U.S. patent application number 12/203946 was filed with the patent office on 2009-07-02 for piperazine derivative having affinity for the histamine h3 receptor.
Invention is credited to Desmond John BEST, Sing Yeung Mak, Barry Sidney Orlek, Geracimos Rassias, Pamela Joan Theobald.
Application Number | 20090170869 12/203946 |
Document ID | / |
Family ID | 39885024 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090170869 |
Kind Code |
A1 |
BEST; Desmond John ; et
al. |
July 2, 2009 |
PIPERAZINE DERIVATIVE HAVING AFFINITY FOR THE HISTAMINE H3
RECEPTOR
Abstract
The present invention relates to
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine ##STR00001## or a pharmaceutically acceptable salt thereof,
in particular the hydrochloride salt thereof and crystalline forms
of the hydrochloride salt; to processes for the preparation of the
compound or its salt; to compositions containing it; and to its use
in the treatment or prophylaxis of neurological or psychiatric
diseases, such as cognitive impairment, fatigue or a sleep
disorder, for example in a mammal such as a human. The compound or
a salt thereof has affinity for and is an antagonist and/or inverse
agonist of the histamine H3 receptor.
Inventors: |
BEST; Desmond John; (Harlow,
GB) ; Mak; Sing Yeung; (Singapore, SG) ;
Orlek; Barry Sidney; (Harlow, GB) ; Rassias;
Geracimos; (Stevenage, GB) ; Theobald; Pamela
Joan; (Harlow, GB) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B482
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
39885024 |
Appl. No.: |
12/203946 |
Filed: |
September 4, 2008 |
Current U.S.
Class: |
514/254.1 ;
544/374 |
Current CPC
Class: |
A61P 25/16 20180101;
A61P 25/20 20180101; A61P 25/00 20180101; A61P 25/28 20180101; C07D
309/12 20130101 |
Class at
Publication: |
514/254.1 ;
544/374 |
International
Class: |
A61K 31/497 20060101
A61K031/497; C07D 405/12 20060101 C07D405/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 6, 2007 |
GB |
0717336.2 |
Aug 15, 2008 |
GB |
0814987.4 |
Claims
1. A compound which is
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine ##STR00020## or a pharmaceutically acceptable salt
thereof.
2. A compound or salt as claimed in claim 1, which is
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
3. Crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, characterised by an X-ray powder diffraction
spectrum having five or more of the following peaks defined as
degrees two-theta angles obtained with a diffractometer using
copper K-alpha X-radiation: 6.4.+-.0.1, 12.7.+-.0.1, 15.4.+-.0.1,
15.7.+-.0.1, 17.1.+-.0.1, 19.1.+-.0.1, 19.7.+-.0.1, 21.9.+-.0.1,
25.5.+-.0.1, 27.0.+-.0.1, and 28.2.+-.0.1 degrees two-theta;
provided that the X-ray powder diffraction spectrum has the
following two peaks: 15.7.+-.0.1 and 25.5.+-.0.1 degrees
two-theta.
4. Crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, as claimed in claim 3, wherein the X-ray
powder diffraction spectrum has eight or more of the peaks defined
in claim 3.
5. Crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, characterised by a solid-form
Fourier-Transform Infrared spectrum substantially the same as that
shown in FIG. 5.
6. Crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, characterised by a .sup.13C solid-state
nuclear magnetic resonance spectrum having the following chemical
shifts for the resonances: 18.5.+-.0.3, 30.4.+-.0.3, 31.8.+-.0.3,
37.6.+-.0.3, 45.8.+-.0.3, 49.4.+-.0.3, 52.3.+-.0.3, 59.2.+-.0.3,
63.6.+-.0.3, 68.4.+-.0.3, 110.3.+-.0.3, 118.8.+-.0.3, 128.4.+-.0.3,
131.2.+-.0.3, 133.9.+-.0.3, 159.1.+-.0.3, and 167.6.+-.0.3 ppm.
7. Crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, characterised by an X-ray powder diffraction
spectrum having five or more of the following peaks defined as
degrees two-theta angles obtained with a diffractometer using
copper K-alpha X-radiation: 6.4.+-.0.1, 12.8.+-.0.1, 15.4.+-.0.1,
19.2.+-.0.1, 19.7.+-.0.1, 20.0.+-.0.1, 21.8.+-.0.1, 21.9.+-.0.1,
23.5.+-.0.1, 24.65.+-.0.1 (or 24.7.+-.0.1), 25.8.+-.0.1, and
27.0.+-.0.1 degrees two-theta; provided that the X-ray powder
diffraction spectrum has the following two peaks: 20.0.+-.0.1
degrees two-theta, and either 24.65.+-.0.1 or 24.7.+-.0.1 degrees
two-theta.
8. Crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, as claimed in claim 7, wherein the X-ray
powder diffraction spectrum has eight or more of the peaks defined
in claim 7.
9. Crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, characterised by a solid-form
Fourier-Transform Infrared spectrum substantially the same as that
shown in FIG. 7.
10. Crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, characterised by a .sup.13C solid-state
nuclear magnetic resonance spectrum having the following chemical
shifts for the resonances: 18.8.+-.0.3, 19.5.+-.0.3, 32.4.+-.0.3,
37.5.+-.0.3, 45.7.+-.0.3, 49.3.+-.0.3, 52.7.+-.0.3, 59.1.+-.0.3,
66.3.+-.0.3, 71.1.+-.0.3, 109.4.+-.0.3, 119.6.+-.0.3, 128.4.+-.0.3,
131.3.+-.0.3, 134.3.+-.0.3, 158.7.+-.0.3, and 167.8.+-.0.3 ppm.
11. A compound or salt as claimed in claim 1, which is
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine.
12. A pharmaceutical composition which comprises a
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier or excipient.
13. A pharmaceutical composition as claimed in claim 12, which is
for oral administration, and which comprises
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
14. A pharmaceutical composition as claimed in claim 12, which is
for oral administration, and which comprises crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride as defined in claim 7.
15. A method of treatment of a neurological disease in a mammal
such as a human in need thereof, which comprises administering to
the mammal an effective amount of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof.
16. A method of treatment of cognitive impairment, or of treatment
or prophylaxis of fatigue or a sleep disorder, in a human in need
thereof, which comprises administering to the mammal a
therapeutically effective amount of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]car-
bonyl}piperazine or a pharmaceutically acceptable salt thereof.
17. A method as claimed in claim 16, wherein the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof is
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
18. A method as claimed in claim 16, which is a method of treatment
of cognitive impairment, or of treatment or prophylaxis of fatigue,
in a human in need thereof.
19. A method as claimed in claim 16, which is a method of treatment
of cognitive impairment in a human in need thereof.
20. A method as claimed in claim 19, wherein the cognitive
impairment is in Alzheimer's disease, dementia, mild cognitive
impairment, or a related neurodegenerative disorder, or wherein the
cognitive impairment is in Parkinson's disease, or wherein the
cognitive impairment is in schizophrenia.
21. A method as claimed in claim 19, wherein the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof is
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
22. A process for the preparation of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a salt thereof, which process comprises: a) reacting
4-(tetrahydro-2H-pyran-4-yloxy)benzoyl chloride with 1-isopropyl
piperazine; or b) reacting 4-(tetrahydro-2H-pyran-4-yloxy)benzoic
acid, or a non-acid-chloride derivative thereof in which the
carboxylic acid group has been activated, with 1-isopropyl
piperazine; and optionally preparing a salt of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine.
23. A process as claimed in claim 22, comprising process step b),
and wherein process step b) comprises activation of
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with a coupling reagent
in a suitable solvent, followed by reaction with 1-isopropyl
piperazine.
24. A process as claimed in claim 23, wherein the coupling reagent
is carbonyl diimidazole.
25. A process as claimed in claim 24, wherein the activation of the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with the carbonyl
diimidazole coupling reagent, and the subsequent reaction with
1-isopropyl piperazine, are both carried out in a reaction solvent
comprising acetonitrile and/or propionitrile.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a novel piperazine
derivative having pharmacological activity, to processes for its
preparation, to compositions containing it, and to its use in the
treatment of neurological or psychiatric disorders such as
cognitive impairment e.g. in Alzheimer's disease.
[0002] The histamine H3 receptor is predominantly expressed in the
mammalian central nervous system (CNS), with minimal expression in
peripheral tissues except on some sympathetic nerves (Leurs et al.,
(1998), Trends Pharmacol. Sci. 19, 177-183). Activation of H3
receptors by selective agonists or histamine results in the
inhibition of neurotransmitter release from a variety of different
nerve populations, including histaminergic and cholinergic neurons
(Schlicker et al., (1994), Fundam. Clin. Pharmacol. 8, 128-137).
Additionally, in vitro and in vivo studies have shown that H3
antagonists can facilitate neurotransmitter release in brain areas
such as the cerebral cortex and hippocampus, relevant to cognition
(Onodera et al., (1998), In: The Histamine H3 receptor, ed. Leurs
and Timmerman, pp 255-267, Elsevier Science B.V.). Moreover, a
number of reports in the literature have demonstrated the cognitive
enhancing properties of H3 antagonists (e.g. thioperamide,
clobenpropit, ciproxifan and GT-2331) in rodent models including
the five choice task, object recognition, elevated plus maze,
acquisition of novel task and passive avoidance (Giovanni et al.,
(1999), Behav. Brain Res. 104, 147-155). The histamine H3 receptor
antagonist GSK189254 inhibited [3H]R-.alpha.-methylhistamine ex
vivo binding in the rat cortex following oral administration to the
rat, and at certain oral doses improved performance of rats in the
following cognition paradigms: passive avoidance, water maze,
object recognition, and attentional set shift (A. D. Medhurst et
al., J. Pharmacol. Exp. Therap., 2007, 321(3), 1032-1045).
[0003] These data suggest that novel H3 antagonists and/or inverse
agonists could be useful for the treatment of cognitive impairments
in neurological diseases such as Alzheimer's disease or a related
neurodegenerative disorder.
[0004] WO 2005/040144 A1 (Glaxo Group Limited) discloses a series
of 1-benzoyl-substituted diazepanyl derivatives having affinity for
and being antagonists and/or inverse agonists of the histamine H3
receptor. Example 10 of WO 2005/040144 A1 discloses
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride:
##STR00002##
[0005] WO 2004/037801 A1 (Janssen Pharmaceutica, N.V.) discloses a
series of piperazinyl and diazepanyl benzamides and benzothiamides
with the ability to modulate the activity of the histamine
receptor, specifically the H3 receptor.
[0006] WO 2004/101546 A1 (Glaxo Group Limited) discloses a number
of (piperidine-4-carbonyl)-piperazine derivatives and
(piperidine-4-carbonyl)-[1,4]-diazepane derivatives having affinity
for and being antagonists and/or inverse agonists of the histamine
H3 receptor.
[0007] WO 03/004480 A2 (Novo Nordisk A/S and Boehringer Ingelheim
International GmbH) discloses a series of substituted piperazines
and diazapanes having binding affinity to the histamine H3
receptor.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides a compound or salt thereof,
which has affinity for, and which is an antagonist and/or inverse
agonist of, the histamine H3 receptor.
[0009] The present invention provides, in a first aspect,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine
##STR00003##
or a salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is an X-ray powder diffraction (XRPD) spectrum of
crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, expressed in terms of two-theta angles (in
degrees), and obtained with a diffractometer using copper K.alpha.
(copper K-alpha) X-radiation, with a step size of 0.01670
two-theta, a time per step of 31.75 sec, and using a sample mounted
on a silicon wafer plate.
[0011] FIG. 2 is an X-ray powder diffraction (XRPD) spectrum of
crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, expressed in terms of two-theta angles (in
degrees), and obtained with a diffractometer using copper K.alpha.
(copper K-alpha) X-radiation, with a step size of 0.01670
two-theta, a time per step of 31.75 sec, and using a sample mounted
on a silicon wafer plate.
[0012] FIG. 3 is an overlay of a portion of the XRPD spectra for
crystalline Form 1 (top) and crystalline Form 2 (bottom) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
[0013] FIG. 4 is a Fourier-Transform Infrared (FT-IR) spectrum for
crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, showing the spectral region from 4000 to 675
cm.sup.-1.
[0014] FIG. 5 is a Fourier-Transform Infrared (FT-IR) spectrum for
crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, showing the spectral region from 2000 to 675
cm.sup.-1.
[0015] FIG. 6 is a Fourier-Transform Infrared (FT-IR) spectrum for
crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, showing the spectral region from 4000 to 675
cm.sup.-1.
[0016] FIG. 7 is a Fourier-Transform Infrared (FT-IR) spectrum for
crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, showing the spectral region from 2000 to 675
cm.sup.-1.
[0017] FIG. 8 is an overlay of the FT-IR spectra for crystalline
Form 1 (top) and crystalline Form 2 (bottom) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, showing the spectral region from 2000 to 675
cm.sup.-1.
[0018] FIG. 9. is an overlay of the .sup.13C solid-state nuclear
magnetic resonance (solid-state NMR) spectra, in ppm, of
crystalline Form 1 (top) and crystalline Form 2 (bottom) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
[0019] FIG. 10 is a scheme showing how the receptor occupancy of a
test compound ("drug candidate") can be measured in vivo by the
reduction in radioligand specific binding to receptors. B.sub.A is
the concentration of available receptor sites. Notice how B.sub.A
changes between baseline, and 10 min, 2.5 hours, and 6 hours after
administration of the test compound, as a consequence of the
presence of different concentrations of the drug candidate in
tissue.
[0020] FIG. 11 is a scheme showing the pig-PET protocol to measure
the H3 receptor occupancy of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention), and
1-(isopropyl)-4-[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonylhexahydro--
1H-1,4-diazepine hydrochloride ("salt B", a comparator
compound).
[0021] FIG. 12, graph A, is a graph showing the average (mean)
plasma concentration over time of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention,
filled circles) and
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride ("salt B", a comparator compound,
filled diamonds), following 50 micrograms/kg intravenous
administration to pigs.
[0022] FIG. 12, graph B, is a graph showing the average (mean)
measured H3 receptor occupancy time course at three time points
during an in vivo pig-PET study, and the k.sub.on-k.sub.off limited
model fitted to it, for "salt A" within the present invention
(measurements as filled circles, and model fit as solid line), and
for "salt B" a comparator compound (measurements as filled
diamonds, and model fit as dashed line), following 50 micrograms/kg
intravenous administration of salt A or salt B to pigs.
[0023] FIG. 13, parts A and B, are graphs showing the average
(mean) plasma concentration over time and average (mean) H3
receptor occupancy time course respectively for "salt A" (only), as
shown in part of FIG. 12 graphs A and B.
[0024] FIG. 13, parts C and D, are graphs showing the average
(mean) plasma concentration over time and average (mean) H3
receptor occupancy time course respectively for "salt B" (only), as
shown in part of FIG. 12 graphs A and B.
[0025] FIG. 14 is a series of graphs showing individual plasma
concentration and H3 receptor occupancy time courses, for each
individual pig studied, which are the data which generated the mean
measurements shown in FIGS. 12 and 13, for
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention, left
hand graphs, n=3), and for
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride ("salt B", a comparator compound,
right hand graphs, n=3).
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides a compound or salt thereof,
which has affinity for, and which is an antagonist and/or inverse
agonist of, the histamine H3 receptor.
[0027] The present invention provides, in a first aspect,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine
##STR00004##
or a salt thereof.
[0028] In preliminary tests comprising oral administration to rats
or pigs,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine, as its hydrochloride salt, has exhibited in rats and
pigs certain time courses (decays over time) of brain histamine H3
receptor occupancy (see the Rat ex vivo binding studies and the
Pig-PET studies hereinafter), which suggest that
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof might have
certain suitable properties for human pharmaceutical use, in
particular in the treatment of cognitive impairment in humans such
as cognitive impairment in Alzheimer's disease.
[0029] In the context of this invention, reference to
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine (the "free base") or a salt thereof encompasses solvates and
hydrates of the free base or the salt thereof.
[0030] In one embodiment, the invention provides
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine (the "free base").
[0031] Because of its potential use in medicine, a salt of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine is preferably a pharmaceutically acceptable salt thereof, in
particular a pharmaceutically acceptable acid addition salt
thereof.
[0032] Pharmaceutically acceptable acid addition salts of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine include hydrobromide (e.g. monohydrobromide), hydrochloride
(e.g. monohydrochloride), sulfate, nitrate, phosphate, succinate,
maleate, formate, acetate, propionate, fumarate, citrate, tartrate,
lactate, benzoate, salicylate, glutamate, aspartate,
p-toluenesulfonate, benzenesulfonate, methanesulfonate,
ethanesulfonate, naphthalenesulfonate (e.g. 2-naphthalenesulfonate)
or hexanoate salts. Such salts can generally be formed by mixing
with the appropriate acid, optionally in a suitable solvent such as
an organic solvent, to give the salt, which can be isolated, for
example by crystallisation and filtration, usually followed by
drying.
[0033] The invention includes within its scope all possible
stoichiometric and non-stoichiometric forms of the salts of the
compound of the invention including hydrates and solvates.
[0034] In one preferred embodiment, the compound or salt is in the
form of a hydrochloride salt,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, typically the monohydrochloride salt.
[0035]
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine hydrochloride, e.g. monohydrochloride, can be a solid
form, particularly a crystalline form, more particularly
crystalline Form 1 or crystalline Form 2.
[0036] The invention therefore also provides crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, (hereinafter
"crystalline Form 1").
[0037] Crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by an X-ray powder diffraction (XRPD) spectrum having five or more,
e.g. eight or more, e.g. all, of the following peaks defined as
degrees two-theta angles obtained with a diffractometer using
copper K.alpha. (copper K-alpha) X-radiation: [0038] 6.4.+-.0.1,
12.7.+-.0.1, 15.4.+-.0.1, 15.7.+-.0.1, 17.1.+-.0.1, 19.1.+-.0.1,
19.7.+-.0.1, 21.9.+-.0.1, 25.5.+-.0.1, 27.0.+-.0.1, and 28.2.+-.0.1
degrees two-theta; [0039] provided that the X-ray powder
diffraction spectrum has the following two peaks: [0040]
15.7.+-.0.1 and 25.5.+-.0.1 degrees two-theta.
[0041] Alternatively or additionally, crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by an X-ray powder diffraction (XRPD) spectrum substantially the
same as that shown in FIG. 1, expressed in terms of two-theta
angles (in degrees) and obtained with a diffractometer using copper
K.alpha. (copper K-alpha) X-radiation.
[0042] In one embodiment, crystalline Form 1, characterised by the
XRPD spectrum peaks defined herein and/or characterised by an XRPD
spectrum substantially the same as that shown in FIG. 1, can be
additionally characterised as having been obtained with a
diffractometer using a step size of 0.0167.degree. two-theta or
less, and/or a time per step of 31.75 sec or more, and/or using a
sample mounted on a silicon wafer plate.
[0043] Alternatively or additionally, crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by a solid-form Fourier-Transform Infrared (FT-IR) spectrum
substantially the same as that shown in FIG. 5. FIG. 5 shows the
FT-IR spectrum of crystalline Form 1 in the spectral region from
2000 to 675 cm.sup.-1. The FT-IR spectrum can e.g. be measured
using a Nicolet Avatar 360 FT-IR spectrometer, and/or can e.g. be
as measured at 4 cm.sup.-1 or 2 cm.sup.-1 resolution. A variation
can be allowed for each peak of about +2 cm.sup.-1.
[0044] Alternatively or additionally, crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by a .sup.13C solid-state nuclear magnetic resonance (solid-state
NMR) spectrum having the following chemical shifts for the
resonances: 18.5.+-.0.3, 30.4.+-.0.3, 31.8.+-.0.3, 37.6.+-.0.3,
45.8.+-.0.3, 49.4.+-.0.3, 52.3.+-.0.3, 59.2.+-.0.3, 63.6.+-.0.3,
68.4.+-.0.3, 110.3.+-.0.3, 118.8.+-.0.3, 128.4.+-.0.3,
131.2.+-.0.3, 133.9.+-.0.3, 159.1.+-.0.3, and 167.6.+-.0.3 ppm.
This solid-state NMR spectrum can for example be obtained at a
frequency of 90.55 MHz for .sup.13C observation, e.g. using a 4-mm
Bruker HFX MAS (magic-angle spinning) probe at a temperature of
296K, and/or e.g. using a spinning speed of 8 kHz. Data can e.g. be
acquired using a cross polarisation sequence with side-band
suppression. A relaxation delay of 10 seconds can be used during
scanning.
[0045] In one embodiment, the hydrochloride salt of the invention
is substantially (e.g. 60% or more or 70% or more or 80% or more by
weight or molarity) in the form of crystalline Form 1 in terms of
crystal form purity.
[0046] The invention also provides crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, (hereinafter
"crystalline Form 2").
[0047] Without being bound by theory, crystalline Form 2 appears to
be more thermodynamically stable than crystalline Form 1, which may
give certain advantages in relation to storage, formulation and/or
use.
[0048] Crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by an X-ray powder diffraction (XRPD) spectrum having five or more,
e.g. eight or more, e.g. all, of the following peaks defined as
degrees two-theta angles obtained with a diffractometer using
copper K.alpha. (copper K-alpha) X-radiation: [0049] 6.4.+-.0.1,
12.8.+-.0.1, 15.4.+-.0.1, 19.2.+-.0.1, 19.7.+-.0.1, 20.0.+-.0.1,
21.8.+-.0.1, 21.9.+-.0.1, 23.5.+-.0.1, 24.65.+-.0.1 (or
24.7.+-.0.1), 25.8.+-.0.1, and 27.0.+-.0.1 degrees two-theta;
[0050] provided that the X-ray powder diffraction spectrum has the
following two peaks: [0051] 20.0.+-.0.1 degrees two-theta, [0052]
and either 24.65.+-.0.1 or 24.7.+-.0.1 degrees two-theta.
[0053] Alternatively or additionally, crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by an X-ray powder diffraction (XRPD) spectrum substantially the
same as that shown in FIG. 2, expressed in terms of two-theta
angles (in degrees) and obtained with a diffractometer using copper
K.alpha. (copper K-alpha) X-radiation.
[0054] In one embodiment, crystalline Form 2, characterised by the
XRPD spectrum peaks defined herein and/or characterised by an XRPD
spectrum substantially the same as that shown in FIG. 2, can be
additionally characterised as having been obtained with a
diffractometer using a step size of 0.0167.degree. two-theta or
less, and/or a time per step of 31.75 sec or more, and/or using a
sample mounted on a silicon wafer plate.
[0055] Alternatively or additionally, crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by a solid-form Fourier-Transform Infrared (FT-IR) spectrum
substantially the same as that shown in FIG. 7. FIG. 7 shows the
FT-IR spectrum of crystalline Form 2 in the spectral region from
2000 to 675 cm.sup.-1. The FT-IR spectrum can e.g. be measured
using a Nicolet Avatar 360 FT-IR spectrometer, and/or can e.g. be
as measured at 4 cm.sup.-1 or 2 cm.sup.-1 resolution. A variation
can be allowed for each peak of .+-.2 cm.sup.-1 such as .+-.1
cm.sup.-1.
[0056] Alternatively or additionally, crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. monohydrochloride, can be characterised
by a .sup.13C solid-state nuclear magnetic resonance (solid-state
NMR) spectrum having the following chemical shifts for the
resonances: 18.8.+-.0.3, 19.5.+-.0.3, 32.4.+-.0.3, 37.5.+-.0.3,
45.7.+-.0.3, 49.3.+-.0.3, 52.7.+-.0.3, 59.1.+-.0.3, 66.3.+-.0.3,
71.1.+-.0.3, 109.4.+-.0.3, 119.6.+-.0.3, 128.4.+-.0.3,
131.3.+-.0.3, 134.3.+-.0.3, 158.7.+-.0.3, and 167.8.+-.0.3 ppm.
This solid-state NMR spectrum can for example be obtained at a
frequency of 90.55 MHz for .sup.13C observation, e.g. using a 4-mm
Bruker HFX MAS (magic-angle spinning) probe at a temperature of
296K, and/or e.g. using a spinning speed of 8 kHz. Data can e.g. be
acquired using a cross polarisation sequence with side-band
suppression. A relaxation delay of 10 seconds can be used during
scanning.
[0057] The hydrochloride salt of the invention can suitably be
substantially (e.g. 70% or more or 80% or more or 90% or more or
95% or more by weight or molarity) in the form of crystalline Form
2 in terms of crystal form purity.
Synthetic Processes
[0058] The present invention also provides a process for the
preparation of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}p-
iperazine or a salt (e.g. pharmaceutically acceptable salt)
thereof, which process comprises: [0059] a) reacting
4-(tetrahydro-2H-pyran-4-yloxy)benzoyl chloride with 1-isopropyl
piperazine; or [0060] b) reacting
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid, or a non-acid-chloride
derivative thereof in which the carboxylic acid group has been
activated, with 1-isopropyl piperazine; [0061] and optionally
preparing a salt (e.g. pharmaceutically acceptable salt) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine.
[0062] Process (a) typically comprises the use of amide formation
conditions in the presence of a suitable base such as triethylamine
or a solid supported base (e.g. diethylaminomethylpolystyrene), in
an appropriate solvent e.g. a non-aqueous organic solvent such as
dichloromethane, at an appropriate temperature, for example from
about -10.degree. C. to about 40.degree. C., such as room
temperature. In a particular embodiment of process (a), a catalytic
amount of N,N-dimethylformamide (DMF) is added to catalyse the
reaction.
[0063] In the synthetic processes, room temperature (ambient
temperature) is usually 12-35.degree. C., for example 18-30.degree.
C. or 18-25.degree. C., such as about 22.degree. C.
[0064] Process (b) typically comprises activation of
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with a coupling
reagent, e.g. in a suitable solvent e.g. a polar aprotic organic
solvent, such as N,N-dimethylformamide, dimethylsulfoxide,
acetonitrile or propionitrile, followed by reaction with
1-isopropyl piperazine.
[0065] In one embodiment, the coupling reagent is an organic
di-substituted carbodiimide, such as
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
or dicyclohexylcarbodiimide (DCC), in which case: the reaction can
optionally be carried out in the presence of 1-hydroxybenzotriazole
(HOBT), and/or the reaction solvent can for example be
N,N-dimethylformamide, and/or the reaction temperature can e.g. be
from about 0.degree. C. to about 40.degree. C., such as room
temperature.
[0066] In process (b), in one embodiment, the coupling reagent is
carbonyl diimidazole, pivaloyl chloride (trimethylacetyl chloride)
or 2-propane phosphonic acid anhydride. However, for activation of
the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with a coupling
reagent, it is preferable, especially for processes carried out on
a medium or large scale, that the coupling reagent is carbonyl
diimidazole (CDI). On a medium or large scale, the use of carbonyl
diimidazole as coupling reagent is thought to give better yields
and/or a cleaner reaction, compared to the use of pivaloyl chloride
(trimethylacetyl chloride) or 2-propane phosphonic acid anhydride
as a coupling reagent.
[0067] In process (b), more particularly, the activation of the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with the carbonyl
diimidazole coupling reagent, and the subsequent reaction with
1-isopropyl piperazine, are both carried out in a reaction solvent
comprising (or, in one particular embodiment, consisting
essentially of) acetonitrile and/or propionitrile, more preferably
acetonitrile.
[0068] When carbonyl diimidazole (CDI) is used as coupling reagent
for activation of the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid,
followed by reaction with the 1-isopropyl piperazine, then the
reaction conditions can in particular be as follows, independently
and/or in any combination: [0069] the carbonyl diimidazole is
typically present in 0.5 to 1.5 mole equivalents (with reference to
the number of moles of the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic
acid), suitably 0.9 to 1.1 mole equivalents, preferably 1.0 to 1.1
mole equivalents, e.g. 1.1 mole equivalents; and/or [0070] the
1-isopropyl piperazine is typically present in 0.5 to 1.5 mole
equivalents (with reference to the number of moles of the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid), suitably 1.0 to 1.25
mole equivalents, preferably 1.1 to 1.2 mole equivalents, e.g. 1.15
or 1.2 mole equivalents; and/or [0071] the reaction (the activation
of the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with CDI, or the
subsequent reaction with 1-isopropyl piperazine, or both) is
typically carried out in a suitable organic solvent such as a polar
aprotic organic solvent, for example a solvent comprising (e.g.
consisting essentially of) acetonitrile, propionitrile,
dimethylsulfoxide, N,N-dimethylformamide (DMF),
N-methylpyrrolidinone (NMP), and/or 1,4-dioxane; preferably the
reaction solvent comprises (e.g. consists essentially of)
acetonitrile and/or propionitrile, more preferably acetonitrile;
and/or [0072] the reaction solvent is typically dry, although a
small percentage of water in the reaction solvent can sometimes be
tolerated; and/or [0073] when the reaction solvent is acetonitrile
or propionitrile, the temperature of the reaction (for either the
activation of the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with
the carbonyl diimidazole, or for the subsequent reaction with the
1-isopropyl piperazine, or for both) can for example be from about
0.degree. C. to the boiling point or reflux temperature of the
solvent. The temperature of the activation reaction can e.g. be in
the range of about 20 to about 40.degree. C. (e.g. about 30.degree.
C.), e.g. followed by reaction with the 1-isopropyl piperazine at a
temperature of from about 20.degree. C. to the boiling point or
reflux temperature of the reaction solvent (e.g. from about 40 to
about 60.degree. C., e.g. about 50.degree. C.); this low activation
reaction temperature can help to maximise yield due to decreased
CDI decomposition, but any surviving excess CDI after the
activation reaction is thought to then be more likely to react with
the later-added 1-isopropyl piperazine to form a
difficult-to-remove impurity which is thought to be
1-isopropyl-piperazin-4-yl-C(O)-imidazole or a salt thereof. Hence,
it is currently thought preferable to activate the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with the carbonyl
diimidazole at a temperature of from about 50.degree. C. to the
boiling point/reflux temperature of the reaction solvent or from
about 60.degree. C. to the boiling/reflux temperature (e.g. about
60 to about 70.degree. C., e.g. 65 to 70.degree. C., e.g. in
acetonitrile solvent), and optionally also to have this temperature
range (from about 50.degree. C. to the boiling point/reflux
temperature, e.g. about 60 to about 70.degree. C.) as the
temperature for the subsequent reaction with the 1-isopropyl
piperazine, e.g. in order to potentially reduce this impurity;
and/or [0074] the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid and
the carbonyl diimidazole are typically reacted together (e.g. with
stirring) for at least 0.5 hours, suitably for at least 2 hours,
e.g. for 0.5 to 5 hours such as 0.5 to 3 hours, e.g. for 2 to 5
hours or 2 to 3 hours, before the 1-isopropyl piperazine is mixed
with the activated 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid;
and/or [0075] the product of activation of the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid by the carbonyl
diimidazole, and the 1-isopropyl piperazine, are typically reacted
together (e.g. with stirring) for at least 0.5 hours (e.g. 0.5 to
24 hours), suitably for at least 1 hour (e.g. 1 to 3 hours), such
as for at least 2 hours (e.g. 2 to 3 hours). General Process for
Preparation of a Salt (e.g. Hydrochloride Salt) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine:
[0076] To prepare, crystallise and isolate a salt (e.g.
hydrochloride) of the compound of the invention, in one embodiment,
at the end of the reaction in which the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid has been activated by a
coupling reagent (e.g. carbonyl diimidazole) followed by reaction
of the activated acid with the 1-isopropyl piperazine, in a
reaction solvent such as acetonitrile or propionitrile, the
following process can be carried out: [0077] the volume of reaction
solvent (e.g. acetonitrile or propionitrile) is reduced under
reduced pressure, e.g. to about 2-5 volumes e.g. about 3 volumes
(e.g. of acetonitrile or propionitrile), and [0078] then a solution
of the appropriate salt-forming acid (e.g. HCl) in a suitable
solvent (e.g. a crystallisation solvent as defined below e.g.
isopropanol) (e.g. to prepare the hydrochloride salt: this can be
HCl in isopropanol, e.g. 5 to 6 N HCl in isopropanol, e.g. ca. 0.9
volumes thereof) is added to the reaction mixture; with preferably
the appropriate salt-forming acid e.g. HCl being added in an amount
of 0.5 to 1.3 mole equivalents such as 0.85 to 1.05 mole
equivalents e.g. 1.0 mole equivalents with respect to the molar
amount of the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid used),
and [0079] preferably, before or after or at the same time as the
addition of the appropriate salt-forming acid, a crystallisation
solvent is added (wherein the crystallisation solvent can e.g.
comprise or be: an alcohol being a C.sub.1-3 alcohol or n-butanol
(including mixtures of alcohols), for example isopropanol,
n-propanol, n-butanol, ethanol, or methanol; a mixture of water and
an alcohol being a C.sub.1-3 alcohol or n-butanol, for example
isopropanol:water, ethanol:water, or methanol:water; isopropyl
acetate; ethyl acetate; a C.sub.3-6 ketone such as methyl isobutyl
ketone (MIBK), methyl ethyl ketone, or acetone; acetonitrile; or
dichloromethane; and wherein suitably the crystallisation solvent
comprises or is an alcohol being a C.sub.1-3 alcohol or n-butanol
(including mixtures of alcohols), or a mixture of water and an
alcohol being a C.sub.1-3 alcohol or n-butanol; such as preferably:
isopropanol, isopropanol:water such as ca. 2-10% e.g. ca. 2-5% e.g.
ca. 5% water in isopropanol, or ethanol:water such as ca. 1-5%
water in ethanol or industrial methylated spirits) (e.g. 6 to 20
volumes, e.g. ca. 12 volumes of the crystallisation solvent can
e.g. be added), and
[0080] the solvent-containing mixture comprising the salt (e.g. HCl
salt) product is at, or is heated to, a temperature of about
50.degree. C. to the boiling point or reflux temperature of the
solvent (e.g. about 50-75.degree. C., e.g. about 60-70.degree. C.,
e.g. about 60-65.degree. C.), and
[0081] the salt (e.g. hydrochloride salt) of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine is allowed or caused to crystallise or recrystallise from
the hot mixture (e.g. by cooling the hot mixture), and [0082] the
crystalline salt (e.g. hydrochloride salt) of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine is isolated from the solvent (e.g. by filtration), and is
usually dried (e.g. by drying under reduced pressure at about
40-60.degree. C. e.g. about 50.degree. C., or e.g. by drying at
room temperature e.g. under suction or a stream of gas such as air
or nitrogen).
[0083] For the hydrochloride (e.g. monohydrochloride) salt of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine, without being bound be theory, it appears, from preliminary
experiments on the above-mentioned types of HCl-salt-formations and
crystallisations, that crystalline Form 1 often tends to be the
kinetic product of the salt-forming process, and that crystalline
Form 2 is the thermodynamic product (i.e. thermodynamically more
stable product). Crystalline Form 1 (or a predominance of
crystalline Form 1) is often formed initially depending on the
conditions, but, depending on the conditions (such as the type of
solvent and the solubility of crystalline Form 1 in it, and/or the
temperature and/or temperature time course, and/or the contact time
of the crystalline Form 1 with the solvent), the crystalline Form 1
can then often convert to crystalline Form 2 to a greater or lesser
extent when in contact with a suitable solvent i.e. a solvent
suitable for converting crystalline Form 1 to crystalline Form
2.
[0084] The invention, in one aspect, therefore provides a process
for preparing crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (e.g. monohydrochloride), comprising:
converting crystalline Form 1 to crystalline Form 2 by contacting
crystalline Form 1 with a suitable conversion solvent, for example
by slurrying crystalline Form 1 in the suitable conversion solvent,
for a sufficient time and/or at a sufficiently-high temperature to
effect conversion of crystalline Form 1 to crystalline Form 2. The
suitable conversion solvent typically comprises (e.g. consists
essentially of) a C.sub.1-3 alcohol or n-butanol or a mixture of
water and an alcohol being a C.sub.1-3 alcohol or n-butanol. The
time and/or temperature required to effect conversion can e.g.
depend on the solvent and the solubility of Form 1 in it. In the
conversion process, the crystalline product of the process is
suitably substantially (e.g. 70% or more or 80% or more or 90% or
more or 95% or more by weight or molarity) in the form of
crystalline Form 2 in terms of crystal form purity.
[0085] In order to prepare crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, [0086] the above-mentioned HCl-salt-formation
process suitably uses a crystallisation solvent such as solvent
comprising a C.sub.1-3 alcohol or n-butanol or a mixture of water
and an alcohol being a C.sub.1-3 alcohol or n-butanol, in
particular isopropanol, n-propanol, n-butanol, ethanol or a mixture
thereof, and [0087] after formation, the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (e.g. monohydrochloride) is allowed or caused
to crystallise from the hot mixture by cooling the hot mixture,
e.g. to about 0 to about 25.degree. C., over a period of 2-4 hours
or less (e.g. over 1.5-3 hours or less, e.g. over ca. 1.5 hours)
measured from the onset of crystallisation, and [0088] the
crystalline hydrochloride salt of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbon-
yl}piperazine comprising crystalline Form 1 is isolated from the
solvent after no more than 6 hours (preferably no more than 4
hours, e.g. no more than 2-3 hours, e.g. ca. 1.5 hours), of contact
time with the solvent measured from the onset of
crystallisation.
[0089] In order to prepare crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, e.g. by conversion of Form 1 into Form 2,
[0090] (i) the above-mentioned HCl-salt-formation process uses a
crystallisation solvent (e.g. an alcohol being a C.sub.1-3 alcohol
or n-butanol (including mixtures of alcohols), or a mixture of
water and an alcohol being a C.sub.1-3 alcohol or n-butanol; in
particular isopropanol, n-propanol, n-butanol, ethanol, methanol,
isopropanol:water, ethanol:water, or methanol:water; preferably
isopropanol, or isopropanol:water such as ca. 2-10% e.g. ca. 2-5%
e.g. ca. 5% water in isopropanol, or ethanol:water such as ca. 1-5%
water in ethanol or industrial methylated spirits); and [0091]
(ii)(a) in the event that the crystallisation solvent is methanol,
isopropanol:water, n-propanol:water, n-butanol:water,
ethanol:water, or methanol:water, then after formation the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}-
piperazine hydrochloride (e.g. monohydrochloride) is allowed or
caused to crystallise or recrystallise from the hot mixture (e.g.
at about 50-75.degree. C., e.g. ca. 60-70.degree. C., e.g. ca.
60-65.degree. C.), by cooling the hot mixture, e.g. to about 0 to
about 30.degree. C., over a period of 4 hours or more (e.g. 5-6
hours or more) measured from the onset of crystallisation
(preferably using gradual cooling); and optionally, before cooling,
by ageing the mixture of the salt and the solvent (e.g. slurry) at
a temperature of about 50.degree. C. to the boiling point or reflux
temperature of the solvent (e.g. at about 50-75.degree. C., e.g.
ca. 60-70.degree. C., e.g. ca. 60-65.degree. C.) for 0.5 hours or
more (e.g. for 1 hour or more, e.g. 1-3 hours, or for 2 hours or
more e.g. ca. 2 hours) measured from the onset of crystallisation;
or [0092] (ii)(b) in the event that the crystallisation solvent is
ethanol, isopropanol, n-propanol or n-butanol, then after formation
the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (e.g. monohydrochloride) is allowed or caused
to crystallise or recrystallise from the hot mixture, and the
mixture of the salt and the solvent (e.g. a slurry) is aged at a
temperature of about 50.degree. C. to the boiling point or reflux
temperature of the solvent (e.g. at about 50-75.degree. C., e.g.
ca. 60-75.degree. C., e.g. ca. 60-70.degree. C.) for 6 hours or
more (e.g. for 10 hours or more, e.g. for 15 hours or more, e.g.
for about 18-24 hours) measured from the onset of crystallisation;
and then the hot mixture is cooled, e.g. to about 0 to about
30.degree. C., e.g. using gradual cooling; and [0093] (iii) the
crystalline hydrochloride salt of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine, substantially (e.g. 80% or more or 90% or more or 95% or
more by weight or molarity) in the form of crystalline Form 2, is
isolated from the solvent (e.g. by filtration), and is usually
dried (e.g. by drying under reduced pressure at about 40-60.degree.
C. e.g. about 50.degree. C., or e.g. by drying at room temperature
e.g. under suction or a stream of gas such as air or nitrogen).
Synthetic Processes, Continued
[0094] 4-(Tetrahydro-2H-pyran-4-yloxy)benzoyl chloride (V) or
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid (IV) may be prepared in
accordance with the following scheme wherein P represents a
suitable protecting group, such as C.sub.1-6 straight-chain alkyl
(e.g. methyl, ethyl, n-propyl or n-butyl) or isopropyl or isobutyl,
or benzyl; such as methyl or ethyl; in particular methyl.
##STR00005##
[0095] Step (i) typically comprises the use of a phosphine such as
triphenylphosphine in a suitable solvent such as tetrahydrofuran,
toluene and/or xylene (wherein "xylene" can be o-xylene, m-xylene,
p-xylene, or a mixture of xylenes), followed by the addition (e.g.
slow and/or dropwise addition) of an azodicarboxylate such as
diethyl azodicarboxylate or diisopropyl azodicarboxylate, at a
suitable temperature, for example, from room temperature to about
80.degree. C., e.g. room temperature. Reaction times (including any
azodicarboxylate addition time) can be e.g. from 0.5 to 72 hours.
When using tetrahydrofuran as reaction solvent, room temperature
can be used, and the reaction time is for example from 3 to 72
hours. When the reaction solvent comprises or consists essentially
of toluene and/or xylene, in particular toluene, a reaction
temperature of about 40 to about 80.degree. C., e.g. about 40 to
about 70.degree. C., e.g. about 55.degree. C., can be used; and/or
a reaction time (including any azodicarboxylate addition time) of
about 0.5 to 6 hours, e.g. 0.5 to 3 hours, e.g. 1-2 hours, can be
used. In a particular embodiment, the reaction solvent comprises or
consists essentially of toluene and/or xylene, preferably toluene,
and reaction step (i) uses triphenylphosphine and diisopropyl
azodicarboxylate; in which case suitably the heated (e.g. ca.
40-70.degree. C.) reaction mixture can be cooled (e.g. to -10 to
25.degree. C., e.g. to ca. 0-5.degree. C., provided that it is not
cooled to the melting point of the solvent or below), e.g. for 0.5
to 2 hours, and then the solid byproduct formed is removed e.g. by
filtration. The use of toluene as reaction solvent helps to
crystallise out the byproduct adduct of triphenylphosphine oxide
and diisopropyl hydrazinedicarboxylate from the solution
(especially when the reaction mixture is seeded with this adduct
e.g. after cooling), which helps to reduce the levels of
triphenylphosphine oxide in the crude product (III).
[0096] When using toluene and/or xylene as a solvent in reaction
step (i), in one embodiment, the reaction product compound of
formula (III) is not isolated. Optionally, in this embodiment, the
toluene and/or xylene solution of the compound of formula (III) is
used directly in the subsequent reaction (deprotection e.g.
hydrolysis) step (ii), in particular when C.sub.1-6 straight-chain
alkyl (e.g. methyl, ethyl, n-propyl or n-butyl) or isopropyl or
isobutyl and the subsequent step (ii) comprises alkaline (e.g. NaOH
or KOH) hydrolysis of the ester.
[0097] According to a further aspect of the invention, there is
provided a process for preparing a compound of formula (III)
##STR00006##
wherein P represents a protecting group such as C.sub.1-6
straight-chain alkyl (e.g. methyl, ethyl, n-propyl or n-butyl) or
isopropyl or isobutyl, or benzyl (in particular C.sub.1-6
straight-chain alkyl or isopropyl, e.g. methyl or ethyl), wherein
the process comprises: [0098] (i) reacting the compound of formula
(I)
##STR00007##
[0098] wherein P represents the protecting group as defined for the
compound of formula (III), with 4-hydroxytetrahydropyran of formula
(II) or a derivative thereof in which its OH group is activated;
[0099] wherein the reaction step (i) is carried out in a reaction
solvent comprising or consisting essentially of toluene and/or
xylene (in particular toluene). [0100] "Xylene" can be o-xylene,
m-xylene, p-xylene, or a mixture of xylenes. [0101] In this process
aspect of the invention using a step (i) reaction solvent
comprising toluene and/or xylene, the reaction conditions for step
(i) can in particular be as described herein for step (i) for the
general synthetic processes. In particular, reaction step (i) can
use triphenylphosphine and diisopropyl azodicarboxylate. For a step
(i) reaction solvent comprising toluene and/or xylene, in
particular toluene, a reaction temperature of about 40 to about
80.degree. C., e.g. about 40 to about 70.degree. C., e.g. about
55.degree. C., can be used; and/or a reaction time (including any
azodicarboxylate addition time) of about 0.5 to 6 hours, e.g. 0.5
to 3 hours, e.g. 1-2 hours, can be used. In a particular
embodiment, when the step (i) reaction solvent comprises toluene
and/or xylene, preferably toluene, and reaction step (i) uses
triphenylphosphine and diisopropyl azodicarboxylate, the heated
(e.g. ca. 40-70.degree. C.) reaction mixture can be cooled (e.g. to
-10 to 25.degree. C., e.g. to ca. 0-5.degree. C., provided that it
is not cooled to the melting point of the solvent or below), e.g.
for 0.5 to 2 hours, and then the solid byproduct (the adduct of
triphenylphosphine oxide and diisopropyl hydrazinedicarboxylate)
formed is removed e.g. by filtration. In particular, the reaction
mixture can be seeded with the adduct of the adduct of
triphenylphosphine oxide and diisopropyl hydrazinedicarboxylate,
e.g. after cooling the reaction mixture. The use of toluene as
reaction solvent helps to crystallise out the byproduct adduct of
triphenylphosphine oxide and diisopropyl hydrazinedicarboxylate
from the solution (especially when the reaction mixture is seeded
with this adduct e.g. after cooling), which helps to reduce the
levels of triphenylphosphine oxide in the crude product (III).
[0102] For the aspect of the invention being a process for
preparing a compound of formula (III), using a step (i) reaction
solvent comprising toluene and/or xylene; there is also provided a
process for preparing a compound of formula (IV), which is
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid, comprising: [0103]
performing step (i) using a reaction solvent comprising or
consisting essentially of toluene and/or xylene, and then, [0104]
(ii) converting the compound of formula (III) to the compound of
formula (IV); e.g. by hydrolysing the ester within the compound of
formula (III) when P represents C.sub.1-6 straight-chain alkyl
(e.g. methyl, ethyl, n-propyl or n-butyl) or isopropyl or isobutyl
(in particular methyl or ethyl), e.g. under alkaline conditions
(e.g. using sodium hydroxide or potassium hydroxide, e.g. aqueous),
or e.g. by hydrogenation when P represents benzyl.
[0105] In this process aspect of the invention, there is also
provided a process for the preparation of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a salt thereof, which process comprises: [0106]
performing step (i) using a reaction solvent comprising or
consisting essentially of toluene and/or xylene; then [0107] (ii)
converting the compound of formula (III) to the compound of formula
(IV), which is 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid, e.g. as
described herein; and then [0108] either a) converting the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid to
4-(tetrahydro-2H-pyran-4-yloxy)benzoyl chloride and then reacting
this with 1-isopropyl piperazine; [0109] or b) reacting the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with 1-isopropyl
piperazine, or converting the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid to a non-acid-chloride
derivative thereof in which the carboxylic acid group has been
activated, and then reacting this with 1-isopropyl piperazine;
[0110] and optionally preparing a salt (e.g. pharmaceutically
acceptable salt) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carb-
onyl}piperazine. Steps a) and/or b) can e.g. be as described
herein.
[0111] Step (ii) is a deprotection reaction. When P represents
C.sub.1-6 straight-chain alkyl (e.g. methyl, ethyl, n-propyl or
n-butyl) or isopropyl or isobutyl (in particular methyl or ethyl),
the reaction typically comprises treatment with a suitable alkali
(e.g. aqueous), such as sodium hydroxide or potassium hydroxide
(e.g. aqueous sodium hydroxide or potassium hydroxide solution), in
a suitable solvent such as methanol (e.g. when P=Me), or ethanol
(e.g. when P=Et), or toluene and/or xylene; e.g. at a suitable
temperature, such as 70-100.degree. C. (e.g. 95.degree. C. or
80.degree. C.) and/or at reflux, e.g. for 1 to 24 hours such as 2-6
hours or 2-3 hours; typically until the hydrolysis is substantially
complete. In a particular embodiment, when the step (ii) reaction
solvent is toluene and/or xylene, and the reaction comprises
treatment with a suitable aqueous alkali such as aqueous sodium
hydroxide or potassium hydroxide solution, the reaction comprises
efficient (e.g. vigorous) stirring or mixing.
[0112] In a particular embodiment, a toluene and/or xylene solution
containing the compound of formula (III), produced in step (i), is
used directly in the subsequent hydrolysis step (ii), i.e. without
isolation of the compound of formula (III), in particular when the
subsequent step (ii) comprises alkaline (e.g. NaOH or KOH)
hydrolysis of the ester. The reaction conditions for steps (i)
and/or (ii) can in particular be as described herein, e.g. reaction
step (i) can use triphenylphosphine and diisopropyl
azodicarboxylate.
[0113] When P represents benzyl, the deprotection reaction (ii) can
comprise hydrogenation.
[0114] According to another aspect of the invention, there is
provided a process for preparing a compound of formula (IV)
##STR00008##
which is 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid, wherein the
process comprises: (i) reacting the compound of formula (I),
wherein P represents C.sub.1-6 straight-chain alkyl (e.g. methyl,
ethyl, n-propyl or n-butyl) or isopropyl or isobutyl (in particular
methyl or ethyl), with 4-hydroxytetrahydropyran of formula (II) or
a derivative thereof in which its OH group is activated, to prepare
a compound of formula (III), wherein P has the same definition as
in the compound of formula (I), and (ii) hydrolysing the ester
within the compound of formula (III), e.g. under alkaline
conditions (e.g. using sodium hydroxide or potassium hydroxide,
e.g. aqueous), to form the compound of formula (IV), wherein the
reaction steps (i) and (ii) are both carried out in a reaction
solvent comprising or consisting essentially of toluene and/or
xylene (in particular toluene). "Xylene" can be o-xylene, m-xylene,
p-xylene, or a mixture of xylenes.
[0115] In a particular embodiment of this process aspect of the
invention, the toluene and/or xylene solution of the compound of
formula (III) produced in step (i) is used directly in the
subsequent hydrolysis step (ii), i.e. without isolation of the
compound of formula (III), in particular when the subsequent step
(ii) comprises alkaline (e.g. NaOH or KOH) hydrolysis of the ester.
The reaction conditions for steps (i) and/or (ii) can in particular
be as described herein, e.g. reaction step (i) can use
triphenylphosphine and diisopropyl azodicarboxylate. In a
particular embodiment of this process aspect of the invention,
there is also provided a process for the preparation of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a salt thereof, which process comprises: [0116]
performing steps (i) and (ii), wherein the reaction steps (i) and
(ii) are both carried out in a reaction solvent comprising or
consisting essentially of toluene and/or xylene, e.g. as described
hereinabove; and then [0117] either a) converting the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid to
4-(tetrahydro-2H-pyran-4-yloxy)benzoyl chloride and then reacting
this with 1-isopropyl piperazine; [0118] or b) reacting the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid with 1-isopropyl
piperazine, or converting the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid to a non-acid-chloride
derivative thereof in which the carboxylic acid group has been
activated, and then reacting this with 1-isopropyl piperazine;
[0119] and optionally preparing a salt (e.g. pharmaceutically
acceptable salt) of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carb-
onyl}piperazine.
[0120] Steps a) and/or b) can e.g. be as described herein.
[0121] Step (iii) typically comprises treatment with suitable
chlorinating agent such as oxalyl chloride or thionyl chloride,
e.g. in a suitable solvent (e.g. non-aqueous organic solvent) such
as dichloromethane or ethyl acetate (suitably dichloromethane), or
(for thionyl chloride) without solvent, at a suitable temperature,
such as room temperature.
[0122] Compounds of formula (I) are either commercially available
(for example, methyl 4-hydroxybenzoate is available from Aldrich),
or they may be prepared from commercially available compounds using
standard methodology. 1-Isopropyl piperazine and
4-hydroxytetrahydropyran are commercially available, e.g. from
Aldrich.
Uses
[0123]
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine or a pharmaceutically acceptable salt thereof has
affinity for and is an antagonist and/or inverse agonist of the
histamine H3 receptor, and for example has potentially useful
therapeutic properties.
[0124] More particularly,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof has potential
use in the treatment or prophylaxis (in particular treatment) of:
[0125] neurological diseases (e.g. in a mammal such as a human);
such as: cognitive impairment(s), cognitive deficit, Alzheimer's
disease, dementia (such as Lewy body dementia or vascular
dementia), age-related memory dysfunction, epilepsy, migraine,
Parkinson's disease, multiple sclerosis (including fatigue),
fatigue (in particular fatigue in multiple sclerosis, fatigue in
depression, fatigue in cancer or in cancer chemotherapy, or chronic
fatigue syndrome) such as cognitive and/or psychological fatigue,
stroke, pain of neuropathic origin (such as neuralgias e.g.
post-herpetic neuralgia, neuritis, neuropathic back pain,
allodynia, etc.), inflammatory pain (in particular chronic
inflammatory pain such as pain in osteoarthritis or pain in
rheumatoid arthritis or inflammatory back pain; or acute
inflammatory pain), or sleep disorders (such as hypersomnolence,
excessive daytime sleepiness, narcolepsy, or sleep deficits
associated with Parkinson's disease, restless leg's syndrome and/or
fatigue, especially in multiple sclerosis); wherein cognitive
impairment(s) can be cognitive impairment(s) in: Alzheimer's
disease, dementia (e.g. Lewy body dementia or vascular dementia),
mild cognitive impairment, or a related neurodegenerative disorder;
or cognitive impairment(s) in Parkinson's disease, or cognitive
impairment(s) in schizophrenia; or [0126] psychiatric disorders
(e.g. in a mammal such as a human); such as: psychotic disorders
(such as schizophrenia or bipolar disorder), attention deficit
hyperactivity disorder (ADHD), depression (including major
depressive disorder), anxiety or addiction; or [0127] other
diseases (e.g. in a mammal such as a human); such as obesity or a
gastro-intestinal disorder.
[0128] Thus the invention also provides
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof, for use as a
therapeutic substance in the treatment or prophylaxis (in
particular treatment) of any of the above disorders; in particular
cognitive impairment(s), e.g. cognitive impairment(s) in a disease
such as Alzheimer's disease, dementia (e.g. Lewy body dementia or
vascular dementia), mild cognitive impairment, or a related
neurodegenerative disorder, or cognitive impairment(s) in
Parkinson's disease, or cognitive impairment(s) in schizophrenia;
or fatigue; or a sleep disorder.
[0129] The invention also provides
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof, for use as a
therapeutic substance in the treatment or prophylaxis (in
particular treatment) of any of the above disorders, in particular
cognitive impairment(s), fatigue or a sleep disorder, in a mammal
(e.g. rodent such as rat, or pig or human) such as a human.
[0130] The invention further provides a method of treatment or
prophylaxis (in particular treatment) of any of the above
disorders, e.g. a neurological disease, in a mammal such as a
human, which comprises administering to the sufferer (the mammal in
need thereof) a therapeutically effective amount of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof.
[0131] The invention further provides a method of treatment or
prophylaxis (in particular treatment) of: [0132] cognitive
impairment(s); e.g. cognitive impairment(s) in a disease such as
Alzheimer's disease, dementia (e.g. Lewy body dementia or vascular
dementia), mild cognitive impairment, or a related
neurodegenerative disorder, or cognitive impairment(s) in
Parkinson's disease, or cognitive impairment(s) in schizophrenia;
[0133] or fatigue (in particular fatigue in multiple sclerosis,
fatigue in depression, fatigue in cancer or in cancer chemotherapy,
or chronic fatigue syndrome); [0134] or a sleep disorder (such as
hypersomnolence, excessive daytime sleepiness, narcolepsy, or sleep
deficits associated with Parkinson's disease, restless leg's
syndrome and/or fatigue); in a mammal (e.g. rodent such as rat, or
pig or human), such as a human, in need thereof, which comprises
administering to the mammal a therapeutically effective amount of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof.
[0135] In another aspect, the invention provides the use of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof in the
manufacture of a medicament for use in the treatment or prophylaxis
(in particular treatment) of any of the above disorders, in
particular a neurological disease and/or in particular cognitive
impairment(s), fatigue or a sleep disorder.
[0136] In particular, the invention provides the use of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof in the
manufacture of a medicament for use in the treatment or prophylaxis
(in particular treatment) of any of the above disorders, in
particular a neurological disease and/or in particular cognitive
impairment(s), fatigue or a sleep disorder, in a mammal (e.g.
rodent such as rat, or pig or human) such as a human.
[0137] More particularly, the invention provides the use of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof in the
manufacture of a medicament: [0138] for use in the treatment or
prophylaxis (in particular treatment) of cognitive impairment(s);
e.g. cognitive impairment(s) in a disease such as Alzheimer's
disease, dementia (e.g. Lewy body dementia or vascular dementia),
mild cognitive impairment, or a related neurodegenerative disorder,
or cognitive impairment(s) in Parkinson's disease, or cognitive
impairment(s) in schizophrenia; [0139] or for use in the treatment
or prophylaxis (in particular treatment) of fatigue (in particular
fatigue in multiple sclerosis, fatigue in depression, fatigue in
cancer or in cancer chemotherapy, or chronic fatigue syndrome);
[0140] or for use in the treatment or prophylaxis (in particular
treatment) of a sleep disorder (such as hypersomnolence, excessive
daytime sleepiness, narcolepsy, or sleep deficits associated with
Parkinson's disease, restless leg's syndrome and/or fatigue);
[0141] e.g. in a mammal (e.g. rodent such as rat, or pig or human)
such as a human.
Pharmaceutical Compositions, Doses, and Dosage Regimens
[0142] When used in therapy, the compound of the invention or a
pharmaceutically acceptable salt thereof is usually formulated in a
pharmaceutical composition. Such compositions can be prepared using
various procedures.
[0143] Thus, the present invention further provides a
pharmaceutical composition for use in the treatment or prophylaxis
(e.g. treatment) of any the above disorders, e.g. a neurological
disease and/or cognitive impairment(s), fatigue or a sleep
disorder, which comprises
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier.
[0144] The present invention further provides a pharmaceutical
composition which comprises
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier.
[0145] A pharmaceutical composition of the invention, which may be
prepared by admixture, for example at ambient temperature and/or
atmospheric pressure, is usually adapted for oral, parenteral or
rectal administration and, as such, may be in the form of a tablet,
a capsule, an oral liquid preparation, a powder, granules, a
lozenge, a reconstitutable powder, an injectable or infusible
solution or suspension, or a suppository.
[0146] An orally administrable pharmaceutical composition, such as
a tablet or capsule, is generally preferred.
[0147] A tablet or capsule for oral administration may be in unit
dose form, and may contain one or more excipients, such as a
binding agent (e.g. povidone, hydroxypropylmethylcellulose or
starch), a filler (e.g. mannitol or lactose), microcrystalline
cellulose, a lubricant e.g. tabletting lubricant (e.g. magnesium
stearate, calcium stearate or stearic acid), a disintegrant e.g.
tablet disintegrant, and/or a pharmaceutically acceptable wetting
agent. A tablet may be coated, e.g. film-coated, e.g. according to
a tablet coating method. A capsule can be a hard or soft capsule,
containing the compound or salt of the invention and the one or
more excipients e.g. in powder or pellet form.
[0148] An oral liquid preparation may be in the form of, for
example, an aqueous or oily suspension, a solution, an emulsion, a
syrup or elixir, or may be in the form of a dry product for
reconstitution with water or other suitable vehicle before use.
Such liquid preparations may contain additive(s) such as suspending
agents, emulsifying agents, non-aqueous vehicles (which may include
edible oils), and/or preservatives, and/or, if desired, flavourings
and/or colorants.
[0149] For parenteral administration, fluid unit dosage forms are
typically prepared utilising the compound of the invention or
pharmaceutically acceptable salt thereof and a sterile vehicle. The
compound or salt, e.g. depending on the vehicle and/or
concentration used, can be either suspended or dissolved in the
vehicle. In preparing solutions, the compound or salt can be
dissolved for injection and filter sterilised before filling into a
suitable vial or ampoule and sealing. Adjuvant(s) such as a local
anaesthetic, preservative(s) and/or buffering agent(s) can be
dissolved in the vehicle. To enhance the stability, the composition
can be frozen after filling into the vial and the water removed
under vacuum. Parenteral suspensions are typically prepared in
substantially the same manner, except that the compound or salt is
suspended in the vehicle instead of being dissolved, and
sterilisation typically is not accomplished by filtration. In one
embodiment, the compound or salt is sterilised, e.g. by exposure to
ethylene oxide, before suspension in a sterile vehicle. In one
embodiment, a surfactant or wetting agent is included in the
composition to facilitate uniform distribution of the compound or
salt.
[0150] The pharmaceutical composition may contain from 0.1% to 99%
by weight of the composition of the active material (i.e. the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof), in
particular from 1 to 60% by weight or from 10 to 60% by weight of
the composition of the active material. For example, this may vary
depending on the route of administration and/or the composition's
intended use(s).
[0151] The total amount of the pharmaceutically acceptable carrier
in the pharmaceutical composition can for example vary depending on
the pharmaceutical composition and/or its intended use and/or the
route of administration. In one embodiment, the total amount of the
pharmaceutically acceptable carrier in the pharmaceutical
composition (e.g. or i.e. the total amount of the one or more
excipients present therein, such as one or more of the excipient
types mentioned herein), is in the range of from 1% to 99.9% by
weight of the composition, for example from 40% to 99% by weight
such as from 40% to 90% by weight of the composition. Additionally
or alternatively, in one embodiment, for a composition (e.g.
composition for oral administration, e.g. tablet or capsule) in
unit dose form, the total amount of the pharmaceutically acceptable
carrier in the unit dose form pharmaceutical composition (e.g. or
i.e. the total amount of the one or more excipients present
therein) can be from 10 mg to 2000 mg, for example from 20 mg to
1500 mg such as from 100 mg to about 1000 mg.
[0152] The dose, e.g. oral dose, of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof, e.g. used
in the treatment or prophylaxis of the aforementioned
disorders/diseases /conditions and/or comprised in a pharmaceutical
composition, can for example vary in the usual way with the
seriousness of the disorders, the weight of the sufferer, and/or
other similar factors. However, as a general guide, in one
embodiment a suitable unit dose (e.g. oral unit dose) of 0.02 to
1000 mg or 0.05 to 1000 mg, for example 0.1 to 200 mg such as 1.0
to 200 mg, and/or for example 0.02 to 200 mg or 0.05 to 200 mg such
as 0.05 to 45 mg or 0.1 to 45 mg, of the compound or the
pharmaceutically acceptable salt of the invention (measured as the
"free base" compound), may be used, for example in a pharmaceutical
composition (e.g. in an oral pharmaceutical composition, and/or
e.g. in a unit dose form) of the invention. In one embodiment, such
a unit dose is for administration once a day, e.g. orally and/or to
a mammal such as a human; alternatively such a unit dose may be for
administration more than once a day, for example two or three times
a day, e.g. orally and/or to a mammal such as a human. Such therapy
may extend for a number of weeks, months or years.
One Embodiment of a Pharmaceutical Dosage Form
[0153] In one embodiment, the invention provides a pharmaceutical
dosage form (e.g. orally-administrable dosage form) comprising:
a)
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}p-
iperazine or a pharmaceutically acceptable salt thereof (e.g.
hydrochloride salt); b) optionally a stabiliser, which reduces
degradation of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the salt thereof in the dosage form when compared to a
dosage form lacking said stabiliser; and c) a pharmaceutically
acceptable excipient.
[0154] In one embodiment of this embodiment, the pharmaceutical
dosage form (e.g. orally-administrable dosage form) comprises a
carrier tablet, which carrier tablet is at least partially (e.g.
partially or wholly, e.g. only partially) covered by a film
comprising:
a)
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}p-
iperazine or a pharmaceutically acceptable salt thereof (e.g.
hydrochloride salt), and b) optionally a stabiliser that reduces
degradation of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the salt thereof in the dosage form, when compared to a
dosage form lacking said stabiliser.
[0155] In this embodiment, the term "carrier tablet" refers to a
pharmaceutically acceptable tablet substantially free of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof.
[0156] In one embodiment, the carrier tablet is formed by direct
compression technology.
[0157] In one embodiment, the carrier tablet comprises: [0158] a
diluent (e.g. in an amount of from 50 to 100%, e.g. from 80 to 98%,
by weight of the carrier tablet), such as microcrystalline
cellulose e.g. microcrystalline cellulose having a nominal mean
particle size of about 50 microns (e.g. Avicel PH-101 .TM.) or 100
microns (e.g. Avicel PH-102 .TM.), or lactose, or mannitol; and/or
[0159] a binding agent (e.g. in an amount of from 0.5 to 15%, e.g.
from 2 to 10%, by weight of the carrier tablet), such as starch
(e.g. corn starch, potato starch or pre-gelatinised starch),
polyvinylpyrrolidone (povidone), or hydroxypropylmethylcellulose;
and/or [0160] a lubricant (e.g. in an amount of from 0.1 to 5%,
e.g. from 0.3 to 3%, by weight of the carrier tablet), such as
magnesium stearate, calcium stearate or stearic acid.
[0161] In one embodiment, the carrier tablet is a tablet comprising
microcrystalline cellulose (e.g. Avicel PH-102 .TM.) (e.g. at 90%
by weight of the carrier tablet), pregelatinized starch (e.g.
Starch 1500 .TM.) (e.g. at 9% by weight of the carrier tablet), and
magnesium stearate (e.g. at 1% by weight of the carrier
tablet).
[0162] The above-mentioned pharmaceutical dosage form comprising
the optional stabiliser, can for example contain from 0.02 mg to 2
mg (e.g. 0.05 mg to 1 mg) of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof (e.g.
hydrochloride salt), when measured as the amount of free base
present.
[0163] In one embodiment, the dosage form does not comprise a
stabiliser.
[0164] In a particular embodiment, the dosage form does comprise a
stabiliser.
[0165] In the above-mentioned pharmaceutical dosage form(s), the
stabiliser can typically comprise citric acid or a salt thereof,
malic acid or a salt thereof, ascorbic acid or a salt thereof,
sodium bicarbonate, optionally butylated hydroxyanisole and/or
butylated hydroxytoluene. In one particular embodiment, the
stabiliser comprises optionally butylated hydroxyanisole, such as
butylated hydroxyanisole, or, more particularly, citric acid or a
salt thereof, such as citric acid. In the dosage form, the molar
ratio of the citric acid or the salt thereof (measured as citric
acid) to the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the salt thereof (measured as the free base) can for
example be from 550:1 to 1:2, such as from 500:1 to 2:3.
[0166] In one embodiment, the carrier tablet is coated with a
carrier tablet film coat, e.g. to a 2-6% weight gain, for example
using a coating not soluble in water (or not soluble in methanol or
ethanol), for example using ethylcellulose (e.g. Surelease.TM.) or
methacrylic acid copolymer (e.g. Eudragit.TM.) as the carrier
tablet film coat. The film covering the carrier tablet and
comprising the compound or salt of the invention and the optional
stabiliser is typically outside of and/or coated onto the carrier
tablet film coat.
[0167] In one embodiment, in the above-mentioned dosage form
comprising the optional stabiliser, there is substantially no
absorption of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof by the
carrier tablet.
[0168] The carrier tablet can in particular have one or more
recesses or depressions. In a particular embodiment, the film
(which at least partially, e.g. only partially, covers the carrier
tablet and which comprises the compound or salt of the invention
and the optional stabiliser) is substantially present within the
one or more recesses or depressions of the carrier tablet.
[0169] In one embodiment, the above-mentioned dosage form (e.g.
comprising a carrier tablet at least partially covered by a film
comprising the compound or salt of the invention and an optional
stabiliser) is further coated with an outer film coating.
[0170] In another aspect of this embodiment, the invention provides
a method for preparing the above-mentioned pharmaceutical dosage
form (comprising a carrier tablet at least partially covered by a
film comprising the compound or salt of the invention and an
optional stabiliser), wherein the method comprises dispensing a
solution or suspension of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof and a
stabiliser (e.g. citric acid or a salt thereof, e.g. present at
about 2-3% w/v) onto a carrier tablet. Any solvent may be used
provided that the stabiliser and any other excipients present in
the film (which is to at least partially cover the carrier tablet)
are soluble in the solvent. The solvent is typically volatile. The
solvent should be pharmaceutically acceptable in any (residual)
quantities in which it appears in the finished dosage form. The
solvent used in the method can include water, and/or an organic
solvent such as methanol, ethanol, acetone, acetic acid and/or
dichloromethane. A mixture of solvents (e.g. water-ethanol) may be
used. In one embodiment, the solvent is methanol.
[0171] In the method for preparing the dosage form, the carrier
tablet and the dispensed solution or suspension may be heated (e.g.
in a forced air oven) to evaporate excess liquid and may result in
the formation of a film upon at least a part of the surface of the
carrier tablet. The dosage form may then optionally be film coated,
e.g. according to known methods, to create an outer film
coating.
[0172] The carrier tablet used in the method for preparing the
dosage form may have a recess or depression that provides a basin
for the solution or suspension of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof and the
stabiliser to land after being dispensed. Typically, biconcave
tablets having recesses on two faces of the tablet are
employed.
[0173] In one optional embodiment, the above-mentioned dosage form
comprising a carrier tablet of the present invention is produced by
an apparatus described in WO 2005/123569, and more particularly is
produced by an apparatus containing a dispensing module for
accurately dispensing a predetermined amount of the solution or
suspension of the
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or the pharmaceutically acceptable salt thereof and the
stabiliser onto the carrier tablets. The apparatus may also have a
holding member for holding the carrier tablets, which may move
continually along the apparatus as the dispensing module dispenses
the solution or suspension onto each of the carrier tablets. The
apparatus may also have a drying system that dries or evaporates
solvent from the solution or suspension deposited on each of the
carrier tablets. The holding member may move continually along the
apparatus as the drying system dries the dosage on each of the
carrier tablets. The drying system may dry the dosage form by use
of heated air, or by infrared or microwave heating. The apparatus
may also have a coating system that applies an outer film coating
over the dosage form.
Combinations
[0174]
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine or a pharmaceutically acceptable salt thereof may be
used in combination with other therapeutic agents. When
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof is intended
for use in the treatment or prophylaxis (in particular treatment)
of Alzheimer's disease, dementia, mild cognitive impairment, or a
related neurodegenerative disorder, in particular in the treatment
or prophylaxis (in particular treatment) of cognitive impairment(s)
in Alzheimer's disease, dementia (e.g. Lewy body dementia or
vascular dementia), mild cognitive impairment, or a related
neurodegenerative disorder, e.g. in a mammal such as a human, it
may be used in combination with medicaments claimed to be useful as
either disease modifying or symptomatic treatments of Alzheimer's
disease, dementia, mild cognitive impairment, or a related
neurodegenerative disorder. Suitable examples of such other
therapeutic agents may be symptomatic agents, for example those
known to modify cholinergic transmission such as M1 muscarinic
receptor agonists or allosteric modulators, M2 muscarinic
antagonists, acetylcholinesterase inhibitors (such as
tetrahydroaminoacridine, donepezil e.g. donepezil hydrochloride,
rivastigmine, or galantamine e.g. galantamine hydrobromide),
nicotinic receptor agonists or allosteric modulators (such as
.alpha.7 agonists or allosteric modulators or .alpha.4.beta.2
agonists or allosteric modulators), PPAR agonists (such as
PPAR.gamma. agonists), 5-HT.sub.4 receptor partial agonists,
5-HT.sub.6 receptor antagonists [such as
3-(phenylsulfonyl)-8-(1-piperazinyl)quinoline or a salt thereof,
e.g. disclosed in WO03/080580 as the hydrochloride salt (Example 2)
and as the free base (Example 16)], 5HT1A receptor antagonists,
NMDA receptor antagonists or modulators (such as memantine e.g.
memantine hydrochloride), or disease modifying agents such as
.beta. or .gamma.-secretase inhibitors.
[0175] When
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof is intended
for use in the treatment of narcolepsy, it may be used in
combination with medicaments claimed to be useful as treatments for
narcolepsy. Suitable examples of such other therapeutic agents
include modafinil, armodafinil and monoamine uptake blockers.
[0176] When
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof is intended
for use in the treatment of schizophrenia, it may be used in
combination with medicaments claimed to be useful as treatments of
schizophrenia including i) antipsychotics including typical
antipsychotics (for example chlorpromazine, thioridazine,
mesoridazine, fluphenazine, perphenazine, prochlorperazine,
trifluoperazine, thiothixine, haloperidol, molindone or loxapine),
atypical antipsychotics (for example clozapine, olanzapine,
risperidone, quetiapine, ziprasidone, amisulpride or aripiprazole),
glycine transporter 1 inhibitors and metabotropic receptor ligands;
ii) drugs for extrapyramidal side effects, for example
anticholinergics (such as benztropine, biperiden, procyclidine, or
trihexyphenidyl) and dopaminergics (such as amantadine); iii)
antidepressants including serotonin reuptake inhibitors (such as
citalopram, escitalopram, fluoxetine, paroxetine, dapoxetine or
sertraline), dual serotonin/noradrenaline reuptake inhibitors (such
as venlafaxine, duloxetine or milnacipran), noradrenaline reuptake
inhibitors (such as reboxetine), tricyclic antidepressants (such as
amitriptyline, clomipramine, imipramine, maprotiline, nortriptyline
or trimipramine), monoamine oxidase inhibitors (such as
isocarboxazide, moclobemide, phenelzine or tranylcypromine), and
others (such as buproprion, mianserin, mirtazepine, nefazodone or
trazodone); iv) anxiolytics including benzodiazepines such as
alprazolam or lorazepam; and v) cognitive enhancers for example
cholinesterase inhibitors (such as tacrine, donepezil, rivastigmine
or galantamine).
[0177] When the compound of the invention or a pharmaceutically
acceptable salt thereof is used in combination with other
therapeutic agents, the compounds may be administered either
sequentially or simultaneously by any convenient route.
[0178] The invention thus provides, in a further aspect, a
combination comprising
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine or a pharmaceutically acceptable salt thereof together with
a further therapeutic agent or agents.
[0179] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation and
thus pharmaceutical formulations comprising a combination as
defined above together with a pharmaceutically acceptable carrier
or excipient comprise a further aspect of the invention. The
individual components of such combinations may be administered
either sequentially or simultaneously in separate or combined
pharmaceutical formulations.
[0180] When the compound of the invention or a pharmaceutically
acceptable salt thereof is used in combination with a second
therapeutic agent active against the same disease state the dose of
each compound may differ from that when the compound is used
alone.
EXPERIMENTAL SECTION
[0181] The following Descriptions and Examples illustrate the
compound of the invention, its hydrochloride salt, preparations
thereof, and intermediates ("Descriptions") of use in the
preparation thereof.
Description 1
Methyl 4-(tetrahydro-2H-pyran-4-yloxy)benzoate (D1)
##STR00009##
[0182] Method A
[0183] A stirred solution of methyl 4-hydroxybenzoate (1.49 g, 9.8
mmol, e.g. available from Aldrich), 4-hydroxytetrahydropyran (1 g,
9.8 mmol, e.g. available from Aldrich) and triphenylphosphine (3.85
g, 14.7 mmol) in tetrahydrofuran (60 ml) at room temperature was
treated dropwise with diethyl azodicarboxylate (2.32 ml, 1.5 mole
equivalents). The reaction was stirred overnight. The solvent was
evaporated off, and the crude product was re-dissolved in ethyl
acetate (50 ml), washed with 5% sodium carbonate solution
(2.times.40 ml), water (3.times.40 ml), brine (40 ml), dried
(magnesium sulfate) and evaporated. The crude product was loaded
onto a silica column and was subjected to flash chromatography,
eluting with a 10% to 30% gradient of ethyl acetate in light
petroleum (40.degree.-60.degree. C.) to yield the title compound
(2.12 g).
Method B
[0184] Diisopropyl azodicarboxylate (7.8 ml, 39.6 mmol, 2 mole
equivalents) was added to a stirred solution of methyl
4-hydroxybenzoate (3.0 g, 19.7 mmol), 4-hydroxytetrahydropyran (2.8
ml, 28.2 mmol, ca. 1.4 mole equivalents) and triphenylphosphine
(10.3 g, 39.3 mmol, 2 mole equivalents) in tetrahydrofuran (120
ml). The reaction mixture was stirred at room temperature for 68
hours. The solvent was then removed in vacuo and the crude residue
was dissolved in ethyl acetate (100 ml). The organic solution was
then washed with a saturated aqueous solution of sodium hydrogen
carbonate (40 ml), water (40 ml) and brine (40 ml). The organic
phase was dried (phase-separating column) and concentrated. The
crude residue was purified by silica gel chromatography eluting
with a gradient of from 0% to 30% ethyl acetate in hexane to yield
the title compound as a pale yellow oil (3.14 g) (.sup.1H NMR in
CDCl.sub.3 suggested the product to be contaminated with a
substantial amount of diisopropyl azodicarboxylate residue).
Method C
[0185] Diisopropyl azodicarboxylate (2.89 ml, 14.68 mmol, 1.5 mole
equivalents) was added to a stirred solution of methyl
4-hydroxybenzoate (1.49 g, 9.80 mmol, 1.0 mole equivalents),
4-hydroxytetrahydropyran (1.00 g, 9.79 mmol, 1.0 mole equivalents)
and triphenylphosphine (3.85 g, 14.68 mmol, 1.5 mole equivalents)
in tetrahydrofuran (60 ml) at 0.degree. C. The reaction mixture was
warmed to room temperature and stirred for 21 hours.
4-Hydroxytetrahydropyran (0.3 ml), triphenylphosphine (1.11 g) and
diisopropyl azodicarboxylate (0.9 ml) were added sequentially to
the reaction mixture at room temperature and stirring was continued
for 2 hours. The solvent was removed in vacuo and the residue was
dissolved in ethyl acetate (50 ml). The organic phase was washed
with a saturated aqueous solution of sodium hydrogen carbonate (30
ml), water (30 ml) and brine (30 ml). The organic phase was dried
(phase-separating column) and concentrated. The crude residue was
purified by silica gel chromatography eluting with a gradient of
from 0% to 30% ethyl acetate in hexane to yield the title compound
as a pale yellow oil (2.19 g) (contaminated with trace amount of
diisopropyl azodicarboxylate residue).
Method D
[0186] To a stirred solution of methyl 4-hydroxybenzoate (30 g, 197
mmol, 1.0 mole equivalents), tetrahydro-4-pyranol (24 ml, 251 mmol,
1.3 mole equivalents) and triphenylphosphine (78 g, 297 mmol, 1.5
mole equivalents) in tetrahydrofuran (600 ml) at room temperature,
was added diisopropyl azodicarboxylate (58 ml, 298 mmol, 1.5 mole
equivalents) over a period of 15 minutes. The reaction mixture was
stirred for 24 hours at room temperature. Another portion of
diisopropyl azodicarboxylate (5 ml) and tetrahydro-4-pyranol (2 ml)
was added and the reaction mixture was stirred for another 2 hours
at room temperature. The reaction mixture was then quenched by the
addition of a saturated aqueous solution of sodium hydrogen
carbonate (500 ml) and ethyl acetate (500 ml). The organic phase
was washed with water (2.times.250 ml), dried (magnesium sulfate),
and was then concentrated in vacuo to give the crude product,
methyl 4-(tetrahydro-2H-pyran-4-yloxy)benzoate, as a thick yellow
oil (182 g).
Description 2
4-(Tetrahydro-2H-pyran-4-yloxy)benzoic acid (D2)
##STR00010##
[0187] Method A
[0188] A stirred solution of methyl
4-(tetrahydro-2H-pyran-4-yloxy)benzoate (2.12 g; prepared as
described in Description 1 Method A) in methanol (20 ml) at room
temperature was treated with 1 M sodium hydroxide solution (17.9
ml, about 2 mole equivalents). The reaction mixture was refluxed
for 4 hours and then cooled to room temperature. The methanol was
evaporated off, and the aqueous mixture was washed with
dichloromethane (3.times.10 ml) and then acidified to pH 2 with
concentrated hydrochloric acid. The aqueous layer was extracted
with ether (100 ml) and the ether solution was washed with water
(3.times.50 ml), brine (50 ml), dried (magnesium sulfate) and
evaporated to yield the title compound (1.27 g, contains ca. 15% of
4-hydroxybenzoic acid by NMR).
[0189] Alternatively, after the reaction is cooled to room
temperature, the methanol is evaporated off, the aqueous mixture is
washed with dichloromethane (3.times.10 ml) and then is acidified
to pH 2 with concentrated hydrochloric acid, and then the product
is filtered off directly.
Method B
[0190] To a stirred solution of methyl
4-(tetrahydro-2H-pyran-4-yloxy)benzoate (3.14 g, prepared as
described in Description 1 Method B) in methanol (28 ml), was added
a 1.0 M aqueous solution of sodium hydroxide (28 ml, 28 mmol). The
reaction mixture was heated at 95.degree. C. for 18 hours and was
then cooled to room temperature. The methanol was removed in vacuo
and the remaining aqueous phase was washed with dichloromethane
(2.times.30 ml). The aqueous layer was then acidified to pH 2 using
a 1.0 M aqueous solution of HCl. The resulting white precipitate
was filtered off and dried (vacuum oven at 40.degree. C. for 3
hours) to yield the title compound (1.52 g).
Method C
[0191] To a solution of methyl
4-(tetrahydro-2H-pyran-4-yloxy)benzoate (2.19 g, prepared as
described in Description 1 Method C) in methanol (20 ml) at room
temperature, was added a 1.0 M aqueous solution of sodium hydroxide
(19 ml, 19 mmol). The reaction mixture was then heated at reflux
for 15 hours. The reaction mixture was then cooled to room
temperature and the methanol was removed in vacuo. The resulting
aqueous phase was washed with dichloromethane (2.times.15 ml) and
was then acidified to pH 2 using a 1.0 M aqueous solution of HCl.
The resulting white precipitate was filtered off and dried (vacuum
oven at 40.degree. C. for 2 hours) to yield the title compound
(1.32 g).
Method D
[0192] To a stirred solution of crude methyl
4-(tetrahydro-2H-pyran-4-yloxy)benzoate (182 g, prepared as
described in Description 1 Method D) in methanol (800 ml), was
added 1.0 M aqueous solution of sodium hydroxide (900 ml, 900
mmol). The reaction mixture was heated at 50.degree. C. for 4 hours
and was then cooled to room temperature. Methanol was removed in
vacuo and the remaining aqueous phase was washed with ethyl acetate
(2.times.400 ml). The aqueous phase was then acidified with 2.5 M
aqueous HCl. The resultant white solid was filtered off to give the
title compound (36.5 g).
Description 3
4-(Tetrahydro-2H-pyran-4-yloxy)benzoic acid (D3)
##STR00011##
[0193] Short Summary Process Description
[0194] All weights, volumes ("vol") and equivalents are relative to
methyl 4-hydroxybenzoate.
[0195] A solution of methyl 4-hydroxybenzoate (1 wt, 1 mole
equivalent), triphenyl phosphine (2.6 wt, 1.5 mole equivalents),
4-hydroxytetrahydropyran (0.75 vol, 1.2 mole equivalents) in
toluene (3.5 vol) under nitrogen is heated to 55.degree. C. and
diisopropyl azodicarboxylate (1.95 vol, 1.5 mole equivalents) is
added dropwise over 60 minutes, maintaining the contents at
60.+-.2.degree. C. Following the addition, the reaction is stirred
for 30 minutes, and then cooled to 0-5.degree. C. The batch is then
seeded with pre-prepared triphenylphosphine oxide-diisopropyl
hydrazinedicarboxylate adduct, and then allowed to stir for a
further 1 hour before filtering. The wet cake is washed with
toluene (2.times.1 vol), and the combined mother liquors are
transferred into a clean vessel. The toluene solution is washed
with 2M sodium hydroxide solution (5 vol) at 0-5.degree. C., and
then 3M sodium hydroxide solution (5 vol) is added and the reaction
is heated to 80.degree. C. The reaction is stirred for at least 2.5
hours, until HPLC shows no starting material. The mixture is then
cooled to 50.degree. C. and toluene (5 vol) and water (5 vol) are
added. The layers are allowed to separate, and the aqueous layer is
washed with toluene (10 vol) and then acidified to pH1 with 2.5M
HCl solution (7.5 vol). The resultant slurry is filtered and the
wet cake is washed with water (2.times.2 vol). The title product is
dried at about 50.degree. C. in a vacuum oven with a nitrogen bleed
to constant probe temperature.
Detailed Process Description
[0196] 1. Added methyl 4-hydroxybenzoate (1 wt, 482.3 g, available
from Fluka) to Vessel 1. [0197] 2. Added 4-hydroxytetrahydropyran
(0.75 vol, 362 mL, 1.2 mole equivalents, available from
Sigma-Aldrich) to Vessel 1. [0198] 3. Added triphenyl phosphine
(2.6 wt, 1253 g, 1.5 mole equivalents) to Vessel 1. [0199] 4.
Purged Vessel 1 with Nitrogen. [0200] 5. Added toluene (3.5 vol,
1690 mL) to Vessel 1. [0201] 6. Heated contents to 55.degree. C.
with stirring. [0202] 7. Added diisopropyl azodicarboxylate (DIAD,
1.95 vol, 940 mL, 1.5 mole equivalents, available from Aldrich) to
Vessel 1 via a peristaltic pump over 2 hours maintaining the
contents temperature at 60.+-.2.degree. C. [0203] 8. Stirred
contents of Vessel 1 at 60.+-.2.degree. C. for 50 min. [0204] 9.
Sampled reaction mixture for HPLC analysis. [0205] 10. Cooled
contents of Vessel 1 to 0-5.degree. C. [0206] 11. Seeded batch with
triphenylphosphine oxide-diisopropyl hydrazinedicarboxylate adduct
(0.001 wt, 0.482 g) [0207] 12. Stirred contents of Vessel 1 for 81
min. [0208] 13. Filtered off byproduct over 5 min on a PTFE
minifilter fitted with Whatman No. 113 wet strengthened filter
paper (rough side up). Used 20 L Buchner flask as receiver. [0209]
14. Washed wet cake with toluene (2.times.ca. 1 vol, 2.times.490
mL) and sucked cake free of solvent. [0210] 15. Combined filtrate
and cake washes were transferred to Vessel 2 via PTFE suck-up line.
[0211] 16. Cooled Vessel 2 contents to 0-5.degree. C. [0212] 17.
Added 2M sodium hydroxide solution (5 vol, 2400 mL) to Vessel 2.
[0213] 18. Stirred contents of Vessel 2 at 0-5.degree. C. for 5 min
before allowing the layers to settle. [0214] 19. Ran the lower
aqueous layer into a labelled Schott bottle. [0215] 20. Added 3M
sodium hydroxide solution (5 vol, 2410 mL) to Vessel 2. [0216] 21.
Heated contents to 80.degree. C., and stirred for 2 hours 45 min.
[0217] 22. Monitored reaction by HPLC until hydrolysis is complete.
[0218] 23. Cooled contents of Vessel 2 to 50.degree. C., and then
added toluene (5 vol, 2410 mL) to Vessel 2. [0219] 24. Added water
(5 vol, 2410 mL) to Vessel 2. [0220] 25. Stirred contents at
50.+-.5.degree. C. for 5 min before allowing the layers to settle.
[0221] 26. Ran the lower aqueous layer into a labelled Schott
bottle for retention. [0222] 27. Ran the upper organic layer into a
labelled Schott bottle for disposal. [0223] 28. Recharged aqueous
layer from labelled Schott bottle to Vessel 2. [0224] 29. Added
toluene (ca. 10 vol, 4900 mL) to Vessel 2. [0225] 30. Stirred
contents at 50.+-.5.degree. C. for 5 min before allowing the layers
to settle. [0226] 31. Ran the lower aqueous layer into a labelled
Schott bottle for retention. [0227] 32. Ran the upper organic layer
into a labelled Schott bottle for disposal. [0228] 33. Recharged
aqueous layer to Vessel 2. [0229] 34. Added 2.5M aqueous
hydrochloric acid (7.5 vol, 3620 mL) via peristaltic pump until pH
1 is achieved. [0230] 35. Stirred the resulting slurry for 15 min.
[0231] 36. Filtered off product on a PTFE mini filter fitted with
Whatman 113 wet strengthened filter paper (rough side up). 10 min
filtration time. [0232] 37. Washed filter cake with water
(2.times.2 vol, 970 mL). [0233] 38. Dried the solid product in
polythene lined steel trays covered with a muslin cloth, under
vacuum and a nitrogen bleed, at 50.degree. C. overnight and at
75.degree. C. for a further 3 days. [0234] 39. Title product was
obtained as an off-white solid (568.9 g).
Analytical Data
[0235] .sup.1H NMR (400 MHz, DMSO-d.sub.6) delta ppm 1.55-1.64 (m,
2H) 1.95-2.03 (m, 2H), 3.49 (ddd, J=11.74, 9.41, 2.57 Hz, 2H), 3.85
(ddd, J=11.80, 4.34, 4.16 Hz, 2H), 4.69 (ddd, J=8.56, 4.65, 4.40
Hz, 1H) 7.03-7.09 (m, 2H) 7.84-7.90 (m, 2H), and 12.31 (br-s,
1H).
[0236] In an alternative to the above process, in step 37, the
filter cake can be washed with toluene, instead of water, before
the 50-75.degree. C. vacuum drying of step 38.
Example 1
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}piper-
azine Hydrochloride (E1)
##STR00012##
[0237] Method A
[0238] A solution of 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid
(200 mg; prepared as described in Description 2 Method A) in
dichloromethane (10 ml) at room temperature was treated with oxalyl
chloride (0.16 ml, about 2 mole equivalents) and 1 drop of 10%
N,N-dimethylformamide in dichloromethane. After 30 minutes, the
solvent was evaporated off and the product was re-evaporated from
dichloromethane (.times.2). The acid chloride product in
dichloromethane was added to a stirred mixture of
diethylaminomethylpolystyrene (844 mg, 3.2 mmol/g, 2.7 mmol, about
3 mole equivalents) and 1-isopropyl piperazine (115 mg, 0.90 mmol,
e.g. available from Aldrich) in dichloromethane (10 ml) at room
temperature. After 30 minutes, the mixture was loaded directly onto
a silica flash column and eluted with a gradient of from 2% to 6%
methanol (containing 10% 0.88 ammonia) in dichloromethane. The
product-containing fractions were evaporated. The product was
redissolved in dichloromethane and treated with excess 4M HCl in
dioxane. The solvent was evaporated and the product was
crystallised with acetone, filtered off, washed with acetone and
dried to yield the title compound (247 mg).
[0239] .sup.1H NMR (D.sub.6-DMSO, 250 MHz) .delta.10.9 to 11.0 (1H,
br), 7.43 (2H, d, J=8.7 Hz), 7.04 (2H, d, J=8.7 Hz), 4.65 (1H, m),
4.18 (2H, br), 3.90-3.81 (2H, m), 3.57-3.41 (7H, m), 3.12-3.00 (2H,
m), 2.02-1.95 (2H, m), 1.66-1.52 (2H, m), 1.28 (6H, d, J=6.6 Hz);
MS (electrospray): m/z (M+H).sup.+ 333;
C.sub.19H.sub.28N.sub.2O.sub.3 requires 332.
Method B
[0240] To a stirred solution of
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid (2.56 g, 11.5 mmol;
which may be prepared as described in Description 2 Method B and/or
Method C) in N,N-dimethylformamide (40 ml) at room temperature, was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(3.32 g, 17.3 mmol). The reaction mixture was stirred for 15
minutes, followed by the addition of 1-isopropyl piperazine (2.50
ml, 17.5 mmol). The resultant mixture was stirred for 20 hours.
Dichloromethane (10 ml) was added to the reaction mixture and the
solvent was removed in vacuo. The pale yellow oil residue was
purified by silica gel chromatography, eluting with a gradient of
from 0% to 10% of (2N ammonia/methanol) in dichloromethane, to
yield the free base as a pale yellow oil (1.80 g) (LCMS (basic):
m/z (M+H).sup.+ 333). The free base was dissolved in
dichloromethane (20 ml), followed by the addition of 4.0 M HCl
solution in dioxane (5 ml). The solvent was then removed in vacuo
to yield the corresponding hydrochloride salt,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, as an off-white solid (1.81 g); LCMS (basic):
m/z (M+H).sup.+ 333.
Method C
[0241] To a stirred solution of
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid (0.205 g, 0.92 mmol;
which may be prepared as described in Description 2 Method C) in
N,N-dimethylformamide (3.5 ml) was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.268
g, 1.39 mmol). The reaction mixture was stirred for 15 minutes at
room temperature, followed by the addition of 1-isopropyl
piperazine (0.20 ml, 1.4 mmol). The resultant mixture was stirred
for 66 hours at room temperature. Dichloromethane (5 ml) was added
to the reaction mixture and the solvent was removed in vacuo. The
pale yellow crude residue was purified by silica gel
chromatography, eluting with a gradient of from 0% to 10% of (2M
ammonia/methanol) in dichloromethane, to yield the free base as a
pale yellow oil (204 mg) (LCMS (basic): m/z (M+H).sup.+ 333). The
free base was dissolved in dichloromethane (5 ml), followed by the
addition of 4.0 M HCl solution in dioxane (1 ml). The solvent was
then removed in vacuo to yield the corresponding hydrochloride
salt,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, as an off-white solid (0.196 g); LCMS
(basic): m/z (M+H).sup.+ 333.
Method D
[0242] To a stirred solution of
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid (36.5 g, 164 mmol,
which may be prepared as described in Description 2 Method D),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (38 g,
198 mmol), 1-hydroxybenzotriazole (31 g, 203 mmol) in
N,N-dimethylformamide (500 ml) at room temperature, was added
1-isopropylpiperazine (26 ml, 182 mmol). The reaction mixture was
stirred at room temperature for 1.5 hours and was then quenched by
the addition of a saturated aqueous solution of sodium hydrogen
carbonate (500 ml) and ethyl acetate (1 litre). The aqueous layer
was extracted with ethyl acetate (400 ml) and the combined organic
extracts were washed with water (2.times.400 ml). The solvent was
removed in vacuo and the residue was taken up in dichloromethane
(150 ml), followed by the addition of a 1.0 M HCl solution in
diethyl ether (200 ml). The resultant white solid was then
separated by filtration and was washed with dichloromethane to give
the corresponding hydrochloride salt. Recrystallisation of this
material from ethanol gave the title compound,
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, as a solid (32 g).
Example 2
Crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine Hydrochloride
##STR00013##
[0243] Short Summary Process Description
[0244] All weights, volumes ("vol") and equivalents are with
respect to 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid.
[0245] 4-(Tetrahydro-2H-pyran-4-yloxy)benzoic acid (1 wt, e.g.
which may be as prepared in Description 3) and carbonyl diimidazole
(CDI) (0.8 wt, 1.1 mole equivalents) are charged to a 20 L vessel.
Acetonitrile (12 volumes) is then added, and the suspension/slurry
is warmed to 30.degree. C. and stirred for about 2 to 2.25 hours.
N-isopropylpiperazine (1-isopropylpiperazine, 0.66 wt, 1.15 mole
equivalents) is added in one charge and the resulting hazy solution
is heated to 50.degree. C. over about 15-30 minutes and then
stirred for about 2 to 2.25 hours. The reaction is monitored by
HPLC. Following completion of the reaction, the mixture is cooled
to 20.degree. C. and any insoluble matter (e.g. any inorganics
carried over in the 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid) is
removed by filtration. The clarified solution is transferred to a
10 L vessel in portions and is concentrated by distillation under
reduced pressure to approximately 3 volumes to remove acetonitrile
(e.g. using 50.degree. C. jacket temperature and 200 mbar pressure
reducing to 100 mbar pressure). Following the distillation,
propan-2-ol (6 volumes) is added and the solution is concentrated
further by distillation under reduced pressure to 5 volumes. After
further propan-2-ol (8 volumes) is added, the solution is heated to
70.degree. C. with stirring and 5-6N HCl in isopropanol (0.9
volumes) is added over at least 10 minutes. Crystallisation
generally commences during the addition. Following the addition the
resulting slurry is ramp-cooled to 20.degree. C. over 1.5 hours.
The product is filtered and the cake is washed with isopropanol (3
volumes). The solvent is sucked free from the cake for at least 2
hours. The product is dried in a vacuum oven at 50.degree. C. to
constant probe temperature over at least 22 hours to give the title
product.
Detailed Process Description
[0246] 1. Added 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid (1 wt,
1000.0 g) to Vessel 1 (20 L). [0247] 2. Added carbonyl diimidazole
(CDI) (0.8 wt, 1.1 mole equivalents, 800.0 g) (obtained from
Fluorochem) to Vessel 1. [0248] 3. Added acetonitrile (12 vol, 12
L) to Vessel 1 and started stirrer (gas evolution observed). [0249]
4. Heated contents of Vessel 1 cautiously to 30.degree. C. and then
stirred for 2 hours 10 minutes. [0250] 5. Added
N-isopropylpiperazine (1-isopropylpiperazine) (obtained from
Fluorochem) (0.66 wt, 1.15 mole equivalents, 666.4 g) to Vessel 1.
[0251] 6. Heated contents of Vessel 1 cautiously to 50.degree. C.,
over about 15-20 minutes, and then stirred for 2 hours 15 minutes.
[0252] 7. Sampled the mixture for HPLC (quench onto butylamine and
reaction deemed complete if ratio of product:butylamide is
>50:1). [0253] 8. Cooled contents of Vessel 1 to 20.degree. C.
[0254] 9. Transferred the solution to Vessel 2 via a 5 micron
Dominic filter to remove insoluble matter. [0255] 10. Rinsed Vessel
1 with acetonitrile (0.2 vol, 200 mL) and used as a line wash into
Vessel 2. [0256] 11. Concentrated the contents of Vessel 2 to 3.0
volumes via distillation under reduced pressure. Started with
50.degree. C. jacket temperature and 200 mbar vacuum, and reduce
pressure gradually to 100 mbar. [0257] 12. Vessel 1 was rinsed with
water and boiled out with methanol to clean. [0258] 13. Added
propan-2-ol (6 volumes, 6 L) to Vessel 2. [0259] 14. Concentrated
the contents of Vessel 2 to 5 volumes via distillation under
reduced pressure. [0260] 15. Added propan-2-ol (8 volumes, 8 L) to
Vessel 2. [0261] 16. Transferred reaction mixture to Vessel 1 for
crystallisation. [0262] 17. Heat the contents of Vessel 1 to
70.degree. C. with stirring. [0263] 18. Added 5-6N hydrochloric
acid in propan-2-ol (0.9 volumes, 900 mL) to Vessel 1 via
peristaltic pump fitted with silicone tubing over at least 10 mins.
[0264] 19. Ramp-cooled the contents of Vessel 1 to 20.degree. C.
over 1.5 hours. [0265] 20. Filtered off product on a PTFE mini
filter 9 fitted with Whatman No. 113 wet strengthened filter paper
(rough side up). [0266] 21. Washed the filter cake with propan-2-ol
(3 volumes, 3 L) and sucked product free of solvent. [0267] 22.
Dried the solid product in polythene lined steel trays covered with
a muslin cloth, to a constant probe temperature, at 50.degree. C.
under vacuum for about 22 hours to give the title compound as a
solid (1515.1 g).
[0268] From analysis, the crystalline Form 1 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride produced by Example 2 is believed not to be an
entirely pure crystal form, and is believed to contain very
approximately 20% of crystalline Form 2. Analyses of the
crystalline Form 1 product produced by Example 2 include the
following: [0269] X-Ray Powder Diffraction (XRPD)
[0270] XRPD data were acquired on a PANalytical X'Pert Pro powder
diffractometer, equipped with an X'Celerator detector. The
acquisition conditions were: radiation: Cu K.alpha. (copper
K-alpha), generator tension: 40 kV, generator current: 45 mA, start
angle: 2.0.degree. 2.theta. (two-theta), end angle: 40.0.degree.
2.theta., step size: 0.0167.degree. 2.theta. (two-theta). The time
per step was 31.750 s. The sample was prepared by mounting a few
milligrams of sample on a Si wafer (zero background) plates,
resulting in a thin layer of powder.
[0271] Some characteristic peak positions and calculated d-spacings
for the crystalline Form 1 product produced by Example 2 are
summarised in the following table (note: these are not the only
peaks seen). These were calculated from the raw data using
Highscore software.
TABLE-US-00001 XRPD - Crystalline Form 1 d-spacing/.ANG.
2.theta./.degree. (.ANG.ngstroms) 6.4 13.9 12.7 7.0 15.4 5.7 15.7
5.6 17.1 5.2 19.1 4.7 19.7 4.5 21.9 4.1 25.5 3.5 27.0 3.3 28.2
3.2
[0272] The XRPD spectrum for crystalline Form 1 as prepared by
Example 2 is shown in FIG. 1. An XRPD overlay spectrum is shown in
FIG. 3, comparing the XRPD peaks of crystalline Form 1 from Example
2 (top) to those of crystalline Form 2 from Example 3 (bottom), for
comparison purposes. The crystalline Form 1 XRPD peaks at
15.7.degree. 2.theta. and 25.5.degree. 2.theta. appear to be
characteristic for crystalline Form 1 in that these peaks do not
appear to be present in the XRPD spectrum of crystalline Form
2.
FT-IR (FT-Infrared) Spectrum
[0273] FT-IR spectrum was acquired over 64 scans at 4 cm.sup.-1
resolution using a Nicolet Avatar 360 FT-IR spectrometer, fitted
with a Diamond/ZnSe ATR Accessory.
[0274] The FT-IR spectrum for crystalline Form 1 as prepared by
Example 2 is shown in FIGS. 4 and 5, showing the spectral regions
from 4000 to 675 cm.sup.-1 and from 2000 to 675 cm.sup.-1
respectively. An FT-IR overlay spectrum, for comparison purposes,
comparing these peaks of crystalline Form 1 from Example 2 to those
of crystalline Form 2 from Example 3, is shown in FIG. 8, showing
the spectral regions from 2000 to 675 cm.sup.-1.
Solid-State NMR Spectrum
[0275] A solid-state NMR spectrum was obtained at a frequency of
90.55 MHz for .sup.13C observation using a 4-mm Bruker HFX MAS
(magic-angle spinning) probe at a temperature of 296K, and a
spinning speed of 8 kHz. Data were acquired using a cross
polarisation sequence with side-band suppression. Several scans
were acquired, with a relaxation delay of 10 seconds.
[0276] Chemical shifts for the resonances observed for crystalline
Form 1 as prepared by Example 2 are listed below (in ppm):
18.5.+-.0.3, 30.4.+-.0.3, 31.8.+-.0.3, 37.6.+-.0.3, 45.8.+-.0.3,
49.4.+-.0.3, 52.3.+-.0.3, 59.2.+-.0.3, 63.6.+-.0.3, 68.4.+-.0.3,
110.3.+-.0.3, 118.8.+-.0.3, 128.4.+-.0.3, 131.2.+-.0.3,
133.9.+-.0.3, 159.1.+-.0.3, and 167.6 ppm.
[0277] Additional resonances were also observed at 19.5.+-.0.3,
71.1.+-.0.3, 109.5.+-.0.3 and 11 9.6.+-.0.3 ppm, and are thought to
correspond to crystalline Form 2 as an impurity.
[0278] The solid-state NMR spectrum for crystalline Form 1 as
prepared by Example 2, as a comparison overlay with that of
crystalline Form 2, is illustrated in FIG. 9.
Example 3
Crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine Hydrochloride
##STR00014##
[0280] 4-(Tetrahydro-2H-pyran-4-yloxy)benzoic acid (1 wt, 20 g;
e.g. may be prepared as described in Description 3) was suspended
in acetonitrile (80 mL). A solution of carbonyl diimidazole (CDI)
(0.8 wt, 16 g, 1.1 mole equivalents) in acetonitrile (80 mL), which
had been pre-warmed to 35-40.degree. C., was added in a single
charge to the suspension at ambient temperature. Acetonitrile line
wash (20 mL, 1 volume) was also added. The reaction mixture was
heated under nitrogen at 60.degree. C. for 1 hour.
N-isopropylpiperazine (1-isopropylpiperazine, 0.66 wt, 13.33 g,
obtained from Fluorochem ACI) was added to the reaction mixture,
and heating was continued for 2 hours. The mixture was concentrated
to approx 2.5 volumes (50 mL) via vacuum distillation to give a
thick mobile oil. Propan-2-ol (240 mL, 12 volumes) was then added,
and the mixture was concentrated by distillation under reduced
pressure (100 mbar) to remove 2 volumes (40 mL). The mixture was
heated to 70.degree. C. with stirring. 5-6N hydrochloric acid in
Propan-2-ol (20 mL, 1 volume) was added to the mixture over 10 min.
No crystallisation occurred until all of the acid had been added.
The reaction mixture was maintained at 70.degree. C. overnight. The
mixture was cooled and the slurry was filtered. The filtered solid
was dried under air suction on the filter over 1 to 1.5 hours. The
title product was obtained as a slightly pink solid (25 g).
[0281] Analyses of the crystalline Form 2 product produced by
Example 3 include the following: [0282] X-Ray Powder Diffraction
(XRPD)
[0283] XRPD data were acquired on a PANalytical X'Pert Pro powder
diffractometer, equipped with an X'Celerator detector. The
acquisition conditions were: radiation: Cu K.alpha. (copper
K-alpha), generator tension: 40 kV, generator current: 45 mA, start
angle: 2.0.degree. 2.theta. (two-theta), end angle: 40.0.degree.
2.theta., step size: 0.0167.degree. 2.theta. (two-theta). The time
per step was 31.750 s. The sample was prepared by mounting a few
milligrams of sample on a Si wafer (zero background) plates,
resulting in a thin layer of powder.
[0284] Some characteristic peak positions and calculated d-spacings
for the crystalline Form 2 product produced by Example 3 are
summarised in the following table (note: these are not the only
peaks seen). These were calculated from the raw data using
Highscore software.
TABLE-US-00002 XRPD - Crystalline Form 2 d-spacing/.ANG.
2.theta./.degree. (.ANG.ngstroms) 6.4 13.8 12.8 6.9 15.4 5.8 19.2
4.6 19.7 4.5 20.0 4.4 21.8 4.1 21.9 4.1 23.5 3.8 24.65 (rounds 3.6
to 24.7) 25.8 3.5 27.0 3.3
[0285] The XRPD spectrum for crystalline Form 2 as prepared by
Example 3 is shown in FIG. 2. An XRPD overlay spectrum is shown in
FIG. 3, comparing the XRPD peaks of crystalline Form 2 from Example
3 (bottom) to those of crystalline Form 1 from Example 2 (top), for
comparison purposes. The crystalline Form 2 XRPD peaks at
20.0.degree. 2.theta. and 24.65.degree. (or 24.7.degree.) 2.theta.
appear to be characteristic for crystalline Form 2 in that these
peaks do not appear to be significantly present in the XRPD
spectrum of crystalline Form 1.
FT-IR (FT-Infrared) Spectrum
[0286] FT-IR spectrum was acquired over 64 scans at 4 cm.sup.-1
resolution using a Nicolet Avatar 360 FT-IR spectrometer, fitted
with a Diamond/ZnSe ATR Accessory.
[0287] The FT-IR spectrum for crystalline Form 2 as prepared by
Example 3 is shown in FIGS. 6 and 7, showing the spectral regions
from 4000 to 675 cm.sup.-1 and from 2000 to 675 cm.sup.-1
respectively. An FT-IR overlay spectrum comparing these Form 2
peaks from Example 3 to those of crystalline Form 1 from Example 2
is shown in FIG. 8, for comparison purposes, showing the spectral
regions from 2000 to 675 cm.sup.-1.
Solid-State NMR Spectrum
[0288] A solid-state NMR spectrum was obtained at a frequency of
90.55 MHz for .sup.13C observation using a 4-mm Bruker HFX MAS
(magic-angle spinning) probe at a temperature of 296K, and a
spinning speed of 8 kHz. Data were acquired using a cross
polarisation sequence with side-band suppression. Several scans
were acquired, with a relaxation delay of 10 seconds.
[0289] Chemical shifts for the resonances observed for crystalline
Form 2 as prepared by Example 3 are listed below (in ppm):
18.8.+-.0.3, 19.5.+-.0.3, 32.4.+-.0.3, 37.5.+-.0.3, 45.7.+-.0.3,
49.3.+-.0.3, 52.7.+-.0.3, 59.1.+-.0.3, 66.3.+-.0.3, 71.1.+-.0.3,
109.4.+-.0.3, 119.6.+-.0.3, 128.4.+-.0.3, 131.3.+-.0.3,
134.3.+-.0.3, 158.7.+-.0.3, and 167.8.+-.0.3 ppm.
[0290] The solid-state NMR spectrum for crystalline Form 2 as
prepared by Example 3, as a comparison overlay with that of
crystalline Form 1 from Example 2, is illustrated in FIG. 9.
Example 4
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}piper-
azine Hydrochloride
##STR00015##
[0292] All weights, volumes and equivalents are with respect to
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid.
[0293] 4-(Tetrahydro-2H-pyran-4-yloxy)benzoic acid (10 g, 1 wt, 1
vol, 1 mole equivalent) is added portion-wise (take care, gas
evolution) over 10 minutes to a stirred solution of carbonyl
diimidazole (CDI, 8.0 g, 0.8 wt, 1.1 mole equivalents) in
acetonitrile (100 mL, 10 vol) under nitrogen at about 65.degree. C.
(jacket temperature at 70.degree. C.). Acetonitrile (1.5 volumes,
15 mL) is used as line wash for the container of the
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid, and the funnel is used
for the addition of the reagent. The resulting suspension/slurry is
stirred at about 65.degree. C. for at least ca. 2 hours (e.g. ca.
2-2.5 hours) before being sampled.
[0294] Reaction progress is monitored by HPLC: the sample is
prepared by quenching a drop of the reaction mixture into 1 mL of
5% butylamine solution in acetonitrile; this allows determination
of residual 4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid by
derivatisation of the activated acid-imidazolide to the
corresponding butylamide. This is generally recorded for
information only since slurry inhomogeneity can give rise to
inconsistent results (typically <2% residual
4-(tetrahydro-2H-pyran-4-yloxy)benzoic acid is satisfactory at this
stage).
[0295] Subsequently 1-isopropylpiperazine (0.667 wt, 6.67 g, 1.15
mole equivalents) is added in one portion at about 65.degree. C.,
followed by a line wash with acetonitrile (0.5 vol, 5 mL). The
resulting hazy solution is kept stirred at about 65.degree. C. for
at least ca. 2 hours (e.g. ca. 2-2.5 hours) before being sampled.
Reaction progress is monitored by HPLC, using the method stated
above.
[0296] The reaction is then allowed to cool and insoluble matter is
removed by filtration. The clarified solution is then concentrated
by vacuum distillation to 2.5 to 3 volumes and 5% water in
isopropanol (5 volumes, 50 mL) is added at ambient temperature. The
solution is then heated to about 65.degree. C. and 5-6N HCl in
isopropanol (0.9 volumes) is added in one charge. Crystallisation
can commence shortly after the addition. The resulting slurry is
aged at 65.degree. C. for 1.5 hours. The slurry is then cooled to
55.degree. C. over ca. 20 min and kept at 55.degree. C. for 1.5
hours, is then cooled to 45.degree. C. over ca. 20 min and kept at
45.degree. C. for 1.5 hours, and is then allowed to cool to ambient
temperature and the solid is filtered off (1 hour in total); the
total cooling time is therefore about 4.5 to 4.75 hours.
[0297] The solid
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, isolated by filtration, is washed with 4
volumes of isopropanol and is dried under vacuum at 50.degree. C.,
for example overnight.
[0298] The above-described process is currently believed to produce
crystalline Form 2 of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
Example 5
Recrystallisation of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine Hydrochloride
##STR00016##
[0299] Short Process Description
[0300] All weights, volumes ("vol") and equivalents are relative to
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride.
[0301] A suspension of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (1 wt, e.g. may be prepared as described in
Example 1 Method D) in ethanol (50 vol) is heated to reflux and
stirred until a solution has formed. This solution is cooled to
65.+-.3.degree. C. and clarified. A line wash of hot ethanol (3
vol) is added the solution is cooled to 58.+-.3.degree. C. A seed
of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (0.01 wt) is added and the resulting
suspension is stirred at 58.+-.3.degree. C. for 30 min. The
suspension is then cooled to 0.+-.3.degree. C. over 2 hours before
being aged at this temperature for 1 hour. The solid is then
filtered off under vacuum and washed with cold ethanol (3 vol). The
product is the dried in vacuo at 40.degree. C. until constant probe
temperature.
Detailed Process Description
[0302] 1. Add
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (1 wt, 140 g, e.g. may be prepared as
described in Example 1 Method D) to Reactor 1. [0303] 2. Purge
reactor with Nitrogen. [0304] 3. Add ethanol (50 vol, 7100 mL).
[0305] 4. Heat to reflux and stir until a solution formed. [0306]
5. This solution is cooled to 65.+-.3.degree. C. [0307] 6. Contents
of Reactor 1 are transferred to Reactor 2 via a peristaltic pump
fitted with silicone tubing and a 5 micron in-line filter. [0308]
7. Add ethanol to Reactor 1 (3 vol, 420 mL) and heat to
65.+-.3.degree. C. [0309] 8. Contents of Reactor 1 are transferred
to Reactor 2 as per step 6. [0310] 9. The solution is cooled to
58.+-.3.degree. C. [0311] 10. A seed of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (0.01 wt) is added and the resulting
suspension is stirred at 58.+-.3.degree. C. for 30 min. [0312] 11.
The suspension is then cooled to 0.+-.3.degree. C. over 2 hours
before being aged at this temperature for 1 hour. [0313] 12. The
solid is then filtered off under vacuum and washed with cold
ethanol (3 vol). [0314] 13. The product is the dried in vacuo at
40.degree. C. until constant probe temperature. [0315] 14. The
product is generally obtained as a white solid.
Biological Data
[0316] A membrane preparation containing histamine H3 receptors may
be prepared in accordance with the following procedures:
(i) Generation of Histamine H3 Cell Line
[0317] DNA encoding the human histamine H3 gene (Huvar, A. et al.
(1999) Mol. Pharmacol. 55(6), 1101-1107) was cloned into a holding
vector, pcDNA3.1 TOPO (InVitrogen) and its cDNA was isolated from
this vector by restriction digestion of plasmid DNA with the
enzymes BamHI and Not-1 and ligated into the inducible expression
vector pGene (InVitrogen) digested with the same enzymes. The
GeneSwitch.TM. system (a system where in transgene expression is
switched off in the absence of an inducer and switched on in the
presence of an inducer) was performed as described in U.S. Pat.
Nos. 5,364,791; 5,874,534; and 5,935,934. Ligated DNA was
transformed into competent DH5.alpha. E. coli host bacterial cells
and plated onto Luria Broth (LB) agar containing Zeocin.TM. (an
antibiotic which allows the selection of cells expressing the sh
ble gene which is present on pGene and pSwitch) at 50 .mu.g
ml.sup.-1. Colonies containing the re-ligated plasmid were
identified by restriction analysis. DNA for transfection into
mammalian cells was prepared from 250 ml cultures of the host
bacterium containing the pGeneH3 plasmid and isolated using a DNA
preparation kit (Qiagen Midi-Prep) as per manufacturers guidelines
(Qiagen).
[0318] CHO K1 cells previously transfected with the pSwitch
regulatory plasmid (InVitrogen) were seeded at 2.times.10e6 cells
per T75 flask in Complete Medium, containing Hams F12 (GIBCOBRL,
Life Technologies) medium supplemented with 10% v/v dialysed foetal
bovine serum, L-glutamine, and hygromycin (100 .mu.g ml.sup.-1), 24
hours prior to use. Plasmid DNA was transfected into the cells
using Lipofectamine plus according to the manufacturers guidelines
(InVitrogen). 48 hours post transfection cells were placed into
complete medium supplemented with 500 .mu.g ml.sup.-1
Zeocin.TM..
[0319] 10-14 days post selection 10 nM Mifepristone (InVitrogen),
was added to the culture medium to induce the expression of the
receptor. 18 hours post induction cells were detached from the
flask using ethylenediamine tetra-acetic acid (EDTA; 1:5000;
InVitrogen), following several washes with phosphate buffered
saline pH 7.4 and resuspended in Sorting Medium containing Minimum
Essential Medium (MEM), without phenol red, and supplemented with
Earles salts and 3% Foetal Clone II (Hyclone). Approximately
1.times.10e7 cells were examined for receptor expression by
staining with a rabbit polyclonal antibody, 4a, raised against the
N-terminal domain of the histamine H3 receptor, incubated on ice
for 60 minutes, followed by two washes in sorting medium. Receptor
bound antibody was detected by incubation of the cells for 60
minutes on ice with a goat anti rabbit antibody, conjugated with
Alexa 488 fluorescence marker (Molecular Probes). Following two
further washes with Sorting Medium, cells were filtered through a
50 .mu.m Filcon.TM. (BD Biosciences) and then analysed on a FACS
Vantage SE Flow Cytometer fitted with an Automatic Cell Deposition
Unit. Control cells were non-induced cells treated in a similar
manner. Positively stained cells were sorted as single cells into
96-well plates, containing Complete Medium containing 500 .mu.g
ml.sup.-1 Zeocin.TM. and allowed to expand before reanalysis for
receptor expression via antibody and ligand binding studies. One
clone, 3H3, was selected for membrane preparation.
(ii) Membrane Preparation from Cultured Cells
[0320] All steps of the protocol are carried out at 4.degree. C.
and with pre-cooled reagents. The cell pellet is resuspended in 10
volumes of homogenisation buffer (50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 mM
ethylenediamine tetra-acetic acid (EDTA), pH 7.4 with KOH
containing 10e-4M leupeptin (acetyl-leucyl-leucyl-arginal; Sigma
L2884) and 25 g/ml bacitracin (Sigma B0125)) supplemented with 1 mM
phenylmethylsulfonyl fluoride (PMSF) and 2.times.10e-6M pepstain A
(Sigma). The cells are then homogenised by 2.times.15 second bursts
in a 1 litre glass Waring blender, followed by centrifugation at
500 g for 20 minutes. The supernatant is then spun at 48,000 g for
30 minutes. The pellet is resuspended in homogenisation buffer
(4.times. the volume of the original cell pellet) by vortexing for
5 seconds, and then being forced by syringe through a 0.6 mm
internal diameter needle. At this point the preparation is
aliquoted into polypropylene tubes and stored at -80.degree. C.
(iii) Generation of Histamine H1 Cell Line
[0321] The human H1 receptor was cloned generally using known
procedures described in the literature [Biochem. Biophys. Res.
Commun. 1994, 201(2), 894]. Chinese hamster ovary cells stably
expressing the human H1 receptor were generated generally according
to known procedures described in the literature [Br. J. Pharmacol.
1996, 117(6), 1071].
[0322] The compound of the invention or a pharmaceutically
acceptable salt thereof may be tested for in vitro biological
activity in accordance with the following assays:
(I) Histamine H3 Functional Antagonist Assay
[0323] For each compound being assayed, in a solid white 384 well
plate, is added:--
(a) 0.5 .mu.l (0.5 ul) of test compound diluted to the required
concentration in DMSO (or 0.5 .mu.l (0.5 ul) DMSO as a control);
(b) 30 .mu.l (30 ul) bead/membrane/GDP mix prepared by mixing Wheat
Germ Agglutinin Polystyrene LeadSeeker.RTM. (WGA PS LS)
scintillation proximity assay (SPA) beads with membrane (prepared
for example in accordance with the methodology described above) and
10 .mu.M (10 uM) final concentration of guanosine 5' diphosphate
(GDP), and diluting in assay buffer (20 mM
N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)+100 mM
NaCl+10 mM MgCl.sub.2, pH7.4 NaOH) to give a final volume of 30
.mu.l (30 ul) which contains 5 .mu.g (5 ug) protein and 0.25 mg
bead per well, and incubating at room temperature for 60 minutes on
a roller; (c) 15 .mu.l (15 ul) of 0.38 nM [.sup.35S]-GTP.gamma.S
([.sup.35S]-GTP-gamma-S) (Amersham; Radioactivity concentration=37
MBq/ml; Specific activity=1160 Ci/mmol), histamine (at a
concentration that results in the final assay concentration of
histamine being EC.sub.80). The plate is sealed and after 2-6
hours, the plate is centrifuged for 5 min at 1500 rpm and counted
on a Viewlux counter using a 613/55 filter for 5 min/plate. Data is
analysed using a 4-parameter logistical equation. Basal activity
used as minimum, i.e. wherein the histamine H3 antagonist
iodophenpropit (30 uM, 0.5 ul) has been added to the well.
(II) Histamine H1 Functional Antagonist Assay
[0324] Adherent Chinese Hamster Ovary (CHO) cells stably expressing
the recombinant human H.sub.1 receptor were maintained in culture
at 37.degree. C. under 5% CO.sub.2 in Alpha Minimum Essential
Medium without ribonucleosides (Gibco Invitrogen) supplemented with
10% dialysed foetal calf serum and 200 mM Glutamine. These cells,
expressing the human H1 receptor, were snap frozen and stored ready
for assay.
[0325] 24 or 72 hours prior to assay the cells were seeded into
black walled clear-base 384-well plates at a density of 12,000 or
4000 cells per well (respectively) and cultured at 37.degree. C.
under 5% CO.sub.2. Cell seeding densities result in a confluent
monolayer of cells at a time point of approximately 24 hours for
1200 cells or 72 hours for 4 000 cells. Media was aspirated off and
the cells were then incubated with HBSS medium
(CaCl.sub.2.2H.sub.2O 1.26 mM, Glucose 5.55 mM, KCl 5.36 mM,
MgSO.sub.4 (anhyd.) 0.81 mM, NaCl 136.89 mM, KH.sub.2PO.sub.4
(anhyd.) 0.41 mM, HEPES 20 mM, NaHCO.sub.3 4.16 mM) containing the
cytoplasmic calcium indicator, Fluo-4 in the acetylmethyl form (4
mM), 2.5 mM Probenecid and 250 uM Brilliant Black (Molecular
Devices) at 37.degree. C. for 60 min. The loaded cells were then
incubated with test compound for 30 min at 37.degree. C. The plates
were then placed into a FLIPR (Molecular Devices, UK) for testing
in antagonist mode, where a pre-determined concentration of
Histamine (approximately 4.times.EC50) was added while cell
fluorescence (.lamda.ex 488 nm, .lamda.em 540 nm) was
monitored.
[0326] Functional antagonism is indicated by a suppression of
histamine induced increase in fluorescence, as measured by the
FLIPR.TM. system (Molecular Devices). By means of concentration
effect curves, functional affinities are determined using standard
pharmacological mathematical analysis.
Results of H3 and H1 Functional Antagonist Assays
[0327] 1-(1-Methyl
ethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}piperazine
hydrochloride (e.g. Example E1) was tested in the histamine H3
functional antagonism assay. The result is expressed as a
functional pK.sub.i (fpK.sub.i) value. A functional pK.sub.i is the
negative logarithm of the antagonist equilibrium dissociation
constant as determined in the H3 functional antagonist assay using
membrane prepared from cultured H3 cells. The result given is an
average of a number of experiments.
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (e.g. Example E1) exhibited antagonism with a
fpK.sub.i of approximately 7.6 (as the mean of 27 experiments),
with a range of fpK.sub.i observed of from 6.9 to 8.2.
[0328] 1-(1-Methyl
ethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}piperazine
hydrochloride (e.g. Example E1) was tested in the histamine H1
functional antagonist assay or a similar H1 functional antagonist
assay. Again, the result is expressed as a functional pK.sub.i
(fpK.sub.i) value and is an average of a number of experiments.
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (e.g. Example E1) exhibited antagonism
approximately <5.6 fpK.sub.i.
Rat Ex Vivo Binding Studies--Rat Brain Histamine H3 Receptor
Occupancy
[0329] Ex vivo binding studies were carried out to determine brain
histamine H3 receptor occupancy in rats at certain time points
after oral administration of a test compound.
[0330] Adult male rats (Lister hooded 200-250 g, Charles River, UK)
received vehicle (1% w/v aqueous methylcellulose) (control) or the
test compound (10 mg/kg) by oral gavage (n=3 per group) and were
sacrificed 1 or 4 hours following oral dosing. The 1 hour and 4
hour studies were not necessarily done on the same day. Terminal
blood samples were collected and brains rapidly removed. Cerebral
cortex tissue was dissected from half of each brain for ex vivo
binding; the other half brain can be used for pharmacokinetic
analysis of brain concentrations of each compound. All dissected
tissue samples were snap-frozen in liquid nitrogen, and stored at
-80.degree. C. until use. The tissues were rapidly thawed and
homogenised in approximately 30 volumes of ice cold assay reaction
buffer. The assay reaction buffer contained 50 mM Tris-HCl (made up
using Trizma pre-set crystals pH 7.7 @25.degree. C., Sigma cat. No.
T8068-250G) and 5 mM EDTA, with a final buffer pH of 7.2 to 7.8,
usually about 7.4. The crude homogenate (600-800 .mu.g/well) was
then used to measure H3 receptor binding using
[.sup.3H]-R-.alpha.-methylhistamine as radioligand. Assays
measuring total binding of [.sup.3H]-R-.alpha.-methylhistamine
consisted of 50 .mu.l assay reaction buffer, 400 .mu.l of
homogenate (corresponding to 600-800 .mu.g/well) and 50 .mu.l of 2
nM R(-).alpha.-Methyl[imidazole-2,5(n)-3H]histamine dihydrochloride
([.sup.3H]-R-.alpha.-methylhistamine; specific activity, 24 Ci
mmol.sup.-1, Amersham Biosciences, catalogue no. TRK1017).
Incubations with [.sup.3H]-R-.alpha.-methylhistamine were for 45
min at 30.degree. C. Non-specific binding of
[.sup.3H]-R-.alpha.-methylhistamine was determined in parallel
using the same assay except that 50 .mu.l of 10 .mu.M imetit (an H3
receptor agonist, e.g. available from Tocris) was used instead of
the 50 .mu.l of assay reaction buffer. The experiments were
terminated by rapid filtration through Whatman GF/B filters
(pre-soaked in 0.3% v/v polyethyleneimine (PEI)), and then the
filters were washed through with 4.times.5 ml of ice cold
harvesting buffer. The harvesting buffer contained 50 mM Tris-HCl
(from Trizma pre-set crystals pH 7.7 @ 25.degree. C.) and 5 mM
MgCl.sub.2, with a final buffer pH of 7.2 to 7.8, usually about
7.4. Filters were dried and added to vials each containing 4 ml
Ultima Gold MV scintillation fluid (Hewlett Packard) and
radioactivity determined by liquid scintillation spectrometry using
a Packard Tri-Carb 2500TR liquid scintillation counter.
[0331] Specific binding of [.sup.3H]-R-.alpha.-methylhistamine to
the H3 receptor was determined by the subtraction of the value
obtained for non-specific binding from the value obtained for total
binding.
[0332] Protein concentrations were determined using the Bradford
assay method (Bio-Rad
[0333] Protein Assay Dye Reagent Concentrate, catalogue no.
500-0006; from Bio-Rad Laboratories GmbH, Heidemannstrasse 164,
80939 Muenchen, Germany; or from Bio-Rad, York, UK) with bovine
serum albumin as a standard. Specific radioactivity in the samples
was corrected for protein (i.e. per microgram of protein).
[0334] The specific binding of [.sup.3H]-R-.alpha.-methylhistamine
to the H3 receptor is expressed as a mean (n=3 rats).+-.SEM
(standard error of the mean), as a percentage of vehicle-treated
control animals. Data is also expressed as inhibition of
[.sup.3H]-R-.alpha.-methylhistamine specific binding to the H3
receptor, as a surrogate measure for H3 receptor occupancy by the
test compound, calculated as 100% minus the % mean specific binding
of [.sup.3H]-R-.alpha.-methylhistamine to the H3 receptor.
Results of Rat Ex Vivo Binding Studies
[0335] For
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]car-
bonyl}piperazine hydrochloride
##STR00017##
within the present invention, dosed orally to rats (n=3) at 10
mg/kg, and with the rats sacrificed 1 hour following oral dosing,
the specific binding of [.sup.3H]-R-.alpha.-methylhistamine to the
H3 receptor was determined to be 40%+2% (% of control). Hence,
inhibition of [.sup.3H]-R-.alpha.-methylhistamine specific binding
to the H3 receptor was about 60%, as a surrogate measure for rat
brain H3 receptor occupancy by the test compound at 1 hour
following oral 10 mg/kg dosing.
[0336] For
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]car-
bonyl}piperazine hydrochloride, within the present invention, dosed
orally to rats (n=3) at 10 mg/kg, and with the rats sacrificed 4
hours following oral dosing, the specific binding of
[.sup.3H]-R-.alpha.-methylhistamine to the H3 receptor was
determined to be 84%+6% (% of control). Hence, inhibition of
[.sup.3H]-R-.alpha.-methylhistamine specific binding to the H3
receptor was about 16%, as a surrogate measure for rat brain H3
receptor occupancy by the test compound at 4 hours following oral
10 mg/kg dosing.
[0337] For
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}hexahydro-1H-1,4-diazepine hydrochloride
##STR00018##
a comparator compound, dosed orally to rats (n=3) at 10 mg/kg, and
with the rats sacrificed 1 hour following oral dosing, the specific
binding of [.sup.3H]-R-.alpha.-methylhistamine to the H3 receptor
was determined to be 40%+4% (% of control). Hence, inhibition of
[.sup.3H]-R-.alpha.-methylhistamine specific binding to the H3
receptor was about 60%, as a surrogate measure for rat brain H3
receptor occupancy by the test compound at 1 hour following oral 10
mg/kg dosing.
[0338] For
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}hexahydro-1H-1,4-diazepine hydrochloride, a comparator compound,
dosed orally to rats (n=3) at 10 mg/kg, and with the rats
sacrificed 4 hours following oral dosing, the specific binding of
[.sup.3H]-R-.alpha.-methylhistamine to the H3 receptor was
determined to be 88%+6% (% of control). Hence, inhibition of
[.sup.3H]-R-.alpha.-methylhistamine specific binding to the H3
receptor was about 12%, as a surrogate measure for rat brain H3
receptor occupancy by the test compound at 4 hours following oral
10 mg/kg dosing.
Histamine H3 Receptor Antagonist "Pig-PET" Studies, Using Positron
Emission Tomography (PET) in the Yorkshire-Landrace Pig: Pig Brain
H3 Receptor Occupancy Profiles Over Time
[0339] These studies, and results arising therefrom, are
illustrated in part by the attached FIGS. 10, 11, 12, 13 and 14,
already briefly described.
Theory of Positron Emission Tomography (PET)
[0340] PET is a nuclear imaging technique that enables the
measurement of the four-dimensional (three space, one time)
distribution of a radiopharmaceutical in the living body. A
bioactive molecule (which binds to the receptor of interest, in the
present case the histamine H3 receptor) is modified by exchanging
one of its atoms by a positron emitting nuclei (e.g. .sup.15O,
.sup.11C, .sup.18F, etc). The radioactive molecule
(radiopharmaceutical) is then intravenously injected into the
subject. In the theory, a positron-emitting atom undergoes
radioactive decay by releasing a positron from its nucleus.
Generally after several interactions with the surroundings the
positron loses kinetic energy and interacts with an electron by
annihilation. The annihilation results in two high energy photons
(2.times.511 keV) emitted at 180.degree. to each other. The high
energy photon-pair generated by the positron-electron annihilation,
emitted at 180.degree., can be detected externally. A ring of
crystal detectors in the PET scanner senses the presence of two
photons generated simultaneously and records data for the two
detectors pair that sensed the two photons, enabling localization
of the activity. This is generally done millions of times during
the course of a PET scan. 3D images are sub-sequentially generated
using tomographic reconstruction techniques. The nature of PET is
intrinsically quantitative therefore the three-dimensional
distribution of the radiopharmaceutical can be expressed in units
of Bq/ml or nM using the specific activity (SA) of the
radiopharmaceutical.
Measuring In Vivo Receptor Occupancy with PET
[0341] Receptor occupancy of a non-radiolabelled test compound
("drug candidate") can be measured indirectly by measuring the
reduction in specific binding of the radioligand as a consequence
of competitive binding (FIG. 10). As FIG. 10 shows, the occupancy
of the drug candidate can be measured indirectly by the reduction
in radioligand specific binding to receptors. B.sub.A is the
concentration of available receptors sites. Notice how B.sub.A
changes between baseline, and 10 min, 2.5 hours, and 6 hours after
administration of the test compound, as a consequence of the
presence of different concentrations of the drug candidate in
tissue.
[0342] A baseline scan is performed where a small mass of the
radioligand (in the .mu.g range such that the self-occupancy of the
radioligand is minimal, <10%) is administered to the subject.
Using regional time activity curves from a target and a reference
region (area devoid of specific binding sites) and a mathematical
model it is possible to estimate the binding potential, BP.sub.ND,
which is proportional to the concentration of available receptor
B.sub.A of each target region. Following the baseline scan the
unlabeled drug candidate is administered to the subject and
sub-sequent scans are acquired at time points of interest and
binding potentials are estimated. The drug candidate occupancy at
different time points is calculated as the percentage change of the
binding potential measurements with respect to baseline (J.
Passchier, A. Gee, A. Willemsen, W. Vaalburg, and A. van Waarde,
"Measuring drug-related receptor occupancy with positron emission
tomography," Methods, 2002, vol. 27, pp. 278-286).
Preclinical PET Studies in the Yorkshire-Landrace Pig
[0343] The H3 receptor occupancy (R.sup.O) time course of: [0344]
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention), and
[0345]
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride ("salt B", a comparator compound)
were measured in the Yorkshire-Landrace pig using the selective H3
receptor antagonist [.sup.11C]GSK189254 and PET.
[0346] [.sup.11C]GSK189254 is
[.sup.11C-N-methyl]-6-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin--
7-yloxy)-nicotinamide, the structure of which is
##STR00019##
and/or a pharmaceutically acceptable salt thereof. See page 5 line
8 to page 6 line 11 of WO 2006/072596 A1 (Glaxo Group Limited),
Example 1 (Compound A) therein, for the preparation of
[.sup.11C]GSK189254 by the reaction of
6-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy)-nicotinamid-
e with [.sup.11C]methyl iodide at 130.degree. C. in
dimethylsulfoxide in the presence of tetrabutylammonium fluoride,
followed by HPLC purification. See e.g. page 7 line 32 to page 9
line 2 of WO 2006/072596 A1 for the use of [.sup.11C]GSK189254 in
(pig) PET imaging studies. Pig-PET Studies Using
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine Hydrochloride ("Salt A")
[0347] Yorkshire-Landrace pigs (n=3, pig weight about 38.+-.1 kg)
underwent four PET scans in the same day, as shown in the pig-PET
protocol illustrated in FIG. 11, under anaesthesia
(ketamine-midazolam induction+isofluorane maintenance).
[0348] The radioligand [.sup.11C]GSK189254 was synthesized
immediately before each PET scan. The crude
[.sup.11C]GSK189254-containing reaction product was subject to HPLC
purification performed using a reverse phase C.sub.18 column
(Waters, X-Terra.RTM. RP18, 19.times.100 mm, 5 mm) at 10 mL/min
flow rate with a mobile phase consisting of 17% of acetonitrile in
a 0.1 N aqueous buffer solution of ammonium formate at pH 4. The
[.sup.11C]GSK189254-containing product fraction collected was
concentrated in vacuo to remove the acetonitrile and reformulated
in 0.9% aqueous sodium chloride solution.
[0349] For each PET scan, the radioligand [.sup.11C]GSK189254, in
the above-mentioned vehicle which was effectively a mixture of
aqueous ammonium formate buffer and saline, was administered
intravenously (i.v.) over about 1 minute as a bolus. Less than 2
micrograms total mass of [.sup.11C]GSK189254 was injected, with a
mean total dose injected of less than 53 ng/kg for a pig weight of
about 38.+-.1 kg. The amount of radioactivity injected into each
pig via [.sup.11C]GSK189254 was generally about 250 to about 400
MBq (ideally about 300 MBq). The volume of
[.sup.11C]GSK189254+vehicle injected per pig depends on the yield
obtained from the last HPLC purification, but generally ranged from
about 2 to about 11 ml.
[0350] Blood and tissue activity concentration data were recorded
for 90 minutes.
[0351] Following the baseline scan, unlabelled
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", 50 .mu.g/kg, 50 micrograms/kg) (1
ml in an aqueous saline vehicle) was administered intravenously by
manual injection in a bolus fashion over approximately 1 minute.
The subsequent PET acquisitions were initiated at 10, 150, and 360
minutes post administration of salt A. The arterial blood samples
were assayed for [.sup.11C]GSK189254 plasma activity and
metabolites and
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine plasma concentrations.
Pig-PET Studies Using
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine Hydrochloride ("Salt B")
[0352] Substantially the same experimental procedure was repeated
using
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride ("salt B", 50 .mu.g/kg, 50
micrograms/kg in a vehicle) in Yorkshire-Landrace pigs (n=3, pig
weight about 38.+-.1 kg).
Binding Potential and Occupancy Measurements
[0353] PET images were aligned to a stereotaxic atlas and regional
time activity curves were obtained for the target regions of
interest (frontal cortex, hippocampus, putamen, caudate,
diencephalon, medial thalamus, lateral thalamus, vermis, pons,
mesencephalon, and medulla oblongata). The simplified reference
tissue model (SRTM) (A. A. Lammertsma and S. P. Hume, "Simplified
reference tissue model for PET receptor studies," Neuroimage, 1996,
vol. 4, pp. 153-158) was fitted to each regional time activity
curve using the cerebellum as the reference region:
C t ( t ) = R 1 C r ( t ) + ( k 2 - R 1 k 2 1 + BP ND ) C r ( t ) -
k 2 t 1 + BP ND ##EQU00001##
where C.sub.t(t) is the activity concentration in the target
region, C.sub.r(t) is the activity concentration in the reference
region (cerebellum), R.sub.1 is the ratio of influx (K.sub.1)
between the target and reference region, k.sub.2 is the
tissue-plasma efflux rate constant in the target region, and
BP.sub.ND is the binding potential of the target region. Moreover
the binding potential can be defined as
BP ND = f ND B A K d ##EQU00002##
where f.sub.ND is the radioligand free fraction in tissue, B.sub.A
is the available concentration of binding sites, and K.sub.d is the
equilibrium dissociation constant of the radioligand-receptor
complex.
[0354] Receptor occupancy can be calculated as the percentage
change in BP.sub.ND between the baseline and post drug scan (J.
Passchier, A. Gee, A. Willemsen, W. Vaalburg, and A. van Waarde,
"Measuring drug-related receptor occupancy with positron emission
tomography," Methods, 2002, vol. 27, pp. 278-286):
Occ = ( BP ND baseline - BP ND drug BP ND baseline ) .times. 100
##EQU00003##
[0355] In the case of [.sup.11C]GSK189254 the cerebellum is not a
true reference region as there exists a small specific signal in
this region. This can be corrected using a population estimate of
the cerebellum binding potential (BP.sub.ND.sup.ref) and the
equation
Occ corrected = Occ ( 1 + BP ND ref 1 + B ND ref Occ )
##EQU00004##
Modelling the Occupancy Profile
[0356] The temporal occupancy profile at 10, 150, and 360 minutes
(Occ(t)) post administration of the drug candidate and the plasma
concentration of the drug candidate (C.sub.p(t)) measured
throughout the PET whole scan were used to derive PK/RO
(pharmacokinetic/receptor occupancy) model parameter estimates
(f.sub.p, k.sub.on and k.sub.off) from an indirect model
(k.sub.on-k.sub.off limited model)
Occ ( t ) t = f p k on ( 1 - Occ ( t ) ) C p ( t ) - k off Occ ( t
) ##EQU00005##
wherein k.sub.on and k.sub.off are respectively the rate constants
defining the speed that the test compound attaches or de-attaches
to or from the H3 receptor, and f.sub.p is the protein-free
fraction in plasma of the test compound (here, salt A or salt
B).
[0357] This model assumes that the rate limiting step is the
receptor-ligand association and dissociation (k.sub.on, k.sub.off)
whilst the plasma-tissue exchange is rapid enough to be considered
in instantaneous equilibrium.
Results of Pig-PET Studies
[0358] FIG. 12, graph A, shows the average (mean) plasma
concentration over time of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention,
filled circles) and
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride ("salt B", a comparator compound,
filled diamonds).
[0359] FIG. 12, graph B, shows the average (mean) measured H3
receptor occupancy time course at three time points; and the
k.sub.on-k.sub.off limited model fitted to it for "salt A" within
the present invention (measurements as filled circles, and model
fit as solid line), and for "salt B" a comparator compound
(measurements as filled diamonds, and model fit as dashed
line).
[0360] FIG. 13, parts A and B, are graphs showing the data from
FIG. 12 for salt A alone such as to show the average (mean) plasma
concentration over time and average (mean) H3 receptor occupancy
time course respectively for "salt A". FIG. 13, parts C and D, are
graphs showing the data from FIG. 12 for salt B alone such as to
show the average (mean) plasma concentration over time and average
(mean) H3 receptor occupancy time course respectively for "salt
B".
[0361] FIG. 14 shows separated individual plasma concentration and
H3 receptor occupancy time courses, for each individual pig
studied, for
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention, left
hand graphs, n=3), and for
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}hexahydr-
o-1H-1,4-diazepine hydrochloride ("salt B", a comparator compound,
right hand graphs, n=3).
[0362] Table 1 shows the estimated parameters k.sub.on, k.sub.off,
K.sub.d (k.sub.off/k.sub.on), and the plasma clearance for each
individual study (i.e. for each individual pig). The last row in
Table 1 ("average model") shows the estimated parameters derived
from the average plasma data and average occupancy (FIGS. 12, 13);
it can be noticed that these "average model" parameters appear to
be in general agreement with the average parameters ("mean")
estimated individually for each scan (second row from the
bottom).
TABLE-US-00003 TABLE 1 Estimated model parameters for each
individual study (each individual pig) and the parameters derived
from average data. Salt A Salt B Plasma Plasma k.sub.on k.sub.off
K.sub.d clearance k.sub.on k.sub.off K.sub.d clearance (mm.sup.-1)
(mm.sup.-1) (ng/ml) (L/hr) (min.sup.-1) (min.sup.-1) (ng/ml) (L/hr)
pig 1 0.0023 0.0038 1.65 2.9 0.0017 0.0033 1.94 3.6 pig 2 0.0033
0.0114 3.45 3.0 0.0024 0.0042 1.75 1.8 pig 3 0.0058 0.0286 4.93 1.8
0.0031 0.0039 1.25 2.8 mean 0.0038 0.0146 3.35 2.5 0.0024 0.0038
1.65 2.8 average model 0.0027 0.0091 3.37 2.3 0.0025 0.0045 1.80
2.9
[0363] Table 2 shows the measured H3 receptor occupancy at three
time points, in each the three individual studies performed (in
each of the three individual pigs studied), for each test compound,
salt A and salt B.
TABLE-US-00004 TABLE 2 Measured H3 receptor occupancy (and mean
occupancy .+-. standard deviation), at 10 minutes, 2.5 hours and 6
hours post administration of the test compound, in the three
individual pigs, for each test compound, salt A and salt B. Salt A
occupancy @ Salt B occupancy @ 10 min 2.5 h 6 h 10 min 2.5 h 6 h
pig 1 61% 43% 30% 51% 19% 30% pig 2 54% 24% 7% 69% 49% 32% pig 3
58% 22% 5% 68% 43% 38% Mean 58% .+-. 4% 30% .+-. 11% 14% .+-. 14%
63% .+-. 10% 37% .+-. 16% 33% .+-. 4%
Conclusion of Pig-PET Studies
[0364] The preliminary measurements obtained in the
Yorkshire-Landrace pig (n=3) by PET and [.sup.11C]GSK189254 appear
to indicate a generally faster reduction in pig brain H3 receptor
occupancy of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride ("salt A", within the present invention), as
corn pared to
1-(isopropyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl-
}hexahydro-1H-1,4-diazepine hydrochloride ("salt B", a comparator
compound), under the conditions tested (i.e. after 50 micrograms/kg
of intravenous administration of the test compound to the pigs, and
under the other stated conditions).
[0365] The apparently faster reduction in pig brain H3 receptor
occupancy for salt A, within the present invention, compared to
comparator salt B, is seen in studies 2 and 3 (i.e. pigs 2 and 3)
tested with salt A, but apparently not in study 1 (i.e. pig 1)
tested with salt A, as illustrated in Table 2 above.
Toxicity Study 1: Toxicity of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine Hydrochloride in a 7-Day, Oral Repeat-Dose Study in Male
Sprague Dawley Rats
Design of Toxicity Study 1
[0366] The objective of this study was to determine the toxicity of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride, and the toxicokinetics of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]-carbonyl}pip-
erazine hydrochloride (measured as the free base), in a 7-day, oral
repeat-dose study in male Sprague Dawley rats.
[0367]
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine hydrochloride was formulated as a suspension in 1%
(w/v) aqueous methylcellulose and administered to male rats (four
per group), at doses of 0 (vehicle, control), 30, 100 or 300
mg/kg/day for 7 days by oral gavage, at a dose volume of 10 mL/kg.
Three male rats were added at each non-zero dose level for
toxicokinetic evaluation. All doses and concentrations, including
analyte concentration in plasma, are expressed in terms of the
parent "free base" compound.
[0368] The following endpoints/parameters were evaluated for
toxicology animals: clinical observations, body weights, food
consumption measurements, selected haematology and selected
clinical chemistry results, liver weights, selected macroscopic and
microscopic observations, and selected hepatic gene expression
analysis. Toxicokinetic evaluation (serial profiling) was performed
on samples collected from satellite animals on Days 1 and 7. For
male rats dosed at 30 or 100 mg/kg/day, histopathology observations
carried out were on kidneys, mesentery and mandibular lymph node
only. For male rats dosed at 0 mg/kg/day (control) and 300
mg/kg/day, histopathology observations carried out were on
adrenals, brain, heart, kidneys, liver, lung, mandibular lymph
node, mesentery, stomach, testes, and thymus (except that
mandibular lymph node observations were for 0 mg/kg/day dosed rats
only).
Summary of Main Results of Toxicity Study 1
[0369] Reduced body weight gain (ca. 0.38.times. control) and food
consumption (ca. 0.80.times. pretreatment) were observed in male
rats at doses of 300 mg/kg/day. [0370] Increased body weight gain
(ca. 1.43.times. mean control) was seen in male rats at 100
mg/kg/day. [0371] A periarterial inflammatory cell infiltrate,
minimal in severity, was noted in the hilar region of the kidneys
of all four male rats treated with doses of 300 mg/kg/day, and was
unilateral (2 of 4 rats) or bilateral (2 of 4 rats). This finding
was not noted in mesenteric arteries, or any other of the arteries
which were examined, and its significance is unclear. [0372] The
kidneys of male rats dosed at 0 (vehicle), 30 and 100 mg/kg/day
were examined; no kidney hilar periarterial inflammatory cell
infiltrate was found at these doses. [0373] Cholesterol
concentrations were slightly reduced (ca. 0.65.times. mean control)
in male rats at 300 mg/kg/day. [0374] Rubbing chin on cage floor,
accompanied by chewing movements, was noted at all non-zero doses
in male rats, predominantly at 300 mg/kg/day. [0375] Certain other
observations in male rats were noted at 300 mg/kg/day or at 100 and
300 mg/kg/day doses, but were generally sporadic, transient and
predominantly slight.
[0376] In male Sprague Dawley rats, oral-repeat-dosing for 7 days
of 30 or 100 mg/kg/day of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (measured as the free base) appears to be well
tolerated. In male Sprague Dawley rats, oral-repeat-dosing for 7
days of 300 mg/kg/day of this salt (measured as the free base)
appears to be moderately well tolerated.
Toxicity Study 2: Toxicity of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine Hydrochloride in a 7-Day, Oral, Dose Range-Finding Study in
Sprague Dawley Rats
[0377]
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine hydrochloride, formulated as a suspension in 1% (w/v)
aqueous methylcellulose, was administered to groups of Sprague
Dawley rats at a dose volume of 10 mL/kg as follows (all doses and
concentrations are expressed in terms of the parent "free base"
compound): [0378] administered to male rats (4 per group) at doses
of 0 (vehicle, control) or 600 mg/kg/day once daily for up to 7
days by oral gavage (only 2 days at 600 mg/kg/day); [0379]
administered to female rats (4 per group) at doses of 0 (vehicle,
control) or 300 or 600 mg/kg/day once daily for up to 7 days by
oral gavage (only 2 days at 600 mg/kg/day).
[0380] Three male rats and three female rats were added at 600
mg/kg/day and 3 female rats were added at 300 mg/kg/day for
toxicokinetic evaluation.
[0381] The following endpoints/parameters were evaluated for
toxicology animals: clinical observations, body weights, food
consumption, selected haematology, selected clinical chemistry,
liver weights, and selected macroscopic and microscopic
observations. Toxicokinetic evaluation was performed on samples
collected on Day 1 (300 and 600 mg/kg/day dosing) and Day 7 (300
mg/kg/day dosing only).
[0382] The dose of 600 mg/kg/day was not tolerated in either male
or female Sprague Dawley rats.
[0383] For the dose of 300 mg/kg/day in female rats: [0384]
clinical signs included chewing movements, rubbing chin on the cage
floor, and certain other clinical signs; [0385] certain haematology
and other clinical chemistry parameters were increased; [0386] urea
and cholesterol concentrations were decreased (urea ca. 0.81.times.
control or mean control, and cholesterol ca. 0.52.times. control or
mean control); and [0387] liver weight was increased (ca.
1.24.times. control or mean control), although there appeared to be
no related macroscopic or microscopic observations.
[0388] Glandular dilation of the fundic region of the stomach, of
minimal severity, was observed in most female rats given doses of
300 mg/kg/day. This change was also present in two controls (one
male and one female) and its significance is unclear at this
stage.
[0389] In female Sprague Dawley rats, oral-repeat-dosing for 7 days
of 300 mg/kg/day of
1-(1-methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbonyl}pipe-
razine hydrochloride (measured as the free base) appears to be
moderately well tolerated.
Preliminary Conclusions from Toxicity Studies 1 and 2
[0390]
1-(1-Methylethyl)-4-{[4-(tetrahydro-2H-pyran-4-yloxy)phenyl]carbony-
l}piperazine hydrochloride appears to be well tolerated or
moderately well tolerated in male and female Sprague Dawley rats
after 7 days of oral-repeat-dosing at doses of up to 300 mg/kg/day
(measured as the free base).
* * * * *