AU599610B2 - Derivatives of quinuclidine - Google Patents

Description

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FORM 10 6 SPRUSON FERGUSON COMMONWEALTH OF AUSTRALI T ,i dxZ PATENTS ACT 1952 COMPLETE SPECIFICATION k, j (ORIGINAL) I FOR OFFICE USE: Class Int. Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: 0 00 0( 0 0 0 0 t 00 4if 0 o *1 o 41i 00 4 0 O 44 0ats Name of Applicant: STATE OF ISRAEL, REPRESENTED BY THE PRIME MINISTER'S OFFICE Address of Applicant: The Israel Institute for Biological Research, Ness-Ziona, Israel Actual Inventor(s): ABRAHAM FISHER, ISHAI KARTON, ELIAHU HELDMAN, AHARON LEVY and YONA GRUNFELD Address for Service: Spruson Ferguson, Patent Attorneys, Level 33 St Martins Tower, 31 Market Street, Sydney, New South Wales, 2000, Australia Complete Specification for the Invention entitled: "DERIVATIVES OF QUINUCLIDINE" The following statement is a full description of this invention, including the best method of performing it known to us SBR/JS/0042T .4

ABSTRACT

Quinuclidine derivatives having the gen~eral formula (I) and geometrical isomers, enantiomers, diastereoisomers, racemates and/or acid addition salts therei~f, whe~rein Z represents the group 1 2 1 2 CR R or two hydrogen atoms; and R and R which may be identical or different, are each alkyl cyclopentyl cyclohexyl aryl or diarylmethylol or alkyl which is substituted by one or more aryl 1 2 groups, or one of R and R may be hydrogen.

9,006 0 0 0'4 0 0 V p 0044 0 4

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FIELD OF THE INVENTION The present invention relates to novel spiro(1,3oxathiolane-5,3')quinuclidines and a novel hydroxymercaptomethylquinuclidine; processes for preparing the novel compounds; pharmaceutical compositions containing the spiro-compounds; and a method for treating diseases of the central nervous system using such spiro-compounds or pharmaceutical compositions.

BACKGROUND OF THE INVENTION o US Patent No. 4,083,985 describes a range of fused-ring 08 0 quinuclidines which are described as psychomotor stimulators, and °o which may be regarded structurally as a quinuclidine nucleus fused to a cyclohexanone, cyclohexenone or delta-lactone moiety.

These compounds are stated to be useful for treating (inter alia) Parkinsons' Disease and depression, and evidently possess anticholinergic activity. There is no indication in this Patent S" that any of these compounds possess cholinergic activity.

o a US Patent No. 4,104,397 describes spiro(1,3-dioxolane- 4,3')quinuclidines which may have one or two alkyl and/or aryl *Sto substituents in the 2-position of the dioxolane ring. The Patent specifically describes the monomethyl, dimethyl and diphenyl compounds. The monomethyl compound is shown to have cholinergic activity and the diphenyl compound to have anticholinergic activity. The nature of the pharmacological activity exhibited ~i rr; iri--r.~x- by the other compounds embraced by this Patent is not described therein.

A chronic deficiency in vivo in central cholinergic function, that is to say in the function of acetylcholine as a neurotransmitter, has been implicated in a variety of neurologic and psychiatric disorders, including senile dementia of Alzheimer's type(SDAT), tardive dyskinesia, Pick's disease, Huntington's chorea, Gilles de la Tourette disease, Friedrich's ataxia, and Down's syndrome. Clinical data indicate that 0 cholinergic transmission may have been compromised in persons affected with these diseases (Fisher and Hlanin, Life Sciences, 27: 1615, 1980).

0000 0 Among these disorders, SDAT is the most widespread eo o o neuropsychiatric disease (for reviews see Schneck et al, Am. J.

o Psychiatry, 139: 165, 1982 and Coyle et al, Science 219: 1184, 0, 1983). The development of an effective treatment for SDAT is one of the most pressing needs facing medicine today. This age- So related disease is becoming increasingly prevalent as the S\o° population of the elderly grows in line with the progressively S 0 higher life expectancy of the older population.

SSDAT is characterized morphologically by an increased number of senile plaques in selected brain areas; biochemiqally by a significant reduction in presynaptic cholinergic markers in the saie brain areas, the cortex and the hippoompus in particular; and behaviorally by a loss of cognitive functions in individual patients.

i, i 4' Since SDAT appears to be associated with brain cholinergic hypofunction, trials have been conducted in which ACh precursors (choline or lecithin), acetylcholinesterase inhibitors (physostigmine or tetrahydroaminoacridine) or direct acting muscarinic agonists (arecoline) have been administered to SDAT patients because of the ability of these agents to elevate, and thus presumably restore cholinergic activity in the brain.

To date, the results have not been conclusive as to the efficacy of treatment with the above-mentioned agents; this is due mainly to to unwanted side-effects, narrow therapeutic window, or lack of therapeutic efficacy.

o oi S~ Su S 04~ aI S So 04*4 4 S 5*4 4 44L 4* 4 .4*4I t 4 41 44 t a~l There is an urgent need for drugs which are effective in the treatment of SDAT. Progress in this area has been hindered by the lack of adequate animal models that can mimic directly the cholinergic abnormality implicated in SDAT, and by a dearth of long- acting central cholinergic agonists which can discriminate among subclasses of receptors, and primarily activate those that are involved in cognitive functions. Most known cholinergic agonists (muscarinic drugs) have undesirable side-effects. A long-lasting, centrally active cholinomimetic drug without peripheral side effects would therefore be most useful. The R D of such drugs would require their evaluation in suitable animal models for SDAT.

In this context, we have recently developed a selective presynaptic cholinergic neurotoxin, ethyloholine aziridinium ion (AF64A), which on intracerebroventricular injection in rats 3

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induces persistent cholinergic hypofunction that mimics the cortical and hippocampal cholinergic deficiency and the cognitive impairments reported in SDAT. This animal model could be extremely useful in developing novel treatment approaches for SDAT. (Fisher et al in Behavorial Models and the Analysis of Drug Action, eds. Spiegelstein and Levy, Elsevier, Amsterdam, 1983, p. 333; Fisher and Hanin, Ann. Rev. Pharmacol. Toxicol., 26: 161-81 (1986).

The availability of centrally active muscarinic to compounds which have long acting central cholinergic activity without significant peripheral adverse side-effects capable of reversing cognitive impairments induced by AF64A in rats, could o4o be extremely useful in treating SDAT and the above-mentioned 0o 0 S <t related disease states.

S0 a o o4o Therapeutically active oxathiolane compounds and their pharmacology are relatively little known. Moreover, the literature is replete with unsuccessful attempts to replace a 4 oa 0 o particular atom or group in a pharmacologically active chemical o 0o compound by a supposedly analogous atom or group, in an attempt o, to improve the therapeutic profile of the original compound.

Thus, in replacing for example an oxygen atom by a sulfur atom 0 a which has twice the atomic mass of oxygen, the result in pharmacological terms cannot be predicted with any degree of certainty.

However, we have now surprisingly discovered, and this discovery forms the basis of the present invention, that if in 4 jl I I_ 1 I I the spiro(dioxolane)quinuclidines of US Patent No. 4,104, 39 7 the oxygen atom of the dioxolane ring which is more remote from the quinuclidine nucleus is replaced by a sulfur atom, and at the same time the ambit of the substituents at the 2-position is extended to include diarylmethylol, and alkyl substituted by aryl, then the most active isomer of the monomethyl compound, while possessing a not l i ssimilar activity (measured by the Guinea-pig ileum induced contraction and muscarinic receptor binding tests) as compared with that of the most active isomer to of the analogue disclosed in the aforementioned US Patent, however exhibits significantly less pronounced side-effects (sialogenic and tremorigenic activity) than the latter compound under similar conditions; and (ii) the most active isomer of the o0 SCo monomethyl compound has interesting potential for the treatment o of SDAT as shown by the tests on animal models mentioned above.

O On the other hand, the vast majority of the other 2o o substituted members of the series containing the sulfur atom in place of oxygen as aforesaid, and in particular those members containing at least one 2-substituent which is alkyl having three o0 or more carbon atoms, cyclopentyl, cyclohexyl, aryl, Sdiarylmethylol or alkyl substituted by aryl, have anticholinergic |t activity, as contrasted with the cholinergic activity of the monomethyl compound.

I t 1 SUMMARY OF THE INVENTION The invention accordingly provides derivatives having the general formula (I) quinuclidine o-Z and geometrical isomers, enantiomers, diastereoisomers, racemates and/or acid addition salts thereof, wherein Z 1 2 1 represents the group >CR R or two hydrogen atoms; and R and 2 R which may be identical or different, are each alkyl, cyclopentyl, cyclohexyl, aryl, or diarylmethylol, or alkyl which 1 2 is substituted by one or more aryl groups, or one of R and R may be hydrogen.

In accordance with one embodiment of the invention, Z 12 1 2 in formula represents the group >CR R one of R and R is 1 2 hydrogen, and the other of R and R is alkyl, cyclopentyl, cyclohexyl, aryl, or diarylmethylol, or alkyl which is substituted by one or more aryl groups.

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.0 r SIn accordance with a further embodiment of the 1 2 invention, Z in formula represents the group >CR R one of 1 2 1 R and R is alkyl, cyclopentyl or cyclohexyl, and the other of R 2 ako and R is alkyl, oyclopentyl, cyclohexyl, aryl, or diarylmethylol, or alkyl which is substituted by one or more aryl groups.

In accordance with still a further embodiment of the 1 2 invention, Z in formula represents the group >CR R one of 1 I R and R is aryl, and the other of R and R is aryl or diarylmethylol, or alkyl which is substituted by one or more aryl groups.

1 2 When Z in formula represents the group >CR R the compounds thus defined have central nervous system activity.

These compounds may be named as 2,2-disubstituted spiro(1,3oxathiolane-5,3')quinuclidines. Non-limiting examples of such compounds are described in the following table: to 9a 9 o be 1 I~ .4 Dw99 0 o o 00 o 94) a 9 9 04 4 4 9 4e 4944 9~f 1 2 one of R and R hydrogen hydrogen hydrogen hydrogen hydrogen hydrogen hydrogen methyl ethyl cyclohexyl phenyl 1 2 the other of R and R methyl ethyl propyl phenyl 1-pyrenepropyl diphenylmethyl diphenylmethylol phenyl phenyl phenyl phenyl (Ia) (Ib) (Ic) The invention also includes the compound of formula (I) wherein Z represents two hydrogen atoms, i.e. 3-hydroxy-3mercaptomethylquinuclidine, from which the compounds of the 1 2 invention in which Z in formula represents the group >CR R may be prepared.

As previously indicated, the invention includes the geometrical isomers, enantiomers, diastereoisomers, racemates and/or acid addition salts, of the compounds of formula rt arises from will be appreciated that geometrical isomerism the fact that in the spiro-eompounds of the xrr r-rLnrrr~-ilr-lr~-rlu-

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invention, R for example, may be either on the same side of the oxathiolane ring as the nitrogen atom of the quinuclidine ring, or on the opposite side. If, in the spiro-compounds of the 1 2 invention, R and R are identical, then there will be a single centre of asymmetry at the 5,3'(spiro) carbon atom, this will also give rise to enantiomers and the racemates thereof. If, on 1 2 the other hand, R and R in the spito-compounds are not identical, then there will be a further centre of asymmetry at the 2-position of the oxathiolane ring, thus giving rise to diastereoisomers and the racemates thereof, in addition to the geometrical isomers already mentioned. It will moreover be appreciated, that in the compound of the present invention which is 3-hydroxy-3-mercaptomethylquinuclidine, there exists no possibility of geometrical isomerism; here also, however, there 9006 is a centre of asymmetry at the 3-position of the quinuclidine 0 0 ring, thus giving rise to enantiomeric and racemic forms.

00 0 0 O 000 The compounds of formula whether as isomeric 0 to mixtures or compounds, or whether as individually isolated geometric or optical isomers, form stable addition salts with I organic or inorganic acids, as for example with hydrochloric S acid. It will be observed that, while for therapeutic application such salts should be pharmaceutically compatible, nevertheless it may be convenient, as for example for purposes of isolation, to employ acid addition salts which are not pharmaceutically compatible, and the invention relates also to the acid addition salts of the latter kind. As will be obvious to those skilled in the art, if the compounds are obtained e.g.

as a result of isolation from their process of preparation in the form of free bases, they may be converted to the acid addition salts by r-action with the appropriate acid, and conversely, the compounds isolated in the form of their acid addition salts may be converted by reaction with a base, such as an alkali metal hydroxide, to the corresponding free bases.

Geometrical isomers are generally isolated by a physical method such as fractional crystallization (of the isomers per se, or of their salts), fractional distillation or to column chromatography (using high or low pressure liquid chromatography techniques) while optical isomers are isolated by forming a salt mixture with an optically active complementary reagent(in the present case an optically active acid), followed by fractionation of the mixture, and isolation of the desired S optical isomers from the salt fractions.

o0 4 Q I oa" In a particular embodiment, the inventior provides the isolated geometrical isomers of the compound wherein one of R 2 1 2 S and R is hydrogen and the other of R and R is methyl(Ia).

These isomers may be differentiated from each other by the fact S that their salts with hydrochloric acid have quite distinct relatively higher and relatively lower melting-points. The hydrochloric acid salt of the mixture of geometrical isomers of S compound (Ia) Which is isolated from the preparative process of the invention, as well as the hydrochloric acid salts of the individual geometrical isomers are also included within the scope of the invention.

9 In accordance with the invention, the compounds of 1 2 formula wherein Z represents the group >CR R are prepared by a process which comprises reacting 3-hydroxy-3mercaptomethylquinuclidine with a carbonyl compound of formula 1 2 R CO-R and isolating the desired product from the reaction mixture. The process is desirably carried out in the presence of an acid catalyst, such as a Lewis acid, as for example boron trifluoride, which compound may be conveniently used in the form of its complex with diethyl ether, otherwise known as boron o1 trifluoride etherate.

The process is also preferably carried out in presence of an inert organic solvent medium, as for example, chloroform or dichloromethane. The temperature at which the proceiss of the a invention is effected is not critical, but it will evidently be o:S advantageous to use as low a temperature as is consistent with a reasonable yield, in order to avoid contamination of the desired 0o9, product with byproducts which could result from decomposition b and/or side reactions at higher temperatures. It is found that when conducting the process of the invention in presence or a *o catalyst such as boron brlfluoride otherate, a temperature within 0

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i the range of about 20 to 30 C is suitable, buit higher or lower |l temperatures may of course be used; a reacticn temperature of about 25 C is preferred. In order also to avoi undesirable contamination due to oxidation, it is preferable to conduct the reaction in ar atmosphere of inert gas suoh as Mtciosen, In a prtbioular embodiment of 6' erefore, a process for preparing the compounds of formula wherein Z 1 2 represents the group >CR R comprises reacting 3-hydroxy-3mereaptopropylquinuclidine with a carbonyl compound of formula 1 2 R -CO-R in an atmosphere of nitrogen, at a temperature in the o o range of about 20 to about 30 C, preferably at about 25 C, in the presence of boron trifluoride etherate as catalyst and in dichloromethane or/and chloroform as the solvent medium, and isolating the desired product from the reaction mixture.

In this particular embodiment of the process, it is O preferred that the reaction ingredients are first mixed in an atmosphere of nitrogen at a temperature between about -10 and 0 0 C, e.g. at about 0 C and the mixture thus obtained is permitted to rise to the reaction temperature.

The invention further provides a process for preparing the compound of formula which is 3-hydroxy-3oQ. mercaptomethylquinuclidine, wherein the epoxide of 3methylnequinuclidinQ is reacted with hydrogen sulfide; in one 0 0* alternative this reaction may be carried out in the presence of a base such as sodium hydroxide, preferably in an aqueous medium, 'Zo while in another alternative the reaction with hydrogen sulfide may be effected e.g. in DMSO (chloroform and/or toluene) as o" solvent medium. The epoxide may itself be prepared by reacting S quinuclidin-3-one with dimethylsulfoxonium methylide.

The preparative processes of the present invention, including the processes by which relevant starting materials are prepared, are illustrated in a preferred embodiment, in the 11 following reaction sch me: c H -S >G 3 I Ila H DM SO C U-1 13 CH= S >0 21 C U H r NaI 44o 4 04 4 4ro 4 4o a0 4 0 0o 4s 4 0440 4"40I 4 01*~ 44 0 044 4 44 c 4' 4' 04' 4 4 4' 4' 44' r *444'

CH

13 CU11= S >o 2 cU- DMS0 c 7 "o M.,,S3

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aq. NaOH or slvent*a 0c:- Q54'1- S lq C? boron 1 2 trifluoride- R -C R etherate CH C1 or/and CHCi 22 3

S

(I)

(DMSO =dime thylsulfoxide) *DlMSO (chloroform and/or toluene) REACTION SCHEME 12 k,

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The spiro-compounds of formula have central nervous system activity. Thus, for example, the compound (Ia) is a muscarinic agonist with a high specificity for the central nervous system. Due to its pharmacological properties, it can be used to activate central cholinergic functions under conditions where the cholinergic system is hypofunctional. It can accordingly be utilized for the treatment of conditions such as senile dementia of Alzheimer's type(SDAT), tardive dyskinesia, Pick's disease, Huntington's chorea, Gilles de la Tourette o disease, Friedrich's ataxia, and Down's syndrome, because all of these are disturbances in which a central cholinergic hypofunction has been implicated to some extent. This compound appears to be of especial value for the treatment of SDAT, since it is effective in reversing memory disorders due to AF64A- Sinduced cholinotoxicity in a suitable animal model for this o disease. In particular, the geometrical isomer of compound (Ia), of which the HC1 salt has the relatively lower melting-point (the cis-isomer), and which has been assigned the code number AF102B, reverses m~o~ impairments in AF6'4A-treated rats as shown in a passive avoidance test, in the Morris Swimming Maze (see Morris, Learning and Motivation, 12: 239-61, 1978) and in the 8-Arm Radial Maze. [The other geometrical isomer of the compound oi which the HC1 salt has the relatively higher melting-point (the trans-isomer, has been assigned the code number AF102A]. In this context, AF64A (3 nmol/2ul/side, icy) induces marked cognitive impairments in a tep-through passive avoidance test, in the Morris Swimming Maze and in the 8-Arm ii Radial Maze, when the rats are analyzed four to eight weeks following treatment (Brandeis et al, in Alzheimer's and Parkinson's Disease: Strategies in Research and Development, eds.

Fisher et al, Plenum Press, New York, in press; Fisher and Hanin, Ann. Rev. Pharmacol. Toxicol., 26: 161-81 (1986).

The beneficial effects of AF102B in the passive avoidance and Morris Swimming Maze tests occur at low doses (0.1 mg./kg., ip or 1 mg./kg., po) and the therapeutic index is 78-780 and >156, respectively. This therapeutic index is wider (o than that found for physostigmine Moreover, the slope of the acute toxicity curve is very steep, and no overt behavioral effects including parasympathomimetic effects, such as salivation or tremors, were detected up to the lethal dose. In this regard, the compound is superior to well known muscarinic agonists Sincluding arecoline and oxotremorine, in which such adverse sideeffects complicate their possible use in SDAT therapy. In addition, AF102B has a long duration of a.ction in the above- *!oo mentioned memory tests.

Interestingly, the compound is well absorbed into the Zlq4 blood following per os administration since its onset of action is short, i.e. around 10-15 minutes. This is evident in the S following pharmacological tests: analgesia, hypothermia, and n t

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lethal dose level, induced by AF102B.

44 When compared to physostigmine (0.1mg./kg.,ip), AF102B is superior since it improves AF64A-induced memory impairments in the 8-Arm Radial Maze, a test in which 14 physostigmine failed under the same experimental conditions.

The lack of adverse side effects induced by AF102B is remarkable also in analgesia tests (in mice) and hypothermia tests (in rats), two pharmacological tests employed to evaluate central muscarinic activity. In these tests only high doses of the compound elicited any significant side effects. Such doses are at least 15-150 or 40 times higher than those required to reverse AF64A-induced memory impairments in rats in the passive avoidance test.

to Biochemical studies revealed that AF102B is a specific centrally active muscarinic Mi-type agonist and is the first such compound known to the inventors. This selectivity became 3 evident when AF102B was evaluated in the displacement of H]- PO 3 3 pirenzepine, H]-PNZ, (an M1-specific antagonist) versus L H]- 0 .d 3 quiniclidinyl benzilate, QNB (an M1 and M2 antagonist) from Srat brain homogenates. [Trends in Pharmacol. Sci.(Suppl), Jan 1984].

4 4 0 In this regard, it was found that M1-type muscarinic Sreceptors found mainly in the cortex and hippocampus are *ao relatively unchanged in SDAT (Mash et al, Science 228:115- S 117, 1985). These two brain areas in SDAT show the most Sprofound presynoptic cholinergic hypofunctions, and histological abnormalities, and are mainly associated with cognitive dysfunctions reported in SDAT.

The specificity of AF102B to muscarinic receptors in

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I I individual patients.

2 The M-agonist type of activity of AF102 can be general and Mi-receptors in particular, is also evident from lack of any significant activity on high-affinity choline transport (from rat brain synoptosomnes) or choline acetyltranferase activity (from rat brain homogenates) The M1-agonist type of activity of AF102B can be responsible at least in part for the high selectivity of AF102B both in vitro and in vivo (especially in reversal of cognitive impairments induced by AF64A).

Mutagenicity studies on AF102B in vitro have revealed S that the compound is not mutagenic up to high concentrations.

These data taken together with the large therapeutic index of this compound make it a potential drug for treatment of SDAT patients, o 0o In SDAT patients, AF102B can be used in combination oo with anticholinesterase inhibitors such as physostigmine or o e0 tetrahydroaminoacridine; in combination with acetylcholine 0,0 precursors such as choline or lecithin; in addition to "nootropic" drugs such as piracetam, aniracetam, oxiracetam or pramiracetam; in addition to compounds that interact with 2+ Ca channels such as 41-aminopyridine or 3,4-diaminopyridine; or in addition to peptides that can have modulatory effects on acetylcholine release, such as somatostatin.

'4 tAF102B, with or without the aforementioned other active substances, can be administered for example, by way of injection in a suitable diluent or carrier, per os, rectally in the form of suppositories, by way of insufflation, by infusion or

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transdermally in a suitable vehicle with or without physostigmine, for example by using the device which is the subject of Israel Patent Application No. 72684 (vide infra).

This compound may also be used in disturbances where cholinergic underactivity is induced by drugs.

The compound in the form of either geometrical isomer, or a mixture of such isomers, is also of use for the treatment of disorders requiring the application of a longlasting cholinergic agent of mild local activity. Such an agent is needed in disorders such as glaucoma, as the compound is not destroyed by the enzyme which deactivates acetylcholine, i.e.

acetyl- and butyryl-cholinesterase. This compound may also be used for the treatment of peripheral cholinergic disorders such 0 as myasthenia gravis, urinary bladder dysfunctions, Adi's disease I 4 S* and Eaton-Lambret disease. and Eao o 00, 0Q 0 rj0 04 0c 00 0000 ';rB 0 4 cc 0094c When in the spiro-compounds of formula R and/or 2 R are propyl or higher alkyl groups, cyclopentyl, cyclohexyl, aryl, diarylmethylol, or alkyl substituted by aryl, the nature of the pharmacological activity of these compounds is changing, insofar as while they still possess central nervous system activity, this activity is now becoming anticholinergic instead of cholinergic. Such anticholinergic compounds can be used to treat disorders due to cholinergic hyperfunction, whether this be spontaneous or drug-induced. These compounds may accordingly be used in the treatment of Parkinson's disease, pseudo- Parkinson's disease, mental depression and as adjuncts of surgery 17 insueau o ke.g.) acropine or scopolamine. iney may also De usea in ophthalmology when prolonged mydriasis is required for diagnostic and therapeutical purposes.

Where the term "pharmaceutical composition" is used in the specification and claims, this is to be understood in the sense that it may be suitable for human and/or veterinary treatment.

According to a further aspect of the invention, therefore, there is provided a pharmaceutical composition which comprises a quinuclidine derivative of formula wherein Z 1 2 represents the group >CR R or a pharmaceutically compatible acid addition salt thereof, together with an inert carrier or diluent. The term "pharmaceutically compatible acid addition salt" as used herein refers to a combination of said quinuclidine ,o derivative with relatively non-toxic inorganic or organic acids.

o 'o Illustrative only of suitable acids are sulfuric, phosphoric, hydrochloric, hydrobromic, hydriodic, sulfamic, methanesulfonic, 0 tQ benzenesulfonic, para-toluenesulfonic, acetic, lactic, succinic, maleic, tartaric, citric, gluconic, ascorbic, benzoic and cinnamic acids. Suitable pharmaceutical carriers and diluents, which comprise both solids and liquids, S may, by way of example only, be selected from corn starch, S lactose, calcium phosphate, stearic acid, polyethylene glycol, water, sesame seed oil, peanut oil, propylene glycol, and so forth. This composition may be in a form suitable for oral, rectal or parenteral administration, or for administration by insufflation, or in particular it may be in a form suitable for 18 -i transdermal administration, and in any event the composition may be in unit dosage form. Exemplary compositions may take the form of tablets, powder, granules, capsules, suspensions, solutions, suppositories, elixirs, ointments and the like.

The pharmaceutical composition may contain as the spiro-compound of formula for example, the compound identified herein- as and in particular the geometrical isomer(AF102B) of which the hydrochloric acid salt has the relatively lower melting-point. For the reasons noted hereinbefore, such a composition may contain as a further ingredient or ingredients, one or more compounds selected from the groun consisting of physostigmine, tetrahydroaminoacridine, choline, lecithin, piracetam, aniracetam, pramiracetam, oxiracetam, 4-aminopyridine, 3, 4 -diaminopyridine .and jo, somatostatin.

o Alternatively, the pharmaceutical composition may 0 contain as the spiro-compound of formula such a compound in 1 2 which one of R and R is selected from the group consisting of Q alkyl containing three or more carbon atoms, cyclopentyl, cyclohexyl, aryl and alkyl substituted by aryl, and the other of 1 2 R and R is as previously defined, and in particular this compound may be one of those identified herein as (Ib) and (Ic).

SWhen the pharmaceutical composition is to be administered transdermally, it is preferred to utilize the drug delivery system according to Israel Patent Application No. 72684, although transdermal administration in accordance with the 19

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invention is not of course limited to this system. Thus, there is also provided in accordance with the invention, a pharmaceutical composition for transdermal administration, which comprises a compound of formula wherein Z represents the 1 2 group >CR R or a pharmaceutically compatible acid addition salt thereof, as well as a low molecular weight fatty acid.

The invention also relates to a method for treating diseases of the central nervous system in mammals(i.e. in humans or in non-human mammals), which comprises administering to the 0 o mammal a compound of formula or a pharmaceutically compatible acid addition salt thereof, wherein Z represents the group 1 2 >CR R which compound may of course be administered in the form of the pharmaceutical composition described hereinbefore. This method for treating diseases of the central nervous system in o mammals may of course also be applied by using the drug delivery system of the Israel Patent Application which has been described So4 above.

SMore specifically, the method for treating diseases of

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the central nervous system in mammals, in accordance w;ith the Spresent invention, may be used for treating diseases due to a deficiency in the central cholinergic system in mammals, in which case the method comprises administering to the mammal the S compound including the geometrical isomers, enantiomers, diastereoisomers, racemates and/or acid addition salts thereof; St the compound (Ia) may be administered in the form of a pharmaceutical compositions, which may be optionally supported for transdermal administration in the form of the device of fl

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Israel Patent Application No. 72684, as has already been described hereinbefore.

In another aspect of the present invention, the method for treating diseases of the central nervous system in mammals may be used for treating diseases due to a cholinergic hyperfunction in mammals, in which case the method comprises administering to the mammal the spiro-compound of formula in 1 2 which one of R and R is selected from the group consisting of alkyl containing three or more carbon atoms, cyclopentyl, 1o S cyclohexyl, aryl and alkyl substituted by aryl, and the other of 1 2 R and R is as previously defined, as for example, either of the compounds (Ib) and including the geometrical isomers, enantiomers, diastereoisomers, racemates and/or acid addition S°C salts of such spiro-compounds of formula these compounds may a C 0o O be administered in the form of pharmaceutical compositions,

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0 which may be optionally supported for transdermal administration o, in the form of the device of Israel Patent Application No. 72684, «as has already been described hereinbefore.

So, In yet a further aspect of the present invention, the 4 t method for treating diseases of the central nervous system may be S6 used for treating senile dementia of Alzheimer's type in humans, 0 in which case the method comprises administering to a patient the geometrical isomer of compound the hydrochloric acid salt of which has the relatively lower melting-point, including the enantiomers, diastereoisomers, racemates and/or acid addition salts of this geometrical isomer; the geometrical isomer of the 21 1 LLIZ-I ~_J I4 compound (Ia) may be administered in the form of pharmaceutical compositions, which may be optionally supported for transdermal administration in the form of the device of Israel Patent Application No. 72684, as has already been described hereinbefore. Optionally, there may be administered in general, together with the aforementioned geometrical isomer of the compound one or more compounds selected from the group consisting of physostigmine, tetrahydroaminoacridine, choline, lecithin, piracetam, aniracetam, pramiracetam, oxiracetam, 4o0 aminopyridine, 3,4-diaminopyridine and somatostatin. In the case of transdermal administration, however, the additional ingredient is preferably one or more compounds selected from the group consisting of physostigmine, tetrahydroaminoacridine, 4aminopyridine and 3,4-diaminopyridine.

For the purpose of definition, it is intended that the expression "method for the treatment of diseases of the central S nervous system", and like expressions, throughout the specification and claims, be taken to include a method for the prevention of drug-induced diseases of the central nervous o system.

As regards suitable dosages for the administration of A the compounds of the invention, some indication has been obtained from the biological tests, the results and other details of which are recorded infra. It would at present appear that for administration per os it is unlikely that a single dosage of more than about 60mg./kg. or less than about 0.lmg./kg. body weight would be suitable, and that a single dosage in the range of about to about 10mg./kg., especially in the range of about 1 to about 5mg./kg., is to be preferred. For parenteral ad]inistration (which includes, for example, intramuscular, intravenous and subcutaneous administration) it at present appears to be unlikely that a single dosage of more than about or less than about 0.O1mg./kg. body weight would be suitable, and that a single dosage in the range of about 0.05 to about 5ng./kg., especially in the range of about 0.1 to about 2mg./kg., is to be preferred. In prescribing a particular form So and rate of administration, the physician will of course take into consideration the usual factors such as the severity of the symptoms, the physical circumstances of the patient, and so forth.

S, Taking into account the usual weight ranges of S patients, the foregoing dosage ranges, and the possiblity that it may be desirable to administer multiple rather than single doses, pharmaceutical compositions in accordance with the invention S4 tI which are adapted for oral or parenteral, administration, may contain the active ingredient (and especially the compound

&LI

P.Q identified herein by the code number AF 102B), for example, in an ti amount in the range of about 0.5 to about 500 mg., preferably about 5 to about 100 mg., more preferably in the range of about to about 50 mg.

444% S BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the NMR(250 MHz) spectrum of 3-hydroxy-3mercaptomethyiquinuclidine.

Fig. 2 shows the NMR(250 MHz) spectrum 0? cis:trans (Ia)-HCl salt [AF1O2J.

Fig. 3 shows the NMR (250 MHz) spectrum of the geometrical isomer of (Ia)-HCl salt identified as AF1O2A.

Fig. 14 shows the NMR(250 MHz) spectrum of the geometrical isomer of (Ia)-HCl salt identified as AF1O2B.

(0 Fig. 5 depicts the structure of AF1O2B, as determined by X-ray crystallography of its HCl salt, showing that AF102B is the cis-isomer.

Fig. 6 shows the IR spectrum (on Nicolet 20X13 FTIfI) of the geometrical isomer of (Ia)-HCl salt identified~ as AF1O2A.

Fig. 7 shows the IR spectrum of' the geometriLcal isomer of (Ia)-HCl salt identified as AF1O2B.

Fig. 8l shows initial and retention-test latency measures of AF611A and CSF-inJected groups, before and after physostigmine or saline.

Fig. 9 shows initial and retention-test latency measures of AF611A- and CSF-injected groups, before arnd after AF1028 or saline.

Fig. 10 shows retention-test latency measurements for (inter alia) AF102B in extinction trials.

Fig. 11 shows retention-test latency measurements for (inter alia) AF102B in "eitinction latent extinction" trials.

ii

I

Fig. 12 shows retention-test latency measures of AF64Aand CSF-injected groups, after second administration-of AF102B or saline.

Fig. 13 5hows retention-test latency measures of AF6'4Aand CSF-injected groups, af ter AF1O02B 1 mg. /kg.,I ip) or saline administrat ion.

Fig. 14 shows latency measures (sec.) Qf AF6IA- and CS1F-injected groups, before and af ter AF 1Q2B (1 mg po) or saline administr ')tion.

Fig. 15 shows retention test latency mneasures (sec.) of AF6'4A- and CSF-injected groups, before and after AF102B (1 mg.-/kg, po) or sal ine administration Fig. 16 shows escape latency measures, in blocks of two tr~tals, of AF6'IA- and CSF-injected groups, after physostigmine administration.

Fig. 17 shows escape latency measures, in bLocks of two trials, of AF64A-~ and CSF-lnjected groups, after AF102B (1 mg./kg., ip) administration, Fig. 18 shows averoge error in th 8-Arm Radial Maze (RAM) of AF6LIA- and CSF-injected rats, after AF1023 (5 mg./kg., ip) administration.

F i~ shows average error in the RAM of AF6 1 4A- and CSF-injected rats, after saline and physostigmine administ~r'ation.

9 04 99 9 4 0

Q

4 4 940494 9 099944 9 4 99 94 4 9494 L

V

DETAILED DESCRIPTION OF THE INVENTION The invention will now be described in further detail with regard to Examples for the preparation of the compounds of the invention, and biological testing of the spiro-compounds of the invention.

EXAMPLE I Preparation of 2-methylspiro(1,3-oxathiolane-5',3)quinuclidine.

[AF102 (cis: trans); AF102A and AF102B] Epoxide of 3-riethylenequinuclidine.

In a 31. three-necked flask fitted with a mechanical stirrer and thermometer, was placed sodium hydride (42g., 0.88 mole, as a 50% disoersion in oil) and 300ml.

o petroleum ether (30-60). The suspension was stirred, the Shydride allowed to settle, the petroleum ether decanted, and with o stirring 1200ml. of dry DMSO was added, followed by o 04 trimethyloxosulfonium iodide (214g., 0.97 mole), which was added I 44« I 0 t portionwise over a period of 15 minutes, and stirring was then continued for an additional 30 minutes. The reaction flask was equipped with a sealed pressure-compensating dropping funnel rtt Scontaining quinuclidin-3-one (100g., 0.8 mole) dissolved in j 300ml. dry DUMSO. This solution was then added to the reaction mixture over a 15 minute period. After stirring for 15 minutes, 4 0 the reaction mixture was heated to 50 C for 2 hcurs, poured into 4 44 11. of cold water and the mixture extracted with 3 x 500ml.

portions of benzene. The combined extracts were washed with 100ml. of saturated aqueous salt solution, dried over anhydrous sodium sulfate, and the solvent evaporated. The oily residue was dissolved in ether and precipitated as the salt with hydrochloric acid by addition of ether saturated with gaseous HC1. The epoxide product was separated by filtration, washed with ether and dried to give 100g. product which was sufficiently pure to use for the next step. R 0.3 on neutral alumina (ethyl r f acetate); M 139 (mass spectra determinations on a VG 7035 o instrument); HC1 salt has m.p. 200.7 202 C.

(ii) In an alternative synthesis that can also be (o scaled up, 2.2kg. of quinuclidin-3-one (HC1 salt) were added to a liter flask and 2 liters of tap water were then added followed 0 by 1kg of Na0H, The mixture was mechanically stirred at 50 C till the solids dissolved. Under these conditions 3 phases are l: o obtained, two upper phases (liquids) and a lower phase (solid).

o 0 The mixture is kept at 60 C. The upper phase is added to 1 liter of toluene. The two lower phases including the solid phase were filtered. The solid was mashed in 1 liter f tluoene that was also used to mash the water phase. The toluene phases were combined, treated with powder'.: charcoal to remove color S'&o (and impurities), dried over MgSO and filtered to yield 3.3kg.

of product inclusive of toluene; a sample of 215g. of solution was evaporatiJd to obtain 106g. of quinuclidin-3-one (free base) as a white solid, which was sufficiently pure for practiaal 46$4 purposes. (Therefore, under these conditions at least a 88-90% extraction yield can be obtained). The toluene solution is dried hy azeotropic distillation; the dried solution can be used i'r the next step.

In a 3 liter three-necked flask were, introduced quinuclidin-3-one (193g., 1.54 mole) in azeotropically-dried toluene solution (weight of solution 563g), trimethylsulfoxonium iodide (380g., 1.72 mole) and NaH 55-60% dispersion in paraffin 1.68 mole) and then the mixture was mechanically stirred. A weak evolution of H followed that ceased after 2-3 minutes.

The reaction flask was cooled in cold water and DMSO dried 1 month on molecular sieves) was added dropwise; (0 100 ml. were added in one step and the remainder added dropwise during 1.5 hours with stirring and cooling the reaction mixture o between 10 and 30 C. The reaction mixture was then heated to o 55 C for 1 hour till reaction was complete, as determined by TLC o* 9 o on neutral alumina (methanol:dichloromethane 5:95). The reaction mixture was poured into 1 liter of cold water and the mixture was extracted with 0.5 1. portions of chloroform. In boo: each extraction the interphase was removed by filtration; 2.4 liters of organic phase were collected and dried over 160 g. of MgSO The NMR (250 MHz) shows about 120 ml. of DMSO (calculation s based on the ratio DMSO/Toluene). Thus, 80% of DMSO was removed by this treatment. TLC shows the product is slightly impure due to DMSO and other compound(s). This solution was partially o S evaporated to 1900 ml. and kept at 0-4 C for 5 days without change. This solution is sufficiently pure to use for the next step.

it I- I i

I

icirr-~-nI~ r~lnlinrl;~ i~ ill~f.rs~ r -i ru* liuai-li~ iili~:c"e 3-hydroxy-3-mercaptomethylquinuclidine.

In a 11. three-necked flask equipped with a magnetic stirrer, an inlet and an outlet for hydrogen sulfide and a thermometer, were placed 80g. NaOH and 390ml. water. The solution was cooled in an ice-bath and gaseous hydrogen sulfide was passed into the stirred solution until it began to bubble out. The product of step (80g., 0.46 mole) was then added, and stirring was continued for 15 minutes. The reaction mixture o was heated to 45 C for 2.5 hours under a slow stream of hydrogen o Ssulfide. The solution was oooled to 0 C, and 10N HC1 was carefully added to bring the pH to 8; the aqueous solution was then extracted with chloroform (6 x 300ml.). The combined extracts were dried over anhydrous sodium sulfate and evaporated to dryness. The resulting solid was dried over phosphorus o* pentoxide in a dessicator to give 32.9g. of crude product. After purification (TLC, neutral alumina, 10:1 dichloromethane S methanol) R 0.5; M (see Fig. 5) 173; base peak 140; NMR 4 f (see Fig. 1) peak at 250MHz(CDC1 2.8 double doublet, -CH 3 -1 2 SH, AB-type spectrum; IR 9 2900-3300 cm. (broad). The max .o compound was used for the next step without any further I purification.

(ii) In an alternative method, the chloroform solution of step above, (1520g.) was introduced into a 3 1.

three-necked flask equipped with a mechanical stirrer, a H S 2 inlet and two traps with concentrated NaOH solution to trap o excess H S. The reaction mixture was kept at 25 5 C and H S 2 2 was bubbled into it for 8.5 hours, after which it was allowed to

*I

stand overnight. The next day, H S was again bubbled in for 2 more hours, when the reaction was complete (TLC) and could be used for the next step without any further purification.

(c) cis:trans] 2-methylspiro( 1 3-oxathiolan- 5, 3 1)quinuc I idine .CAF 1O2 In a 0.51. three-necked flask equipped with a magnetic stirrer, an inlet and an outlet for nitrogen gas and a thermometer, were placed the product of step (32g., 0.18 mole), 200ml. dichloromethane (dried over molecular sieves) and tO freshly distilled acetaldehyde (110ml., 1.97 mole). The solution was cooled in an ice-bath under nitrogen gas, and boron trifluoride etherate (60ml., 0.40 mole) was added over a period o of 30 minutes. The mixture was stirred three hours at 25 C, 0 cooled to 0 C, and then treated with 10% aqueous NaOH solution until it was alkaline to litmus. The alkaline solution was extracted with chloroform (4 x 400ml.), and the combined extracts ,go: were dried over anhydrous sodium sulfate and evaporated. The 0 *4 Soily residue was dissolved in ether and precipitated by addition of gaseous HC1. The product hydrochloride was separated by 2 s o filtration, washed with ether and dried, to give 26.5g.(yield: as a mixture of two geometric isomers in a ratio of 0.8 1.2 R (TLC, neutral alumina, chloroform) 0.7; IR (using f max a Perkin-Elmer 457 grating infrared spectrophotometer)C-0 1200- -1 T S t 1250, 1150 cm. ;M 199; high resolution molecular weight determination: calc. for C H NOS: 199.1020, found: 199.1017.

17 NMR of the HC1 salt is shown in Fig. 2. Resolution of the spectrum enables the existence of both isomers to be seen.

The location of the in the oxothiolane ring is evident from the chemical shifts of the respective Ha and lib protons. These protons in the isomeric mixture and also in each of the isomers appear at higher field than their possible chemical shifts if they had been attached to as in the analogous 1,3-dioxolane structure. The product can be recrystallized from ethyl acetate (600ml. per or from acetone (220ml. per The isomeric mixture of hydrochlorides can be separated into its components by fractional recrystallization in S redistilled dry acetone. Melting points as well as NMR spectra of 250 MHz can be used as an indication of purity for each isomer. Such a separation will be described in step o° a (ii) In an alternative method, AF102 in free base Sform is prepared directly from the solution of crude Sthioalcolhol. Thus, such a solution from step (ii) containing 240g. of thioalcolhol (1.38 mole) was added to a 0 liter 3-necked flask, cooled in cold water to 10 C and then S, acetaldehyde (680 ml.; freshly distilled on para-toluene sulfonic tg acid) was added during 30 mins. and the temperature was kept at S.o 20 5 C during this time. The solution was then cooled to C and stirred for another 30 mins. BF etherate (450 ml) was 3 added dropwise during 30 mins. and the solution was kept ar 0 S 5 C and stirred for an additional 30 mins. A solution of NaOH (1 liter) was added dropwise and the reaction temperature o was kept at 20 5 C during this addition.

The mixture was filtered and the water solution was 31 ii---lg i; III_ 1. I~l_ iii liextracted with 1 liter of chloroform. The chloroform extracts were combined, dried over MgSO and evaporated. The oil obtained 4 o could be distilled at ca. 90 C/1 mm. Hg to obtain AF102 (cis:trans), or alternatively diluted with 2.5 1. toluene. In the latter case, 5-10g. of solid which deposited on the walls of the flask were removed and HC1 (gaseous) was bubbled into the toluene solution till the organic solution was acidic to pH paper; the solid' cake was filtered off, washed with toluene, o dissolved in 3/4 1. isopropanol at 50 60 C and filtered to (O remove insoluble impurities. The filtrate was evaporated with the addition of 1 1. toluene to obtain 168g. of crude product AF102 (cis:trans) (HC1) with some slight impurity as shown by TLC.

This product (crude yield 51%) can be purified further as described below in step Separation of AF102 into its components AFIO2A and AF102B.

A 1:1 isomeric mixture of hydrochlorides was crystallized from 4.71. of redistilled dry acetone. The product which precipitated was crystallized again (see Table), to yield o after four crystallizations 1.9g. of pure AF102A, m.p. 240-242 C.

Concentration of the mother liquor (see Table) resulted in an oily product which was purified on an alumina column(as a free 4 base, 1% methanol in dichloromethane) to give mainly (10:1) the o isomer AF102B, m.p. 176-179 C as the HC1 salt.

*4480 IIIII~I~YIU~r~ i _i -L I TABLE: Separation of AF102A and AF102B as HC1 salts.

0 23g.1:1 mixture 190-2 C).

11,71. acetone 4I days) concentration to 21. 13.3g. M.P. 201-5oC 1(31. acetone) concentration lo to 11.

concn. to 0. 651.

2.7g., m.p. 195-BoG 1. 8g., m.p. 190-L4oC 1. 5g., M.P. 1900C mother- liquor A\i 7.5g., m.p. 205-10oQ acetone) mother- liquor 5 \3 N/ l4.5g., m.p. 215QC mother- liquor C evaporation of mrother-liquor *~column purification free base 2. 8g.,m. p 220oC* mother- liquor D 71) 1.9g. ,m.p.2 1 10-2oC AF102A**(HCI) 11 on alumina M.p.176-179oC AF102B***(precipitated as HOl salt)

U

*isomer ratio 10:1 Spure isomer (NMR) isomer ratio about 1:10 (WMR) AF102A(HC1): NMR (see Fig. 250 MHz (CDCI )r5. 24 (quartet, peak of Rl which is a quartet since it is coupled to R CH AF1O2B,(HGl): NMR (Fig. 4I) 250 MHz CDC1 5. 17 quartet peak 2 3 1 of R which is a quartet since it is coupled to R CH 3 EXAMPLE II Preparation of 2-diphenylmethylspiro(l,3-oxathiolane- 5,3')quinuclidine.

In a 100ml. three-necked flask equipped with a magnetic stirrer, an inlet and outlet for nitrogen and a thermometer, were placed 3-hydroxy-3-mercaptomethylquinuclidine 0.045 mole), dichloromethane (dried over molecular sieves) and diphenylacetaldehyde (15ml., 0.085 mole). The solution was cooled in an ice bath, and kept under nitrogen while distilled boron trifluoride etherate (20ml., 0.13 mole) was added over a 0 period of 30 minutes. The mixture was stirred two hours at 25 C, 0 cooled to 0 C and then treated with 10% aqueous sodium hydroxide until the solution was alkaline to litmus. The basic solution s Sr was extracted with benzene (4 x 0OOml.), and the combined extracts were dried over anhydrous sodium sulfate and evaporated.

4o4: The oily residue was dissolved in ether and precipitated as the hydrochloric acid salt by addition of gaseous HC1. The product was further purified on a neutral alumina column as free base 0 using 1:10 petroleum ether(40-60 ethyl acetate as the eluent.

"io Under these conditions, 2g. of the title compound were isolated.

M 351.

EXAMPLE III S 2-methyl-2-phenylspiro(1,3-oxathiolane-5,3')quinuclidine.

This compound was prepared by a similar method to Examples 1 and 2, by reacting acetophenone with 3-hydroxy-3mercaptomethylquinuclidine. The yield was relatively low M 275.

lr.- *rr-nii~~v^lcx*ln*'i"ii Various other compounds within the ambit of formula (I) can be prepared by the exemplified method 'by using the 1 1 2 1 2 appropriate aldehyde R -CHO or ketone R -CO-R where R and R are as defined with respect to formula As has already been mentioned, the spiro-compounds of formula including their pharmaceutically compatible acid addition salts may be formulated, together with conventional pharmaceutical inert carriers, diluents, adjuvants and so forth, as pharmaceutical compositions, which may be in a form suitable to for oral, rectal or parenteral administration, or for administration by insufflation, or they may be in a form suitable for transdermal administration, and in any event the composition oS' ,may be in unit dosage form.

0 01 o* The spiro-compounds of formula or such S pharmaceutical compositions containing them, may be administered to humans, as well as to laboratory animals such as rodents, cats, dogs and monkeys, as for example by peripheral or intracerebral or intracerebroventricular injections(for animals), by infusion, through the skin, or per os, It is evident that the o dosage and the route will need to be adjusted according to the specific biological use, and in particular according to the nature of the disease or disorder to be treated and its severity.

For the reasons which have already been noted hereinbefore, such pharmaceutical compositions may, in the case of I*I..IUI^~IIUJII~"*L' .ii li i.ii ~-~-Y~IYPIIIYI- 11111111_11 contain as an additional ingredient, one or more compounds selected from the group consisting of physosti.,nine, tetrahydroaminoacridine, choline, lecithin, piracetam, aniracetam, pramiracetam, oxiracetam, 4-aminopyridine, 3,4diaminopyridine and somatostatin.

As has also been previously mentioned in some detail above, when the pharmaceutical compositions are to be administered transdermally, it is preferred to utilize the drug delivery system according to Israel Patent Application No. 72684.

(o Biological Testing of the Spiro-Compounds of the Invention.

For AF102A and AF102B the general toxicity profile was Sfirst established. This study phase was routinely divided into os two parts, i.e. "dose range finding" and LD determination. In 0 Sthe former, usually pairs of mice were administered widely spaced dosages and the animals were subsequently observed. The type, time of onset and duration of reactions to treatment were recorded. LD determinations were based on the preliminary findings of the range-finding studies. Under conditions of this S study, the test material solutions were administered to at least o five dose-level groups of at least five animals of the same sex S per group and at geometrically-spaced dosages. On the basis of mortality, the LD value and 95% confidence interval were calculated according to Well's method (Weil, Biometrics, 8: 24 9 283, 1952).

AF 102A LD mice; oral administration.

Test conditions male mice, 20 24Ig.

N constant volume-dosage of 20m1./1cg.

I~o Dose kg per os) 16Q 139 120 10J4 78 Mortality (number dead/number in group) (b) OCOTest 0 d 0 0 a 1 140 04 LD (95% 102 (89 117) mg./kg.

LO) mice; intravenous administration.

conditions male mice, 20 211 g.

N =6/gr ou p variabli volume-dosage of 9.0 11.5 ml./kg.

Dose (mg./kg. i-v-)

Q

42.5 110.0 37.9 36.Q Dose mortality (number deod/number In group) I '15.06/6 S '1/6 ii 0.02/6 37.9 3/6 36.0 2/6 0 0 4 4 0 ~50 0 LE) M11 (35.5 415.9) mTg./ka.

rel1atively narrow ratio of abo ,t "1 2. 5 between the LD values of the i-v. and oral routes of administration suggests an efficient, rapid and apparently unaltered absorption Z,o of the test mraterial by the enteric route.

signs observed.

Signs in reaction to treatment In mice and listed below were mostly confined to decedents, as survivors of treatment groups, administered respectively doses which caused only partial lethality, rarely demonstrated any of the reported affects in decedents. Undoubtedly, this finding points to a rather steep toxic-lethal slope of the test material.

Furthermore, there was essentially no obvious difference in effects observed between orally and intravenously treated animals, with the exception of time of onset, duration and time of death. Examination to assess pupillary changes were o carried out only in surviving animals.

Signs are listed in order of their appearance: slight transient decrease of spontaneous motor activity (only in orally treated animals' severe tremors, particularly of head region (within 2-3 mins. or 10 sees. after oral or intravenous administration, respectively) dyspnoea convulsive seizures (elonia) gasping, cyanosis o singular clonic-tonic convulsive seizure folloed by death.

Death in mice to which were administered approximate LD doses, occurred about 10-15 mins. after oral administration and at about 30 sees. after intravenous injection, respectively.

In surviving anirals, transient partial analgesic activity in response to tail-pricking could be detected. Li1kewise, even among survivors in which one of the above-described signs was shown, full recovery was extremely rapid. Another feature worthwhile mentioning is the finding of slight to moderate mydriasis after pupillary examination, in surviving animals of the intravenously-treated group.

It should be pointed out that none of the test animals exhibited effects considered characteristic of parasympatho- or cholinomimetic activity, i.e. continuous generalized tremors, salivation, lachrymation, or diarrhoea and miosis.

Rats to which were administered an oral dose of (0 100mg./kg. showed the following: 10 mins, post-administration spontaneous motor f activity was slightly reduced 15 mins. p.a. mydriasis, with pupillary size being about o*0 three times that seen in controls 0 0009 20 mii3. sudden onset of a brief clonic convulsive sa seizure which 5 mins. later changed into periodic head twitching S and tremors 30 mins. animals appeared somnolent, accompanied by partial loss of righting reflex, dyspnoeic and cyanotic, with S very slight salivation and lachrymation.

All of the above signs refer mostly to the one animal, out of the group of three, which was found dead at 40 mins. postadminisctration. In the remaining two rats, all of the abovedisplayed effects were much less in their relative intensity, and full recovery occurred after several hours.

39 0s 1

I^-

AF 102B LD mice; oral administration.

Test conditions male mice, 20 24g.

N constant volume-dosage of 1

I

I

lo i Dose (mg./kg. per os) 113 101 80 71 64 Mortality (number dead/number in group) 0 (b) Test LD (95% 92 (74 116) mg./kg.

LD mice; intravenous administration.

conditic u male mice, 20 24 g.

N 6/group variable volume-dosage of 9.0 11.5 ml./kg.

Dose (mg./kg. i.v.) 39 37 33 31 Mortality (number dead/number in group) 6/6 6/6 5/6 1/6 3/6 LD (95% 33 (31 35) mg./kg.

The relatively narrow ratio of about 1: 2.5 between the LD values of the i.v. and oral routes of administration suggests an efficient, rapid and apparently unaltered absorption of the test material by the enteric route.

3o Signs observed.

Inasmuch as both LD values after i.v. and per os administration of AF102B were sligh-ly lower than those obtained with AF102A, the general signs of reaction to treatment with the former were essentially similar to those reported for the latter, with the mere difference being confined to a slightly enhanced intensity in the case of AF102B.

It should be pointed out that, as with AF102A, none of the test animals exhibited effects considered characteristic of parasympatho- or cholinomimetic activity, i.e. continuous generalized tremors, salivation, lachrymation, or diarrhoea and miosis. This feature is in contrast to the cholinergically tO active compound of US 4,104,397, namely cis-2-methylspiro(1,3dioxolane-4, 3')quinuclidine, hereinafter referred to under the code number AF30, which did exhibit such side effects.

0 *o Intraperitoneal Toxicity.of AF102B in Rats 000 a The acute intraperitoneal toxicity of AF 102B (HC1) was 0 00 investigated in five groups of five male and five female rats of the Charles River strain at dosages with the range 20.0 187.0 mg./kg. The test material was prepared at various concentrations 0 *1 in saline and was administered at a constant volume-dosage of ml/kg. Mortality and signs of reaction to treatment were Srecorded during a 14-day period of observation. Early decedents and animals killed on day 15 were subjected to necropsy. It was S found that deaths occurred at the three highest dose levels (61, 107, 187 within twenty minutes after dosing.

The principal signs of reaction to treatment observed in both decedent and surviving animals srwea spastic and tonic convulsions, tremors, tachypnoea and dysi. .ta, decreased motor activity, slight to very strong salivation and urination. In 4I 1 surviving animals all signs of reaction to treatment were resolved within two hours after dosing. Surviving animals generally made expected bodyweight gains over the study period.

At necropsy the gross observations related to treatment were seen only in decedents. Congested blood vessels were observed in the brain of the high dosage group. No abnormalities were detected at necropsy of surviving animals. LD (95% 77.6 (60.1 o 100.2); slope 83 Acute Oral Toxicity of AF 102B in Rats to The acute oral toxicity of AF 102B (HC1) was investigated in four groups of five male and five female rats (Charles River CD strain). The test material was prepared at various concentrations in saline and was administered at a constant volume-dosage of 5 ml./kg. Dosages were selected according to the availability of the test material. Mortality and signs of reaction to treatment were recorded during a 14-day period. Early decedents and surviving animals were subjected to necropsy on Day 15. It was found that deaths occurred at all four dose-levels studied, within three hours 30 minutes after 7 3 o dosing.

The principal signs of reaction to treatment observed in both decedent and surviving animals were clonic convulsions, tremor, salivation, urogenital staining, diarrhoea and pigmented orbital secretion. In surviving animals, all clinical signs of reaction to treatment were resolved by twenty four hours after dosing. The main treatment-related finding of decedent animals at necropsy was salivation. Internally, a single case of haemorrhagic contents and occasional congested gastri.c mucosa was observed. Necropsy of surviving animals did hot reveal any abnormalities. Surviving animals generally made expected bodyweight gains over the study period. The oral LD of AF 102B is estimated to be greater than 156 mg./kg.

AF102B: Summary of Acute Toxicity in Studies of Mice and Rats (LD 95% Confidence Limits).

Animal iv ip po Specie ip :Mice 33(31-35) 92(74-116) D 04 o Rats -77.6(60. 1-100.2) >156 S o0 *All mice died at 80mg./kg., ip.

@000 Analgesic (Antinociception) Activity in Mice of AF102B 0 The potential analgesic activity of the test material AF102-B was assessed in groups of male and female mice, by use of two primary analgesic tests: Tail Clip and Phenylquinone Writhing. The test material was administered either by the oral or the intraperitoneal route at three different dose levels and the analgesic effect was compared with that of two reference compounds. In preliminary studies codeine phosphate administered s.c. one hour before testing) and oxotremorine (either 0.1mg./kg., administered p.o. one hour before testing, or 0.05mg./kg., administered i.p. 30 minutes before testing), were selected as analgesic and cholinomimetic reference compounds, respectively. Under the conditions of this study, AF102-B exhibited antinociceptive activity only at dose levels greater than 20mg./kg. p.o. and 10mg./kg. i.p. This dose-dependent antinociceptive effect was not fully developed at 60mg./kg. p.o.

and 40mg./kg. which were the highest non-lethal dose levels tested in this study and remained obviously weaker than those of codeine phosphate and oxotremorine.

Potential hypothermia-inducing activity of AF102B in rats C> The potential hypothermia-inducing activity of the test material AF102B was assessed in groups of 8 male rats, comparatively with two reference materials. The rectal temperature was recorded before and at different times after os administration of the test material, either by the oral or the ao 00 intra-peritoneal route at three different dose levels. In 000 .a preliminary studies chlorpromazine (10mg./kg. administered i.p.) and oxotremorine (either 3.2mg./kg., administered p.o. or 1.2 mg./kg. administered were selected as neuroleptic and cholinomimetic reference material, respectively. Under the Sconditions of this study, AF102B exerted hypothermia inducing t activity only at dose levels greater than 40mg./kg. and S 15mg./kg. At the maximum dose-levels of 80mg./kg. p.o.

9 1 and 60mg./kg. i.p. (the highest tested dose-levels which did not induce convulsions), AF102B induced a decrease in the rectal temperature which was similar in its amplitude to those induced by chlorpromazine and oxotremorine. Slight diarrhea was the single cholinomimetic manifestation induced by AF102B at these p.maximum dose-levels, while oxotremorine induced a full cholinomimetic syndrome associated with tonic-clonic convulsions when this reference material was administered i.p.

Mutagenic activity of AF102B AF102B was examined for mutagenic activity in five histidine-dependent auxotrophs of Salmonella typhimurium, strains TA-1535, TA-100, TA1538, TA-98 and TA-1537, using pour-plate assays. The procedures used complied with the OECD guidelines 471, adopted May 1983. The studies, which were conducted in the O absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels selected following a preliminary toxicity test in strain TA-98. Each test was conducted in duplicate and was carried out on two separate occasions. Positive controls such as the known mutagens sodium 2 o'azide, 4-nitro-o-phenylenediamine (NPD), ICR-191 and 2aminoanthracene, were used under the same experimental Sconditions. No increases in reversion to prototrophy were obtained with any of the five bacterial strains at the compound levels tested, either in the presence or absence of S-9 mix. No inhibition of growth observed as thinning or absence of the background lawn of non-revertant cells occurred in any strains following exposure to the test material at 1000ug. per plate. It is concluded that AF102B is devoid of mutagenic activity under the conditions of this study.

Binding affinities of putative cholinergic agonists to muscarinic receptors A) Scatchard plot: i i T r PNZ is considered as a specific M antagonist [See 1 volume dedicated to this topic in Trends Pharmacol. .Sci. (Suppl) Jan 19841. Its affinity to the receptor is not as'high as that of QNB, therefore the apparent Kd should be higher than that of QNB.

Indeed the Kd of PNZ, determined by us, was 13.0 nM compared with 0.048 nM found for QNB. The Kd value found by us for PNZ is consistent with the published data.

3 B) Displacement of bound H]-PNZ by the tested compound: The advantage of using PNZ lies in its selectivity toward M -receptor. It should be expected that compounds which are more selective toward M receptors will displace it more 1 3 efficiently. The displacement of H]-PNZ by the muscarinic antagonist, atropine and the muscarinic agonist, oxotremorine -7 results in IC 's of 5 x 10 M and 8 x 10 M, respectively and they are both higher than the concentrations needed to displace 3 H]-QNB. These results are not surprising in the light of the higher Kd of pirenzepine. The ratio of these two IC 's to the IC 's of the same compound for the displacement of 3 H]-QNB can be used as reference for preferential binding of other tested compounds of pirezepine binding sites M receptors).

1 The following table shows the IC 's of the tested 3 50 3 compounds for the displacement of H]-PNZ or HI-QNB. It c.an 3 be seen that the tested compounds displaced HI-PNZ more 3 efficiently than H]-QNB. However, AF102B was more selective for M receptors by one order of magnitude than the rest of the 1 1 2 tested compounds, as expressed by the ratio IC IC 46 L TABLE 1 displacing as IC The potency of putative cholinergic compounds in IIJ-PNZ from central muscarinic receptors (expressed Compound IC [MI IC IC IC 50 50 oxotremorine 8 x 10-7 3 x 10-6 0.27 AF102B 4x 10 10 0.04 AF'102A 3 x 10 7 x 10 0. 43 *approximately twice as potent as AF 30 (cis) 1 3 IC IC for displacement of H]-PNZ 2 3 IC IC for displacement of HI-QNB [Muscarinic receptor assay was performed according to Yamamura and Snyder, PNAS US 71: 1725, (197'4).] 44 4 00 0 0 4 4 4 4, 4 4 04~ 4 04 44 4 TABLE 2: Guinea-pig ileum induced contractions COMPOUND Type of activity EC (M) IC (MI*

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Acetylcholine AF102B AF1O02A S (Ib) (Ic) agonist agonist agonist agonist antagonist antagonist -8 5.0 x 6 41.0 x 6 4.0 x 2 x 10-8 2 x 10-6 *This dose is contractions of the IC the ileum.

for inhibiting acetylcholine-induned 0- I -Mmd A A A From TABLES 1 and 2 it is evident that AF102B is a potent muscarinic agonist, whereas its geometrical isomer AF102A is almost one order of magnitude less active; moreover, from TABLE 1 it is evident that AF102B is a selective M agonist.

1 BEHAVIORAL STUDIES.

Summary of Results Behavioral studies in rats treated icv with AF64A (3 nmole/2ul/side) revealed marked cognitive dysfunctions in a stepthrough passive avoidance test. These memory impairments could to be reversed by physostigmine and by AF102B.

This reversal, as noted in the following table, occurs o" °.at low doses of physostigmine (0.06 mg./kg., ip) and AF102B (0.1 o o'O and 1 mg./kg., ip and 1 mg./kg., po). Therefore the "therapeutic index" of AF102B is 78-780. This "therapeutic index" is o. definitely wider than is found in physostigmine (5-17).

o 0 a t€

I

Ia 1 6 a ala Reversal, of AF6 1 IA-Induced Cognitive Impairments Ly Physostipmine and AF10213 and Their Relative Toxicity in Rats.

Physostigmine AF102B (mg./kg.) 2D ipp0 AF64IA Passive 0.06 1 1 Avoidance Morris Swimming 0.1 worsens 1 Maze (a 8-Arm Radial 0.1 (NS) Arm Maze LD 1-2 78 >156 Therapeutic 10-17 78-78Q >156 aIndex (NS =non-significant) S*This number is derived by dividing the LD50 by the dose used in the Radial Arm Maze (ip administration).

Moreover, it is important to note that the slope of the acute toxicity curve of A5F102B is steep and no overt behavioral ~O signs including parasympathomimetic effects, such as salivation and tremors, were detected up to the lethal dose. Thus, the '1 range of the "sign free" dose is rather wide, emphasizing the potential. use of this drug for treatment of SDAT.

In addition, in the extinction studies performed In the AF64lA-treated rats, a very long beneficial effeot of AF102B was irunr~-ccu.;;... 3D1.IPL found, indicating that this compound has long-term duration effects on cognitive functions (Figs. 10 and 11).

It is also of great importance to note that AF64A (3 nmole/2ul/side) induced marked memory impairments in the Morris Swimming Maze test (Morris, loc. cit.), which AF102B (1 mg./kg., ip) counteracted, whereas physostigmine (0.1 mg./kg., ip) had a negative effect (Figs. 16 and 17). Interestingly, the beneficial effects of AF102B in this test were on spatial memory dysfunctions induced by AF64A. In this regard, it is noteworthy to that a main memory dysfunction in SDAT patients is impairment of spatial memory. Moreover, it is also of great importance to note that AF64A (3 nmole/2 ul/side) induced memory impairments in the 8-Arm Radial Arm Maze (RAM) which AF102B (5 mg./kg., ip) counteracted, whereas physostigmine (0.1 mg./kg.) had no o significant effect (Figs. 18 and 19).

0. Experimental Bilateral icy injections of AF64A produced cognitive impairments in rats in a passive avoidance-step through test; the Morris Swimming Maze; and the Radial Arm Maze; and Q the possibility cf reversal of the AF64A-produced effects by o 0 physostigmine as a reference compound, and by AF102B.

o 0 QAF64A Preparaition AF64A was freshly prepared each d:y in 10 mM concentration and then diluted in artificial cerebrospinal fluid (CSF) to the proper concentration for injection. The composition of the artificial CSF pH 7.1-7.3 solution was: NaCI

KCI

MgC .66H 0 2 2 Dextrose CaCI .2H 0 2 2 mM 1117 2.9 1.6 2.2 1.7 Step-through Passive Avoidance: Experiment 1 The effect of physcstigmine on the performance and 24 hours retention of an inhibitory learning (passive avoidance-step (0 through) task, in AF64A and CSF injected rats was investigated, using a post-training drug treatment paradigm.

Male Sprague-Dawley(raised by Charles River) rats, 110 days old, weighing 230-360 were all allowed free access to food and water. Prior to surgical procedure, rats were anesthetized with Equithesin (0.3 ml./ 100 g. ip). Bilateral injections were made by stereotaxic application of AF64A or o o o t 0 vehicle into the lateral cerebroventricles (icv) (AP 0.8 mm.

from bregma, L-1.5 mm. from bregma and 4.7 mm. from skull surface). 23 rats were infused with 3 mole of AF64A in a volume o '0 of 2ul*, into each lateral ventricle (group and 20 control rats were infused similarly with the same volume of CSF (group Altogether 13 rats were operated. The infusion was made by a 28-ga. injection oannula. the rate of injection was kept constant at 0.25 ul/ min. The injection cannula was left in 0 plece for 4 mins. after injection to allow diffusion of the solutions into the ventricles.

27-28 days after injection#*, each group of rats was post-operatively subdivided randomly into 2 groups of 10 rats each; subgroup 1 was assigned to the physostigmine treatment and 14 subgroup 2 was treated with saline. Each rat was individually piaced in a small lighted front compartment of a two-compartment box. After a 60 seo. familiarization/adaptation period, the door separating the two compartments was opened and a clock activated.

The rat's latency to enter the large dark compartment of the box (to step through) was measured. Immediately following entry into the dark compartment, the rat was subjected to an inescapable scrambled foot shock applied to the grid floor (0.6mA for 3 sees.). 60 seas after the termination of the shock, at the eid )c of the training procedure, the rat was removed from the de-k compartment and physostigmine dissolved in saline (0.06 mg./kg,) or saline placebo were administered ip. Rats were then returned to their home cage, Retention of the passive avoidance task was measured 241 hrs. after training by again placing the rat in the lighted front compartment and after a 60 see. adaptation period, measuring the latency to enter the dark compartment. The test ~ession ended when a rat entered the dark compartment, or after 600 sees. had elapsed. Animals that failed to step through within 600 sees. were removed from the apparatus and a 600 seas, ao~ latency was recorded for them, *This dose was found effective in impairing the passive avoidance step-through performance of rats.

#*The 27-day interval was found to be effective in impairing the passive avoidance step-through performance in 3 nmole/2UI AF6 1 4Ainjected rats.

Results: Mortality and Body Weight.

Immediately following surgery the AF6LA-injected animals appeared unrespoisiVe to environmental stimuli. A few of the animals (2 out of 20 in the AF64A-ojented group and 6 out of 1

C,

in the CSF-iiijected group). showed and 12.5i%, respectively) which was dosing. Within 4~8 hours of dosing mortality of 13-1' of the AF6'A-injected a loss of body weight (2% observed 7 day.s following there was a cumulative group.

Results: Passive Avoidance Test.

The initial latency measures and the retention-test latency measures were analyzed by a two-way ANOVA, Injection (AF64lA/CSF) VS. Treatment (physostigmine/saline), Table presents the mean S.E.M. of the initial latency measures, while Table (ii) fo presents the mean S. E. M. of the rete~itiv-n-test latency measures4

I

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2

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o o

II

0 01 Ii.

ao ]nitial-test Injection Pre-Treatment Physostigmine (0.06 mg./kg.) Saline Table latency measures (secs.) AF64IA 3 nmole/2u1 25.50 5.68 29.70

CSF

2u1 /-7.93' 33.50 9.6 6.0+7- 3.73 Table (ii) Retention-te t latencZ measurc5 (seps.) Injection AF64A GSF Treatment 3 nmole/2ult 2ul Thysostigmine 45 +T7TY -576. 10 22. 69 (0.06 rng./kg.) Saline 24J7-90 52.26 556,60 28.1111 No significant differences were found during the training trial between any of the groups tested (see Fig. 8): F(1,36) 0.81, p>0.05.

The step-through latency of the AF64A-injected group was significantly shorter, F(1,36) 20.18, p<0.01 during the 24hr. retention test than the CSF-injected group's latency. In addition, the step-through latency of the physostigmine-treated group was significantly longer, F(1,36) 5.91, p<0.05, during the 24hr. retention-test than the saline-treated group's latency.

(O Retention of a step-through passive avoidance response of the AF64A-injected group was significantly improved by physostigmine administration while the retention of the CSF-injected group was not affected by this treatment (see Fig. F(1,36) 408, p o 0.05. Shiffe's contrasts of the step through latency measures S during the retention test revealed a significant difference Sbetween the saline-treated AF64A an, CSF-injec ted groups (p<0.05) The three other controsts were not significant although the latency of the salinetreated AF6 4 A-injected group appears to be shorter than the latency of the physostigmine- -i0 treio;ed AF64A-injected group.

S Step-through Passive Avoidance: Experiment 2 The effects of AF1Q2B on the performance and 24 hou retention of an inhibitory learning (passive avoidance-stepthrough) task, in AF64A and CSF injected rats were investigated, using a post-treatment drug paradigm.

The surgery procedure was identical to that of

I

Experiment 1 except '.hat the weight of the operated rats was 250-325 2? rats were infused with 3 nmole of AF64A in a volume of 2ul, into each ventricle (group and 20 control rats were infused with a volume of 2ul of CSF into each lateral ventricle (group Altogether 40 rats were operated.

The behavioral testing procedure comprised 4 phases.

Pretest: 28 male Sprague-Dawley naive rats, 90-110 days old, weighing 270-310 g. were randomly divided into 4 groups of 7 rats each; 3 groups were assigned to AF102B dissolved in saline Streatments 1 or 5 mg./kg.) and the fourth group was treated with saline. The training and test-retention procedure was identical to that in Experiment 1 except that at the end of the training procedure different doses of AF102B or saline placebo were administered ip.

S Phase 1: 27-28 days after injection each group of rats was S postoperatively subdivided randomly into 2 groups of 10 rats Seach; subgroup 1 was assigned to the AF102B treatment and S subgroup 2 was treated with saline. The training and the testretention procedure was identical to that of Experiment 1 except o that at the end of the training procedure, the rat was removed from the dark compartment and doses of AF102B dissolved in saline (1 mg./kg.) or saline were administered ip.

Phase 2: 6 days after the retention test, rats were subjected to 6 daily sessions of extinction procedure. The rat was placed in the lighted front compartment and the latency to enter the dark compartment was measured. This procedure was identical to the retention test's procedure in Phase 1.

Phase 3: After the extinction procedure, rats were subjected to a latent extinction procedure. The rat was placed in the lighted front compartment and after a 60 sec. adaptation period, was pushed into the dark compartment and kept there for 60 secs.

This procedure was followed once a day for three days. After the latent-extinction procedure, rats were subjected to an "extinction latent extinction" procedure. The rat was placed in the lighted front compartment and after a 60 sec. adaptation period, the latency to enter the dark compartment was measured.

to The test session ended when a rat entered the dark compartment.

Animals that failed to step through within 600 sees. were pushed into the dark compartment and a 600 sees. latency was recorded for them. This procedure was followed once a day for 4 days. At the end of the 4th session, immediately following entry into the dark compartment, the rat was subjected to a training and retention-test procedure identical with that in Phase 1 except that the two sub-groups treated in Phase 1 with AF102B were now treated with saline placebo and vice-versa. (It should be notea that this treatment took place 2 months after the AF64A/CSF 0° injection).

Results: Mortality and Body Weight.

Immediately following surgery the AF64A-injected animals appeared unresponsive to environmental stimuli. A few of the animals (2 out of 20 in the AF64A-injected group) showed a 2% loss of body weight which was observed 7 days following dosing. Within 48 hours of dosing no mortality occurred in any of the groups.

Results: Passive Avoidance Test.

Pretest.

The initial latency measures and the' retention-test latency measures were analyzed by a one-way ANOVA. Table (iii) presents the mean S.E.M. of the initial latency measures, while Table (iv) presents the mean S.E.M. of the retentiontest latency measures.

Table (iii) Initial latency measures (secs.) to AF102B AF102B AF102B Saline 0.1 mg./kg. 1 mg./kg. 5 mg./kg.

16.71+/-4.77 11.86+/-1.23 19.71+/-5.39 20.00+/-5.64 Table (iv) oOO Retention-test latency measures (secs.) AF102B AF102B AF102B Saline t 0. 1 mg./kg. 1 mg./kg. 5 mg./kg.

0 0 330.57+/-75.55 3 48.14+/-81.66 551.71+/-32.92 513.00+/-56.39 0 1I No significant differences were found during the training trial between any of the groups tested, F(3,24) 0.57; p>0.05. In addition, no significant differences were found during the 24 hr. retention-test between the different doses of p; AF102B, F(3,2 1 2.61; p>0.05, although the retention latency of the 0.1 mg./kg. and the 1 mg./kg/ groups tends to be shorter than the latency of the 5 mg./kg. and the saline groups.

Phase 1.

The initial latency measures and the retention-test latency measures were analyzed hy a two-way ANOVA, Injection 57 *1 1 *0

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i I 4 I I I

II

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(AF6'4A/CSF) vs. Treatment (AF1O2B/Saline) Table presents the mean S.E.M. of the initial latency measures, while Table (vi) presents the mean S.E.M. of' the retention-test latency measures.

Table (v) Initial-test latency measures (secs.) Injection AF64IA CSF Treatment 3 nmole/2u1 2u1 AF102B 18.40+/-3.y74 15.30+/-2.83 (0 mg./kg.) Saline 2 2. 66+ 4 3 15.20+/-2.10 Table (vi) Retention-test latency peasures (secs.) Injection AF6'4A CSF Treatment 3 nmole/2u1 2ul AF102B 514. 10+/-38.21 4 47 6 o+/T-58 9

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(1 mg./kg.) Saline 182. 414/-2 9 9 0 5714.30+/-16.39

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No significant differences were found, during the ac) training trial, between any of the groups tested (see Fig. 9).

F(1, 35) 2.16, p>0.05 for the injection variable (AF6 1 4A/CSF) main effect. For the other main effect (the two groups to be treated with AF102B or saline) :F(1,35) =0.29; p>0.05.

The step-through latency of the AF6tIA-injected group was significantly shorter, F(1,35) 13.89; p<0.01, during the 241 hr. retention test than the CSF-injected group's latency. In was significantly longer, F(1,35) 4.98; p<0.05, during the 24 hr. retention test than the saline-treated group's latency.

Reteftion of a step-through passive avoidance response of the AF64A-injected group was significantly improved by AF102B administration, while that of the CSF-injected group was significantly impaired (see Fig. F(1,35) 31.18, p<0.01.

Shiffe's contrasts of the step through latency measures during the retention test revealed that all the between-groups (o differences were significant (0.01 p 0.05) except the AF64A AF102B vs. CSF AF102 difference.

Phase 2.

9o e o The retention-test latency measures during the .'".extcttion period was analyzed by a three-way ANOVA (6x2x2) with or one repeated variable (Trials) and two non-repeated variables (Injection AF64A/CSF and Treatment AF102B/CSF). Fig. shows that the step-through latency of the AF64A-injected group was significantly shorter, F(1,36) 16.83; p<0.01, than the CSFinjected group's latency. In addition, the step-through latency of the AF102B-treated group was significantly longer, F(1,36) 31.45, p<0.01, than the saline-treated 'group's latency.

Retention of a step through-passive avoidance response of both the AF6'4A and the CSF-injected groups, during extinction, was significantly inproved by AF102B administration. F(1,36) 15.80, p<0.01. Shiffe's contrasts of the step-through latency measures during the extinction period revealed that all the between-groups -tLYY~ ui ui ereciue were sigrinican p kpS.u I) except tne A' 4A AFU'IU2 vs. CSF AF102B difference.

The trials variable main effect during extinction was significant, F(5,180) 2.68, p<0.05. Contrasts analysis showed that the retention latency of the second extinction trial was significantly longer than the latency of the 6th trial (p<0.05).

There were no other significant differences.

The interaction between treatment and trials was significant, F(5,180) 3.30, p<0.05. Significant simple main effects contrasts were found between the AF102B-treated groups and the saline-treated groups during all the extinction trials except the first trial. In addition, the retention latency Sextinction curve was significantly different from that of the o o 0 So saline-treated groups The retention latency of the AF102B-treated groups increased from the first to the fourth trial and then decreased. The retention latency of the salinetreated groups decreased from the first trial to the sixth trial.

Phase 3.

A. The retention-test latency measures during the "extinction latent extinction" period was analyzed by a threeway ANOVA 4 x2x2) with one repeated variable (Trials) and two non-repeated variables (Injection AF64A/CSF and Treatment AF102B/CSF). No significant differences were found during "extinction latent extinction" period between any of the conditions tested, in the retention-test latency measures, although the retention latency of the saline-treated AF64Ainjected group appears to be shorter than the retention latency of the three other groups (see Fig. 11).

B. The retention-test latency measures of the second administration of AF102B/Saline were analyzed by a two-way ANOVA, Injection (AF64A/CSF) vs. Treatment (AF102B/Saline). Table (vii) presents the mean S.E.M. of the retention-test latency measures.

Table (vii) Retention-test latency measures (secs.) 1 o of second administration of AF102B/Saline Injection AF64IA CSF Treatment 3 nmole/2ul 2ul AF1 02 (1 mg./kg.) 580. 600 (previosly Saline) Saline (previously AF102B) 600 600 00 0 60') 6 0 00 0 60 00 6 0 L 0' No significant differences were found following the second AF102B/Saline administration between any of the groups tested (p>O.05) (see Fig. 12). The retention latency of the Saline-treated AF64A-injected group, previously treated with AF102B, remained in the same level, while the retention latency of the AF102B-treated AF64A-injected group, previosly treated with saline, increased and reached the level of the CSF-injected groups.

BEHAVORIAL EXPERIMENTS The present work investigated further .the possibility of reversal of the AF6 4 A-induced effects by various doses of AF102B in the passive avoidance task and in two other tasks, namely, the Morris Swimming Maze and the 8-Arm Radial Maze.

Passive Avoidance Task Experiment 1 In tnis experiment we investigated the effect of AF102B (0.1mg./kg., ip) administration on the performance and 211 hours o 0 retention of an inhibitory learning (passive avoidance-step through) task in AF64A and CSF-injected rats, using a post- 0 a 0 "o training drug treatment paradigm.

Q..0 Surgery 0 Male Sprague-Dawley (raised by Charles River) rats, o o 110 days old, weighing 265-340 g. were injected icy. 10 rats were infused with 3 nmole of AF64A in a volume of 2 ul, into each lateral ventricle (group and 10 control rats were infused with CSF (group Altogether 20 rats were operated.

SBehavioral Testing 0 o The behavioral testing procedure comprised two phases, 1 and 2. In a pretest training procedure each rat was individually placed in a small lighted front compartment of a two-compartment box. After a 60 sees. familiarization/adaptation period, the door separating the two compartments was opened and a clock activated. The rat's latency to enter the largo dark compartment of the box (to step-through) was measured.

62 1 1 Immediately following entry into the dark compartment, the rat was subjected to an inescapable scrambled foot shock applied to the grid floor (0.6mA for 2 sees.). Sixty sees. after the termination of the shock, at the end of the training procedure, the rat was removed from the dark compartment and saline placebo was administered i.p. Rats were then returned to their home cage. Retention of the passive avoidance task was measured 24 hr. after training by again placing the rat in the lighted front compartment, and after a 60 sees. adaptation period, measuring S the latency to enter the dark compartment. The test session ended when a rat entered the dark compartment or after 600 sees. had elapsed. Animals that failed to step through within 600 sees.

S, were removed from the apparatus and a 600 sees. latency was an 4 o recorded for them.

4 4 Phase 1 27-29 Days after AF64A or CSF injection, each group of rats was subjected to the same training and the test-retention procedure as in the pretest except that: a) in the training procedure, the rat was treated i.p. with saline placebo; b) the ,O duration of the shook was 3 sees.

S Phase 2 S13-80 Days after the retention test (40-47 days after AF64A of CSF injection) the rats were subjected to a second passive avoidance test, Each rat was placed in the lighted front compartment and after a 60 sees. adaptation period, the latency to measure the dark compartment was measured. Animalsa that failed to step through within 600 secs. were pushed into the dark compartment and a 600 secs. latency was recorded for them.

Rlats were subjected to training and retention-test procedures Identical to the procedures in phase 1 except that groups treated with saline were now treated with AF102B (0.1 rng./kg., ip Results Mortality and Body Weight Immediately following surgery the AF611A-inJected .O animals appeared unresponsive to environmental stimuli. One animal (out of 20 AF6 1 JA-injected animals), showed a 2% loss of ,-;body Weight, and two animals did not show an increase in body ~weight 7 days following injection, No mortality ocoured in any of 0 the groups.

ft 0 0 Passive Avoidance Tost; Pretest No sIgnirlcant differences were found during the :.~training trial between any of the groups tested tr(3,24) OU p> 0Q 1.

Comparison wa11s mnad e between the retention-test latencies obtained after administration of AF'1O28 (0.1mg./k., (second running) and the retention-test latencies ebtained after silline-administration (f'irist running). The retoention- test latency measures were annalyzed by a 2-woy ANOVA (2x2) with one repeated variable (Treatment+Running) and one non-repeated variable [Injection (AF6'IA/CSF)].

The retention latency of tY if AF6'4A-injected group was s' ill significantly shorter [F(1,18) ,29.48; p<0.001] than the CSF--injected group's latency (see Fig. 13). In addition, the retention latency of the AF1O2B-treated groups (second running) was significantly longer [F(1,18) 33.71; p<0.001) than the saline-treated group's latency (first running), Retention of a step through-Passive Avoidance response of the AF6LA in jec ted group was significantly improved by AF'f02B administration W. A1,18) 33.27; p<0.001) while that of the CSF-injected group did not significantly change by this treatment. Indeed, since golatencies of CSF treated rats were already clov t6- 600 secs., no change was really expected. Shiffee's contrasts revealed that~ AF6'4A AF102B vs. AF6 1 4A saline difference and AF64IA p'saline vs. CSF saline, difference were significant (p<0.001).

It is pointed out, however, that th,4 improvement of th~f AF6'IAinjected Firoup by AF102B administration was recorded, in this, case, oil,/ a second shock, a fact which might have influenced 0 the improvement in retention.

Experiment 2 -tole! Th iq cxperiment investigpted the effect of AF1102B administration on the performance and 24) hours retention of an inhibitory learning (Passive Avoidanice step-through) task, in AF6'IA- and CSF-injected rats, using post-training treatment paradigm.

f Method Surgery The surgery procedure was identical to the surgery procedure in Experiment 1, except: 1) the weight of the operated rats was 265-320 2) 10 rats were infused with 3 nmole of AF64A in a volume of 2ul, into each lateral ventricle (group 1) and 10 control rats were infused with 2 volumes of 2ul of CSF into each lateral ventricle (group Altogether 20 rats were operated.

(O Behavioral Testing The behavioral testing procedure comprised 2 phases.

Phase 1 The training and test-retention procedures of phase 1 o were identical to the procedures of phase 1 in Experiment 1 Sexcept that: 1) each group of rats was post-operatively 9404 subdivided; and 2) at the end of the training procedure the rat 0 0 f was removed from the dark compartment and AF102B (1mg./kg., or saline were administered.

o P e o Phase 2 4CL 4 4 44L 44 19-22 Days after the retention test (49 days after AF64A or CSF injection) the saline-treated rats only were subjected to a second running. The training and test-retention procedures were identical to the procedures in phase 2 of Experiment 1 except that the two subgroups treated in phase 1 with sali., were now treated with AF102B (Img./kg., Results Pha,se 1 The initial latency measures of the AF6LA- and CSFinjected groups were analyzed by a t-test for independent samples. The following tables present respectively the means of the initial latency measures and the means+/ S.E.M. of the retention-test latency measures.

"Initial-test latency measures (sees.) A F6 U1A CSF 3 nmole/2ul/side 2u1/side 19.0 2.18 18. 86 2. 07 Retention-test laec measures.(sees.) I n jec t io n AF6 1 4A CSF Treatment 3 nmole/2u1/side 2u1/side AF102B 524.55 45.60 Saline 16 8. 90 2 8. 91 5118. 90 4 8.51 No significant differences were found during the training trial, between the AF611A- and COSE-injected groups (see 67 Fig. 14), t(57) 0.05; p>0.05. The first analysis of the retention latency measures showed that the step-through latency of the AF64A-injected group was significantly shorter [F(1,36) 37.01; p<0.001 (see Fig. 14) during the 24 hr. retention test than the CSF-injected group's latency.

The second analysis of the retention latency measures showed that the step-through latency of the AF64A-injected group was significantly shorter [F(1,35) 37.93; p<O.001 (see Fig.

14) during the 24 hr. retention test than the CSF-injected o( group's latency. In addition, the step-through latency of the AF102B-treated group was significantly longer [F(1,35) 28.46; p<0.001] than the saline treated group's latency. Retention of a step-,through passive avoidance response of the AF64A-injected group was significantly improved by AF102B administration [F(1,35) 13.94; p<O.0011 while there was no significant difference between the AF102B-treated-CSF-injected group and the saline-treated-CSF-injected group. Shiffee contrasts revealed that the AF64A AF102B vs. AF64A saline difference was significant (p<0.001).

-o Phase 2 In a first analysis, the retention-test latency measures before the shock administration in the second running were compared to the retettion-test latency measures during the first running. The retention-test latency measures were analyzed by a 2-way ANOVA (2x2) with one repeated variable (running) and one non-repeated variable [injection (AF64A/CSF)]. The retention latency of the AFI6A-injected group was still significantly 68 1 shorter running, There was p>0.05) p>0.05).

[F(1,18) 49.51; p<0.001) 19-22 days after the than the CSF-injected group's latency (see Fig.

neither a significant running effect [F(1,18) nor a significant interaction effect [F(1,18) first 0.91; 1.28; In the second analysis the retention-test latency measures after the shock administration during the second running w compared to the retention-test latency measures during the first running. The retention-test latency measures were analyzed by a S 2-way ANOVA (2x2) with one repeated variable (running treatment) and one non-repeated variable [injection (AF64A/CSF)]. The retention latency of the AF64A-injected group was still significantly shorter [F(1,18) 40.81, p<0.001) than the 9 Q Sa" CSF-injected group's latency (see FIg, 15). In addition, the O 09 retention latency of the AF102B treated group (second running) S was significantly longer [F(1,18) 65.71; p<0.001] than the o o latency of the saline-treated group (first running). Retention of Sa step through-passive avoidance response of the AF64A-injected 0094 group was significantly improved by AF102B administration EF(1,18) 40.81; p<0.0011 while that of the CSF-injected S group did not significantly change by this treatment. Shiffee contracts revealed that the difference of AF64A AF102B vs.

S AF6 1 1A saline was significant.

ere Morris Swimming Maze Cholinergic deficiency, as well as treatments with i: nn~ur r IL1_ Y-X l I -II1~1~IYYIC~-*-~IICYII1----(anticholinergic drugs, were shown to impair memory and learning processes associated with spatial orientation (Sutherland et al, J. Comp. Physiol. Psychol. 96: 563-73, 1982). In this respect, the water-maze (Morris, Learning and Motivation, 12: 239-61, 1978) seemed to be a suitable behavioral paradigm for the detection of cognitive impairments induced by AF64A in rats, and of their reversal by cholinergic drugs. Since physostigmine is currently one of the few cholinergic drugs used in Alzheimer's ratients, it was selected for preliminary testing of its effects IO on AF64A treated rats.

38 male Sprarue Dawley rats (raised by Charles River) 5-6 months old with an average weight of 500 g. were used. The o o rats were housed in groups of 5 and were given free access to 0 0 0 o food and water. Testing was carried out in a round white metal tub 1.4 m. diameter and 0.4 m. deep. The tub was filled with o 00 oo,, water, made opaque by milk powder to the level of 18 cm. A platform of 12 cm. diameter and at a height of 16 cm. was placed in the tub 2 cm. below water level. The platform was covered with gauze to prevent the animal from slipping back into the Swater after reaching the platform.

Prior to testing, the rat was placed on the platform for 120 secs., placed gently into the water, facing the wall of the pool, at one of four starting locations (north, south, east or west) around the pool's perimeter. Within each block of four trials, each rat was started at each of the four starting locations, with the sequence of starting locations randomly selected. Testing was carried out on 2 consecutive days with each rat submitted daily to eight trials. During trials 1-12 the platform was located in the center of the south-east quadrant and during trials 13-16 the platform was transferred to the center of the north-west quadrant. If in a particular trial the rat found the platform, it was permitted to remain on it for 60 secs.

before starting the next trial. If a rat failed to find the platform, the trial was terminated after a cut-offf time of 120 sees., and subsequently was put on it for 60 secs. before tO starting the next trial. The latency to find the platform was measured.

For drug testing, rats were injected with AF64A or CSF j (3nmole/2ul/side) as described in Experiment 2, above. The rats i were subjected first to a step-through learning procedure (see ao. Experiment 1, above) and were 3-3.5 months after injection when tested in the water maze. The rats were divided into 4 groups; 18 CSF-injected rats were treated either with physostigmine (0.1 mg./kg. ip)(10 rats) or with saline (8 rats). 20 AF64A-injected rats were divided similarly. The rats were injected with G physostigmine immediately before testing on each day.

Results.

The escape latency was analyzed by a 3-way ANOVA (4x2x2) with one repeated variable (trials) and two non-repeated variables (Injection AF64A/CSF and Treatment physostigmine/saline). Table (viii) presents the escape-latency of the groups tested. Fig 16 depicts that AF64A injected rats showed an increase in escape latency (in seconds) relative to CSF I treated rats. This effect is highly significant, F(1,34) 14.88, p<0.001. The initial escape-latency for all four groups was similar. However, the escape-latency of the CSF-treated rats decreased faster than the escape-latency of the AF64A rats.

Physostigmine apparently impaired the performance of both AF64A and CSF-injected groups, compared with non-treated AF64A and CSFinjected groups but this result was not statistically significant. The trials effect was statistically significant, F(15,510) 5.9, p<0.001. In addition, a significant interaction [o between trials and physostigmine was found, F(15,510) 4.3, p<0.001; physostigmine inhibited the decrease of the escapeoa, latency curve, regardless of the injected group.

O 0 V When similar Morris Swimming Maze experiments were o 0 carried out on AF102B (1 mg./kg., ip), it was found that this i compound definitely improved the memory deficits induced by AF64A (Fig. 17), whereas physostigmine (0.1 mg./kg., ip) had a negative effect. The beneficial effects of AF102B in this test were on spatial memory dysfunctions induced by AF64A. It is noteworthy that in this respect, a main memory dysfunction in -eo SDAT patients is impairment of spatial memory. Details of these experiments are given in the description which follows.

Morris Water Maze: Method and Results a.Subjects Forty male Sprague-Dawley rats (obtained from Charles River Breeding Laboratories 5-6 months old, weighing 1150- 580 g. during the experiment served as subjects. Rats were housed in groups of 5 and were given free access to food and water. months before behavioral testing, rats were injected with AF64A (3 nmole/2ul/side) (20 rats-group 1) or CSF (20 rats-group 2) as described in the surgery procedure.

t o b)Drug Administration Each of the injected groups was randomly subdivided into two equal size treatment groups. At the end of the training procedure, in the first and second day of behavioral testing, one 000 of the AF64A-injected subgroups and one of the CSF-injected o o subgroups (n=20) were treated with AF102B dissolved in saline 0 4 o o* (1mg./kg., while the other two subgroups (n=20) were 0 0, treated with saline.

AF102B (1mg./k i.p.) 0 04 The escape latency of this treatment was analysed by a 4 00 -C 3-way ANOVA (4x2x2) with one repeated variable (trials) and two non-repeated variables Linjection (AF64A/CSF) and treatment 6 (AF102B/saline)l. Fig. 17 depicts that AF64A-injected rats o showed a significant increase in escape latency relative to CSFinjected rats [F(1,36) 11,26; p<0.005]. In addition, AF102Btreated rats showed a significant decrease in escape latency relative to saline treated rats [F(1,36) 4.89; p<0.05] regardless of the groups injected. The trials effect was 73 -1 ~i _i statistically significant [F(5,180) 33.34; p<O.001]; in addition, a significant interaction between trials and injection was found [F(5,180) 5,53; p<0,001); the escape latency of the CSF-injected rats decreased faster than the escape latency of the AF64A-injected rats.

8-Arm Radial Arm Maze Introduction In the 8-Arm Radial Arm Maze, AF61A (3 nmole/2ul/side) induces memory impairments. In this study, the effects of AF102B 1o and physostigmine were evaluated in AF64'A- and CSF-treated rats.

Experiments were carried out on the effect of AF1028 ip), as well as on physostigmine (0.1 mg./kg., ip), in both cases on trained rats with 2 hours delay.

S? Method 0* In all experiments Sprague Dawley rats were used (Charles River Breeding Laboratories they were housed individually and were deprived of food until reaching 85% of their free feeding weight. The room was illuminated 12 hours per S day (6:00 to 18:00) and behavorial training and testing sessions o were carried out during the day. After reaching 85% of free feeding weight, rats received 3 food pellets (Labena) per day and had free access to water. Two days before training began, animals were familiarized with 115 mg. precision pellets (Bioserv Inc.) which were later used for reinforcement in the maze.

The same surgery procedure was used as in the passive avoidance and Morris Swimming Maze tests.

711 Procedure A group of 20 rats, which were first used in a passive avoidance test, were trained in the new maze 7 weeks post injection. Following 6 days of training a period of 2 hours delay was inserted. Rats were allowed to collect 4 pellets, then they were returned from the maze to their cages. After 4 hours delay they were put back in the maze until they collected the remaining 4 pellets or until 5 minutes had elapsed.

Experiment 1: l The same group of rats was injected with AF102D ip) or saline (1 ml./kg., ip), immediately following picking the first 4 baits. The effects on performance were tested 2 hours post injection. Each rat received both treatments twice. A 1 day interval was given between the AF102B treatment and the saline treatment and two days between the sets of treatments.

Experiment 2: A week later, the same group of rats was injected with physostigmine (0.1mg./kg., ip) or saline (1 ml./kg., ip), Co immediately following picking the first 4 baits. The effects on performance were tested 2 hours post injection.

Results and Discussion Experiment 1: The treatment with AF102B (5mg./kg., ip) immediately following completion of the first four choices had a significant effect on performance (Fig. 18). Analysis of the results in a 2 factors mixed design: repeated measures on one factor showed a significant (p<0.001, F 17.5) decrease in the mean number of errors for the AF64A injected rats when compared to their controls (AF64A injected rats treated with saline). A significant effect (p<0.005, F 14.5) was found between trials (treatment with AF102B vs. saline) and conditions (AF64A icy injections vs. CSF icy injections). The AF102B treatment improved performance only when rats were previously injected icy o with AF64A.

Experiment 2: Treatment with physostigmine (0.1mg./kg., ip) had. no o« significant effect on the performance of the rats (p>O0.5, F S 4 .03) (fiR. 19) although there is a tendency for an apparent a Simprovement following physostigmine. However, the differences between the AF64 1 A injected rats and the CSF injected rats remained significant (p<0.005, F 11.4).

SConclusions The Radial Arm Maze procedure has been extremely i o successful in proving that: there is a significant difference in the performance of AF6 1 4A as compared with the CSFtreated rats, and memory impairment could be reversed by AF102B (5mg./kg., ip).

SUMMARY OF BEHAVORIAL 2rUDIES The table which follows summarizes the relevant data regarding AF1O2B, oxotremorine and pirenzepine, illustrating the outstanding properties of the compound of the invention as an apparent M -type Egonist. Although experiments were not 1 performed in all the tests in the same species (mice or rats) and under the same experimental conditions, a striking parallelism in the relative activity profiles does appear between pirenzepine (M -antagonist) ard AF102B (M -agonist). For both compounds, S 1 1 memory impairments (induced by pirenzopine) and reversal of AF64A-induced memory impairments (caused uy AF102B) occur at much lower doses than needed to antagonize central effects induced by xotremorine (in the case of pirenzepine) or to induce the same o" kind of central side effects (in the case of AF102B).

SOQ

4444 Thus, cognitive functions mediated by an M -antagonist (pirenzepine) or an M -agonist (AF102B) are more sensitive to gtt 4 r, 41 41 such cholinergic interventions than other central effects such as tremoeq or antineclception. This finding is extremely important since it can be used to explain why AF102B has such a remarkable selectivity and why it can be considered as an excellent candidate for a drug in SDAT.

t 1.

Table: The relative acbivity o f pirenzepine, AF 102B and oxotremorine in various in vivo and i~n vitro tests.

Test Test Pirenzepine (PNZ) (M -antagonist) ug/mouse ,icv A1102B (M -agonist) 1 rng./kg. ,rats* to 4004 44 *4 0 4 44 00 0 00*. 0 4 04 o o 0 4* 0040 0 0 0* 04 0 passive avoidance 0. 1(a) 0. 1-1 ip(b) 1 po(b) tremor 5.8(c) >60-80 ip**(d) >78 ip(.1) >100 po(d) antinociception 11.6(c) 60 po**(d) ip**(d) Oxotrp~morine AF102B FC ,M 3 -7 II FI-PNZ-binciing 8 x 10 -6 4x 10 -M-N-binding 3 x 10 notes: 0 t4 66t*uriless otherwise indicated minimum effective dose of PWZ producing impaired passive avoidance learning.

effective dose of AF102B produ~cing reversal of AF6 (3 nmol/slde, icv)-induced Tmpir'ed passive avoidance learning \'the minimum effective dose can be lower for the po route -of administration), oc) dose of PNZ required to reduce the effect of oxotremorino ip) by 50% [Caulfield et alt, J. Pharm. Phairmnool.

131-2 (198,3)].

:3o tremors and antinocio eption (ailgesia) induced by AC1025 in mice and rats; these effects are observed around the oT.

of the compound (antinociception is performed 41Y thr, test in mice).

While the invention has been particularly described with respect to certain presently preferred ernbodiemnnts, it will be appreciated by those skilled in the art that many modifications and variations can be made. Accordingly, the invention is not to be construed as limited by these embodiments, rather it is to be defined only by the claims which follow.

I

4 47 t79 L. ii :I r

Claims (37)

1. Quinuclidine derivatives3 having the general formula (I) a nd geometrical isomers, enartiomers,. d 1astereo isomers, racem'ates and/or acid addition salts thereof, wherein Z represents the group >Cfl1R2 or two hydrogen atoms; R1 is selected 1from the group consisting of hydrogen, al kyl, cyclopentyl, oyclohexyl, aryl, diarylmethylol, and alkyl which is substituted by one or more aryl groups, and H2 IS selected Sfrom the group consi sting Qf al kyl oyc lopentyl cyoiohex yl, aryl, diarylmethylol,0 and alkyl which is substituted by one or *Imore aryl groups.

2. Quinuolidine derivatives according to claim 1, wherein Z represents the group >C~p 4 2 H 1 is hydrogen, and li i s selected from the group consisting of al kyl, cyclopentyl, cyclohexyl aryl dioirylmethylol anid alkyl wich is substituted by one or more aryl groups.

3. Quinuclidine derivatives according to olaim 1, wherein Z represents the group >cR1R2, R1iIs selected from the group consisting of alkyl, cyclopentyl, cyelohexyl and aryl, arid RJ2 is selected from the group consisting of alkyl, oc 'kloperityl oy~slohexyl, aryl, diarylmethylol, and alkyl which is substituted by one or more aryl. groups. 4, A quinuclidine derivative as defined in claim 2, wherein R1 is hydrogen and R2 is selected from the group consisting of methyl, ethyl, propyl, phenyl, 1-pyrenepropyl, diphenylmethyl and diphenylmethylol. A quinuclidine derivative as defined in claim 3, 1 2 wherein H is phenyl and H is selected from the group consisting of methyl, ethyl, cyclohexyl and phenyl.

6. A quinuclidine derivative as defined in claim 4, wherein H1 is hydrogen and H2 is methyl, including the individual geometrical isomers thereof.

7. The geometrical isomer o' the compound defined in claim 1 2w S 6, of which the hydrochloric acid salt has the relatively lower Smelting-point (the cs-isomer), and the hydrochloric acid salt 4 4t om 'erca reom r. S. The geometrical Isomer of' the compound defined in claim 6, of which the hydrochloric acid salt has the relatively higher melting-point (the trans-isomer), and the hydrochlorio acid salt the reof.

9. A process for preparing quinuclidine derivatives as defined in claim 1, and wherein Z represents the group >CR H12, which comprises reacting 3-hydroxY-3-mer aptomethylquinuolidine with a Tarbonyl compound of formula R1-CO-R2, andd isolating the desired product fhrom the reaction mixture. desired product from the reaction mixture. A process according to claim 9, wherein the reaction is carried out in the presence of an inert organic solvent.

11. A process according to claim 10, wherein the inert organic solvent comprises one or more members selected from the group consisting of dichloromethane and chloroform.

12. A process according to claim 9 or 10, which is carried out. in the presence of an acid catalyst. A process according to claim 9, which comprises co'f ect ing the reaction in an atmosphere of nitrogen, at a temperature in the range of about 20 to about 300C, in tho presence of boron trifluoride etherate as catalyst and in a "solvent medium which comprises one or more members selected from 0 c0 o f the group consisting of dichloromethane and chloroform, and 0 t isolating the desired product from the reaction mixture. 4 t

14. A process according to claim 13, wherein the reaction ingredients are first mixed in an atmosphere of nitrogen at a temperature between about -10 and +200C, and the mixture thus obtained is permitted to rise to the reaction temperature. t A process according to claim 9, wherein following isolation at least one of the following steps is carried out, 0 t namely, the desired product is separated into its geometrical isomers, it is converted from free base to its acid addition salt, and(or) it is converted from its acid addition salt to the free base.

16. A quinuclidine derivative as defined in claim 1, wherein Z represents two hydrogen atoms.

17. A process for preparing the compound defined in claim 16, wherein the epoxide of 3-methylenequinuclidine is reacted with hydrogen sul fide. 1R. A quinuclidine derivative as defined in claim 1 and substantially as hereinbefore descrited.

19. A quinuclidine derivative as defined in claim 1 and substantially as hereinbefore described in any of the Examples. A process according to claim 9 and substantially as 'hereinbefore described.

21. A process according to claim 8 and substantially as hereinbefore described in any of the Examples.

22. A process for preparing a quinuclidine derivative as defined in claim II, wherein Ri is hydrogen and R2 is methyl, the individual geometrical isomers thereof, and the hydrochloric acid salts of these compounds, substantially as hereinbefore described with particular reference to the Examples. I 23. A quinuclidine derivative as defined in claim 1, when prepared by a process as hereinbefore described and claimed. 83 L C 2i|. A pharmaceutical composition which comprises a quinuclidine derivative of formula as defined in claim 1, and wherein Z represents the group >CR1R2, or a pharmaceutically compatible acid addition salt thereof, together with an inert carrier or diluent. A pharmaceutical composition according to claim 21, which is in a form suitable for oral, rectal or parenteral administration, or for administration by insufflation.

26. A pharmaceutical composition according to claim 21, which is in a form suitable for transdermal administration.

27. A pharmaceutical composition according to claim 24, <o a S, which is in unit dosage form. SO 0

28. A pharmaceutical composition for transdermal o administration, which comprises a quinuclidine derivative of formula as defined in claim 1, wherein Z represents the group 4 *t >CR 1 R 2 or a pharmaceutically compatible acid addition salt thereof, as well as a low molecular weight fatty acid. S 29, A pharmaceutical composition according to claim 24, S wherein the quinuclidine derivative of formula is that defined in claim 7. A pharmaceutical composition according to claim 29, which contains additionally one or more compounds selected from the group consisting of physostigmine, tetrahydroaminoacridine, choline, lecithin, piracetam, aniracetam, pramiracetam, oxiracetam, 4-aminopyridine, 3,4 -diaminopyridine and somatostatin.

31. A pharmaceutical composition which is in unit dosage form and which comprises a quinuclidine derivative of formula (I) as defined in claim 1, wherein Z represents the group >CR1R2, or a pharmaceutically compatible acid addition salt tnereof, in an amount in the range of about 0.5 to about 500 mg., together with an inert carrier or diluent. Q a 0 0

32. A pharmaceutical composition according to claim 31 and which comprises the said quinuclidine derivative, or a 4 t pharmaceutically compatible acid addition salt thereof, in an amount in the range of about 5 to about 100 mg. of 4i

33. A pharmaceutical composition according to claim 32 and which comprises the said quinuclidine derivative, or a I tI pharmaceutically compatible acid addition salt thereof, in an amount in the range of about 10 to about 50 mg. 31. A pharmaceutical composition according claim 31, wherein the quinuelidine derivative of formula is that defined in claim 7. uri-~rri~t~c- A pharmaceutical composition according to claim 34, which contains additionally one or more compounds selected from the group consisting of physostigmine, tetrahydroaminoacridine, choline, lecithin, piracetam, aniracetam, pramiracetam, oxiracetam, 4-aminopyridine, 3,4-diaminoapyridine and somatostatin.

36. A pharmaceutical composition according to any one of claims 31 to 35, which is adapted for oral administration.

37. A pharmaceutical composition according to any one of claims 31 to 35, which is adapted for parenteral administration.

38. A pharmaceutical composition which comprises a quinuclidine derivative as defined in claim 2 wherein R 1 hydrogen and R 2 methyl, and including geometrical isomers, enantiomers, racemates and/or pharmaceutically compatible acid addition salts thereof, together with an inert carrier or diluent,

39. A pharmaceutical composition which comprises a quinuclidine o derivative as defined in claim 1 wherein Z represents the group >CR R 2 R is selected from te group consisting of alkyl containing three or .oo more carbon atoms, cyclopentyl, cyclohexyl, aryl, diarylmethylol, and alkyl o substituted by aryl, and R is selected from the group consisting of hydrogen, alkyl, cyclopentyl, cyclohexyl, aryl, diarylmethylol, and alkyl substituted by aryl, or a pharmaceutically compatible acid addition salt thereof, together with an inert carrier or diluent.

40. A pharmaceutical composition which comprises the geometrical isomer of the compound defined In claim 6, of which the hydrochloric acid salt has the relatively lower melting-point (the is-isomer), or a pharmaceutically compatible acid addition salt thereof, together with an inert carrier or diluent.

41. A method for treating diseases of the central nervous system, in a patient requiring said treatment, which method comprises administering to said patient an effective amount of a compound according to any one of claims 1 to 8 or of a pharmaceutical composition according to any one of claims 24 to 29,

42. The method of claim 41, wherein the compound or pharmaceutical composition is administered transdermally.

43. A method of treating diseases due to central cholinergic system deficiencies In a patient requiring said treatment, which method comprises administering to said patient an effective amount of a compound according to claim 2, wherein R is hydrogen and R Is methyl or of a I/ TR pharmaceutical composition according to claim 38. S TLH/860c 86- T'

44. The method of claim 43, wherein the compound or pharmaceutical composition is administered transdermally. A method of treating diseases due to cholinergic hyperfunction in a patient requiring -aid treatment, which method comprises administering to said patient an effective amount of a compound according to claim 1, 1 2 2 wherein Z represents the group >CR R 2 R is selected from the group consisting of alkyl containing three or more carbon atoms, cyclopentyl, cyclohexyl, aryl, diarylmethylol, and alkyl substituted by aryl, and R 1 is selected from the group consisting of hydrogen, alkyl, cyclopentyl, cyclohexyl, aryl, diarylmethylol, and alkyl substituted by aryl, or a pharmaceutically compatible acid addition salt thereof, or of a composition according to claim 39.

46. The method of claim 45, wherein the compound or composition is Sadministered transdermally. S47. The method of claim 45 or 46 wherein R is hydrogen and R 2 1 2 o is dlphenylmethyl,

48. The method of claim 45 or 46 wherein R is methyl and R is phenyl, *49. A method of treating senile dementia of Alzhelmer's type, in a patient requiring said treatment, which method comprises administering to said patient an effective amount of a geometrical isomer of the compound defined in claim 6, of which the hydrochloric acid salt has the relatively ower melting-point (the ca-isomer), or a pharmaceutically compatible acid addition salt thereof, or of a pharmaceutical composition according to claim The method according to claim 49 wherein said Isomer is co-administered with one or more compounds selected from the group i consisting of physostigmine, tetrahydroamlnoacridine, chol'ne, lecithin, S piracetam, aniracetam, pramiracetam, oxiracetam, 4-aminopyridine, o 3,4-diaminopyridine and somatostatin. J 51. The method of claim 49 or 50 wherein the compound or composition is administered transdermally.

52. The method according to claim 49 or 50 wherein said isomer is administered orelly in an amount in the range of about 0.1 to 60 mg,/kg. body weight.

53. The method as defined in claim 52, whereln said amount lies in the range of about 0.5 to about 10 mg./kg, body weight. SL I) 54. The method as defined in claim 53, wherein said amount lies in \;/the range of about 1 to about 5 mg./kg. body weight. TLH/860c 87 i, it i- 1 P The method as defined in claim 52, wherein administration is by means of a pharmaceutical composition in unit dosage form which contains the said quinuclidine derivative in an amount in the range of about 0.5 to about 500 mg., together with an inert carrier or diluent.

56. The method as defined in claim 49 or 50, wherein the compound or composition is administered parenterally, in an amount in the range of about 0.01 to about 40 mg./kg. body weight.

57. The method as defined in claim 56, wherein said amount lies in the range of about 0.05 to about 5 mg./kg, body weight.

58. The method as defined in claim 57, wherein said amount lies in the range of about 0.1 to about 2 mg./kg, body weight.

59. The method as defined in claim 56, wherein administration is by means of a pharmaceutical composition in unit dosage form which contains the said quinuclidine derivative in an amount In the range of about 0,5 to a about 500 mg., together with an inert carrier or diluent, a a 0t CC C0C DATED this The State of Israel, the Israel TWENTY-THIRD day of NOVEMBER 1989 Represented by The Prime Ministers Office, Institute for Biological Research C0 0 0 CC 0 g 4t Patent Attorneys for the Applicant SPRUSON FERGUSON TLH/860c 88