|Title of Invention||
A SUBSTITUTED TRIAZOLO-PYRIDAZINE DERIVATIVE, PHARMACEUTICAL COMPOSITIONS MADE THEREFROM, AND THEIR USE IN THERAPY
|Abstract||A compound of formula (1, 1- Dimethylethyl-6-(2-ethyl- 2H-1, 2, 4- triazol- 3-ylmethoxy) -3- (2-fluorophenyl) - 1,2,4-triazolo[4,3-b]pyridazine, and pharmaceutically acceptable salts thereof, are selective ligands for GABA<sub>A</sub> receptors, in particular having high affinity for the α2 and/or α3 subunit thereof, and are accordingly of benefit in the treatment and/or prevention of disorders of the central nervous system, including anxiety and convulsions.|
The present invention relates to a substituted triazolo-pyridazine derivative and to its use in therapy. More particularly, this invention is concerned with a particular substituted l,2,4-triazolo[4,3-6]pyridazine derivative which is a GABAA receptor ligand and is therefore useful in the therapy of deleterious mental states.
Receptors for the major inhibitory neurotransmitter, gamma-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 mammalian family has grown to include at least six a subunits, four (3 subunits, three y subunits, one 5 subunit, one s 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-gated ion channel, insight into the extent of subtype diversity is still at an early stage. It has been indicated that an a 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, 5, e and p subunits also exist, but are 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 for the possible existence of more than 10,000 pentameric subunit combinations. Moreover, this calculation overlooks the additional 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 others, a1β2y2, a2p2/3y2, a3βy2/3, a2βyl, a5β3y2/3, a6py2, a6β5 and a4β5. Subtype assemblies containing an al 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 a2 and a3 subunits respectively are thought to account for about 25% and 17% of GABAA receptors in the 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 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, BZl and BZ2, on the basis of radioligand binding studies. The BZl subtype has been shown to be pharmacologically equivalent to a GABAA receptor comprising the al subunit in combination with a (3 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.
Two other major populations are the α2βY2 and α3βy2/3 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 subtype assemblies. The physiological role of these subtypes has hitherto been unclear because no sufficiently selective agonists or antagonists were known.
It is now believed that agents acting as BZ agonists at α1βY2, α2βY2 or α3βY2 subunits will possess desirable anxiolytic properties. Compounds 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 a 1-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 act at the BZl binding site is mediated through GABAA receptors containing the al subunit. Accordingly, it is considered that GABAA receptor agonists which interact more favourably with the a2 and/or a3 subunit than with al will be effective in the treatment of anxiety with a reduced propensity to cause sedation. Also, agents which are antagonists 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 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; 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; neurodegeneration arising from cerebral ischemia; attention deficit hyperactivity disorder; and disorders of circadian rhythm, e.g. in subjects suffering from the effects of jet lag or shift work.
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 postoperative nausea and vomiting; 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,
WO 98/04559 describes a class of substituted and 7,8-ring fused l,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 particular triazolo-pyridazine derivative, and pharmaceutically acceptable salts thereof, 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 a2 and/or a3 subunit of the human GABAA receptor. The compounds of this invention interact more favourably with the a2 and/or a3 subunit than with the al subunit. Indeed, the compounds of the invention exhibit functional selectivity in terms of a selective efficacy for the a2 and/or a3 subunit relative to the a 1 subunit.
The compounds of the present invention are GABAA receptor subtype ligands having a binding affinity (Kj) for the a2 and/or a3 subunlt, as measured in the assay described hereinbelow, of less than 1 nM. Furthermore, the compounds in accordance with this invention exhibit functional selectivity in terms of a selective efficacy for the a2 and/or a3 subunit relative to the al subunit. Moreover, the compounds according to the present invention possess interesting pharmacokinetic properties, notably in terms of improved oral bioavailability.
or a pharmaceutical ly acceptable salt thereof.
The compounds in accordance with the present invention are encompassed within the generic scope of WO 98/0455.* There is, however, no specific disclosure therein of the compound of formula I as depicted above, or pharmaceutical ly acceptable salts thereof
For use in medicine, the salts of the compound of formula I above will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compound of formula I or of its pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compound of formula I include acid addition salts which may, for example, be formed by mixing a solution of the compound of formula 1 with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, 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.
Also provided by the present invention is 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 the compound of formula I as depicted 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 epilepsy or a related disorder) which comprises administering to a patient in need of such treatment an effective amount of the compound of formula I as depicted above or a pharmaceutically acceptable salt thereof
The binding affinity (KI) of the compounds according to the present invention for the a3 subunit of the human GABAA receptor is conveniently as measured in the assay described hereinbelow. The a3 subunit binding affinity (Kj) of the compounds of the invention is less than 1 nM.
The compounds according to the present invention elicit a selective potentiation of the GABA EC20 response in stably transfected recombinant cell lines expressing the a3 subunit of the human GABAA receptor relative to the potentiation of the GABA EC20 response elicited in stably transfected recombinant cell lines expressing the al subunit of the human GABAA receptor*
The potentiation of the GABA EC20 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 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 demonstrated by a positive response in the elevated 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 confirmed by an appropriate result obtained from the response sensitivity (chain-pulling) test (cf Bayley et al,, J. Psychopharmacol., 1996,10, 206-213).
The compounds according to the present invention may also exhibit anticonvulsant activity. This can be demonstrated by the ability to block pentylenetetrazole-induced seizures in rats and mice, following a protocol analogous to that described by Bristow et ai in J. Pharmacol Exp. Ther., 1996,279,492-501.
Since they elicit behavioural effects, the compounds of the invention plainly are brain-penetrant; In other words, these compounds are capable of crossing the so-called "blood-brain barrier". Advantageously, the compounds of the invention are capable of exerting their beneficial therapeutic action following administration by the oral route.
The invention also provides pharmaceutical compositions comprising one or more compounds of this invention in association with a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, 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 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 ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. 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 or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be
delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
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. In accordance with the other aspect of the present invention, there is provided a process for the preparation of 7-(l,l-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-6]pyridazine, which comprises:
reacting a compound of general formula A wherein
wherein R is L1, which represents a suitable leaving group selected from halo preferably chloro, or OH, or 2-ethyl substituted pendant triazole ring and Rl is fluorophenyl or halogen atom with a compound B selected from (a) silver nitrate or ammonium persulfate; (b) substituted fluorophenyl ring of formula C wherein M represents -B(0H)2 or -Sn(Alk)3 in which Alk represents a C1-,6 alkyl
wherein further, (i) when compound B is silver nitrate or ammonium persulfate, compound A has to be with R equal to 2-ethyl substituted pendant triazole ring and Rl representing fluorophenyl as substitutent ; (ii) when compound B is a substituted fluorophenyl ring of formula C compound A has to be with R equal to 2-ethyl substituted pendant triazole ring and Rl is halogen atom as substitutent; (iii) when compound B is substituted pendant triazole ring of formula D wherein R2 represents OH or halogen atom R in compound A has to be either OH or halogen atom and Rl has to be fluorophenyl in presence of solvents and under process conditions such as herein described. Further, the reaction between compound B with substituted pendant triazole ring of formula D wherein R2 represents OH and compound A having R as halogen atom and Rl as fluorophenyl may be carried out in l-methyl-2-pyrrolidinone, in the presence of sodium hydroxide, at a temperature in the region of 0°C.
The compound of formula 1 as depicted above may be prepared by a process which comprises reacting a compound of formula III with the compound of formula IV:
wherein L1 represents a suitable leaving group.
The leaving group L1 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, in the presence of a base. Typically, the solvent is N',N', dimethylformamide, and the base is a strong base such a sodium hydride. Preferably, the solvent is dimethyl sulfoxide, and the base is cesium carbonate. More preferably, the solvent is l-methyl-2-pyrrolidinone, and the base is sodium hydroxide, in which case the reaction is advantageously performed at a temperature in the region of OC,
The intermediates of formula III above may be prepared by reacting a compound of formula V with a substantially equimolar amount of the hydrazine derivative of formula VI:
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.
Alternatively, the intermediates of formula III above may be prepared by reacting a hydrazine derivative of formula VII with the aldehyde derivative of formula VIII:
The reaction between compounds VII and VIII is conveniently effected under acidic conditions, for example in the presence of a mineral acid such as hydrochloric acid, Cyclization of the resulting SchifFs base intermediate may then conveniently be carried out by treatment 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.
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 isobutyl alcohol at an elevated temperature, e.g. a temperature in the region of 90nC, or 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.
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:
wherein L1 is as defined above.
Suitable values for the reactive carboxyiate moiety Q include esters, for example
C1-4 alkyl esters; acid anhydrides, for example mixed anhydrides with C1-4 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 under basic conditions, e.g. in the presence of triethylamine, suitably in an inert solvent such as diethyl ether, and typically at a temperature in the region of 0°C. Cyclization of the resulting compound of formula X may then conveniently be carried out by treatment with 1,2-dibromo-l,l,2,2-tetrachloroethane and triphenylphosphine, in the presence of a base such as triethylamine, suitably in an inert solvent such as acetonitrile, and typically at a temperature in the region of 0°C.
In a preferred method, the reaction between compounds VII and IX may be effected by mixing the reactants in a solvent such as l-methyl-2-pyrrolidinone at a temperature in the region of OC; cyclization of the compound of formula X thereby obtained may then
be accomplished in situ by heating the reaction mixture at a temperature in the region of 130C.
The reaction between compound V and hydrazine hydrate or compound VI will, as indicated above, possibly give rise to a mixture of isomeric products depending upon whether the hydrazine nitrogen atom displaces the leaving group L or L . Thus, in addition to the required product of formula III or VII, the alternative isomer will possibly be obtained to some extent. For this reason it might be necessary to separate the resulting mixture of isomers by conventional methods such as chromatography.
In another procedure, the compound of formula I as depicted above may be prepared by a process which comprises reacting the compound of formula XI (or its 1,2,4-triazolo[4,3-6]pyridazin"6-one tautomer) with a compound of formula XII:
wherein L3 represents a suitable leaving group.
The leaving group L is suitably a halogen atom, typically chloro or bromo.
The reaction between compounds XI and XII is conveniently effected by stirring the reactants in a suitable solvent, typically MA^-dimethylformamide, in the presence of a strong base such as sodium hydride.
The intermediate 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.
In a further procedure, the compound of formula I as depicted above may be prepared by a process which comprises reacting trimethylacetic acid with the compound of formula XIII:
in the presence of silver nitrate and ammonium persulphate.
The reaction is conveniently carried out in a suitable solvent, for example in water or aqueous acetonitrile, optionally under acidic conditions, e.g. using trifluoroacetic acid or sulphuric acid, typically at an elevated temperature.
The intermediate of formula XIII corresponds to the compound of formula I as depicted above wherein the tert-butyl substituent at the 7-position is absent, and the intermediate XIII may therefore be prepared by methods analogous to those described above for preparing the compound of formula I.
In a still further procedure, the compound of formula I as depicted above may be prepared by a process which comprises reacting a compound of formula XIV with a compound of formula XV:
wherein M represents -B(0H)2 or -Sn(Alk)3 In which Alk represents a C1-6 alkyl group, typically «-butyl, and L4 represents a suitable leaving group; in the presence of a transition metal catalyst.
The leaving group L4 is suitably a halogen atom, e.g. bromo.
A suitable transition metal catalyst of use in the reaction between compounds XIV
and XV comprises dichlorobis(triphenylphosphine)-palladium(II) or
The reaction between compounds XIV and XV is conveniently effected in an inert solvent such as N,N-dimethylformamide, typically at an elevated temperature.
The intermediates of formula XIV may be prepared by reacting the compound of formula IV as defined above with a compound of formula XVI:
wherein L1 and L2 are as defined above; in the presence of silver nitrate and ammonium persulphate; under conditions analogous to those described above for the reaction between trimethylacetic acid and compound XIII. Where L and L are both chloro in compound XVII, the reaction is advantageously effected in the presence of trifluoroacetic acid.
Where they are not commercially available, the starting materials of formula VI, VIII, IX, XII, XV, XVI and XVII may be prepared by methods analogous to those described in the accompanying Examples, or by standard methods well known from the art.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
Four anhydrous polymorphs, two solvates and a dihydrate of 7-(l,l-dimethylethyl)' 6-(2-ethyl-2H-l,2,4-triazol-3-yimethoxy)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-b]pyridazine have been synthesised and characterised. All the polymorphs and solvates revert to the most thermodynamically stable form, polymorph A (for formation and characterisation see Example 3), after stirring as a suspension in water. A dihydrate of polymorph A is stable, but only at elevated humidities.
The following Examples illustrate the preparation of compounds according to the invention.
The compounds in accordance with this invention potently inhibit the binding of [3H]-fluma2enil to the benzodiazepine binding site of human GABAA receptors containing the a2 or a3 subunit stably expressed in Ltk' cells.
• Phosphate buffered saline (PBS).
Assay buffer: 10 mM KH2PO4, 100 mM KCl, pH 7.4 at room temperature.
• [3HJ-Flumazenil (18 nM for a1β3Y2 cells; 18 nM for a2β3Y2 cells; 10 nM for a3P3y2
cells) in assay buffer.
□ Flunitrazepam 100 µM in assay buffer.
Cells resuspended in assay buffer (1 tray to 10 ml).
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 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.
Can be carried out in deep 96-well plates or in tubes. Each tube contains; J 300 µl of assay buffer.
G50 \x\ of [3H]-flumazenil (final concentration for alpβ3Y2: L8 nM; for a2β3Y2: 1.8 nM; for a 3β3Y2: 1.0 nM).
50 µ\ 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 µM final concentration. IJ100 |il of cells.
Assays are incubated for 1 hour at 40°C, then filtered using either a 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 scinfillation 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 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 Kj can be calculated for each test compound.
The compound of the accompanying Examples was tested in the above assay, and was found to possess a KI value for displacement of [3H]-flumazenil from the a2 and/or a3 subunit of the human GABAA receptor of less than 1 nM.
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 (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 one minute. This caused the reaction mixture to become milky in appearance. A solution of ammonium persulphate (230 g, LO mol) in water (0.63 1) was then added over 20-30 minutes. The internal temperature rose to approximately 85°C. During the addition the product formed as a sticky precipitate. Upon complete addition the reaction was stirred for an additional 10 minutes, 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 temperature below 10°C. The aqueous was extracted with dichloromethane (3 x 300 ml). The combined extracts were dried (MgS04), filtered and evaporated to give 55.8 g of crude product as an oil. This was purified by silica gel chromatography using 0-15% ethyl acetate in hexane as eluent to give 37,31 g (53%) of the desired compound. Data for the title compound: 1H NMR (360 MHz, do-DMSO) 5 1.50 (9H, s), 7.48 (1H, s); MS (ES+) m/e 205 [MH]+ 207 [MH]+
A mixture of 3,6-dichloro-4-(l,l-dimethylethyl)pyridazine (20 g, 0.097 mol), 2-
fluorobenzhydrazide (22.6 g, 0.145 mol) and triethylamine hydrochloride (20 g, 0.0145 mol) in dioxan (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 on silica gel eluting with 0%->25% ethyl acetate/dichloromethane to give the title compound (12.95 g, 44%) as a white solid. Data
for the title compound: 1H NMR (360 MHz, CDCI3) 5 1.57 (9H, s), 126-135 (2H, m), 7.53-7,60 (1H, m), 7.89-7.93 (1H, m), 8.17 (1H, s); MS (ES+) m/e 305 [MH]\ 307 [MH]^
To a solution of 1,2,4-triazole (10 g, 0.145 mol) in DMF (150 ml) at room temperature was added sodium hydride (6.4 g of a 60% disp. in oil, 0.16 mol) in portions over 15 min. When the addition was complete, the reaction mixture was allowed to cool to room temperature, then cooled in an ice-bath and iodoethane (14 ml, 0.174 mol) added dropwise over 10 min. The reaction mixture was allowed to warm to room temperaure and after stirring for 3 h the solvents were removed under high vacuum to leave a residue which was partitioned between water (300 ml) and ethyl acetate (3 x 300 ml). The combined organic layers were washed with saturated brine and dried (MgS04), filtered and concentrated under vacuum to leave an oily residue which was purified by distillation (120°C @ -20 mmHg) to give l-ethyL-1,2,4-triazole contaminated with -15% DMF (2.4 g). The crude product (2.4 g, 0.025 mol) was dissolved in dry THF (35 ml), cooled to -40°C and n-butylIithium (16.2 ml of a 1,6 M solution in hexane, 0.026 mol) was added slowly over 20 min keeping the temperature constant. DMF (2.03 ml, 0.026 mol) was then added and after 15 min the reaction mixture was allowed to warm slowly to room temperature over 2 h. To the reaction mixture was added methanol (20 ml) followed by sodium borohydride (1 g, 0.026 mol) and the solution was allowed to stir for 14 h. The solvents were removed under vacuum and the residue was partitioned between brine (50 ml) and dichloromethane (6 x 50 ml). The combined organic layers were dried (MgS04), filtered and concentrated under vacuum to leave a residue which was purified by silica gel chromatography using 0-5% methanol in dichlorometane as eluent to give the title compound as an off-white solid (0.5 g, 3%). Data for the title compound: 1H NMR (250 MHz, CDCI3) 5 .48 (3H, t, J = 7.3 Hz), 4.25 (2H, q, J = 7.3 Hz), 4.75 (2H, s), 5.14 (1H, br s),7.78(lH, s).
To a solution of (2-ethyl-2H-l,2,4-triazol-3-yl)methanol (0.094 g, 0.74 mmol) and 6-chloro-7-(l,l-dimethylethyl)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-B]pyridazine (0.15 g,
0.49 mmol) in DMF (10 ml) was added sodium hydride (0.024 g of a 60% dispersion in oil, 1,1 mol eq.) and the reaction mixture was stirred at room temperature for 30 minutes. After this time, the reaction mixture was diluted with water (80 ml) and the solid that precipitated was collected by filtration and washed several times with water in the sinter funnel The solid was recrystallised from ethyl acetate/hexane to give pure title compound (0.085 g, 44%). Data for the title compound: 1HNMR (250 MHz, CDCI3) 5 1.40-1.47 (12H, m), 4.14 (2H, t, J = 7.3 Hz), 5.26 (2H, s), 7.26-7.38 (2H, m), 7.53-7.58 (1H, m), 7.86-7.90 (1H, m), 7.93 (1H, s), 7.99 (1H, s); MS (ES^) m/e 396 [MH]^ Anal. Found C, 61.02; H, 5,45; N, 24.75%. C20H22FN7O requires C, 60.75; H, 5.61; N, 24.79%.
7-(1,l-Dimethvlethvn-6-(2-ethvl-2H-L2.4-triazol-3-vlmethoxvV3-(2-fluorophenvn-1.2.4-triazQlor4,3-6]pvridazine: Alternative Synthetic Route
a) 3,6-Dichloro-4-( 1.1 -dimethylethyL)pyridazine
A mixture of dichloropyridazine (100 g, 0.67 mol), trimethylacetic acid (96 g, 0.94 mol) and water (800 ml) in a 10 1 flask with overhead stirring was warmed to 55 C resulting in a two-phase solution. A solution of AgNO3 (11.4 g, 0.134 mol) in water (125 ml) was added in one portion resulting in an opaque solution. Trifluoroacetic acid (10.3 ml, 0.134 mol) was added in one portion. Ammonium persulfate (245 g, 1.07 mol) was dissolved in water (500 ml) and added dropwise to the suspension over 45-60 min resulting in an exotherm (typically temperature increases to between 75-80C and can be controlled by rate of persulfate addition). The temperature was maintained at 75 DC for a further 1 h and then cooled to room temperature. The reaction mixture was extracted with isobutyl alcohol (1 1) and the aqueous layer discarded. The organic layer was washed with water (250 ml) and the aqueous cut discarded. HPLC assay yield is 134 g (97%). The isobutyl alcohol solution was used as is in the next step.
Hydrazine hydrate (95 ml, 1.95 mol) was added to the isobutyl alcohol solution from step a in a 3 1 flask and heated at 90C for 20 h. The reaction mixture was cooled to room
temperature and the lower aqueous layer discarded. The reaction mixture was washed with water (450 ml) and the aqueous cut discarded. The reaction mixture was distilled under reduced pressure until the product started to crystallise and then l-methyl-2-pyrrolidinone (NMP) (550 ml) was added. Distillation was continued to remove the last of the isobutyl alcohol. The solution was used as is in the next step.
c) 6-Chloro-7-(1,l-dimethvlethyl)-3-(2-fluorophenvl)1,2,4-triazolo[4.3-B]Pvridazine 2-Fluorobenzoyl chloride (103 g, 0,65 mol) was added dropwise to the cooled (ODC) NMP solution from step b maintaining the internal temperature d) l-Ethyl-1,2,4-triazole
1,2,4-Triazole (100,0 g, 1.45 mol) in anhydrous THF (950 ml) was cooled to OC and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (220 g, 1.45 mol) was added in one portion. The reaction mixture was stirred for 30 min until complete dissolution was observed. Whilst maintaining the ice/water cooling bath, iodoethane (317 g, 2.03 mol) was added dropwise over a 15 min period resulting in an internal temperature rise to 30DC. The reaction was stirred at room temperature for 16 h, after which the DBU hydroiodide was removed by filtration. The filtrate solution was used as is in the next step.
The stirred solution from the preceding step was cooled to -75 DC internal temperature in a solid C02/acetone slurry bath. Hexyllithium (458 ml of 33% solution in hexanes) was added dropwise over 25 min keeping the internal temperature below -55C. The reaction mixture was aged for 30 min (back to -75DC) and then neat DMF (108 ml, 1.39 mol) was added dropwise over 10 min maintaining internal temperature below -60C. The reaction mixture was aged at -70C for 90 min before the cooling bath was removed and the reaction mixture allowed to warm to OTIC over 30 min. Industrial
methylated spirits (340 ml) was added over 10 min. Sodium borohydride (26.3 g, 0.695 mol) was then added portionwise maintaining internal temperature below 6C. After the addition the reaction mixture was allowed to warm to room temperature and stirred for 1 h at that temperature. The reaction was quenched by the careful addition of 2M H2SO4 (200 ml) and then stirred at room temperature for 20 h. The reaction mixture was concentrated to 675 ml and sodium sulfate (135 g) was added in one portion. The reaction mixture was warmed to 35°C and stirred for 15 min. The solution was extracted with warm (45C) isobutyl alcohol (2 x 675 ml). The combined organic fractions were concentrated under reduced pressure 450 ml at which point the product crystallised. Heptane (1.125 1) was added and the slurry concentrated under reduced pressure to remove most of the isobutyl alcohol. Heptane was added to give a final slurry volume of 680 ml. After cooling to one, filtration gave the title compound (137 g, 74% from 1,2,4-triazole).
6-Chloro-7'(l,l-dimethylethyl)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-6]pyridazine (255 g, 0.819 mol), (2-ethyl-2H-1,2,4-triazol-3-yl)methanol (125 g, 0.983 mol) and cesium carbonate (640 g, 1.966 mol) were charged to a 10 1 flask equipped with an overhead stirrer. Dimethyl sulfoxide (2.5 1) was added in one portion and the reaction mixture stirred at room temperature for 20 h. The product had crystallised. The reaction mixture was maintained at Method B
6-Chloro-7-(l,l-dimethylethyl)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-6]pyridazine (10 g, 32.12 mmol) and (2-ethyl-2H-l,2,4-triazol-3-yl)methanol (5.01 g, 38.55 mmol) were charged to a 500 ml flask equipped with an overhead stirrer. NMP (100 ml) was added in one portion and the reaction mixture stirred until complete dissolution was achieved. The
reaction mixture was cooled to OC and 48% w/w sodium hydroxide solution (4.02 g, 46 mmol) was added in one portion. After stirring for 1 h at OC, the product had crystallised. Water (100 ml) was added dropwise over 15 min and the slurry aged for 30 min. The product was isolated by filtration and the cake washed with water (100 ml). Drying at 50nC in vacuo gave the title compound (12.50 g, 98%) as a white solid.
Formation and Characterisation of Polymorphs and Solvates of 7-(1.1-dimethvlethYL)-6-(2-ethyl-2H-K2,4-triazo^3-vlmethoxvV3-(2-fluorophenvl)1,2.4-triazolo[43-61pvridazine
7-( 1,1 -Dimethylethyl)-6-(2-ethyl-2i/-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-6]pyridazine (hereinafter referred to as Compound I) was recrystallised from a selection of organic solvents and the resultant solids were dried in vacuo overnight at 60 GC unless otherwise stated. Each batch was then characterised by optical microscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and X-ray powder diffraction (XRPD). Four distinct anhydrous polymorphs, a hydrate and two solvates have been characterised, as summarised in Table 1.
Polymorph A consists of irregular or elongated rectangular birefringent crystals. It shows one major endotherm by DSC at ca. 186DC due to melting. In the case of some of the samples prepared this melt displays several events happening within it, typified by shoulders on the melting endotherm. Polymorph A is anhydrous and displays no losses by TGA. It has a unique XRPD diffractogram, typified by two peaks at 26 « 7.3 i i.
Polymorph B consists of irregular or elongated rectangular birefringent crystals. Polymorph B displays one major endotherm by DSC at ca. 181 DC due to melting. It is anhydrous and displays no weight loss by TGA. It has a unique XRPD diffractogram.
Polymorph C consists of acicular, birefringent crystals. The DSC thermograms of polymorph C show an endotherm at ca. 170DC, an exotherm at ca. 173GC, a small endotherm at ca. 181 DC, and an endotherm due to melting at ca. 186C. No losses are observed by thermogravimetric analysis.
Polymorph D consists of acicular birefringent crystals. The XRPD diffractogram of polymorph D is similar in pattern to that of polymorph C. However, significant differences are observed, notably additional peaks at 26 = 9.861, 15.113, 18.015 and 22.224. The DSC trace of polymorph D displays a very broad exotherm at ca, 108C, followed by an endotherm and exotherm at 170DC and 173 DC, as for polymorph C. The major melting is observed at ca. 181 DC, and several events are seen to occur within the melting endotherm, with minor melting at 186C. No losses are observed by TGA.
The DSC thermogram of the methanol solvate shows one endotherm at ca, 157C due to loss of methanol, and one endotherm at 186C due to melting. A gradual loss is observed by TGA up to ca. 150°C, at which point a step loss is observed, coincident with
the endotherm observed by DSC. This step loss is in some instances composed of more than one event, and the amount varies among recrystallised samples and does not appear to correspond to a stoichiometric solvate. However, methanol vapour sorption studies on polymorph A show the presence of a distinct hemisolvate. The XRPD diffractogram of the solvate is unique.
The ethanol solvate is typified by its DSC thermogram which shows one endotherm at ca. 111 C due to loss of ethanol, and an endotherm at ca, 186C due to melting. TGA shows the loss to be variable in different recrystallised samples, between ca, 4 and 6.5%, and does not correspond to a stoichiometric solvate. The XRPD diffractogram is unique.
Hydrate of Polymorph A
Figure 1 shows the adsorption/desorption isotherm of Compound I polymorph A at 25 C. These moisture vapour sorption studies show formation of a dihydrate above 80% relative humidity (RH) at 25C. Hysteresis indicative of hydrate formation is seen to occur, with desorption occurring below ca. 60%, RH.
Figure 2 shows side-by-side XRPD diffractograms of anhydrous Compound I polymorph A and of the dihydrate of polymorph A. The XRPD diffractogram of the dihydrate, obtained on a wetted sample of polymorph A, demonstrates the distinct differences observed between the two forms. The major changes in diffraction can be seen to be loss of the peak at 20 = 11.2°, and the appearance of two major peaks at 20 - 11.9° and 12.3°.
Conversion of Compound I Polymorphs to Polymorph A
On slurrying in water all of the polymorphs and solvates decribed above convert to polymorph A over a period of 1-4 days, indicating that this is the most stable form at room temperature. This conversion is slow due to the low aqueous solubility of Compound I in relation to the large excess of solid compound slurried.
X-Ray Powder Diffraction Data
Figure 3 shows side-by-side XRPD diffractograms of anhydrous Compound I polymorphs A, B, C and D, of the methanol and ethanol solvates, and of the dihydrate of polymorph A. Numerical data associated therewith are presented below. Compound I Polymorph A: X-Ray Powder Diffraction Data
1. A compound of formula 7-(l,l-Dimethylethyl)-6^(2^ethyl-2//-l,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-fe]pyridazine, pharmaceutically acceptable salts thereof useful for the manufacture of a medicament for the treatment and/or prevention of anxiety.
2. A compound as claimed in claim 1 is a Polymorph as characterised herein.
3. A pharmaceutical composition comprising 7-(l, 1-dimethylethyl)-6-(2-ethyl-2H-l,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-l,2,4'triazolo[4,3-6]pyridazine in association with a pharmaceutically acceptable carrier,
4. A process for the preparation of 7-(l,l-dimethylethyl)-6-(2-ethyl-2H-l,2,4-trxazol-3-ylmethoxy)-3-(2-fluorophenyl)-l,2,4-triazolo[4,3-6]pyridazine, which comprises:
reacting a compound of general formula 'A' wherein
wherein R is L1, which represents a suitable leaving group selected from halo preferably chloro, or OH, or 2-ethyl substituted pendant triazole ring and Rl is fluorophenyl or halogen atom with a compound *B' selected from (a) silver nitrate or ammonium persulfate; (b) substituted fluorophenyl
wherein further, (i) when compound B is silver nitrate or ammonium persulfate, compound A has to be with R equal to 2-ethyl substituted pendant triazole ring and Rl representing fluorophenyl as substitutent ; (ii) when compound B is a substituted fluorophenyl ring of formula C compound A has to be with R equal to 2-ethyl substituted pendant triazole ring and Rl is halogen atom as substitutent; (iii) when compound B is substituted pendant triazole ring of formula D wherein R2 represents OH or halogen atom R in compound A has to be either OH or halogen atom and Rl has to be fluorophenyl in presence of solvents and under process conditions such as herein described.
5. A process as claimed in claim 4 wherein reaction between compound B with substituted pendant triazole ring of formula D wherein R2 represents OH and compound A having R as halogen atom and Rl as fluorophenyl is carried out in l-methyl-2-pyrrolidinone, in the presence of sodium hydroxide, at a temperature in the region of 0°C.
6. A compound of formula 7-(l,l-Dimethylethyl)-6-(2-ethyl-2H-
6]pyridazine, substantially as herein before described and illustrated with
reference to the accompanying examples and drawings.
7. A pharmaceutical Composition substantially as herein before
described and illustrated with reference to the accompanying examples
8. A process for the preparation of a compound of formula 7-(l,l-Dimethylethyl)-6-(2-ethyl-2H-l,2,4-triazol-3-ylmethoxy)-3-(2-fluoro phenyl)-l,2,4-triazolo[4,3-b]pyridazine, substantially as herein before described and illustrated with reference to the accompanying examples and drawings.
|Indian Patent Application Number||IN/PCT/2000/823/CHE|
|PG Journal Number||07/2008|
|Date of Filing||13-Dec-2000|
|Name of Patentee||M/S. MERCK SHARP & DOHME LIMITED|
|Applicant Address||Hertford Road, Hoddesdon, Hertfordshire EN11 9BU,|
|PCT International Classification Number||C07D 487/04|
|PCT International Application Number||PCT/GB1999/001897|
|PCT International Filing date||1999-06-15|