AU2306500A - Triazolo-pyridazine derivatives as ligands for gaba receptors - Google Patents

Description

WO 00/47582 PCT/GBOO/00308 TRIAZOLO-PYRIDAZINE DERIVATIVES AS LIGANDS FOR GABA RECEPTORS The present invention relates to a class of substituted triazolo 5 pyridazine derivatives and to their use in therapy. More particularly, this invention is concerned with substituted 1,2,4-triazolo[4,3-b]pyridazine derivatives which are ligands for GABAA receptors and are therefore useful in the therapy of deleterious mental states. Receptors for the major inhibitory neurotransmitter, gamma 10 aminobutyric acid (GABA), are divided into two main classes: (1) GABAA receptors, which are members of the ligand-gated ion channel superfamily; and (2) GABAB receptors, which may be members of the G-protein linked receptor superfamily. Since the first cDNAs encoding individual GABAA receptor subunits were cloned the number of known members of the 15 mammalian family has grown to include at least six a subunits, four p subunits, three y subunits, one 8 subunit, one 6 subunit and two p subunits. Although knowledge of the diversity of the GABAA receptor gene family represents a huge step forward in our understanding of this ligand 20 gated ion channel, insight into the extent of subtype diversity is still at an early stage. It has been indicated that an c subunit, a P subunit and a y subunit constitute the minimum requirement for forming a fully functional GABAA receptor expressed by transiently transfecting cDNAs into cells. As indicated above, 6, E and p subunits also exist, but are 25 present only to a minor extent in GABAA receptor populations. Studies of receptor size and visualisation by electron microscopy conclude that, like other members of the ligand-gated ion channel family, the native GABAA receptor exists in pentameric form. The selection of at least one a, one P and one y subunit from a repertoire of seventeen allows 30 for the possible existence of more than 10,000 pentameric subunit combinations. Moreover, this calculation overlooks the additional WO 00/47582 PCT/GBOO/00308 -2 permutations that would be possible if the arrangement of subunits around the ion channel had no constraints (i.e. there could be 120 possible variants for a receptor composed of five different subunits). Receptor subtype assemblies which do exist include, amongst many 5 others, clp2y2, a2p23y2, a3py2/3, a2pyl, c5p3y2/3, a6py2, a6PS and c4p8. Subtype assemblies containing an cl subunit are present in most areas of the brain and are thought to account for over 40% of GABAA receptors in the rat. Subtype assemblies containing c2 and a3 subunits respectively are thought to account for about 25% and 17% of GABAA receptors in the 10 rat. Subtype assemblies containing an a5 subunit are expressed predominantly in the hippocampus and cortex and are thought to represent about 4% of GABAA receptors in the rat. A characteristic property of all known GABAA receptors is the presence of a number of modulatory sites, one of which is the 15 benzodiazepine (BZ) binding site. The BZ binding site is the most explored of the GABAA receptor modulatory sites, and is the site through which anxiolytic drugs such as diazepam and temazepam exert their effect. Before the cloning of the GABAA receptor gene family, the benzodiazepine binding site was historically subdivided into two subtypes, BZ1 and BZ2, 20 on the basis of radioligand binding studies. The BZ1 subtype has been shown to be pharmacologically equivalent to a GABAA receptor comprising the al subunit in combination with a P subunit and y2. This is the most abundant GABAA receptor subtype, and is believed to represent almost half of all GABAA receptors in the brain. 25 Two other major populations are the a2py2 and a3py2/3 subtypes. Together these constitute approximately a further 35% of the total GABAA receptor repertoire. Pharmacologically this combination appears to be equivalent to the BZ2 subtype as defined previously by radioligand binding, although the BZ2 subtype may also include certain a5-containing 30 subtype assemblies. The physiological role of these subtypes has hitherto WO 00/47582 PCT/GB0O/00308 -3 been unclear because no sufficiently selective agonists or antagonists were known. It is now believed that agents acting as BZ agonists at alpy2, a2py2 or c3py2 subunits will possess desirable anxiolytic properties. Compounds 5 which are modulators of the benzodiazepine binding site of the GABAA receptor by acting as BZ agonists are referred to hereinafter as "GABAA receptor agonists". The al-selective GABAA receptor agonists alpidem and zolpidem are clinically prescribed as hypnotic agents, suggesting that at least some of the sedation associated with known anxiolytic drugs which 10 act at the BZ1 binding site is mediated through GABAA receptors containing the c subunit. Accordingly, it is considered that GABAA receptor agonists which interact more favourably with the a2 and/or ca3 subunit than with al will be effective in the treatment of anxiety with a reduced propensity to cause sedation. Also, agents which are antagonists 15 or inverse agonists at al might be employed to reverse sedation or hypnosis caused by al agonists. The compounds of the present invention, being selective ligands for GABAA receptors, are therefore of use in the treatment and/or prevention of a variety of disorders of the central nervous system. Such disorders 20 include anxiety disorders, such as panic disorder with or without agoraphobia, agoraphobia without history of panic disorder, animal and other phobias including social phobias, obsessive-compulsive disorder, stress disorders including post-traumatic and acute stress disorder, and generalized or substance-induced anxiety disorder; neuroses; convulsions; 25 migraine; depressive or bipolar disorders, for example single-episode or recurrent major depressive disorder, dysthymic disorder, bipolar I and bipolar II manic disorders, and cyclothymic disorder; psychotic disorders including schizophrenia; neurode generation arising from cerebral ischemia; attention deficit hyperactivity disorder; and disorders of 30 circadian rhythm, e.g. in subjects suffering from the effects of jet lag or shift work.

WO 00/47582 PCT/GBOO/00308 -4 Further disorders for which selective ligands for GABAA receptors may be of benefit include pain and nociception; emesis, including acute, delayed and anticipatory emesis, in particular emesis induced by chemotherapy or radiation, as well as post-operative nausea and vomiting; 5 eating disorders including anorexia nervosa and bulimia nervosa; premenstrual syndrome; muscle spasm or spasticity, e.g. in paraplegic patients; and hearing loss. Selective ligands for GABAA receptors may also be effective as pre-medication prior to anaesthesia or minor procedures such as endoscopy, including gastric endoscopy. 10 WO 98/04559 describes a class of substituted and 7,8-ring fused 1,2,4-triazolo[4,3-b]pyridazine derivatives which are stated to be selective ligands for GABAA receptors beneficial in the treatment and/or prevention of neurological disorders including anxiety and convulsions. The present invention provides a class of triazolo-pyridazine 15 derivatives which possess desirable binding properties at various GABAA receptor subtypes. The compounds in accordance with the present invention have good affinity as ligands for the ca2 and/or a3 subunit of the human GABAA receptor. The compounds of this invention may interact more favourably with the a2 and/or a3 subunit than with the al subunit. 20 Desirably, the compounds of the invention will exhibit functional selectivity in terms of a selective efficacy for the a2 and/or c3 subunit relative to the al subunit. The compounds of the present invention are GABAA receptor subtype ligands having a binding affinity (Ki) for the ca2 and/or a3 subunit, 25 as measured in the assay described hereinbelow, of 100 nM or less, typically of 50 nM or less, and ideally of 10 nM or less. The compounds in accordance with this invention may possess at least a 2-fold, suitably at least a 5-fold, and advantageously at least a 10-fold, selective affinity for the a2 and/or a3 subunit relative to the al subunit. However, compounds 30 which are not selective in terms of their binding affinity for the ca2 and/or a3 subunit relative to the al subunit are also encompassed within the WO 00/47582 PCT/GBOO/00308 -5 scope of the present invention; such compounds will desirably exhibit functional selectivity in terms of a selective efficacy for the a2 and/or a3 subunit relative to the cl subunit. Moreover, the compounds according to the present invention may possess interesting pharmacokinetic properties, 5 notably in terms of improved oral bioavailability. The present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof: N-N R I\ Z NR 0"' NO N R (I) 10 wherein Z represents tert-butyl, cyclobutyl, phenyl or pyrrolidin-1-yl; R' represents hydrogen, methyl, methoxy or fluoro;

R

2 represents hydrogen or fluoro;

R

3 represents hydrogen, methyl or ethyl; 15 R 4 represents trifluoromethyl, chloromethyl, or a group of formula

-CH

2 ORa or -CH 2 NRbRc; Ra represents hydrogen, methyl or tert-butyldimethylsilanyl; and Rb and Rc both represent methyl; or Rb and Rc together represent the residue of an azetidine, 3,3-difluoroazetidine, pyrrolidine, morpholine 20 or N-methylpiperazine moiety. Certain compounds in accordance with the present invention are encompassed within the generic scope of WO 98/04559. There is, however, WO 00/47582 PCT/GBOO/00308 -6 no specific disclosure therein of compounds corresponding to those of formula I as defined above. For use in medicine, the salts of the compounds of formula I will be pharmaceutically acceptable salts. Other salts may, however, be useful in 5 the preparation of the compounds of formula I or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of formula I include acid addition salts which may, for example, be formed by mixing a solution of the compound of formula I with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, 10 sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In the compounds of formula I above, the moiety Z suitably represents tert-butyl, phenyl or pyrrolidin-1-yl. 15 Suitably, R1 represents hydrogen or fluoro. Suitably, R 2 represents hydrogen. Suitably, R 3 represents methyl or ethyl. Suitably, Ra represents hydrogen or methyl. Particular sub-classes of compounds according to the invention are 20 represented by the compounds of formula IIA and IIB, and pharmaceutically acceptable salts thereof: WO 00/47582 PCT/GBO0/00308 -7 R1 R N-N N-N N R2 N R o 0 N N RN N N R /R R3 (IIA) (IIB) wherein Z, R1, R 2 , R 3 and R4 are as defined above. Specific compounds within the scope of the present invention 5 include: N- [5-(3,7-diphenyl- 1,2,4-triazolo [4, 3-b]pyridazin-6-yloxymethyl)-2-methyl 2H-1,2,4-triazol-3-ylmethyl]-N,N-dimethylamine; [5-(3,7-diphenyl-1,2,4-triazolo[4,3-b]pyridazin-6-yloxvmethyl)-1-methyl 1H- 1,2,4-triazol-3-yl] methanol; 10 N-[5-(3,7-diphenyl-1,2,4-triazolo[4,3-b]pyridazin-6-yloxymethyl)-1-methyl 1H-1,2,4-triazol-3-ylmethyl]-N,N-dimethylamine; [1-methyl-5-(3-phenyl-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazin-6 yloxymethyl)- 1H- 1,2,4-triazol- 3-yl] methanol; 6-(5-chloromethyl-2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-phenyl-7 15 (pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine; N-[5-(3-phenyl-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazin-6 yloxymethyl)-1-methyl-1H-1,2,4-triazol-3-ylmethyl]-N,N-dimethylamine; 6-[2-methyl-5-(morpholin-4-ylmethyl)-2H-1,2,4-triazol-3-ylmethoxy]-3 phenyl-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine; 20 6-[5-(azetidin-1-ylmethyl)-2-methyl-2H-1,2,4-triazol-3-ylmethoxy]-3 phenyl-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine; WO 00/47582 PCT/GBOO/00308 -8 6- [2-methyl-5-(4-methylpiperazin- 1-ylmethyl)-2H- 1,2,4-triazol-3 ylmethoxy]-3-phenyl-7-(pyrrolidin- 1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 6- [5-(3,3-difluoroazetidin- 1-ylmethyl)-2-methyl-2H- 1,2,4-triazol-3 ylmethoxy] -3-phenyl-7-(pyrrolidin- 1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 5 6- [2-methyl-5-(pyrrolidin- 1-ylmethyl)-2H- 1,2,4-triazol-3-ylmethoxy] -3 phenyl-7-(pyrrolidin- 1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 7-tert-butyl-6- [5-(tert-butyldimethylsilanyloxymethyl)-2-methyl-2H- 1,2,4 triazol-3-ylmethoxy] -3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; [5-(7-tert-butyl-3-(2-fluorophenyl)- 1,2,4-triazolo [4,3-b]pyridazin-6 10 yloxymethyl)- 1-methyl- 1H- 1,2,4-triazol-3-yl] methanol; 7 -tert-butyl-3-(2-fluorophenyl)-6-(5-methoxymethyl-2-methyl-2H- 1,2,4 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 7 -tert-butyl-3-(2-fluorophenyl)-6-(5-trifluoromethyl- 1H- 1,2,4-triazol-3 ylmethoxy)- 1,2,4-triazolo [4,3-b]pyridazine; 15 7 -tert-butyl-3-(2-fluorophenyl)-6-(2-methyl-5-trifluoromethyl-2H- 1,2,4 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 7 -tert-butyl-3-(2-fluorophenyl)-6-(1-methyl-5-trifluoromethyl- 1H- 1,2,4 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 7-tert-butyl-3-(2-fluorophenyl)-6-(2-ethyl-5-trifluoromethyl-2H- 1,2,4 20 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 7 -tert-butyl-3-(2-fluorophenyl)-6-(1-ethyl-5-trifluoromethyl- 1H- 1,2,4 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; and pharmaceutically acceptable salts thereof. Also provided by the present invention is a method for the 25 treatment and/or prevention of anxiety which comprises administering to a patient in need of such treatment an effective amount of a compound of formula I as defined above or a pharmaceutically acceptable salt thereof. Further provided by the present invention is a method for the treatment and/or prevention of convulsions (e.g. in a patient suffering from 30 epilepsy or a related disorder) which comprises administering to a patient WO 00/47582 PCT/GBOO/00308 -9 in need of such treatment an effective amount of a compound of formula I as defined above or a pharmaceutically acceptable salt thereof. The binding affinity (Ki) of the compounds according to the present invention for the cc3 subunit of the human GABAA receptor is conveniently 5 as measured in the assay described hereinbelow. The a3 subunit binding affinity (Ki) of the compounds of the invention is ideally 10 nM or less, preferably 2 nM or less, and more preferably 1 nM or less. The compounds according to the present invention will ideally elicit at least a 40%, preferably at least a 50%, and more preferably at least a 10 60%, potentiation of the GABA EC 2 o response in stably transfected recombinant cell lines expressing the a3 subunit of the human GABAA receptor. Moreover, the compounds of the invention will ideally elicit at most a 30%, preferably at most a 20%, and more preferably at most a 10%, potentiation of the GABA EC 2 o response in stably transfected recombinant 15 cell lines expressing the al subunit of the human GABAA receptor. The potentiation of the GABA EC 2 o response in stably transfected cell lines expressing the a3 and al subunits of the human GABAA receptor can conveniently be measured by procedures analogous to the protocol described in Wafford et al., Mol. Pharmacol., 1996, 50, 670-678. The 20 procedure will suitably be carried out utilising cultures of stably transfected eukaryotic cells, typically of stably transfected mouse Ltk fibroblast cells. The compounds according to the present invention exhibit anxiolytic activity, as may be demonstrated by a positive response in the elevated 25 plus maze and conditioned suppression of drinking tests (cf. Dawson et al., Psychopharmacology, 1995, 121, 109-117). Moreover, the compounds of the invention are substantially non-sedating, as may be confirmed by an appropriate result obtained from the response sensitivity (chain-pulling) test (cf. Bayley et al., J. Psychopharmacol., 1996, 10, 206-213). 30 The compounds according to the present invention may also exhibit anticonvulsant activity. This can be demonstrated by the ability to block WO 00/47582 PCT/GBOO/00308 - 10 pentylenetetrazole-induced seizures in rats and mice, following a protocol analogous to that described by Bristow et al. in J. Pharmacol. Exp. Ther., 1996, 279, 492-501. In order to elicit their behavioural effects, the compounds of the 5 invention will ideally be brain-penetrant; in other words, these compounds will be capable of crossing the so-called "blood-brain barrier". Preferably, the compounds of the invention will be capable of exerting their beneficial therapeutic action following administration by the oral route. The invention also provides pharmaceutical compositions 10 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, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, 15 parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. 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 20 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 pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active 25 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 30 present invention. Typical unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets WO 00/47582 PCT/GBOO/00308 - 11 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 5 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 10 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 15 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. 20 In the treatment of anxiety, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, and especially about 0.05 to 5 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day. The compounds of formula I as defined above may be prepared by a 25 process which comprises reacting a compound of formula III with a compound of formula IV: WO 00/47582 PCT/GBOO/00308 - 12 OH N N -- ' N 3 / \ N NN Z R 2 N R ~-N NR L (III) (IV) wherein Z, R', R 2 , R 3 and R 4 are as defined above, and Li represents a suitable leaving group. 5 The leaving group L' is typically a halogen atom, especially chloro. The reaction between compounds III and IV is conveniently effected by stirring the reactants in a suitable solvent, typically NN-dimethyl formamide, in the presence of a strong base such as sodium hydride. The intermediates of formula III above may be prepared by reacting 10 a compound of formula V with a substantially equimolar amount of a hydrazine derivative of formula VI: L 2 R1 0 N I NHNH, Z N z 4 L R 2 (V) (VI) 15 wherein Z, R1, R 2 and L' are as defined above, and L 2 represents a suitable leaving group; followed, if necessary, by separation of the resulting mixture of isomers by conventional means. The leaving group L 2 is typically a halogen atom, especially chloro. In the intermediates of formula V, the leaving groups Li and L 2 may be 20 the same or different, but are suitably the same, preferably both chloro.

WO 00/47582 PCT/GBOO/00308 - 13 The reaction between compounds V and VI is conveniently effected by heating the reactants in the presence of a proton source such as triethylamine hydrochloride, typically at reflux in an inert solvent such as xylene or 1,4-dioxane. 5 Alternatively, the intermediates of formula III above may be prepared by reacting a hydrazine derivative of formula VII with an aldehyde derivative of formula VIII:

NHNH

2 R CHO z N (VII) (ViII) 10 wherein Z, R1, R 2 and L 1 are as defined above; followed by cyclization of the intermediate Schiffs base thereby obtained. The reaction between compounds VII and VIII is conveniently effected under acidic conditions, for example in the presence of a mineral 15 acid such as hydrochloric acid. Cyclization of the resulting Schiffs base intermediate may then conveniently be carried out by treatment with lead(IV) acetate in acetic acid or with iron(III) chloride in a suitable solvent, e.g. an alcoholic solvent such as ethanol, at an elevated temperature, typically at a temperature in the region of 60-70*C. 20 The intermediates of formula VII above may be prepared by reacting the appropriate compound of formula V as defined above with hydrazine hydrate, typically in 1,4-dioxane at the reflux temperature of the solvent; followed, if necessary, by separation of the resulting mixture of isomers by conventional means.

WO 00/47582 PCT/GBOO/00308 - 14 In an alternative approach, the intermediates of formula III above may be prepared by reacting the hydrazine derivative of formula VII as defined above with a compound of formula IX: Q R 2 5 (IX) wherein R1 and R 2 are as defined above, and Q represents a reactive carboxylate moiety; followed by cyclization of the hydrazide derivative of formula X thereby obtained: 10 0 R NHN H N 2> R N z LI (X) wherein Z, R 1 , R 2 and Li are as defined above. Suitable values for the reactive carboxylate moiety Q include esters, 15 for example C.4 alkyl esters; acid anhydrides, for example mixed anhydrides with C 1 4 alkanoic acids; acid halides, for example acid chlorides; and acylimidazoles. Suitably, Q represents an acid chloride moiety. The reaction between compounds VII and IX is conveniently effected 20 under basic conditions, e.g. in the presence of triethvlamine, suitably in an inert solvent such as diethyl ether, and typically at a temperature in the WO 00/47582 PCT/GBOO/00308 - 15 region of 0 0 C. Cyclization of the resulting compound of formula X may then conveniently be carried out by treatment with 1,2-dibromo-1,1,2,2 tetrachloroethane and triphenylphosphine, in the presence of a base such as triethylamine, suitably in an inert solvent such as acetonitrile, and 5 typically at a temperature in the region of 0*C. The reaction between compound V and hydrazine hydrate or compound VI will, as indicated above, usually give rise to a mixture of isomeric products depending upon whether the hydrazine nitrogen atom displaces the leaving group L 1 or L 2 . Thus, in addition to the required 10 product of formula III, the isomeric compound wherein the moiety Z is attached at the 8-position will usually be obtained to some extent; and likewise for compound VII. For this reason it will generally be necessary to separate the resulting mixture of isomers by conventional methods such as chromatography. 15 In another procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula XI (or its 1,2,4-triazolo[4,3-b]pyridazin-6-one tautomer) with a compound of formula XII: N-N R I \ NI NN Z NN Z "NR2 N R3 OH R4 20 (XI) (XII) wherein Z, R1, R 2 , R 3 and R 4 are as defined above, and L 3 represents a suitable leaving group. The leaving group L is suitably a halogen atom, typically chloro or 25 bromo.

WO 00/47582 PCT/GBOO/00308 - 16 The reaction between compounds XI and XII is conveniently effected by stirring the reactants in a suitable solvent, typically NN dimethylformamide, in the presence of a strong base such as sodium hydride. 5 The intermediates of formula XI above may conveniently be prepared by reacting a compound of formula III as defined above with an alkali metal hydroxide, e.g. sodium hydroxide. The reaction is conveniently effected in an inert solvent such as aqueous 1,4-dioxane, ideally at the reflux temperature of the solvent. 10 In a further procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula Z-CO 2 H with a compound of formula XIII: N-N R 1 NI 0 N R

RR

3 (XIII) 15 wherein Z, Ri, R 2 , R 3 and R4 are as defined above; in the presence of silver nitrate and ammonium persulphate. The reaction is conveniently carried out in a suitable solvent, for example water or aqueous acetonitrile, optionally under acidic conditions, 20 e.g. using sulphuric acid, typically at an elevated temperature. The intermediates of formula XIII correspond to the compounds of formula I as defined above wherein Z is hydrogen, and they may therefore WO 00/47582 PCT/GBOO/00308 - 17 be prepared by methods analogous to those described above for preparing the corresponding compounds of formula I. In a still further procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound 5 of formula XIV with a compound of formula XV: N-N N I' R z N N N N R 3 R 4 (XIV) (XV) wherein Z, R', R 2 , R 3 and R 4 are as defined above, M represents -B(OH) 2 or 10 -Sn(Alk) 3 in which Alk represents a C 1 .6 alkyl group, typically n-butyl, and

L

4 represents a suitable leaving group; in the presence of a transition metal catalyst. The leaving group L 4 is suitably a halogen atom, e.g. bromo. A suitable transition metal catalyst of use in the reaction between 15 compounds XIV and XV comprises dichlorobis(triphenylphosphine) palladium(II) or tetrakis (triphenylphosp hine)p alladium(0). The reaction between compounds XIV and XV is conveniently effected in an inert solvent such as N,N-dimethylformamide, typically at an elevated temperature. 20 The intermediates of formula XIV may be prepared by reacting a compound of formula IV as defined above with a compound of formula XVI: WO 00/47582 PCT/GBOO/00308 - 18 N-N L4 N I I ZN z L (XVI) wherein Z, L' and L 4 are as defined above; under conditions analogous to those described above for the reaction between compounds III and IV. 5 In a yet further procedure, the compounds of formula I wherein R 4 represents trifluoromethyl and R 3 is hydrogen may be prepared by a process which comprises reacting a compound of formula XVII with the hydrazide of formula XVIII: N-N R Z 2 NI 0 Z N

F

3 C NHNH, 0) CN 10 (XVII) (XVIII) wherein Z, R 1 and R 2 are as defined above. The reaction is conveniently accomplished by treating compound XVII with sodium methoxide in methanol, neutralising with acetic acid, 15 adding hydrazide XVIII and heating in a solvent such as methanol at a temperature typically in the region of 60 C. The intermediates of formula XVII above may be prepared by reacting the appropriate compound of formula XI (or its 1,2,4-triazolo[4,3 b]pyridazin-6-one tautomer) as defined above with a compound of formula WO 00/47582 PCT/GBOO/00308 - 19 L 3

-CH

2 CN, wherein L is as defined above, under conditions analogous to those described above for the reaction between compounds XI and XII. The hydrazide of formula XVIII above may be prepared as described in Magn. Reson. Chein., 1990, 28, 331-336. 5 Where they are not commercially available, the starting materials of formula IV, V, VI, VIII, IX, XII, XV and XVI may be prepared by methods analogous to those described in the accompanying Examples, or by standard methods well known from the art. It will be understood that any compound of formula I initially 10 obtained from any of the above processes may, where appropriate, subsequently be elaborated into a further compound of formula I by techniques known from the art. For example, a compound of formula I initially obtained wherein R 3 is hydrogen may be converted into a corresponding compound wherein R 3 represents methyl or ethyl by 15 standard alkylation procedures, typically by treatment with iodomethane or iodoethane in the presence of sodium hydride and NN-dimethyl formamide. A compound of formula I initially obtained wherein R4 represents a group of formula -CH 2 ORa and Ra represents tert butyldimethylsilanyl may be converted into the corresponding compound 20 wherein Ra is hydrogen using conventional protodesilylation procedures, typically by heating in ethanolic hydrochloric acid. The resulting compound of formula I wherein R 4 represents -CH 2 OH may be converted into the corresponding compound wherein R 4 represents

-CH

2 ORa and Ra is methyl by methylating with iodomethane in the 25 presence of sodium hydride and NN-dimethylformamide. Alternatively, a compound of formula I wherein R 4 represents -CH 2 OH may be converted into the corresponding compound wherein R 4 represents chloromethyl by stirring in phosphorus oxychloride. In addition, a compound of formula I wherein R 4 represents -CH 2 OH may be converted into the corresponding 30 compound wherein R 4 represents -CH 2 NRbRc in a two-step process which comprises oxidising the -CH 2 OH moiety to -CHO by treatment with oxalyl WO 00/47582 PCT/GBOO/00308 - 20 chloride and dimethylsulphoxide in the presence of triethylamine and dichloromethane; followed by treatment of the resulting aldehyde derivative with the appropriate amine of formula H-NRbRc in the presence of a reducing agent such as sodium cyanoborohydride. A compound of 5 formula I wherein R 4 represents chloromethyl may be converted into a corresponding compound wherein R 4 represents -CH 2 NRbRc by treatment with the appropriate amine of formula H-NRbRc, typically with heating in the presence of sodium hydride and NN-dimethylacetamide. During any of the above synthetic sequences it may be necessary 10 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. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 15 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. The compounds in accordance with this invention potently inhibit 20 the binding of [ 3 H]-flumazenil to the benzodiazepine binding site of human GABAA receptors containing the cc2 or a3 subunit stably expressed in Ltk cells. Reagents 25 o Phosphate buffered saline (PBS). " Assay buffer: 10 mM KH 2

PO

4 , 100 mM KCl, pH 7.4 at room temperature. e [ 3 H]-Flumazenil (18 nM for alp3y2 cells; 18 nM for a2p3y2 cells; 10 nM for a3p3y2 cells) in assay buffer. o Flunitrazepam 100 [tM in assay buffer. 30 o Cells resuspended in assay buffer (1 tray to 10 ml).

WO 00/47582 PCT/GBOO/00308 - 21 Harvesting Cells Supernatant is removed from cells. PBS (approximately 20 ml) is added. The cells are scraped and placed in a 50 ml centrifuge tube. The procedure is repeated with a further 10 ml of PBS to ensure that most of 5 the cells are removed. The cells are pelleted by centrifuging for 20 min at 3000 rpm in a benchtop centrifuge, and then frozen if desired. The pellets are resuspended in 10 ml of buffer per tray (25 cm x 25 cm) of cells. Assay 10 Can be carried out in deep 96-well plates or in tubes. Each tube contains: * 300 ptl of assay buffer. * 50 l of [ 3 H]-flumazenil (final concentration for alp3y2: 1.8 nM; for c2p3y2: 1.8 nM; for ca3p3y2: 1.0 nM). 15 * 50 pl of buffer or solvent carrier (e.g. 10% DMSO) if compounds are dissolved in 10% DMSO (total); test compound or flunitrazepam (to determine non-specific binding), 10 pM final concentration. * 100 pl of cells. Assays are incubated for 1 hour at 40 0 C, then filtered using either a 20 Tomtec or Brandel cell harvester onto GF/B filters followed by 3 x 3 ml washes with ice cold assay buffer. Filters are dried and counted by liquid scintillation counting. Expected values for total binding are 3000-4000 dpm for total counts and less than 200 dpm for non-specific binding if using liquid scintillation counting, or 1500-2000 dpm for total counts and 25 less than 200 dpm for non-specific binding if counting with meltilex solid scintillant. Binding parameters are determined by non-linear least squares regression analysis, from which the inhibition constant Ki can be calculated for each test compound. The compounds of the accompanying Examples were tested in the 30 above assay, and all were found to possess a Ki value for displacement of WO 00/47582 PCT/GBO0/00308 - 22 [ 3 H]-flumazenil from the u.2 and/or a3 subunit of the human GABAA receptor of 100 nM or less. EXAMPLE 1 5 N-[5-(3,7-Diphenvl-1,2,4-triazolo[4,3-bllpyridazin-6-vloxvmethvl)-2-methvl 2H-1,2,4-triazol-3-vlmethyll-N,N-dimethylamine a) 4-Phenyl-1,2-dihydropyridazine-3,6-dione 10 Phenylmaleic anhydride (30 g, 0.17 mol), sodium acetate trihydrate (28 g, 0.21 mol) and hydrazine monohydrate (10 ml, 0.21 mol) were heated together at reflux in 40% acetic acid (600 ml) for 18 h. The mixture was cooled at 7'C for 2 h, then filtered. The solid was washed with diethyl ether and dried in vacuo to give 11 g (34%) of the title compound: 1H NMR 15 (250 MHz, DMSO-d 6 ) 8 7.16 (1H, br s), 7.44 (5H, m), 7.80 (2H, br s); MS (ES+) m/e 189 [MH+]. b) 3,6-Dichloro-4-phenvlpyridazine 4-Phenyl-1,2-dihydropyridazine-3,6-dione (3.4 g, 18 mmol) was 20 heated at reflux in phosphorus oxychloride (70 ml) for 6 h. The solution was concentrated in vacuo, then the residue was dissolved in dichloromethane (100 ml) and was neutralised by the addition of cold 10% aqueous sodium hydrogen carbonate (150 ml). The aqueous phase was washed with dichloromethane (2 x 50 ml), then the combined organic 25 layers were washed with saturated aqueous sodium chloride (50 ml), dried (Na2SO 4 ), and concentrated in vacuo to yield 3.9 g (97%) of the title compound: 1H NMR (250 MHz, DMSO-d) 6 7.54-7.66 (5H, in), 8.14 (1H, s); MS (ES+) m/e 225/227/229 [MH+].

WO 00/47582 PCT/GBOO/00308 - 23 c) 6-Chloro-3,7-diphenvl-1,2,4-triazolo[4,3-blpvridazine 3,6-Dichloro-4-phenylpyridazine (2.9 g, 13 mmol), benzoic hydrazide (1.9 g, 21 mmol) and triethylammonium chloride (2.0 g, 14 mmol) were heated together at reflux in xylene (150 ml) for three days. More benzoic 5 hydrazide (0.88 g, 6.5 mmol) was added and the mixture was heated as before for another day. The solvent was removed in vacuo, and the residue was purified by flash chromatography (silica gel, 0-50% EtOAc/CH 2 Cl 2 ) to afford 1.4 g (36%) of the title compound as a solid: 1H NMR (250 MHz, CDCl 3 ) 8 7.55 (8H, m), 8.12 (1H, s), 8.50 (2H, in); MS (ES+) m/e 307/309 10 [MH+]. d) 5-(tert-Butvldimethvlsilanvloxvmethvl)-2-methyl-2H-1.2,4-triazole-3 carbaldehyde To 3-(tert-butyldimethylsilanyloxymethyl)-1-methyl-1H-1,2,4 15 triazole (EP-A-0421210) (0.50 g, 2.2 mmol) in THF (10 ml) was added butyllithium (1.5 ml of a 1.6 M solution in hexanes, 2.3 mmol) dropwise at -40'C with stirring. The solution was stirred at -40'C under nitrogen for 15 min, then DMF (0.18 ml, 0.17 g, 2.3 mmol) was added, and the mixture was allowed to warm to room temperature over 90 min. Saturated 20 aqueous ammonium chloride (5 ml) was added, then the organic phase was separated, diluted with ethyl acetate (10 ml), dried (MgSO 4 ) and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 50% EtOAc/hexane) to give 0.29 g (56%) of the title compound as a colourless solid: 1H NMR (360 MHz, CDCla) 8 0.14 (6H, s), 0.93 (9H, 25 s), 4.18 (3H, s), 4.80 (2H, s), 9.98 (1H, s); MS (ES+) m/e 288 [M + MeOH]+. e) N-[5-(tert-Butvldimethvlsilanyloxvmethyl)-2-methyl-2H-1,2,4-triazol-3 vlmethyll-N,N-dimethylamine To 5-(tert-butyldimethvlsilanvloxymethyl)-2-methyl-2H-1,2,4 30 triazole-3-carbaldehyde (0.23 g, 0.88 mmol) in methanol (15 ml) were added 3 A molecular sieves (180 mg) and dimethylamine (0.49 ml of a 2 M WO 00/47582 PCT/GBOO/00308 - 24 solution in methanol, 0.98 mmol) at room temperature with stirring. The mixture was stirred at room temperature under nitrogen for 1 h. Sodium cyanoborohydride (63 mg, 1.0 mmol) was added and the resultant mixture was stirred at room temperature under nitrogen for 3 h, with the pH of the 5 solution being maintained at 5 by the addition of methanolic hydrogen chloride as necessary (methyl orange indicator). The solution was diluted with water (70 ml) and saturated aqueous sodium hydrogen carbonate was added until the pH was approximately 8. The solution was then washed with dichloromethane (3 x 50 ml). The organic layers were combined, 10 washed with saturated aqueous sodium chloride (50 ml), dried (MgSO 4 ) and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-5% MeOH/CH2Cl 2 ) to afford 0.20 g (81%) of the title compound as a solid: 1H NMR (400 MHz, CDCl 3 ) 6 0.12 (6H, s), 0.92 (9H, s), 2.26 (6H, s), 3.57 (2H, s), 3.90 (3H, s), 4.75 (2H, s); MS (ES+) 15 m/e 285 [MH+]. f) N-(5-Hydroxvmethyl-2-methyl-2H-1,2,4-triazol-3-vlmethyl)-NN dimethylamine To N-[5-(tert-butyldimethylsilanyloxymethyl)-2-methyl-2H-1,2,4 20 triazol-3-ylmethyl]-NN-dimethylamine (0.19 g, 0.69 mmol) in ethanol (3 ml) was added 4 N sodium hydroxide solution (0.34 ml), and the solution was stirred at 45*C for 18 h. The solvent was removed in vacuo and the residue was purified by flash chromatography (silica gel, 10% MeOH/CH 2 Cl 2 ) to yield 71 mg (61%) of the title compound as a colourless 25 solid: 1H NMR (360 MHz, CDCl 3 ) 5 2.26 (6H, s), 3.57 (2H, s), 3.90 (3H, s), 4.68 (2H, s); MS (ES+) m/e 171 [MH+]. g) N-[5-(3,7-Diphenvl-1,2,4-triazolo [4,3-bllpyridazin-6-vloxvmethvl)-2 methvl-2H- 1.2. 4-triazol-3-vlmethvll -A, A

T

-Ndimethylamine hydrochloride 30 To N-(5-hydroxymethyl-2-methyl-2H-1,2,4-triazol-3-ylmethyl)-NA

T

dimethylamine (71 mg, 0.42 mmol) in DMF (5 ml) was added sodium WO 00/47582 PCT/GBOO/00308 - 25 hydride (33 mg of a 60% dispersion in mineral oil, 0.83 mmol), and the resultant slurry was stirred at room temperature under nitrogen for 5 min. 6- Chloro- 3,7-diphenyl- 1,2,4-triazolo [4, 3-b]pyridazine (from step c) (0.11 g, 0.35 mmol) was added and the mixture was stirred as before for 5 5 min. Water (50 ml) was added and the resultant precipitate was filtered off, and was then purified by flash chromatography (silica gel, 0-5% MeOH/CH 2 Cl 2 ). The product was taken up in methanolic hydrogen chloride (2 ml), concentrated in vacuo, and recrystallised from ethanol/diethyl ether, to give 9.0 mg (5%) of the title compound as a white 10 solid: 'H NMR (400 MHz, DMSO-d 6 ) 8 2.81 (6H, s), 3.92 (3H, s), 4.53 (2H, s), 5.59 (2H, s), 7.50 (2H, in), 7.58 (1H, m), 7.64 (3H, in), 7.75 (2H), 8.44 (1H, s), 8.49 (2H, in), 10.52 (1H, br s); MS (ES+) m/e 441 [MH+]. EXAMPLE 2 15 5-(3, 7-Diphenvl- 1,2,4-triazolo [4.3- b]pyridazin-6-vloxvmethyl)- 1 -methyl 1H- 1, 2,4-triazol-3-vll methanol a) [5-(tert-Butyldimethvlsilanyloxymethvl)-2-methvl-2H- 12,4-triazol-3 20 vllmethanol To 3-(tert-butyldimethylsilanyloxymethyl)-1-methyl-1H-1,2,4 triazole (EP-A-0421210) (3.5 g, 0.015 mol) in THF (70 ml) was added n-butyllithium (10.2 ml of 1.6 M solution in hexanes, 0.016 mol) dropwise with stirring at -40'C. The solution was stirred at -40*C under nitrogen 25 for 15 min, then DMF (1.3 ml, 1.2 g, 0.016 mol) was added and the mixture was allowed to warm to room temperature over 90 min. Methanol (20 ml) was added, and the solution was cooled to 0*C. Sodium borohydride (0.64 g, 0.017 mol) was added and the slurry was stirred for 5 min, during which time it cleared to a solution. Saturated aqueous sodium chloride (50 ml) 30 was added, then the mixture was filtered to remove insoluble material. The organic phase of the filtrate was dried (MgSO4), and concentrated in WO 00/47582 PCT/GBOO/00308 - 26 vacuo. The residue was taken up in dichloromethane (50 ml), filtered to remove insoluble material, and the filtrate was concentrated in vacuo to yield 3.3 g (83%) of the title compound as a colourless solid: 'H NMR (250 MHz, CDCl 3 ) 5 0.11 (6H, s), 0.91 (9H, s), 3.89 (3H, s), 4.68 (2H, s), 4.72 5 (1H, br s); MS (ES+) m/e 258 [MH+]. b) 6-[5-(tert-Butvldimethylsilanvloxymethyl)-2-methyl-2H-1,2,4-triazol-3 vlmethoxyl-3,7-diphenyl-1,2,4-triazolo[4,3-blpyridazine This was prepared in 72% yield using the procedure described in 10 Example 1, step g, with [5-(tert-butyldimethylsilanyloxymethyl)-2-methyl 2H- 1,2,4-triazol-3-yl] methanol being used instead of N-(5-hydroxymethyl 2-methyl-2H-1,2,4-triazol-3-ylmethyl)-N,N-dimethylamine. Data for the title compound: 'H NMR (250 MHz, CDCl 3 ) 6 0.11 (6H, s), 0.91 (9H, s), 3.70 (3H, s), 4.73 (2H, s), 5.64 (2H, s), 7.46-7.59 (8H, in), 8.06 (1H, s), 8.44 15 (2H, m); MS (ES+) m/e 528 [MH+]. c) [5-(3,7-Diphenyl-1,2,4-triazolo f4,3-blpyridazin-6-vloxymethyl)-1-methyl 1H- 1,2.4-triazol-3-vll methanol 6-[5-(tert-Butyldimethylsilanyloxymethyl)-2-methyl-2H-1,2,4 20 triazol-3-ylmethoxy]-3,7-diphenyl- 1,2,4-triazolo[4,3-b]pyridazine (0.58 g, 1.1 mmol) was stirred in a mixture of ethanol (10 ml) and 2 N hydrochloric acid (20 ml) at 60*C for 1 h. The solution was allowed to cool to room temperature, and the pH was adjusted to >7 with saturated sodium hydrogen carbonate solution, precipitating the title compound as a 25 colourless solid (0.41 g, 90%). The material was recrystallised to analytical purity from ethanol. Data for the title compound: 'H NMR (360 MHz, CDCl 3 ) 8 2.49 (1H, t, J 5.8 Hz), 3.71 (3H, s), 4.73 (2H, d, J 5.8 Hz), 5.64 (2H, s), 7.47-7.60 (8H, in), 8.06 (1H, s), 8.43 (2H, in); MS (ES+) m/e 414 [MH+]. 30 WO 00/47582 PCT/GBOO/00308 - 27 EXAMPLE 3 N- r5-(3.7-Diphenyl- 1, 2,4-triazolo [4,3-bllpvridazin-6-vloxvmethvl)- 1-methyl 1H- 1.2,4-triazol-3-vlmethvll-NN-dimethvlamine 5 To oxalyl chloride (63 pil, 92 mg, 0.73 mmol) in dichloromethane (15 ml) was added DMSO (110 pl, 1.5 mmol) with stirring at -78 0 C, and the resultant solution was stirred at -78*C under nitrogen for 30 min. [5-(3,7 Diphenyl-1,2,4-triazolo[4,3-b]pyridazin-6-yloxymethyl)-1-methyl-1H-1,2,4 triazol-3-yl]methanol (from Example 2, step c) (0.25 g, 0.61 mmol) was 10 added, and the mixture was stirred as before for 90 min. Triethylamine (0.42 ml, 0.31 g, 3.0 mmol) was added and the solution was stirred at -78*C under nitrogen for 15 min, then allowed to warm to room temperature over 90 min. Water was added with vigorous stirring, then the aqueous phase was washed with dichloromethane (2 x 15 ml). The 15 organic layers were combined, dried (MgSO 4 ), and concentrated in vacuo. The residue was dissolved in methanol (10 ml), then 3 A molecular sieves (0.20 g) and dimethylamine (0.33 ml of a 2 M solution in methanol, 0.67 mmol) were added. The slurry was stirred at room temperature for 30 min under nitrogen, then sodium cyanoborohydride (58 mg, 0.93 mmol) was 20 added. The slurry was stirred as before for 24 h, maintaining the pH of the solution at 5 by the addition of methanolic hydrogen chloride as necessary (methyl orange indicator). Water (50 ml) was added, precipitating a solid. The slurry was washed with dichloromethane (3 x 30 ml), then the organic layers were combined, dried over magnesium sulfate 25 and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0-7% MeOH/CH 2 Cl 2 ). The product was triturated under diethyl ether, to afford 18 mg (6%) of the title compound as a colourless solid: 1 H NMR (400 MHz, CDCl 3 ) 5 2.33 (6H, s), 3.54 (2H, s), 3.69 (3H, s), 5.64 (2H, s), 7.48-7.61 (8H, m), 8.08 (1H, s), 8.46 (2H, in): 30 MS (ES+) m/e 441 [MH+].

WO 00/47582 PCT/GBOO/00308 - 28 EXAMPLE 4 [1-Methyl-5-(3-phenvl-7-(pyrrolidin- 1-vl)- 1,2,4-triazolo [4,3-blpyridazin-6 vloxvmethvl)- 1H- 1,2,4-triazol-3-vll methanol 5 a) 4-Bromo-1.2-dihydropyridazine-3,6-dione A mixture of bromomaleic anhydride (50 g, 280 mmol) and sodium acetate (76.5 g, 560 mmol) in 40% acetic acid/water (750 ml) was treated with hydrazine monohydrate (16.5 ml, 340 mmol) at room temperature 10 under nitrogen. The brown solution was stirred and heated at 100'C for 18 hours. After cooling the mixture was poured into water (1 1) and extracted with ethyl acetate (6 x 500 ml). The combined extracts were dried (MgSO 4 ), filtered and evaporated to afford 20 g (37%) of the title compound as an orange solid: 1H NMR (250 MHz, DMSO-d 6 ) 8 7.68 (1H, br 15 s); MS (ES+) m/e 191/193 [MH]+. This material was used without further purification. b) 4-Bromo-3.6-dichloropvridazine A solution of 4-bromo-1,2-dihydropyridazine-3,6-dione (10 g, 52 20 mmol) in phosphorus oxychloride (100 ml) was stirred and heated at 100 0 C under nitrogen for 16 h. After cooling the excess phosphorus oxychloride was removed in vacuo. The residue was azeotroped with toluene (x2), then taken up in dichloromethane/water. The mixture was carefully basified with solid sodium hydrogen carbonate. It was necessary to further dilute 25 the mixture to get two clear layers. The two layers were separated and the aqueous was extracted with dichloromethane (x3). The combined extracts were dried (Na 2

SO

4 ), filtered and evaporated. The residue was purified by chromatography (silica gel, CH 2 Cl 2 ) to afford 5.0 g (42%) of the title compound as a colourless solid: 'H NMR (250 MHz. CDCl1) 8 7.68 30 (1H, br s); MS (ES+) m/e 228/230 [MH]+.

WO 00/47582 PCT/GBOO/00308 - 29 c) 3,6-Dichloro-4-(pvrrolidin-1-yl)pyridazine To a slurry of 4-bromo-3,6-dichloropyridazine (115 g, 0.51 mol) and potassium carbonate (209 g, 1.5 mol) in DMF (1 1) was added pyrrolidine (46 ml, 0.56 mol) at 0*C with stirring. The mixture was allowed to warm 5 to room temperature and then stirred under nitrogen overnight. Water (1.5 1) was added and the resultant slurry was filtered. The residue was washed thoroughly with water and diethyl ether, yielding the title compound (110 g, 100%) as a fine white powder: 1H NMR (250 MHz, CDC1 3 ) 6 2.03 (4H, in), 3.64 (4H, in), 6.46 (1H, s); MS (ES+) m/e 218/220 10 [MH]+. d) [6-Chloro-5-(pvrrolidin- 1-vl)pyridazin-3-vllhvdrazine A solution of 3,6-dichloro-4-(pyrrolidin-1-yl)pyridazine (53 g, 0.24 mol) and hydrazine monohydrate (105 ml, 2.2 mol) in 1,4-dioxane (820 ml) 15 was heated at reflux overnight. The solution was allowed to cool, and dichloromethane was added, precipitating a solid. This was filtered off, affording the title compound as a pale yellow solid (14 g, 27%). The filtrate was concentrated in vacuo, and the residue was purified by flash chromatography (silica gel, 2-5% MeOH/CH 2 Cl 2 containing 0.1% 20 concentrated aqueous ammonia) yielding a further 5 g (4%) of the title compound: 'H NMR (250 MHz, CDCl 3 ) 6 1.98 (4H, in), 3.26 (2H, br s), 3.57 (4H, in), 6.11 (1H, s), 6.41 (1H, br s); MS (ES+) m/e 214/216 [MH]+. e) N-Benzylidene-N'-[6-chloro-5-(pyrrolidin-1-vl')pvridazin-3-vllhydrazine 25 To a solution of [6-chloro-5-(pyrrolidin-1-yl)pyridazin-3-yl]hydrazine (14 g, 0.063 mol) in 0.1 N hydrochloric acid (600 ml) was added benzaldehyde (6.4 ml, 0.063 mol) dropwise at room temperature. The mixture was stirred at 60'C for 15 min, yielding a thick slurry. The pH of the solvent was adjusted to -11 with 4 N aqueous sodium hydroxide 30 solution, and the precipitate was filtered off. The residue was washed with water, ethanol and diethyl ether, yielding 14 g (73%) of the title WO 00/47582 PCT/GBOO/00308 - 30 compound as a white solid: 1H NMR (360 MHz, DMSO-d 6 ) 8 1.93 (4H, in), 3.58 (4H, in), 6.57 (1H, s), 7.35 (1H, in), 7.39 (2H, in), 7.66 (2H, d, J 7.1 Hz), 8.06 (1H, s), 11.16 (1H, s); MS (ES+) m/e 302/304 [MH]+. 5 f) 6-Chloro-3-phenvl-7-(pyrrolidin-1-vl)-1,2.4-triazolo[4,3-blpvridazin A slurry of N-benzylidene-N'-[6-chloro-5-(pyrrolidin-1-yl)pyridazin 3-yl]hydrazine (14 g, 0.046 mol) and lead(IV) acetate (24 g, 0.055 mol) in acetic acid (300 ml) was stirred at 60 0 C under nitrogen overnight. The solvent was removed in vacuo, and the residue was purified by flash 10 chromatography (silica gel, 0-10% MeOH/EtOAc) to give 4.1 g (30%) of the title compound as a yellow solid: 'H NMR (250 MHz, CDCl 3 ) 6 2.05 (4H, in), 3.56 (4H, m), 7.05 (1H, s), 7.51 (3H, in), 8.42 (2H, m); MS (ES+) m/e 300/302 [MH]+. 15 g) 6-[5-(tert-Butvldimethylsilanvloxvmethvl)-2-methyl-2H-1.2.4-triazol-3 Vlmethoxvl-3-phenyl-7-(pvrrolidin-1-vl)-1,2,4-triazolo f4,3-b]pvridazine This was prepared in 75% yield using the procedure described in Example 1, step g, with [5-(tert-butyldimethylsilanyloxymethyl)-2-methyl 2H- 1,2,4-triazol-3-yl] methanol being used instead of N-(5-hydroxymethyl 20 2-methyl-2H-1,2,4-triazol-3-ylmethyl)-NN-dimethylamine, and with 6 chloro-3-phenyl-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine being used instead of 6-chloro-3,7-diphenyl- 1,2,4-triazolo [4,3- b]pyridazine. Data for the title compound: 1H NMR (250 MHz, CDCl 3 ) 8 0.11 (6H, s), 0.91 (9H, s), 1.92 (4H, in), 3.39 (4H, in), 3.99 (3H, s), 4.76 (2H, s), 5.54 (2H, s), 6.60 25 (1H, s), 7.41-7.53 (3H, in), 8.30 (2H, in); MS (ES+) m/e 521 [MH+]. h) [1-Methvl-5-(3-phenvl-7-(pvrrolidin-1-vl)-1,2,4-triazolo[4,3-blpvridazin 6-vloxymethyl)- 1H- 1,2,4-triazol- 3-vll methanol This was prepared in 92% yield following the procedure described in 30 Example 2, step c, but using 6-[5-(tert-butyldimethylsilanyloxymethyl)-2 methyl-2H-1,2,4-triazol-3-ylmethoxy]-3-phenyl-7-(pyrrolidin- 1-yl)-1,2,4- WO 00/47582 PCT/GBOO/00308 - 31 triazolo[4,3-b]pyridazine instead of 6-[5-(tert-butyldimethylsilanyloxy methyl)-2-methyl-2H-1,2,4-triazol-3-ylmethoxy]-3,7-diphenyl-1,2,4 triazolo[4,3-b]pyridazine. Data for the title compound: 1H NMR (360 MHz, DMSO-d 6 ) 8 1.90 (4H, m), 3.47 (4H, in), 4.40 (2H, s), 5.23 (1H, br s), 5.64 5 (2H, s), 6.83 (1H, s), 7.47-7.59 (3H, m), 8.31 (2H, in); MS (ES+) m/e 407 [MH+]. EXAMPLE 5 10 6-(5-Chloromethyl-2-methyl-2H-1,2,4-triazol-3-vlmethoxv)-3-phenvl-7 (pyrrolidin-1-vl)-1,2,4-triazolo[4,3-bipyridazine [1-Methyl-5-(3-phenyl-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3 b]pyridazin-6-yloxymethyl)- 1H- 1,2,4-triazol-3-yl] methanol (from Example 4, step h) (1.0 g, 2.5 mmol) was stirred in phosphorus oxychloride (50 ml) 15 at room temperature for 18 h. The solvent was removed in vacuo, and the residue was partitioned between dichloromethane (50 ml) and water (50 ml). The aqueous phase was washed with more dichloromethane (2 x 50 ml), then the organic layers were combined, dried (MgSO 4 ) and concentrated in vacuo. The crude product was eluted through a plug of 20 silica with 7.5% methanol in dichloromethane, yielding 1.2 g (100%) of the title compound as a colourless solid. An analytically pure sample was obtained after recrystallisation from ethanol/ethyl acetate/hexane. Data for the title compound: 'H NMR (360 MHz, DMSO-d 6 ) 8 1.96 (4H, m), 3.68 (4H, m), 3.92 (3H, s), 4.72 (2H, s), 5.73 (2H, s), 6.81 (1H, s), 7.68 (3H, m), 25 8.29 (2H, m); MS (ES+) m/e 425/427 [MH+]. EXAMPLE 6 N-[5-(3-Phenvl-7-(pyrrolidin-1-vl)-1,2,4-triazolo[4,3-b]pyridazin-6 30 vloxymethyl)-1-methyl-1H-1,2,4-triazol-3-vlmethyll-NN-dimethylamine To dimethylamine hydrochloride (73 mg, 0.90 mmol) in WO 00/47582 PCT/GBOO/00308 - 32 N,N-dimethylacetamide (2 ml) was added sodium hydride (60% dispersion in mineral oil; 88 mg, 2.2 mmol) and the resultant slurry was stirred at room temperature under nitrogen for 90 min. 6-(5-Chloromethyl--2 methyl-2H- 1,2, 4 -triazol-3-ylmethoxy)-3-phenyl-7-(pyrrolidin- 1-yl)- 1,2,4 5 triazolo[4,3-b]pyridazine (from Example 5) was added (99 mg, 0.23 mmol) and the mixture was stirred at 50*C under nitrogen for 18 h. Water (15 ml) was added, and the solution was washed with dichloromethane (3 x 10 ml). The combined organic layers were washed with water (1 x 10 ml), dried (MgSO 4 ), and concentrated in vacuo. The residual oil was triturated 10 under diethyl ether/hexane to give a solid. This was recrystallised from ethyl acetate/ethanol/hexane, yielding 13 mg (13%) of the title compound as colourless crystals: 1H NMR (400 MHz, CDCl 3 ) 3 1.97 (4H, in), 2.34 (6H, s), 3.46 (4H, in), 3.57 (2H, s), 3.93 (3H, s), 5.56 (2H, s), 6.72 (1H, s), 7.44 7.54 (3H, in), 8.33 (2H, in); MS (ES+) m/e 434 [MH+], 218 [MH2+]/2. 15 EXAMPLE 7 6-r2-Methl-5-(morpholin-4-vlmethyl)-2H-1.2,4-triazol-3-vlmethoxvl-3 phenyl-7-(pyrrolidin-1-vl)-1,2,4-triazolof4.3-blvpvridazine hydrochloride 20 This was prepared in 31% yield following the procedure described in Example 6, except using morpholine instead of dimethylamine hydrochloride, and dissolving the free base in methanolic hydrogen chloride prior to recrystallisation. Data for the title compound: 1H NMR (360 MHz, DMSO-d6) 6 1.94 (4H, in), 3.63 (4H, in), 3.83 (8H, br in), 3.97 25 (3H, s), 4.40 (2H, s), 5.76 (2H, s), 6.83 (1H, s), 7.64 (3H, in), 8.27 (2H, in); MS (ES+) m/e 476 [MH+], 239 [MH 2 +]/2. EXAMPLE 8 30 6-r5-(Azetidin-1-vlmethyl)-2-methyl-2H-1.2.4-triazol-3-vlmethoxyl-3 phenvl-7-(pvrrolidin-1-vl)-1,2,4-triazolo f4.3-blpvridazine WO 00/47582 PCT/GBOO/00308 - 33 This was prepared in 23% yield following the procedure described in Example 6, but using azetidine instead of dimethylamine hydrochloride. Data for the title compound: 1H NMR (360 MHz. DMSO-d 6 ) 6 1.95 (4H, in), 2.10 (2H, quintet, J 7.0 Hz), 3.33 (4H, t, J 7.0 Hz), 3.44 (4H, in), 3.65 (2H, 5 s), 3.93 (3H, s), 5.54 (2H, s), 6.68 (1H, s), 7.45-7.53 (3H, m), 8.30 (2H, m); MS (ES+) m/e 446 [MH+], 224 [MH 2 +]/2. EXAMPLE 9 10 6-[2-Methvl-5-(4-methylpiperazin-1-vlmethyl)-2H-1,2,4-triazol-3 vlmethoxyl-3-phenvl-7- (pyrrolidin-1-vl)-1,2,4-triazolo f4,3-bllpvridazine This was prepared in 38% yield following the procedure described in Example 6, but using 1-methylpiperazine instead of dimethylamine hydrochloride. Data for the title compound: 1H NMR (360 MHz, CDCl 3 ) 6 15 1.95 (4H, m), 2.28 (3H, s), 2.48 (4H, br s), 2.63 (4H, br s), 3.43 (4H, m), 3.66 (2H, s), 3.95 (3H, s), 5.56 (2H, s), 6.68 (1H, s), 7.43-7.53 (3H, m), 8.33 (2H, m); MS (ES+) m/e 489 [MH+], 245 [MH 2 +]/2. EXAMPLE 10 20 6-[5-(3,3-Difluoroazetidin-1-vlmethyl)-2-methyl-2H-1,2,4-triazol-3 ylmethoxy]l-3-phenvl-7-(ovrrolidin-1-vl)-1,2,4-triazolof4,3-blpvridazine a) 1-Benzhydrylazetidin-3-one 25 To 1-benzhydrylazetidin-3-ol (10 g, 0.042 mol) and triethylamine (58 ml, 42 g, 0.42 mol) in DMSO (100 ml) at 15*C was added sulfur trioxide pyridine complex (40 g, 0.25 mol) in DMSO (50 ml) over 5 min. The reaction was stirred at room temperature under nitrogen for 3.5 h, and was then poured onto a mixture of crushed ice (200 g) and saturated 30 ammonium chloride solution (100 ml). The slurry was extracted with ethyl acetate (3 x 200 ml), then the combined organic layers were washed WO 00/47582 PCT/GBO/00308 - 34 with saturated aqueous ammonium chloride (200 ml) and water (200 ml), dried (MgSO 4 ), and concentrated in vacuo to leave 6.4 g (64%) of the title compound as a pale yellow solid: IH NMR (250 MHz, CDCl 3 ) 6 4.00 (4H, s), 4.59 (1H, s), 7.18-7.34 (6H, in), 7.47 (4H, in). 5 b) 1-Benzhvdryl-3,3-difluoroazetidine To 1-benzhydrylazetidin-3-one (5.0 g, 0.021 mol) in benzene (50 ml) was added diethylaminosulfur trifluoride (7.8 ml, 0.063 mol), and the mixture was stirred at room temperature under nitrogen overnight. The 10 solution was partitioned between water (100 ml) and ethyl acetate (100 ml), then the organic layer was washed with water (100 ml) and saturated sodium chloride solution (1 x 100 ml), dried (MgSO 4 ) and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 10% EtOAc/hexane) and was then recrystallised from hexane, yielding 3.0 g 15 (54%) of the title compound as a colourless solid: 1H NMR (250 MHz, CDCl 3 ) 8 3.51 (4H, t, J 12 Hz), 4.44 (1H, s), 7.16-7.32 (6H, in), 7.42 (4H, in). c) 3,3-Difluoroazetidine hydrochloride 20 1-Benzhydryl-3,3-difluoroazetidine (3.0 g, 0.011 mol) was hydrogenated in a mixture of methanol (70 ml) and 2 N hydrochloric acid (7 ml) over palladium hydroxide catalyst (20% on activated carbon, 600 mg), at 50 psi for 18 h. The catalyst was filtered off and the filtrate was concentrated in vacuo. The residue was triturated under diethyl ether, 25 yielding 1.3 g (88%) of the title compound as a pale yellow solid: 1H NMR (250 MHz, DMSO-d 6 ) 8 4.50 (2H, t, J 12 Hz), 10.05 (2H, br s). d) 6-[5-(3,3-Difluoroazetidin-1-vlmethvl)-2-methvl-2H-1,2.4-triazol-3 vlmethoxvl-3-phenvl--7-(ovrrolidin-1 -vI)-1,2,4-triazolo[4,3-blpvridazine 30 This was prepared in 20% yield using the procedure described in Example 6, but using 3,3-difluoroazetidine hydrochloride instead of WO 00/47582 PCT/GB0O/00308 - 35 dimethylamine hydrochloride. Data for the title compound: IH NMR (360 MHz, CD Cl 3 ) 6 1.96 (4H, in), 3.44 (4H, in), 3.75 (4H, t, J 12 Hz), 3.84 (2H, s), 3.95 (3H, s), 5.55 (2H, s), 6.69 (1H, s), 7.49 (3H, in), 8.30 (2H, in); MS (ES+) m/e 463 [M-F]+. 5 EXAMPLE 11 6-[2-Methyl-5-(pyrrolidin-1-vlmethyl)-2H-1,2, 4 -triazol-3-vlmethoxyl-3 phenyl-7-(pvrrolidin-1-vl)-1,2,4-triazolo [4,3-blpvridazine 10 This was prepared in 3% yield using the procedure described in Example 6, but using pyrrolidine instead of dimethylamine hydrochloride. Data for the title compound: 1H NMR (400 MHz, DMSO-d 6 ) 6 1.81 (2H, in), 1.92 (6H, in), 3.09 (2H, in), 3.40 (2H, in), 3.56 (4H, in), 3.96 (3H, s), 4.46 (2H, s), 5.74 (2H, s), 6.86 (1H, s), 7.59 (3H, in), 8.25 (2H, in); MS (ES) m/e 15 460 [MH]+, 230 [MH 2 +]/2. EXAMPLE 12 7-tert-Butvl-6-r5-(tert-butvldimethylsilanvloxvmethvl)-2-methvl-2H-1,2,4 20 triazol-3-vlmethoxy-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-blpyridazine a) 4-tert-Butyl-3,6-dichloropyridazine Concentrated sulphuric acid (53.6 ml, 1.0 mol) was added carefully to a stirred suspension of 3,6-dichloropyridazine (50.0 g, 0.34 mol) in water 25 (1.25 1). This mixture was then heated to 70*C (internal temperature) before the addition of trimethylacetic acid (47.5 ml, 0.41 mol). A solution of silver nitrate (11.4 g, 0.07 mol) in water (20 ml) was then added over approximately 1 min. This caused the reaction mixture to become milky in appearance. A solution of ammonium persulphate (230 g, 1.0 mol) in 30 water (0.63 1) was then added over 20-30 min. The internal temperature rose to approximately 85 0 C. During the addition the product formed as a WO 00/47582 PCT/GBOO/00308 -36 sticky precipitate. Upon complete addition the reaction was stirred for an additional 10 min, then allowed to cool to room temperature. The mixture was then poured onto ice and basified with concentrated aqueous ammonia, with the addition of more ice as required to keep the 5 temperature below 10 0 C. The aqueous was extracted with dichloromethane (3 x 300 ml). The combined extracts were dried (MgSO 4 ), filtered and evaporated to give 55.8 g of crude product as an oil. This was purified by flash chromatography (silica gel, 0-15% EtOAc/hexane) to give 37.31 g (53%) of the title compound: 'H NMR (360 MHz, DMSO-d 6 ) 8 1.50 10 (9H, s), 7.48 (1H, s); MS (ES+) m/e 205/207 [MH]+. b) 7-tert-Butvl-6-chloro-3-(2-fluorophenvl)-1,2,4-triazolo f4,3-blpyridazine A mixture of 4-tert-butyl-3,6-dichloropyridazine (20 g, 0.097 mol), 2 fluorobenzhydrazide (22.6 g, 0.145 mol) and triethylamine hydrochloride 15 (20 g, 0.0145 mol) in dioxane (1.2 1) was stirred and heated at reflux under a stream of nitrogen for 4 days. Upon cooling the volatiles were removed in vacuo and the residue was triturated with dichloromethane (200 ml), filtered and concentrated under vacuum. The residue was purified by chromatography (silica gel, 0-25% EtOAc/CH 2 Cl 2 ) to give 12.95 g (44%) of 20 the title compound as a white solid: 1 H NMR (360 MHz, CDCl 3 ) 6 1.57 (9H, s), 7.26-7.35 (2H, in), 7.53-7.60 (1H, in), 7.89-7.93 (1H, in), 8.17 (1H, s), MS (ES+) m/e 305/307 [MH]+. c) 7-tert-Butvl-6-[5-(tert-butvldimethvlsilanvloxvmethyl)-2-methyl-'2H 25 1,2,4-triazol-3-vlmethoxv1-3-(2-fluorophenvl)-1,2,4-triazolo f4,3 b1pyridazine To a stirred solution of 7-tert-butyl-6-chloro-3-(2-fluorophenyl)-1,2,4 triazolo[4,3-b]pyridazine (1.0087 g, 3.31 mmol) in anhydrous DMF (22 ml) under nitrogen was added [5-(tert-butyldimethylsilanyloxymethyl)-2 30 methyl-2H-1,2,4-triazol-3-yl]methanol (from Example 2, step a) (1.0224 g, 3.97 mmol) then sodium hydride (60% dispersion in oil; 0.1589 g, 3.97 WO 00/47582 PCT/GBOO/00308 - 37 mmol) portionwise over 2 min. The mixture was stirred for 30 min at room temperature. Water (78 ml) was added and the resulting solid was collected by filtration, washed with water, then hexane, and dried at 60 0 C under vacuum to leave 1.6129 g (93%) of the title compound as a white 5 solid: mp 121-123 0 C (CH2Cl2-EtOAc-hexane); 1H NMR (360 MHz, CDCl 3 ) 8 0.09 (6H, s), 0.90 (9H, s), 1.40 (9H, s), 3.79 (3H, s), 4.73 (2H, s), 5.49 (2H, s), 7.27 (1H, in), 7.33 (1H, t, J 7.6 Hz), 7.56 (1H, in), 7.87 (1H, td, J 7.4, J' 1.7 Hz), 7.98 (1H, s); MS (ES+) m/e 526 [MH]+. Anal. Found C, 59.31; H, 6.91; N, 18.53. C 26

H

3

GFN

7 0 2 Si requires C, 59.40; H, 6.90; N, 18.65%. 10 EXAMPLE 13 [5-(7-tert-Butyl-3-(2-fluorophenvl)-1,2,4-triazolo[4,3-blpyridazin-6 vloxvmethyl)- 1-methyl- 1H- 1,2,4-triazol-3-vllmethanol 15 This was prepared in 93% yield using a similar procedure to that described in Example 2, step c, but using 7-tert-butyl-6-[5-(tert butyldimethylsilanyloxymethyl)-2-methyl-2H-1,2,4-triazol-3-ylmethoxy]-3 (2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine instead of 6-[5-(tert butyldimethylsilanyloxymethyl)-2-methyl-2H-1,2,4-triazol-3-ylmethoxy] 20 3,7-diphenyl- 1, 2,4-triazolo [4,3- b]pyridazine. Data for title compound: mp 225-230'C (CH 2 Cl 2 -EtOAc); 'H NMR (360 MHz, CDCl 3 ) 8 1.41 (9H, s), 2.34 (1H, br t), 3.80 (3H, s), 4.72 (2H, d, J 5.6 Hz), 5.50 (2H, s), 7.28 (1H, m), 7.35 (1H, t, J 7.6, J'1.0 Hz), 7.57 (1H, in), 7.87 (1H, td, J 7.4, J'1.8 Hz), 7.99 (1H, s); MS (ES+) m/e 412 [MH]+. Anal. Found C, 58.14; H, 5.24; N, 25 23.70. C 2 0

H

2 2

FN

7 0 2 requires C, 58.39; H, 5.39; N, 23.83%. EXAMPLE 14 7 -tert-Butvl-3-(2-fluorophenvl)--(5-methoxmethvl-2-methvl2H-1.2,4 30 triazol-3-vlmethoxv)-1,2,4-triazolo f4,3-blpvridazine WO 00/47582 PCT/GBOO/00308 - 38 To a stirred mixture of [5-(7-tert-butyl-3-(2-fluorophenyl)-1,2,4 triazolo[4,3-b]pyridazin-6-yloxymethyl)-1-methyl-1H-1,2,4-triazol-3 yl]methanol (from Example 13) (0.1017 g, 0.247 mmol) in anhydrous DMF (5 ml) under nitrogen was added sodium hydride (60% dispersion in oil; 5 10.9 mg, 0.273 mmol). The mixture was stirred at room temperature for 5 min, then iodomethane (18.5 ptl, 0.297 mmol) was added dropwise. The mixture was stirred for a further 3 h, adding more sodium hydride (60% dispersion in oil; 1.1 mg, 0.028 mmol) after 45 min. Water (20 ml) was added and the mixture was filtered, washing the solid with more water. 10 Saturated aqueous NaCl was added to the filtrate and this was extracted with ethyl acetate (3 x 40 ml). The combined organic extracts were dried (Na 2

SO

4 ) and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 3% MeOH/CH 2 Cl 2 ) to give 80.0 mg (76%) of the title compound as a white solid: mp 138-140*C (CH2Cl2-EtOAc-isohexane); 15 'H NMR (360 MHz, CDCl 3 ) 5 1.40 (9H, s), 3.47 (3H, s), 3.82 (3H, s), 4.51 (2H, s), 5.50 (2H, s), 7.27 (1H, in), 7.35 (1H, t, J 7.5, J'0.9 Hz), 7.56 (1H, in), 7.88 (1H, td, J 7.4, J'1.8 Hz), 7.99 (1H, s); MS (ES+) m/e 426 [MH]+. Anal. Found C, 59.02; H, 5.48; N, 22.74. C 2 1

H

2 4

FN

7 0 2 requires C, 59.28; H, 5.69; N, 23.05%. 20 EXAMPLE 15 7 -tert-Butvl-3-(2-fluorophenvl)-6-(5-trifluoromethvl-1H-1,2,4-triazol-3 Vlmethoxv)-1,2,4-triazolo[4,3-bipyridazine 25 a) 7-tert-Butyl-3-(2-fluorophenvl)-1,2,4-triazolo[4,3-blpyridazin-6-one To a stirred solution of 7-tert-butyl-6-chloro-3-(2-fluorophenyl)-1,2,4 triazolo[4,3-b]pyridazine (from Example 12, step b) (1.0468 g, 3.43 mmol) in 1,4-dioxane (60 ml) and water (12 ml) was added 4 M aqueous NaOH 30 (4.29 ml, 17.2 mmol), and the solution was heated at reflux for 23 h. The solvent was removed in vacuo and the residue was partitioned between WO 00/47582 PCT/GBOO/00308 - 39 water (200 ml) and diethyl ether (100 ml). The aqueous layer was then acidified to pH 3 with 5 M aqueous HCl. The resulting precipitated solid was collected by filtration, washed with water, then hexane, and dried at 60*C under vacuum to give 0.8990 g (91%) of the title compound as a white 5 solid: 1 H NMR (360 MHz, DMSO-d 6 ) 6 1.40 (9H, s), 7.40-7.48 (2H, m), 7.65 (1H, in), 7.84 (1H, td, J 7.3, J'1.8 Hz), 8.00 (1H, s), 12.64 (1H, br s); MS (ES+) m/e 287 [MH]+. b) 7-tert-Butvl-6-cvanomethoxy-3-(2-fluorophenvl)-1,2,4-triazolo[4,3 10 blpyridazine To a stirred solution of 7-tert-butyl-3-(2-fluorophenyl)-1,2,4 triazolo[4,3-b]pyridazin-6-one (0.8953 g, 3.13 mmol) in anhydrous DMF (47 ml) under nitrogen was added sodium hydride (60% dispersion in oil, 0.1878 g, 4.70 mmol) and the mixture was stirred at room temperature for 15 25 min, then at 80 0 C for 25 min. After allowing to cool, bromoacetonitrile (0.327 ml, 4.69 mmol) was added dropwise and the mixture was stirred at room temperature for 18 h. Water (160 ml) was then added and the resulting precipitated solid was collected by filtration, washed with water, then hexane, and dried at 60'C under vacuum to afford 0.8506 g (84%) of 20 the title compound as a white solid: 1 H NMR (360 MHz, CDCl 3 ) 6 1.45 (9H, s), 5.07 (2H, s), 7.30 (1H, in), 7.36 (1H, td, J 7.6, J'0.9 Hz), 7.57 (1H, m), 7.93 (1H, td, J 7.3, J'1.8 Hz), 8.04 (1H, s); MS (ES+) m/e 326 [MH]+. c) 7-tert-Butvl-3-(2-fluorophenvl)-6-(5-trifluoromethvl-1H-1,2,4-triazol-3 25 vlmethoxv)-1,2,4-triazolo[4,3-blpyridazine To a stirred solution of 7-tert-butyl-6-cyanomethoxy-3-(2 fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (0.5455 g, 1.68 mmol) in anhydrous methanol (7 ml) under nitrogen was added a 0.4 M solution of sodium methoxide in methanol (0.126 ml, 0.0504 mmol), and the mixture 30 was stirred at room temperature for 5 h. The mixture was neutralised by adding acetic acid (2.9 1d, 0.051 mmol), then trifluoroacetic hydrazide WO 00/47582 PCT/GBOO/00308 - 40 (Fritz, H. et al., Magn. Reson. Chen., 1990, 28, 331-336) (0.2246 g, 1.75 mmol) in anhydrous methanol (2 ml) was added, and the mixture was heated at 60'C for 18 h. The solvent was removed in vacuo and the residue was purified by flash chromatography (silica gel, 5% 5 MeOH/CH 2 Cl 2 ) to leave 0.6893 g (94%) of the title compound as a white solid: mp 206-217 0 C (CHCl3-EtOAc-isohexane); 1 H NMR (360 MHz, CDCl 3 ) 8 1.34 (9H, s), 5.70 (2H, s), 7.13 (1H, in), 7.22 (1H, t, J 7.5 Hz), 7.45 (1H, in), 7.72 (1H, in), 8.83 (1H, s); MS (ES+) m/e 436 [MHJ+. Anal. Found C, 52.70; H, 3.78; N, 22.31. C 19

H

17

F

4

N

7 0 requires C, 52.42; H, 3.94; N, 10 22.52%. EXAMPLE 16 7 -tert-Butyl-3-(2-fluorophenyl)-6-(2-methvl-5-trifluoromethyl-.2H-1,2,4 15 triazol-3-vlmethoxv)-1,2,4-triazolo[4,3-blpyridazine and 7-tert-Butvl-3-(2 fluorophenvl)-6-(1-methvl-5-trifluoromethvl-1H-1,2,4-triazol-3-vlmethoxv) 1,2,4-triazolo[4,3-blipyridazine To a stirred mixture of sodium hydride (60% dispersion in oil; 32.7 mg, 0.818 mmol) and iodomethane (50.7 ptl, 0.814 mmol) in anhydrous 20 DMF (4 ml), cooled under nitrogen to -3 0 C, was added dropwise, over 11 min, a solution of 7-tert-butyl-3-(2-fluorophenyl)-6-(5-trifluoromethyl-1H 1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine (from Example 15) (0.2953 g, 0.678 mmol) in anhydrous DMF (6 ml). The mixture was stirred at -2*C for 15 min, then allowed to warm to 18*C over 2 h, and 25 stirred at this temperature for a further 45 min. The mixture was partitioned between ethyl acetate (40 ml) and brine (40 ml). The aqueous layer was extracted further with ethyl acetate (40 ml), and the combined organic extracts were dried (Na2SO 4 ) and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 2% 30 MeOH/CH 2 Cl 2 ) to afford 0.2405 g (79%) of a 4:1 mixture of the two title WO 00/47582 PCT/GBOO/00308 - 41 compounds as a white solid. These were separated by flash chromatography (silica gel, 70-100% EtOAc/isohexane). 7-tert-Butyl-3-(2-fluorophenvl)-6-(2-methyl-5-trifluoromethvl-2r-1,2,4 triazol-3-vlmethoxv)-1,2,4-triazolo[4,3-blipyridazine: mp 139-141*C 5 (CH2Cl2-EtOAc-isohexane); 1H NMR (400 MHz, CDCl 3 ) 8 1.45 (9H, s), 4.04 (3H, s), 5.46 (2H, s), 7.25 (1H, in), 7.32 (1H, td, J 7.6, J'0.9 Hz), 7.53 (1H, in), 7.92 (1H, td, J7.5, J'1.7 Hz), 7.96 (1H, s); MS (ES+) m/e 450 [MH]+. Anal. Found C, 52.94; H, 4.11; N, 21.19. C 20

H

19

F

4

N

7 0.0.3H 2 0 requires C, 52.82; H, 4.34; N, 21.56%. 10 7 -tert-Butyl-3-(2-fluorophenvl)-6-(1-methyl-5-trifluoromethvl-1H-1.2,4 triazol-3-vlmethoxv)-1,2,4-triazolof4,3-blpyridazine: mp 160-162 0 C (CH2Cl2-EtOAc-isohexane); 1H NMR (400 MHz, CDCl 3 ) 5 1.44 (9H, s), 3.85 (3H, s), 5.54 (2H, s), 7.25 (1H, in), 7.33 (1H, td, J 7.6, J'1.0 Hz), 7.54 (1H, in), 7.84 (1H, td, J 7.4, J'1.7 Hz), 7.99 (1H, s); MS (ES+) m/e 450 [MH]+. 15 Anal. Found C, 53.43; H, 4.19; N, 21.89. C 2 oHi 9

F

4

N

7 0 requires C, 53.45; H, 4.26; N, 21.82%. EXAMPLE 17 20 7-tert-Butvl-3-(2-fluorophenyl)-6-(2-ethyl-5-trifluoromethyl-2H-1.2.4 triazol-3-vlmethoxy)-1,2,4-triazolo[4,3-blpyridazine and 7-tert-Butvl-3-(2 fluorophenvl)-6-(1-ethyl-5-trifluoromethyl- 1H- 1,2,4-triazol- 3-vlimethoxy) 1,2,4-triazolo [4,3-blpyridazine These were prepared in an overall yield of 92% following a similar 25 procedure to that described in Example 16, but using iodoethane instead of iodomethane. 7 -tert-Butyl-3-(2-fluorophenyl)-6-(2-ethvl-5-trifluoromethvl-2H-1,2,4 triazol-3-vlmethoxv)-1,2,4-triazolo[4,3-blpyridazine: mp 132-140 0 C (CH2Cl2-EtOAc-isohexane); 'H NMR (360 MHz, CDCl 3 ) 6 1.45 (9H, s), 1.49 30 (3H, t, J 7.3 Hz), 4.33 (2H, d, J 7.3 Hz), 5.48 (2H, s), 7.25 (1H, in), 7.32 WO 00/47582 PCT/GBOO/00308 - 42 (1H, td, J 7.6, J'1.0 Hz), 7.53 (1H, m), 7.93 (1H, td, J 7.4, J'1.7 Hz), 7.96 (1H, s); MS (ES+) m/e 464 [MH]+. 7-tert-Butyl-3-(2-fluorophenvl)-6-(1-ethyl- 5-trifluoromethyl- 1H- 1,2,4 triazol-3-vlmethoxy)-1,2,4-triazolo f4,3-blpyridazine: mp 175-178 0 C 5 (CH2Cl2-EtOAc-isohexane); 1 H NMR (360 MHz, CDC1 3 ) 5 1.42 (9H, s), 1.45 (3H, t, J 7.3 Hz), 3.85 (2H, d, J 7.3 Hz), 5.56 (2H, s), 7.28 (1H, in), 7.36 (1H, td, J7.5, J'1.0 Hz), 7.57 (1H, in), 7.88 (1H, td, J 7.5, J'1.8 Hz), 8.01 (1H, s); MS (ES+) m/e 464 [MH]+. Anal. Found C, 54.44; H, 4.52; N, 21.03.

C

2 1

H

2 1

F

4

N

7 0 requires C, 54.43; H, 4.57; N, 21.16%.

Claims (10)

1. A compound of formula I, or a pharmaceutically acceptable salt thereof: 5 N-N R N2 Z R 0 I NON N R 3 R 4 (I) wherein Z represents tert-butyl, cyclobutyl, phenyl or pyrrolidin-1-yl; RI represents hydrogen, methyl, methoxy or fluoro; 10 R 2 represents hydrogen or fluoro; R 3 represents hydrogen, methyl or ethyl; R4 represents trifluoromethyl, chloromethyl, or a group of formula -CH 2 ORa or -CH 2 NRbRc; Ra represents hydrogen, methyl or tert-butyldimethylsilanyl; and 15 Rb and Rc both represent methyl; or Rb and Rc together represent the residue of an azetidine, 3,3-difluoroazetidine, pyrrolidine, morpholine or N-methylpiperazine moiety.

2. A compound as claimed in claim 1 represented by formula 20 IIA, and pharmaceutically acceptable salts thereof: WO 00/47582 PCT/GBOO/00308 -44 R N-N N R2 z N 0" N N-R -N R4/ (IIA) wherein Z, R 1 , R 2 , R 3 and R 4 are as defined in claim 1. 5

3. A compound as claimed in claim 1 represented by formula IIB, and pharmaceutically acceptable salts thereof: R N-N N R" Ij z N 0 -1N N N R (IIB) 10 wherein Z, RI, R 2 , R 3 and R4 are as defined in claim 1. WO 00/47582 PCT/GBOO/00308 -45

4. A compound as claimed in any one of the preceding claims wherein R 1 represents hydrogen or fluoro.

5. A compound as claimed in any one of the preceding claims 5 wherein R 3 represents methyl or ethyl.

6. A compound selected from: N- [5- (3,7-diphenyl- 1,2,4-triazolo [4,3- b]pyridazin-6-yloxymethyl) -2-methyl 2H-1,2,4-triazol-3-ylmethyl]-N,N-dimethylamine; 10 [5-(3,7-diphenyl-1,2,4-triazolo[4,3-b]pyridazin-6-yloxymethyl)- 1-methyl 1H- 1,2,4-triazol-3-yl] methanol; N-[5-(3,7-diphenyl- 1,2,4-triazolo[4,3-b]pyridazin-6-yloxymethyl)-1-methyl 1H-1,2,4-triazol-3-ylmethyl]-NN-dimethylamine; [1-methyl-5-(3-phenyl-7-(pyrrolidin- 1-yl)-1,2,4-triazolo[4,3-b]pyridazin-6 15 yloxymethyl) -1H- 1,2,4-triazol- 3-yl] methanol; 6-(5-chloromethyl-2-methyl-2H- 1,2,4-triazol-3-ylmethoxy)-3-phenyl-7 (pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine; N-[5-(3-phenyl-7-(pyrrolidin-1-yl)- 1,2,4-triazolo[4,3-b]pyridazin-6 yloxymethyl)- 1-methyl-1H- 1,2,4-triazol-3-ylmethyl]-NN-dimethylamine; 20 6-[2-methyl-5-(morpholin-4-ylmethyl)-2H-1,2,4-triazol-3-ylmethoxy]-3 phenyl-7-(pyrrolidin-1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 6-[5-(azetidin-1-ylmethyl)-2-methyl-2H-1,2,4-triazol-3-ylmethoxy]-3 phenyl-7-(pyrrolidin-1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 6-[ 2 -methyl-5-(4-methylpiperazin-1-ylmethyl)-2H-1,2,4-triazol-3 25 ylmethoxy]-3-phenyl-7-(pyrrolidin- 1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 6-[5-(3,3-difluoroazetidin-1-ylmethyl)-2-methyl-2H-1,2,4-triazol-3 ylmethoxy]-3-phenyl-7-(pyrrolidin- 1-yl)-1,2,4-triazolo[4,3-b]pyridazine; 6-[2-methyl-5-(pyrrolidin-1-ylmethyl)-2H-1,2,4-triazol-3-ylmethoxy]-3 phenvl-7-(pyrrolidin-1-yl)- 1,2,4-triazolo[4,3-b]pyridazine; 30 7-tert-butyl-6-[5-(tert-butyldimethylsilanyloxymethyl)-2-methyl-2H- 1,2,4 triazol-3-ylmethoxy]-3-(2-fluorophenyl)- 1,2,4-triazolo[4,3-b]pyridazine; WO 00/47582 PCT/GB00/00308 - 46 [5-(7-tert-butyl-3-(2-fluorophenyl)- 1,2,4-triazolo[4,3-b]pyridazin-6 yloxymethyl)- 1-methyl- 1H- 1,2,4-triazol- 3-yl]methanol; 7 -tert-butyl-3-(2-fluorophenyl)-6-(5-methoxymethyl-2-methyl-2H- 1,2,4 triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine; 5 7-tert-butyl-3-(2-fluorophenyl)-6-(5-trifluoromethyl- 1H-1, 2 ,4-triazol-3 ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;

7-tert-butyl-3-(2-fluorophenyl)-6-(2-methyl-5-trifluoromethyl-2H- 1,2,4 triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine; 7-tert-butyl-3-(2-fluorophenyl)-6-(1-methyl-5-trifluoromethyl- 1H-1,2,4 10 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 7-tert-butyl-3-(2-fluorophenyl)-6-(2-ethyl-5-trifluoromethyl-2H- 1,2,4 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 7 -tert-butyl-3-(2-fluorophenyl)-6-(1-ethyl-5-trifluoromethyl- 1H- 1,2,4 triazol-3-ylmethoxy)- 1,2,4-triazolo[4,3-b]pyridazine; 15 and pharmaceutically acceptable salts thereof. 7. A pharmaceutical composition comprising a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable carrier. 20

8. The use of a compound as claimed in any one of claims 1 to 6 for the manufacture of a medicament for the treatment and/or prevention of anxiety. 25

9. A process for the preparation of a compound as claimed in claim 1, which comprises: (A) reacting a compound of formula III with a compound of formula IV: WO 00/47582 PCT/GBOO/00308 - 47 OH N-N RO N N ( Z4 NR 2 4 NR L R (III) (IV) wherein Z, R1, R 2 , R 3 and R 4 are as defined in claim 1, and L 1 represents a suitable leaving group; or 5 (B) reacting a compound of formula XI (or its 1,2,4-triazolo[4,3 b]pyridazin-6-one tautomer) with a compound of formula XII: N-N R L 3 / \ NfN Z N 2R3 OH R4 (XI) (XII) 10 wherein Z, R 1 , R 2 , R 3 and R 4 are as defined in claim 1, and L 3 represents a suitable leaving group; or (C) reacting a compound of formula Z-CO 2 H with a compound of formula XIII: WO 00/47582 PCT/GBOO/00308 - 48 N-N R 1N R 0 NQN N R R 4 (XIII) wherein Z, R1, R 2 , R 3 and R 4 are as defined in claim 1; in the presence of silver nitrate and ammonium persulphate; or 5 (D) reacting a compound of formula XIV with a compound of formula XV: N-N N R 2 NQN R N R 3 (XIV) (XV) 10 wherein Z, R1, R 2 , R 3 and R 4 are as defined in claim 1, M represents -B(OH) 2 or -Sn(Alk) 3 in which Alk represents a Cl- 6 alkyl group, and L4 represents a suitable leaving group; in the presence of a transition metal catalyst; or WO 00/47582 PCT/GBOO/00308 - 49 (E) reacting a compound of formula XVII with the hydrazide of formula XVIII: N-N R N 0 Z R F 3 C NHNH 2 0) CN (XVII) (XVIII) 5 wherein Z, R 1 and R 2 are as defined in claim 1; and (F) subsequently, where required, converting a compound of formula I initially obtained into a further compound of formula I by conventional methods. 10

10. A method for the treatment and/or prevention of anxiety which comprises administering to a patient in need of such treatment an effective amount of a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof. 15