This invention relates to a class of tricyclic compounds, which are useful as tachy inin receptor antagonists.

The tachykinins are a group of naturally- occurring peptides found widely distributed throughout mammalian tissues, both within the central nervous system and in the peripheral nervous and circulatory systems. The structures of three known mammalian tachykinins are as follows: Substance P: Arg-Pro-Lys-Pro-Glri-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ Neurokinin A:

His-Lys-Thr-Asp-Ser-Phe-Val-Gly- eu-Met-NH ₂ Neurokinin B: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH ₂ Substance P is believed inter alia to be involved in the neurotransmission of pain sensations [Otsuka et al, "Role of Substance P as a Sensory Transmitter in Spinal Cord and Sympathetic Ganglia" in 1982 Substance P in the Nervous System, Ciba Foundation Symposium 91, 13-34 (published by Pitman) and Otsuka and Yanagisawa, "Does Substance P Act as a Pain Transmitter?" TIPS (Dec. 1987) 8.506-510], specifically in the transmission of pain in migraine (B.E.B. Sandberg et al, J. Med Chem, (1982) 25. 1009) and in arthritis [Levine et al, in Science (1984) 226 547-549]. These peptides have also been implicated in gastrointestinal (GI) disorders and diseases of the GI tract such as inflammatory bowel disease [Mantyh et al in Neuroscience (1988) 25. (3) 817- 37 and D. Regoli in "Trends in Cluster Headache" Ed.

Sicuteri et al Elsevier Scientific Publishers, Amsterdam

(1987) page 85)].. It is also hypothesised that there is a neurogenic mechanism for arthritis in which substance P may play a role [Kidd et al "A Neurogenic Mechanism for Symmetrical Arthritis" in The Lancet, 11 November 1989 and Grόnblad et al "Neuropeptides in Synovium of Patients with Rheumatoid Arthritis and Osteoarthritis" in J. Rheumatol. (1988) 15(12) 1807-10]. Therefore, substance P is believed to be involved in the inflammatory response in diseases such as rheumatoid arthritis and osteoarthritis [O'Byrne et al in Arthritis and Rheumatism (1990) 3_3.1023-8]. Other disease areas where tachykinin antagonists are believed to be useful are allergic conditions [Hamelet et al Can. J. Pharmacol. Physiol. (1988) 6_6 1361-7] , iiomunoregulation [Lotz et al Science

(1988) 241 1218-21 and Ki ball et al, J. Immunol. (1988) 141 (10) 3564-9 ^" ], vasodilation, bronchospasm, reflex or neuronal control of the viscera [Mantyh et al, PNAS (1988) J35.3235-9] and, possibly by arresting or slowing β-amyloid-mediated neurodegenerative changes [Yankner et al, Science (1990) 250- 279-82], in senile dementia of the Alzheimer type, Alzheimer's disease and Down's Syndrome. Substance P may also play a role in demyelinating diseases such as multiple sclerosis and amyotrophic lateral sclerosis [J. Luber-Narod et. al., poster to be presented at C.I.N.P. XV IIth Congress, 28th June-2nd July, 1992, in press], and in disorders of bladder function such as bladder detrusor hyper-reflexia (Lancet. 16th May, 1992, 1239). It has furthermore been suggested that tachykinins have utility in the following disorders: depression, dysthymic disorders, chronic obstructive airways disease, hypersensitivity disorders such as poison ivy, vasospastic diseases such as angina and

Reynauld's disease, fibrosing and collagen diseases such as scleroderma and eosinophillic fascioliasis, reflex sympathetic dystrophy such as shoulder/hand syndrome, addiction disorders such as alcoholism, stress related somatic disorders, neuropathy, neuralgia, disorders related to immune enhancement or suppression such as systemic lupus erythmatosis (European patent application no. 0 436 334), opthalmic disease such as conjuctivitis, vernal conjunctivitis, and the like, and cutaneous diseases such as contact dermatitis, atropic dermatitis, urticaria, and other eczematoid dermatitis (European patent application no. 0 394 989).

In view of their metabolic instability, peptide derivatives are likely to be of limited utility as therapeutic agents. ^" It is for this reason that non- peptide tachykinin receptor antagonists are sought.

In essence, this invention provides a class of potent non-peptide tachykinin receptor antagonists. By virtue of their non-peptide nature, the compounds of the present invention do not suffer from the shortcomings, in terms of metabolic instability, of known peptide-based tachykinin receptor antagonists.

The present invention provides a compound of formula (I) , or a salt or prodrug thereof:

( I) wherein

Q represents a group

where represents a bond, 0, S, -CH CH -, -CH=CH- or a group NR ⁶ , where R is H or C^-gallyl and one or both of the phenyl rings may be replaced by a heteroaryl moiety;

X and Y each represent H or X and Y together form a group =0;

Z represents 0, S or NR ⁸ , where R ⁸ represents H or Ci-salkyl; ¹ and R ² independently represent H; C^-β alkyl optionally substituted by hydroxy, cyano, C0R ^a , COOR ^a , C0NR ^a R ^b , COC ₁ _ alkylNR ^a R ^b , CONR ^a C ₁ _ ₄ alkylCONR ^a R ^b or NR ^a R , (where R ^a and R ^b each independently represent H, C±-6 alkyl, phenyl (optionally substituted by one or more of Ci-galkyl, Ci-galkoxy, halo and trifluoromethyl) or phenyl(Cι_ ₄ alkyl) (optionally substituted in the phenyl ring by one or more of C±-Q alkyl, C^-g alkoxy, halo and trifluoromethyl)); phenyl(Cι_ alkyl), (optionally substituted by one or more of Cι_g alkyl, Cι_g alkoxy, halo and trifluoromethyl in the phenyl ring) ; C _g alkylene; COCx-galkylhalo; C0R ^a ; C00R ; C0NHR ^a ; COC ₁ _ ₄ alkylNR ^a R ^b ; or CONR ^a C ₁ _ ₄ alkylCONR ^a R ^b ; (where R ^a and R ^b are as previously defined) or R ¹ and R together form a chain (CH ₂ )p where p is 4 or 5 and where one non¬ terminal methylene group may optionally be replaced by an oxygen atom or a group NR ^X , where R ^x is H or C±-β alkyl; R ³ represents H or C-^-galkyl;

R ⁴ represents H, Cχ_ alkyl or phenyl (optionally substituted by one or more of C- _^ -g alkyl, C ₂ .g alkenyl, C ₂ _g alkynyl, halo, cyano, nitro, trifluoromethyl, trimethylsilyl, SR ^C , SOR ^c , S0 ₂ R ^c , OR ^c , NR ^c R ^d , NR ^c COR ^d , NR ^c COOR ^d , COOR ^c or CONR ^c R ^d , where R ^c and R ^d each independently represent H, Cι_,g alkyl, phenyl or trifluoromethyl) ;

R ⁵ represents (CH )qpheny1, wherein q is 0, 1, 2 or 3 which may optionally be substituted in the phenyl ring by one or more of C-^- alkyl, C ₂ _g alkenyl, C ₂ -g alkynyl, halo, cyano, nitro, trifluoromethyl, trimethylsilyl, SR ^C , SOR ^c , S0 ₂ R ^c , OR ^c , NR ^c R ^d , NR ^c C0R ^d , NR ^c COOR ^d , COOR ^c or CONR ^c R ^d , where R ^c and R ^d are as above defined; each R ⁷ and R ⁸ independently represents C- _^ -g alkyl, C _g alkenyl, C _g alkynyl, halo, cyano, nitro, trifluoromethyl, trimethylsilyl, SR ^C , SOR ^c , S0 ₂ R ^c , OR ^c , NR ^c R ^d , NR ^c COR ^d , NR ^c COOR ^d , COOR ^c or CONR ^c R ^d , where R ^c and R are as above defined; and m and n independently represent 0, 1, 2 , 3 or

4.

The alkyl, alkenyl and alkynyl groups referred to with respect to any of the above formulae may represent straight, branched or cyclic groups. Thus, for example, suitable alkyl groups include methyl, ethyl, n- or iso-propyl, n-, sec-, iso- or tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and cycloalkyl- alkyl groups such as cyclopropylmethyl; suitable alkenyl groups include vinyl and allyl; and suitable alkynyl groups include propargyl.

The term "halo" as used herein includes fluoro, chloro, bromo and iodo, especially chloro and fluoro.

- 6 -

When one or both of the phenyl rings of Q is/are replaced by a heteroaryl moiety, this will suitably be a pyridyl moiety.

A particular subgroup of compounds according to formula (I) is represented by compounds wherein Q represents a group

where W ¹ is a bond, O, S or a group NR ⁶ , where R ⁶ is H or

C _X _ ₆ alkyl;

R ¹ and R ² each independently represent H, C^_g alkyl, phenyl(C-ι_ ₄ alkyl) , COR ,1 ^J 0 COOR ¹⁰ or CONHR ¹⁰ , where R ¹⁰ is C- _^ - alkyl or phenyl;

R ⁴ represents H, Cι_g alkyl or phenyl (optionally substituted by one or more of C-^-g alkyl, C ₂ _ ₆ alkenyl, C _g alkynyl, halo, cyano, nitro, trifluoromethyl, SCH ₃ , S0CH ₃ , S0 ₂ CH ₃ , 0R ^C , NR ^c R ^d , NR ^c C0R ^d , NR ^c COOR ^d , COOR ^c or CONR ^c R ^d (where R ^c and R ^d are as above defined) ; and

R ⁵ represents (CH )qphenyl, wherein q is 0, 1, 2 or 3 which may optionally be substituted in the phenyl ring by one or more of Ci-g alkyl, C ₂ _g alkenyl, C __g alkynyl, halo, cyano, nitro, trifluoromethyl, SCH ₃ , SOCH ₃ , S0 ₂ CH ₃ , OR ^c , NR ^c R ^d , NR ^C C0R ^d , NR ^c COOR ^d , COOR ^c or C0NR ^c R ^d (where R ^c and R ^d are as above defined) ; wherein any phenyl (including benzyl) ring in the definitions of R ¹ or R ² (including R ¹⁰ ) above may be optionally substituted by one or more of Cι_g alkyl, C- _^ -g alkoxy, halo or trifluoromethyl.

In the compounds of the invention, W suitably represents a bond- CH ₂ CH ₂ or HC=CH.

Preferably Q is 10,ll-dihydro-5H-dibenzo[a,d] cyclohepten-5-yl, 5H-dibenzo[a,d]cyclohepten-5-yl or 9- fluorenyl.

Preferably X and Y each represent H.

Preferably Z represents oxa.

Suitable values for the groups R ¹ and R include H, C±-Q alkyl, especially methyl; Cι_galkyl substituted by, for example, cyano, hydroxy, NH ₂ , C0 ₂ C ₁ _galkyl and CONH ₂ .

Suitable values for the group R ³ include H and methyl, preferably H.

Preferably R ⁴ represents H. Suitably R ⁵ represents (CH )gphenyl where q is

0, 1 or 2 and the phenyl is substituted. Suitable phenyl substituents include methyl, methoxy, nitro, cyano, halo and trifluoromethyl. Preferably R represents a substituted phenyl group. More preferably R ⁵ represents 3,5-dimethylphenyl or 3,5-bistrifluoromethylphenyl.

Preferably m and n each represent O.

A preferred sub-group of compounds according to the invention is represented by formula (la)

( I σ)

wherein R , R ² and Z are as defined for formula (I) above;

Q ¹ represents 10,ll-dihydro-5H-dibenzo[a,d] cyclohepten-5-yl, 5H-dibenzo[a,d]cylcohepten-5-yl or 9- fluorenyl.

R ²⁰ and R ²¹ each independently represent H, Cι_ alkyl, C _g alkenyl, C ₂ _g alkynyl, halo, cyano, nitro, trifluoromethyl, trimethylsilyl, SR ^C , SOR ^c , S0 ₂ R ^c , OR ^c , NR-TR ¹ -, NR ^c C0R ^d , NR ^c C00R ^d , COOR ^c or C0NR ^c R ^d , where R ^c and R are as above defined; and salts and prodrugs thereof. Particularly preferred are compounds of formula

(la) wherein R ²⁰ and R ²¹ are other than hydrogen and are located in the 3- and 5-positions. Most preferably R ²⁰ and R - ^* each represent methyl or trifluoromethyl.

For use in medicine, the salts of the compounds of formula I will be non-toxic pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their non-toxic pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, oxalic acid, fumaric acid, p-toluenesulphonic acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Salts of amine groups may also comprise quaternary ammonium salts in which the a ino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety. Thus, for example, when both R ¹ and R ² are other than hydrogen, the nitrogen atom to which they are attached may be further substituted to give a quaternary ammonium salt. Furthermore, where the compounds of the invention carry

an acidic moiety, suitable pharmaceutically acceptable salts thereof m y include metal salts such as alkali metal salts, e.g. sodium or potassium salts; and alkaline earth metal salts, e.g. calcium or magnesium salts. The present invention includes within its scope prodrugs of the compounds of formula (I) above. In general, such prodrugs will be functional derivatives of the compounds of formula (I) which are readily convertible in vivo into the required compound of formula (I) . Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

The compounds according to the invention may exist both as enantiomers and as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

The invention also provides pharmaceutical compositions comprising one or more compounds of this invention in association with a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, or suppositories, for oral, parenteral or rectal administration. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-

toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate. The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium

carboxymethylcellulose, methylcellulose, polyvinyl- pyrrolidone or gelatin.

The present invention further provides a process for preparing a pharmaceutical composition comprising a compound of formula (I) , which process comprises bringing a compound of formula (I) into association with a pharmaceutically acceptable carrier or excipient.

The compounds of the present invention are of value in the treatment of a wide variety of clinical conditions which are characterised by the presence of an excess of tachykinin, in particular substance P, activity. These may include disorders of the central nervous system such as anxiety, depression, psychosis and schizophrenia; neurbdegenerative disorders such as dementia, including senile dementia of the Alzheimer type, Alzheimer's disease and Down's syndrome; demyelinating diseases such as MS and ALS and other neuropathological disorders such as peripheral neuropathy, for example, diabetic or chemotherapy-induced neuropathy, and postherpetic and other neuralgias; respiratory diseases such as chronic obstrucutive airways disease, bronchopneumonia, bronchospasm and asthma; inflammatory diseases such as inflammatory bowel disease, psoriasis, fibrositis, osteoarthritis and rheumatoid arthritis; allergies such as eczema and rhinitis; hypersensitivity disorders such as poison ivy; ophthalmic diseases such as conjunctivitis, vernal conjunctivitis, and the like; cutaneous diseases such as contact dermatitis, atropic dermatitis, urticaria, and other eczematoid dermatitis; addiction disorders such as alcoholism; stress related somatic disorders; reflex sympathetic dystrophy such as shoulder/hand syndrome; dysthymic disorders; adverse immunological reactions such

as rejection of transplanted tissues and disorders related to immune ^enhancement or suppression such as systemic lupus erythematosis; gastrointestinal (GI) disorders and diseases of the GI tract such as disorders associated with the neuronal control of viscera such as ulcerative colitis, Crohn's disease and incontinence; disorders of bladder function such as bladder detrusor hyper-reflexia; fibrosing and collagen diseases such as scleroderma and eosinophilic fascioliasis; disorders of blood flow caused by vasodilation and vasospastic diseases such as angina, migraine and Reynaud's disease; and pain or nociception, for example, that attributable to or associated with any of the foregoing conditions, especially the transmission of pain in migraine. For example, the compounds of formula (I) may suitably be used in the treatment of disorders of the central nervous system such as anxiety, psychosis and schizophrenia; neurodegenerative disorders such as senile dementia of the Alzheimer type, Alzheimer's disease and Down's syndrome; respiratory diseases such as bronchospas and asthma; inflammatory diseases such as inflammatory bowel disease, osteoarthritis and rheumatoid arthritis; adverse immunological reactions such as rejection of transplanted tissues; gastrointestinal (GI) disorders and diseases of the GI tract such as disorders associated with the neuronal control of viscera such as ulcerative colitis, Crohn's disease and incontinence; disorders of blood flow caused by vasodilation; and pain or nociception, for example, that attributable to or associated with any of the foregoing conditions or the transmission of pain in migraine.

The compounds of formula (I) are particularly useful in the treatment of pain or nociception and/or inflammation and disorders associated therewith such as.

for example, neuropathy, such as diabetic and chemotherapy-induced neuropathy, postherpetic and other neuralgias, asthma, osteroarthritis, rheumatoid arthritis and migraine. The present invention further provides a compound of formula (I) for use in the manufacture of a medicament for the treatment of physiological disorders associated with an excess of tachykinins, especially substance P. The present invention also provides a method for the the treatment or prevention of physiological disorders associated with an excess of tachykinins, especially substance P, which method comprises administration to a patient in need thereof of a tachykinin reducing amount of a compound or composition of this invention.

In the treatment of conditions involving actions of tachykinins released physiologically in response to noxious or other stimuli, a suitable dosage level is about 0.001 to 50 g/kg per day, such as 0.001 to 25 mg/kg per day, preferably about 0.005 to 10 mg/kg per day, and especially about 0.005 to 5 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once daily.

The compounds according to the invention wherein Z is O or S may be prepared by reaction of a compound of formula (II)

(N) wherein , R ¹ , R ² , R ³ , X and Y, are defined as for formula (I) and Z is O or S, with a compound of formula HalCHR ⁴ R ⁵ , where R ⁴ and R ⁵ are as defined for formula (I) and Hal is halo, such as bromo, chloro or iodo, in the presence of a base. The reaction is conveniently carried out in a suitable organic solvent, such as an ether, for example, tetrahydrofura .

Suitable bases of use in the reaction include alkali or alkaline earth metal hydrides, for example, sodium hydride.

The compounds of formula (I) prepared to the above described process may, if necessary or desired be converted to other compounds of formula (I) . Thus, for eaxmple, the compounds of the invention wherein Z is a group NR ⁸ and X and Y together represent =0 may be prepared from the compounds of formula (II) wherein Z is O and X and Y together represent =0 by reaction with a compound of formula HNR ⁸ CHR ⁴ R ⁵ in the presence of a coupling agent, such as dicyclohexylcarbodiimide. The reaction is conveniently effected in a suitable organic solvent, such as an ether, for example, diethyl ether or tetrahydrofuran.

The compounds according to the invention wherein Z is NR ⁸ and X and Y are hydrogen may be prepared

- 15 -

from the corresponding compounds of formula (I) wherein X and Y together represent =0, by reduction.

Suitable reducing agents of use in the reaction include borane and metal hydrides, such as lithium aluminium hydride. The reaction is conveniently effected in a suitable organic solvent, such as an ether, for example, tetrahydrofuran.

Compounds of formula (II) wherein Z is O and X and Y together represent a group =0 may be prepared, for example, from intermediates of formula (III)

Ph

( I I I ) wherein Q and R ³ are as above defined and Ph represents phenyl, by hydrolysis.

The reaction is conveniently effected by heating a solution of the compound of formula (III) in concentrated hydrochloric acid at reflux.

Alternatively, compounds of formula (II) wherein Z is O, X and Y are =0 and R ³ is H may be prepared from intermediates of formula (IVA)

( IV) wherein Q, R ¹ and R ² are as above defined, and R ⁴⁰ and R ⁴¹ both represent H (IVA) , by decarboxylation.

The reaction is conveniently effected by heating the compound of formula (IVA) in a concentrated mineral acid, such as concentrated hydrochloric acid, to a temperature of about 90-120*C, such as about 100*C. Compounds of formula (II) wherein Z is O and X and Y are =0 may also be prepared by conventional procedures for the preparation of a ino acids which are well documented and are described, for example, in Chemistry and Biochemistry of the Amino Acids, ed. G. C. Barrett, Chapman and Hall, 1985.

Compounds of formula (II) wherein Z is S may be prepared from the corresponding compounds of formula (II) wherein Z is 0 by treating the latter compound with Lawesson's reagent or phosphorus pentasulphide in a suitable solvent, e.g. pyridine, at ambient or elevated temperature, suitably at the reflux temperature of the chosen solvent.

Compounds of formula (II) wherein X and Y represent H may be prepared from the corresponding compounds of formula (II) wherein X and Y together represent =0, by reduction.

Suitable reducing agents include metal hydrides, such as lithium aluminium hydride. The reaction is conveniently effected in a suitable organic

solvent, such as an ether, for example, tetrahydrofur n, suitably at elevated temperature, such as the reflux temperature of the solvent.

Intermediates of formula (III) may be prepared from compounds of formula (V)

( v ) wherein R ³ is as defined for formula (I) , by reaction with a compound of formula Q-Hal wherein Hal is halo, such as bromo, chloro or iodo, in the presence of a base.

Suitable bases of use in the reaction include metal hydroxides, for example, sodium hydroxide. The reaction is conveniently effected in a mixture of water and a suitable organic solvent, such as a hydrocarbon, for example, toluene, in the presence of a phase transfer catalyst, such as benzyltrimethyl ammonium chloride. Compounds of formula (V) are commercially available or may be prepared by procedures readily apparent to one skilled in the art.

Compounds of formula (IVA) may be prepared from the corresponding compounds of formula (IV) wherein and R ⁴¹ each represnt alkyl (IVB) , by saponification. The saponification is conveniently effected using an alkali metal hydroxide, such as sodium hydroxide, in an aqueous solvent, such as aqueous alcohol, for example, aqueous methanol, preferably at elevated temperature, e.g. the reflux temperature of the chosen solvent.

Compounds of formula (IVB) may be prepared by reaction of a compound of formula Q-Hal as above defined with a malonate derivative of formula R ⁴¹ 0 ₂ CCH(NR ¹ R ² )C0 ₂ R ⁴⁰ , which malonate derivatives are commercially available or may be prepared from commercially avaible compounds by conventional means known to those skilled in the art.

Compounds of formula Q-Hal are commercially available or may be prepared by conventional procedures known to those skilled in the art.

Compounds of formula (I) may also be prepared from other compounds of formula (I) . Thus, for example, compounds of formula (I) wherein one or both of R 1 ^x and R2 ^*~ represent hydrogen may be reacted with an optionally substituted alkylating or an acylating agent to produce compounds of formula (I) wherein one or both of R ^*- and R ^*4, represent an optionally substituted alkyl or an acyl group. Suitable procedures are described in the accompanying examples, or will be readily apparent to one skilled in the art.

Conversely, compounds of formula (I) wherein one or both of R ¹ and R ² represent, for example, an acyl or a benzyl group, may be converted to compounds of formula (I) wherein one or both of R ¹ and R ² represent H by, for example, hydrolysis or catalytic hydrogenation. Suitable reagents and conditions are decribed in the accompanying examples, or will be readily apparent to one skilled in the art of organic chemistry.

Intermediates of formula (II) are novel compounds. Intermediates of formula (II) and the preparation thereof represent further aspects of the present invention.

Where the above-described process for the preparation of the compounds according to the invention

gives rise to mixtures of stereoisomers these isomers may, if desired, be separated, suitably by conventional techniques such as preparative chromatography.

The novel compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The novel compounds may, for example, be resolved into their component enantiomers by standard techniques, such as the formation of diastereomeric pairs by salt formation with an optically active acid, such as (-)-di-p-toluoyl-d- tartaric acid and/or (+)-di-p-toluoyl-1-tartaric acid followed by fractional crystallization and regeneration of the free base. The novel compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary.

During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J.F.W. McO ie, Plenum Press, 1973; and T.W. Greene and P.G.M. Wutts, Protective Groups in Organic Synthesis. John Wiley & Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.

The following Examples illustrate the preparation of compounds according to the invention.

EXAMPLE 1: l-(5H-Dibenzora,d1cyclohepten-5-yl)-2-(3,5- dimethylbenzyloxy)ethylamine, oxalate salt a) Through a cooled (-15°C) solution of dibenzosuberenol (20g) and calcium chloride (20g) in toluene (750ml) was bubbled hydrogen chloride gas for 15 minutes. The solution was warmed to room temperature over 1 hour and hydrogen chloride gas bubbled through for a further 20 minutes. After 16 hours the solution was filtered and the filtrate evaporated in υacuo to leave 5-chloro-5H-dibenzora,d]cycloheptene as a yellow crystalline solid. ^~ H NMR (360MHz, CDClg) δ 6.25 (1H, bs),

7.14 (2H, s), 7.39-7.45 (8H, m). b) To a stirred suspension of diethyl acβtamidomalonate (21.2g) in tetrahydrofuran (150ml) was slowly added sodium hydride (3.4g, 80% dispersion in oil) under an atmosphere of nitrogen. After lb to this solution was added a solution of 5- chloro-5H-dibenzo[a,d]cycloheptene (20.3g, Example la) in tetrahydrofuran (100ml). The solution was heated to reflux for 18h, cooled to room temperature and to this was added a saturated solution of NH^Cl and ethyl acetate. The organic phase was dried (MgSO^), concentrated in υacuo and the residue chromatographed on silica (eluting with ethyl acetate.petroleum ether bp 60-80 ° C ( 1 :4 ) . This gave 2-diethyl( 5H- chbenzo[a,d1cyclohepten-5-yl)-2-acetamidomalonate as a yellow solid. Η NMR (360MHz, CDCI3) δ 1.02-1.04 (6H, m), 1.6 (3H, s), 3.82-3.91 (2H, m), 4.0-4.13 (2H, m), 5.78 (1H, s), 6.39 (1H, s),

6.79 (2H, s), 7.21-7.36 (6H, m), 7.54-7.56 (2H, m).

- 21 - c) A mixture of diethyl 2-(5H-dibenzo[a,d]cyclohepten-5-yl)- 2-acetamidomalonate (20.2g, Example lb) and sodium hydroxide (4g) in methanol (75ml) and water (75ml) was heated to reflux for 8h. To the cooled solution was added IM-HCl until pH = 6. The solution was concentrated in υacuo and acidified to pH = 2 by addition of IM-HC The solid which formed was collected by filtration and heated together with 6M-HC1 for 3h at 100°C. The cooled solution was filtered and the residue dissolved in hot water, refiltered and to the filtrate was added aqueous ammonia until pH 7-8. The solid which formed on cooling to room temperature was collected by filtration and dried in υacuo to give 5H-dibenzo[a,dlcyclohepten-5-ylglycine. Η NMR (360MHz, d ₄ MeOH), δ 4.08-4.12 (1H, m), 4.35-4.39 (1H, m), 7.0-7.11 (2H, m), 7.24-7.30 (3H, m), 7.34-7.45 (5H, ). d) 5-H-DibenzQ_a,d]cyclohepten-5-ylglycine (4.8g, Example lc) was added in portions to a solution of lithium aluminium hydride (54ml of 1.0M solution) in 30ml dry THF at 0°C. The reaction was allowed to stand at room temperature overnight, quenched with 2N sodium hydroxide and poured through Celite. The filtrate was washed with water, dried over MgSO^ and solvent removed in υacuo to leave a residue which was induced to crystallised in ethyl acetate or dichloromethane to give 5H- dibenzo[a,dlcycloheptene glycinol. ^~ H NMR (360MHz, d^ MeOH) δ 3.05-3.17 (2H, m), 3.29-3.33 (1H, m), 3.89-3.94 (1H, m), 6.90-6.97 (2H, m), 7.23-7.38 (8H, m).

e) A mixture of 5H-dibenzo[a,d]cycloheptene glycinol (1.13g, Example Id) and di-tert-butyldicarbonate (l.Og) in 10ml CH2CI2 was stirred at room temperature for 1.5h. Solvent was removed in υacuo to give N-te rt-butoxycarbonyl (5H- dibenzo[a,d]cycloheptene) glycinol which could be induced to crystallise in diethyl ether. Η NMR (360MHz, CDCI3) δ 1.24 (9H, s), 2.43 (1H, brs), 3.1-3.3 (1H, m), 3.3-3.5 (1H, m), 4.1-4.2 (1H, m), 4.2-4.4 (1H, m), 4.4-4.6 (1H, m), 6.9-7.1 (2H, m), 7.2-7.4 (8H, m). f) To a solution of N-έer.-butoxycarbonyl (5H- dibenzo[a,d]cycloheptene) glycinol (1.49g, Example le) and 3,5- dimethylbenzyl bromide (0.92g) in 4ml DMF-THF (1:1) under nitrogen at 0°C was added sodium hydride (130mg, 80% dispersion). The reaction was stirred at room temperature for 2h then p-_rtitione4***between aqueous NH^Cl-ethyl acetate. The organic phase was dried (MgSO^) and solvent removed in υacuo. The residue was chromatographed on silica (5% ethyl acetate- petroleum ether eluant) to give N-£β-£-butoxycarbonyl (5H- dibenzo[a,d3cycloheptene) glycinol-3,5-dimethylbenzyl ether, mp 116-118°C (petroleum ether). ^~ H NMR (360MHz, CDCI3) 1.15

(9H, s), 2.36 (6H, s), 2.90-2.93 (1H, m), 3.18-3.21 (1H, m), 4.16- 4.37 (4H, m), 4.79-4.82 (1H, m), 6.88-6.94 (4H, m), 7.05-7.08 (1H, ), 7.21-7.32 (8H, m). g) A solution of N-terέ-butoxycarbonyl (5H- dibenzo[a,d]cycloheρtene) glycinol-3,5-dimethylbenzyl ether

(0.55g, Example If) in 2ml dry CH2CI2 was treated with

trifluoroacetic acid (2ml) and stirred for 20 minutes. Solvent and other volatiles were removed in υacuo. The residue was dissolved in ethyl acetate, washed with aqueous Na2CU , dried (MgSO^) and solvent removed in υacuo. The residue was dissolved in ethanol and treated with oxalic acid to give the title compound, mp 175-177°C (ethanol-diethyl ether). ^~ H NMR (360MHz, DMSO-d ₆ ) 2.27 (6H, s), 2.85-2.87 (1H, m), 3.17-3.19 (1H, m), 3.58-3.40 (1H, m), 4.15-4.40 (3H, m), 6.88 (2H, s), 6.92 (1H, s), 6.99-7.08 (2H, m), 7.28-7.48 (8H, m).

EXAMPLE 2: N-Acetamido-l-(5H-dibenzo[a,d]cyclohepten-5-yl)- 2-(3,5-dimethylbenzyloxy)ethylamine

A solution of the product of Example 1 (0.35g) and acetic anhydride (0.178ml) in 3ml pyridine was allowed to stand overnight. The re ^~ *tion was partitioned between ethyl acetate and IN HCl. The organic phase was dried (MgSO^) and solvent removed in υacuo. The residue was chromatographed (20% ethyl acetate-petroleum ether) to give the title compound, mp 115-117°C (ethyl acetate-petroleum ether). ^~ H NMR (360MHz, CDC1 ₃ ) 1.54 (6H, s), 2.34 (3H, s), 2.92-2.94 (1H, s), 3.19-3.23

(1H, m), 4.20-4.31 (3H, m), 4.65-4.8 (1H, m), 5.5-5.6 (1H, m), 6.90-6.96 (4H, m), 7.01-7.05 (1H, m), 7.21-7.34 (8H, m). Found: C, 81.3; H, 7.3; N, 3.2: Calculated for C ₂ 8H ₂ 9N0 ₂ C, 81.72; H, 7.10; N, 3.40%.

EXAMPLE 3: l-(10.11-Dihvdro-5H-dibenzora,d1cvclohepten-5- yl]-2-(3,5-dimethylbei-zyloxy)ethylamine, oxalate salt

1212

-24-

5-Chlorodibenzosuberane was treated in an analogous manner to that described in Example 1 to yield the title compound, mp 197-200°C. Η NMR (360MHz,DMSO-d ₆ ) δ 2.27 (6H, m), 2.8-3.1 (1H, br m), 3.15-3.25 (1H, m), 3.3-3.5 (3H, m), 4.1-4.2 (2H, m), 4.3-4.35 (1H, m), 4.4-4.5 (1H, m), 6.93 (3H, s),

7.1-7.3 (8H, m). Found: C, 71.83; H, 6.77; N, 2.97; Calculated for

^C 25 ^H 29 ^NO - ^C 2 ^H 2°4 ^C ' ^72U ' ^H > ^6*95 ' ^N ' ^2Λ2% '

EXAMPLE 4: N-Acetamido-l-(10.11-dihydro-5H- dibenzo[a,d1cyclohepten-5-yl)-2-(3,5- dimethylbenzyloxy)ethyl ^~ rnine

The product of Example 3 was acetylated in an analogous manner to that described in Example 2 to give the title compound, mp 109-111°C. -H NMR (360MHz, CDClg) δ 1.60 (3H, s), 2.35 (6H, g*, 2.84-2.91 (2H, m), 3.37 (2H, s), 3.46-3.68

(2H, m), 4.14-4.17 (1H, m), 4.35-4.44 (2H, m), 4.84-4.90 (1H, m),

5.66-5.71 (1H, m), 6.98-7.25 (11H, m). Found: C, 71.83; H, 6.77;

N, 2.97; Calculated for C ₂ 5H ₂₉ NO.C ₂ H ₂ θ4 C, 72.14; H, 6.95;

N, 3.12%.

EXAMPLE 5: N.N-Dimethyl-l-(10,ll-dihydro-5H- dibenzo[a,d]cyclohepten-5-yl)-2-(3,5- dimethylbenzylo ^~ - ^~ )^thyl ^~ -mine, oxalate salt

To a solution of the product of Example 3 (320mg) in 2ml methanol at 0°C was added acetic acid (0.25ml) followed by sodium borohydride (O.lg) and aqueous formaldehyde (0.167ml of 37 wt %). Solvent was removed after 3 days and the residue

-25- purified by chromatography (1:1 ethyl acetate-petroleum ether eluant). The purified free base was dissolved in diethyl ether and oxalic acid in methanol was added. Solvent was removed to give the title compound, mp 68-69°C (ethyl acetate-petroleum ether). Η NMR (360MHz, DMSO-d ₆ ) 2.24 (6H, s), 2.49 (6H, s), 2.8-3.0 (2H, m), 3.32-3.50 (4H, m), 3.8-4.4 (4H, br m), 6.82 (2H, s), 6.88 (1H, s), 7.0-7.3 (8H, m). Found: C, 69.34; H, 6.71; N, 2.64: Calculated for C ₂₈ H ₃₃ N0.1.5 (C ₂ H ₂ 0 ₄ ) C, 69.64; H, 6.78; N, 2.62%.

EXAMPLE 6: l-(10,ll-Dihvdro-5H-dibenzora,d1cyclohepten-5- yl)-2-(3,5-bistrifluoromethylbei-zyloxy)ethyl amine, oxalate salt

Prepared in an analogous manner to that described for the product of Example 3. mp 175-176°C. -H NMR (360MHz, DMSO-d ₆ ) 2.8-3.0 (2H, m), 3.3-3.6 (3H, m), 4.1-4.3 (2H, m), 4.5-

4.7 (2H, m), 7.0-7.4 (8H, m), 8.0-8.1 (3H, m).

EXAMPLE 7: N-Carbomethoxymethyl-l-(10,ll-dihydro-5H- dibenzof a,d]cyclohepten-5-yl)-2-(3,5- bistrifluoromethylbenzyloxy)ethylamine, oxalate salt

A mixture of the title product of Example 6, methyl bromoacetate (one equivalent) and triethylamine (one equivalent) were refluxed in THF for 22h. The reaction was partitioned between diethyl ether-water and the organic phase separated and dried (MgS0 ₄ ). Solvent was removed in υacuo and the residue redissolved in ethanol. Oxalic acid was added,

solvent removed in υacuo, and the residue recrystallised to give the title compound, mp 160-165°C (ethyl acetate-petroleum ether). -H NMR (360MHz, DMSO-d ₆ ) 2.8-3.0 (2H, m), 3.2 (2H, s), 3.4-3.5 (6H, m), 3.6-3.65 (IH, m), 3.9-3.95 (IH, m), 4.5 (3H, s), 7.00-7.2 (8H, m), 7.9 (3H, s).

EXAMPLE 8: N-Carbamoylmethyl-l-(10,ll-dihydro-5H- dibenzo[a,d1cyclohepten-5-yl)-2-(3,5- bistrifluoromethylbenzyloxy)ethylaτrιine, oxalate salt A solution of the product of Example 6 in 5ml methanol saturated with ammonia was sealed in a reaction vessel and left at 0°C for 48h. Solvent was removed in υacuo, and the residue dissolved in ethanol. Oxalic acid and water were added to give the title compound, mp 120-122°C. ^~ H NMR (360MHz, DMSO- d ₆ ) 2.8-3.0 (2H, m_H 3.3-3.7 (6H, m), 4.2-4.6 (4H, m), 7.00-7.4

(8H, m), 7.9 (IH, s), 7.95 (2H, s).,

EXAMPLE 9: 3,5-Dimethylbenzyl-2-(9-fluoroenyl)glycinate a) A solution of N-(diphenylmethylene)glycine ethyl ester (5g) in tetrahydrofuran (15ml) was added dropwise to a cooled solution of lithium diisopropylamide (1 mol eq) in tetrahydrofuran (20ml) and hexane (11ml) at -70°C. After lh at -70°C, a solution of 9-bromofluorene (4.58g) in tetrahydrofuran (15ml) was added. After stirring the solution for lh at -70°C and then at room temperature (16h), the solution was diluted with saturated ammonium chloride solution (500ml) and diethyl

ether (500ml). The organic phase was washed with saturated brine and dried (MgSO^). After removal of the solvent in υacuo the residue was purified on a silica column to give ethyl N- (diphenylmethylene)-(9-fluoroenyl)glycinate, 2.2g. b) The product of part (a) (2.2g) was heated at reflux for 16h with 5.5M-hydrochloric acid (30ml). The cooled solution was washed with diethyl ether and the aqueous phase evaporated to dryness. The residue was recrystallised from acetone-water to give 2-(9-fluorenyl)glycine hydrochloride, 0.7g. c) To a solution of the product of part (b) (0.7g) and sodium carbonate (0.95g) in dioxan (5ml) and water (10ml) was added di-t-butyldicarbonate (0.664g). After stirring the solution for 16h at room temperature, water (50ml) and diethyl ether (60ml) were added. To the aqueous phase was added solid citric acid to pH 3 and the product extracted into ethyl acetate (3 x 30ml).

The combined organic phases were washed with water, saturated brine, dried (MgSO^) and evaporated to dryness to give N-t-buto ^~ τycarbonyl-2-(9-fluorenyl) ^~ ^lvcine 0.75g. d) To a solution of the product of part (c) (0.750g) in methanol (10ml) was added a solution of cesium carbonate

(348mg) in water (5ml). The solvent was removed by evaporation and the residue dried by repeated evaporation from dimethylformamide (3 x 30ml). To the residue dissolved in dimethylformamide (50ml) was added 3,5-dimethylbenzyl bromide (0.636g) and the solution stirred at room temperature

for 16h. The solvent was removed in υacuo and the residue partitioned between dichloromethane and water. The organic phase was washed with saturated brine, dried (MgS0 ₄ ) and evaporated in υacuo. The residue was chromato graphed on silica gel eluting with mixtures of ethyl acetate in petroleum ether bp 60-80°C to give 3,5-dimethylbenzyl-N-t-butoxycarbonyl- 2-(9-fLuorenyl)glycinate. Trifluoroacetic acid (3ml) was added to the ester (0.2g) and after 40 minutes the solvent was removed by evaporation and a solution of 4-toluenesulfonic acid (84mg) in ethanol (1ml) added to give 3,5-dimethylbenzyl-2-(9- fluorenyl)glycinate, 4-toluenesulphonic acid salt, mp 150-151°C. Found: C, 70.19; H, 5.74; N, 2.61. C ₂₄ H ₂₃ N0 ₂ .C ₇ H ₈ S0 ₃ requires C, 70.30; H, 5.90; N, 2.64%. m/z (FAB ⁺ ) = 358 (M+H).

EXAMPLE 10 : -8 ,5 -Dimethylbenzyl N-acetyl-2-(9 - fluorenvDglvcinate

3 , 5 -Dime thylbenzyl N-t-butoxycarbo nyl-2-(9- fluorenyDglycinate (Example 9d, 0.5g) was dissolved in trifluoroacetic acid (10ml) for 40 minutes then evaporated in υacuo. To a solution of the residue in pyridine (10ml) was added acetic anhydride (1ml) for 16h. The solvent was removed in υacuo and the residue crystallised from diethyl ether/petroleum ether bp 60-80°C to give the title compound mp 162-164°C. Found: C, 78.19; H, 6.26; N, 3.45. C ₂₆ H ₂₅ N0 ₃ requires C,. 78.17; H, 6.30; N, 3.50%. m/z (CI ⁺ ) = 400 (M+H), (CD = 398 (M-

H).

- 29 -

The following examples illustrate pharmaceutical compositions according to the invention.

EXAMPLE 11A Tablets containing 1-25mo of compound

Compound of formula (I) Microcrystalline cellulose Modified food corn starch Lactose

Magnesium Stearate

EXAMPLE 11B Tablets containing 26-100πιg of compound

Amount mα Compound of formula ^" (I)

Microcrystalline cellulose

Modified food corn starch

Lactose

Magnesium Stearate The compound of formula (I) , cellulose, lactose and a portion of the corn starch are mixed and granulated with

10% corn starch paste. The resulting granulation is sieved, dried and blended with the remainder of the corn starch and the magnesium stearate. The resulting granulation is then compressed into tablets containing l.Omg, 2.0mg, 25.0mg, 26.0mg, 50.0mg and lOOmg of the active compound per tablet.

EXAMPLE 12 Parenteral injection Amount irig

Compound of formula (I) 1 to lOOmg

Citric Acid Monohydrate 0.75mg

Sodium Phosphate 4.5mg

Sodium Chloride 9mg

Water for injection to 1ml

The sodium phosphate, citric acid monohydrate and sodium chloride are dissolved in a portion of the water. The compound of formula (I) is dissolved or suspended in the solution and made up to volume.

EXAMPLE 13 Topical formulation

Amount mg

Compound of formula (I) 1-lOg Emulsifying Wax 30g

Liquid paraffin 20g

White Soft Paraffin to lOOg

The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The compound of formula (I) is added and stirring continued until dispersed. The mixture is then cooled until solid.

SUBSTANCE P ANTAGONISM ASSAY

A. Receptor Expression in Monkey Kidney Cell Line (COS)

To express the cloned human neurokinin-1- receptor (NK1R) transiently in COS, the cDNA for the human NK1R was cloned into the expression vector pCDM9 which was derived from pCDM8 (INVITROGEN) by inserting the ampicillin resistance gene (nucleotide 1973 to 2964 from BLUESCRIPT SK+ (trademark, STRATAGENE, La Jolla, CA, USA)) into the Sac II site. Transfection of 20 ug of the plasmid DNA into 10 million COS cells was achieved by electroporation in 800 μl of transfection buffer (135 mM NaCl, 1.2 mM CaCl ₂ , 1.2 mM MgCl ₂ , 2.4 mM K ₂ HP0 , 0.6 mM KH ₂ P0 ₄ , 10 mM glucose, 10 mM N-2-hydroxyethyl-piperazine- N ^« -2-ethane sulphonic acid (HEPES) pH 7.4) at 260 V and 950 μF using the IBI GENEZAPPER (trademark IBI, New

Haven, CT, USA) . The cells were incubated in 10% fetal calf serum, 2 mM glutamine, lOOU/ml penicillin- streptomycin, and 90% DMEM media (GIBCO, Grand Island, NY, USA) in 5% C0 ₂ at 37 ^* C for three days before the binding assay.

B. Stable Expression in Chinese Hamster Ovarian Cell Line To establish a stable cell line expressing cloned human NKIR, the cDNA was subcloned into the vector pRcCMV (INVITROGEN) . Transfection of 20 ug of the plasmid DNA into CHO cells was achieved by electroporation in 800 μl of transfection buffer supplemented with 0.625 mg/ml Herring sperm DNA at 300 V and 950 μF using the IBI GENEZAPPER (IBI) .' The transfected cells were incubated in CHO media [10% fetal calf serum, 100 U/ml penicillin- streptomycin, 2 mM glutamine, 1/500 hypoxanthine- thymidine (ATCC) , 90% IMDM media (JRH BIOSCIENCES, Lenexa, KS, USA), 0.7 mg/ml G418 (GIBCO)] in 5% C0 ₂ at 37"C until colonies were visible. Each colony was separated and propagated. The cell clone with the highest number of human NKIR was selected for subsequent applications such as drug screening.

C. Assay Protocol using COS or CHO

The binding assay of human NKIR expressed in either COS or CHO cells is based on the use of ¹²⁵ I-substance P ( ¹²⁵ I-SP, from DU PONT, Boston, MA) as a radioactively labeled ligand which competes with unlabeled substance P or any other ligand for binding to the human NKIR.

Monolayer cell cultures of COS or CHO were dissociated by the non-enzymatic solution (SPECIALTY MEDIA, Lavellette, NJ) and resuspended in appropriate volume of the binding buffer (50 mM Tris pH 7.5, 5 mM MnCl ₂ , 150 mM NaCl, 0.04

- 32 -

mg/ml bacitracin, 0.004 mg/ml leupeptin, 0.2 mg/ml BSA, 0.01 mM phosphoramidon) such that 200 μl of the cell suspension would give rise to about 10,000 cpm of specific ¹²⁵ I-SP binding (approximately 50,000 to 200,000 cells) . In the binding assay, 200 ul of cells were added to a tube containing 20 ul of 1.5 to 2.5 nM of ¹²⁵ I-SP and 20 μl of unlabeled substance P or any other test compound. The tubes were incubated at 4*C or at room temperature for 1 hour with gentle shaking. The bound radioactivity was separated from unbound radioactivity by GF/C filter (BRANDEL, Gaithersburg, MD) which was pre- wetted with 0.1% polyethylenimine. The filter was washed with 3 ml of wash buffer (50 mM Tris pH 7.5, 5 mM MnCl ₂ , 150 mM NaCl) three times and its radioactivity was determined by gamma counter.

The activation of phospholiphase C by NKIR may also be measured in- CHO cells expressing the human NKIR by determining the accumulation of inositol onophosphate which is a degradation product of IP ₃ . CHO cells are seeded in 12-well plate at 250,000 cells per well. After incubating in CHO media for 4 days, cells are loaded with 5μCi of ³ H-myoinositol in 1 ml of media per well by overnight incubation. The extracellular radioactivity is removed by washing with phosphate buffered saline. LiCl is added to the well at final concentration of 10 mM with or without the test compound, and incubation is continued at 37 ^* C for 15 min. Substance P is added to the well at final concentration of 0.3nM to activate the human NKIR. After 30 min of incubation at 37'C, the medium is removed and 0.1 N HCl is added. Each well is sonicated at 4*C and extracted with CHCl ₃ /methanol (1:1). The aqueous phase is applied to a 1 ml Dowex AG 1X8 ion exchange column. The column is washed with 0.1 N formic acid followed by 0.025 M ammonium formate-0.1 N formic acid.

33 -

The inositol monophosphate is eluted with 0.2 M ammonium formate-0.1 N formic acid and quantitated by beta counter.

The data in Table 1 were obtained for compounds of formula (I) :

TABLE 1