH-pJ

0.6

0.6

6 60 aAs determined b As determined

the above conformat for

incubations

in pituitary

cell

results

change.

contains

insertion

- See experimental

culture

suggest

ional

LbRI1

thus,

by hemipituitary

vs.

standard

that

the

Perhaps

a bend or of the

of a preferred

low energy

than

bond.

While

by the

equipotent

the

amide

been ruled some type activity

out

of bend found

additional

in the (Table

now-modified with

which

the

the

it

may more readily

in the --in vitro

6-7 amide

bond proved

at

link

junction

(lo),

acids

is the

greater

seems possible activity

of the

antagonist

to substitution

1020

content

LH-FUI analogue of

(11).

residual

1 and 10 (12). overall

An

lipophilicity

by HPLC retontion

more readily phase

the

seems to have

in view

amino

reversed

interact

and,

is more flexible

position

this

folding

lipophilic

intolerant

0.10

Momany model(s)

, as ascertained

II

that

rcsuits

0.02

may have reduced

especially

analogues

structure

proposed

of an NO-Me-Leu7

analogue

to the

greater

se

that

interpretation

bend region

A substantially

w;1s noted

substituted

6-7

site

likely,

analogues among these

6-7

II);

is

c 0.001

an unfavorable

the CHgS substitution

activity

region

cyclic

correlation

observed times

for

per

the

at the

since

a g-turn

induces

that

at this

conformer

in this

is

turn

linkage

Pot*ncyb LH-P&l - 1

section.

6-7 pseudolinkage

hairpin

Relative

section.

- See experimental

pertinent

pseudo

FSI? SEM (*I

[Tyr ~Gly5-61LH-lW

COMMUNICATIONS

OF PSEUDDPEPTIDES RELATED T O LH-FJi

A, w/m1 of medium

100 1000 10000

ho

RESEARCH

LHa

DOS.? Peptide ngJm1 of medium

BIOPHYSICAL

substrate

column

although

dcpcndoncc

of

analogucs even with

the

CX~OSCS

still tho

speculative. pscudopcptidcs

(Table

V).

a 6-D-residue

While

Vol.

97, No.

3, 1980

BIOCHEMICAL

TABLE V.

AND

Peptide ngfm1 of medium

Pnalogue

[D-pG1ul, D-Phe'. D-Trp’, Gly $Leu 6-‘lLN-F.H

[D-pGlu', D-Ser

LAP lx-lw nglml of medium

10 100 1000 10,000

.6 .6 .6 .6 .6

6 450 366 415 380 268

10,000 50,000

.6 .6 .6 .6 .6

7 532 594 553 563 226

23 144 82 108 97 16

10 30 100 1000

.6 .6 .6 .6 .6

-2 636 251 164 19 -20

10 72 31 27 2s 13

-16 526 26 25 9 5

D-Phe', D-Trp' $Leu6-'ILki-PH

D-Phe’, D-Trp3j6 Pro’4 GlytW2 g-'o]&gH

$D-Phel-',

aA~ determined b As determined

D-Trp'%"-RN

VI),

Peptide its

with

parents

is unknown Both assayed

for

administering

124 <.OOl 1462 563 672 803 373

.OS ns ns IIS

ns ns ns .05

127 5339 4265 4600 4988 2081

185 1282 250 542 871 371

ns ns ns .05

.OOl ,001 ,001 .OOl

375 7241 3852 2420 360 -283

207 953 1251 305 287 165

.05 ,001 .OOl ,001

2 e.001 41 10 ,001 24 ,001 5 ,001 11 .OOl

-15 7871 773 386 -23 -117

< 0.001

c 0.001

153 c.001 231 225 ,001 156 ,001 75 .OOl 96 .OOl

0.47

(.30-.71)

0.60

(0.40-1.0)

were

considerably

activity; the least

retained

moreover, [AC-Pro

1-2

in the

LH-IUi suggest bond at l-2 l-2

position

a role

in these

more than

compared

QD-Phe

as active

at the

more expendable. SO%

to the

, D-Trp

3D6]

LH-KH

hemipituitary

assay.

the possible

and 9-10 after

structure

for

importance

agonist

activity.

substitution activity

of relationships

time.

antagonists their

plays

467 7856 4400 7091 7061 5807

substitution

of the peptide

linkage

at this

bonds

structure,

lipophilicity

Relative Poteneyb [DpGlu', D-Phe’, D-Tq3’61ui-Rli = 1

P

section.

of Pro JI G~YNH~‘-~’

function

pseudopeptide

amide

to be at

P

section.

pseudopeptide

linked

SFm (+I

A. nsln1 of medium

6 ~001 23 74 ns 20 ns 53 "S 15 . 001

- see experimental

biological

proved

increased

standard

9-10

and those

carbonyl

Whether

vs.

and 9-10

the

(VIII)

results

of the

l-2

amide

antagonist These

culture

--in vitro

corresponding

the

cell

the

VII

.6 .6 .6 .6 .6 - see experimntal

30 100 1000 10.000 incub%rions

in pituitary

(peptide

of

_

by hemipituitary

COMMUNICATIONS

FSI? SEM ('1

A. w/m1 of medium

[D-pGlu',

[AC-Pro

RESEARCH

--IN VITRO ANTAGONIST ACTIVITY OF PSSUCOPEPTIDES RELATED T O LH-RH. DOW

Pseudopeptide

BIOPHYSICAL

displaying --in vivo

250 ug

substantial

antiovulatory

peptide/animal

in vitro A-

activity on

the

1021

activity

in female

day of proestrus.

were adult

rats

next by

Vol.

97, No.

3, 1980

Surprisingly

BIOCHEMICAL

both of the pseudopeptide

at which

the corresponding

have been fully to determine degradation

active

such findings analogues

are unique

peptide

analogues,

in the tight

antagonists

antagonists

that

even when the replacement

involves

of the replacement the question

terminal

role. positions

can yield

of such substitutions

Appreciation for

support

Karten

for

of this helpful

technical

through

discussions.

Mr. Benjamin Van Osdol, for expert

retention

active

We

injection

of full

for

orally

and this

Ms. Georgeann Reynolds

changes. again

once

in peptides

may have a

at N and C

tends

to support peptide

active

NO l-HD-8-2882

are indebted

potency,

and polarity

or blocking

1022

poor

(in vivo).

biological

steric

analogues

assistance.

are involved

and the possible

to the NIH Contraceptive Contract

modified

the problems

which perhaps

amide replacements

in the search

is expressed work

between

backbone elements

highly

and --in vivo

of amide bonds in LH-RH analogue

as a mode of retarding

by endo- and exopeptidases

between --in vitro

subcutaneous

that

non-

lipophilic

does appear to be critical

The observation

or

preferred

highly

Other

(in vitro)

substantial

as to whether

problem,

whether

to distinguish

the replacement with

is in progress

Work

By the use of suitably

at the LH-RH receptor

suggest

(11).

and to establish

of the analogues

is not incompatible

functional

(14).

after

at dosages

mechanisms,

lack of correlation

of the analogue

Our results

components,

lipophilicity

bending

transportation

by unexpected

it may be possible

and global

assay

to LH-RH pseudopeptides.

of LH-RH analogue

COMMUNICATIONS

amide linkages

may be due to a transport

to extracellular

results

raises

this

have shown a similar

of local

with

RESEARCH

were inactive

in the rat antiovulatory

to what extent

binding

BIOPHYSICAL

analogues

analogues

of the amide surrogates

reversible

‘111~ site

AND

the use

degradation

analogues.

Development

Branch

and to Dr. Marvin

to Mr. Scott

Mack,

and Ms. Karen Von Dessonneck

Vol.

97, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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