Comparative studies of thyrotropin releasing hormone diazomethyl ketone and chloromethyl ketone analogs

Vol. 98, No. 1,1981 January

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

15, 1981

Pages 234-241

COMPARATIVE STUDIES OF THYROTROPIN RELEASING HORMONE DIAZOMETHYL KETONE AND CHLOROMETHYL KETONE ANALOGS James L. Balk,

Robert

B. Johnston

and John T. Pelton

Department of Chemistry University of Nebraska - Lincoln Lincoln, Nebraska 68588 Received

December

1,198O SIJWARY

Diazomethyl ketone and chloromethyl ketone analogs of thyrotropin releasing hormones have been synthesized and studied for their inhibitory effects on thyrotropin releasing hormone-induced release of radioactive 1251-labelled hormones from the thyroid gland of eight-week old male LongEvans rats. When Long-Evans rats were pretreated with thyrotropin releasing hormone diazomethyl ketone (TRH-DMK) or the chloromethyl ketone derivative (TRH-CMK), a dose-related inhibition of thyrotropin releasing hormone-induced 125 I release was observed which could be partially reversed by thyrotropin stimulating hormone (TSH). The diazomethyl ketone was a more effective inhibitor than the chloromethyl ketone. These compounds may act as an active-site directed antagonists whose effects are unique to the hypothalamo-pituitary-thyroid system.

INTRODUCTION The hormones weight

isolation

has established peptides

in

releasing

hormone

biological

active

synthesis

and chemical

of

the

the (TRH)

analogs

important

biochemical

of

hypothalamic role

releasing

of small

molecular

neuroendocrine

system

(1,2).

Since

is a relatively

simple

molecule

compared

an obvious

model

peptides, to

identification

it

study

is the

relationships

between

of

thyrotropin

choice specific

to other for

the

changes

Abbreviations: TRH, thyroid releasing hormone; TRH-OH, the free acid derivative of thyroid releasing hormone which the proline residue has a carboxylic acid group; TRH-OMe, the derivative of thyroid releasing hormone which the prolinamide is replaced by proline methyl ester; TRH-DMK, the derivative of thyroid releasing hormone which the amide is replaced in a diazomethyl group; TRH-CMK, the derivative of thyroid releasing hormone which the amide is replaced by a chloromethyl group. 0006-291X/81/010234-08$01.00/0 Copyright 0 1981 by Academic Press, Inc. AN rights of reproducrion in any form reserved.

234

Vol.98,No.1,1981

BIOCHEMICAL

in molecular

structure

conducted

on the

ting

an amino

acid

that

preserving ent

the

amino

the

antagonists

a superactive

structural

of

features

introduction react

of

are known

of the

site

In this

for

to

unique

reactive

dependent

upon

constitu-

have been synthebut

to obtain here

retain

the molecule groups

site.

ketone

(7,8,9).

was to

functional

receptor

chemical

the by the

which

may

Inhibitors

and diazomethyl

paper

as to study involved

diazomethyl

for ketones)

in

vivo

from

the

thyroid

permits

the

of TRH could

of the

by which

the possible

provide

these

that

of TRH-induced

the than

release

could at

the

compounds

may

of TRH.

of TRH-diazomethyl

evidence

useful

which

and breakdown

synthesis

the

convenient

TRH interacts

effects

synthesis

for

of diazo-

two inhibitors

of TRH is more effective

inhibition

group

The availability

analogs

and present

ketone

in the

the

TRH.

mechanism

in the

we report ketone,

of

studies

the

as a blocking

property

ketone

comparative

as well

TRH serves

ketone

investigate

TRH-chloromethyl

hormones

in

and chloromethyl

have on key enzymes

ketone

activity

and to modify

(ha lomethyl

diazomethyl

probes

be employed

related

have

of the three

described

pituitary

residue This

ketone

receptor

studies

(10,ll).

synthesis

chemical

the

highly

chain

difficult

TRH molecule

interactions

terminal.

methyl

are

chemically

have

([NT(3-Me)-histidine]*-TRH),

experiments

with

The pyroglutamyl amino

the

specific

enzyme-substrate

agonist

hormone

of the

specifically

with agonist

releasing

The rationale

side

analogs

These

of TRH is

of the

studies

of TRH by substitu-

(3,4,5,6).

activity

features

Several

of the

analogs

COMMUNICATIONS

Extensive

relationship

acid

biological

essential

including

sized,

amino

RESEARCH

activity.

structure-function

or

acids.

BIOPHYSICAL

and of biological

been

established

AND

more the of

ketone

and

structurally chloromethyl 1251-labelled

gland.

MATERIALS

AND METHODS

Preparation of TRH-CMK and TRH-DMK: TRH-OH (pGluHisProOH) was synthesized

235

by coupling

the

dipeptide

free

Vol. 98, No. 1,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

acid, pGluHisOH, with neutralized ProOMe with dicyclohexylcarbodiimide in dimethylformamide and triethylamine. The resulting ester was saponified (NaOH/MeOH) and purified by Dowex 50 (H+), 8X, 50-100 mesh by the batch The free acid exhibited no biological activity in the McKenzie technique. in vivo bioassay (12), The amide (pGluHisProNH,) obtained from the ester by treatment with methanolic ammonia was as active as an authenic sample of TRH in the McKenzie assay. Thin layer chromatography with silica G showed one Pauly (+), ninhydrin (-) spot (Rf = 0.53) for pGluHisProOMe; MeOH/CHCls (2:l). Rf for the TRH-OMe = 0.38; 8uOH:AcOH:H20:Pyr (60:12:48:40). TRH-OH (pGluHisProOH) gave a Pauly (t), ninhydrin (-) spot (R = 0.27) with the same system. IR spectrum (Perkin-Elmer 621 Grating IRf for TRH-OMe (pGluHisProOMe) had absorption frequences. (a) broad band 3700 cm-' - 2200 cm" (c) carbonyl (NH stretch); (b) peak at 3224 cm-'* stretch at 1690 cm-l* and (d) a series of peaks at 1600 ci-* 1455 cm -' 1400 cm -l, 1265 cm -I, and 1100 cm -' which are charact&istic fob pGluHisProNHz, pGluHisProOH, and pGluHisProOMe. Analysis of a TRH-OH acid hydrolysate (Beckman Model 120°C amino acid analyzer) gave a molar ratio of the histidine:glutamic acid:proline:1.37:1.00:1.13, respectively. Electron impact low resolution mass spectrum for TRH (probe temp. 280" C) from aanionalysis of TRH-OMe showed a molecular ion at m/e 362.13 and is consistent with that reported by Boler, Chan, and Folkers (13). The molecular ion as well as the dipeptide, pGluHis, (m/e = 121.19) indicates that the chemical structure represents TRH. TRH-diazomethyl ketone (TRH-DMK) and TRH-chloromethyl ketone were synthesized by reacting the mixed anhydride of TRH-OH (isobutyl-chloroformate/Et,N:) with ethereal diazomethane (14). TRH-chloromethyl ketone (TRH-CMK) was prepared by reaction of TRH-DMK with 4.2 M HCl in dioxane. The IR spectrum of TRH-DMK showed an absorption frequency at 2100 cm ' (diazo group) and a carbonyl stretch at 1634 cm-', whereas the IR spectrum of TRH-CMK showed a carbonyl stretch at 1742 cm-'. The 60 MHz proton-NMR spectrum of TRH-CMK showed a methylene singlet (-Q-&Cl) at 6 = 4.25 ppm (020). The low resolution electron-impact mass spectrum of TRH-CMK showed m/e at 245.61 (M+-CHzCl), m/e 49.50 (CHzCl), and a M+ (molecular ion) at m/e 396.12 (0.15 % RA). TRH In Vivo Assay for TRH, TRH-DMK, and TRH-CMK Biological Activity: releasing activity was determined from the amount of ""I-labelled thyroid hormone released into peripheral blood in response to TRH injected by a modification of the McKenzie technique (12). Twenty-four hours before bioassay the animals (approximately 200 g male Long-Evans rats, low iodine diet (Remington diet, Nutritional Biochemical Corp., Cleveland, Ohio) with distilled water ad lib) were injected intraperitoneally (ip) with 15uCi Nal*?, andone hour before injection of TRH, TRH-OH, or 0.85% NaCl, the animals (ether anesthesia) were pretreated with various compounds or saline. One hour following pretreatment with 0.85% NaCl, TRHDMK, or TRH-CMK, 0.2 ml blood was taken from either the tail vein or jugular and immediately after the blood sample was withdrawn TRH, TRH-OH, Three hours following preor 0.85% N&l at various doses was injected. treatment a second 0.2 ml blood sample was collected from the same anatomical site. The lz51 content at the first and second blood samples was evaluated with a Beckman y-spectrometer, and the net cpm released calculated by difference. The protocol was designed so that animals were challenged with compounds for which a known response was expected to serve as controls. Four animals per dose-level were used to obtain a precision index of 0.25. RESULTS AND DISCUSSION The

TRH dose-response

curve

obtained

236

from

animals

pretreated

with

Vol.98,

BIOCHEMICAL

No.1.1981

AND

3

4

BIOPHYSICAL

567810

MICROGRAMS

OF

20

TRH

OR

RESEARCH

COMMUNICATIONS

304050

TRW-OH

INJECTED

FIGURE 1: Curve 1. Approximately 200 g male Long-Evans rats (4 per dose level) were pretreated as indicated followed by treatment with 3,6, 10, 20, and 40 ug of TRH one hour later. Curve 1. Pretreated with 0.85% NaCl. Curve 2. Pretreated with 40 pg of TRRH-DMK. Curve 3. Pretreated with -RH-DMK and 5 mU TSH at one-and-one-half hours. Curve 4. Pretreated with physiological saline at "zero" time followed by treatment with 3, 6, 10, 20, and 40 pg of TRH-OH one hour later.

40 ug of TRH-DMK and given

TRH (Fig.

of animals

saline

that

of

lowed

pretreated line

induced tion

4 (TRH-OH)

by treatment

40 ug level

caused

in vivo

TRH-OH at

relief

Animals challenged after

into

animals

with

with

40 pg of

pretreated

with

difference

of 1400 cpm (or

A 37% increase

saline

(1600

(e.g.,

with (see

and treated

similarly

47% decrease)

cpm -f 2200 cpm)

237

with

fol-

saline

TRH-DMK at the

line

resulted

Injec-

various

doses

3). (curve

released

2, a

Figure

2)

1, Figure

Acpm 125 I release when

and

Acpm 1251 (two

to 3000 cpm for

(curve in

T3 and T4).

TRH-

40 ug of TRH-DMK resulted

TRH-CMK

one hour

that than

of the

of 1600 cpm as compared

TRH treatment)

than

but greater

Therefore,

injection

pretreated

TRH after

less

3020 cpm to 812 cpm) of the

following

40 ug of

l),

pretreated

hormone

of TRH-DMK inhibition

pretreated

were

(from

hour

TRH (line

one hour.

of 125 I-labelled

of 5 mil of TSH one-half

in partial

animals

a 73% inhibition

release

2) has a slope

and given

whereby

with

of TRH at one hour

hours

with

1, line

animals

2).

A net

occurred.

TRH-CMR pretreated

BIOCHEMICAL

Vol. 98, No. 1,198l

1001 I

2

AND

3

45

MICROCIRAMS

BIOPHYSICAL

ST810

OF

TRH

RESEARCH

20 OR

30

TRH-OH

COMMUNICATIONS

4050

INJECTED

FIGURE 2: Curve 1: Approximately 200 g male Long-Evans rats (4 per dose level) were pretreated as indicated followed by treatment with 3, 6, 10,20, and 40 pg of, TRH one hour later. Curve 1. Pretreated with 0.85% NaCl. Curve 2. Pretreated with 40 pg of TRH-DMK. Curve 3. Pretreated with 40 ,,g of TRH-DMK and 5 mU TSH at one-and-one-half hours. Curve 4. Pretreated with physiological saline at "zero" time followed by treatment with 3, 6, 10, 20, and 40 ug of TRH-OH one hour later.

animals

were

Figure

injected

5 mU TSH into

in Table

TRH dose-response

decrease

I show the effect

curve.

An

A cpm 125 I release

in

NaCl,

40 ug,

bitory

effect

jugular

vein

of increasing

derivative

of the

may be due chemically

to

is

TRH-induced

its

more

of biologically

animals

(see

curve

3,

special

case of TRH where that

it

TSH partially

release

related

General

the

diazomethyl

relieves

than the

ketone

chloromethyl

inhibition

238

of

with

the

inhi-

is dose-related.

thyroid

the

TRH-CMK

gland.

This

to TRH or to its

being

of diazomethyl

been developed. can be prepared ketone. both

a

0.85%

the

than

of preparation

have not

caused

Therefore,

inhibitor

structure

TRH-DMK

challenged

TRH-CMK)

from

methods

peptides

is more active

were

potent

doses of TRH-DMK on of

pretreatment.

a more 1251

active

dose

of TRH-DMK (or

closely

more reactive.

ketones

interest

when

doses

of various

increasing

and 80 ug TRH-DMK during

The TRH-DMK derivative

that

the

2). The results

the

with

In this it

is of

The observation

substances

suggests

Vol. 98, No. 1, 1981

BIOCHEMICAL

AND

BIOPHYSICAL

Table TRH-INDUCED

'25 I RELEASE

Pretreatment (zero time)

0.85% 0.85% 0.85% 0.85%

(at

40

pg TRH-DMK pg TRH-DMK

0.85% NaCl 4 pg TRH-DMK 40 pg TRH-DMK 80

pg TRH-DMK

40 80

0.85% NaCl ug TRH-CMK ug TRH-CMK pg TRH-CMK

*

that

Clcpm 124 I released

these

inhibitors

presumably The acts

TRH TRH TRH

40 40 40 40

pg pg I.g pg

TRH TRH TRH TRH

80 80 80 80

1-19 pg ug pg

TRH TRH TRH TRH

40 40 40 40

pg TRH vg TRH pg TRH ug TRH

80 80 80 80

pg pg pg r-g

at the

pituitary

simplest

site

for

interaction

of the functional

Although tially

interact

113 T 16 129 + 20

476 + 51 401 T 32 296 T 16

276 i 21 716 693 516 501

other

TRH.

these

than

results

Nonspecific ketone)

+ T T i

32 29 16 22

the thyroid

group. to

Since

site

239

that

this

level

which

at

is

inhibitor

ketones

(e.g., show

any

is not due to a nonspecific

which for

a chemical to

the

do not

the inhibitor

affinity

possess receptor

at

those

is

chloromethyl

observed

have a high

inhibitors the

19

597 + 53 476 T 49

the inhibition

must

with

212 T 29 216 z

of

comparable

these

479 + 56 396 T 49

level.

therefore,

inhibitor

29+ 5 129 T 30 567 7 49 698 z 52

at a site

chloromethyl

concentrations,

hours)

Dev.

interpretation

at a receptor

the

three

TRH TRH TRH TRH

+ 1 Std.

inhibition;

acts,

cpm '*' I released

A

at

pg pg pg

are acting

tosylphenylalanine

low

*

4 40 80

pg TRH-CMK ug TRH-CMK

4 40 80

VARIOUS DOSES OF TRH-DMK OR TRH-CMK

AT

Treatment one hour)

0.85% NaCl 4 pg TRH-CMK

COMMUNICATIONS

I

0.85% NaCl

NaCl NaCl NaCl NaCl

0.85% NaCl 4 pg TRH-DMK 80

RESEARCH

form

is active the

its

releasing

site

group

at very factor

of action. which

a covalent

can potencomplex,

our

Vol. 98, No. 1,198l

BIOCHEMICAL

experiments like

do not provide

enzymes

with

proteins

possess

covalent

interaction

ity

chain.

side

ligands with

of

Since

the

into

if

positions

This at

potential

the

would active

effects

ication

which

would

investigate

release,

currently

being of these

such

for

other

associated to some

inhibitor

is

reactive

protein

for

investigating

the

a biochemical

basis

to form

control sites

the

can be introduced

the

at receptor

covalent

of cell of cell

interfor

ccmmunsurfaces.

of TRH-CMK and TRH-DMK on TRH-induced

on TSH and prolactin in

often

of

The rationale

of

as [N'(3-Me)His12-TRH-DMK

also

binding

some of these

specific

a

of an affin-

inhibitor

groups

as provide

compounds

analogs

type

tool

In vivo

are

of the

then

with

from

are

site

proteins

in

a possibility.

molecule,

synthesized.

by radioimmunoassay corresponding

effects

donors

chemical

inhibitors the

analogs

result

group

general

a powerful as well

by use of specific

To further

this

to interact

site

a functional

can be important

reactive

provide

receptor

bond

Un-

receptor

With

remains

of the active

occurs.

residues,

attachment

site

sufficiently

may be in a position

action

effects

the covalent

COMMUNICATIONS

an interaction

residues.

hydrogen

of

RESEARCH

catalytic

bonding

and study

that

complexes.

I

hydrogen

at the active

preparation

groups

type

such

have to be with

groups, group

various

and

and since

nucleophilic

assumption

125

would

BIOPHYSICAL

that

affinity affinity

to proteins,

nucleophilic

evidence

both only

AND

and

progress

in

(or

vitro

studies

release as well

biologically

are

whereby

the

are being

as the

active

-TRH-CMK)

evaluated

preparation

of

peptides.

ACKNWLECM34ENTS We would like to acknowledge Dr. E.F. Ellington and Dr. D.R. Zimmerman of the Department of Animal Science and Dr. E.B. Barnawell, School of Life Sciences, for their assistance and advice during the course of this research. We also wish to thank Ken Wingrove for his financial support of this research endeavor and Paul Mattern, Department of Agronomy, for his help in the characterization of these compounds.

REFERENCES 1.

Guillemin,

R.;

Burgus,

R.,

and Vale,

240

W. (1971),

"The

Hypothalamic

Vol.98,No.

2. 3. 4.

1,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Hypophysiotropic Thyrotropin Releasing Factor", in Vitamins and Hormones 29, pp l-39. Schally, A.V., Arimura, A., and Kastin, A. (1973) Science 179, 341. Sievertssoro, H., Chang, J.K., Folkers, K., and Bowers, C.Y. (1972) Journal Medicinal Chemistry 15, 219. Rivier, J. and Vale, W., et al., (1972) Journal Medicinal Chemistry

15, 479. 5.

6. 7. 8. 9.

10. ::: 13. 14.

Grant, G. and Vale, W. (1973) Endocrinology 92, 1629. Rivier, C. and Vale, W. (1974) Endocrinology 95, 978. Lybeck, H., Leppaluoto, J., Verkkunen, D., et al., (1973) Neuroendocrinology 12, 366. Sievertsson, H., Castensson, S. (1975) Biochem. Biophys. Research Communications 66, 1401-1407. Bowers, C.Y., Sievertsson, H., Chang, J., et al., (1975), In: Thyroid Research, Proceedings of the 7th Int'l. Thyroid Conference, pp. l-3, Eds., Jacob Robbins and Lewis Braverman. Mares, M. and Shaw, E. (1963) Federation Proceedings 22, 528. Shaw, E. and Ruscia, J. (1971) Archs. Biochem. Biophys. 145, 484. McKenzie, J.M. (1958) Endocrinology 63, 372. Folkers, K. (1971) Journal Medicinal Chem. Boler, J., Chang, J.K., 15, 479. Anderson, G.W. (1967) Journal Am. Chem. Sot. 89, 5012.

241