U.S. patent application number 10/324968 was filed with the patent office on 2003-08-21 for triazolo-pyridazine derivatives as ligands for gaba receptors.
This patent application is currently assigned to Merck & Co., Inc.. Invention is credited to Carling, William Robert, Castro Pineiro, Jose Luis, Lewis, Richard Thomas, Moore, Kevin William, Street, Leslie Joseph.
Application Number | 20030158203 10/324968 |
Document ID | / |
Family ID | 27269629 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030158203 |
Kind Code |
A1 |
Carling, William Robert ; et
al. |
August 21, 2003 |
Triazolo-pyridazine derivatives as ligands for GABA receptors
Abstract
A class of substituted 1,2,4-triazolo[4,3-b]pyridazine
derivatives, possessing a difluoro- or trifluoro-substituted phenyl
ring at the 3-position, a triazolyl-methoxy moiety at the
6-position, and a tert-butyl group at the 7-position, are selective
ligands for GABA.sub.A receptors, in particular having high
affinity for .alpha.2 and/or .alpha.3 subunit thereof, and are
accordingly of benefit in the treatment and/or prevention of
disorders of the central nervous system, including anxiety and
convulsions.
Inventors: |
Carling, William Robert;
(Bishops Stortford, GB) ; Castro Pineiro, Jose Luis;
(Bishops Stortford, GB) ; Lewis, Richard Thomas;
(Bishops Stortford, GB) ; Moore, Kevin William;
(Buntingford, GB) ; Street, Leslie Joseph; (Little
Hallingbury, GB) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Assignee: |
Merck & Co., Inc.
|
Family ID: |
27269629 |
Appl. No.: |
10/324968 |
Filed: |
December 20, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10324968 |
Dec 20, 2002 |
|
|
|
09890000 |
Jul 23, 2001 |
|
|
|
6500828 |
|
|
|
|
09890000 |
Jul 23, 2001 |
|
|
|
PCT/GB00/00183 |
Jan 19, 2000 |
|
|
|
09890000 |
Jul 23, 2001 |
|
|
|
09674894 |
Nov 7, 2000 |
|
|
|
6479506 |
|
|
|
|
Current U.S.
Class: |
514/248 ;
544/236 |
Current CPC
Class: |
A61P 25/18 20180101;
A61P 25/22 20180101; A61P 25/24 20180101; A61P 9/10 20180101; C07D
487/04 20130101; A61P 25/00 20180101; A61P 25/06 20180101 |
Class at
Publication: |
514/248 ;
544/236 |
International
Class: |
A61K 031/502; C07D
487/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 1999 |
GB |
9901743.6 |
Jan 27, 1999 |
GB |
9901744.4 |
May 27, 1999 |
GB |
9912429.9 |
Claims
1. A compound of formula I, or a pharmaceutically acceptable salt
thereof: 15wherein Y represents hydrogen and Z represents fluoro,
or Y represents fluoro and Z represents hydrogen or fluoro; and
R.sup.1 represents methyl or ethyl.
2. A compound of formula I as depicted in claim 1, or a
pharmaceutically acceptable salt thereof, wherein Y and Z both
represent fluoro; and R.sup.1 represents methyl or ethyl.
3. A compound as claimed in claim 1 represented by formula IA, and
pharmaceutically acceptable salts thereof: 16wherein Y, Z and
R.sup.1 are as defined in claim 1.
4. A compound as claimed in claim 3 represented by formula IIA, and
pharmaceutically acceptable salts thereof: 17wherein R.sup.1 is as
defined in claim 1.
5. A compound as claimed in claim 3 represented by formula IIB, and
pharmaceutically acceptable salts thereof: 18wherein R.sup.1 is as
defined in claim 1.
6. A compound as claimed in claim 3 represented by formula IIC, and
pharmaceutically acceptable salts thereof: 19wherein R.sup.1 is as
defined in claim 1.
7. A compound as claimed in any one of the preceding claims wherein
R.sup.1 represents methyl.
8. A compound as claimed in any one of claims 1 to 6 wherein
R.sup.1 represents ethyl.
9. A compound selected from:
3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)--
6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol--
3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine; and pharmaceutically
acceptable salts thereof.
10. A compound selected from:
3-(2,6-difluorophenyl)-7-(1,1-dimethylethyl)-
-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
and pharmaceutically acceptable salts thereof.
11. A compound selected from:
7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-tr-
iazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazi-
ne;
7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3-
,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;
7-(1,1-dimethylethyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6--
trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; and
pharmaceutically acceptable salts thereof.
12. 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.
13. The use of a compound of formula I as defined in claim 1 or a
pharmaceutically acceptable salt thereof for the manufacture of a
medicament for the treatment and/or prevention of anxiety.
14. 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: 20 wherein Y, Z and R.sup.1 are as defined
in claim 1, and L.sup.1 represents a suitable leaving group; or (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: 21 wherein Y, Z and R.sup.1 are as defined in claim 1,
and L.sup.3 represents a suitable leaving group; or (C) reacting
trimethylacetic acid with a compound of formula XIII: 22 wherein Y,
Z and R.sup.1 are as defined in claim 1; in the presence of silver
nitrate and ammonium persulphate; or (D) reacting a compound of
formula XIV with a compound of formula XV: 23 wherein Y, Z and
R.sup.1 are as defined in claim 1, M represents --B(OH).sub.2 or
--Sn(Alk).sub.3 in which Alk represents a C.sub.1-6 alkyl group,
and L.sup.4 represents a suitable leaving group; in the presence of
a transition metal catalyst.
15. 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.
Description
[0001] The present invention relates to a class of substituted
triazolo-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
GABA.sub.A receptors and are therefore useful in the therapy of
deleterious mental states.
[0002] Receptors for the major inhibitory neurotransmitter,
gamma-aminobutyric acid (GABA), are divided into two main classes:
(1) GABA.sub.A receptors, which are members of the ligand-gated ion
channel superfamily; and (2) GABA.sub.B receptors, which may be
members of the G-protein linked receptor superfamily. Since the
first cDNAs encoding individual GABA.sub.A receptor subunits were
cloned the number of known members of the mammalian family has
grown to include at least six a subunits, four .beta. subunits,
three .gamma. subunits, one .delta. subunit, one .epsilon. subunit
and two p subunits.
[0003] Although knowledge of the diversity of the GABA.sub.A
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 .beta. subunit and a .gamma. subunit
constitute the minimum requirement for forming a fully functional
GABA.sub.A receptor expressed by transiently transfecting cDNAs
into cells. As indicated above, .delta.,.epsilon. and .rho.
subunits also exist, but are present only to a minor extent in
GABA.sub.A receptor populations.
[0004] Studies of receptor size and visualisation by electron
microscopy conclude that, like other members of the ligand-gated
ion channel family, the native GABA.sub.A receptor exists in
pentameric form. The selection of at least one .alpha., one .beta.
and one .gamma. 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).
[0005] Receptor subtype assemblies which do exist include, amongst
many others, .alpha.1.beta.2.gamma.2, .alpha.2.beta.2/3.gamma.2,
.alpha.3.beta..gamma.2/3, .alpha.2.beta..gamma.1,
.alpha.5.beta.3.gamma.2- /3, .alpha.6.beta..gamma.2,
.alpha.6.beta..delta. and .alpha.4.beta..delta.. Subtype assemblies
containing an .alpha.1 subunit are present in most areas of the
brain and are thought to account for over 40% of GABA.sub.A
receptors in the rat. Subtype assemblies containing .alpha.2 and
.alpha.3 subunits respectively are thought to account for about 25%
and 17% of GABA.sub.A receptors in the rat. Subtype assemblies
containing an .alpha.5 subunit are expressed predominantly in the
hippocampus and cortex and are thought to represent about 4% of
GABA.sub.A receptors in the rat.
[0006] A characteristic property of all known GABA.sub.A 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 GABA.sub.A receptor modulatory sites, and is
the site through which anxiolytic drugs such as diazepam and
temazepam exert their effect. Before the cloning of the GABA.sub.A
receptor gene family, the benzodiazepine binding site was
historically subdivided into two subtypes, BZ1 and BZ2, on the
basis of radioligand binding studies. The BZ1 subtype has been
shown to be pharmacologically equivalent to a GABA.sub.A receptor
comprising the .alpha.1 subunit in combination with a .beta.
subunit and .gamma.2. This is the most abundant GABA.sub.A receptor
subtype, and is believed to represent almost half of all GABA.sub.A
receptors in the brain.
[0007] Two other major populations are the .alpha.2.beta..gamma.2
and .alpha.3.beta..gamma.2/3 subtypes. Together these constitute
approximately a further 35% of the total GABA.sub.A 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
.alpha.5-containing subtype assemblies. The physiological role of
these subtypes has hitherto been unclear because no sufficiently
selective agonists or antagonists were known.
[0008] It is now believed that agents acting as BZ agonists at
.alpha.1.beta..gamma.2, .alpha.2.beta..gamma.2 or
.alpha.3.beta..gamma.2 subunits will possess desirable anxiolytic
properties. Compounds which are modulators of the benzodiazepine
binding site of the GABA.sub.A receptor by acting as BZ agonists
are referred to hereinafter as "GABA.sub.A receptor agonists". The
.alpha.1-selective GABA.sub.A 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 BZ1 binding site is mediated through
GABA.sub.A receptors containing the .alpha.1 subunit. Accordingly,
it is considered that GABA.sub.A receptor agonists which interact
more favourably with the .alpha.2 and/or .alpha.3 subunit than with
.alpha.1 will be effective in the treatment of anxiety with a
reduced propensity to cause sedation. Also, agents which are
antagonists or inverse agonists at .alpha.1 might be employed to
reverse sedation or hypnosis caused by .alpha.1 agonists.
[0009] The compounds of the present invention, being selective
ligands for GABA.sub.A 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.
[0010] Further disorders for which selective ligands for GABA.sub.A
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; 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 GABA.sub.A receptors may also be effective as
pre-medication prior to anaesthesia or minor procedures such as
endoscopy, including gastric endoscopy.
[0011] 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 GABA.sub.A receptors beneficial in the
treatment and/or prevention of neurological disorders including
anxiety and convulsions.
[0012] The present invention provides a class of
triazolo-pyridazine derivatives which possess desirable binding
properties at various GABA.sub.A receptor subtypes. The compounds
in accordance with the present invention have good affinity as
ligands for the .alpha.2 and/or .alpha.3 subunit of the human
GABA.sub.A receptor. The compounds of this invention interact more
favourably with the .alpha.2 and/or .alpha.3 subunit than with the
.alpha.1 subunit. Indeed, the compounds of the invention exhibit
functional selectivity in terms of a selective efficacy for the
.alpha.2 and/or .alpha.3 subunit relative to the .alpha.1
subunit.
[0013] The compounds of the present invention are GABA.sub.A
receptor subtype ligands having a binding affinity (K.sub.i) for
the .alpha.2 and/or .alpha.3 subunit, 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 .alpha.2
and/or .alpha.3 subunit relative to the .alpha.1 subunit. Moreover,
the compounds according to the present invention possess
interesting pharmacokinetic properties, notably in terms of
improved oral bioavailability.
[0014] The present invention provides a compound of formula I, or a
pharmaceutically acceptable salt thereof: 1
[0015] wherein
[0016] Y represents hydrogen and Z represents fluoro, or Y
represents fluoro and Z represents hydrogen or fluoro; and
[0017] R.sup.1 represents methyl or ethyl.
[0018] The compounds in accordance with the present invention are
encompassed within the generic scope of WO 98/04559. There is,
however, no specific disclosure therein of compounds corresponding
to those of formula I as defined above.
[0019] For use in medicine, the salts of the compounds of formula I
above will be pharmaceutically acceptable salts. Other salts may,
however, be useful in 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,
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.
[0020] The present invention also provides a compound of formula I
as depicted above, or a pharmaceutically acceptable salt thereof,
wherein Y and Z both represent fluoro; and R.sup.1 represents
methyl or ethyl.
[0021] A particular sub-class of the compounds in accordance with
the invention is represented by the compounds of formula IA, and
pharmaceutically acceptable salts thereof: 2
[0022] wherein Y, Z and R.sup.1 are as defined above.
[0023] Specific sub-classes of the compounds in accordance with the
invention are represented by the compounds of formula IIA, IIB and
IIC, and pharmaceutically acceptable salts thereof: 3 4
[0024] wherein R.sup.1 is as defined above.
[0025] In one embodiment of the compounds according to the
invention, the moiety R.sup.1 represents methyl.
[0026] In another embodiment of the compounds according to the
invention, the moiety R.sup.1 represents ethyl.
[0027] Specific compounds within the scope of the present invention
include:
[0028]
3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-t-
riazol-3-ylmethoxy)-1,2,4-triazolo [4,3-b]pyridazine;
[0029]
3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-tr-
iazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
[0030]
3-(2,6-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-tr-
iazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
[0031]
7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-
,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;
[0032]
7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(-
2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;
[0033]
7-(1,1-dimethylethyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-3-(-
2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;
[0034] and pharmaceutically acceptable salts thereof.
[0035] 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 a compound of formula I as defined above or a
pharmaceutically acceptable salt thereof.
[0036] 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 a compound of formula I as defined above or a
pharmaceutically acceptable salt thereof.
[0037] The binding affinity (K.sub.i) of the compounds according to
the present invention for the .alpha.3 subunit of the human
GABA.sub.A receptor is conveniently as measured in the assay
described hereinbelow. The .alpha.3 subunit binding affinity
(K.sub.i) of the compounds of the invention is less than 1 nM.
[0038] The compounds according to the present invention elicit a
selective potentiation of the GABA EC.sub.20 response in stably
transfected recombinant cell lines expressing the .alpha.3 subunit
of the human GABA.sub.A receptor relative to the potentiation of
the GABA EC.sub.20 response elicited in stably transfected
recombinant cell lines expressing the .alpha.1 subunit of the human
GABA.sub.A receptor.
[0039] The potentiation of the GABA EC.sub.20 response in stably
transfected cell lines expressing the .alpha.3 and .alpha.1
subunits of the human GABA.sub.A receptor can conveniently be
measured by procedures analogous to the protocol described in
Wafford et al., Mol. Pharinacol., 1996, 50, 670-678. The procedure
will suitably be carried out utilising cultures of stably
transfected eukaryotic cells, typically of stably transfected mouse
Ltk.sup.-fibroblast cells.
[0040] The compounds according to the present invention exhibit
anxiolytic activity, as may be 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 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).
[0041] 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 al. in J. Pharmacol. Exp. Ther., 1996, 279, 492-501.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] The compounds of formula I as defined above may be prepared
by a process which comprises reacting a compound of formula III
with a compound of formula IV: 5
[0047] wherein Y, Z and R.sup.1 are as defined above, and L.sup.1
represents a suitable leaving group.
[0048] The leaving group L.sup.1 is typically a halogen atom,
especially chloro.
[0049] 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 as sodium
hydride. In one preferred embodiment, the solvent is
dimethylsulfoxide, and the base is caesium carbonate. In another
preferred embodiment, the solvent is 1-methyl-2-pyrrolidinone, and
the base is sodium hydroxide, in which case the reaction is
advantageously performed at a temperature in the region of
0.degree. C.
[0050] The intermediates of formula III above may be prepared by
reacting a compound of formula V with a substantially equimolar
amount of a hydrazine derivative of formula VI: 6
[0051] wherein Y, Z and L.sup.1 are as defined above, and L.sup.2
represents a suitable leaving group; followed, if necessary, by
separation of the resulting mixture of isomers by conventional
means.
[0052] The leaving group L.sup.2 is typically a halogen atom,
especially chloro. In the intermediates of formula V, the leaving
groups L.sup.1 and L.sup.2 may be the same or different, but are
suitably the same, preferably both chloro.
[0053] 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.
[0054] 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: 7
[0055] wherein Y, Z and L.sup.1 are as defined above; followed by
cyclization of the intermediate Schiff's base thereby obtained.
[0056] 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 Schiff's 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 80.degree.
C.
[0057] 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
90.degree. C., or in 1,4-dioxane or ethanol at the reflux
temperature of the solvent; followed, if necessary, by separation
of the resulting mixture of isomers by conventional means.
[0058] 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: 8
[0059] wherein Y and Z are as defined above, and Q represents a
reactive carboxylate moiety; followed, if necessary, by cyclization
of the hydrazide derivative of formula X thereby obtained: 9
[0060] wherein Y, Z and L.sup.1 are as defined above.
[0061] Suitable values for the reactive carboxylate moiety Q
include esters, for example C.sub.1-4 alkyl esters; acid
anhydrides, for example mixed anhydrides with C.sub.1-4 alkanoic
acids; acid halides, for example acid chlorides; and
acylimidazoles. Suitably, Q represents an acid chloride moiety.
[0062] The reaction between compounds VII and IX is conveniently
effected by heating in a solvent such as 1-methyl-2-pyrrolidinone
to a temperature typically in the region of 160.degree. C.
[0063] Alternatively, the reaction between compounds VII and IX may
be 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.degree. 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 typically at a temperature in the
region of 0.degree. C.
[0064] 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.sup.1 or L.sup.2. Thus,
in addition to the required product of formula III, the isomeric
compound wherein the hydrazine moiety displaces the leaving group
L.sup.1 will possibly be obtained to some extent; and likewise for
compound VII. For this reason it might be necessary to separate the
resulting mixture of isomers by conventional methods such as
chromatography.
[0065] 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: 10
[0066] wherein Y, Z and R.sup.1 are as defined above, and L.sup.3
represents a suitable leaving group.
[0067] The leaving group L.sup.3 is suitably a halogen atom,
typically chloro or bromo.
[0068] The reaction between compounds XI and XII is conveniently
effected by stirring the reactants in a suitable solvent, typically
N,N-dimethylformamide, in the presence of a strong base such as
sodium hydride.
[0069] 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.
[0070] In a further procedure, the compounds of formula I as
defined above may be prepared by a process which comprises reacting
trimethylacetic acid with a compound of formula XIII: 11
[0071] wherein Y, Z and R.sup.1 are as defined above; in the
presence of silver nitrate and ammonium persulphate.
[0072] The reaction is conveniently carried out in a suitable
solvent, for example water or aqueous acetonitrile, optionally
under acidic conditions, e.g. using trifluoroacetic acid or
sulphuric acid, typically at an elevated temperature.
[0073] The intermediates of formula XIII correspond to the
compounds of formula I as defined above wherein the tert-butyl
substituent at the 7-position is absent, and they may therefore be
prepared by methods analogous to those described above for
preparing the corresponding compounds of formula I.
[0074] In a still further procedure, the compounds of formula I as
defined above may be prepared by a process which comprises reacting
a compound of formula XIV with a compound of formula XV: 12
[0075] wherein Y, Z and R.sup.1 are as defined above, M represents
--B(OH).sub.2 or --Sn(Alk)3 in which Alk represents a C.sub.1-6
alkyl group, typically n-butyl, and L.sup.4 represents a suitable
leaving group; in the presence of a transition metal catalyst.
[0076] The leaving group L.sup.4 is suitably a halogen atom, e.g.
bromo.
[0077] A suitable transition metal catalyst of use in the reaction
between compounds XIV and XV comprises
dichlorobis(triphenylphosphine)-palladium(- II) or
tetrakis(triphenylphosphine)palladium(0).
[0078] The reaction between compounds XIV and XV is conveniently
effected in an inert solvent such as N,N-dimethylformamide,
typically at an elevated temperature.
[0079] The intermediates of formula XIV may be prepared by reacting
a compound of formula IV as defined above with a compound of
formula XVI: 13
[0080] wherein L.sup.1 and L.sup.4 are as defined above; under
conditions analogous to those described above for the reaction
between compounds III and IV.
[0081] Where R.sup.1 is methyl, the relevant intermediate of
formula IV above may be prepared by the procedures described in
EP-A-0421210, or by methods analogous thereto. Where R.sup.1 is
ethyl, the relevant intermediate of formula IV may conveniently be
prepared by the method described in the accompanying Examples.
[0082] The intermediates of formula V above may be prepared by
reacting trimethylacetic acid with a compound of formula XVII:
14
[0083] wherein L.sup.1 and L.sup.2 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.
[0084] 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.
[0085] 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.
[0086] The following Examples illustrate the preparation of
compounds according to the invention.
[0087] The compounds in accordance with this invention potently
inhibit the binding of [.sup.3H]-flumazenil to the benzodiazepine
binding site of human GABA.sub.A receptors containing the .alpha.2
or .alpha.3 subunit stably expressed in Ltk.sup.-cells.
[0088] Reagents
[0089] Phosphate buffered saline (PBS).
[0090] Assay buffer: 10 mM KH.sub.2PO.sub.4, 100 mM KCl, pH 7.4 at
room temperature.
[0091] [.sup.3H]-Flumazenil (18 nM for .alpha.1.beta.3.gamma.2
cells; 18 nM for .alpha.2.beta.3.gamma.2 cells; 10 nM for
.alpha.3.beta.3.gamma.2 cells) in assay buffer.
[0092] Flunitrazepam 100 .mu.M in assay buffer.
[0093] Cells resuspended in assay buffer (1 tray to 10 ml).
[0094] Harvesting Cells
[0095] 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.times.25 cm) of cells.
[0096] Assay
[0097] Can be carried out in deep 96-well plates or in tubes. Each
tube contains:
[0098] 300 .mu.l of assay buffer.
[0099] 50 .mu.L of [.sup.3H]-flumazenil (final concentration for
.alpha.2.beta.3.gamma.2: 1.8 nM; for .alpha.2.beta.3.gamma.2: 1.8
nM; for .alpha.3.beta.3.gamma.2: 1.0 nM).
[0100] 50 .mu.l 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 .mu.M final
concentration.
[0101] 100 .mu.l of cells.
[0102] Assays are incubated for 1 hour at 40.degree. C., then
filtered using either a Tomtec or Brandel cell harvester onto GF/B
filters followed by 3.times.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 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 K.sub.i can be calculated for each test
compound.
[0103] The compounds of the accompanying Examples were tested in
the above assay, and all were found to possess a K.sub.i value for
displacement of [.sup.3H]-flumazenil from the .alpha.2 and/or
.alpha.3 subunit of the human GABA.sub.A receptor of less than 1
nM.
EXAMPLE 1
[0104]
3-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-t-
riazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine
[0105] a) 3,6-Dichloro-4-(1,1-dimethylethyl)pyridazine
[0106] Concentrated sulfuric 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 l). This mixture was then heated to
70.degree. 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, 1.0 mol) in water (0.63 l) was then added over 20-30 minutes.
The internal temperature rose to approximately 85.degree. 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.degree. C. The aqueous was extracted with
dichloromethane (3.times.300 ml). The combined extracts were dried
(MgSO.sub.4), 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: .sup.1H NMR
(360 MHz, d.sub.6-DMSO) .delta. 1.50 (9H, s), 7.48 (1H, s); MS
(ES.sup.+) m/e 205 [MH].sup.+, 207 [MH].sup.+a.
[0107] b) 3-Chloro-4-(1,1-dimethylethyl)-6-hydrazinylpyridazine
[0108] To a solution of
3,6-dichloro-4-(1,1-dimethylethyl)pyridazine (2.0 g, 9.76 mmol) in
ethanol (30 ml) was added hydrazine hydrate (0.34 ml, 10.9 mmol)
dropwise. The reaction mixture was heated at reflux for 18 h under
an atmosphere of nitrogen. The solvent was removed under high
vacuum to leave a residue to which was added 5N hydrochloric acid
(50 ml). The solution obtained was washed with dichloromethane (20
ml) and the aqueous layer was poured on to a mixture of ice and
aqueous ammonia. The resultant solid was collected by filtration
and dried under vacuum to yield the title compound (1.2 g). Data
for the title compound: .sup.1H NMR (360 MHz, DMSO) .delta. 1.39
(3H, t, J=7.3 Hz), 4.35 (2H), 7.07 (1H, s), 8.07 (1H, s); MS
(ES.sup.+) m/e 201, 203 [MH].sup.+.
[0109] c)
6-Chloro-3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-tria-
zolo[4,3-b]pyridazine
[0110] To a slurry of
3-chloro-4-(1,1-dimethylethyl)-6-hydrazinylpyridazin- e (1.3 g, 6.5
mmol) in 0.1N hydrochloric acid (60 ml) was added
2,5-difluorobenzaldehyde (0.70 ml, 6.5 mmol) and the reaction
mixture was stirred at room temperature for 30 minutes and then
heated to 60.degree. C. for 40 minutes. The reaction mixture was
allowed to cool and the resultant solid was collected by
filtration, dried and dissolved in ethanol (60 ml). To this
solution was added iron(III) chloride hexahydrate (5.4 g, 32.5
mmol) in ethanol (15 ml) dropwise over 10 minutes at 80.degree. C.
The reaction mixture was stirred at 80.degree. C. for 2 h, allowed
to cool and the solvent removed by evaporation under vacuum. The
residue was dissolved in dichloromethane (100 ml) and washed with
water (3.times.100 ml), brine, dried (MgSO.sub.4), filtered and
concentrated under vacuum to yield the title compound (0.75 g).
Data for the title compound: .sup.1H NMR (250 MHz, CDCl.sub.3)
.delta. 1.57 (9H, s), 7.22-7.29 (2H, m), 7.65-7.71 (1H, m), 8.19
(1H, s); MS (ES.sup.+) m/e 323 [MH].sup.+.
[0111] d)
3-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,-
4-triazol-3-ylmethoxy)-1,2,4-triazolo [4,3-b]pyridazine
[0112] To a solution of (2-methyl-2H-1,2,4-triazol-3-yl)methanol
(0.069 g, 0.4 mmol) and
6-chloro-3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-
-triazolo[4,3-b]pyridazine (0.10 g, 0.31 mmol) in DMF (10 ml) was
added sodium hydride (0.015 g of a 60% dispersion in oil, 1.2 mol
eq.) and the reaction mixture was stirred at room temperature for
40 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 (0.088 g, 65%). Data for the title
compound: .sup.1H NMR (360 MHz, CDCl.sub.3) .delta. 1.41 (9H, s),
3.89 (3H, s), 5.54 (2H, s), 7.23-7.26 (2H, m), 7.64 (1H, m), 7.91
(1H, s), 8.00 (1H, s); MS (ES.sup.+) m/e 400 [MH].sup.+. Anal.
Found C, 57.36; H, 4.61; N, 24.60%. C.sub.19H.sub.19F.sub.2N.sub.7O
requires C, 57.14; H, 4.79; N, 24.55%.
EXAMPLE 2
[0113]
3-(2.5-Difluorophenyl)-7-(1.1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-tr-
iazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine
[0114] a) (2-Ethyl-2H-1.2,4-triazol-3-yl)methanol
[0115] 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) was added dropwise over 10 mins. The reaction mixture
was allowed to warm to room temperature 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.times.300 ml). The combined organic layers were washed with
saturated brine and dried (MgSO.sub.4), filtered and concentrated
under vacuum to leave an oily residue which was purified by
distillation (120.degree. C. @.about.20 mmHg) to give
1-ethyltriazole contaminated with .about.15% DMF (2.4 g). The crude
product (2.4 g, 0.025 mol) was dissolved in dry THF (35 ml), cooled
to -40.degree. C. and n-butyllithium (16.2 ml of a 1.6 molar
solution in hexane, 0.026 mol) was added slowly over 20 mins
keeping the temperature constant. DMF (2.03 ml, 0.026 mol) was then
added and after 15 mins 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.times.50 ml). The combined
organic layers were dried (MgSO.sub.4), filtered and concentrated
under vacuum to leave a residue which was purified by silica gel
chromatography using 0-5% methanol in dichloromethane as eluent to
give the title compound as an off-white solid (0.5 g, 3%). Data for
the title compound: .sup.1H NMR (250 MHz, CDCl.sub.3) .delta. 1.48
(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 (1H, s).
[0116] b)
3-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-
-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine
[0117] This compound was prepared using the procedures described in
Example 1 Steps a), b), c) and d) with
(2-ethyl-2H-1,2,4-triazol-3-yl)met- hanol used instead of
(2-methyl-2H-1,2,4-triazol-3-yl)methanol in Step c). Data for the
title compound: .sup.1H NMR (360 MHz, CDCl.sub.3) .delta. 1.40 (9H,
s), 1.47 (3H, t, J=7.3 Hz), 4.20 (2H, q, J=14.6 & 7.3 Hz), 5.54
(2H, s), 7.23-7.27 (2H, m), 7.65 (1H, m), 7.94 (1H, s), 8.00 (1H,
s); MS (ES.sup.+) m/e 414 [MH].sup.+. Anal. Found C, 58.17; H,
5.01; N, 23.79%. C.sub.20H.sub.21F.sub.2N.sub.7O requires C, 58.10;
H, 5.12; N, 23.72%.
EXAMPLE 3
[0118]
3-(2,6-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-tr-
iazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine
[0119] a)
6-Chloro-3-(2.6-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-tria-
zolo[4,3-b]pyridazine
[0120] A mixture of 3,6-dichloro-4-(1,1-dimethylethyl)pyridazine (2
g, 9.75 mmol), 2,6-difluorobenzoic acid hydrazide (2.52 g, 14.6
mmol) (WO 95/24403) and triethylamine hydrochloride (2.01 g, 14.6
mmol) in 1,4-dioxane (10 ml) was stirred and heated at reflux for
3.5 days. Upon cooling, the volatiles were removed in vacuo and the
residue was triturated with dichloromethane. Any undissolved solid
was removed by filtration. The residue was purified by
chromatography on silica gel eluting with 0%.fwdarw.25% ethyl
acetate in dichloromethane to give the required product (0.42 g).
Data for the title compound: .sup.1H NMR (250 MHz, CDCl.sub.3)
.delta. 1.57 (9H, s), 7.09-7.16 (2H, m), 7.51-7.63 (1H, m), 8.19
(1H, s); MS (ES.sup.+) m/e 323 [MH].sup.+.
[0121] b)
3-(2,6-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-
-triazol-3-ylmethoxy)-1,2,4-triazolo [4,3-b]pyridazine
[0122] This compound was prepared from
6-chloro-3-(2,6-difluorophenyl)-7-(-
1,1-dimethylethyl)-1,2,4-triazolo[4,3-b]pyridazine and
(2-ethyl-2H-1,2,4-triazol-3-yl)methanol following the procedure
(sodium hydride, DMF) described in WO 98/04559. Data for the title
compound: m.p. 182.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.39-1.45 (12H, m), 4.10-4.16 (2H, m), 5.46 (2H, s),
7.09-7.15 (2H, m), 7.52-7.59 (1H, m), 7.92 (1H, s), 8.00 (1H, s);
MS (ES.sup.+) m/e 414 [MH].sup.+; Anal. Found: C, 58.19; H, 5.05;
N, 23.80%. C.sub.20H.sub.21F.sub.2N.sub.7O requires: C, 58.10; H,
5.12; N, 23.72%.
EXAMPLE 4
[0123]
7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1.2,4-triazol-3-ylmethoxy)-3-(2-
,3.6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
[0124] a)
6-Chloro-7-(1,1-dimethylethyl)-3-(2,3,6-trifluorophenyl)-1,2,4-t-
riazolo[4,3-b]pyridazine
[0125] 2,3,6-Trifluorobenzoyl chloride (4.0 g) was added dropwise
to a cooled (15.degree. C.) solution of
3-chloro-4-(1,1-dimethylethyl)-6-hydra- zinylpyridazine (4 g) in
dry 1-methyl-2-pyrrolidinone (50 ml). After the addition the
reaction mixture was heated at 160.degree. C. for 24 h. The
reaction mixture was cooled to room temperature, diluted with ethyl
acetate (200 ml), and washed twice with water (200 ml). The organic
phase was separated, dried (sodium sulfate), and evaporated at
reduced pressure. The residue was crystallised from dichloromethane
on dilution with diethyl ether to afford the title compound (5.3 g)
as a colourless solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
1.52 (9H, s), 7.50 (1H, m), 7.92 (1H, m), 8.45 (1H, s); MS
(ES.sup.+) m/z 341/343 [MH].sup.+.
[0126] b) (2-Ethyl-2H-1,2,4-triazol-3-yl)methanol: Alternative
Procedure
[0127] 1,2,4-Triazole (100.0 g, 1.45 mol) in anhydrous THF (950 ml)
was cooled to 0.degree. C. 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 30.degree. C. The reaction was
stirred at room temperature for 16 h, after which the DBU
hydroiodide was removed by filtration. The filtrate was cooled to
-75.degree. C. under an atmosphere of dry nitrogen. Hexyllithium
(458 ml of 33% solution in hexanes) was added dropwise over 25 min
keeping the internal temperature below -55.degree. C. The reaction
mixture was aged for 30 min (back to -75.degree. C.) and then dry
N,N-dimethylformamide (108 ml, 1.39 mol) was added dropwise over 10
min maintaining internal temperature below -60.degree. C. The
reaction mixture was aged at -70.degree. C. for 90 min, then
allowed to warm to 0.degree. C. over 30 min. Ethanol (340 ml) was
added over 10 min. Sodium borohydride (26.3 g, 0.695 mol) was then
added portionwise maintaining the internal temperature below
6.degree. C. After the addition the reaction mixture was allowed to
warm to room temperature and stirred for 1 h. 2M H.sub.2SO.sub.4
(200 ml) was then added slowly with caution and the mixture 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.degree. C. and stirred for 15
min. The solution was extracted with warm (45.degree. C.) isobutyl
alcohol (2.times.675 ml). The combined organic fractions were
concentrated under reduced pressure to a volume of approximately
450 ml at which point the product crystallised. Heptane (1.125 l)
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 0.degree. C.,
filtration gave the title compound (137 g, 74% from
1,2,4-triazole). .sup.1H NMR as for Example 2 Step a).
[0128] c)
7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-
-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4.3-b]pyridazine
[0129] To the product of Step a) (2.60 g) and the foregoing product
(1.0 g) in dry dimethylsulfoxide (10 ml) was added caesium
carbonate (3.05 g), and the mixture stirred at 50.degree. C. under
an atmosphere of dry nitrogen for 24 hours. On cooling to room
temperature, the mixture was partitioned between ethyl acetate and
water. The organic phase was separated, washed with water,
evaporated at reduced pressure, and the residue chromatographed on
silica gel (eluent 2.5% methanol-dichloromethane). The product was
crystallised from ethyl acetate/diethyl ether/isohexane, to afford
the title compound as a colourless solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.26 (3H, t, J=7.1 Hz), 1.38 (9H, s), 4.10
(2H, q, J=7.1 Hz), 5.53 (2H, s), 7.46 (1H, m), 7.88 (1H, m), 7.94
(1H, s), 8.18 (1H, s); MS (ES.sup.+) m/z 432 [MH].sup.+.
EXAMPLE 5
[0130]
7-(1,1-Dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(-
2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
[0131] To the product of Example 4 Step a) (2.67 g) and
(2-methyl-2H-1,2,4-triazol-3-yl)methanol (prepared as described in
EP-A-421210) (0.93 g) in dry dimethylsulfoxide (10 ml) was added
caesium carbonate (3.14 g), and the mixture stirred at 50.degree.
C. under an atmosphere of dry nitrogen for 24 hours. On cooling to
room temperature, the mixture was partitioned between ethyl acetate
and water. The organic phase was separated, washed with water,
evaporated at reduced pressure, and the residue chromatographed on
silica gel (eluent 2.5% methanol-dichloromethane). The product was
crystallised from ethyl acetate/diethyl ether/isohexane, to afford
the title compound as a colourless solid. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.39 (9H, s), 3.74 (3H, s), 5.50 (2H, s),
7.46 (1H, m), 7.88 (1H, m), 7.90 (1H, s), 8.17 (1H, s); MS
(ES.sup.+) m/z 418 [MH].sup.+.
EXAMPLE 6
[0132]
7-(1,1-Dimethylethyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-3-(-
2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
[0133] To the product of Example 4 Step a) (0.559 g) and
(1-methyl-1H-1,2,4-triazol-3-yl)methanol (prepared as described in
EP-A-421210) (0.20 g) in dry dimethylsulfoxide (2 ml) was added
caesium carbonate (0.67 g), and the mixture stirred at 60.degree.
C. under an atmosphere of dry nitrogen for 48 hours. On cooling to
room temperature, the mixture was partitioned between ethyl acetate
and water. The organic phase was separated, washed with water,
evaporated at reduced pressure, and the residue chromatographed on
silica gel (eluent 2% methanol-dichloromethane). The product was
crystallised from ethyl acetate/diethyl ether, to afford the title
compound as a colourless solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 1.39 (9H, s), 3.85 (3H, s), 5.30 (2H, s), 7.46 (1H, m),
7.86 (1H, m), 8.14 (1H, s), 8.46 (1H, s); MS (ES.sup.+) m/z 418
[MH].sup.+.
* * * * *