2,7-dimethylthiazolo[4,5-d]pyradazine-4-(5H)-thione: A corticotrophin-releasing hormone type 1 receptor agonist

Article abstract of DOI:10.1071/CH99015

Thiazoles, including 2,7-dimethylthiazolo[4,5-d]pyradazine-4-(5H)-thione (4b) and the corresponding 5-phenylthiazolo[4,5-d]pyradazine-4-methylthiol (5a), were synthesized as part of an ongoing investigation into corticotrophin-releasing hormone (CRH) type

Full text of DOI:10.1071/CH99015

C S I R O P U B L I S H I N G
Australian Journal
of Chemistry
Volume 53, 2000
© CSIRO 2000
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Aust. J. Chem., 2000, 53, 905–908
2,7-Dimethylthiazolo[4,5-d]pyradazine-4-(5H)-thione:
A Corticotrophin-Releasing Hormone Type 1 Receptor Agonist
Adam McCluskey,A,B Matthew Finn,A Maria Bowman,C Roger SmithC and Paul A. KellerD
A Medicinal Chemistry Group, Chemistry, School of Biological and Chemical Sciences,
The University of Newcastle, N.S.W. 2308.
B Author to whom correspondence should be addressed (e-mail: amcclusk@mail.newcastle.edu.au).
C Department of Endocrinology, Mothers & Babies Health Research Centre,
John Hunter Hospital, N.S.W. 2310.
D Department of Chemistry, The University of Wollongong, Wollongong, N.S.W. 2522.
Dedicated to Dr John Zdysiewicz on the occasion of his retirement
Thiazoles, including 2,7-dimethylthiazolo[4,5-d]pyradazine-4-(5H)-thione (4b) and the corresponding 5-
phenylthiazolo[4,5-d]pyradazine-4-methylthiol (5a), were synthesized as part of an ongoing investigation into
corticotrophin-releasing hormone (CRH) type 1 receptor activity. Subsequent screening indicated the successful
discovery of receptor agonists. Assay results indicated a 52 and 3% increase in β-endorphin release after the
administration of 100 µM (4b) and (5a), respectively. It is believed that this represents the first evidence of this class
of compounds displaying CRH type 1 receptor agonist activity.
Keywords: Thiazoles; agonist; CRH; β-endorphin.
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Introduction
CRH has recently been the target of a series of drug-
discovery programs,13,15 both of academic and industrial
interest. Until now, these programs have resulted in the
development of a series of small-molecule antagonists
(pyrimidines, pyrazidines, pyrazolopyrimidines, etc)16 of
CRH type 1. Antalarmin, Fig. 1, is perhaps the best known of
the CRH1 receptor antagonists, being both potent and
selective.16
Corticotrophin-releasing hormone (CRH), sometimes
known as CRF, is a 41-residue peptide, originally isolated
from ovine hypothalamus based on its ability to stimulate
adrenocorticotrophin (ACTH) and β-endorphin release from
cultured anterior-pituitary cells.1 CRH is the principal
neuroregulator of the basal and stress-induced secretion of
ACTH, β-endorphin, and other proopiomelanocortin-related
peptides from the anterior pituitary.2
In addition to its endocrine role in the regulation of the
hypothalamic-pituitary-adrenal axis, CRH is implicated in a
variety of other central and peripheral functions including
food
intake,3
thermoregulation,4
reproduction,5
inflammation,6 and cardiovascular function.7 CRH exerts its
effects by binding to a range of high-affinity membrane
receptors, of the seven transmembrane-domain family, that
are coupled, via G-coupled proteins, to second messenger-
signalling mechanisms which vary with the receptor type
and tissue.8–12 CRH has been linked to many central nervous
system disorders, anorexia nervosa, Alzheimer’s disease and
inflammatory disorders such as arthritis.13 In the brain, CRH
Recently, sulfur-containing compounds have drawn much
attention due to their significant biological and
pharmacological activities.17–22 Dury17 reported that 5-
amido-, and 5-acetylamino-4-chloropyridazinones reacted
with hydrogen sulfide to give thiazolo[4,5-d]pyridazines,
while Simiti et. al.19 reported the preparation of this
heterocyclic system from thiazoles and hydrazines.
Thiazolo[4,5-d]pyridazinones bearing C–2 amino groups
were first synthesized by Takaya et. al.20 via cyclization of
5-amino-6-chloropyridazinones with carbon disulfide,
followed by S-methylation with methyl iodide and then
amination with amines under heating. More recently,
Furukawa et. al.21,22 have developed a new route, which
produces
a
wide spectrum of autonomic and
electrophysiological effects.14 It activates the sympathetic
nervous system, with consequential increases in epinephrine,
norepinephrine and glucose levels, increased heart rate and
increased mean arterial blood pressure.13 CRH, when
injected peripherally, causes vasodilatation which reduces
blood pressure. Additionally, we have shown that levels of
plasma CRH rise exponentially in human pregnancy.12 We
have also shown that this increase can be used to predict
idiopathic pre-term labour.
Manuscript received 9 February 1999
© CSIRO 2000
10.1071/CH99015
0004-9425/00/110905

906
A. McCluskey et al.
entails heating 5-amino-4-chloropyridazinones with an
excess of methyl dithiocarbamates, yielding the
corresponding 2-aryl-aminothiazolo[4,5-d]pyridazin-7(6)-
ones.
Herein we wish to report a new lead, in the discovery of a
thiazolo[4,5-d]pyradazine-derived agonist for CRH1
receptors.
release of β-endorphin; characteristic of its antagonistic
effects. However, both (4b) and (5a), when tested at the same
concentration (100 µM), resulted in a 52 and 3% increase in
β-endorphin levels, respectively, relative to the CRH
stimulated levels. The latter result is unlikely to be
significant and is probably a function of experimental error.
Experiments with (5a) at higher concentrations failed to
show any concentration dependent response in this assay
(data not shown).
Results and Discussion
As part of ongoing studies into the prevention of pre-term
delivery in humans we have synthesized a range of small
heterocyclic molecules, including those based on the
thiazolo[4,5-d]pyridazine skeleton, as shown in Scheme 1.23
The syntheses were conducted essentially according to
the method of Makki.24 Starting from the commercially
available ethyl 5-acetyl-2-phenylthiazole-4-carboxylate (1a)
or ethyl 5-acetyl-2-methylthiazole-4-carboxylate (1b), and
treatment with hydrazine hydrate (1.1 equiv., in refluxing
ethanol, 2 h), afforded the corresponding substituted methyl
4-oxothiazolo[4,5-d]pyridazines (2a and 2b). Chlorination of
the enol with POCl3 proceeded smoothly, giving (3a and 3b)
and allowing subsequent displacement of the chlorine atom.
This displacement could be readily accomplished in a two-
stage procedure with thiourea and base. Alternatively,
treatment of (2b) with Lawesson’s reagent in refluxing
toluene gave thiol analogue (4b), quantitatively.25
Fig. 2. % Change in β-endorphin release after addition of test
compounds: (a) effects of Antalarmin at different doses on the CRH
(10 nM) stimulation of β-endorphin in cultured AtT20 cells; (b) effect
of added thiazoles (4b) and (5a) (at 100 µM concentration) on β-
endorphin concentrations in the presence of 10 nM CRH. A negative
change in β-endorphin levels indicates an antagonistic effect.
Conversely a positive change in β-endorphin levels indicates agonist
effect. Data is the average of two experiments conducted in duplicate.
Treatment of a refluxing xylene mixture of (3a) with
sodium thiomethoxide, resulted in the desired S-methyl
analogue (5a) without the potential complication of N-
methylation.
In order to assess the agonist/antagonistic effects of the
compounds synthesized, we made use of a β-endorphin
stimulation assay in mouse AtT20 cells which express type 1
receptors (see experimental).
As can be seen from Fig. 2, a 100 µM concentration of
antalarmin results in a 54% decrease in the CRH-stimulated
Conclusion
We believe that these thiazoles represent the first evidence of
the potential of small molecules to act as agonists of CRH1
receptors. We also believe that the results presented herein

2,7-Dimethylthiazolo[4,5-d]pyradazine-4-(5H)-thione
907
removed under vacuum. The residual solid was purified by silica-gel
will allow a greater understanding of CRH receptors and
should act as a stepping-stone to more potent analogues that
will be of use as pharmacological tools.
flash chromatography using CHCl /MeOH (19:1) as eluent and then
3
recrystallized from dimethyl sulfoxide (DMSO) to yield (4b), as pale
yellow needles (0.197 g, 100%), m.p. 320–322°C (Found: C, 42.3; H,
1
3.4; N, 21.6%. Calc. for C H N S : C, 42.6; H, 3.6; N, 21.3%).
H
7
7 3 2
13
Acknowledgments
n.m.r. (d -DMSO) δ 2.90, s, 3H, CH ; 2.60, s, 3H, CH . C n.m.r. (d -
6
3
3
6
DMSO) δ 158.6, 155.8, 148.6, 145.8, 136.6, 21.5, 20.1.
Adam McCluskey gratefully acknowledges the financial
assistance from the Clive & Vera Ramaciotti Foundation,
Australian Research Council and the University of
Newcastle. Roger Smith wishes to acknowledge support
from the National Health and Medical Health Research
Council.
7-Methyl-2-phenylthiazolo[4,5-d]pyridazine-4-thiomethane (5a)
A xylene solution (25 ml) of (3a) (0.261 g, 1 mmol) and sodium
thiomethoxide (0.067 g, 0.95 mmol) was heated under reflux under a
nitrogen atmosphere for 2 h, cooled and the solvent removed under
vacuum. Recrystallization from DMSO yielded tan coloured crystals
(0.205 g, 79%), m.p. 150–152°C (Found: C, 56.8; H, 3.7; N, 15.3%.
1
Calc. for C
H
N S : C, 57.1; H, 4.1; N, 15.4%). H n.m.r. (CDCl ) δ
13 11
3
2
3
Experimental
13
7.50–7.70, m, 5H, Ph; 2.95, s, 3H, CH ; 2.85, s, 3H, CH . C n.m.r.
(CDCl ) δ 174.1, 151.7, 149.1, 133.2, 132.7, 130.0, 128.9, 125.0, 22.5,
3
3
General Methods
3
1
13
21.5.
H and C nuclear magnetic resonance (n.m.r.) spectra were recorded
at 300.13 and 75.47 MHz respectively, on a Bruker Advance 300 MHz
spectrometer. Residual protonated solvent peaks were used as internal
standards. Melting points are uncorrected and were measured using a
Buchi melting point apparatus. Flash chromatography was performed
Biochemistry
β-Endorphin Stimulation Experiments
The cells were washed with Dulbecco’s modified Eagle’s medium
(DMEM) containing bovine serum albumin (0.2%, w/v) (Sigma
Chemical Company Ltd, St. Louis, U.S.A.)] and then incubated for 90
26
according to the method of Still et. al. using silica gel (Aldrich
Chemical Co. 200–400 mesh, 60Å). All solvents were distilled from
glass prior to use. Ethyl 5-acetyl-2-phenylthiazole-4-carboxylate (1a)
and ethyl 5-acetyl-2-methylthiazole-4-carboxylate (1b) were purchased
from the Maybridge Chemical Co. and used without further
purification.
min at 37°C in a humidified atmosphere of 5% CO in air. The
2
supernatant was removed and the cells were treated with 0.5 ml of
incubation medium containing the specified drug together with 10 nM
CRH (Peninsula Laboratories, Belmont, CA, U.S.A.). Following a 60
min incubation, the supernatant was collected and frozen at –80°C until
assayed.
Synthesis
AtT20 cells were cultured in multiwell plates and stimulated with
CRH (10 nM), test compounds plus 10 nM CRH, or 10 nM CRH plus
antalarmin. The cells were incubated and the level of β-endorphin
7-Methyl-2-phenyl-4-oxothiazolo[4,5-d]pyridazine (2a)
A solution of ethyl 5-acetyl-2-phenylthiazole-4-carboxylate (1a) (2.75
g, 10 mmol) in ethanol (25 ml) was heated under reflux with hydrazine
monohydrate (1.00 g) for 2 h. The ethanol was removed under vacuum,
27
stimulated in AtT20 cells, which express type 1 receptors, were
briefly recorded. A decrease in β-endorphin levels (i.e. negative) is
indicative of antagonistic activity (cf. dose response with antalarmin, a
known potent CRH type 1 antagonist) (Fig. 2); and an increase in β-
endorphin levels (i.e. positive) is indicative of agonist activity. Note in
Fig. 2, that the CRH stimulated β-endorphin release (i.e. the
concentration of β-endorphin measure above the basal level) has been
normalized to 0%, the percent increase or decrease is then reported
relative to the stimulated level.
and the resulting solid residue recrystallized from ethanol to yield white
1
needles (2.163 g, 89%), m.p. 316–318°C. H n.m.r. (CDCl ) δ 12.90, s,
3
13
1H, NH; 7.70–8.25, m, 5H, Ph; 2.64, s, 3H, CH . C n.m.r. (CDCl ) δ
3
3
174.5, 160.2, 143.4, 142.2, 136.3, 135.8, 133.7, 131.4, 115.3, 24.5.
2,7-Dimethyl-4-oxothiazolo[4,5-d]pyridazine (2b)
As for (2a), starting from ethyl 5-acetyl-2-methylthiazole-4-
carboxylate (1b) (2.13 g, 10 mmol). Recrystallization from ethanol
afforded white needles (1.81 g, 100%), m.p. 254–255°C. H n.m.r.
1
AtT20 Cell Cultures
13
(CDCl ) δ 12.94, s, 1H, NH; 2.95, s, 3H, CH ; 2.60, s, 3H, CH .
C
3
3
3
AtT20 mouse anterior-pituitary tumour cells were grown and
subcultured in DMEM (4500 mg glucose/l, with glutamine) (Gibco,
n.m.r. (CDCl ) δ 171.7, 157.4, 148.6, 140.9, 112.3, 21.6, 20.7.
3
Grand Island, NY, U.S.A.), buffered with Hepes and NaHCO (BDH
3
4-Chloro-7-methyl-2-phenylthiazolo[4,5-d]pyridazine (3a)
Chemicals) at pH 7.4, supplemented with 10% foetal bovine serum
(Cytosystems, N.S.W., Australia), 5% horse serum (Gibco) and
A suspension of (2a) (0.243 g, 1 mmol) in freshly distilled POCl (5 ml)
3
–1
–1
was heated at reflux for 1 h. The excess POCl was then removed under
3
penicillin-streptomycin (100 IU ml , 100 mg ml ). Cells were plated
in 24-well plates (nunclon–22, falcon–23) at an initial density of 50,000
cells/well and were used 3 days after subculturing (~60% confluency).
vacuum. The residual syrup was poured slowly onto finely crushed ice
(50 g) and extracted with ether (10 × 15 ml), the solvent removed and
the residual solid recrystallized from methanol to yield yellow needles
1
(0.173 g, 66%) m.p. 218–220°C. H n.m.r. (CDCl ) δ 7.65–8.25, m, 5H,
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