WO2001049690A1

WO2001049690A1 - 9-azabicyclo[4.2.1]non-2-ene derivatives as ligands for nicotinic acetylcholine receptors

Info

Publication number: WO2001049690A1
Application number: PCT/GB2001/000040
Authority: WO
Inventors: Timothy Charles Gallagher
Original assignee: University Of Bristol
Priority date: 2000-01-07
Filing date: 2001-01-05
Publication date: 2001-07-12
Also published as: AU2531101A; GB0000257D0

Abstract

9-Azabicyclo[4.2.1]non-2-ene derivatives, which are ligands for nicotinic acetylcholine receptors (nAChRs), are disclosed. The compounds show a capacity to differentiate between different subtypes of nAChRs. Pharmaceutical compositions containing such compounds and a method of using such compounds in the treatment of a disease or disorder which is responsive to the activity of nAChR modulators are also disclosed.

Description

9-AZABICYCLQΓ4.2.ΠNQN-2-ENE DERIVATIVES AS LIGANDS FOR

NICOTINIC ACETYLCHOLINE RECEPTORS

The present invention relates to 9-azabicyclo[4.2.1]non-2-ene derivatives which are ligands for nicotinic acetylcholine receptors. It further relates to a method of making such derivatives, to a pharmaceutical composition comprising at least one of such derivatives as a pharmaceutically-active ingredient and to a method of treating a disease in a living animal body, including human, using a therapeutically-effective amount of at least one of such derivatives. The present invention yet further relates to novel intermediate compounds useful for making the derivatives of the invention.

Nicotinic acetylcholine receptors (nAChRs) are widely distributed in the vertebrate central nervous system. With a few recently reported exceptions (e.g., Frazier et al., J. Neurosci, 18 (1998), 8228-8235 and Alkondon et al., Brain Res. 810 (1998), 257-63) most nAChRs in the brain do not appear to mediate synaptic transmission. Instead, their primary function may be modulatory (Role L.W. and Berg D.K., Neuron, 16 (1996), 1077-1085).

One locus for modulation is the nerve terminal where presynaptic nAChRs act to promote transmitter release and hence influence resting tone or synaptic efficacy. Presynaptic nAChRs facilitate the release of many neurotransmitters, such as acetylcholine, dopamine, serotonin and norepinephrine, in numerous brain regions.

The presynaptic nicotinic modulation of [³H]dopamine release from striatal preparations has been widely used as a model system for examining native nAChR responses and for evaluating novel ligands and is pertinent to physiological and pathological processes such as reward, motor control and cognition.

Many diseases and disorders are associated with decreased cholinergic function. For instance, lack of stimulation of nAChRs is thought to be connected, in some way, with dysfunction of the central and autonomic nervous systems (inter alia, dementia of the Alzheimer's type, Parkinson's disease, Tourette's syndrome, cognitive disorders, drug dependencies and addictions and analgesia). Ligands which have the ability to modulate the activity of nAChRs, therefore, may be useful in treating conditions such as those mentioned above and others that are caused by malfunctioning of the nicotinic cholinergic system.

There have been several recent disclosures of potentially useful ligands for nAChRs. Among these, the following can be mentioned.

WO 94/22868 discloses derivatives of the naturally-occurring compound epibatidine which was isolated from the skins of the Ecuadorian poison frog, Epipedobates tricolor. The derivatives having 7-azabicyclo[2.2.1]heptane and heptene structure are disclosed as having analgesic or anti-inflammatory activity.

WO 96/25160 discloses several 1-azabicycloheptane and octane derivatives.

WO 97/30998 discloses carbamic acid esters of 1 -azabicyclo[2.2.2]octane.

WO 98/54181 relates to 8-azabicyclo[3.2.1]octane and oct-2-ene derivatives.

WO 98/54182 relates to 9-azabicyclo[3.3.1]nonane and non-2-ene derivatives.

WO 99/35131 discloses many different aryl fused azapolycyclic compounds.

WO 99/03859 discloses many different spiroazabicyclic heterocyclic compounds.

WO 99/32117 discloses many pyridine-containing azabicycloheptane, octene and nonane derivatives.

In accordance with the present invention there is provided a 9- azabicyclo[4.2.1]non-2-ene compound of the general formula I

wherein:

R¹ is hydrogen or an aliphatic hydrocarbyl group; R² is an optionally-substituted carbocyclic or heterocyclic aromatic group optionally linked through a linker group selected from alkylene, alkenylene and alkynylene; R³, R⁴, R⁵, R⁶ and R⁷ are each independently selected from hydrogen, optionally-substituted hydrocarbyl, OR⁸ in which R⁸ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(O)R⁹ in which R⁹ is an optionally-substituted aliphatic hydrocarbyl group and n is 0, 1 or 2 or is hydrogen and n is 0, -NR¹⁰R¹¹ in which R¹⁰ and R¹¹ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, halo, -C(O)R¹² in which R¹² is an optionally- substituted aliphatic hydrocarbyl group, -C(O)OR¹³ in which R¹³ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, CN, - NO₂, and -C(O)NR¹⁴R¹⁵ in which R¹⁴ and R¹⁵ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, any of its enantiomers or any mixture thereof or a pharmaceutically- acceptable salt thereof with the proviso that when R¹ is hydrogen and R² is a 2-chloropyrid-5-yl group at least one of the groups R³, R⁴, R⁵, R⁶ and R⁷ must be other than hydrogen. The compounds of the present invention are capable of modulating nAChRs. Furthermore, the compounds disclosed herein show a capacity to differentiate between different subtypes of nAChRs. The present invention, therefore, provides a pharmaceutical composition comprising a therapeutically-effective amount of a compound of the invention as described herein, or an enantiomer thereof or a pharmaceutically-acceptable salt thereof together with at least one pharmaceutically-acceptable carrier or diluent. A further aspect of the invention provides the use of a compound of the invention for the manufacture of a medicament for the treatment of a disease of the living animal body, including human, which disease is responsive to the activity of nAChR modulators. A yet further aspect of the invention provides a method of treating a disease of a living animal body, including a human, which disease is responsive to the activity of nAChR modulators which method comprises administering to the living animal body a therapeutically-effective amount of a compound according to the invention.

Compounds of the invention have the general formula I given above. The group R¹ in the formula I is hydrogen or an aliphatic hydrocarbyl group. The term "aliphatic hydrocarbyl group" refers to straight or branched chain or cyclic groups (and mixtures of these) containing hydrogen and carbon containing typically from 1 to 10 carbon atoms and preferably from 1 to 6 carbon atoms. Preferably, R¹ is hydrogen or a methyl group.

The group R² in formula I above is an optionally-substituted carbocyclic or heterocyclic aromatic group which may be linked to the azabicyclononene structure via a linker group selected from alkylene, alkenylene and alkynylene groups. The term "carbocyclic aromatic group" refers to aromatic groups having a carbon ring structure, typically containing 6 to 10 ring carbon atoms such as phenyl, 1 -naphthyl and 2-naphthyl groups. The term "heterocyclic aromatic group" refers to cyclic aromatic groups in which one or more heteroatoms, such as nitrogen, oxygen or sulphur, are present in the ring structure. Typically, these will contain 5 to 10 atoms in the ring and preferred examples are those containing one or more nitrogen atoms in the ring such as pyridyl, for example 2-pyridyl, 3-pyridyl and 4-pyridyl; pyrimidinyl, for example 2-pyrimidinyl, 4-pyrimidinyl and 5-pyrimidinyl; pyrazinyl; pyridazinyl, for example, 3-pyridazinyl; quinolyl, for example 3-quinolyl; and indolyl, for example 2-indolyl; pyrrolyl; imidazolyl; and pyrazolyl.

The term "optionally-substituted" as is used to describe the aromatic groups of R² refers to the optional presence of substituent groups attached directly to the aromatic ring atoms. Typically, from 1 to 3 substituent groups may be attached to the aromatic ring structure. Examples of substituent groups include, but are not limited to optionally-substituted hydrocarbyl, for instance an aliphatic hydrocarbyl group such as 1-6C alkyl or 1-6C alkylene or an aromatic hydrocarbyl group such as phenyl, halo, OR¹⁶ in which R¹⁶ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(O)_nR¹⁷ in which R¹⁷ is an optionally-substituted aliphatic hydrocarbyl group and n has a value of 0, 1 or 2 or R¹⁷ is hydrogen and n is 0, -NR¹⁸R¹⁹, in which R¹⁸ and R ⁹ are each independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, -C(O)R²⁰ in which R²⁰ is an optionally-substituted hydrocarbyl group, -C(O)OR²¹ in which R²¹ is hydrogen or an optionally- substituted hydrocarbyl group, -CN, -NO₂ and -C(O)NR²²R²³ in which R²² and R²³ are independently hydrogen or an optionally-substituted hydrocarbyl group. The term "optionally-substituted hydrocarbyl group" refers to a hydrocarbyl group such as an aliphatic or aromatic hydrocarbon radical optionally-substituted by one or more substituents such as halo, hydroxy, 1 to 6C alkoxy, amino, nitro and carboxy.

The aromatic group R² may be attached directly by a bond to the azabicyclononene structure or may be linked to the azabicyclononene structure through a linker group as mentioned above. Specific examples of linker groups that may be used include methylene, ethylene, ethenylene and ethynylene groups.

Preferably the group R² is selected from phenyl, naphthyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolyl and indolyl each of which may be substituted by one or more substituent groups selected from halo, e.g., chloro, fluoro, bromo or iodo, and oxyalkyl containing from 1 to 6 carbon atoms in the alkyl group, e.g., methoxy, which may be linked to the azabicyclononene structure through a linker group, as described above.

The groups R³, R⁴, R⁵, R⁶ and R⁷ in general formula I above are each independently selected from hydrogen, optionally-substituted hydrocarbyl, for instance an aliphatic hydrocarbyl group such as 1-6C alkyl or 1-6C alkylene or an aromatic hydrocarbyl group such as phenyl, OR⁸ in which R⁸ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(0)R⁹ in which R⁹ is an optionally-substituted aliphatic hydrocarbyl group and n is 0, 1 or 2 or is hydrogen and n is 0, -NR¹⁰R¹¹ in which R¹⁰ and R¹¹ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, halo, - C(O)R¹² in which R¹² is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, -C(O)OR¹³ in which R¹³ is hydrogen or an optionally- substituted aliphatic hydrocarbyl group, CN, NO₂ and -C(O)NR¹⁴R¹⁵ in which R¹⁴ and R¹⁵ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group. An optionally-substituted hydrocarbyl group, as mentioned here, is a group as defined above. According to a preferred embodiment each of R³, R⁴, R⁵, R⁶ and R⁷ is hydrogen.

Compounds according to the present invention as described above which form a preferred embodiment are those having the general formula I wherein: R¹ is an aliphatic hydrocarbyl group;

R² is an optionally-substituted carbocyclic or heterocyclic aromatic group optionally linked through a linker group selected from alkylene, alkenylene and alkynylene; R³, R⁴, R⁵, R⁶ and R⁷ are each independently selected from hydrogen, optionally-substituted hydrocarbyl, OR⁸ in which R⁸ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(O)R⁹ in which R⁹ is an optionally-substituted aliphatic hydrocarbyl group and n is 0, 1 or 2 or is hydrogen and n is 0, -NR¹⁰R¹¹ in which R¹⁰ and R¹¹ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, halo, -C(O)R¹² in which R¹² is an optionally- substituted aliphatic hydrocarbyl group, -C(O)OR¹³ in which R¹³ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, CN, - NO₂, and -C(O)NR¹⁴R¹⁵ in which R¹⁴ and R¹⁵ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, or wherein: R¹ is H and

R², which is optionally linked through a linker group selected from alkylene, alkenylene and alkynylene, is selected from an optionally- substituted carbocyclic aromatic group, a heterocyclic aromatic group which is unsubstituted, a substituted bicyclic heterocyclic aromatic group, a substituted monocyclic heterocyclic group containing five ring atoms; a substituted monocyclic heterocyclic group containing six ring atoms comprising at least two heteroatoms in the ring; a substituted pyridyl group attached at the 2-, 3- or 4- position on the pyridyl; a substituted 5-pyridyl which is substituted at at least one of ring positions 3, 4 and 6 of the pyridyl and optionally-substituted at the ring position 2 of the pyridyl or is a substituted 5-pyridyl which is substituted only at the ring position 2 of the pyridyl by a group selected from fluoro, bromo, iodo, OR¹⁶, S(O)_nR¹⁷, -NR¹⁸R¹⁹, -C(O)R²⁰, C(O)OR²¹, CN, NO₂ and -C(O)NR²²R²³, in which the groups R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ and n are as defined above;

R³, R⁴, R⁵, R⁶ and R⁷ are each independently selected from hydrogen, optionally-substituted hydrocarbyl, OR⁸ in which R⁸ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(O)R⁹ in which R⁹ is an optionally-substituted aliphatic hydrocarbyl group and n is 0, 1 or 2 or is hydrogen and n is 0, -NR¹⁰R¹¹ in which R¹⁰ and R¹¹ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, halo, -C(O)R¹² in which R¹² is an optionally- substituted aliphatic hydrocarbyl group, -C(O)OR¹³ in which R¹³ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, CN, - NO₂, and -C(O)NR¹⁴R¹⁵ in which R¹⁴ and R¹⁵ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, any of its enantiomers or any mixture thereof or a pharmaceutically- acceptable salt thereof. Specific examples of compounds according to the present invention include the compounds of the formula I in which groups R¹ and R² are as follows: (each of groups R³-R⁵ in all cases is H).

Cod No Bl Bf

1 H 3-pyridyl

2 H phenyl

3 H 2-pyridyl

4 CH₃ phenyl

5 H 4-pyridyl

6 H 5-pyrimidinyl

7 H o-methoxyphenyl

8 H m-methoxyphenyl

9 H β-methoxyphenyl 10 H 3-pyridazinyl

11 H 3-pyrazinyl

12 H 3-quinolyl

13 H 2-(2-pyridyl)-ethenyl

14 H 2-naphthyl

15 H 1 -naphthyl

16 H p_-chlorophenyl

17 H 2-indolyl

The compounds 1 to 17 have been prepared and isolated by us as the hydrochloride and the trifluoroacetate salts.

It will be understood, from the general formula I above, that the compounds of the invention may contain one or more chiral centres. The compounds may, therefore, be in the form of racemic mixtures or may be in the form of a single enantiomer or a composition that includes not less than 90% by weight of a single enantiomer. The compounds of the present invention may be in the form of a pharmaceutically-acceptable salt. Such salt anions that may be employed in this respect are well-known to those skilled in the art and include, for example, hydrochloride, chloride, sulphate, carbonate, citrate, lactate and phosphate.

The compounds of the present invention may be prepared by a method comprising the steps of:

(1) reacting a compound of formula II

in which R³, R⁴, R⁵, R⁶ and R⁷ are as defined above with the compound R²-Li, where R² is as defined above in an anhydrous organic solvent in the presence of ZnCI₂ and Pd(P Ph₃) in an inert atmosphere to give a compound having the formula III

and

(2) treating the compound of the formula III above to hydrolysis under acid conditions to give the product compound of the formula I.

We have found that a compound of the formula I, in which R² is a 4- pyridinyl group, can be prepared by a modified procedure by reacting a compound of the formula II, in which R³, R⁴, R⁵, R⁶ and R⁷ are as defined above, with 3-bromo-4-lithio-pyridine in an anhydrous organic solvent in the presence of ZnCI₂ and Pd(PPh₃) in an inert atmosphere to give a compound of the formula Ilia

wherein R^a is a 3-bromopyridin-4-yl group and then subjecting the compound of the formula Ilia above to reducing conditions to convert the R^a group to a 4-pyridinyl group. Following this reduction, the compound is subjected to acid hydrolysis as mentioned above to give the desired product compound of the formula I wherein R² is a 4-pyridinyl group.

The vinyl triflate compound II, according to the method described above, is subjected to coupling using Pd(O)-catalysis and an organozinc species generated in situ from a lithium-R² species and ZnCI₂ to give the N- vinyloxycarbonyl intermediate III.

The coupling reaction is carried out under an inert atmosphere, for example dry nitrogen, in an anhydrous organic solvent, such as anhydrous tetrahydrofuran. The starting vinyl triflate compound II may be obtained by subjecting a compound of the formula P, which is available in three steps from cis-1 ,5-cyclooctanediol (Kulkarni, S.U. et al., Heterocycles 1982, 18, 321. Wiseman J.R. et al., J.Org.Chem. 1986, 51 , 2485),

to N-demethylation with vinyl chloroformate to give the carbamate Q

which may then be converted to the vinyl triflate using the Comins reagent (Comins D.L.; Dehghani A. Tetrahedron Lett. 1992, 33, 6299, O'Neill l.A. et al., Synlett 1995, 151).

The resulting N-vinyloxycarbonyl intermediate III may be hydrolysed in the presence of aqueous acid, for example dilute hydrochloric acid, to produce the product compound of the formula I in which R¹ is H.

The product compound may be purified by a process which initially involves conversion to the protected N-tert. butyl intermediate IV

by reaction of the compound of formula I with di-tert-butyldicarbonate in the presence of triethylamine.

The protected intermediate IV is amenable to purification, such as by flash chromatography, and, after its purification, can be converted back to the biologically-active product compound by acid hydrolysis.

The intermediates of formulas III and IV are believed to be novel and, thus, form a further aspect of the invention.

As mentioned above, compounds of the formula I have use as pharmaceutically-active ingredients in the treatment of an animal body, including the human body, suffering from a disease or disorder which is responsive to the activity of nAChR modulators. The dosage administered to the animal body in need of therapy will, of course, depend on the actual active compound used, the mode of treatment and the type of treatment desired. The active compound may, of course, be administered on its own or in the form of an appropriate medicinal composition containing, for instance, an appropriate pharmaceutically-acceptable carrier or diluent. Other substances can, of course, also be employed in such medicinal compositions, such as antioxidants and stabilisers the use of which is well know to persons skilled in the art.

EXAMPLES

EXAMPLE 1 : 2-Phenyl-9-azabicvclor4.2.1lnon-2-ene hydrochloride salt

Step l :

Preparation of 2-Phenyl-9-vinyloxycarbonyl-9-azabicvclor4.2.11non-2-ene

To a solution of iodobenzene (153mg, 0.75mmol) in dry THF (5cm³) at -78°C under a nitrogen atmosphere was added nBuLi (1.6M in hexanes, 0.46cm³, 0.75mmol) dropwise and the solution stirred at -78°C for 20 minutes. Zinc chloride (101mg, 0.75mmol) in dry THF (5cm³) was added and the solution allowed to warm to room temperature. Vinyl triflate (95mg, 0.277mmol) in dry THF (5cm³) was then added via a cannula followed by Pd(PPh₃)₄ (16mg, 0.014mmol) in dry THF (5cm³) also via a cannula. The solution was heated to reflux for 3 hours then allowed to cool before being quenched with saturated aqueous ammonium chloride solution (30cm³). The organic layer was separated and the aqueous layer washed with diethyl ether (3 x 20cm³). The combined organic extracts were dried (Na₂SO₄) and the solvent removed in vacuo. The residue was purified by flash chromatography (60 H silica) to give, by elution with ethyl acetate: light petroleum, 1:9, a colourless solid. Recrystallisation from toluene gave the title compound as colourless crystals (37mg, 50%).m.p. 49-51°C (Found M⁺ 269.1414, Cι₇H₁₉N₂0₂ requires M⁺ 269.1416); v_maχ (CHCI₃ solution/cm^'1) 1706 (S), 1427 (S), 1149 (S), δ_H (300 MHz; CDCI₃) 7.58-7.18 (6 H, m, ArH and CH=CH₂), 5.84 (1 H, t, J=5.9 Hz, CH of vinyl), 5.00 (1 H, d, J=8.2 Hz, bridgehead CH), 4.78 (0.5 H, dd, J=14.0, 1.5 Hz, trans CHjpCH), 4.62-4.52 (1.5 H, m, trans CH₂=CH and bridgehead CH), 4.44 (0.5 H, dd, J=6.2, 1.5 Hz, cis CH CH), 4.38 (0.5 H, dd, J=6.2, 1.5 Hz, cis CH^CH), 2.50-1.65 (8 H, m, ChU); m/z (El) 269 (M⁺, 14%), 226 (78), 198 (28), 183 (26), 86 (72), 84 (100). Step 2:

Preparation of 2-Phenyl-9-butoxycarbonyl-9-azabicyclo[4.2. Hnon-2-ene

To a solution of 2-(2-phenyl)-9-vinyloxycarbonyl-9-azabicyclo[4.2.1]non-2- ene (37mg, 0.137mmol) in dioxane (4cm³) was added water (1.0cm³) and concentrated hydrochloric acid (0.2cm³). The mixture was heated to reflux for 22 hours then allowed to cool. The majority of the dioxane was evaporated in vacuo and the residue diluted with water (5cm³), cooled to 0°C and made basic by the dropwise addition of 5M NaOH solution. The basic solution was extracted with dichloromethane (5 x 5cm³) and the combined organic phases evaporated in vacuo. The residue was taken up in 2:1 THF:water (7.5cm³) and triethylamine (0.037cm³, 0.26mmol) was added followed by di-tert- butyldicarbonate (87mg, 0.40mmol). The resulting solution was left standing at room temperature for 30 hours after which time the THF was evaporated in vacuo and the residue diluted with water (5cm³) and extracted with dichloromethane (5 x 5cm³). The combined organic phases were dried (Na₂SO₄) and then evaporated in vacuo to give a yellow oil which was purified by flash chromatography to give, by elution with ethyl acetate: light petroleum, 1:3, the title compound (32mg, 78%) as a clear oil. (Found M⁺ 299.1873, C₁₉H₂₅NO₂ requires M⁺ 299.1885); v_max (thin film/cm-¹) 1691 (S), 1406 (S), 1110 (M), δ_H (300 MHz; CDCI₃) 7.53 (1 H, m, ArH), 7.38-7.18 (4 H, m, ArH), 5.81 (1 H, m, CH of vinyl), 4.98 (0.3 H, d, J=7.4 Hz, bridgehead CH), 4.84 (0.7 H, d, J=7.4 Hz, bridgehead CH), 4.46 (0.6 H, m, bridgehead CH), 4.37 (0.4 H, m, bridgehead CH), 2.42-1.61 (8 H, m, CHa) 1.48 (3.5 H, s, ^lBu), 1.32 (5.5 H, s, ^lBu); m/z (El) 299 (M⁺, 57%), 274 (55), 258 (38), 250 (13), 243 (33), 84 (100). Step 3:

Preparation of 2-Phenyl-9-azabicvclor4.2.11non-2-ene hydrochloride salt

2-(2-Pyridinyl)-9-tert-butoxycarbonyl-9-azabicyclo[4.2.1]non-2-ene (148mg, 0.494mmol) in dioxane (9cm³) and 2M aqueous HCI (18cm³) stirred at room temperature for 14 hours, solvent removed on a freeze drier. Residue purified by flash chromatography with 60 H silica gel to give, by elution with 0-»5-»10% methanol in chloroform, the title compound as a colourless glass (90mg, 77%). (Found M⁺ 199.1367, Cι₃Hι₆N₂ requires M⁺ 199.1361); δ_H (270 MHz; CD₃OD) 7.37-7.24 (5 H, m, ArH), 6.21 (1 H, m, C=CH), 4.65 (1 H, m, CH), 4.28 (1 H, m, CH), 2.72-1.85 (8 H, m, CH₂); m/z (Cl) 215 (M⁺, 100%), 158 (8), 96 (30), 85 (38).

EXAMPLE 2: 2-(2-Pyridinyl)-9-azabicvclof4.2.nnon-2-ene hydrochloride salt

Step l :

Preparation of 2-(2-Pyridinyl)-9-vinyloxycarbonyl-9-azabicyclof4.2.11non-2-ene To a solution of 2-bromopyridine (136mg, 0.855mmol) in dry THF (7cm³) cooled to -78°C under a nitrogen atmosphere was added nBuLi (1.6M in hexanes, 0.53cm³, 0.855mmol) dropwise and the solution stirred at -78°C for 20 minutes. Zinc chloride (117mg, 0.855mmol) in dry THF (3cm³) was added and the solution allowed to warm to room temperature. Vinyl triflate (108mg, 0.317mmol) in dry THF (7cm³) was then added via a cannula followed by a solution of Pd(PPh₃) (11.6mg, 0.0095mmol) in dry THF (3cm³) also via a cannula. The solution was heated to reflux for 1 hour. After this time the mixture was allowed to cool then quenched with saturated aqueous ammonium chloride solution (30cm³). The organic layer was separated and the aqueous layer extracted with diethyl ether (3 * 20cm³). The combined organic phases were dried (Na₂SO ) and the solvent removed in vacuo. The residue was purified by flash chromatography to give, by elution with ethyl acetate: light petroleum, 1 :3, the title compound (72mg, 84%) as a pale yellow oil. (Found MH⁺ 271.1442, Cι₆Hι₈N₂O₂ requires MH⁺ 271.1446); v_max (thin film/cm^"1) 1713 (S), 1420 (S), 1147 (S), δ_H (300 MHz; CDCI₃) 8.57 (1 H, m, ArH), 7.78-7.62 (1.5 H, m, ArH), 7.46 (0.5 H, d, J=8.1Hz, ArH), 7.25-7.10 (2 H, m, ArH and CH=CH₂), 6.50 (0.4 H, m, CH-vinyl), 6.42 (0.6 H, m, CH-vinyl), 5.44 (0.6 H, d, J=9.2 Hz, bridgehead CH), 5.29 (0.4 H, d, J=9.0 Hz, bridgehead CH), 4.78 (0.5 H, dd, J=14.0, 1.6 Hz, trans CJ±^CH), 4.62-4.52 (1 H, m, bridgehead CH), 4.43 (0.5 H, dd, J=6.3, 1.5 Hz, cis CH₂=CH), 4.28 (0.5 H, dd, J=13.9, 1.3 Hz, trans CH CH), 4.24 (0.5 H, dd, J=6.3, 1.2 Hz, cis CH₂=CH), 2.60-1.65 (8 H, m, ChU); m/z (Cl) 271 (M⁺, +H, 49%), 227 (100), 184 (7), 157 (14), 84 (18). Step 2:

Preparation of 2-(2-Pyridinyl) -9-tert-butoxycarbonyl-9-azabicyclor4.2.11non-2-ene

To a solution of 2-(2-Pyridinyl)-9-vinyloxycarbonyl-9-azabicyclo[4.2.1]non- 2-ene (72mg, 0.267mmol) in dioxane (8cm³) was added water (2cm³) and concentrated hydrochloric acid (0.4cm³). The mixture was heated to reflux for 22 hours then allowed to cool. The majority of the dioxane was evaporated in vacuo and the residue diluted with water (5cm³), cooled to 0°C and made basic by the dropwise addition of 5M NaOH solution. The basic solution was extracted with dichloromethane (5 x 10cm³) and the combined organic phases evaporated under reduced pressure. The residue was taken up in 2:1 THF:water (20cm³) and triethylamine (0.074cm³, 0.53mmol) was added followed by di-tert-butyldicarbonate (170mg, 0.78mmol). The resulting solution was left standing at room temperature for 30 hours after which time the THF was evaporated in vacuo and the residue diluted with water (5cm³) and extracted with dichloromethane (5 x 10cm³). The combined organic phases were dried (Na₂SO₄) and then evaporated under reduced pressure to give a yellow oil which was purified by flash chromatography to give, by elution with ethyl acetate: light petroleum, 1 :1 , an off-white solid. This was then recrystallised from n-hexane to give the title compound (47mg, 58%) as colourless crystals, m.p. 93-94°C. (Found M⁺ 300.1835, Cι₈H₂ N₂O₂ requires M⁺ 300.1837); v_ma_X (CHCI₃ solution/cm^'1) 1683 (S), 1415 (S), δ_H (300 MHz; CDCI₃) 8.53 (1 H, m, ArH), 7.67-7.58 (1.3 H, m, ArH), 7.42 (0.7 H, d, J=7.8 Hz, ArH), 7.15-7.07 (1 H, m, ArH), 6.44 (0.3 H, m, CH of vinyl), 6.36 (0.7 H, m, CH of vinyl), 5.26 (1 H, d, J=8.8 Hz, bridgehead CH) 4.46 (0.6 H, m, bridgehead CH), 4.38 (0.4 H, m, bridgehead CH), 2.59 (8 H, m, Cl±>), 1.46 (3 H, s, ^lBu), 1.17 (6 H, s, *Bu); m/z (El) 300 (M\ 22%), 281 (10), 244 (74), 235 (41), 227 (45), 84 (100). Step 3

Preparation of 2-(2-Pyridinyl) -9-azabicvclor4.2.1lnon-2-ene hydrochloride salt To 2-(2-Pryidinyl)-9-tert-butoxycarbonyl-9-azabicyclo[4.2.1 ]non-2-ene (47mg, 0.156mmol) dissolved in dioxane (300μl) was added 2M aqueous hydrochloric acid (8cm³). The solution was stirred vigorously at room temperature for 17 hours then freeze dried to give the title compound as a colourless glass in quantitative yield. (Found M⁺ 200.1310, Cι₃HιβN₂ requires M⁺ 200.1313); δ_H (300 MHz; CD₃OD) 8.76 (1 H, m, ArH), 8.59 (1 H, t, J=8.0 Hz, ArH), 8.17 (1 H, d, J=8.2 Hz, ArH), 7.97 (1 H, m, ArH), 6.95 (1 H, m, CH of vinyl), 4.90 (1 H, d, J=8.5 Hz, bridgehead CH), 4.38 (1 H, m, bridgehead CH), 2.88-2.62 (3 H, m, ChU), 2.44-1.94 (5 H, m, CFU); m/z (El) 200 (M\ 69%), 171 (66), 157 (38), 144 (43), 131 (54) 105 (100), 77 (40).

EXAMPLE 3: 2-(4-Pyridinyl)-9-azabicvclo.4.2.11non-2-ene hydrochloride salt Step 1:

Preparation of 2-(3-bromo-4-pyridinyl)-9-vinyloxycarbonyl-9- azabicyclor4.2. Hnon-2-ene

To diisopropylamine (324μl, 2.32mmol) in dry THF (5cm³) at -78°C was added n-butyllithium (1.08M in hexanes, 2.14cm³, 2.32mmol) dropwise. The solution stirred at -78°C for 10 minutes then removed from the cooling bath and allowed to warm to room temperature. A volume of this LDA solution (7.05cm³) was taken and added dropwise over 10 minutes to a solution of 3- bromopyridine (186μl, 1.935mmol) in dry THF (5cm³) stirring at -100°C and the solution stirred at -100°C for 25 minutes. After this time zinc chloride (0.5M in THF, 3.87cm³, 1.935mmol) was added dropwise and the solution allowed to warm to room temperature. Vinyl triflate (220mg, 0.64mmol) in dry THF (6cm³) was added via a cannula followed by Pd(PPh₃)₄ (37mg, 0.032mmol) in dry THF (3cm³) also via a cannula. The solution was heated to reflux for VΛ hours after which time it was allowed to cool and quenched with saturated aqueous ammonium chloride solution (20cm³) and extracted into diethyl ether (3 20cm³). The combined organic phases were dried (Na₂SO ) and the solvent removed in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution with 25- 50% ethyl acetate in petrol, the title compound as a colourless solid which was recrystallised from hexane (174mg, 77%) m.p. 80-82°C. (Found MH⁺ 349.0456, C₁₆H₁₇N₂O₂Br requires MH⁺ 349.0552); v_maχ (CHCI₃ solution/cm^"1) 2970 (M), 1699 (S), 1420 (S), 1194 (S), 910 (S), δ_H (270 MHz; CDCI₃) 8.70 (1 H, m, ArH), 8.46 (1 H, m, ArH), 7.50 (0.5 H, d, J=5 0 Hz, ArH), 7.22 (0.5 H, m, HC =CH₂), 7.18 (0.5 H, m, HC =CH₂), 7.12 (0.5 H, d, J=4.6 Hz, ArH), 5.79 (1 H, M, CH =C), 4.80 (0.5 H, dd, J=14.1 , 1.3 Hz, CH=CH₂), 4.67 (1 H, m, CH), 4.56 (1 H, m, CH), 4.47 (0.5 H, dd, J=6.3,.1.6 Hz, CH^tU), 4.30 (0.5 H, dd, J=6.3, 1.3 Hz, CH=CJH₂), 4.24 (0.5 H, dd, J=13.8, 1.6 Hz, CH^hb), 2.64-1.62 (8 H, m, CH₂); m/z (Cl) 351 (MH⁺, 90% ⁸¹Br), 349 (MH⁺ 100%, ⁷⁹Br), 305 (95), 307 (100), 227 (50), 85 (82). Step 2:

Preparation of 2-(4-Pyridinyl) -9-vinyloxycarbonyl-9-azabicvclor4.2.nnon-2-ene Freshly cut lithium (106mg, 15.32mmol) and naphthalene (1.99g, 15.57mmol) in dry THF (15cm³) under a nitrogen atmosphere stirred at room temperature for 2 hours. 0.95cm³ of this solution removed and transferred to a clean dry flask. Zinc chloride (0.5M in THF, 0.97cm³, 0.485mmol) added dropwise over 10 minutes. 2-(3-Bromo-4-pyridinyl)-9-vinyloxycarbonyl-9- azabicyclo[4.2.1]non-2-ene (85mg, 0.243mmol) in dry THF (4cm³) added and the suspension heated to reflux for 1 hour. The mixture was allowed to cool and quenched with water (10cm³). The organic phase was separated and the aqueous phase extracted with dichloromethane (2 x 10cm³) and ethyl acetate (3 x 10cm³). The combined organic residues were dried (Na₂SO₄) and then the solvent removed in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution 50→100% ethyl acetate: in petrol, the title compound as a colourless oil (55mg, 83%). (Found MH⁺ 271.1458, Cι₆H₁₈N₂O₂ requires MH⁺ 271.1447); v_max (CHCI₃ solution/cm- ¹) 2964 (M), 1711 (S), 1425 (S), 1149 (S), 1597 (M), 1648 (M), δ_H (270 MHz; CDCI₃) 8.55 (2 H, m, ArH), 7.55 (1 H, m, ArH), 7.31 (1 H, m, ArH), 7.24 (0.5 H, dd, J=14.2, 6.3 Hz, CH=CH₂), 7.18 (0.5 H, dd, J=14.2, 6.3 Hz, CH=CH₂), 6.10 (1 H, m, CH=C), 4.96 (1 H, m, CH), 4.80 (0.5 H, dd, J=13.8, 1.6 Hz, CH=CH2), 4.60 (1 H, m, CH), 4.50 (0.5 H, dd, J=13.8, 1.6 Hz, CH^tb), 4.46 (0.5 H, dd, J=6.3, 1.6 Hz, CH=CH₂), 4.37 (0.5 H, dd, J=6.3, 1.6 Hz, CH=CH₂), 2.56-1.64 (8 H, m, CH₂); m/z (Cl) 271 (MH\ 66%), 227 (100), 199 (30), 184 (34).

Step 3

Preparation of 2-(4-PyridinvD -9-tert-butoxycarbonyl-9-azabicvclor4.2.nnon-2- ene hydrochloride salt

To a solution of 2-(4-Pyridinyl)-9-vinyloxycarbonyl-9-azabicyclo[4.2.1]non- 2-ene (90mg, 0.267mmol) in dioxane (8cm³) was added water (2cm³) and concentrated hydrochloric acid (0.4cm³). The mixture was heated for 22 hours then allowed to cool. The majority of the dioxane was evaporated in vacuo and the residue diluted with water (5cm³), cooled to 0°C and made basic by the dropwise addition of 5M NaOH solution. The basic solution was extracted with ethyl acetate (5 x 10cm³) and the combined organic phases evaporated in vacuo. The residue was taken up in 2:1 THF:water (20cm³) and triethylamine (0.093cm³, 0.66mmol) was added followed by di-tert- butyldicarbonate (212mg, 0.98mmol). The resulting solution was left standing at room temperature for 44 hours after which time the THF was evaporated under reduced pressure and the residue diluted with water (5cm³) and extracted with dichloromethane (3 10cm³). The combined organic phases were dried (Na₂SO ) and the solvent removed in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution with 50→100% ethyl acetate in petrol, the title compound as a colourless oil (63mg, 64%). (Found MH⁺ 301.1923, C₁₈H₂₄N₂O2 requires 301.1916); v_maχ (CHCI₃ solution) 2980 (S), 1681 (S), 1413 (S), 1168 (S), 1118 (S); δ_H (270 MHz, CD₂CL₂) 8.48 (2 H, d, J=4.6 Hz, ArH), 7.43 (1 H, d, J=5.9 Hz, ArH), 7.23 (1 H, d, J=5.9 Hz, ArH), 6.04 (1 H, m, CH=C), 4.85 (0.6 H, d, J=9.2 Hz, CH), 4.78 (0.4 H, d, J=8.6 Hz, CH), 4.38 (1 H, m, CH), 2.48-1.58 (8 H, m, CH₂), 1.44 (4 H, s, 'Bu), m/z (Cl) 301 (MH⁺, 1%), 277 (2), 244 (50), 200 (100), 171 (60) 144 (55). Step 4

Preparation of 2-(4-Pyridinyl) -9-azabicyclo[4.2.11non-2-ene hydrochloride salt 2-(4-Pyridinyl)-9-tert-butoxycarbonyl-9-azabicyclo[4.2.1]non-2-ene (8mg, 26μmol), dioxane (75μl) and 2M aqueous HCI (2cm³) stirred at room temperature for 14 hours, solvent removed on freeze drier to give the title compound as a colourless glass in quantitative yield. (Found MH⁺ 201.1392, Cι₃H₁₆N₂ requires MH⁺ 201.1392) δ_H (270 MHz; CD₃NO₂) 10.47 (1 H, broad s, NH), 9.79 (1 H, broad s, NH), 8.71 (2 H, broad s, ArH), 8.04 (2 H, broad s, Ar H), 6.86 (1 H, m, CH=CH₂), 4.84 (1 H, m, CH), 4.42 (1 H, m, CH), 2.86-1.91 (8H, m, CH₂); m/z (Cl) 201 (MH⁺, 100%), 184 (20), 171 (6), 159 (8), 132 (8) 94 (12).

EXAMPLE 4: 2-(5-Pyrimidinyl)-9-azabicyclo[4.2.nnon-2-ene hydrochloride salt

Step 1 : Preparation of 2-(5-Pyrimidinyl)-9-vinyloxycarbonyl-9- azabicvclo[4.2. Hnon-2-ene

To a stirred solution of 5-bromopyrimidine (219mg, 1.38mmol) in dry THF (10cm³) cooled to -78°C under a nitrogen atmosphere was added nBuLi (1.08M in hexanes, 1.28cm³, 1.38mmol) dropwise and the solution stirred at -78°C for 20 minutes. Zinc chloride (0.5M in THF, 2.76cm³, 1.38mmol) was added and the solution allowed to warm to room temperature. Vinyl triflate (175mg, 0.513mmol) in dry THF (10cm³) was then added via a cannula followed by a solution of Pd(PPh₃)₄ (30mg, 0.025mmol) in dry THF (3cm³) also via a cannula. The solution was heated to reflux for 2 hours. After this time the mixture was allowed to cool then quenched with saturated aqueous ammonium chloride solution (20cm³). The organic layer was separated and the aqueous layer extracted with dichloromethane (2 x 20cm³). The combined organic phases were dried (Na₂SO ) and the solvent removed under in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution with 50→100% ethyl acetate in petrol, the title compound as a colourless solid which was recrystallised from toluene (107mg, 77%) m.p. 85-86°C. (Found MH⁺ 272.1391 , C₁₅H₁₇N₃θ₂ requires MH⁺ 272.1399) v_max (CHCI₃ solution/cm^'1) 2972 (M), 1705 (S), 1648 (M), 1424 (S), 1148 (S); δ_H (270 MHz, CDCI₃) 9.12 (1 H, s, ArH), 8.95 (1 H, s, ArH), 8.80 (1 H, s, ArH), 7.22 (1 H, m, CH=CH₂), 5.97 (1 H, m, CH=C), 4.89 (1 H, m, CH), 4.82 (0.5 H, dd, J=13.8, 1.6 Hz, CH=CH₂), 4.61 (1.5 H, m, CH=Cj-J₂ and CH), 4.48 (0.5 H, dd, J=6.6, 1.6 Hz, CH=CH₂), 4.43 (0.5 H, dd, J=6.8, 1.6 Hz, CH^ϋ), 2.58-1.68 (8 H, m, CH₂); m/z (Cl) 272 (MH⁺, 65%), 228 (100), 185 (35), 143 (42), 119 (52). Step 2: Preparation of 2-(5-Pyrimidinyl)-9-tert-butoxycarbonyl-9- azabicyclor4.2. Hnon-2-ene

To a solution of 2-(5-Pyrimidinyl)-9-vinyloxycarbonyl-9- azabicyclo[4.2.1]non-2-ene (200mg, 0.738mmol) in dioxane (12cm³) was added water (3cm³) and concentrated hydrochloric acid (0.6cm³). The mixture was heated to reflux for 22 hours then allowed to cool. The majority of the dioxane was evaporated in vacuo and the residue diluted with water (5cm³), cooled to 0°C and made basic by the dropwise addition of 5M NaOH solution. The basic solution was extracted with dichloromethane (5 x 10cm³) and the combined organic phases evaporated in vacuo. The residue was taken up in 2:1 THF:water (40cm³) and triethylamine (0.208cm³, 1.476mmol) was added followed by di-tert-butyldicarbonate (477mg, 2.214mmol). The resulting solution was left standing at room temperature for 16 hours after which time the THF was evaporated in vacuo and the residue diluted with water (5cm³) and extracted with dichloromethane (3 x 10cm³). The combined organic phases were dried (Na₂SO ) and solvent removed in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution with 25→50% ethyl acetate in petrol, the title compound as a colourless solid which was recrystallised from hexane (158mg, 71%), m.p. 91- 92°C. (Found M⁺ 301.1789, Ci7H₂₃N₃O₂ requires 301.1790); v_maχ (CHCI₃ solution/cm^"1) 2982 (S), 1686 (S), 1413 (S), 1172 (S), 1119 (S), δ_H (270 MHz; CDCI₃) 9.10 (1 H, s, ArH), 8.95 (1 H, s, ArH), 8.74 (1 H, s, ArH), 5.97 (0.5 H, m, CH=C), 5.90 (0.5 H, m, CH=C), 4.83 (0.5 H, d, J=8.3 Hz, CH), 4.75 (0.5 H, d, J=8.3 Hz) 4.50 (0.5 H, m, CH), 4.39 (0.5 H, m, CH), 2.56-1.61 (8 H, m, CH₂), 1.49 (4 H, s, ^lBu), 1.34 (5 H, s, ^lBu); m/z (El) 301 (M⁺, 1%), 277 (50), 83 (100), 57 (28). Step 3

Preparation of 2-(5-Pyrimidinyl)-9-azabicyclof4.2.11non-2-ene hydrochloride salt

2-(5-Pyrimidinyl)-9-tert-butoxycarbonyl-9-azabicyclo[4.2.1]non-2-ene (50mg, 0.16mmol) dioxane (1cm³) and 2M aqueous HCI (8cm³) stirred at room temperature for 14 hours, solvent removed on a freeze drier to give the title compound as a colourless glass in quantitative yield. (Found MH⁺ 202.1346, Cι₂H₁₅N₃ requires 202.1345); δ_H (270 MHz; CD₃NO₂), 9.96 (1 H, broad s, NH), 9.70 (1 H, broad s, NH), 9.57 (2 H, s, ArHO, 9.43 (1 H, s, ArH), 6.67 (1 H, m, CH=C), 4.86 (1 H, m, CH), 4.46 (1 H, m, CH), 2.86-1.92 (8 H, m, CH₂); m/z (Cl) 202 (MH⁺, 100%), 185 (10), 173 (9), 159 (9), 133 (8) 82 (9).

EXAMPLE 5: 2-(2-methoxyphenyl)-9-azabicvclo| .2.1lnon-2-ene hydrochloride salt

Step 1 : Preparation of 2-(2-methoxyphenyl)-9-vinyloxycarbonyl-9- azabicvclof4.2.1 lnon-2-ene

To a stirred solution of 2-bromoanisole (126μL, 1.01 mmol) in dry THF (8cm³) cooled to -78°C under a nitrogen atmosphere was added nBuLi (1.56M in hexanes, 0.65cm³, 1.01 mmol) dropwise and the solution stirred at -78°C for 20 minutes. Zinc chloride (0.5M in THF, 2.02cm³, 1.01 mmol) was added and the solution allowed to warm to room temperature. Triflate (230mg, 0.674mmol) in dry THF (8cm³) was then added via a cannula followed by a solution of Pd(PPh₃) (39mg, 0.034mmol) in dry THF (5cm³) also via a cannula. The solution was heated to reflux for 1 hour. After this time the mixture was allowed to cool then quenched with saturated aqueous ammonium chloride solution (20cm³). The organic layer was separated and the aqueous layer extracted with dichloromethane (2 20cm³). The combined organic phases were dried (Na₂SO₄) and the solvent removed under in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution with 0->25% ethyl acetate in petrol, the title compound as a colourless solid which was recrystallised from ethanol (190mg, 94%) m.p. 105-106°C. (Found M⁺ 299.1524, Cι₈H₂ιNO₃ requires 299.1521) v_max (CHCI₃ solution/cm-¹) 3009 (M), 1704 (M), 1508 (M), 1432 (M), 1245 (S); δ_H (270 MHz, CDCI3), 7.49 (1 H, d, J=8.6 Hz, ArH), 7.34 (1 H, d, J=8.6 Hz, ArH), 7.25 (1 H, m, CH₂=CH), 6.86 (2 H, d, J=8.6 Hz, ArH), 5.76 (1 H, m, CH), 4.97 (1 H, d, J=8.6 Hz, bridgehead CH), 4.78 (0.5 H, d, J=13.9 Hz, CH=CH₂), 4.64 (0.5 H, d, J=13.9 Hz, CH=CH₂) 4.56 (1 H, m, bridgehead CH), 4.43 (0.5 H, d, J=6.3 Hz, CH=CH₂), 4.40 (0.5 H, d, J=6.3 Hz, CH=CH₂)3.81 (1.5 H, s, OMe), 3.80 (1.5 H, s, OMe), 2.45-1.56 (8 H, m, CHa); m/z (El) 299 (M⁺, 76%), 256 (76), 228 (35), 213 (50), 127 (62), 118 (100). Step 2: Preparation of 2-(2-Methoxyphenyl)-9-tert-butoxycarbonyl-9- azabicvclof4.2.1 ]non-2-ene

To a solution of 2-(2-Methoxyphenyl)-9-vinyloxycarbonyl-9- azabicyclo[4.2.1]non-2-ene (170mg, 0.568mmol) in dioxane (10cm³) was added water (2.5cm³) and concentrated hydrochloric acid (0.5cm³). The mixture was heated to reflux for 16 hours then allowed to cool. The majority of the dioxane was evaporated in vacuo and the residue diluted with water (5cm³), cooled to 0°C and made basic by the dropwise addition of 5M NaOH solution. The basic solution was extracted with ethyl acetate (5 x 10cm³) and the combined organic phases evaporated in vacuo. The residue was taken up in 2:1 THF:water (30cm³) and triethylamine (0.159cm³, 1.136mmol) was added followed by di-tert-butyldicarbonate (363mg, 1.704mmol). The resulting solution was left standing at room temperature for 16 hours after which time the THF was evaporated in vacuo and the residue diluted with water (5cm³) and extracted with dichloromethane (3 x 10cm³). The combined organic phases were dried (Na₂SO ) and solvent removed in vacuo. The residue was purified by flash chromatography with 60 H silica gel to give, by elution with 0->25% ethyl acetate in petrol, the title compound as a colourless solid which was recrystallised from hexane (135mg, 73%), m.p. 84-85°C. (Found M⁺ 329.1990, C₂₀H₂₇NO₃ requires 329.1977); v_max (CHGI₃ solution/ cm^-1) 3029 (M), 1678 (S), 1488 (M), 1412 (S), 1252 (M), 1117 (M), δ_H (300 MHz; CDCI₃) 7.23-7.11 (2 H, m, ArH), 6.93-6.80 (2 H, m, ArH), 5.67 (1 H, m, CH), 4.66 (1 H, d, J=8.6 Hz, bridgehead CH), 4.42 (1 H, m, bridgehead CH), 3.81 (3 H, s, OMe), 2.52-1.56 (8 H, m, Chb), 1.26 (9 H, s, ^lBu); m/z (El) 329 (M⁺, 2%), 273 (32), 229 (100), 186 (60), 115 (78). Step 3

Preparation of 2-(2-Methoxyphenyl)-9-azabicvclof4.2.nnon-2-ene hydrochloride salt

2-(2-Methoxyphenyl)-9-tert-butoxycarbonyl-9-azabicyclo[4.2.1]non-2-ene (15mg, 0.45mmol) dioxane (3cm³) and 2M aqueous HCI (4cm³) stirred at room temperature for 14 hours, solvent removed on a freeze drier to give the title compound as a colourless glass in quantitative yield. (Found MH⁺ 230.1538, C₁₅H₁₉NO requires 230.1544); δ_H (270 MHz; CD₃CN), 7.25 (1 H, m, ArH), 6.87 (3 H, m, ArH), 6.15 (1 H, m, CIH), 4.58 (1 H, m, bridgehead CH), 4.22 (1 H, m, bridgehead CH), 3.78 (3 H, s, OMe), 2.64-1.71 (8 H, m, Chb), m/z 230 (MH\ 45%), 63 (97), 62 (100).

EXPERIMENTAL METHODS

ASSAY 1

Several compounds, including compounds of the invention, were assayed to obtain their binding affinities against four different nAChR subtypes associated with different tissue preparations.

The compounds assayed were as follows:

(±)-epibatidine - comparative

(±) - anatoxin - comparative

(±) - UB-165 (2-(2-chloro-5-pyridinyl)-9-azabicyclo[4.2.1 ]non-2-ene)

Cpd 1 - 2-(5-pyridinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 2 - 2-phenyl-9-azabicyclo[4.2.1]non-2-ene hydrochloride salt

Cpd 3 - 2-(2-pyridinyl)-9-azabicyclo[4.2.1]non-2-ene hydrochloride salt

Cpd 4 - N-methyl-2-phenyl-9-azabicyclo[4.2.1]non-2-ene

The different nAChR subtypes used in the Experiment were as follows: nAChR Subtype Tissue Origin α4β2 - rat brain membrane α3βx - human receptor expressed in a cell line α7 - rat brain membrane αlβlδε - rat muscle

MATERIALS AND METHODS

MATERIALS Cell Culture

SH-SY5Y cells were from ECACC (Porton Down, Salisbury, Wilts. UK) and were cultured as described by Murphy et al., (1991) J. Neurochem. 57: 2116-2123. Cell culture media were provided by Gibco BRL (Paisley, Renfrewshire, Scotland) and tissue culture plasticware was obtained from Beckton Dickinson UK Ltd. (Oxford, UK) and Sterilin (Stone, Staffs. UK). Drugs and Reagents

(-)-[³H]-Nicotine (3.0 TBq/mmol in ethanol) and (+)-[³H]-epibatidine (2.1 TBq/mmol in ethanol) were provided by Dupont NEN (Stevenage, Herts. UK) and stored at -20°C. Na¹²⁵l from Amersham International was used to iodinate αbungarotoxin (αBgt) to a specific activity of 26 TBq/mmol. (+)- Epibatidine was purchased from RBI (Nateck, MA, USA) and (±)-anatoxin-a was obtained from Tocris Cookson (Bristol, UK). Racemic UB-165 (Wright et al., 1997, Biorg.Med.Chem.Lett. 7:2867-2870). All other drugs and reagents were provided by Sigma (Poole, Dorset, UK).

METHODS Tissue Preparations

Rat Brain Membranes

P2 membranes from whole rat brain (minus cerebellum) were prepared as previously described (Davies et al., 1999, Neuropharmacol. 38:679-690). Briefly, brains were homogenised (10% w/v) in ice cold 0.32M sucrose, pH 7.4, containing 1mM EDTA, 0.1 mM PMSF and 0.01% NaN₃, and centrifuged at 1000g for 10 minutes. The supernatant fraction (S1) was decanted and retained on ice. The pellet (P1) was resuspended in ice cold 0.32M sucrose (5ml/g original wt), and recentrifuged at 1000g for 10 minutes. The supernatant was combined with S1 and centrifuged at 12000g for 30 minutes. The pellet (P2) was resuspended (2.5ml/g original wt) in Phosphate Buffer (50mM potassium phosphate, pH 7.4, containing 1mM EDTA, 0.1 mM PMSF and 0.01% NaN₃), and washed twice by centrifugation at 12000g for 30 minutes. The washed pellet was resuspended in Phosphate Buffer (2.5ml/g original wt) and stored in 5ml aliquots at -20°C. Rat Muscle Extract Preparation

A Triton X-100 extract of muscle from the hind limbs of Wistar rats was prepared as previously described (Garcha et al., 1993. Psychopharmacol 110:347-354). SH-SY5Y Cell Membrane Preparation

SH-SY5Y cells, grown to confluency in 175cm² flasks, were briefly washed with warm phosphate-buffered saline (PBS; 150mM NaCI, 8mM K₂HPO₄, 2mM KH₂PO₄ pH 7.4, 37°C) and scraped into cold Phosphate Buffer. Cells were washed by centrifugation for 3 minutes at 500g and resuspended in 10ml of ice cold Phosphate Buffer. The suspension was homogenised for 10 seconds using a Ultraturax and centrifuged for 30 minutes at 45,000g. The pellet was resuspended in Phosphate Buffer (0.5ml per original flask).

Radioligand Binding Assays

(-H³Hl-Nicotine Competition Binding Assays, Rat Brain Membranes

P2 membranes (250μg protein) were incubated in a total volume of 250μg in HEPES buffer (20mM HEPES, pH 7.4, containing 118mM NaCI, 4.8mM KCI, 2.5mM CaCI₂, 200mM Tris, 0.1 mM PMSF, 0.01% (w/v) NaN₃ Romm et al., 1990) with 10nM (-)-[³H]-nicotine and serial dilutions of test drugs. Nonspecific binding was determined in the presence of 100μM (-)-nicotine. Samples were incubated for 30 minutes at room temperature followed by 1 H at 4°C. The reaction was terminated by filtration through Whatman GFA/E filter paper (presoaked overnight in 0.3% polyethyleneimine (PEI) in PBS), using a Brandel Cell Harvester. Filters were counted for radioactivity in 5ml Optiphase "Safe" scintillant in a Packard Tricarb 1600 scintillation counter (counting efficiency 45%). (-)-.³H1-Epibatidine Competition Binding Assays. SH-SY5Y Cells

SH-SY5Y membranes (30μg protein) were incubated in a total volume of 2ml in 50mM Phosphate Buffer with 150pM (±)-[³H]-epibatidine and serial dilutions of test drugs. Non-specific binding was determined in the presence of 100μM (-)-nicotine. Samples were incubated for 2 hours at 37°C. They were filtered and counted for radioactivity as described above. [¹²⁵l1-αBgt Competition Binding Assays, Rat Brain Membranes

P2 membranes (250μg protein) were incubated in a total volume of 250μl in 50mM Phosphate Buffer with 1nM [¹²⁵l]-αBgt and serial dilutions of test drugs (Davies et al., 1999, Supra). Non-specific binding was determined in the presence of 10μM αBgt. Samples were incubated for 3 hours at 37°C. Ice cold PBS (0.5ml) was then added, and the samples were centrifuged for 3 minutes at 10,000g. Pellets were washed by resuspension in 1.25ml PBS and centrifugation as before. The resultant pellets were counted for radioactivity in a Packard Cobra II auto-gamma counter. f¹²⁵l1-αBgt Competition Binding Assays, Rat Muscle Extract

Rat muscle extract (1.5mg protein) was incubated in a total volume of 500μl in 2.5mM sodium phosphate buffer, pH 7.4, with 1 nM [¹²⁵l]-αBgt and serial dilutions of test drugs (Garcha et al., 1993, Supra). Non-specific binding was determined in the presence of 10μM αBgt. Samples were incubated for 2 hours at 37°C. Bound radioligand was separated by filtration through Whatman GF/C filters (presoaked in 0.3% PEI in PBS overnight) using a Millipore vacuum manifold. Filters were washed three times with 3ml cold PBS supplemented with 0.1% BSA, and counted for radioactivity in a Packard Cobra II auto-gamma counter.

DATA ANALYSIS

Competition Binding

IC50 values were calculated by fitting data points to the Hill equation, using the non-linear least squares curve fitting facility of Sigma Plot V2.0 for windows, K-i values were derived from IC₅₀ values according to the method of Cheng and Prusoff (1973), Biochem Pharmacol 22:3089-3108, assuming K_<j values of 10nM and 1nM for [³H]nicotine and [¹²⁵l]αBgt binding to rat brain membranes and 0.12nM for [¹²⁵l]αBgt binding to rat muscle, respectively.

RESULTS

Competition Binding Assays

Compounds of the invention were compared with (±)-anatoxin-a, (±)- epibatidine and (±) UB-165 for their ability to displace the binding of a number of nicotinic radioligands. The majority of high affinity [³H]-nicotine binding sites in rat brain are considered to represent the α4β2 subtype (Zoli et al., 1998 J.Neurosci 18:4461-4472). (±)-Anatoxin-a and (±)-epibatidine inhibited [³H]-nicotine binding with Kι values of 1.25 ± O.OOδnM respectively while (±) UB-165 displayed an intermediate potency, with a Ki value of 0.27 ± 0.05nM. The Kι values for the compounds screened are shown below in Table 1. (+)- Anatoxin-a and (±)-epibatidine competed with [¹²⁵l]-αBgt for binding to putative α7-type nAChR in rat brain membranes with Kj values of 1840 ± 250nM and 233 ± 69nM respectively. (±) UB-165 exhibited slightly lower potency than ±- anatoxin-a, having a Kι value of 2790 ± 370nM.

[³H]-Epibatidine labels cc3βx containing nAChRs in human neuorblastoma SH-SY5Y cells (Wang et al., 1996 J.Biol Chem 271 :17656-17685). The concentration of [³H]-epibatidine used (150pM) was chosen to preferentially label putative α3β2 nAChR, but Kj values have not been derived because of the inherent nAChR heterogeneity. (±)-UB-165 was also examined for its ability to displace [¹²⁵l]- Bgt from rat muscle extract: its Kι value of 990 ± 240nM was similar to that of (±)-epibatidine (Kj =610 ± 160nM), whereas (±)- anatoxin-a was the most potent competing ligand with a Kj value of 85 ± 41nM.

The ability of each of these ligands to discriminate between nAChR subtypes was expressed as an affinity ratio, relative to the value at the rat brain [³H]nicotine binding site, which was defined as 1.

TABLE 1 - AFFINITIES FOR RADIOLIGAND BINDING SITES CORRESPONDING TO VARIOUS nAChR SUBTYPES

Compound 2, although less potent than UB-165 having regard to the binding constant values is relatively much more potent at the α3βx subtype than compared with compounds 3 and 5.

The results indicate that the compounds of the invention differentiate between different nAChR subtypes.

ASSAY 2

Several compounds were assayed to obtain their binding affinities against four different nAChR subtypes.

The compounds assayed were as follows:

(±)-epibatidine - comparative

(±) - anatoxin-a - comparative

(+) - UB-165 (2-(2-chloro-5-pyridinyl)-9-azabicyclo[4.2.1]non-2-ene)

Cpd 1 2-(5-pyridinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 2 2-phenyl-9-azabicyclo[4.2.1]non-2-ene

Cpd 3 2-(2-pyridinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 4 N-methyl-2-phenyl-9-azabicyclo[4.2.1]non-2-ene

Cpd 5 2-(4-pyridinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 6 2-(5-pyrimidinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 7 2-o-methoxyphenyl-9-azabicyclo[4.2.1]non-2-ene

Cpd 8 2-m-methoxyphenyl-9-azabicyclo[4.2.1]non-2-ene

Cpd 9 2-p_-methoxyphenyl-9-azabicyclo[4.2.1]non-2-ene

Cpd 10 2-(3-pyridazinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 11 2-(3-pyrazinyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 12 2-(3-quinolyl)-9-azabicyclo[4.2.1]non-2-ene

Cpd 13 2-(2-(2-pyridinyl)ethenyl)-9-azabicyclo[4.2.1]non-2-ene

Epiboxidine

All compounds 1 to 3, which are all racemic, were isolated, after synthesis, and screened as the corresponding hydrochloride salt. Epibatidine, Anatoxin-a and Epiboxidine are standard ligands for nAChR subtypes.

The different nAChR subtypes against which the compounds were assayed were: nAChR Subtype Tissue Origin α4β2 - rat brain membrane α3β4 - expressed by a modified L929 cell line α7 - rat brain membrane αlβlδγ - rat muscle

The Ki values for the compounds screened are shown below in Table 2. TABLE 2: Affinity values of ligands at nAChR subtypes

Claims

1. A 9-azabicyclo[4.2.1]non-2-ene compound of the general formula I

wherein:

R 1¹ is hydrogen or an aliphatic hydrocarbyl group;

R² is an optionally-substituted carbocyclic or heterocyclic aromatic group optionally linked through a linker group selected from alkylene, alkenylene; R³, R⁴, R⁵, R⁶ and R⁷ are each independently selected from hydrogen, optionally-substituted hydrocarbyl, OR⁸ in which R⁸ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(O)R⁹ in which R⁹ is an optionally-substituted aliphatic hydrocarbyl group and n is 0, 1 or 2 or is hydrogen and n is 0, -NR¹⁰R¹¹ in which R¹⁰ and R¹¹ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, halo, -C(O)R¹² in which R¹² is an optionally- substituted aliphatic hydrocarbyl group, -C(0)OR¹³ in which R¹³ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, CN, - NO₂, and -C(O)NR¹⁴R¹⁵ in which R¹⁴ and R¹⁵ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, any of its enantiomers or any mixture thereof or a pharmaceutically- acGeptable salt thereof with the proviso that when R¹ is hydrogen and R² is a 2-chloropyrid-5-yl group at least one of the groups R³, R⁴, R⁵, R⁶ and R⁷ must be other than hydrogen.

2. A compound according to claim 1 , wherein R² is selected from optionally-substituted phenyl, pyridyl and pyrimidyl groups optionally linked through a linker group selected from methylene, ethenylene and ethynylene.

3. A compound according to claim 2, wherein R¹ is a phenyl, pyridyl or pyrimidyl group any of which may be substituted by one or more substituent groups selected from halo, OR¹⁶ in which R¹⁶ is hydrogen or an optionally-substituted aliphatic hydrocarbyl group, S(O)_nR¹⁷ in which R¹⁷ is an optionally-substituted aliphatic hydrocarbyl group and n is 0, 1 or 2 or is hydrogen and n is 0, -NR¹⁸R¹⁹ in which R¹⁸ and R¹⁹ are independently hydrogen or an optionally-substituted aliphatic hydrocarbyl group, -C(O)R²⁰ in which R²⁰ is an optionally-substituted hydrocarbyl group, -C(O)OR²¹ in which R²¹ is hydrogen or an optionally-substituted hydrocarbyl group, CN, -NO₂ and -C(O)NR²²R²³ in which R²² and R²³ are independently hydrogen or an optionally- substituted hydrocarbyl group.

4. A compound according to claim 3, wherein R² is a phenyl, pyridyl or pyrimidyl group any of which may be substituted by one or more substituent groups selected from halo and oxyalkyl containing 1 to 6 carbon atoms in the alkyl group.

5. A compound according to any of claims 1 to 4, wherein each of R³, R⁴, R⁵, R⁶ and R⁷ is hydrogen.

6. A compound according to any one of claims 1 to 5, wherein R¹ is hydrogen or methyl. A method of making a compound of formula I as defined in any one of claims 1 to 6 which comprises the steps of (1) reacting a compound of formula II

in which R³, R⁴, R⁵, R⁶ and R⁷ are as defined in claim 1 with the compound R²-Li, where R² is as defined in claim 1 , in an anhydrous organic solvent in the presence of ZnCI₂ and Pd(P Ph₃) in an inert atmosphere to give a compound having the formula III

and

(2) treating the compound of the formula III above to hydrolysis under acid conditions to give the product compound of the formula I. A method of making a compound of the formula I as defined in claim 1 wherein R² is 4-pyridinyl group which comprises the steps of reacting a compound of the formula II

in which R³, R⁴, R⁵, R⁶ and R⁷ are as defined in claim 1 with the 3-bromo-4-lithibpyridine in an anhydrous organic solvent in the presence of ZnCI₂ and Pd(PPh₃)₄ in an inert atmosphere to give a compound having the formula Ilia

wherein R^a is a 3-bromopyridin-4-yl group and then subjecting the compound of the formula Ilia above to reducing conditions to convert the R¹ group to a 4-pyridinyl group and then treating the resulting compound to hydrolysis under acid conditions to give the product compound of the formula I wherein R² is a 4-pyridinyl group.

9. A method according to either claim 7 or claim 8 wherein, the compound of the formula I is further reacted with di-tert-butyldicarbonate in the presence of triethylamine to produce a compound of the formula IV.

purifying the compound IV and then treating the compound of formula IV to acid hydrolysis to give the product compound of the formula I.

10. A pharmaceutical composition comprising a therapeutically-effective amount of a compound of any one of claims 1 to 6, or an enantiomer thereof or a pharmaceutically acceptable salt thereof together with at least one pharmaceutically-acceptable carrier or diluent.

11. The use of a compound according to any one of the claims 1 to 6 for the manufacture of a medicament for the treatment of a disease of a living animal body, including a human, which disease is responsive to the activity of nicotinic acetylcholine receptor modulators.

12. A method of treating a disease of a living animal body, including a human, which disease is responsive to the activity of nicotinic acetylcholine receptor modulators which method comprises administering to the living animal body a therapeutically-effective amount of a compound according to any one of claims 1 to 6.

3. A compound of the formula V

wherein R², R³, R⁴, R⁵, R⁶ and R⁷ are each as defined above in claim 1 and wherein Q is selected from the groups o and

H,C=CHOC- t-BuO C