Synthetic analogs of luteinizing hormone releasing hormone (LH-RH) substituted in position 6 and 10

Vol. 60, No. 1, 1974

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SYNTHETIC ANALOGS OF LUTEINIZING HORI~IONERELEASING HORMONE (LGRH) SUBSTITUTED IN POSITION 6 AND 10 M. Fujino, Central

T. Fukuda,

S. Shinagawa, S. Kobayashi, I. and R. Nakayama Division, Takeda Chemical Industries, Higashiyodogawa, Osaka, Japan

Research

J. H. Seely, of Antibiotics North

Division

Received

July

Yamazaki Ltd.,

W. F. White and R. H. Rippel and Natural Products, Abbott Chicago, Illinois 60064

Laboratories,

22,1974

SUklMARY : A series of peptide analogs of luteinizing hormone releasing hormone (LH-RH), altered at position 6 and 10, was synthesized and evaluated to induce ovulation in --in vivo for the ability the diestrous rat and --in vitro for ability to release pituitary luteinizing hormone and follicle stimulating hormone. All the analogs with D-amino acid substitutions at position 6, even those with large bulky side chain, exhibited an amazingly high potency compared with the parent hormone, LH-RH. On the basis of the biological activities, structure-activity relationships in the central part of this molecule were discussed in detail.

This logical

paper

is

effects

luteinizing

part

of a continuing

of amino

hormone

acid

with

based

D-Ala

5, i.e.,

in

on the position

hormone (1)).

highly

when evaluated Coy --et

Copyright All rights

al.(4)

We have

potent

the

(LH-RH,

des-Gly

6 and a hydrophobic

D-Ala?-LH-RH-ethylamide CIVI,

in

CIIII possessed

reported

0 1974 by Academic Press, of reproduction in any form

inc. resewed.

molecule

recently

surprizingly

amino

similar

intense

406

results

of

reported

acid

[III,

index

bio-

that

lo-LH-RH-ethylamide in

[I](2), position

des-GlylO-CPhe5,

and des-Gly10-CIle5,

by ovulation-inducing have

on the

pGlu-His-Trp-Ser-

-CD-Ala?-LH-RH-ethylamide

des-Gly"

ethylamide

replacement

releasing

Tyr-Gly-Leu-Arg-Pro-Gly-NH2 analogs

investigation

D-Ala%-LH-RHbiological

(3). on the

activity

Independently, --in

vivo

LH-

Vol. 60, No. 1, 1974

release

activity

previously of

although

the

In of the

Monahan

D-Pro

--et al.

and D-Leu)

explained

studies

standard,

on the

having

D-amino This

of seven

paper

synthesis acids

the

des-Gly

des-Gly"

[:1X1,

that

CVIIII,

des-Gly10-CIle5,

D-Leu'l-IH-RH-

CXIIII.

C-terminal

fragments,

acidsl,were

prepared

The essential has been

of the

protecting

cA5=Tyr,

described

by acylation

of H-Leu-Arg(N02)-Pro-NHCH

C-terminal

strategy

[XII]

for

making

previously

(3).

H-A6-Leu-Arg(N02)-Pro-NHCH2CH3

esters

group

fragments Phe or Ile]

on the

were (3)

with 2

CH

3

amino

coupled by the

acid6]-

des-Gly"-[Phe5,

10-CD-Ser6]-LH-RH-ethylamide

series

potency

-CD-norvaline'l-LH-RH-ethylamide

and des-Gly

this

10.

biological

D-Phe%-LH-RH-ethylamide

in

of LH-RH

at position

des-Gly"-[Phe5,

peptides

ovulation-

of LH-RH analogs

[Xl,

of peptides.

and des-

systematic

ethylamide

Synthesis

site.

10-CD-a-amino-n-butyric

-CD-Leu6]-LH-RH-ethylamide

D-Leu'l-LH-RH-ethylamide

binding

high

us to

and the

of

unpublished

6 and ethylamine

of LH-RH,

[VII,

al.,

evaluation

synthesis

LH-RH

relationships

very

led

and biological

describes

et

exhibited

findings

at position

than

CDL-Leu'l-LH-RH

Fukuda

in

consideration at the

8 and 20 times

These

new analogs

des-Glyl'

analogs

approximately

LH-RH-ethylamide [VIII,

these

potent

on structure-activity

(T.

respectively.

studies

- 450% of the

by steric

we had synthesized

that

activity,

reported

less

interaction

-[DL-Leu']-I&RH-ethylamide

inducing

350

were

this

and/or

independent

and found

al.(5)

in --in vivo and --in vitro assays, a D-amino acid with a bulky side chain

LH-RH molecule,

data)

--et

CV] exhibited

interactions

our

RESEARCH COMMUNICATIONS

hormone

having

Monahan

intermolecular

Gly"

parent

6 (D-Val,

itself.

II.

CD-Ala']-LH-RH

analogs

position

AND BIOPHYSKAL

of compound

that

potency

the

BIOCHEMKAL

with

activated by removal

The resulting

pGlu-His-Trp-Ser-A5-OH

HONB/DCC method

407

acid

and followed

function.

The

lA6=D-amino

Z- or BOG-amino (2,6),

the

(7)

to minimize

Vol. 60, No. 1, 1974

undesirable

BIOCHEMICAL

racemization

AND BIOPHYSICAL

during

the

protected

peptides

were

Amberlite

XAD-2 in

a manner

synthesis

of other

LH-RH analogs

protected

peptides

were

in

60% formic

the

method

were

acid

to

obtained amino

purified that

were acid

intermediates

similar

and the

final

which

is

stannous

All

homogeneous

thus

characterization

obtained

analogs the

of the in

of

a manner

the

and gave

are listed

chloride

a modification

on CMC in

(6,7,8).

products

our

mono-

peptides

chromatography

for

for

with

The crude

chromatographically

on

The purified

to reduction

previously

The data

described

(6,8).

--et a1.(9).

The crude

chromatography

to that

subjected

described

reactions.

by column

by column

ratios.

coupling

2 hr at 80 - 85"

of Hayakawa

further

similar

for

purified

RESEARCH COMMUNICATIONS

Table

correct key I and II,

respectively. Biological activity

evaluations of these

Table

I.

and discussion.

synthetic

Physicochemical

analogs

The ovulation-inducing was determined

Properties

of

in

the

diestrous

Intermediates

R-A6-Leu-Arg(N02)-Pro-NHCH2CHj

Compound R-A6

Z-D-AbuC)

Formulaa) (cont.,

Cd E3 solvent)

Rfb)

C28H5108N9*0.5H20

111-114

-51.2"(0.5,

DE?)

0.58

(+12~51’8~9

105-107

-51.2y1.0,

MeOH)

0.57

Z-D-Leu

C33H5308N9

133-134

-47.3"(0.5,

MeOH)

0.59

Z-D-Phe

C36H5108'Tg

162-164

-64.2O(l.O,

MeOH)

0.67

BOC-D-Ser

C27H4q09~Tq*H20

136-139

-17.6O(l.O,

DJb'IF')

0.45

Z-D-NvaC)

a) All compounds listed gave the correct analytical values (C, H, N). b) Solvent system (Merck's precoated silica gel plate F 254): CHC13c) Abu'= a-amino-n-butyric acid, Nva = norvaline MeOH-AcOH (9:1:0.5).

408

BIOCHEMICAL

Vol. 60, No. 1, 1974

Table

II.

Chemical

AND BIOPHYSICAL

and Physical

RESEARCH COMMUNICATIONS

Properties

of LH-RH Analogs

[a]g3-43.6"(c 0.5, 5% AcOH); TLCa) Rf20. 063, Rf30.67, 0.61, Rf50.6Y; Amino acid anal. b' His 0.97, Arg 0.97, Trp 1.00 Ser 0.94, Glu 1.00, Pro 1.01, Abu 1.03, Leu 1.03, Tyr 1.03 (86%) 4 .

hT:Rf

-39.8"(c Ser

0.81,

Glu 1.00,

0.5, 5% A,,;;

Amino acid Pro 1.01,

TLCa) Rf20.063, Rf30.68, His 0.92, Arg 0.96, Trp 0.92, anal. Nva 1.04, Leu 1.01, Tyr 1.00 (84.5%)')

-32.7"(c 0.55, 5% AcOH); TLCa) Rf20.070, Rf30.65, $nal"ff VIII: [al$ Rf 0.65, Rf50.72; Amino acid anal. b, His 1.00, Arg 0.96 Trp 0.96, Ser 1.00, Glu 1.00, Pro 1.04, Leu 2.00, Tyr 1.00 (82%) 4 . Rf30.6Y, [a]g5-38.5'=(c 0.5, 5% AcOH); TLCa) Rf20.082, .@%-= Rf 0.72, Kf50.74; Amino acid anal.b) His 1.00, Arg 1.04, Wp 0.98, c) Ser 0.92, Glu 1.00, Pro 1.00, Leu 2.04, Phe 1.01, Ethylamine 1.02(87%), Ca3;4-50.30(c

Ser

0.6,

Rf50.73; Amino 0.98, Glu 1.01, Pro

5% AC,,];

acid 1.00,

TLCa) Rf20.082, Rf30,6Y, anal. His 0.98, Arg 1.00, Trp 0.97, Leu 2.02, Ile 0.98 (89%):)

-71.8O(c 0.2, 5% AC,,]; TLCa) Rf20.082, Rf30.70, Analog XII: [aI; Rf40.64, Rf50.74; Amino acid anal. His 1.01, Arg 0.97 Trp 0.92, Ser 0.98, Glu 1.00, Pro 0.99, Leu 1.00, Phe 2.03 (87%) 4 . Analog XIII:CalE7 -46.4O(c 0.5, 5% AC,,]; TLCa) Rf20.038, Rf30.64, Rf 0.42, Rf50.65; Amino acid anal. His 1.00, Arg 1.04 Trp 1.00, Ser 1.82, Glu 1.00, Pro 1.00, Leu 1.04, Tyr 0.96 (76%) CS. a) Solvent systems and sheets employed were: Rf2(silica gel), EtOAcpyridine-AcOH-H20 (60:20:6:10); Rf'(silica gel), EtOAc-BuOH-AcOH-H20 (1:l:l:l); Rf4(cellulose), n-BUOY-AcOH-H20 (4:l:l); Rf5(silica gel), n-BuOH-pyridine-AcOH-H20 (30:20:6:24). b) Acid hydrolysate (5.7 E of thioglycolic acid)(l4). HCl, 105O, 30 hr.,. in the presence c) Peptide content.

rat

(SC,

the

injection)

--in vitro modification hemisected

by the

method

of Yamazaki

LH- and FSH-releasing of the anterior

method pituitaries

activities

of Mittler (12).

409

end Nakayama were

and Meites The details

measured

(11)

using of these

(10)

and

by a rat assays

Vol. 60, No. 1, 1974

Table

III.

BIOCHEMICAL

Structures

AND BIOPHYSICAL

and Relative

RESEARCH COMMUNICATIONS

Activities

of Synthetic

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 -5 6 Compound No. LH-RH Ia) II lot lot IIIb) IVb) Vb)

5

Substituted 6

residue 10

10

Ovulation-inducing activity ($)

in vitro LH -FSH release release($y;

100 lb) 2

NHCH2CH3 NHCH2CH3

D-Ala Phe Ile

VI VII VIIIe)lot

D-Ala D-Ala D-Ala

NHCH2CH3 NHCH2CH3 NECH2CH5 NHCH2CH3 NHCH2CH3

g;:; D-Leu

1 2

lot IX X XIf) XII XIII

LH-RH Analogs

672 8,270 5,120 5,810 1,340 2,830

100 300 180

280 300

270 160 570

300 350 475

6,520d)

180

170 go-400 160-290 280 275

5,810 8,270

6,900 D.-Leu D-Leu D-Leu D-Phe D-Ser

Phe Ile Phe

NHCH2CH3 NHCH2CH3 NHCH2CH3 NHCH2CH3

100

4,580 1,990 2,900 5,400 6,140

90 67 100 120-520

85 110 140 100

et al., Ref. 4. a) See Fujino, et al., Ref. 3. b) See Fujino, cl) The relative c) Abu = a-amino-n-butyric acid, Nva = norvaline. which were reduced from potencies were calculated from the ED50 values e) The the data of five or six different dosages (5-10 rats each). first synthesis of this analog in our collaborative project was carried The details of out by Dr. John Seely using the solid phase method. the synthesis of this compound by the solid phase method will be published f) The synthesis of this analog was carried separately by Dr. Seely. out by the solid phase method: Cal, 24-31.50(c 0.55, 5% AcOH)(M. Fujino and C. Kitada, unpublished data).

have all

been the

a large

described analogs

bulky

previously

having side

chain

D-amino

(8). acids

possessed 410

As can be seen at position

6,

an amazingly

high

from

Table

even those potency

III, with

when

Vd. 60, No. 1, 1974

evaluated of

by the

a series

corresponding

the

spatial

6 is relatively

analogs

analogs

D-Ala

effect

analogs

of the

site.

closely

side-chain in

comparable These

(3).

of the

the

as the

the

Moreover,

data

to

interaction

of the

are

those

very

of

that

acid

in

position

peptide

potencies

[D-Leu']-analogs

potencies

indicate

D-amino

Our ovulation-inducing

as well

RESEARCH COMMUNICATIONS

index.

were

unimportant

binding

AND BIOPHYSICAL

ovulationcinducing

of D-Leu

the

the

BIOCHEMICAL

with

of

CD-Ala']-

different

from

the

activity reported by Monahan --et al.(5), --in vivo LH-releasing whereas our --in vitro results are in good agreement with Monahan's The reasons for this apparent discrepancy are not known at data. the

present

compound

II

LH levels

time.

However,

or VIII

show a 50 - 80 fold

in

paration),

both

rats

which

activities.

is

This

by a comparative --in

vivo

good

time

Moreover,

indicate

that

delayed

intense

may result

from

their

to

increased

af isolated are lower

than It,

of

those

obtained

by the

appears

in

position

freedom

in

the

a conformational

central

results

that

duration

6 might

giving

exhibited of the

feedback

of the rise

411

analogs

incubation influences are

induction

result

LH peak.

digestion

In the

introduction

a

of these

acid?-analogs

ovulation

generally

portion change

[D-amino

above

enzymatic

receptor.

much

test.

of a D-amino in

molecule. to a better

the

of the

generally

to

pre-

determined

mentioned

catalytic

the

in

may be resolved

potencies

the

integrated

ovulation-inducing

--et a1.(5)

have

where

activities

residue

in

tissue

the

administration

resistance at

LH-RH and

et al.,

discrepancy

ovulation-inducing

the

however,

the

an increased

greater

in

H. Rippel

Monahan

acid%-analogs

oompting

increase

15 min after

affinity

pituitary

minimal,

since

and also

the

and/or

the

our unpublished

Therefore,

studies

with

that

only

CD-amino

LH-release

(R.

agreement

study,

at

level

and sheep

indicates

LH-release

peptides.

dose

restriction This fit

acid of

could at

the

result

Vol. 60, No. 1,1974

receptor

site(s)

of the

it

connection, very

BIOCHEMICAL

is

methyl This

group

active

to of

note

the

pituitary.

that

Arnold

In this --et a1,(13)

[Sarcosine'l-LH-RH,

nitrogen

prevent

RESEARCH COMMUNICATIONS

the

between attainment

which

found has a

5 and 6.

residues

of a biological

conformation.

Takeda

Laboratories this

peptide could

We wish

Acknowledgment: of

organ,

activity

on the

substitution

target

interesting

low biological

AND BIOPHYSICAL

Chemical for

Industries, their

to thank Ltd.

encouragement

Drs.

E. Ohmura

and Dr.

W. J.

and suggestions

and K. Morita Close

of Abbott

throughout

work.

References 1.

2.

3.

4.

5.

6.

7.

a*

Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V., Biochem. Biophys. Res. Commun., 4$, 1334 (1971). Fujino, M., Shinagawa, S., Yamazaki, I., Kobayashi, S., Obayashi, M ., Fukuda, T., Nakayama, R., White, W. and Rippel, R., Arch. Biochem. Biophys., &, 488 (1973). Fujino, M., Yamazaki, I., Kobayashi, S., Fukuda, T., Shinagawa, S ., Nakayama, R., White, W. and Rippel, R., Biochem. Biophys. Res. Commun., z, 1248 (1974). Coy, D. H., Coy, E. J., Schally, A. V., Martinez, J. V., Hirotsu, Y. and Arimura, A., Biochem. Biophys. Res. Commun., 2, 335 (1974). Anderson, H. and Vale,W., Biochemistry, Monahan, M., Amoss, M., l2, 4616 (1973); Monahan, M., Vale, W., Rivier, C., Grant, G. and Guillemin, R., Abs. 35th Annual Meeting of Endocrine Society, 1973, P* 145, Shinagawa, S. and Fujino, M., Chem. Pharm. Bull.(Tokyo), submitted for publication. Fujino, M., Kobayashi, S., Fukuda, T., Obayashi, M., Shinagawa, NO. 7 (1974) S. and Nishimura, O., Chem. Pharm. BulP.(Tokyo), in press. Fujino, M., Shinagawa, S., Obayashi, M., Kobayashi, S., Fukuda, T ., Yamazaki, I., Nakayama, R., White, W. and Rippel, R., J. Med. Chem., l.6, 1144 (1973). 412

Vol. 60, No. 1, 1974

9.

10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Hayakawa, T., Fujiwara., Y. and Noguchi, J., Buil. Chem. Sot, Japan, 5, 1205 (1967). Gakkai Zasshi, Yamazaki, I. and Nakayama, R., Nippon Naibumpi Lt.9, 189 (1973). Mittler, J. C. and Meites, J., Froc. Sot. Exp. Biol. Med., 117, 309 (1964). W. and Flouret, G., White, W., Hedlund, M., Rippel, R., Arnold, Endocrinology, 93, 96 (1973). Arnold, W., Flouret, G., Morgan, R., Rippel, R. and White, W., J. Med. Chem., x, 314 (1973). Matsubara, H. .and Sasaki, K., Biochem. Biophys. Res. Commun., a, 176 (1969).

413