Novel 2,3-Dihalo-4,4-bis-(aryl)-but-2-enoic acid -amides

Field of the Invention

01. The present invention relates to novel 2,3-Dihalo-4,4-bis-(2,4-aryl)-but-2-enoic acid-amides , the salts thereof , their preparation and their use as non-peptide CCK ligands, opiate agonists and particularly pharmaceutical medicinal applications thereof.

Background of the Invention

2. Cholecystokinins (CCKs) act as anti-opioid peptides. CCK was initially described as a regulatory hormone found in endocrine cells of the gastro-intestinal (GI) tract. Some CCKs share a common amino acid sequence with gastrin, which is involved in control of gastric acid and pepsin secretion. CCKs have also been found throughout the central nervous system (CNS), where they are believed to act as a neurotransmitter and/or modulator of many important functions. There are various known structures of CCK, identified with reference to the number of amino acids they comprise. For example, CCK-8 is a naturally-occurring predominating CCK peptide and, having only eight amino acids, is the smallest fully-active sequence, although small amounts of CCK-4 may also be present.

3. The gut hormone CCK exerts various actions on the GI tract, including the regulation of growth. The hormone has been reported to induce hypertrophy and hyperplasia of the pancreas and to enhance chemically-induced pancreatic carcinogenesis in animals. CCK plays an important role in the invasiveness through the production of matrix metalloproteinase-9 (MMP-9) in human pancreatic cancer cell lines. Stimulation of endogenous CCK secretion through the induction of deficiency of intraintestinal proteases and bile salts by trypsin-inhibiting nutrients, bile salt-binding drugs or surgical intervention is also capable of stimulating growth and tumour development in the rat. In humans, factors suggested to increase the risk of pancreatic cancer, such as a high-fat and high-protein diet or gastrectomy, are known to stimulate plasma CCK secretion. Receptors for CCK have been demonstrated on human pancreatic adenocarcinomas, and CCK has been demonstrated to enhance the growth of xenografted pancreatic cancer and to inhibit growth of gastric and bile duct cancer.

4. There are two subtypes of CCK receptor which were initially termed as type-A and type-B, reflecting their preferential localisation in the alimentary tract and in the brain, respectively. Recently, these receptors have been re-named as CCKl and CCK2, respectively, although the original designation is used hereinbelow with respect to the present invention. The molecular cloning of two CCK receptor subtypes, one from rat and human pancreas and one from human brain, has confirmed the pharmacological classification of CCK receptors. Both CCKl and CCK2 receptors belong to the family of G-protein coupled receptors. However, the differential distribution of CCKl and CCK2 receptors in the peripheral vs. central nervous system is not absolute, and CCKl receptors have been shown to be present in discrete regions of the CNS, including the spinal cord, particularly in primates.

5. The functions of the CCKl receptors in the brain is poorly understood, whereas the CCK2 receptor is known to mediate anxiety, panic attacks, satiety and pain. Therefore, antagonists to CCK and to gastrin have been useful for preventing and treating CCK-related and/or gastrin-related disorders of the GI and CNS of animals, especially of humans. Just as there is some overlap in the biological activities of CCK and gastrin, antagonists also tend to have affinity for both receptors. In a practical sense, however, there is enough selectivity for the respective receptors that greater activity against specific CCK- or gastrin-related disorders can often also be identified.

6. Selective CCK antagonists are themselves useful in treating CCK-related disorders of the appetite regulatory systems of animals as well as in potentiating and prolonging opiate-mediated analgesia, thus having utility in the treatment of pain, while selective gastrin antagonists are useful in the modulation of CNS behaviour as a palliative for gastrointestinal neoplasms, and in the treatment and prevention of gastrin-related disorders of the GI system in humans and animals, such as peptic ulcers, Zollinger-Ellison syndrome, antral G cell hyperplasia and other conditions in which reduced gastrin activity is of therapeutic value. Also, since CCK and gastrin also have trophic effects on certain tumours, antagonists of CCK and gastrin are useful in treating these tumours.

7. Various chemical classes of CCK-receptor antagonists have been reported. These include pyrazolidinones showing good selectivity for CCKB receptors (Howbert, J.J.et. al.; Diphenylpyrazolidinone and benzodiazepine cholecystokinin antagonists: A case of convergent evolution in medicinal chemistry., Bioorg. Med. Chem. Lett. 1993, 3, 875-880.), ureidoacetamides which are potent and selective ligands for CCKs/gastrin receptors (WO 91/113874), ureidophenoxyacetanilides (Takeda, Y.et. al.; Synthesis of phenoxyacetic acid derivatives as highly potent antagonists of gastrin/ cholecystokinin-B receptors, Chem. Pharm Bull. 1998, 46 , 951-961), ureidomethylcarbamoylphenylketones (Hagishita, S.; et. al., Ureido-methylcarbamoyl-phenylketones as selective CCKB receptor antagonists. Bioorg. MedChem. 1997, 5, 1695-1714), and ureidobenzodiazepine derivatives (Evans, B.E.; et. al., Design of potent, orally effective, non peptidal antagonists of the peptide hormone cholecystokinin, Proc. Natl. Acad. Sci. USA 1986, 83, 4918-4922).

8. It is an object of the present invention to provide novel 2,3-Dihalo-4,4-bis-(aryl)-but-2-enoic acid -amide derivatives, which preferably act as CCK and/or opiod ligands, and pharmaceutical formulations thereof

9. According to the present invention there is provided a compound of formula (I):

10. Wherein X is selected from hydrogen, a halogen, pseudohalogen, a hydroxyl group, a substituted or unsubstituted cyclic and heterocyclic moiety, substituted or unsubstituted, linear or branched alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy, alkenylcarbonyl, alkenyioxycarbonyl, alkynyl, alkynyloxy, alkynylcarbonyl, alkynyloxycarbonyl, aryl, benzyl, arlyoxy, arylcarbonyl, aryloxycarbonyl and sulphur equivalents of said oxy, carbonyl and oxycarbonyl moieties or as defined for Aryl and R.

11. R is selected from hydrogen, a halogen, an amide, an ester, a urea, a carbamate, a carbonyl derivative, a substituted or unsubstituted cyclic and heterocyclic moiety, a phenyl group, aryl, substituted or unsubstituted, linear or branched alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy, alkenylcarbonyl, alkenyioxycarbonyl, alkynyl, alkynyloxy, alkynylcarbonyl, alkynyloxycarbonyl, aryl, benzyl, arlyoxy, arylcarbonyl, aryloxycarbonyl and sulphur equivalents of said oxy, carbonyl and oxycarbonyl moieties, and as in Aryl and X defined.

12. Aryl is selected fi-om H, phenyl, substituted phenyl, aryl, hetero aryl, methyl, alkyl, substituted aryl, ortho-, meta- and para- substituted aryl, bis- and tris- substituted aryl , poly substituted aryl, benzyl. C1-18 straight, branched or cyclic, saturated, unsaturated and aromatic hydrocarbyl groups, which aromatic groups may be heterocyclic, cyclic or acyclic and which may optionally be substituted by alkyl, alkoxy, or halo; or R1 and R2, when taken together with the N-atom to which they are bonded, may form an N-containing saturated, unsaturated or partially unsaturated ring system comprising 3 to 10 ring atoms selected from C, N and O, optionally substituted at any position of the ring by a substituent selected from a halogen, a substituted or unsubstituted cyclic and heterocyclic moiety, substituted or unsubstituted, linear or branched alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl, alkenyloxy, alkenylcarbonyl, alkenyloxycarbonyl, alkynyl, alkynyloxy, alkynylcarbonyl, alkynyloxycarbonyl, aryl, benzyl, arlyoxy, arylcarbonyl, aryloxycarbonyl, sulphur equivalents of said oxy, carbonyl and oxycarbonyl moieties, and as defined for the other substituents and any possible combination thereof.

13. Preferred substituents for R of amide and esters are alkyl, cycloalkyl, phenyl, substituted phenyl formed by alkyl or alkoxy, cycloalkyl, phenyl, benzyl, phenyl (C24) alkenyl, phenoxy, benzyloxy, halo, 0x0 or alkyloxycarbonyl.

14. Suitable substituents on the aromatic system are methyl, halogen, nitro, trifluormethyl nitrilo, bromo, chloro, halo, benzyl, phenyl, alkoxycarbonyl and 0x0. Preferably, said aromatic system is substituted.

15. Preferably, X is H, halo (F, Br, CI, I) CF3, CN or methyl, and is most preferably chloro.

16. Preferably, R is cycloalkyl or heterocyclic, most preferred is cyclopentyl amino, benzyl pirarazinyl for CCK antagonists and, simple alkyl for opiate agonists.

17. More preferably, X is chlorine and aryl is substituted a nono-substituted phenyl.

18. It will be understood that formula (I) is intended to embrace all possible isomers, the amide are Z isomers. As the molecule is symmetrical no optical isomers are present.

In addition, the present invention includes within its scope prodrugs of the compounds of formula (I) and in particular salts of the di-amino derivative of which, one amine formed the amide and the other tertiary amine easily formed salts. HCl salts are most preferred and salts of other organic acids including amino acids and lactate and tartrates are all essential for a good water solubility. 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. Bungaard, Elsevier, 1985.

19. The scope of the invention also extends to salts, particularly physiologically
acceptable salts and hydrates of the compounds of formula (II).

Rl is benzyl, alkyl any substituted piperazine

20. The pharmaceutically acceptable salts of the compounds of formula (I) include the conventional salts or the quartemary ammonium salts of the compounds of formula
(II) formed, eg, from inorganic or organic acids. The pharmaceutically acceptable
salts of formula (I) also include those formed from a base, such as an alkali or
alkaline earth metal hydroxide, or an organic base, such as an amine or a
quartemary ammonium hydroxide.

21. Highly preferred compounds in accordance with the invention are:-

22. Exp 5 and Exp 11 are CCK antagonists and the alkyl amide Exp 1 and 2 have opiate agonistic properties. The best amide is containing a n-propyl group.

23. The present invention also resides in the use of a compound of the first aspect as a CCK receptor ligand and/or as a CCK antagonist. Preferably, said use is as a selective CCKl or CCK2 ligand. Most preferred are mixed antagonists as reported in Lattmann et al.

[Lattmann, E., Arayarat, P. and Singh, H. (2000) Review article: Small organic molecules as cholecystokinin antagonists.Science (KKU), 28, 288-299

Lattmann, E., Billington, D.C., Poyner, D.R., Howitt, S.B. and Offel M. (2001)
Synthesis and evaluation of Asperlicin analogues as non-peptidal Cholecystokinin-antagonists.

Drug Design and Discovery, 17, 219-230
Lattmann, E., Billington, D.C., Poyner, D.R., Howitt, S.B. and Offel, M. (2001)

Solid phase synthesis of 3-alkylated 1,4-benzodiazepines as non-peptidal Cholecystokinin antagonists. Pharm. Pharm. Lett. 11, 5-8

Lattmann, E., Billington, D.C., Poyner, D.R., Arayarat, P., Howitt, S.B., Lawrence, S. and Offel, M. (2002)

Combinatorial solid phase synthesis of multiply-substituted 1,4-benzodiazepines and affinity studies on the CCK2 receptor (Part 1). Drug Design and Discovery, 18, 9-21.

Lattmann, E., Sattayasai, J., Billington, D.C., Poyner, D.R., Puapairoj, P., Tiamkao, S.,
Airarat, W., Singh, H. and Offel, M. (2002)

Synthesis and evaluation of Ni-substituted-3-propyl-l,4-benzodiazepine-2-ones as
Cholecystokinin (CCK2)-receptor ligands. J. Pharm. Pharm. 54, 827-834

Lattmann, E., Arayarat, P. (2003)

Review article: From CNS-drugs to anti-neoplastic agents: Cholecystokinin (CCK)-
antagonists as modem anti-cancer agents. Science (KKU) 2003, 31,178-193

Lattmann, E., Sattayasai, J., Boonprakob, J., Lattmann, P., Singh, H. (2005)
Synthesis and evaluation of N-(5-methyl-3-oxol,2-diphenyl-2,3-dihydro-lH-pyrazol-4yl)-
N-phenylureas as Cholecystokinin antagonists

Drug Res. /Arzneimittelforschung 55,251-258 Eric Lattmann, Harjit Singh, Yodchai Boonprakob, Pomthip Lattmann, Jintana Sattayasai (2006) Synthesis and evaluation of N-(3oxo-2,3-dihydro-lHpyrazol-4-yl)-lH-indole-carboxamide as cholecystokinin antagonists, J. Pharm. Pharm. 2006, 58, 1-9 Michael Offel, Pomthip Lattmann, Harjit Singh, David C. Billington, Yodchai Bunprakob, Jintana Sattayasai, Eric Lattmann (2006) Synthesis of substituted 3-anilino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepinones and their evaluation as cholecystokinin ligands Archiv der Pharmazie - Chemistry in Life Science, 339, 163-173

Eric Lattmann, Jintana Sattayasai, Pomthip Lattmann, David C. Billington, Carl H. Schwalbe, Jordchai Boonprakob, Wanchai Airarat, Harjit Singh, and Michael Offel (2007) Anti-depressant and anti-nociceptive effects of l,4-Benzodiazepine-2-ones based Cholecystokinin (CCK2) antagonists, DrugDiscov Ther 1(1): 45-56

Eric Lattmann, Jintana Sattayasai, Yodchai Boonprakob, Harjit Singh, Pomthip Lattmann and Simon Dunn (2008)

Cholecystokinin antagonists (part 1): Antinociceptive, anxiolytic and antidepressant effects of N-(5-methyl-3-oxo-1,2-diphenyl-2,3-dihydro-1 H -pyrazol-4-yl)-N'-phenylureas and carboxamides (2008) DrugDiscov Ther. 2, (3) 156-167.

Eric Lattmann, Yodchai Boonprakob and Jintana Sattayasai (2008) Part 2. Long term in vivo/ in vitro evaluation of the Cholecystokinin antagonists: N-(5-methyl-3-oxo-1,2-diphenyl-2,3-dihydro-l H -pyrazol-4-yl)-N'-phenylurea MPP and carboxamide MPM (2008) Drug Discov Ther. 2, (6) 344-352.]

24 The ability of the compounds of formula (I) to antagonise CCK by acting as CCK-receptor ligands makes these compounds useful as pharmacological agents for vertebrates, especially humans, for the treatment and prevention of disorders wherein CCK, gastrin, and opiod receptor may be involved.

25 The ability of the compounds of formula (I) to agonise opiates by acting as mu, delta or kappa ligands makes these compounds useful as pharmacological agents for vertebrates, especially humans, for the treatment and prevention of disorders wherein opiates and opiate related mechanisms are involved.

26 The ability of a combination of compounds of formula (I) to potentiate biological effects makes these compounds useful as pharmacological agents for mammals, especially humans, in the treatment and prevention of a variety of disorders.

27 Therefore the present invention in a second aspect resides in a method of treatment of a mammal afflicted with a CCK-related condition, or prophylaxis in a mammal at risk of a CCK-related condition by administration of a therapeutically effective amount of a compound of the first aspect of the invention. By definition opiate based mechanisms are CCK related.

28 The invention also resides in a pharmaceutical formulation comprising a compound of said first aspect in admixture with a pharmaceutically acceptable carrier therefor.

29 The invention further resides in the use of a compound of the first aspect in the preparation of a medicament, particularly a medicament for the treatment or prophylaxis of a CCK-related disorder.

30 Examples of CCK-related conditions include GI disorders, especially such as irritable bowel syndrome, gastro-oesophageal reflux disease or ulcers, excess pancreatic or gastric secretion, acute pancreitis, or motility disorders; CNS disorders caused by CCK interactions with dopamine, such as neuroleptic disorders, tardive dyskinesia, Parkinson's disease, psychosis or Gilles de la Tourette syndrome; disorders of appetite regulatory systems; Zollinger-Ellison syndrome; antral G cell hyperplasia; or pain (potentiation of opiate analgesia)._The molecules in this present invention could also be used to prevent or treat eating disorders such as anorexia and others and muscle wasting disorders such as cachexia, sarcopenia and others.

31 The treatment of opiate-resistant severe clinical pain may represent the most important of the CNS applications, but other applications based on the interaction between CCK and dopamine in forebrain could also deserve clinical exploration.

Dopamine is collocated with CCK and via the mediation of dopamine these agents may proof useful in the treatment of Parkinson disease.

32 The compounds of the invention may further be useful in the treatment or prevention of additional central nervous system disorders including neurological and psychiatric disorders. Example of such central nervous system disorders include anxiety disorders and panic disorders, wherein CCK is involved. Additional examples of central nervous system disorders include panic syndrome, anticipatory anxiety, phobic anxiety, panic anxiety, chronic anxiety and endogeneous anxiety.

33. Extensive research with respect to the use as antidepressants, anxiolytics, analgesics is published in the literature by E. Lattmann et al.

34. The compounds of the invention may further be useful in the treatment of proliferative disorders. Examples of such proliferative disorders include small cell adenocarcinomas and primary tumours of the central nervous system glial and neuronal cells. Example of such adenocarcinomas and tumours include, but are not limited to, tumours of the lower oesophagus, stomach, intestine, colon and lung, including small cell lung carcinoma, breast cancer and others.

35. Most effective are the lipophilic molecules as agents to treat brain cancers and the salts of formula II to treat GI cancers.

36. The compounds of the invention may be further useful for preventing or treating a variety of inflammatory diseases such as Chron's disease, inflammatory bowel disease, arthritis etc.

37. The compounds of the invention may further be useful for preventing or treating the withdrawal response produced by chronic treatment or abuse of drugs or alcohol. Such drugs include, but are not limited to, cocaine, alcohol or nicotine.

38. CCK antagonists potentiate the analgesic activity of opiates ( Lattmann et al).

39. The compounds of the invention may also be useful as neuroprotective agents, for example, in the treatment and/or prevention of neuro-degenerative disorders arising as consequence of such pathological conditions as stroke, hypoglycaemia, cerebral palsy, transient cerebral ischaemic attack, cerebral ischaemia during cardiac pulmonary surgery or cardiac arrest, perinatal asphyxia, epilepsy, Huntingdon's chorea, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, olivo-pontocerebellar atrophy, anoxia such as from drowning, spinal cord and head injury, and poisoning by neurotoxins, including environmental neurotoxins.

40. The dosage administered to a patient will normally be determined by the prescribing physician and will generally vary according to the age, weight and response of the individual patient, as well as the severity of the patient's symptoms. However, in most instances, an effective therapeutic daily dosage will be in the range of from about 0.05 mg/kg to about 50 mg/kg of body weight and, preferably, of from 0.5 mg/kg to about 20 mg/kg of body weight administered in single or divided doses. In some cases, however, it may be necessary to use dosages outside these limits. The dose may be administered orally, intravenously, subcutaneously, intra-peritoneally and by other routes.
Pain modulation requires generally the lower dose range, while the use as an anticancer agent will be in the higher dose range.

41. In the treatment of irritable bowel syndrome, for instance, 0.001 to 100 mg/kg of a CCK antagonist might be administered orally (p.o.), divided into two doses per day
(b.i.d.). In treating delayed gastric emptying, the dosage range would probably be the same, although the drug might be administered either intravenously (i.v.) or orally, with the i.v. dose probably tending to be slightly lower due to a better availability. Acute pancreitis might be treated preferentially in an i.v. form,
whereas spasm and/or reflex oesophageal, chronic pancreitis, post-vagotomy diarrhoea, anorexia or pain associated with biliary dyskinesia might indicate a p.o. form of administration.

42. In the effective treatment of panic syndrome, panic disorder, anxiety disorder and the liKE, , preferably about 0.001 mg/kg to about 100 mg/kg of CCK antagonist may be administered orally (p.o.), in single or divided doses per day. Other routes of administration are also suitable.

43. For directly introducing analgesia, anaesthesia or loss of pain sensation, the effective dosage range is preferably from about 1 ng/kg to about 100 mg/kg by intraperitoneal administration. Oral administration is an alternative route, as well as others.

44. While it is possible for an active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation. The formulations, both for veterinary and for human medical use, of the present invention comprise an active ingredient in association with a pharmaceutical ly acceptable carrier therefore and optionally other therapeutic ingredient(s). The carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof

45. Conveniently, unit doses of a formulation contain between 0.1 mg and 1 g of the active ingredient. Preferably, the formulation is suitable for administration from one to six, such as two to four, times per day. For topical administration, the active ingredient preferably comprises from 1% to 2% by weight of the formulation but the active ingredient may comprise as much as 10% w/w. Formulations suitable for nasal or buccal administration, such as the self-propelling powder-dispensing formulations described hereinafter, may comprise 0.1 to 20% w/w, for example about 2% w/w of active ingredient.

46. The formulations include those in a form suitable for oral, ophthalmic, rectal, parenteral (including subcutaneous, vaginal, intraperitoneal, intramuscular and intravenous), intra-articular, topical, nasal or buccal administration.

47. Formulations of the present invention suitable for oral administration may be in the form of discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be in the form of a bolus, electuary or paste. For such formulations, a range of dilutions of the active ingredient in the vehicle is suitable, such as from 1% to 99%, preferably 5% to 50% and more preferably 10% to 25% dilution. Depending upon the level of dilution, the formulation will be either a liquid at room temperature (in the region of about 20°C) or a low-melting solid.

48. Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and a carrier such as cocoa butter, or in the form of an enema.

49. Formulations suitable for parenteral administration comprise a solution, suspension or emulsion, as described above, conveniently a sterile aqueous preparation of the active ingredient that is preferably isotonic with the blood of the recipient.

50. Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of the active ingredient, which may be in a microcrystalline form, for example, in the form of an aqueous microcrystalline suspension or as a micellar dispersion or suspension. Liposomal formulations or biodegradable polymer systems may also be used to present the active ingredient particularly for both intra-articular and ophthalmic administration.

51. Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions or applications; oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops. For example, for ophthalmic administration, the active ingredient may be presented in the form of aqueous eye drops, as for example, a 0.1-1.0% solution.

52. Drops according to the present invention may comprise sterile aqueous or oily solutions. Preservatives, bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric salts (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

53. Lotions according to the present invention include those suitable for application to the eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide or preservative prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol, or a softener or moisturiser such as glycerol or an oil such as castor oil or arachis oil.

54. Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient in a base for external application. The base may comprise one or more of a hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil such as a vegetable oil, eg almond, com, arachis, castor or olive oil; wool fat or its derivatives; or a fatty acid ester of a fatty acid together with an alcohol such as propylene glycol or macrogols. The formulation may also comprise a suitable surface-active agent, such as an anionic, cationic or non-ionic surfactant such as a glycol or polyoxyethylene derivatives thereof Suspending agents such as natural gums may be incorporated, optionally with other inorganic materials, such as silicaceous silicas, and other ingredients such as lanolin.

55. Formulations suitable for administration to the nose or buccal cavity include those suitable for inhalation or insufflation, and include powder, self-propelling and spray formulations such as aerosols and atomisers. The formulations, when dispersed, preferably have a particle size in the range of 10 to 200μ.

56. Such formulations may be in the form of a finely comminuted powder for pulmonary administration from a powder inhalation device or self-propelling powder-dispensing formulations, where the active ingredient, as a finely comminuted powder, may comprise up to 99.9% w/w of the formulation.

57. Self-propelling powder-dispensing formulations preferably comprise dispersed particles of solid active ingredient, and a liquid propellant having a boiling point of below 18°C at atmospheric pressure. Generally, the propellant constitutes 50 to 99.9% w/w of the formulation whilst the active ingredient constitutes 0.1 to 20% w/w. for example, about 2% w/w, of the formulation.

58. The pharmaceutically acceptable carrier in such self-propelling formulations may include other constituents in addition to the propellant, in particular a surfactant or a solid diluent or both. Surfactants are desirable since they prevent agglomeration of the particles of active ingredient and maintain the active ingredient in suspension. Especially valuable are liquid non-ionic surfactants and solid anionic surfactants or mixtures thereof Suitable liquid non-ionic surfactants are those having a hydrophile-lipophile balance (HLB, see Journal of the Society of Cosmetic Chemists Vol. 1 pp. 311-326 (1949)) of below 10, in particular esters and partial esters of fatty acids with aliphatic polyhydric alcohols. The liquid non-ionic surfactant may constitute from 0.01 up to 20% w/w of the formulation, though preferably it constitutes below 1% w/w of the formulation. Suitable solid anionic surfactants include alkali metal, ammonium and amine salts of dialkyl sulphosuccinate and alkyl benzene sulphonic acid. The solid anionic surfactants may constitute-from 0.01 up to 20% w/w of the formulation, though preferably below 1% w/w of the composition. Solid diluents may be advantageously incorporated in such self-propelling formulations where the density of the active ingredient differs substantially from the density of the propellant; also, they help to maintain the active ingredient in suspension. The solid diluent is in the form of a fine powder, preferably having a particle size of the same order as that of the particles of the active ingredient. Suitable solid diluents include sodium chloride, sodium sulphate and sugars.

59. Formulations of the present invention may also be in the form of a self-propelling formulation wherein the active ingredient is present in solution. Such self-propelling formulations may comprise the active ingredient, propellant and co-solvent, and advantageously an antioxidant stabiliser. Suitable co-solvents are lower alkyl alcohols and mixtures thereof The co-solvent may constitute 5 to 40% w/w of the formulation, though preferably less than 20% w/w of the formulation. Antioxidant stabilisers may be incorporated in such solution-formulations to inhibit deterioration of the active ingredient and are conveniently alkali metal ascorbates or bisulphites. They are preferably present in an amount of up to 0.25% w/w of the formulation.

60. Formulations of the present invention may also be in the form of an aqueous or dilute alcoholic solution, optionally a sterile solution, of the active ingredient for use in a nebuliser or atomiser, wherein an accelerated air stream is used to produce a fine mist consisting of small droplets of the solution. Such formulations usually contain a flavouring agent such as saccharin sodium and a volatile oil. A buffering agent such as sodium metabisulphite and a surface-active agent may also be included in such a formulation, which should also contain a preservative such as methylhydroxybenzoate.

61. Other formulations suitable for nasal administration include a powder, having a particle size of 20 to 500 microns, which is administered in the manner in which snuff is taken, ie by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.

62. In addition to the aforementioned ingredients, the formulations of this invention may include one or more additional ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives eg methylhydroxybenzoate (including anti-oxidants), emulsifying agents and the like. A particularly preferred carrier or diluent for use in the formulations of this invention is a lower alkyl ester of a C18 to C24 mono-unsaturated fatty acid, such as oleic acid, for example ethyl oleate. Other suitable carriers or diluents include capric or caprylic esters or triglycerides, or mixtures thereof, such as those caprylic/capric triglycerides sold under the trade name Miglyol, eg Miglyol 810.

63. Because these compounds antagonise the function of CCK in animals, they may also be used as feed additives to increase the food intake of animals, such as in a daily dosage of from about 0.05 to 50 mg/kg of body weight.

64. The invention will now be further described by the way of example only.

65. Remarkable are the similarities between but-2-enoic acid amides and ureidobenzodiazepines, which was the rationale behind this discovery programme. 2-substituted benzodiazepines act as opiate agonists and 3-substituted benzodiazepines are known as CCK antagonists. The Z configurated unsaturated amides mimic this 7 membered ring system, and this template was applied to the design of CCK antagonists and opiate agonists.

66. Figure 1 shows the structural similarities of the antagonist L-365,260 and Exp 8, which appeared to be the best amide to mimic Merck's compounds. _

67. Molecular modelling of L-365,260 and Exp 8 to show the overlapping structural features from the top and the side. The bisarylated amides do not contain a chiral centre unlike the benzodiazepines.

EXAMPLES

68. Example A-G

Synthesis of 2,3-dichIoro-4,4-di- p -tolyl-but-2-enoic acid amides.

The bisarylated acids were synthesised according to the methods prescribed in the
patent entitled novel tetramic acids by using excess of the arene.

Selected examples A-D of the building blocks are outlined in this experimental
section.

69. Synthesis of but-2-enoic acids.

A range of compounds were reacted with mucochloric acid in the presence of aluminium chloride at room temperature for 72 hours to form various bis-subsubstituted-aryl-but-2-enoic acids as shown in figure 2.

70. The outlined acids were prepared in high yields and they were used to synthesise
combinatorial libraries with 12 amines to give a combinatorial library of 60 amides.

Exp A and Exp B had previously been synthesised by Langley and were the starting point for the formation of these compounds. Exp F = p-nitrophenyl, Exp G = dimethoxy phenyl were formed in poor yields.

71. The series D gave the best CCK binding affinity, while donor substituents, such as based on Exp E resulted in the best opiate agonist activity.

73. The the bis-arylated but-2-enoic acids A-E were refluxed with thionyl chloride to form an acid chloride of the corresponding compound. Amines such as isobutyl-and cyclopropylamine were instantly added to give the desired amides. Reactions were allowed to proceed at both RT in ether and 60 °C in THF for 30 and 90 minutes and the progress of the reaction was monitored by TLC analysis.

EVALUATION

74. The formation of these amides was used originally to mimic Merck's CCK antagonist L-365,260 which led to a full SAR analysis with respect to CCK antagonists and additionally opiate agonists.

75. The molecules were formed as stable white crystalline compounds in good yields and as the bis-arylated template contains a symmetry plain, a non-chiral ligand is resembled.

76. The second methyl group in the xylene series B1-B12 reduced or diminished the bio- activity, a halogen enhanced generally the binding affinity for boths molecular targets.

77. Donor substituents, such as methoxy group, as obtained for the E series, resulted in an enhanced opiate activity.

78. From a comparison of cLogP values, the compounds with the p-methyl
group occurred:

• the optimum clog p,

• the best binding affinity and

• potentially best brain / membrane penetrating properties.

79. In receptor binding studies the 2 best compounds were confirmed as mixed CCK antagonist with a binding affinity of 1 nM and 13 nM.

80. The synthesised compounds of the A series are outlined as typical examples in Fig 3.

81. Binding affinity, with respect to the cholecystokinin receptor, is outlined in fig 4.

82. Functional studies in transfected cells, monitoring the effects of second messenger, are outlined in fig 5.

83. Ex vivo studies, analysing the effects on isolated tissues preparations, were outlined in Fig 6.

84. The results of the nociceptic pharmacological testing are displayed in figure 7.

85. Using a radiolabeled receptor binding assay, the IC50 were calculated for the cyclopentyl-derivative ( Example 5) and the piperazinyl derivative (Exp 11), as 11 nM and 15 nM for the CCKA receptor, respectively.

86. Using brain tissue, a binding affinity towards the CCK-B receptor was determined for the selected biarylated amides Exp 5 and Exp 1 l of 50nM and 62nM.

87. Subsequently, a second messenger based assay was applied to confirm the ability of the ligands to act as CCK-A antagonists.

Figure 5 . Functional in vitro assay.

88. The CCK-standard proglumide and the most preferred example 5 and 11 were
evaluated in duplicate using a standardised 96 well plate discovery kit from
DiscoveryX. The functional in vitro test resulted in milimolar activity for the
standard and low micromolar activity for the selected bisarylated amides.

89. Luminescence was stimulated by using sulphated CCK-8 and these effects were
antagonised by .the standard and the selected test molecules.

90. Further in vitro studies were applied using isolated tissue preparations.

91. In vitro studies

In the GPI assay, electrically induced contractions were reduced by increasing the concentration of test molecules and subsequently IC 50 were calculated.

92. The effects of Expl on electrically induced ileum twitches. Increased test
concentrations from O.lnM-SOOnM, SOOOnM (IX)and reversed by +100nM
naloxone (X.).

Fig 6. Ex vivo effects using a GPI preparation.

93. These effects were reversed by the opiate antagonist naloxone and the experimental details are described in the example section.

94. No problems with respect to water (Tyrode solution) solubility of these lipophilic
agents was observed.

95. No toxicity towards the guinea pig intestine was observed, which fully responded to acetyl-choline after many hours.

96. In this series of bisarylated amides a structure -activity -relationship (SAR) with
respect to CCK antagonism and opiate agonism was elaborated.

98. The potentiation of antinociception at a 0.5 mg/kg dose of the test molecule and 40 mg/kg tramadol were studied in mice.

99. The essav is a novel research tool

• CCK antagonists potentiate the anti-nocipeptive effects of tramadol

• The anti-nociceptive effects of full opiate agonists were reduced at low doses in the presence of the partial opiate agonist tramadol.

100. DMSO served as vehicle and a dose of 40mg/kg Tramadol as positive standard. The Toluene based examples of CCK antagonists Expl-12 and their potentiation of the analgesic effect of tramadol are outlined.

101. The ureidopyrazol served as internal standard, STD, and occurred 24% MPE. Exp 5 and Exp 11 at a dose of 0.5mg/kg showed a higher MPE with 27% and 32%, respectively.

102. General Synthesis methods

The majority of chemicals used were obtained from the laboratory and chemical stores.

The remainder were ordered from Aldrich Catalogue Handbook of Fine Chemicals and Lancaster 1999/2000/2001.

103. Mass spectrometric analyses was obtained by Atmospheric Pressure Chemical
lonisation (APCI), negative or positive mode, using a Hewlett-Packard 5989b
quadrupole instrument. This was connected to an electrospray 59987A unit with
automatic injection (Hewlett-Packard 1100 series autosampler). Samples were
dissolved in HPLC grade methanol, toluene or acetonitrile.

104 Both Proton and Carbon NMR spectra were obtained on a Brucker AC 250
instrument, operating at 250 MHz, calibrated with the solvent reference peak or
TMS.

IR spectra were plotted from KBr discs on a Mattson 300 FTIR Spectrophotometer. Melting points were recorded from a Stuart Scientific Melting Point (SMPl) and are uncorrected.

105 Analytical Thin Layer Chromatography was obtained using aluminium sheets, silica gel60 F254 and visualized using ultraviolet light.

Preparative chromatography was performed on 250 μm, 20 x 20 cm silica gel TLC plates from Aldrich.

106 Small scale solution syntheses were carried out on a carousel reaction station (RR 98030), comprising a 12-place carousel reaction station and reflux head, and 12 x flexible tubing from Radleys, on a RCT basic hotplate from IKA Labortechnik with IKATRON ETS D3 temperature controller or using heating blocks (TECHNE Dri-block DB-3A).

107 Example A-E for the formation of bisarylated acids Example A: 2,3-Dichloro-4,4-di-p-tolyl-but-2-enoic acid.

The experimental details were described in and in addition 10% THF served as co-solvent, which activated the Lewis acid aluminium chloride.

Yield = 75%; Melting Point: 189-191 "C; Rf (80% ether / 20% petrol ether) = 0.06
MS (APCI(-)): 218/220 (M-1), 297/299 (M-) m/z

•H NMR (CDCI3) 250 MHz: 5 = 11.35-11.61 (bs, COOH), 7.13-7.33 (m, ArH, 8H), 6.65
(s, CH-CCl), 2.41 (s, 6H, CH3) p.p.m.

'C NMR (CDCI3-APT) 250 MHz: 5 = 167.6 (COOH), 154.0 (CCl-CH), 137.2, 136.5
(4xArC), 129.22, 129.15 (8xAr-C), 122.4 (CCl-COOH), 53.5 (CH-CCl), 21.2 (CH3, 2xC) p.p.m.

IR (KBr-disc) \) max: 3457, 3017, 2974, 2934, 2881, 2658, 2496, 1700, 1614, 1558, 1502, 1418, 1270, 1006, 926, 824, 705, 672 cm''.

108. Example B: 23-Dichloro-4,4-bis-(2,4-dimethyl-phenyl)-but-2-enoic acid Yield = 71%; Melting Point: 175-177 °C; Rf (80% ether / 20% petrol ether) = 0.08 MS (APCI(+)): 289/291 (M-1), 325/327 (M-) m/z 'H NMR (CDCI3) 250 MHz: 6 = 9.49-10.53 (bs, COOH), 6.89-7.08 (m, ArH, 6H), 2.41 (s, CH3, m -position), 2.19 (s, CH3, o-position) p.p.m. 13C NMR (CDCI3) 250 MHz: 8 = 166.9 (COOH), 155.4 (CCl-CH), 136.9, 136.4, 135.7 (6xArC), 131.3, 128.4, 126.7 (6xAr-C), 122.4 (CCl-COOH), 49.1 (CH-CCl), 21.0 (CH3, 2xC, p-position), 19.3, (CH3, 2xC, o-position) p.p.m.
IR (KBr-disc) v max: 3483, 2971, 2933, 2870, 2637, 2489, 1696, 1566, 1508, 1412, 1255, 1113,1005,913,816,699 cm-'.

109 Exp C: 2,3-Dichloro-4,4-diphenyl-but-2-enoic acid Yield = 33%; Melting Point: 169-172 °C; Rf (80% ether / 20% petrol ether) = 0.05 MS (APCI(+)): 190/192 (M-1), 269/271 (M-) m/z'H NMR (CDCI3) 250 MHz: 5 = 9.24-10.39 (bs, COOH), 7.26-7.74 (m, ArH, lOH), 6.78(s, CH) p.p.m.13 C NMR (CDCI3) 250 MHz: 167.6 (COOH), 155.5 (CCl-CH), 139.4 (2xArC), 129.3(4xArC), 128.5 (4xArC), 127.4 (2xArC), 123.8 (CCl-COOH), 54.0 (CH) p.p.m.IR (KBr-disc) v max: 3432, 3032, 2933, 2862, 2638, 2500, 1689, 1557, 1491, 1407, 1257, 1111, 1027, 998, 917, 693 cm-1.

110. Exp D: 2,3-Dichloro-4,4-bis-(4-chIoro-phenyl)-but-2-enoic acid Yield = 51 %; Melting Point: 155-158 °C; Rf (80% ether / 20% petrol ether) = 0.04 MS (APCI(+)): 258/260/262 (M-1), 337/339/341 (M-) m/z 1 H NMR (CDCI3) 250 MHz: 5 = 9.99-10.51 (COOH), 7.28-7.41 (d, ArH, 4H), 7.19-7.25 (d, ArH, 4H), 6.69 (s, CH-CCl) p.p.m.
'C NMR (CDCI3) 250 MHz: 5 = 163.0 (COOH), 151.8 (CCI-CH), 137.8 (2xArC), 132.0 (2xArC), 130.6 (2xArC), 129.8 (2xArC), 129.2 (2xArC), 128.6 (2xArC), 125.1 (CCl-COOH), 52.1 (CH) p.p.m. IR (KBr-disc) max: 3458, 2938, 2854, 2641, 2493, 1773, 1687, 1629, 1564, 1493, 1413, 1261, 1097, 1013, 903, 822, 697 cm"'.

111. Exp E: 2,3-Dichloro-4,4-bis-(4-methoxy-phenyl)-but-2-enoic acid Yield = 47%; Melting Point: 114-115 °C; Rf (80% ether / 20% petrol ether) = 0.05 MS (APCI(+)): 250/252 (M-1), 329/331 (M-) m/z 1H NMR (CDCl3) 250 MHz: 6 - 11.42-11.68 (bs, COOH), 7.11-7.40 (d, ArH, 4H), 6.89-7.01 (d, ArH, 4H), 6.59 (s, CH), 3.92 (s, CH3, 6H) p.p.m. 13C NMR (CDCl3) 250 MHz: 6 = 166.7 (COOH), 158.8 (2xArC), 154.0 (CCl-CH), 131.6 (2xArC), 130.3 (4xArC), 122.0 (CCl-COOH), 113.8 (4xArC), 55.3 (CH3, 2xC), 52.6 (CH-CCl) p.p.m.IR (KBr-disc) \) max: 3433, 3018, 2964, 2931, 2843, 2663, 2515, 2381, 1698, 1611, 1564, 1520, 1420, 1256, 1182, 1038, 914, 827, 699 cm"'.

112. Example 1-12 for the formation of bisarylated amides(1.49 mmol) of the building block of example A-E was added to 5ml of the thionyl chloride and refluxed for 30 minutes. The excess thionyl chloride was distilled off, leaving a medium brown viscous oil. 2 ml ether was added and the content of the beaker was stirred, after which 2 equivalents of the appropriate amine (3.00 mmol) were added. The solution was stirred for a further 10 minutes. The reaction was monitored using TLC. 10 ml water was added to the resulting product and washed twice with 20 ml ether. After work up, the product which slowly precipitated out of the ether solution was collected by filtration. The sample was dried in a vacuum dessicator.

113. Exp 1: 2,3-Dichloro-4,4-di-p-toIyI-but-2-enoic acid dimethylamide. Yield = 67%; Melting Point: 114-116 C; Rf (80% ether / 20% petrol ether) = 0.39 MS (APCI(+)): 270/272 (M+), 362/364 (M+1) m/z 1H NMR (CDCI3) 250 MHz: 5 = 7.11-7.26 (m, Ar-H, 8H), 5.45 (s, CH), 3.05 (s, N-CH3), 2.39 (s, N-CH3), 2.41 (s, Ar-CH3, 6H) p.p.m.13CNMR(CDCl3)250MHz:6= 163.2 (C=0), 145.7 (CH-CCl), 137.0, 136.2, 129.1, 129.0
(Ar-C), 54.0 (CH-CCl), 38.3 (NH-CH3), 34.9 (NH-CH3), 21.1 (Ar-C-CH3, 2xC) p.p.m.IR (KBr-disc) \) max: 3425, 3022, 2924, 2850, 2363, 2340, 1653, 1514, 1398, 1259, 1180, 672 cm"'.

114. Exp 2: 2,3-Dichloro-4,4-di-p-tolyl-but-2-enoic acid n-propylamide. Yield = 69%; Melting Point: 118-122 °C; Rf (80% ether / 20% petrol ether) = 0.49 MS (APCI(+)): 270/272 (M+), 362/364 (M+1) m/z 1H NMR (CDCI3) 250. MHz: 6 = 7.11-7.26 (m, Ar-H, 8H), 5.45 (s, CH), 3.25 (s, N-CH2), 2.19 (m, CH2), 2.41 (s, Ar-CH3, 6H, 1.41 (t, CH3, 3H) p.p.m. 13C NMR (CDCl3) 250 MHz: 5= 163.2 (C=0), 145.7 (CH-CCl), 137.0, 136.2, 129.1, 129.0 (Ar-C), 54.0 (CH-CCl), 38.3 (NH-CH3), 34.9 (NH-CH3), 21.1 (Ar-C-CHa, 2xC) p.p.m. IR (KBr-disc) t> max: 3425, 3022, 2924, 2850, 2363, 2340, 1653, 1514, 1398, 1259, 1180, 672 cm-'.

115. Exp 3: 2,3-Dichloro-4,4-di-p-tolyl-but-2-enoic acid cyclopropylamide.Yield = 82%; Melting Point: 130-132 °C; Rf (80% ether / 20% petrol ether) = 0.42 MS (APCI(+)): 302/304 (M+1), 374/376 (M+) m/z 'H NMR (CDCI3) 250 MHz: 8 = 7.07-7.41 (m, ArH, 8H), 6.78 (s, CH), 6.46-6.61 (bs, NH), 2.71-2.93 (m, NH-CH), 2.40 (s, CH3, 6H), 1.88-1.99 (m, NH-CH-CH2), 1.50-1.74 (m, NH-CH-CH2) p.p.m. 13C NMR (CDCl3) 250 MHz: 5 = 163.1 (C=0), 147.0 (CH-CCl), 137.1 (ArC), 136.8 (ArC), 124.1 (CO-CCl), 52.7 (CH-CCl), 23.3 (NH-CH), 21.1 (CH3, 2xC), 6.8 (CH2, 2xC) p.p.m. IR (KBr-disc) X) max: 3281, 3084, 3020, 2919, 2362, 2335, 1908, 1638, 1512, 1446, 1285, 1181, 1115, 1028, 823, 649 cm-1.

116. Exp 4: 2,3-DichIoro-4,4-di-p-tolyl-but-2-enoic acid isobutyl-amide.Yield = 84%;Melting Point: 138-140 °C;Rf (80% ether / 20% petrol ether) = 0.42 MS (APCI(+)): 318/320 (M+1), 390/392 (M+) m/z 'H NMR (CDCI3) 250 MHz: 5 = 7.01-7.23 (m, ArH, 8H), 6.71 (s, CCl-CH), 6.28-6.43 (bs, NH), 3.02-3.18 (d, CH2-CH), 2.32 (s, 6H, C-CH3), 1.77-1.98 (m, J= 4.9 Hz, 2H, CH-CH3), 0.86-1.01 (m, 6H, CH-CH3) p.p.m. 13C NMR (CDCI3) 250 MHz: 6 = 161.8 (C=0), 146.2 (CH-CCl), 137.1, 136.7 (4xArC), 129.1, 129.0 (8xAr-C), 124.6 (CO-CCl), 52.8 (CH-CCl), 47.5 (NH-CH2), 28.4 (CH-CH2),21.1 (Ar-C-CH3), 20.1 (CH3) p.p.m. IR (KBr-disc) u max: 3293, 2967, 2929, 2872, 1642, 1549, 1515, 1464, 1278, 1118, 732 cm"'.

117. Exp 5: 2,3-Dichloro-4,4-di-p-tolyl-but-2-enoic acid cyclopentyiamide. Yield = 76%; Melting Point: 156-158 °C; Rf (80% ether / 20% petrol ether) = 0.43 MS (APCI(+)): 330/332 (M+1), 402/404(M+) m/z'H NMR (CDCI3) 250 MHz: 6 - 7.12-7.23 (m, ArH, 8H), 6.70 (s, CH), 6.22-6.39 (bs, NH),4.16-4.38 (m, CH, J= 6.8 Hz), 2.40 (s, CH3, 6H), 1.96-2.12 (m, CH2), 1.62-1.78 (m, CH2,4H), 1.37-1.56 (m, CH2) p.p.m. 13C NMR (CDCI3) 250 MHz: 5= 161.3 (C=0), 145.7 (CH-CCl), 137.1 (ArC), 136.8 (ArC),129.1 (4xArC), 129.0 (4xArC), 124.7 (CO-CCl), 52.9 (CH-CCl), 52.1 (NH-CH), 32.9 (NH-CH-CH2, 2xC), 23.7 (NH-CH-CH2-CH2, 2xC), 21.1 (CH3) p.p.m. IR (KBr-disc)) max: 3437, 3279, 3056, 2026, 2960, 2864, 2362, 2340, 1637, 1536, 1510,1440,1314, 1270,1178, 1113, 817, 732, cm"'.

118. Exp 6: 2,3-Dichloro-4,4-di-p-tolyI-but-2-enoic acid cyclohexyiamide.Yield - 68%; Melting Point: 158-160 °C; Rf (80% ether / 20% petrol ether) = 0.45MS (APCI(+)): 344/346 (M+1), 416/418 (M+) m/z'H NMR (CDCI3) 250 MHz: 6 = 7.01-7.51 (m, ArH, 8H), 6.70 (s, CCI-CH), 6.21-6.38 (bs,NH), 3.80-3.98 (m, IH, NH-CH), 2.42 (s, 6H, CH3), 1.09-2.06 (m, lOH, CH2) p.p.m.'C NMR (CDCI3) 250 MHz: 5 = 160.8 (C=0), 142.8 (CH-C-Cl), 137.1 136.7 (4xArC),129.0, 129.1 (8xAr-C), 124.7 (CO-CCl), 52.8 (NH-CH), 49.2 (CCl-C-H), 32.7 (NH-CH-CH2, 2xC), 25.4 (NH-CH-CH2-CH2, 2xC), 24.7 (NH-CH-CH2-CH2-CH2), 21.1 (CH3)p.p.m.IR (KBr-disc) v max: 3272, 2932, 2863, 1642, 1554, 1513, 1450, 1321, 1117, 890, 815,726 cm-'.

119. Exp 7: 2,3-Dichloro-4,4-di-p-tolyl-but-2-enoic acid cyclohexyl-isopropyl-amide.
Yield = 62%; Mehing Point: 156-159 °C; Rf (80% ether / 20% petrol ether) = 0.45 MS (APCI(+)): 458/460 (M+1) m/z1H NMR (CDCl3) 250 MHz: 5 = 7.05-7.27 (m, ArH, 8H), 5.51-5.54 (d, CH), 3.28-3.49 (m,N-CH), 2.50-2.71 (m, N-CH-CH2), 2.33-2.42 (m, CH3, 6H), 0.58-1.99 (m, overlapping,CH2 & CH3, 16H) p.p.m.13C NMR (CDCI3) 250 MHz: 6 = 162.9 (C=0), 147.0 (2xArC), 137.1 (CH-CCl), 136.8,136.7, 136.5, 136.2 (4xArC), 129.5 (2xArC), 129.2 (2xArC), 128.9 (2xArC), 128.2(2xArC), 125.2 (CO-CCl), 60.2 (N-CH-CH2), 53.2 (N-CH-CH3), 47.4 (CH-CCl), 30.7, 30.8(CH2, 4xC), 25.5, 25.6 (CH2, 2xC), 25.1 (CH2), 20.6, 20.1 (Ar-CHj, 2xC), 19.5 (CH-CH3,2xC) p.p.m.
IR (KBr-disc) D max: 3443, 2994, 2948, 2858, 2368, 2341, 1910, 1647, 1511, 1439, 1357,1244,1112,985,822,670 cm"'.

120. Exp 8: 2,3-Dichloro-4,4-di-p-tolyI-but-2-enoic acid methyl-m-toiyl-amide.Yield - 65%; Melting Point: 125-127 "C; Rf (80% ether / 20% petrol ether) = 0.36MS (APCI(+)): 438/440 (M+1) m/z 'H NMR (CDCI3) 250 MHz: 6 = 7.03-7.42 (m, ArH, l0H), 6.90-6.97 (m, ArH), 6.76 (s, ArH), 5.66 (s, CH), 3.46 (s, N-CH3), 2.45 (s, Ar-CH3, 9H) p.p.m.13CNMR(CDCl3)250MHz:5= 163.8 (C=0), 141.8 (CH-CCl), 139.4 (ArC), 138.9 (ArC),136.7 (2xArC), 136.4 (2xArC), 129.2 (4xArC), 129.1 (4xArC), 129.0 (ArC), 128.7 (ArC),127.2 (ArC), 124.5 (CO-CCl), 123.8 (ArC), 53.5 (CH-CCl), 38.0 (N-CH3), 21.1 (CH3,2xC) p.p.m. IR (KBr-disc) M max: 3445, 3056, 3020, 2961, 2921, 2362, 2345, 1650, 1605, 1511, 1462, 1368, 1305, 1112, 812, 777, 696 cm"'.

121. Exp 9: 2,3-dichloro-4,4-di-p-tolyl-but-2-enoic acid amides.Yield - 71%; Melting Point: 165-167 °C; Rf (80% ether / 20% petrol ether) = 0.38MS (APCI(+)): 352/354 (M+1), 424/426 (M+) m/z 'H NMR (DMSO-d6) 250 MHz: 5 = 6.86-7.61 (m, ArH, 13H), 6.72 (m, 2H, CH & NH), 4.49-4.56 (d, 2H, CH2X 2.21 (s, 6H, CH3) p.p.m.13C NMR (CDCI3) 250 MHz: 5 = 161.6 (C=0), 146.8 (CH-CCl), 137.1 (ArC), 137.0(2xArC), 136.8 (2xArC), 129.1 (3xArC), 129.08 (2xArC), 128.9 (2xArC), 127.9 (2xArC),127.8 (2xArC), 127.8 (2xArC), 124.2 (CO-CCl), 52.8 (CCl-CH), 44.3 (CH2), 21.1 (CH3,2xC) p.p.m.IR (KBr-disc) D max: 3312, 3059, 3022, 2953, 2921, 2857, 2366, 2343, 1902, 1645, 1580,1530, 1507, 1447, 1424, 1282, 1259, 1020, 818,690 cm'.122. Exp 10: 2,3-Dichloro-4,4-di-p-tolyI-but-2-enoic acid benzyl-methyl-amide.

Yield = 59%; Melting Point: 120-122 °C; Rf (80% ether / 20% petrol ether) = 0.37
MS (APCI(+)): 366/368 (M+), 438/440 (M+1) m/z'H NMR (CDCI3) 250 MHz: 5 = 7.02-7.75 (m, ArH, 13H), 5.50 (s, CH), 4.66 (s, CH2),2.81 (s, N-CH3), 2.41-2.52 (m, Ar-CH3, 6H) p.p.m.13C NMR (CDCI3) 250 MHz: 6 - 164.1 (C=0), 141.8 (2xArC), 137.5, (CH-CCl), 136.9 (ArC), 136.1, 136.0 (2xArC), 129.15, 129.1 (2xArC), 128.9 (2xArC), 128,8 (2xArC), 128.7 (2xArC), 128.3 (2xArC), 127.9 (2xArC), 127.5 (ArC), 123.2 (CO-CCl), 53.9 (CH), 50.6 (CH2), 35.6 (N-CH3), 21.1 (CH3) p.p.m.IR (KBr-disc) v max: 3443, 3271, 3019, 2921, 2855, 2360, 2337, 1643, 1506, 1404, 1267, 1183, 1135, 1020, 895, 821, 706 cm-'.123. Exp 11: l-(4-Benzyl-piperazin-l-yl)-2,3-dichloro-4,4-di-p-tolyl-but-2-en-l-one.

Yield = 72%; Melting Point: 182-184 °C;Rf (80% ether / 20% petrol ether) - 0.36
MS (APCI(+)): 493/495 (M+1) m/z'H NMR (CDCI3) 250 MHz: 5 = 7.41-7.62 (m, ArH, 5H), 7.00-7.27 (m, ArH, 8H), 5.43 (s,CH), 3.01-4.18 (m, overlapping CH2, 8H), 2.41 (s, CH3, 6H) p.p.m.'C NMR (CDCI3) 250 MHz: 5 = 162.2 (C=0), 147.1 (CH-CCl), 139.7 (2xArC), 137.4(ArC), 131.3 (4xArC), 130.5 (2xArC), 129.5 (4xArC), 129.4 (2xArC), 128.8 (ArC), 123.0(CO-CCl), 61.2 (Ar-CH2), 46.8 (CH-CCl), 43.3 (Ar-CH2-N-CH2, 2xC), 38.4 (CO-N-CH2,2xC), 21.2 (CH3, 2xC) p.p.m.IR (KBr-disc) v max: 3524, 3439, 3027, 2915, 2536, 2457, 2372, 1642, 1511, 1429, 1282,951,820,755,702 cm"'.

124. Biological Evaluation - [125] l-CCK-8 receptor binding essay:

CCKA and CCKB receptor binding assays were performed, by using guinea pig cerebral cortex (CCKB) or rat pancreas (CCKA). Male guinea pig brain tissues were prepared according to the modified method described by Saita et al, [(1994), Characterization of YM022: its CCKB/gastrin receptor binding profile and antagonism to CCK-8-induced Ca2+ mobilization., Eur. J. Pharmacol.,269, 249-254]. Pancreatic membranes were prepared in a similar way but by Charpentier et al, [(1988), Cyclic cholecystokinin analogues with high selectivity for central receptors., Proc Natl Acad Sci USA, 85, 1968-1972]. The in vivo CCK binding assay: Tissues were homogenized in ice cold sucrose (0.32 M, 25 ml) for 15 strokes at 500 rpm and centrifuged at 13000 rpm for 10 mins.

The supernatant was re-centrifuged at 13000 rpm for 20 mins. The resulting pellet was re-dispersed to the required volume of buffer at 500 rpm and stored in aliquots at 70°C. Binding was achieved using a radioligand 125-Bolton-Hunter labeled CCK, NEN at 25 pM. The samples were incubated {with membranes (0.1 mg/ml)} in 20 mM Hepes, ImM EGTA, 5 mM MgCl2 , 150 mm NaCl, 0.25 mg/ml bacitracin at pH 6.5 for 2 hrs at RT and then suspended by centrifugation at 1100 rpm for 5 minutes. The membrane pellets were washed twice with water and the bound radioactivity was measured in a Packard Cobra Auto-gamma counter (B5005). All binding assays were carried out with L-363, 260 as an internal non-specific standard. Controls (no compound) were also added. All samples were made in duplicate and repeated twice. All compounds were initially screened for percentage inhibition at 10 |i.m. Samples showing an average inhibition of <35% were diluted to lμm and re-screened and if active diluted again.

125. The in vivo assays were extensively described in Lattmann et al.

126. The in vitro assay is based on the standard GPI assay which was electrically stimulated, the compounds reversed the contraction and the effect was reduced by the administration of the opiate antagonist naloxone.

127. In vitro bioassays

The myenteric plexus-longitudinal muscle from the guinea pig ileaum (GPI) (Kosterlitz and Waterfield, 1975), mouse vas deferens (MVD) (Hughes et al., 1975) and rabbit vas deferens (LVD) (Oka et al., 1981) were prepared as described. The preparation were suspended in an organ bath containing 4 ml Krebs solution. The bath fluid was kept at 37 ± 1 °C and gassed continuoualy with 95% O2 and 5% CO2. The rsting tension was maintained at 250 mg for the MVD and 500 mg for the GPl and LDV. The composition of Krebs solution was (in mM) NaCl 119, KCI 4.7, CaCl2 2.55, KH2PO4 1.6, MgS04 1.18, NaHCO3 25, glucose 11 and mepyamine maleate 0.00013. Hexamethonium bromide 70 μM and choline chloride 20 μM were added to the Krebs solution for the GPL MgS04 was not included in the Krebs solution for the MVD. After equilibrium for 45 min, longitudinal contractions were evoked by field stimulation through Pt-electrodes at the upper and lower ends of the bath. The parameters of stimulation were as follows. For the MVD, the trains were consisted of 4 pulses at intervals Of 200 ms (40 V, 1.0 ms duration, 15 s interval). The contractions were recorded with a force displacememt transducer and an autoequilibrium recorder. The agonist pptencies of the compounds were obtained from dose-response curves by calculating the concentration of the compounds that height of the contractions by 50% (IC50).

128. The antagonist equilibrium constant, Ke values were determined by the single dose method of Kosterlitz and Watt (1968). Ke = a(DR - 1), where a is the molar concentration of antagonist and DR is the dose-ratio. DR = M3/(M2 -Ml), where Ml is the concentration of test compound which would be expected to have the same depressant effect produced by antagonist on the twitch in the absence of the antagonist. M2 is the concentration of test compound which would have a depressant effect equal to the combined actions of the antagonist and the test compound (in higher concentration, M3) in the absence of the antagonist.

129. Antinociceptive assay

Antinociceptive activity was examined by using the mouse hot plate assay (Chao and Tsoh, 1956) and the mouse writhing assay (Koster et al., 1959). In the mouse hot plate assay, female mice were placed on a zinc plate heated to 55 ± 1 °C. The latency time of licking hind-paw was recorded as nociceptive response. Each mouse was tested twice before drug administration and the latencies were averaged to obtaine the baseline latency. Only mice with baseline latencies between 1—20 s were chosen for further studies. The efficiency of antinociceptive activity was defined as doubling of the baseline latency ( Huang et al., 1984). The antinociceptive activity of drugs was measured 5 min after intraperitoneal ly injection. In the writhing assay, the male mice were received 0.2 ml of 1% acetic acid intraperitoneally and they were observed for 15 min for writhing. Different doses of test compounds, morphine or fentanyl were subcutaneously administered before acetic acid administration. The absence of writhing response was scored as antinociception. Ten animal were used at each dose group.

CLAIMS

1 A compound of formula (I):

wherein

X is selected as previously specified

R is selected from an ester or amine or functionality as previously specified Aryl is selected as previously specified or as defined in X, Rl and aryl.

2 A compound, as claimed in claim 1, wherein substituents are as defined, represent examples of novel chemical entities.

3 A compound as claimed in claim 1 having the one of following formulae are novel agents

4 The use of a compound as claimed before are novel ligands, in particular as CCK receptor ligands, CCK antagonist and or opiate agonist.

5 The agents are useful as novel medicines as claimed, wherein said compound is a selective CCKl , CCK2 or mixed CCK ligand.

6 The agents are useful as novel medicines as claimed, wherein said compound is a selective mu, delta, or kappy agonist.

7 The agents are useful as novel medicines as claimed, wherein said compound is a selective analgesic and potentiating the action of analgesics.

8 The agents are useful as novel medicines as claimed, wherein said compound is a selective anticancer agent.

9 The agents are useful as novel medicines as claimed, wherein said compound is a selective agent exerting its actions by other mechanisms.

10 A method of a research tool: The application of tramadol in an assay to select potentiators, CCK ligands and opiates by reducing their biological effect.

11 A method of treatment of a mammal afflicted with a CCK-related condition, or prophylaxis in a mammal at risk of a CCK-related condition by administration of a therapeutically effective amount of a compound as claimed in any one of claims.

12 The use of a compound in accordance with any one of claims in the preparation of a medicament, for the treatment or prophylaxis of a CCK-related condition.

13 The method of previous claims, wherein said CCK-related conditions is a GI disorder, a CNS disorder caused by CCK interactions with dopamine, other CNS disorder; oncologic disorder, disorder of appetite regulatory systems; Zollinger-Ellison syndrome; antral G cell hyperplasia; or pain.

14 The method or use of a claim, wherein said GI disorder is selected from irritable bowel syndrome, gastro-oesophageal reflux disease or ulcers, excess pancreatic or gastric secretion, acute pancreitis, or motility disorders; said CNS disorder is selected from neuroleptic disorders, tardive dyskinesia, Parkinson's disease, psychosis or Gilles de la Tourette syndrome, said other CNS disorder is selected from anxiety disorders and panic disorders and said oncologic disorder is selected from small cell adenocarcinomas and primary tumours of the central nervous system glial and neuronal cells.

15 The method or use of these novel compounds as CCK antagonists and supported by other pharmacological mechanisms, to protect against stress related aging.

16 The use of a compound to potentiate the analgesic effects of SE/ NE reuptake inhibitors.

17 The use of these agents to potentiate the analgesic effect of opiates and the use of these agents as opiates.

18 Derived from the CCK antagonism and complex interactions with dopamine, the use of these agents as a medicines to treat Parkinson's disease.

19 Formulations of these agents with other known medicines as described for a specified given dose range.

20 The enhancement of anticancer agents, such as Pt compounds, in other than CCK related cancers.