U.S. patent application number 12/742302 was filed with the patent office on 2010-11-11 for amino pyrazole compound.
This patent application is currently assigned to ELI LILLY AND COMPANY. Invention is credited to Timothy Paul Burkholder, Joshua Ryan Clayton, Liandong Ma.
Application Number | 20100286139 12/742302 |
Document ID | / |
Family ID | 41630090 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286139 |
Kind Code |
A1 |
Burkholder; Timothy Paul ;
et al. |
November 11, 2010 |
AMINO PYRAZOLE COMPOUND
Abstract
The present invention provides amino pyrazole compounds useful
in the treatment of chronic myeloproliferative disorders and
various cancers, e.g., glioblastoma, breast cancer, multiple
myeloma, prostate cancer, and leukemias.
Inventors: |
Burkholder; Timothy Paul;
(Carmel, IN) ; Ma; Liandong; (Carmel, IN) ;
Clayton; Joshua Ryan; (Fishers, IN) |
Correspondence
Address: |
ELI LILLY & COMPANY
PATENT DIVISION, P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Assignee: |
ELI LILLY AND COMPANY
Indianapolis
IN
|
Family ID: |
41630090 |
Appl. No.: |
12/742302 |
Filed: |
December 8, 2009 |
PCT Filed: |
December 8, 2009 |
PCT NO: |
PCT/US09/67056 |
371 Date: |
May 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61122854 |
Dec 16, 2008 |
|
|
|
Current U.S.
Class: |
514/233.2 ;
544/117 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; C07D 487/04 20130101; A61P 35/02 20180101;
A61P 25/00 20180101 |
Class at
Publication: |
514/233.2 ;
544/117 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; C07D 413/14 20060101 C07D413/14; A61P 35/00 20060101
A61P035/00 |
Claims
1.
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(m-
orpholinomethyl)imidazo[1,2-b]pyridazin-6-amine, or a
pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 which is
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine.
3. A compound according to claim 1 which is
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine hydrochloride.
4. A method of treating chronic myeloproliferative disorders
selected from the group consisting of polycythemia vera, essential
thrombocytosis, and myelosclerosis with myeloid metaplasia in a
mammal comprising administering to a mammal in need of such
treatment an effective amount of
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(m-
orpholinomethyl)imidazo[1,2-b]pyridazin-6-amine, or a
pharmaceutically acceptable salt thereof.
5. A method of treating glioblastoma, breast cancer, multiple
myeloma, prostate cancer, and leukemias, such as atypical chronic
myeloid leukemia, primary and secondary acute myeloid leukemia,
T-lineage and B-lineage acute lymphoblastic leukemia,
myelodysplasia syndrome, and myeloproliferative disorders in a
patient comprising administering to a patient in need of such
treatment an effective amount of
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine, or a pharmaceutically
acceptable salt thereof.
6. A pharmaceutical composition comprising a compound according to
claim 3 and a pharmaceutically acceptable carrier, diluent or
excipient.
7. A compound according to claim 3 for use as a medicament.
8. A compound according to claim 3 for use in the treatment of
glioblastoma, breast cancer, multiple myeloma, prostate cancer, and
leukemias, T-lineage and B-lineage acute lymphoblastic leukemia,
myelodysplasia syndrome, and myeloproliferative disorders.
9. A compound for use according to claim 8, or a pharmaceutically
acceptable salt thereof, in the treatment of chronic
myeloproliferative disorders.
10. A method of treating conditions associated with activity of
mutant JAK2 in a patient in need thereof, which comprises
administering to said patient a compound according to claim 3.
11. A pharmaceutical composition comprising a compound according to
claim 1, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier, diluent or excipient.
12. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, for use as a medicament.
13. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, for use in the treatment of glioblastoma,
breast cancer, multiple myeloma, prostate cancer, and leukemias,
T-lineage and B-lineage acute lymphoblastic leukemia,
myelodysplasia syndrome, and myeloproliferative disorders.
14. A compound for use according to claim 13, or a pharmaceutically
acceptable salt thereof, in the treatment of chronic
myeloproliferative disorders.
15. A method of treating conditions associated with activity of
mutant JAK2 in a patient in need thereof, which comprises
administering to said patient a compound according to claim 1, or a
pharmaceutically acceptable salt thereof.
Description
[0001] Janus kinase 2 (JAK2) is a member of the tyrosine kinase
family which is involved in cytokine signaling. JAK2 has a pivotal
role in the erythropoietin (EPO) signaling pathway, including
erythrocyte differentiation and Stat5 activation. Recent studies
have demonstrated that patients with chronic myeloproliferative
disorders such as polycythemia vera, essential thrombocytosis, and
myelosclerosis with myeloid metaplasia and thrombotic disorders
such as activated protein C resistance, splanchnic vein thrombosis,
Budd-Chiari Syndrome, and portal vein thrombosis frequently have
acquired activating mutations in JAK2. The mutation, a
valine-to-phenylalanine substitution at amino acid position 617,
leads to constitutive tyrosine phosphorylation activity, by an
unknown mechanism. The constitutive activity of mutant JAK2 leads
to increased levels of phosphorylated JAK2, pSTAT5, and STAT5
transcriptional activity, which leads to the pathogenesis of
myeloproliferative disorders and leukemias, such as atypical
chronic myeloid leukemia. In addition, JAK2 is activated by
interleukin-6-depedent autocrine loop or other genetic alterations
in solid and hematologic tumors, e.g., glioblastoma, breast cancer,
multiple myeloma, prostate cancer, primary and secondary acute
myeloid leukemia, T-lineage and B-lineage acute lymphoblastic
leukemia, myelodysplasia syndrome.
[0002] Various amino pyrazole tyrosine kinase inhibitors have been
reported. See for example, WO06087538 and WO2007064797.
[0003] However, there is still a need for further compounds that
inhibit tyrosine kinases such as JAK2. The present invention
provides a novel amino pyrazole compound believed to have clinical
use for treatment of myeloproliferative disorders in which the JAK2
signaling pathway is activated or in which JAK/STAT signaling is
dysregulated.
[0004] The present invention provides
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof.
[0005] The present invention provides a method of treating chronic
myeloproliferative disorders selected from the group consisting of
polycythemia vera, essential thrombocytosis, and myelosclerosis
with myeloid metaplasia in a mammal comprising administering to a
mammal in need of such treatment an effective amount of
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof.
[0006] The present invention also provides a method of treating
glioblastoma, breast cancer, multiple myeloma, prostate cancer, and
leukemias, such as atypical chronic myeloid leukemia, primary and
secondary acute myeloid leukemia, T-lineage and B-lineage acute
lymphoblastic leukemia, myelodysplasia syndrome, and
myeloproliferative disorders in a patient comprising administering
to a patient in need of such treatment an effective amount of
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof.
[0007] The present invention also provides a pharmaceutical
composition comprising
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable carrier,
diluent or excipient.
[0008] This invention also provides
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable carrier,
diluent or excipient in combination with another therapeutic
ingredient.
[0009] This invention also provides
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof for use as a medicament. Additionally, this
invention provides use of
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof in the manufacture of a medicament for
treating chronic myeloproliferative disorders. In particular these
chronic myeloproliferative disorders are selected from the group
consisting of polycythemia vera, essential thrombocytosis, and
myelosclerosis with myeloid metaplasia. Furthermore, this invention
provides a pharmaceutical composition for treating chronic
myeloproliferative disorders selected from the group consisting of
polycythemia vera, essential thrombocytosis, and myelosclerosis
with myeloid metaplasia comprising
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine or a pharmaceutically
acceptable salt thereof as an active ingredient.
[0010] It will be understood by the skilled reader that the
compound of the present invention is capable of forming salts. The
compound of the present invention is an amine, and accordingly
reacts with any of a number of inorganic and organic acids to form
pharmaceutically acceptable acid addition salts. Such
pharmaceutically acceptable acid addition salts and common
methodology for preparing them are well known in the art. See,
e.g., P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS:
PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); L. D.
Bighley, S. M. Berge, D. C. Monkhouse, in "Encyclopedia of
Pharmaceutical Technology`. Eds. J. Swarbrick and J. C. Boylan,
Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995, pp.
453-499; S. M. Berge, et al., "Pharmaceutical Salts, "Journal of
Pharmaceutical Sciences, Vol 66, No. 1, January 1977.
[0011] The following preparations and examples are named using
ChemDraw Ultra, Version 10.0.
Scheme 1
Preparation 1
1-(4-Methoxybenzyl)-5-methyl-1H-pyrazol-3-amine
Method 1
[0012] In a 1 L round bottom flask, combine
5-amino-3-methylpyrazole (22.8 g, 234.8 mmol) and
N-methylpyrrolidone (200 mL). Cool flask to 0.degree. C. and place
under nitrogen. Add sodium hydroxide (9.39 g, 1.0 equivalent
(equiv.)) to the flask and stir for 30 minutes (min). Add a
solution of alpha-chloro-4-methoxytoluene (31 mL, 1.0 equiv.) in
N-methylpyrrolidone (100 mL) to the flask drop-wise. Let the
reaction warm to room temperature (RT) slowly overnight. Dilute the
reaction with water, and extract with ethyl acetate (EA). Wash the
organics with aqueous saturated sodium chloride. Concentrate in
vacuo. Purify on a plug of silica (hexane.fwdarw.2:1
hexane:EA.fwdarw.3:2 hexane:EA.fwdarw.1:1 hexane:EA.fwdarw.1:2
hexane:EA.fwdarw.EA). Concentrate the desired fractions to give the
title compound (10.8 g, 21%). LCMS (4 min)=218.0 (M+1).
Method 2
A. (E)-tert-Butyl 2-(4-methoxybenzylidene)hydrazinecarboxylate
[0013] Add 4-methoxybenzaldehyde (400 g, 2.94 mol) over 20 min to a
solution of tert-butyl carbazate (400 g, 2.94 mol) in toluene (750
mL) at 50.degree. C. Heat to reflux over a period of 1 hour (h),
collecting water in an azeotrope with the toluene. After no further
water is collected, cool to 60.degree. C. Add hexanes until the
product precipitates from solution. Cool the bath further to
20.degree. C. Collect the solids by filtration and dry using a
nitrogen press to afford the title compound (750.5 g, 91%). .sup.1H
NMR [400 MHz, dimethyl sulfoxide-d.sub.6 (DMSO-d.sub.6)] .delta.
10.6-10.8 (bs, 1H), 7.88-8.0 (S, 1H), 7.5-7.55 (d, 2H), 6.95-7.0
(d, 2H), 1.45 (s, 9H). ES/MS (m/z): 249 [M-H].
B. tert-Butyl 2-(4-methoxybenzyl)hydrazinecarboxylate
[0014] Add 10% palladium on carbon (water wet, 20 g) slurried in EA
(100 mL) to a sealed pressure reactor via vacuum transfer. Rinse
transfer line with a minimal amount of EA. Charge (E)-tert-butyl
2-(4-methoxybenzylidene)hydrazinecarboxylate (320 g, 1.28 mol)
dissolved in tetrahydrofuran (THF, 1000 mL) via vacuum transfer and
rinse line with a minimal amount of THF. Pressurize the reactor to
50 PSI with H.sub.2 and mix the contents of the reactor at
20.+-.10.degree. C. Continue the reaction, maintaining the hydrogen
pressure at 50 PSI, until no further hydrogen uptake is observed.
Filter the reaction solution to remove the catalyst and wash the
catalyst filter-cake with THF (500 mL). Add the wash to the
reaction filtrate. Concentrate the solution in vacuo to obtain the
title compound (337 g, 86%) as an oil. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.1-8.3 (s, 1H), 7.1-7.3 (d, 2H), 6.8-6.9 (d,
2H), 4.4-4.6 (bs, 1H), 3.7-3.8 (s, 2H), 3.6-3.7 (s, 3H), 1.3-1.5
(s, 9H).
C. (4-Methoxybenzyl)hydrazine dihydrochloride
[0015] To a solution of 4 N hydrogen chloride in dioxane (2000 mL,
8.00 mol HCl), add tert-butyl
2-(4-methoxybenzyl)hydrazinecarboxylate (324 g, 1.09 mol) dissolved
in a minimal amount of dioxane, slowly over a period of 1 h. A
precipitate gradually forms. Allow the solution to stir 16 h at
20.+-.5.degree. C. Collect the solids by filtration. Slurry the
solids in heptane (2000 mL) and isolate the solids by filtration.
Dry the solids using a nitrogen press to give the title compound
(242.3 g, 1.08 mol, 98%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 8.2-9.0 (bs, 5H), 7.3-7.4 (d, 2H), 6.8-7.0 (d, 2H), 4.0 (s,
2H), 3.7 (s, 3H).
D. 1-(4-Methoxybenzyl)-5-methyl-1H-pyrazol-3-amine and
1-(4-methoxybenzyl)-3-methyl-1H-pyrazol-5-amine
[0016] Combine potassium tert-butoxide (191.89 g, 1.71 mol) and THF
(2000 mL) at 22.degree. C. Mix until a homogeneous solution is
obtained. Cool to 5.degree. C. Add a premixed solution of
acetonitrile (84.25 g, 2.05 mol) and methyl acetate (126.7 g, 1.71
mol) to the potassium tert-butoxide solution over 45 min
maintaining a temperature less than 10.degree. C. After the
addition is complete, allow the reaction to warm to 20.+-.5.degree.
C. and stir for about 2 h. Add (4-methoxybenzyl)hydrazine
dihydrochloride (250 g) portion-wise to the reaction over about 5
min, followed by 4 N hydrogen chloride in dioxane (262.5 g, 1.00
mol) at a rate that maintains the temperature <30.degree. C.
When the addition is complete, allow to stir at 25.+-.5.degree. C.
for about 16 h. Isolate the solids by filtration and wash with THF
(500 mL). Slurry the crude solids in dichloromethane (DCM, 4 L) and
water (2 L) adjusting the pH to >10 with 5 N NaOH. Allow the
layers to settle and collect the organic phase. Wash the aqueous
phase with DCM (2 L). Combine the organic phases and dry over
anhydrous sodium sulfate and concentrate the solution to a solid in
vacuo to afford 165 g of the crude. Heat the crude in isopropyl
acetate (660 mL) to reflux to dissolve as many solids as possible.
Cool to 33.degree. C. and add hexane (600 mL) slowly over 1 h. Cool
to 10.degree. C. and maintain the temperature at 10.degree. C. for
10 min. Isolate the solids by filtration, wash with hexane (200
mL), and dry using a nitrogen press to afford a mixture of the
title compounds (91.5 g, 0.4 mol, 47%). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.2-7.3 (d, 2H), 6.7-6.9 (d, 2H), 5.1 (bs,
2H), 5.0 (s, 1H), 4.9 (s, 2H), 3.6-3.8 (s, 3H), 1.9 (s, 3H).
Note: these intermediates can be separated by chromatography;
however in this case, they are isolated as a mixture and can be
used in the final sequence below which involves removal of the
benzyl protection group resulting in the same product.
Preparation 2
2-Chloro-1-(4-chloro-2-fluorophenyl)ethanone
[0017] In a 1 L round bottom flask combine
4'-chloro-2'-fluoroacetophenone (40 g, 231.8 mmol), heptane (120
mL), and methanol (16 mL). Cool to 0.degree. C. and place under
nitrogen. Dissolve sulfuryl chloride (21.5 mL, 1.15 equiv.) in
heptane (120 mL) and charge to an addition funnel. Add drop-wise to
the reaction over 60 min. Stir for 2.5 h at 0.degree. C.; a white
precipitate forms during this time. Charge the addition funnel with
1 M sodium bicarbonate (400 mL) then add to the reaction drop-wise.
After all gas evolution stops, filter the biphasic suspension to
collect the title compound (38.18 g, 80%) as white needles. .sup.1H
NMR (DMSO-d.sub.6) .delta. 5.00 (d, 2H, J=2.5 Hz), 7.43 (m, 1H),
7.63 (m, 1H), 7.89 (t, 1H, J=8.4 Hz).
Preparation 3
(E)-N'-(6-Chloropyridazin-3-yl)-N,N-dimethylacetimidamide
[0018] In a 2 L round bottom flask combine
3-chloro-6-pyridazinamine (43.2 g, 333.5 mmol), toluene (500 mL),
and N,N-dimethylacetamide dimethyl acetal (67.8 mL, 1.25 equiv.).
Attach a reflux condenser then heat to reflux for 2 h. Let cool to
RT. Concentrate in vacuo. Triturate the crude material with hexanes
and filter to isolate the title compound (60.4 g, 91%) as a light
tan solid. MS=199.0 (M+1).
Preparation 4
(4-Chloro-2-fluorophenyl)(6-chloro-2-methylimidazo[1,2-b]pyridazin-3-yl)me-
thanone
[0019] In a round bottom flask combine
(E)-N'-(6-chloropyridazin-3-yl)-N,N-dimethylacetimidamide (36.61 g,
184.3 mmol), 2-chloro-1-(4-chloro-2-fluorophenyl)ethanone (38.15 g,
1 equiv.), and dimethylformamide (150 mL). Place under nitrogen
then heat at 120.degree. C. for 4 h. Let cool to RT and stir
overnight. Dilute with EA (1 L) and water (500 mL). Extract
organics three times with water followed by aqueous saturated
sodium chloride aqueous. Dry organics over anhydrous magnesium
sulfate. Filter and concentrate in vacuo. Purify by silica plug
(hexane.fwdarw.4:1 hexane:EA .fwdarw.3:1 hexane:EA.fwdarw.2:1
hexane:EA.fwdarw.1:1 hexane:EA) and isolate the title compound
(33.8 g, 57%) as a light green solid. LCMS (4 min=324.0, 326.0,
M+1).
Preparation 5
2-((6-Chloro-3-(4-chloro-2-fluorobenzoyl)-2-methylimidazo[1,2-b]pyridazin--
8-yl)methyl)isoindoline-1,3-dione
[0020] Combine
(4-chloro-2-fluorophenyl)(6-chloro-2-methylimidazo[1,2-b]pyridazin-3-yl)m-
ethanone (5.6 g, 17.3 mmol), N-phthaloylglycine (6.0 g, 1.7
equiv.), acetonitrile (60 mL), water (15 mL), trifluoroacetic acid
(0.26 mL, 0.2 equiv.), and silver nitrate (294 mg, 0.1 equiv.) in a
round bottom flask with attached addition funnel and place under
nitrogen. Heat to 70.degree. C. and maintain at this temperature
for 15 min. Dissolve ammonium persulfate (7.1 g, 1.8 equiv.) in
water (15 mL) and charge to an addition funnel. Add drop-wise to
the reaction flask over approximately 20 min. Heat reaction at
70.degree. C. for 1 h. A precipitate forms during this time; filter
via Buchner funnel to isolate the title compound crude (7.3 g, 87%)
as an off-white solid. LCMS (4 min)=483.0, 485.0, M+1).
Preparation 6
(8-(Aminomethyl)-6-chloro-2-methylimidazo[1,2-b]pyridazin-3-yl)(4-chloro-2-
-fluorophenyl)methanone
[0021] Combine
2-((6-chloro-3-(4-chloro-2-fluorobenzoyl)-2-methylimidazo[1,2-b]pyridazin-
-8-yl)methyl)isoindoline-1,3-dione (7.30 g, 15.1 mmol), ethanol
(200 mL), and hydrazine (1.45 mL, 3 equiv.) in a round bottom flask
and place under nitrogen. Stir for 2 days at RT. Heat for 2 h at
50.degree. C. then concentrate the reaction in vacuo. Dilute with
EA. Wash the organics with 1N HCl (aq) to pull product into the
aqueous layer. Make the aqueous layer basic with 1N NaOH (aq) and
extract with EA. Wash the EA layer with aqueous saturated sodium
chloride, and dry over anhydrous magnesium sulfate. Filter and
concentrate in vacuo to give the title compound crude (1.2 g, 23%)
as a light green solid. MS=355.0, 353.0 (M+1).
Preparation 7
(4-Chloro-2-fluorophenyl)(6-chloro-2-methyl-8-(morpholinomethyl)imidazo[1,-
2-b]pyridazin-3-yl)methanone
[0022] Combine
(8-(aminomethyl)-6-chloro-2-methylimidazo[1,2-b]pyridazin-3-yl)(4-chloro--
2-fluorophenyl)methanone (1.15 g, 3.3 mmol), water (12 mL),
potassium carbonate (495 mg, 1.1 equiv.), and 2-bromoethyl ether
(0.47 mL, 1.1 equiv) in a 20 mL microwave reaction vessel. Seal
with a crimp cap then heat in a microwave reactor at 120.degree. C.
for 20 min. Cool to RT and partition between EA and water. Wash EA
layer with aqueous saturated sodium chloride, and dry over
anhydrous magnesium sulfate. Filter and concentrate in vacuo.
Purify on silica gel (4:1 hexane:EA.fwdarw.2:1 hexane:EA.fwdarw.1:1
hexane:EA) to give the title compound (0.43 g, 31%) as a light
yellow foam. LCMS (4 min)=423.0, 425.0, M+1.
Preparation 8
(4-Chloro-2-fluorophenyl)(6-chloro-2-methyl-8-(morpholinomethyl)imidazo[1,-
2-b]pyridazin-3-yl)methanol
[0023] Combine
(4-chloro-2-fluorophenyl)(6-chloro-2-methyl-8-(morpholinomethyl)-imidazo[-
1,2-b]pyridazin-3-yl)methanone (0.43 g, 1.0 mmol) and methanol (15
mL) in a round bottom flask. Place under nitrogen and cool to
0.degree. C. Add sodium borohydride (58 mg, 1.5 equiv.) in one
portion. Stir for 5 min at this temperature then remove cooling
bath and let warm to RT. After 15 min, quench the reaction with
water then extract with EA. Wash the organics with water followed
by aqueous saturated sodium chloride. Dry the organics over
anhydrous magnesium sulfate. Filter and concentrate in vacuo to
give the title compound (0.4 g, 93%). LCMS (4 min)=425.0, 427.0,
M+1.
Preparation 9
4-((6-Chloro-3-(4-chloro-2-fluorobenzyl)-2-methylimidazo[1,2-b]pyridazin-8-
-yl)methyl)morpholine
[0024] Combine
(4-chloro-2-fluorophenyl)(6-chloro-2-methyl-8-(morpholinomethyl)-imidazo[-
1,2-b]pyridazin-3-yl)methanol (0.4 g, 0.94 mmol),
1,2-dichloroethane (25 mL), triethylsilane (0.45 mL, 3 equiv.), and
trifluoroacetic acid (0.57 mL, 8 equiv.) in a round bottom flask
and place under nitrogen. Heat at 70.degree. C. overnight.
Concentrate reaction in vacuo. Load onto a Varian MegaElut.RTM. 10
gram SCX ion exchange cartridge (prewashed with methanol). Elute
with methanol to remove non-basic impurities. Elute with 2 M
ammonia in methanol. Concentrate in vacuo to give the title
compound (0.36 g, 94%). LCMS (4 min)=409.0, 411.0, M+1.
Preparation 10
3-(4-Chloro-2-fluorobenzyl)-N-(1-(4-methoxybenzyl)-5-methyl-1H-pyrazol-3-y-
l)-2-methyl-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine
[0025] Combine
4-((6-chloro-3-(4-chloro-2-fluorobenzyl)-2-methylimidazo[1,2-b]pyridazin--
8-yl)methyl)morpholin (0.36 g, 0.88 mmol),
1-(4-methoxybenzyl)-5-methyl-1H-pyrazol-3-amine (0.248 g, 1.3
equiv.), potassium carbonate (0.30 g, 2.5 equiv.),
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.076 g, 0.15
equiv.), water (2 mL), and 1,4-dioxane (20 mL) in a round bottom
flask. Degas thoroughly with nitrogen then add
bis(dibenzylideneacetone)palladium (0.10 g, 0.2 equiv.). Attach a
reflux condenser and place under nitrogen. Heat the reaction at
reflux overnight. Pass reaction through a Celite plug. Wash the
plug with EA. Transfer to a separatory funnel and wash with water.
Wash the organic layer with aqueous sodium chloride, and dry over
anhydrous magnesium sulfate. Filter and concentrate in vacuo.
Purify on silica gel (EA.fwdarw.10% methanol:EA) to give the title
compound (0.447 g, 86%) as a pale yellow solid. LCMS (4 min)=590.2,
591.2, M+1.
EXAMPLE 1
##STR00001##
[0026]
3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-
-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine
[0027] Combine
3-(4-chloro-2-fluorobenzyl)-N-(1-(4-methoxybenzyl)-5-methyl-1H-pyrazol-3--
yl)-2-methyl-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine
(0.447 g, 0.76 mmol) and trifluoroacetic acid (10 mL) in a 20 mL
microwave reactor tube. Seal with a crimp cap then heat in a
microwave reactor at 120.degree. C. for 20 min. Partition between
EA and water that is made basic with excess NaOH aqueous. Wash the
organic phase three times with NaOH aqueous followed by aqueous
saturated sodium chloride. Dry over anhydrous magnesium sulfate.
Filter and concentrate in vacuo. Purify on silica gel
(EA.fwdarw.10% methanol:EA) to give the title compound (0.246 g,
0.52 mmol) as a pale yellow solid. LCMS (8 min)=470.0, M+1.
EXAMPLE 2
##STR00002##
[0028]
3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-
-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine
hydrochloride
[0029] Combine
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine (0.1 g, 0.21 mmol) and
1,4-dioxane (10 mL) in a pear flask and place under nitrogen. Add
hydrogen chloride (4 M in 1,4-dioxane, 0.053 mL, 1.0 equiv.) and
let stir at RT under nitrogen for 1.5 h. Concentrate in vacuo then
evaporate under vacuum from absolute ethanol two times. Dry
overnight in a vacuum oven (60.degree. C.) to give the title
compound (0.11 g, 102%). LCMS (8 min)=470.0, M+1.
Scheme 2
Preparation 11
(E)-N'-(6-chloropyridazin-3-yl)-N,N-dimethylacetimidamide
[0030] Combine 6-chloropyridazin-3-amine (1.500 kg, 11.58 mol);
1,1-dimethoxy-N,N-dimethylethanamine (2.313 kg, 17.37 mol) and
cyclopentyl methyl ether (8.25 L) then heat to 98.degree. C. while
distilling off the resulting methanol byproduct. After 4 h, the
reaction mixture is cooled to ambient temperature and heptanes
(11.2 L) is added to the reaction solution for crystallizing the
product. The title compound is collected by filtration and is
dried. (1.494 kg, 64.95%; mp=73.degree. C.)
Preparation 12
2-chloro-1-(4-chloro-2-fluorophenyl)ethanone
[0031] Stir a mixture of heptanes (1.5 L), methanol (0.4 L), and
1-(4-chloro-2-fluorophenyl)ethanone (1 kg, 5.81 mol) with cooling
to <5.degree. C. Add sulfuryl chloride (0.608 L, 1.02 kg, 7.55
mol) as a heptanes (1.5 L) solution drop-wise to the mixture
keeping the reaction temperature <15.degree. C. during the
addition. After 2 h quench the reaction at ambient temperature to a
pH of 6 with sodium hydroxide (5N, 2.0 L). Extract the reaction
mixture with methylene chloride (2 L) and concentrate the extract
to form a white solid. Filter and dry the solid.
Preparation 13
(4-chloro-2-fluorophenyl)(6-chloro-2-methylimidazo[1,2-b]pyridazin-3-yl)me-
thanone
[0032] Combine 2-chloro-1-(4-chloro-2-fluorophenyl)ethanone (1.5
kg, 5.44 mol), and
(E)-N'-(6-chloropyridazin-3-yl)-N,N-dimethylacetimidamide (1.19 kg,
5.72 mol) in DMF (10.14 L) and heat at 120.degree. C. for 5 h.
After cooling, add water (30 L) and stir to crystallize the
product. Collect the product by filtration and rinse the cake with
water (2.times.12 L) and heptanes (2.times.10 L) then dry under
vacuum to obtain title compound. (1.490 kg, 84.44%; mp=160.degree.
C., M+=324).
Preparation 14
(4-chloro-2-fluorophenyl)(6-chloro-2-methyl-8-(morpholinomethyl)imidazo[1,-
2-b]pyridazin-3-yl)methanone
[0033] Add ethanol (12 L),
(4-chloro-2-fluorophenyl)(6-chloro-2-methylimidazo[1,2-b]pyridazin-3-yl)m-
ethanone (897.70 g, 2.77 mol) and
bis(2,4-pentanedionato)-oxovanadium (IV) (146.81 g, 553.67 mmol) to
a reaction vessel with a nitrogen atmosphere. Add an ethanol (6 L)
solution of 4-methylmorpholine 4-oxide (3.89 kg, 33.21 mol)
drop-wise over 150 min keeping the reaction temperature at
23-33.degree. C.; then heat the reaction at 40.degree. C. for 48 h.
Cool the reaction and concentrate by removal of solvent (13 L).
Filter the resulting mixture, rinse the filter cake with hexane (1
L) and then dry. (728 g, 66.25%; mp 145-147.degree. C.;
M+=423).
Preparation 15
4-((6-chloro-3-(4-chloro-2-fluorobenzyl)-2-methylimidazo[1,2-b]pyridazin-8-
-yl)methyl)morpholine hydrochloride
[0034] At 26.degree. C., combine triethylsilane (110 g, 946 mmol)
and
(4-chloro-2-fluorophenyl)(6-chloro-2-methyl-8-(morpholinomethyl)imidazo[1-
,2-b]pyridazin-3-yl)methanone (50.1 g, 117.06 mmol) to form a
solution. Add trifluoro acetic acid (150 mL, 1.98 mol) to the
reaction mixture then heat at 78.degree. C. for 24 h. Cool the
reaction to ambient temperature and separate the mixture to remove
the top layer. Dissolve the bottom layer with ethyl acetate (1 L)
and adjust the pH to 11 with sodium hydroxide (4 N, 500 mL).
Separate the organic layer and add HCl (4 M in ethyl ether) to the
organic layer to form the HCl salt. Filter and dry the HCl salt.
(100 g (96%); mp=237-238.degree. C.; M+=409).
Preparation 16
3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morph-
olinomethyl)imidazo[1,2-b]pyridazin-6-amine hydrochloride and
freebase
[0035] Prepare active catalyst by combining palladium chloride (160
mg, 0.90 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(1.10 g, 1.84 mmol) in DMF (25 mL) and warming to form a solution.
Add the preformed catalyst to a solution of
3-methyl-1H-pyrazol-5-amine (3.0 g, 29.65 mmol),
4-((6-chloro-3-(4-chloro-2-fluorobenzyl)-2-methylimidazo[1,2-b]pyridazin--
8-yl)methyl)morpholine hydrochloride (9.0 g, 20.19 mmol), potassium
bicarbonate (6.0 g, 59.93 mmol) in DMF (65 mL) and heat to
150.degree. C. for 1 h. Cool the reaction to 60.degree. C. and add
mercaptopropyl functionalized silica (500 mg) and stir for 1 h then
filter to remove the silica. Cool to ambient temperature, add
2-methyltetrahydrofuran (125 mL) and extract with water to remove
DMF. Add HCl to the organic solution to form the
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl-
)-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine hydrochloride
salt. Add the HCl salt (1.1 g) to sodium hydroxide (10 mL, 1N) in
n-butanol (10 mL) and stir. Filter the resulting mixture to obtain
0.22 g of the free base, imidazo[1,2-b]pyridazin-6-amine,
3-[(4-chloro-2-fluorophenyl)methyl]-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-
-8-(4-morpholinylmethyl), (22% yield, M+1.=470).
EXAMPLE 3
Formulation of
3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine
[0036] Optionally pass
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine and excipients through
an appropriate screen. Combine and blend
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine, Pregelatinized
Starch, and Pregelatinized Starch with 5% Dimethicone using an
appropriate tumble bin (with or without intensifier bar) or other
suitable mixing equipment. Alternately, add dimethicone during
blending via a liquid addition system. Fill the blended powder into
capsules using suitable encapsulation equipment. Monitor weight
uniformity and appropriate in-process parameters during the filling
process. Optionally dedust the final capsules or polish by either
manual or automated processes.
JAK2 EPO-TF1/pSTAT5 Cell-Based Assay
Cellomics ArrayScan.RTM. HCS
[0037] JAK2 EPO-TF1/pSTAT5 cell-based assay mimics the constitutive
activation of JAK2-STAT5 in erythroid progenitor cells, which
drives the overproduction of red blood cells, a marker of
polycythemia vera (PV).
[0038] TF-1 (human erythroid leukemia) cells are maintained in RPMI
1640 (RPMI-1640 was developed by Moore et. al. at Roswell Park
Memorial Institute. The formulation is based on the RPMI-1630
series of media utilizing a bicarbonate buffering system and
alterations in the amounts of amino acids and vitamins.) with 10%
fetal bovine serum (FBS), 0.075% sodium bicarbonate, 1 mM sodium
pyruvate, 1.times. antibiotic/antimycotic (Invitrogen, Carlsbad,
Calif.) and 0.45% glucose. The medium is supplemented with GM-CSF
(granulocyte-macrophage colony-stimulating factor) at a final
concentration of 2 ng/mL. Cells are kept at 37.degree. C. with 5%
CO.sub.2. Cells are starved in serum free medium to remove
endogenous growth factors. TF-1 cells are counted and cells are
collected to seed 2.times.10.sup.7 cells per 96-well plate at a
density of 2.times.10.sup.5 cells per well. The cells are rinsed
twice with unsupplemented RPMI 1640 (RPMI 1640 with 0.075% sodium
bicarbonate, 1 mM sodium pyruvate, 1.times. antibiotic/antimycotic,
and 0.45% glucose) before suspending cells at a final concentration
of 5.times.10.sup.5 cells/mL in RPMI with 0.6% FBS. The diluted
cells are added back to tissue culture flasks and incubated
overnight at 37.degree. C. Test compounds are prepared in 100% DMSO
at 10 mM concentration. Compounds are serially diluted 1:3 with
100% DMSO in a 10 point-200.times. concentration-response range (4
mM-200 nM). In a separate 96 deep well plate 2.5 .mu.L of
200.times. compound solution is added to 125 .mu.L of complete RPMI
1640 media with 10% FBS for a 4.times. concentration compound
plate.
[0039] To perform the assay, serum-starved cells are collected and
washed once with unsupplemented RPMI 1640 medium. Cells are
suspended in 10% FBS complete RPMI medium for a final concentration
of 8.times.10.sup.5 cells/mL. An aliquot of 250 .mu.L of diluted
cells (2.times.10.sup.5 cells) are added to each well in the
4.times. concentration compound plate. Cells are mixed by vortexing
and the plate is incubated in a 37.degree. C. water bath for 10
min. A fresh 4.times. working solution of Erythropoietin (EPO) at
6.4 Units/mL is prepared by using pre-warmed 10% FBS complete RPMI
1640 medium. After the cells are treated with compound for 10 min,
125 .mu.L of EPO medium is added into each well and the plate is
vortexed. Cells are incubated in a 37.degree. C. water bath for 20
min and mixed every 5 min during the incubation time. Final 10
point concentration-response range is 20 .mu.M-1 nM at a final
concentration of DMSO at 0.5% and EPO at 1.6 U/mL. After cell
treatment, 500 .mu.L of 1% formaldehyde solution (made freshly with
phosphate-buffered saline (PBS) and kept warm at 37.degree. C.) is
added to each well. Plates are sealed and inverted 8-10 times to
mix. Plates are placed in a 37.degree. C. water bath for 10 min.
After incubation, cell plates are spun at 1200 rpm for 5 min at
room temperature (RT). The supernatant is aspirated, leaving 100
.mu.L of cells (2.times.10.sup.5 cells). The cells are vortexed and
washed twice with 800 .mu.L of PBS by repeating the spin steps and
leaving 100 .mu.L containing .about.2.times.10.sup.5 cells after
the final wash. An aliquot of 800 .mu.L of cold 90% methanol is
added to the cells and placed at -20.degree. C. overnight. Plates
are spun and methanol is removed. Cells are washed with FACS buffer
(PBS with 5% FBS and 0.02% sodium azide). An aliquot of 200 .mu.L
of 1 to 10 dilution of Mouse anti-pSTAT5 (pY694) Alexa Fluor
647.RTM. in fluorescence activated cell sorting (FACS) buffer is
added to the cells. Cells are mixed well and incubated at RT in the
dark for 2 h. Cells are washed once with PBS and 100 .mu.L of cells
are left. A working solution of 2 mg/mL Hoechst (Acros Organics,
Morris Plains, N.J.) is prepared with PBS. An aliquot of 200 .mu.L
is added to each well and cells are incubated at RT in the dark for
10 min. Cells are washed with PBS, and 50 .mu.L of Cytofix (BD
Biosciences, San Jose, Calif.) is added to the cells. The cells are
transferred to 96 well black tissue culture plates and sealed. The
plates are spun down. Mean fluorescent intensity data are collected
and analyzed using Cellomics Arrayscan.RTM. VTi. Compound treatment
is compared to the vehicle to determine percent inhibition data.
The minimum significant ratio (MSR) between two test compounds with
different IC.sub.50s is determined to be 2.2. The relative
IC.sub.50 is calculated using a 4 parameter logistic curve fitting
analysis with ActivityBase 4.0. For
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine, the IC.sub.50=0.033
.mu.M, n=4. The results of this assay demonstrate that
3-(4-chloro-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morpho-
linomethyl)imidazo[1,2-b]pyridazin-6-amine is a potent JAK2
inhibitor.
JAK3 IL-2-NK-92/pSTAT5 Cell-Based Assay
Cellomics ArrayScan.RTM. HCS
[0040] IL-2 activates the JAK3 pathway in natural killer (NK) cells
to drive the NK and CD8 lymphocyte proliferation. Therefore, IL-2
stimulated NK92/pSTAT5 cell-based assay enables the evaluation of
the JAK3 cellular activity of JAK2 compounds in vitro.
[0041] NK-92 (natural killer) cells (ATCC, Manassas, Va.) are
maintained in minimum essential medium (MEM) Alpha with 15% fetal
bovine serum, 15% Horse Serum and 1.times. antibiotic/antimycotic
(Invitrogen, Carlsbad, Calif.). The medium is supplemented with
IL-2 (R&D systems, Minneapolis, Minn.) for a final
concentration of 4 ng/mL. Cells are kept at 37.degree. C. with 5%
CO.sub.2. Cells are starved in serum free medium to remove
endogenous growth factors. NK-92 cells are counted and collected to
seed 2.times.10.sup.7 cells per 96-well plate at a density of
2.times.10.sup.5 cells per well. The cells are rinsed twice with
unsupplemented MEM Alpha (MEM Alpha) before suspending cells at a
final concentration of 8.times.10.sup.5 cells/mL in MEM Alpha with
0.6% serum (0.3% FBS, 0.3% horse serum). The diluted cells are
added back to tissue culture flasks and incubated overnight at
37.degree. C. Test compounds are prepared in 100% DMSO at 10 mM
concentration. Compounds are serially diluted 1:3 with 100% DMSO in
a 10 point-200.times. concentration-response range (4 mM-200 nM).
In a separate 96 deep well plate 2.5 .mu.L of 200.times. compound
solution is added to 125 .mu.L of 10% FBS complete RPMI 1640 medium
for a 4.times. concentration compound plate.
[0042] To perform the assay, serum-starved cells are collected and
washed once with unsupplemented RPMI 1640 medium. Cells are
suspended in 10% FBS complete RPMI 1640 medium for a final
concentration of 8.times.10.sup.5 cells/mL. An aliquot of 250 .mu.L
of diluted cells (2.times.10.sup.5 cells) is added to each well in
the 4.times. concentration compound plate. Cells are mixed by
vortexing and the plate is incubated in a 37.degree. C. water bath
for 10 min. A fresh 4.times. working solution of IL-2 at 2 ng/mL is
prepared using pre-warmed 10% FBS complete RPMI medium. After the
cells are treated with compound for 10 min, 125 .mu.L of IL-2
medium is added into each well. Cells are mixed by vortexing. Cells
are incubated in a 37.degree. C. water bath for 20 min and mixed
every 5 min during the incubation time. Final 10 point
concentration-response range is 20 .mu.M-1 nM at a final
concentration of DMSO at 0.5% and IL-2 at 0.5 ng/mL. After cell
treatment, 500 .mu.L of 1% formaldehyde solution (made freshly with
phosphate-buffered saline (PBS) and kept warm at 37.degree. C.) is
added to each well. Plates are sealed and inverted 8-10 times to
mix. Plates are placed in a 37.degree. C. water bath for 10 min.
After incubation, cell plates are spun at 1200 rpm for 5 min at RT.
The supernatant is aspirated, leaving 100 .mu.L of cells
(2.times.10.sup.5 cells). The cells are vortexed and washed twice
with 800 .mu.L of PBS by repeating the spin steps and leaving 100
.mu.L containing .about.2.times.10.sup.5 cells after the final
wash. An aliquot of 800 .mu.L of cold 90% methanol is added to the
cells and placed at -20.degree. C. overnight. Plates are spun and
methanol is removed. Cells are washed with FACS buffer (PBS with 5%
FBS and 0.02% sodium azide). An aliquot of 200 .mu.L of 1 to 10
dilution of Mouse anti-pSTAT5 (pY694) Alexa Fluor 647.RTM. in
fluorescence activated cell sorting (FACS) buffer is added to the
cells. Cells are mixed well and incubated at RT in the dark for 2
h. Cells are washed once with PBS and 100 uL of cells are left. A
working solution of 2 mg/mL Hoechst (Acros Organics, Morris Plains,
N.J.) is prepared with PBS. An aliquot of 200 .mu.L is added to
each well and cells are incubated at RT in the dark for 10 min.
Cells are washed with PBS, and 50 .mu.L of Cytofix.RTM. (BD
Biosciences, San Jose, Calif.) is added to the cells. The cells are
transferred to 96 well black tissue culture plates and sealed. The
plates are spun down. Mean fluorescent intensity data are collected
and analyzed using Cellomics Arrayscan.RTM. VTi. Compound treatment
is compared to the vehicle to determine percent inhibition data.
The MSR is determined to be 2.06. The relative IC.sub.50 is
calculated using a 4 parameter logistic curve fitting analysis with
ActivityBase 4.0. For
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine, the IC.sub.50=0.94
.mu.M, n=4. The results of the JAK3 IL2-NK92-pSTAT5 cell-based
assay demonstrate that
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8--
(morpholinomethyl)imidazo-[1,2-b]pyridazin-6-amine is a less potent
inhibitor of JAK3 (when compared to the results of the JAK2
EPO-TF1/pSTAT5 cell based assay with an IC.sub.50=0.033 uM). From
these results, the ratio of JAK3/JAK2, the IC.sub.50 was determined
to be 28.5 fold, which demonstrates
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine is a selective JAK2
inhibitor over JAK3.
Ba/F3JAK2V617F Cell-Based Assay
Cellomics ArrayScan.RTM. HCS
[0043] JAK2 target inhibition has been evaluated in Ba/F3
expressing JAK2 V617F by Western blot as reported in Wernig et al.
(Wernig G, et al. Efficacy of TG101348, a selective JAK2 inhibitor,
in treatment of a murine model of JAK2V617F-induced polycythemia
vera, Cancer Cell, April; 13(4):311-20). A medium throughput
Cellomics assay was established to evaluate the JAK2 target
inhibition in Ba/F3 cells expressing JAK2V617F. This assay enables
the discovery of an effective therapeutic agent to treat disorders
associated with JAK2V617F mutation.
[0044] Ba/F3 (murine pro-B) cells expressing JAK2V617F maintained
in RPMI 1640 with 10% FBS, 0.07% sodium bicarbonate, 1 mM sodium
pyruvate, 1.times. antibiotic/antimycotic (Invitrogen, Carlsbad,
Calif.) and 0.45% glucose (Sigma, St Louis, Mo.). Cells are kept at
37.degree. C. with 5% CO.sub.2. The test compound is prepared in
100% DMSO at 10 mM concentration. The compound is serially diluted
1:3 with 100% DMSO in a 10 point 200.times. concentration-response
range (4 mM-200 nM.). In a separate 96 deep well plate 2.5 .mu.L of
200.times. compound solution is added to 125 .mu.L of complete RPMI
1640 media with 10% FBS for a 4.times. concentration compound
plate.
[0045] To perform the assay, cells are collected and washed twice
with unsupplemented RPMI 1640. Cells are then suspended in 10% FBS
completed RPMI medium for a final concentration of
4.times.10.sup.5/mL. Next, 500 mL of cells (2.times.10.sup.5 cells)
are transferred into 96 deep well plates. Finally, 2.5 .mu.L (1:200
dilution) of compound stock solution are added to the cells and are
incubated with cells in a 37.degree. C. water bath for 60 min.
[0046] After cell treatment, 500 .mu.L of 1% formaldehyde solution
(made freshly with phosphate-buffered saline (PBS) and kept warm at
37.degree. C.) is added to each well. Plates are sealed and
inverted 8-10 times to mix. Plates are placed in a 37.degree. C.
water bath for 10 min. After incubation, cell plates are spun at
1200 rpm for 5 min at RT. The supernatant is aspirated, leaving 100
.mu.L of cells (2.times.10.sup.5 cells). The cells are vortexed and
washed twice with 800 .mu.L of PBS by repeating the spin steps and
leaving 100 .mu.L containing .about.2.times.10.sup.5 cells after
the final wash. An aliquot of 800 .mu.L of cold 90% methanol is
added to the cells and placed at -20.degree. C. overnight. Plates
are spun and methanol is removed. Cells are washed with FACS buffer
(PBS with 5% FBS and 0.0% sodium azide). An aliquot of 200 .mu.L of
1 to 10 dilution of Mouse anti-pSTAT5 (pY694) Alexa Fluor 647.RTM.
in fluorescence activated cell sorting (FACS) buffer is added to
the cells. Cells are mixed well and incubated at RT in the dark for
2 h. Cells are washed once with PBS and 100 .mu.L of cells are
left. A working solution of 2 mg/mL Hoechst (Acros Organics, Morris
Plains, N.J.) is prepared with PBS. An aliquot of 200 .mu.L is
added to each well and cells are incubated at RT in the dark for 10
min. Cells are washed with PBS, and 50 .mu.L of Cytofix.RTM. (BD
Biosciences, San Jose, Calif.) is added to the cells. The cells are
transferred to 96 well black tissue culture plates and sealed. The
plates are spun down. Mean fluorescent intensity data are collected
and analyzed using Cellomics Arrayscan.RTM. VTi. Compound treatment
is compared to the vehicle to determine percent inhibition data.
The relative IC.sub.50 is calculated using a 4 parameter logistic
curve fitting analysis with ActivityBase 4.0. For
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl-
)-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine, the
IC.sub.50=0.03 .mu.M. The results of this assay demonstrate that
3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holinomethyl)imidazo[1,2-b]pyridazin-6-amine effectively inhibits
the JAK2V617F target in Ba/F3 cells expressing JAK2V617F gene.
[0047] The compounds of the present invention are preferably
formulated as pharmaceutical compositions administered by a variety
of routes. Most preferably, such compositions are for oral
administration. Such pharmaceutical compositions and processes for
preparing same are well known in the art. See, e.g., REMINGTON: THE
SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro, et al., eds.,
19.sup.th ed., Mack Publishing Co., 1995).
[0048] The compounds of the present invention are generally
effective over a wide dosage range. For example, dosages per day
normally fall within the range of about 1 mg to about 1000 mg total
daily dose, preferably 500 mg to 1000 mg total daily dose, more
preferably 600 mg to 1000 mg total daily dose. In some instances
dosage levels below the lower limit of the aforesaid range may be
more than adequate, while in other cases still larger doses may be
employed. The above dosage range is not intended to limit the scope
of the invention in any way. It will be understood that the amount
of the compound actually administered will be determined by a
physician, in the light of the relevant circumstances, including
the condition to be treated, the chosen route of administration,
the actual compound or compounds administered, the age, weight, and
response of the individual patient, and the severity of the
patient's symptoms.
* * * * *