Vol. 187, No. 3, 1992 September

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1389-l 394

30, 1992

Steric

and

Electronic

Requirements

Receptor-Stimulated in the

CNS

Leander

Merritt’,

Muscarinic

Phosphoinositide

in a Series

Dan Ngur, Scott Roknich,

for

Charles

Turnover

Arecoline

Bioisosteres

H. Mitch*, Steven J. Quimby’,

Per Sauerberg#,

Department

of

William

of Medicinal

S. Messer,

and Biological

John S. Ward’,

Jr.+ and Wayne

Hoss

Chemistry

and Center for Drug Design and Development The University

of Toledo, College of Pharmacy

Toledo,

Ohio

43606

“Eli Lilly and Company Lilly Corporate

Center,

Indianapolis,

#Pharmaceuticals

August

A series important which

6,

derivatives

for activating

muscarinic

methyl

ester

hexyloxythiophene

compound

active as an Ml/M3 On the other reduced

moiety

was utilized

limited

ring were highly

brain.

to form H-bonds.

to stimulate

derivatives

from previous

showed

oxadiazole

had lower

without

work to be

that the N-S bonds of the

The compounds (PI)

polarities capability

for stimulating

in the

and

size to the thiadiazole

were compared

turnover

to the N’s.

in the N-O bonds

was of comparable

the H-bond

in (2b),

with the hexyloxy-

with the ability to form H-bonds

the thiophene

lb was more active than 2b-4b

compared

known

MNDO calculations

phosphoinositide

Arecoline

effects

by hexyloxy-1,2,5-oxadiazole (4b) were

polarized

hand, the smaller

ability to form H-bonds, ability

in the CNS.

replaced

(1 b) (Hexyloxy-TZTP),

partial agonist.

and had large C-S bond polarities abilities

Denmark

to assess steric and electronic

receptors was

(3b) or hexyloxypyrazine

1,2,5-thiadiazole alkoxythiadiazole

Park, 2760 MBlov,

1992

of arecoline

the

46285

Research

Novo Nordisk A/S, Novo Nordisk Received

IN

and the pyrazine with respect

hippocampus

the PI turnover

had

to their

of the rat

response.

The

data suggest that the ability to form H-bonds is an important factor for the ability of 1 0 1992 kademic Press, Inc. to stimulate Ml muscarinic receptors in the CNS.

+To whom correspondence

should be addressed. 0006-291X/92

1389

$4.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

BIOCHEMICAL

187, No. 3, 1992

CH3

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CH3

bH,

1

2

Arecoline

$3

$;I

&H,

N c!H,

R a -&He

3

b-C643

4 Scheme 1

Muscarinic

agonists

with

Mf selectivity

are

of current

interest

as potential

agents for dementias including Alzheimer’s disease in which there is a A series of alkoxy-I ,2,5-thiadiazole derivatives of arecoline cholinergic deficit. (Alkoxy-TZTP’s), including la and 1 b, were shown to be Mf -selective partial agonists

therapeutic

for muscarinic

receptors

hippocampus

in a biphasic

than

116

AF-DX

concentrations thiadiazole

corresponding

muscarinic

for

the Mf

(2-4)

activity

PI turnover

here

to the nitrogen-sulfur

reduces activity

inhibiting

heterocycles

that

PI turnover

bonds

is more stimulated

substitution

in which

there

tissues

(15,16)

of the

is lowered

in 1 and/or

of the compounds.

has been reported

in peripheral

stimulate

(Mt/M4-selective)

We report

of some of these compounds receptor

The compounds

pirenzepine

(M2/Mqselective)

of hexyloxy-TZTP.

hydrogen-bonds,

evaluation

manner;

ring of 1 with other

in the bonds form

in the CNS (1,2).

in the potent by low 1,2,5polarity

lack of ability to Pharmacological

using functional

measures

of

(Scheme 1).

Methods Materials The drugs used in the present study were obtained from the following sources: Alkoxy-1,2,5-thiadiazole and other derivatives of arecoline, Eli Lilly, Indianapolis, In. and Novo Nordisk, Denmark (15,16); carbachol, atropine sulfate, and all other chemicals, Sigma Chemical Co., St. Louis, MO. Dowex AGl-X8 (100-200 mesh, formate form) ion-exchange resin was purchased from BioRad, Rockville Centre, N.Y. Myo-[23H]-inositol (15 Ci/mmol) was obtained from American Radiolabeled Chemicals, Inc. St. Louis, Mo. Young male Long-Evans rats were purchased from Harlan-Sprague Dawley, Indianapolis, In. Phosphoinositide Turnover The methods were modifications of those described by Brown et al., (3) and by Berridge et al., (4). Young adult male rats were sacrificed by cervical dislocation, and their brains were removed and placed in Krebs-Hensleit buffer (KHB) (NaCI, 118 mM; KCI 4.7 mM; CaC12 1.3 mM; K2HP04, 1.2 mM; Mg SO4, 1.2 mM; NaHC03, 25 mM; and

1390

Vol.

187,

No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

glucose 11.7 mM) which had been previously equilibrated with 95% C2/5% CO2 to a final pH of 7.4 at room temperature. The brain region to be examined was dissected according to the method of Glowinski and lversen (5). The tissue was cross chopped at 300 urn on a Mcllwain tissue chopper at room temperature. The tissue slices from three rats were resuspended in 40 ml of gassed (95% 02/5% C02) KHB and incubated at 37°C in a shaking water bath (60 rpm) for 45 min. The buffer was replaced with warm gassed KHB at 15 min intervals. At the end of incubation, the slices were transferred to a graduated conical centrifuge tube and centrifuged at 300 x g for 15 set at room temperature. Aliquots (25 ul) of tissue slices were pipetted into flat-bottomed plastic vials (5 ml capacity) containing approximately 0.3 mM [3H]-lnositol (15 Cilmmole) and 10 mM LiCl in 245 ul of buffer. The vials were then gassed, capped and incubated at 37°C in a shaking water bath (60 rpm) for 30 min at which time agonist (or buffer for the determination of basal levels) then was added (30 ul), and incubation was continued for a further 45 min. The 45 min incubation period was selected on the basis of the time course of labelled inositol phosphates accumulation under these conditions. The incubations were stopped by the addition of 0.94 ml of CHCl3/MeOH (1:2, V/V) followed by 0.31 ml of CHCl3 and 0.31 ml of H20. The samples were vortexed and spun at 1000 x g for 10 min to separate the phases. Aliquots (750 ul) of the upper aqueous phase were removed for determination of [3H]-inositol phosphates ([3H]-IPs). In some cases 200 ul aliquots of the organic phase were removed, dried overnight and counted in 5 ml of scintillation cocktail for determination of [3H]-inositol incorporation into phospholipids. Immediately before each experiment, [3H]-inositol was purified by drying under Np and passing through a 1 ml column of Dowex AGl-X8 (formate form) to remove contaminants. The amount of [3H]-IPs formed in the assay was determined essentially according to Wreggett and Irvine (6) except that the separation of inositol phosphates was carried out by using an Amersham Super Separator Manifold. Total [3H]-IPs were determined by the “batch” method in which 750 ul aliquots of the aqueous phase obtained as described above were diluted to 3 ml with distilled H20. The entire sample was loaded onto the ACCELL QMA anion-exchange SEP-PAK (Waters Associates) cartridge, which had been converted previously into its formate form by washing first with 10 ml of a solution of 1 .O M ammonium formate in 0.1 M formic acid, followed by 20 ml of distilled water. The cartridge was then washed with 10 ml of distilled water, followed by 5 mM-disodium tetraborate. Radiolabelled inositol phosphates were eluted then with 1 ml of 0.6 M ammonium formate/0.06 M formic acid/5mM disodium tetraborate (pH 4.75) and 0.50 ml of this eluate was counted in 5 ml of liquiscint scintillation cocktail. Molecular Calculations Conformational minima for protonated arecoline, 1 and related compounds were obtained from W.C. Still’s program MacroModel (Version 2.0) using the modified MM2 (85) forcefield implemented in the program running on a Vax 6220 minicomputer (7). Minimum energy conformers were calculated using the multiconformer submode by rotation about the carbon-carbon bond linking the carbomethoxy group to the rings as well as selected carbon-carbon bonds in the rings in 30” increments over the full 360” circle, minimizing the resulting structures to a final RMS gradient of co.005 kJ/A. MacroModel files generated from conformational minima were used as input files for M.J. Dewar’s MNDO methodology (8) implemented on the program MOPAC. Refined geometries, charge densities and other molecular parameters were generated within MOPAC.

Results Molecular

Calculations

Results from MNDO level calculations that the 1,2,5-thiadiazole

implemented

ring in la (as well as related 1391

on the program

MOPAC

indicated

compounds)

is highly

polarized

Vol.

187, No. 3, 1992 Table

1.

BIOCHEMICAL

Charge

AND

BIOPHYSICAL

RESEARCH

distributions

in bioisosteric

A=N; X=S)

Atomic Charges Buryl-O-C=A ______ X ______ A ____ -0.399 0.560 -0.328

Comwund

derivatives

COMMUNICATIONS

of arecolinea Volume (IQ

la

(1,2,5thiadiazole;

2a

(1,2,.5-oxadiazole; A=N; X=0)

-0.123

3a

(thiophene;

A=C; X=S)

-0.333 0.418

-0.218

82.0

4a

(pyrazine;

A=N; X=C=C)

-0.254 0.0879 -0.157 -0.0428

84.1

70.8

0.0658 -0.0595

63.1

a MNDO calculations were performed on butoxy compounds in all cases.

with the S bearing (data

not shown)

distribution

a partial atoms

bearing

in the thiophene

ring of 2a nor the pyrazine With

positive

respect

oxadiazole

to size,

Nonbonded 1,2,5-thiadiazole the pyrazine hydrogen-bond

negative

the

pairs

1,2,5-thiadiazole than the thiophene volumes

(see Table

but not in the

1).

ring

in 1 is larger

than

the

ring in 3 or the pyrazine

are available

for hydrogen-bonding

1,2,5-

ring in 4.

as acceptors

ring of 1, and to a lesser extent in the 1,2,.5-oxadiazole acceptors

The charge

1,2,5-oxadiazole

of these ring systems.

ring of 4, but not the thiophene

oxygens can be acceptors.

ring of 3. The alkoxy oxygens

in each of the compounds

l-4 and also arecoline

in the

ring of 2 and can serve as in which both

Because of the greater negative charges on the nitrogens

Pigand Fiaure

charges

similar

and the alkoxy oxygen

ring of 4a.

the molecular electron

partial

and both nitrogen

ring of 3a was

ring in 2 but smaller

Table I compares

charge

of the

(PM)

1. Stimulation of phosphoinositide turnover by several arecoline derivatives in the rat hippocampus. Data represent the mean (t s.e.m.) from three experiments, each performed in triplicate. Two concentrations (10 and 50 uM) are shown for the 1,2$thiadiazole (1 b), the 1,2,5-oxadiazole (2b), the thiophene (3b) and the pyrazine (4b) with the reference compound carbachol at 100 PM.

1392

Vol.

187,

BIOCHEMICAL

No. 3, 1992

1,2,5thiadiazole

of 1 compared

1,2,5thiadiazole

ring would

Phosphoinositide

turnover

The compounds turnover

AND BIOPHYSICAL

with the 1,2,5-oxadiazole

be better

hydrogen-bond

from the hippocampus

1, 2b, 3b and 4b are all weaker

of 2, the nitrogens

in the

acceptors.

1 b, 2b. 3b and 4b were compared

in slices prepared

RESEARCH COMMUNICATIONS

for their abilities

of the rat brain.

than 1 b at stimulating

to stimulate

As shown

the PI turnover

PI

in Figure

response

in the

hippocampus.

Discussion The

PI turnover

10,ll).

response

In addition,

hippocampus, The results

m3 and m5 receptors,

have been shown of the present

the nitrogens turnover

response

PI turnover

receptors

of which when

for activity at Mt receptors that the bond polarities

Similar

findings

of quinuclidine

both smaller

and larger heterocyclic

There

evidence

is recent

the mRNA’s

to stimulate

of 3b suggests

of 1 b (1,2).

derivatives

with Ml

(13).

have

that two conserved

they conceivably

it has been

in the thiadiazole

shown

ability of

The lower PI

since

activities. that conserved

aspartate

segment of the rat It is of interest in

(109 and 112) are in positions donors for muscarinic

that the hydrogen-bond

ring of 1 is important

much

of 1,2,4-

may also be important

mutagenesis

residues

could serve as hydrogen-bond

In conclusion, nitrogens

serine

in cell lines.

acceptor

in studies

residues (99 and 105) within the third putative membrane spanning Ml muscarinic receptor are important for receptor binding (14). this regard

in the

alone do not contribute

Steric factors

from site-directed

are present

in the CNS.

been obtained

rings have reduced

in the CNS (9,

expressed

study show clearly that the hydrogen-bond

in 1 are important

to the activity oxadiazole

has been associated

where

ligands.

acceptor

ability

of the

for its activity at Mt receptors

in the

CNS. It is suggested that one or both nitrogens form hydrogen-bonds with serine hydroxyl groups near aspartate 105, which is known to be important for binding at Mt receptors. Acknowledgments The authors

would

Bymaster

for their

Research

Laboratories

like to express

helpful

discussions

their gratitude

to Harlan E. Shannon

and Frank P.

of the data.

This work was supported

and by NIH grants NS 23929,

NS 25765 and NS 01493.

by Lilly

References 1.

Sauerberg, P., Olesen, P.H., Nielsen, S., Sheardown, M.J., Honor& T., Mitch, C.. Ward, J., Pike, A., Bymaster. F.P., Sawyer, B., Shannon, H.E. (1992) J. Med. Chem., 35, 2274-2283. 1393

Vol.

187, No. 3, 1992 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Periyasamy, S., Messer, W.S., Jr., Rocknich, S., Sauerberg, P. and Hoss, W., (1992), submitted for publication. Brown, E., Kendall, D.A. and Nahorski, S.R., (1984) J. Neurochem., 42, 13791387. Berridge, M.J., Downes, C.P. and Hanley, M.R., (1982) Biochem. J., 206, 587595. Glowinski, J. and Iversen, L.L., (1966) J. Neurochem., 13, 655-669. Wreggett, K.A. and Irvine, RF., (1987) Biochem. J., 245, 655660. Lipton, M. and Still, W.C., (1988) J. Comp. Chem., 9, 343-355. Dewar, M.J.S. and Thiel, W., (1977) J. Am. Chem. Sot., 99, 4899. Gil, D.W. and Wolfe, B.B., (1985) J. Pharmacol. Exp. Therap., 232, 608-616. Fisher, S.K. and Snider, R.M., (1987) Molec. Pharmacol., 32, 81-90. Fisher, S.K. and Agranoff, B.W., (1987) J. Neurochem., 48, 999-1017. Orlek, B.S., Blaney, F.E., Brown, F., Clark, M.S.G., Hadley, M.S., Hatcher, J., Riley, G.J., Rosenberg, H.E., Wadsworth, H.J., and Wyman, P., (1991) J. Med. Chem., 34, 2726-2735. Saunders, J., Cassidy, M., Freedman, S.B., Harley, E.A., Iversen, L.L., Kneen, C., MacLeod, A.M., Merchant, K.J., Snow, R.J. and Baker, R., (1990) J. Med. Chem., 33, 1128-I 138. Fraser, C.M., Wang, CD., Robinson, D.A., Gocayne, J.D. and Venter, J.C., (1989) Mol. Pharmacol., 36, 840-847. Sauerberg, P., Olesen, P.H., Suzdak, P.D., Sheardown, M.J., Mitch, C.H., Quimby, S.J., Ward, J.S., Bymaster, F.P., Sawyer, B.D. and Shannon, H.E. (1992) Bioorganic and Med. Chem. Lett., in press. Ward, J.S., Merritt, L., Klimkowski, V.J., Lamb, M.L., Mitch, C.H., Bymaster, F.P., Sawyer, B.D., Shannon, H.E., Olesen, P.H., Honore, T., Sheardown, M.J. and Sauerberg, P. (1992), submitted for publication.

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