U.S. patent application number 17/092107 was filed with the patent office on 2021-06-24 for compounds that expand hematopoietic stem cells.
The applicant listed for this patent is NOVARTIS AG. Invention is credited to Anthony Edward Boitano, Michael Cooke, Shifeng Pan, Peter G. Schultz, John Edward Tellew, Yongqin Wan, Xing Wang.
Application Number | 20210187033 17/092107 |
Document ID | / |
Family ID | 1000005434770 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210187033 |
Kind Code |
A1 |
Boitano; Anthony Edward ; et
al. |
June 24, 2021 |
COMPOUNDS THAT EXPAND HEMATOPOIETIC STEM CELLS
Abstract
The present invention relates to compounds and compositions for
expanding the number of CD34+ cells for transplantation. The
invention further relates to a cell population comprising expanded
hematopoietic stem cells (HSCs) and its use in autologous or
allogeneic transplantation for the treatment of patients with
inherited immunodeficient and autoimmune diseases and diverse
hematopoietic disorders to reconstitute the hematopoietic cell
lineages and immune system defense.
Inventors: |
Boitano; Anthony Edward;
(Waban, MA) ; Cooke; Michael; (Brookline, MA)
; Pan; Shifeng; (San Diego, CA) ; Schultz; Peter
G.; (La Jolla, CA) ; Tellew; John Edward; (La
Jolla, CA) ; Wan; Yongqin; (San Diego, CA) ;
Wang; Xing; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVARTIS AG |
Basel |
|
CH |
|
|
Family ID: |
1000005434770 |
Appl. No.: |
17/092107 |
Filed: |
November 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15409292 |
Jan 18, 2017 |
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17092107 |
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13857939 |
Apr 5, 2013 |
9580426 |
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15409292 |
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12608946 |
Oct 29, 2009 |
8927281 |
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13857939 |
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61109821 |
Oct 30, 2008 |
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61242765 |
Sep 15, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 487/04 20130101;
A61K 35/28 20130101; C12N 5/0647 20130101; C12N 2501/60 20130101;
C07D 473/34 20130101; A61K 2035/124 20130101; C07D 495/04 20130101;
A61K 38/00 20130101; C07D 473/16 20130101; A61K 9/0019 20130101;
C12N 2501/38 20130101; A61K 31/52 20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 38/00 20060101 A61K038/00; C07D 487/04 20060101
C07D487/04; C12N 5/0789 20060101 C12N005/0789; C07D 473/34 20060101
C07D473/34; C07D 473/16 20060101 C07D473/16; C07D 495/04 20060101
C07D495/04; A61K 9/00 20060101 A61K009/00; A61K 31/52 20060101
A61K031/52 |
Claims
1. A composition comprising a cell population of expanded
hematopoietic stem cells derived from one or two cord blood units;
and an organic compound that inhibits the activity of aryl
hydrocarbon receptor (AHR antagonist); wherein the composition
contains a total number of at least 10.sup.8 cells, wherein 20-100%
of the total cells are CD34+ cells; and wherein the AHR antagonist
is of Formula I: ##STR00175## wherein: G.sub.1 is selected from N
and CR.sub.3; G.sub.2, G.sub.3 and G.sub.4 are independently
selected from CH and N; with the proviso that at least 1 of G.sub.3
and G.sub.4 is N; with the proviso that G.sub.1 and G.sub.2 are not
both N; L is selected from --NR.sub.5a(CH.sub.2).sub.0-3,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl; R.sub.1 is selected from hydrogen,
phenyl, thiophenyl, furanyl, 1H-benzoimidazolyl, isoquinolinyl,
1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, 1H-pyrazolyl,
pyridinyl, 1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl,
1H-pyrrolyl and thiazolyl; wherein said phenyl, thiophenyl,
furanyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl or
thiazolyl of R.sub.1 can be optionally substituted by 1 to 3
radicals independently selected from cyano, hydroxy,
C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkoxy,
hydroxy, amino, --C(O)R.sub.6a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.8a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen; R.sub.2 is selected from
--S(O).sub.2NR.sub.6aR.sub.6b, --NR.sub.9aC(O)R.sub.9b,
--NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl, 1H-pyrrolopyridin-3-yl,
1H-indolyl, thiophenyl, pyridinyl, 1H-1,2,4-triazolyl,
2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl and 1H-indazolyl; wherein
R.sub.6a, R.sub.6b and R.sub.6c are independently selected from
hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl or 1H-indazolyl of R.sub.2 is
optionally substituted with 1 to 3 radicals independently selected
from hydroxy, halo, methyl, methoxy, amino,
--O(CH.sub.2).sub.nNR.sub.7aR.sub.7b,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; R.sub.3 is
selected from hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is
selected from C.sub.1-10alkyl, prop-1-en-2-yl, cyclohexyl,
cyclopropyl, 2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl,
benzhydryl, tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said alkyl, cyclopropyl, cyclohexyl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from hydroxy, C.sub.1-4alkyl and
halo-substituted-C.sub.1-4 alkyl; or a salt thereof.
2. The composition of claim 1, wherein 0.1-40% of the total cells
in the composition are CD34+ Thy1+ cells.
3. The composition of claim 1, wherein 20-80% of the total cells in
the composition are CD34+ CD45RA+ cells.
4. The composition of claim 1, wherein 10-95% of the total cells in
the composition are CD38+ cells.
5. The composition of claim 1, wherein 5-70% of the total cells in
the composition are CD133+ cells.
6. The composition of claim 1, wherein the composition comprises a
pharmaceutically acceptable medium.
7. The composition of claim 1, wherein the AHR antagonist is of any
one of Formulae Ia, Ib, Ic, Id and Ie: ##STR00176## or a salt
thereof.
8. The composition of claim 1, wherein L is selected from
--NR.sub.5a(CH.sub.2).sub.0-3--,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH(CH.sub.3)CH.sub.2-- and --NR.sub.5aCH.sub.2CH(OH)--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and methyl; and R.sub.1 is selected from hydrogen, phenyl,
thiophen-2-yl, thiophen-3-yl, furan-3-yl, 1H-benzo[d]imidazol-1-yl,
isoquinolin-4-yl, 1H-imidazo[4,5-b]pyridin-1-yl,
benzo[b]thiophen-3-yl, pyrimidin-5-yl, 1H-pyrazol-4-yl,
pyridin-2-yl, pyridin-4-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl,
pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, 1H-pyrrol-2-yl and
thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl,
furan-3-yl, 1H-benzo[d]imidazol-1-yl, isoquinolin-4-yl,
1H-imidazo[4,5-b]pyridin-1-yl, benzo[b]thiophen-3-yl,
pyrimidin-5-yl, pyridinyl-2-yl, pyridin-4-yl, 1H-imidazol-1-yl,
pyrrolidin-1-yl, pyrazin-2-yl, pyridinyl-3-yl, pyridazin-4-yl,
1H-pyrrol-2-yl or thiazol-5-yl of R.sub.1 can be optionally
substituted by 1 to 3 radicals independently selected from cyano,
hydroxy, C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, --S(O).sub.0-2R.sub.8a and
--C(O)OR.sub.8a; or wherein R.sub.5a and R.sub.5b are independently
selected from hydrogen and C.sub.1-4alkyl; and R.sub.1 and R.sub.3
are not both hydrogen.
9. The composition of claim 1, wherein L is
--NR.sub.5a(CH.sub.2).sub.0-3--.
10. The composition of claim 9, wherein L is
--NR.sub.5a(CH.sub.2).sub.1-3--.
11. The composition of claim 1, wherein R.sub.2 is selected from
urea, phenyl, 1H-indol-2-yl, 1H-indol-3-yl, thiophen-3-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1H-benzo[d]imidazol-5-yl and 1H-imidazol-4-yl; wherein said phenyl,
1H-indol-2-yl, 1H-indol-3-yl, thiophen-3-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
1H-pyrazol-4-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl or
1H-benzo[d]imidazol-5-yl of R.sub.2 is optionally substituted with
hydroxy, methoxy, methyl, halo, amino and amino-sulfonyl.
12. The composition of claim 1, wherein R.sub.3 is selected from
hydrogen, methyl and biphenyl; and R.sub.4 is selected from
isopropyl, methyl, ethyl, prop-1-en-2-yl, isobutyl, cyclohexyl,
sec-butyl, (S)-sec-butyl, (R)-sec-butyl, 1-hydroxypropan-2-yl,
(S)-1-hydroxypropan-2-yl, (R)-1-hydroxypropan-2-yl, nonan-2-yl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl
and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-1-yl)ethyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl or benzyl can be optionally
substituted with 1 to 3 radicals independently selected from methyl
and trifluoromethyl.
13. The composition of claim 1, wherein the AHR antagonist is
selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethy-
l)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydro-2H-pyran-3-yl)-9H-purin-
-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-(trifluoromethyl)benzyl)-9H-purin-6--
ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isobutyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6-ylamino)e-
thyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-
-6-amine;
2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3-yl)ethyl)-
-9H-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H-purin-6-
-amine;
N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-puri-
n-6-amine;
4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-phenyl-9H-purin-6-ylamino)ethyl) phenol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl) phenol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H-purin-6-amine-
;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)
phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl--
9H-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pe-
ntanoate;
4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6-ylamino)ethyl)phen-
ol; ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotin-
ate;
4-(2-((9-isopropyl-2-(5-methoxypyridin-3-yl)-9H-purin-6-yl)amino)ethy-
l) phenol;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonitril-
e;
4-(2-(2-(1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-((9-isopropyl-2-(4-methylthiophen-3-yl)-9H-purin-6-yl)amino)et-
hyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methoxyphe-
nol;
N-[2-(6-methoxy-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-
-9H-purin-6-amine;
N-[2-(5-methyl-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-p-
urin-6-amine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)imidazo-
lidin-2-one;
N-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pyridin-
-2-amine;
9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-2-(pyridin-3-yl)-9-
H-purin-6-amine;
N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]ethyl}-9-(propan-2-yl)-2-(-
pyridin-3-yl)-9H-purin-6-amine;
1-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)imidazolidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-2-(1-benzothiophen-3-yl)-9-(pr-
opan-2-yl)-9H-purin-6-amine;
N-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)pyridin-2-amine;
2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-9H-
-purin-6-amine;
2-(1-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(pr-
opan-2-yl)-9H-purin-6-amine;
(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)u-
rea;
N-[2-(1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin--
6-amine;
N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)ph-
enyl)methane-sulfonamide;
4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol; 4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)
propyl)phenol;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheny-
l sulfamate;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrrol-2-yl)-9H-purin-6-ylamino)ethyl)
phenol;
4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(1H-benzo[d]imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(2-(2,4-dimethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-yl-
amino)ethyl)phenol;
4-(2-(9-isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)ethyl)
phenol;
5-(9-sec-butyl-6-(4-hydroxy-3-methylphenethylamino)-9H-purin-2-yl-
)nicotinonitrile;
N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9-
H-purin-6-amine;
9-isopropyl-N-(2-(5-methyl-1H-pyrazol-3-yl)ethyl)-2-(pyridin-3-yl)-9H-pur-
in-6-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin-6--
ylamino)ethyl)phenol;
5-(6-(2-(1H-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2-yl)nicotinonitr-
ile;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-pu-
rin-6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin--
6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3--
yl)-9H-purin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-p-
urin-6-amine;
5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidi-
n-4-ylamino)ethyl)phenol;
4-(2-(6-(benzo[b]thiophen-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-
-ylamino)ethyl)phenol;
(R)-4-(2-(2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-yl-
amino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-3-methylphen-
ol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitri-
le;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinoni-
trile;
4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimid-
in-4-ylamino)ethyl)phenol;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitrile;
4-(2-(9-isopropyl-2-(6-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(isoquinolin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol;
3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(5-chloro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-
-purin-6-amine;
N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-m-
ethylphenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
(S)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-pu-
rin-6-ylamino)ethyl)phenol;
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-y-
lamino)ethyl)phenol;
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propan-1-ol;
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(R)--N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofur-
an-3-yl)-9H-purin-6-amine;
4-(2-(2-(3H-imidazo[4,5-b]pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
4-(2-(2-(1H-imidazo[4,5-b]pyridin-1-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-yla-
mino)ethyl)phenol;
4-(2-(2-(4,5-dimethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(pyridin-3-yl)ethyl)-9H-purin-6-
-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)-1-h-
ydroxyethyl)phenol;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-1H-indol-3-yl)ethyl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-1H-indol-3-y-
l)ethyl)-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-1-en-2-yl)-9H-
-purin-6-amine;
5-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pyrid-
in-2-ol;
N-(2-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethyl)-2-(5-fluoropyridin-3-y-
l)-9-isopropyl-9H-purin-6-amine;
N-(2-(6-(2-(diethylamino)ethoxy)-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin--
3-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(2-methyl-1H-imidazol-1-yl)-9H-p-
urin-6-amine;
4-(2-(2-(2-ethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(2-propyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)-
ethyl) phenol;
3-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-6-ol;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-p-
urin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(7-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(4-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
N-(2-(7-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(4-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4--
ylamino)ethyl)phenol;
N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9-
H-purin-6-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(1-hydroxypropan-2-yl)-9H-purin-6-ylamin-
o)ethyl)-2-methylphenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-cyclohexyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
and
1-(2-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino)-9H-purin--
9-yl)ethyl)pyrrolidin-2-one; or a salt thereof.
14. The composition of claim 1, wherein the AHR antagonist is
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol.
15. The composition of claim 1, wherein the AHR antagonist is
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol.
16-30. (canceled)
31. An ex vivo method for increasing a number of hematopoietic stem
and progenitor cells, comprising contacting the hematopoietic stem
and progenitor cells with an agent capable of antagonizing activity
and/or expression of aryl hydrocarbon receptor.
32. The method of claim 31, wherein the hematopoietic stem cells
are derived from a human source.
33. The method of claim 31, wherein the hematopoietic stem cells
are derived from bone marrow or umbilical cord blood.
34. The method of claim 31, wherein said agent capable of
down-regulating the activity of aryl hydrocarbon receptor is (i) an
organic compound, (ii) a small interfering RNA molecule inhibiting
the expression of aryl hydrocarbon receptor, or (iii) an antisense
oligonucleotide inhibiting the expression of aryl hydrocarbon
receptor.
35. A method of expanding hematopoietic stem cells, comprising (a)
providing a starting cell population comprising hematopoietic stem
cells and (b) culturing said starting cell population ex vivo in
presence of an agent capable of antagonizing activity and/or
expression of aryl hydrocarbon receptor, under suitable conditions
for expanding hematopoietic stem cells.
36. The method of claim 35, wherein said conditions for
hematopoietic stem cell expansion include presence of a sufficient
amount of IL6, Flt3-L, TPO and SCF.
37. The method of claim 35, wherein said culturing is for about 3
days to about 90 days.
38. The method of claim 35, wherein said culturing is for about 7
to about 35 days.
39. The method of claim 35, wherein said culturing is for a time
sufficient for a 10 to 50000 fold expansion of CD34+ cells.
40. The method of claim 35, wherein said culturing is for a time
sufficient for a 10 to 10000 fold expansion of CD34+ cells.
41. The method of claim 35, wherein said starting cell population
is derived from umbilical cord blood cells.
42. The method of claim 35, wherein said starting cell population
is derived from one or two cord blood units.
43. The method of claim 35, wherein said starting cell population
is derived from mobilized peripheral blood cells.
44. The method of claim 35, wherein said starting cell population
is enriched in CD34+ cells.
45. The method of claim 35, wherein said starting cell population
consists essentially of CD34+ cells purified from one or two cord
blood units.
46. The method of claim 35, further comprising: (c) resuspending
the cell population resulting from step (b) in a pharmaceutically
acceptable medium suitable for administration to a mammalian
host.
47. The method of claim 35, wherein said agent capable of
antagonizing the activity of aryl hydrocarbon receptor is (i) an
organic compound, (ii) a small interfering RNA molecule inhibiting
the expression of aryl hydrocarbon receptor, or (iii) an antisense
oligonucleotide inhibiting the expression of aryl hydrocarbon
receptor.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/409,292 filed 18 Jan. 2017, which is a divisional
application of U.S. application Ser. No. 13/857,939 filed 5 Apr.,
2013, which is a divisional application of U.S. application Ser.
No. 12/608,946 filed 29 Oct. 2009, which claims the benefit of U.S.
Provisional Patent Application No. 61/109,821 filed 30 Oct. 2008
and U.S. Provisional Patent Application No. 61/242,765 filed 15
Sep. 2009. The full disclosures of these applications are
incorporated herein by reference in their entirety and for all
purposes.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to compounds and compositions
for expanding the number of CD34+ cells for transplantation. The
invention further relates to a cell population comprising expanded
hematopoietic stem cells (HSCs) and its use in autologous or
allogeneic transplantation for the treatment of patients with
inherited immunodeficient and autoimmune diseases and diverse
hematopoietic disorders to reconstitute the hematopoietic cell
lineages and immune system defense.
Background
[0003] Hematopoietic stem cells (HSCs) are capable of regenerating
all blood products throughout the life of an individual, balancing
their self-renewal with progeny differentiation. Hematopoietic stem
cells have therapeutic potential as a result of their capacity to
restore blood and immune cells in transplant recipients.
Furthermore, HSCs have the potential to generate cells for other
tissues such as brain, muscle and liver. Human autologous and
allogeneic bone marrow transplantation methods are currently used
as therapies for leukemia, lymphoma, and other life-threatening
diseases. For these procedures, a large number of stem cells must
be isolated to ensure that there are enough HSCs for engraftment.
The number of HSCs available for treatment is a clinical
limitation.
[0004] The present invention relates to compounds and compositions
for expanding hematopoietic stem cell populations and uses
thereof.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides a compound of
Formula I
##STR00001##
[0006] in which:
[0007] G.sub.1 is selected from N and CR.sub.3;
[0008] G.sub.2, G.sub.3 and G.sub.4 are independently selected from
CH and N; with the proviso that at least 1 of G.sub.3 and G.sub.4
is N; with the proviso that G.sub.1 and G.sub.2 are not both N;
[0009] L is selected from --NR.sub.5a(CH.sub.2).sub.0-3-- (0-3
herein means 0, 1, 2 or 3), --NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl;
[0010] R.sub.1 is selected from hydrogen, phenyl, thiophenyl,
furanyl, 1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl
and thiazolyl; wherein said phenyl, thiophenyl, furanyl,
1H-benzoimidazolyl, isoquinolinyl, 1H-imidazopyridinyl,
benzothiophenyl, pyrimidinyl, 1H-pyrazolyl, pyridinyl,
1H-imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, 1H-pyrrolyl or
thiazolyl of R.sub.1 can be optionally substituted by 1 to 3
radicals independently selected from cyano, hydroxy,
C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4 alkoxy,
hydroxy, amino, --C(O)R.sub.8a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.8a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen;
[0011] R.sub.2 is selected from --S(O).sub.2NR.sub.6aR.sub.6b,
--NR.sub.9aC(O)R.sub.9b, --NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl and 1H-indazolyl; wherein
R.sub.6a, R.sub.6b and R.sub.6c are independently selected from
hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolopyridin-3-yl, 1H-indolyl, thiophenyl, pyridinyl,
1H-1,2,4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl,
2-oxo-2,3-dihydro-1H-benzoimidazolyl or 1H-indazolyl of R.sub.2 is
optionally substituted with 1 to 3 radicals independently selected
from hydroxy, halo, methyl, methoxy, amino,
--O(CH.sub.2).sub.nNR.sub.7aR.sub.7b,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl;
[0012] R.sub.3 is selected from hydrogen, C.sub.1-4alkyl and
biphenyl; and
[0013] R.sub.4 is selected from C.sub.1-10alkyl, prop-1-en-2-yl,
cyclohexyl, cyclopropyl, 2-(2-oxopyrrolidin-1-yl)ethyl,
oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said alkyl, cyclopropyl, cyclohexyl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from hydroxy, C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or the N-oxide derivatives,
prodrug derivatives, protected derivatives, individual isomers and
mixture of isomers thereof; or the salts (preferably the
pharmaceutically acceptable salts) and solvates (e.g. hydrates) of
such compounds.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIG. 1 discloses the PXRD pattern of solid form modification
A of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol.
[0015] FIGS. 2 to 12 disclose the PXRD patterns of solid forms of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol salts, respectively the nitrate, mesylate, tosylate,
hydrochloride, sulphate, besylate, esylate, hydrobromide, orotate,
fumarate and napadysilate salts.
[0016] FIG. 13 discloses the DSC pattern of the amorphous form of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0017] "Alkyl" as a group and as a structural element of other
groups, for example halo-substituted-alkyl and alkoxy, can be
either straight-chained or branched. For example, alkyl includes
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
t-butyl, etc. C.sub.1-4-alkoxy includes methoxy, ethoxy, and the
like. Halo-substituted alkyl includes trifluoromethyl,
pentafluoroethyl, and the like.
[0018] "Aryl" means a monocyclic or fused bicyclic aromatic ring
assembly containing six to ten ring carbon atoms. For example, aryl
may be phenyl or naphthyl, preferably phenyl. "Arylene" means a
divalent radical derived from an aryl group.
[0019] "Heteroaryl" is as defined for aryl where one or more of the
ring members are a heteroatom or moiety selected from --O--,
--N.dbd., --NR--, --C(O)--, --S--, --S(O)-- or --S(O).sub.2--,
wherein R is hydrogen, C.sub.1-4alkyl or a nitrogen protecting
group. For example heteroaryl includes pyridyl, indolyl, indazolyl,
quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl,
benzothiopyranyl, benzo[1,3]dioxole, imidazolyl, benzo-imidazolyl,
pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl,
pyrazolyl, thienyl, etc.
[0020] "Cycloalkyl" means a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly
containing the number of ring atoms indicated. For example,
C.sub.3-10cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, etc. "Heterocycloalkyl" means cycloalkyl, as defined in
this application, provided that one or more of the ring carbons
indicated, are replaced by a moiety selected
from --O--, --N.dbd., --NR--, --C(O)--, --S--, --S(O)-- or
--S(O).sub.2--, wherein R is hydrogen, C.sub.1-4alkyl or a nitrogen
protecting group. For example, C.sub.3-8heterocycloalkyl as used in
this application to describe compounds of the invention includes
morpholino, pyrrolidinyl, piperazinyl, piperidinyl, piperidinylone,
2-Oxo-pyrrolidin-1-yl, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.
[0021] "Halogen" (or halo) preferably represents chloro or fluoro,
but may also be bromo or iodo.
[0022] "Hematopoietic stem cells" (HSCs) as used herein refer to
immature blood cells having the capacity to self-renew and to
differentiate into more mature blood cells comprising granulocytes
(e.g., promyelocytes, neutrophils, eosinophils, basophils),
erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes
(e.g., megakaryoblasts, platelet producing megakaryocytes,
platelets), and monocytes (e.g., monocytes, macrophages). HSCs are
interchangeably described as stem cells throughout the
specification. It is known in the art that such cells may or may
not include CD34.sup.+ cells. CD34.sup.+ cells are immature cells
that express the CD34 cell surface marker. CD34+ cells are believed
to include a subpopulation of cells with the stem cell properties
defined above. It is well known in the art that HSCs include
pluripotent stem cells, multipotent stem cells (e.g., a lymphoid
stem cell), and/or stem cells committed to specific hematopoietic
lineages. The stem cells committed to specific hematopoietic
lineages may be of T cell lineage, B cell lineage, dendritic cell
lineage, Langerhans cell lineage and/or lymphoid tissue-specific
macrophage cell lineage. In addition, HSCs also refer to long term
HSC (LT-HSC) and short term HSC (ST-HSC). ST-HSCs are more active
and more proliferative than LT-HSCs. However, LT-HSC have unlimited
self renewal (i.e., they survive throughout adulthood), whereas
ST-HSC have limited self renewal (i.e., they survive for only a
limited period of time). Any of these HSCs can be used in any of
the methods described herein. Optionally, ST-HSCs are useful
because they are highly proliferative and thus, quickly increase
the number of HSCs and their progeny. Hematopoietic stem cells are
optionally obtained from blood products. A blood product includes a
product obtained from the body or an organ of the body containing
cells of hematopoietic origin. Such sources include un-fractionated
bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph
and spleen. All of the aforementioned crude or un-fractionated
blood products can be enriched for cells having hematopoietic stem
cell characteristics in ways known to those of skill in the
art.
[0023] "Treat", "treating" and "treatment" refer to a method of
alleviating or abating a disease and/or its attendant symptoms.
[0024] "Expansion" in the context of cells refers to increase in
the number of a characteristic cell type, or cell types, from an
initial cell population of cells, which may or may not be
identical. The initial cells used for expansion may not be the same
as the cells generated from expansion.
[0025] "Cell population" refers to eukaryotic mammalian, preferably
human, cells isolated from biological sources, for example, blood
product or tissues and derived from more than one cell.
[0026] "Enriched" when used in the context of cell population
refers to a cell population selected based on the presence of one
or more markers, for example, CD34+.
[0027] The term "CD34+ cells" refers to cells that express at their
surface CD34 marker. CD34+ cells can be detected and counted using
for example flow cytometry and fluorescently labeled anti-CD34
antibodies.
[0028] "Enriched in CD34+ cells" means that a cell population has
been selected based on the presence of CD34 marker. Accordingly,
the percentage of CD34+ cells in the cell population after
selection method is higher than the percentage of CD34+ cells in
the initial cell population before selecting step based on CD34
markers. For example, CD34+ cells may represent at least 50%, 60%,
70%, 80% or at least 90% of the cells in a cell population enriched
in CD34+ cells.
[0029] "Cord blood unit" refers to the blood collected from
umbilical cord of a single birth.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The present invention relates to methods and compositions
for expanding HSC populations using an agent capable of
down-regulating the activity and/or expression of aryl hydrocarbon
receptor and/or a downstream effector of aryl hydrocarbon receptor
pathway.
[0031] In one embodiment, said agent capable of down-regulating the
activity and/or expression of aryl hydrocarbon receptor is a
compound of Formula I.
[0032] In one embodiment, with reference to compounds of Formula I,
are compounds selected from Formulae Ia, Ib, Ic, Id and Ie:
##STR00002##
[0033] in which:
[0034] L is selected from --NR.sub.5a(CH.sub.2).sub.0-3--,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH.sub.2CH(OH)-- and --NR.sub.5aCH(CH.sub.3)CH.sub.2--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and C.sub.1-4alkyl; wherein the right side of the L moiety
as shown is attached to R.sub.2, for example:
--NR.sub.5a(CH.sub.2).sub.0-3--R.sub.2,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--R.sub.2,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--R.sub.2,
--NR.sub.5a(CH.sub.2).sub.2S--R.sub.2,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--R.sub.2,
--NR.sub.5aCH.sub.2CH(OH)--R.sub.2 and
--NR.sub.5aCH(CH.sub.3)CH.sub.2--R.sub.2.
[0035] R.sub.1 is selected from hydrogen, phenyl, thiophen-2-yl,
thiophen-3-yl, furan-3-yl, 1H-benzo[d]imidazol-1-yl,
isoquinolin-4-yl, 1H-imidazo[4,5-b]pyridin-1-yl,
benzo[b]thiophen-3-yl, pyrimidin-5-yl, 1H-pyrazol-4-yl,
pyridin-2-yl, pyridin-4-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl,
pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, 1H-pyrrol-2-yl and
thiazol-5-yl;
[0036] wherein said phenyl, thiophen-2-yl, thiophen-3-yl,
furan-3-yl, 1H-benzo[d]imidazol-1-yl, isoquinolin-4-yl,
1H-imidazo[4,5-b]pyridin-1-yl, benzo[b]thiophen-3-yl,
pyrimidin-5-yl, pyridinyl-2-yl, pyridin-4-yl, 1H-imidazol-1-yl,
pyrrolidin-1-yl, pyrazin-2-yl, pyridinyl-3-yl, pyridazin-4-yl,
1H-pyrrol-2-yl or thiazol-5-yl of R.sub.1 can be optionally
substituted by 1 to 3 radicals independently selected from cyano,
hydroxy, C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, --S(O).sub.0-2R.sub.8a and
--C(O)OR.sub.8a; wherein R.sub.8a and R.sub.8b are independently
selected from hydrogen and C.sub.1-4alkyl; with the proviso that
R.sub.1 and R.sub.3 are not both hydrogen;
[0037] R.sub.2 is selected from --NR.sub.6aC(O)NR.sub.6bR.sub.6c,
phenyl, 1H-pyrrolo[2,3-b]pyridin-3-yl,
1H-pyrrolo[2,3-b]pyridin-5-yl, 1H-indol-3-yl, thiophen-3-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl and 1H-indazol-3-yl;
wherein R.sub.6a, R.sub.6b and R.sub.6c are independently selected
from hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-pyrrolo[2,3-b]pyridin-3-yl, 1H-pyrrolo[2,3-b]pyridin-5-yl,
1H-indol-3-yl, thiophen-3-yl pyridin-2-yl, pyridin-3-yl,
pyridin-4-yl, 1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl,
1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-TH-benzo[d]imidazol-5-yl or 1H-indazol-3-yl of
R.sub.2 is optionally substituted with 1 to 3 radicals
independently selected from hydroxy, halo, methoxy, amino,
--OS(O).sub.2NR.sub.7aR.sub.7b and --NR.sub.7aS(O).sub.2R.sub.7b;
wherein R.sub.7a and R.sub.7b are independently selected from
hydrogen and C.sub.1-4alkyl;
[0038] R.sub.3 is selected from hydrogen, C.sub.1-4alkyl and
biphenyl; and
[0039] R.sub.4 is selected from isopropyl, methyl, ethyl,
prop-1-en-2-yl, isobutyl, cyclohexyl, sec-butyl, (S)-sec-butyl,
(R)-sec-butyl, 1-hydroxypropan-2-yl, (S)-1-hydroxypropan-2-yl,
(R)-1-hydroxypropan-2-yl, nonan-2-yl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl
and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-1-yl)ethyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl or benzyl can be optionally
substituted with 1 to 3 radicals independently selected from
C.sub.1-4alkyl and halo-substituted-C.sub.1-4alkyl.
[0040] In another embodiment, L is selected from
--NR.sub.5a(CH.sub.2).sub.0-3--,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH(CH.sub.3)CH.sub.2-- and --NR.sub.5aCH.sub.2CH(OH)--;
wherein R.sub.5a and R.sub.5b are independently selected from
hydrogen and methyl; and R.sub.1 is selected from hydrogen, phenyl,
thiophen-2-yl, thiophen-3-yl, furan-3-yl, 1H-benzo[d]imidazol-1-yl,
isoquinolin-4-yl, 1H-imidazo[4,5-b]pyridin-1-yl,
benzo[b]thiophen-3-yl, pyrimidin-5-yl, 1H-pyrazol-4-yl,
pyridin-2-yl, pyridin-4-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl,
pyrazin-2-yl, pyridin-3-yl, pyridazin-4-yl, 1H-pyrrol-2-yl and
thiazol-5-yl; wherein said phenyl, thiophen-2-yl, thiophen-3-yl,
furan-3-yl, 1H-benzo[d]imidazol-1-yl, isoquinolin-4-yl,
1H-imidazo[4,5-b]pyridin-1-yl, benzo[b]thiophen-3-yl,
pyrimidin-5-yl, pyridinyl-2-yl, pyridin-4-yl, 1H-imidazol-1-yl,
pyrrolidin-1-yl, pyrazin-2-yl, pyridinyl-3-yl, pyridazin-4-yl,
1H-pyrrol-2-yl or thiazol-5-yl of R.sub.1 can be optionally
substituted by 1 to 3 radicals independently selected from cyano,
hydroxy, C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, --S(O).sub.0-2R.sub.8a and
--C(O)OR.sub.8a; wherein R.sub.8a and R.sub.8b are independently
selected from hydrogen and C.sub.1-4alkyl; with the proviso that
R.sub.1 and R.sub.3 are not both hydrogen.
[0041] In another embodiment, when L is
--NR.sub.5a(CH.sub.2).sub.0-3, it is preferably
--NR.sub.5a(CH.sub.2).sub.1-3 (where 1-3 herein 1, 2 or 3).
[0042] In another embodiment, R.sub.2 is selected from urea,
phenyl, 1H-indol-2-yl, 1H-indol-3-yl, thiophen-3-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1H-benzo[d]imidazol-5-yl and 1H-imidazol-4-yl; wherein said phenyl,
1H-indol-2-yl, 1H-indol-3-yl, thiophen-3-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
1H-pyrazol-4-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl or
1H-benzo[d]imidazol-5-yl of R.sub.2 is optionally substituted with
hydroxy, methoxy, methyl, halo, amino and amino-sulfonyl.
[0043] In another embodiment, R.sub.3 is selected from hydrogen,
methyl and biphenyl; and R.sub.4 is selected from isopropyl,
methyl, ethyl, prop-1-en-2-yl, isobutyl, cyclohexyl, sec-butyl,
(S)-sec-butyl, (R)-sec-butyl, 1-hydroxypropan-2-yl,
(S)-1-hydroxypropan-2-yl, (R)-1-hydroxypropan-2-yl, nonan-2-yl,
2-(2-oxopyrrolidin-1-yl)ethyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, tetrahydro-2H-pyran-2-yl, phenyl, tetrahydrofuran-3-yl
and benzyl; wherein said cyclohexyl, 2-(2-oxopyrrolidin-1-yl)ethyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-4-yl,
phenyl, tetrahydrofuran-3-yl or benzyl can be optionally
substituted with 1 to 3 radicals independently selected from methyl
and trifluoromethyl.
[0044] In another embodiment are compounds selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydro-2H-pyran-3-yl)-9H-
-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-(trifluoromethyl)benzyl)-9H-purin-6--
ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isobutyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6-ylamino)e-
thyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-
-6-amine;
2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3-yl)ethyl)-
-9H-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H-purin-6-
-amine;
N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-puri-
n-6-amine;
4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-phenyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H-purin-6-amine-
; N-benzyl-8-(biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-
-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pe-
ntanoate;
N-(2-(2-(2-(2-(4-(1-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphene-
thylamino)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy-
)ethyl)acetamide;
4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinate;
ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinate-
;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)ph-
enol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonit-
rile;
4-(2-(9-isopropyl-2-(pyrrolidin-1-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methoxyphe-
nol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methox-
yphenol;
N-[2-(6-methoxy-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-
-yl)-9H-purin-6-amine;
N-[2-(5-methyl-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-p-
urin-6-amine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)imidazo-
lidin-2-one;
N-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pyridin-
-2-amine;
9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-2-(pyridin-3-yl)-9-
H-purin-6-amine;
N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]ethyl}-9-(propan-2-yl)-2-(-
pyridin-3-yl)-9H-purin-6-amine;
1-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)imidazolidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-2-(1-benzothiophen-3-yl)-9-(pr-
opan-2-yl)-9H-purin-6-amine;
N-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)pyridin-2-amine;
2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-9H-
-purin-6-amine;
2-(1-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(pr-
opan-2-yl)-9H-purin-6-amine;
(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)u-
rea;
5-({[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}met-
hyl)-2,3-dihydro-1H-1,3-benzodiazol-2-one;
N-[2-(1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-am-
ine;
N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl-
)methane-sulfonamide;
4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)propyl)pheno-
l;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)-N-methylnicotin-
amide;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)-
ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl
sulfamate;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrrol-2-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6-ylamino)ethyl)phenol-
;
4-(2-(2-(1H-benzo[d]imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(2-(2,4-dimethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-yla-
mino)ethyl)phenol;
4-(2-(9-isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
5-(9-sec-butyl-6-(4-hydroxy-3-methylphenethylamino)-9H-purin-2-yl)-
nicotinonitrile;
N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9-
H-purin-6-amine;
9-isopropyl-N-(2-(5-methyl-1H-pyrazol-3-yl)ethyl)-2-(pyridin-3-yl)-9H-pur-
in-6-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin-6--
ylamino)ethyl)phenol;
5-(6-(2-(1H-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2-yl)nicotinonitr-
ile;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-pu-
rin-6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin--
6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3--
yl)-9H-purin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-p-
urin-6-amine;
5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidi-
n-4-ylamino)ethyl)phenol;
4-(2-(6-(benzo[b]thiophen-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-
-ylamino)ethyl)phenol;
(R)-4-(2-(2-(5-fluoropyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-yl-
amino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-3-methylphen-
ol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitri-
le;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinoni-
trile;
4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimid-
in-4-ylamino)ethyl)phenol;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)picolinonitrile;
4-(2-(9-isopropyl-2-(6-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(isoquinolin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol;
3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(5-chloro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-
-purin-6-amine;
N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-m-
ethylphenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
(S)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-pu-
rin-6-ylamino)ethyl)phenol;
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-y-
lamino)ethyl)phenol;
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propan-1-ol;
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(R)--N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofur-
an-3-yl)-9H-purin-6-amine;
4-(2-(2-(3H-imidazo[4,5-b]pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
4-(2-(2-(1H-imidazo[4,5-b]pyridin-1-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-yla-
mino)ethyl)phenol;
4-(2-(2-(4,5-dimethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)et-
hyl)phenol;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(pyridin-3-yl)ethyl)-9H-purin-6-
-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)-1-h-
ydroxyethyl)phenol;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-1H-indol-3-yl)ethyl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-1H-indol-3-y-
l)ethyl)-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-1-en-2-yl)-9H-
-purin-6-amine;
5-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pyrid-
in-2-ol;
N-(2-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethyl)-2-(5-fluoropyridin-3-y-
l)-9-isopropyl-9H-purin-6-amine;
N-(2-(6-(2-(diethylamino)ethoxy)-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin--
3-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(2-methyl-1H-imidazol-1-yl)-9H-p-
urin-6-amine;
4-(2-(2-(2-ethyl-1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(2-propyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)-
ethyl)phenol;
3-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-6-ol;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-p-
urin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(7-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(4-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
N-(2-(7-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(4-methyl-1H-indol-3-yl)ethyl)--
9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4--
ylamino)ethyl)phenol;
9-isopropyl-2-(pyridin-3-yl)-N-(2-(pyridin-4-yl)ethyl)-9H-purin-6-amine;
N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9-
H-purin-6-amine;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(1-hydroxypropan-2-yl)-9H-purin-6-ylamin-
o)ethyl)-2-methylphenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-cyclohexyl-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
and
1-(2-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino)-9H-purin--
9-yl)ethyl)pyrrolidin-2-one. Compounds of Formula I are detailed in
the Examples and Table I, infra.
[0045] In another embodiment are compounds of Formula Ia:
##STR00003##
[0046] in which:
[0047] L is selected from --NR.sub.5a(CH.sub.2).sub.0-3--,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH(CH.sub.3)CH.sub.2--, --(CH.sub.2).sub.3--,
--CH.sub.2OCH.sub.2--, --CH.sub.2NR.sub.5aCH.sub.2--,
--NR.sub.5aC(O)CH.sub.2-- and --NR.sub.5aY--; wherein R.sub.5a and
R.sub.5b are independently selected from hydrogen and
C.sub.1-4alkyl; and Y is a 5 member heteroaryl ring containing up
to 3 heteroatoms selected from O, N and S;
[0048] R.sub.1 is selected from hydrogen, phenyl, thiophen-2-yl,
thiophen-3-yl, furan-2-yl, furan-3-yl, benzo[b]thiophen-2-yl,
benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl,
pyrimidin-4-yl, pyrimidin-5-yl, 1H-pyrazol-4-yl, 1H-pyrazol-3-yl,
pyridin-2-yl, pyridazin-3-yl, pyridin-4-yl, 1H-imidazol-1-yl,
pyrrolidin-1-yl, pyrazin-2-yl, pyridin-3-yl, 1H-pyrazol-1-yl,
pyridazin-4-yl, 1H-indol-2-yl, thiazol-4-yl, 1H-indol-3-yl,
1H-pyrrol-2-yl and thiazol-5-yl; wherein said phenyl,
thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl,
benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl,
benzofuran-3-yl, pyrimidin-4-yl, pyrimidin-5-yl, 1H-pyrazol-4-yl,
1H-pyrazol-3-yl, pyridinyl-2-yl, pyridazin-3-yl, pyridin-4-yl,
1H-imidazol-1-yl, pyrrolidin-1-yl, pyrazin-2-yl, pyridinyl-3-yl,
1H-pyrazol-1-yl, pyridazin-4-yl, 1H-indol-2-yl, thiazol-4-yl,
1H-indol-3-yl, H-pyrrol-2-yl or thiazol-5-yl of R.sub.1 can be
optionally substituted by 1 to 3 radicals independently selected
from cyano, hydroxy, C.sub.1-4alkyl, C.sub.1-4alkoxy, halo,
halo-substituted-C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkoxy,
hydroxy, amino, --C(O)R.sub.8a, --S(O).sub.0-2R.sub.8a,
--C(O)OR.sub.8a and --C(O)NR.sub.8aR.sub.8b; wherein R.sub.8a and
R.sub.8b are independently selected from hydrogen and
C.sub.1-4alkyl; with the proviso that R.sub.1 and R.sub.3 are not
both hydrogen;
[0049] R.sub.2 is selected from --S(O).sub.2NR6aR6b,
--NR.sub.9aC(O)R.sub.9b, --NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl,
1H-indol-2-yl, 1H-indol-3-yl, benzo[b]thiophen-2-yl,
benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl,
thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl,
piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
1H-pyrazol-4-yl, 3-oxopiperazin-1-yl,
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl and 1H-imidazol-4-yl;
wherein R.sub.6a, R.sub.6b and R.sub.6c are independently selected
from hydrogen and C.sub.1-4alkyl; wherein said phenyl,
1H-indol-2-yl, 1H-indol-3-yl, benzo[b]thiophen-2-yl,
benzo[b]thiophen-3-yl, benzofuran-2-yl, benzofuran-3-yl,
thiophen-2-yl, thiophen-3-yl or furan-2-yl, furan-3-yl,
piperidin-4-yl, piperidin-3-yl, piperidin-2-yl, piperidin-1-yl,
pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 1H-1,2,4-triazol-3-yl,
1H-1,2,4-triazol-5-yl, 2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl,
3-oxopiperazin-1-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl or 1H-imidazol-4-yl of
R.sub.2 is optionally substituted with 1 to 3 radicals
independently selected from hydroxy, halo, methyl, methoxy, amino,
--S(O).sub.2NR.sub.7aR.sub.7b, --OS(O).sub.2NR.sub.7aR.sub.7b and
--NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are
independently selected from hydrogen and C.sub.1-4alkyl; or a
single radical selected from
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyloxy-
,
2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta-
namido)ethoxy)ethoxy and
2-(4-(4-hex-5-ynamidobenzoyl)phenylamino)-2-oxoethoxy;
[0050] R.sub.3 is selected from hydrogen, C.sub.1-4alkyl and
biphenyl; and
[0051] R.sub.4 is selected from isopropyl, isobutyl, sec-butyl,
1-hydroxypropan-2-yl, cyclopropyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said cyclopropyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from C.sub.1-4alkyl and
halo-substituted-C.sub.1-4alkyl; or the N-oxide derivatives,
prodrug derivatives, protected derivatives, individual isomers and
mixture of isomers thereof; or the pharmaceutically acceptable
salts and solvates (e.g. hydrates) of such compounds.
[0052] In a further embodiment, with reference to compounds of
Formula Ia, L is selected from --NR.sub.5a(CH.sub.2).sub.0-3--,
--NR.sub.5aCH(C(O)OCH.sub.3)CH.sub.2--,
--NR.sub.5a(CH.sub.2).sub.2NR.sub.5b--,
--NR.sub.5a(CH.sub.2).sub.2S--,
--NR.sub.5aCH.sub.2CH(CH.sub.3)CH.sub.2--,
--NR.sub.5aCH(CH.sub.3)CH.sub.2--, --(CH.sub.2).sub.3--,
--CH.sub.2OCH.sub.2--, --CH.sub.2NR.sub.5aCH.sub.2--,
--NR.sub.5aC(O)CH.sub.2-- and --NR.sub.5aY--; wherein R.sub.5a and
R.sub.5b are independently selected from hydrogen and methyl; Y is
selected from isoxazole and 1,3,4-oxadiazole.
[0053] In another embodiment, when L is
--NR.sub.5a(CH.sub.2).sub.0-3, it is preferably
--NR.sub.5a(CH.sub.2).sub.1-3 (where 1-3 herein means 1, 2 or
3).
[0054] In another embodiment, R.sub.1 is selected from hydrogen,
phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl, furan-2-yl,
benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl, pyridin-2-yl,
pyrrolidin-1-yl, 1H-pyrazol-4-yl, pyrazin-2-yl, pyridazin-3-yl,
pyridazin-4-yl, 1H-pyrazol-1-yl, 1H-pyrazol-3-yl, 1H-imidazol-1-yl,
thiazol-4-yl, 1H-pyrrol-2-yl, thiazol-5-yl, and pyridin-3-yl;
wherein said phenyl, thiophen-3-yl, thiophen-2-yl, furan-3-yl,
furan-2-yl, benzo[b]thiophen-3-yl, pyrimidin-5-yl, pyridin-4-yl,
pyridin-2-yl, pyrrolidin-1-yl, 1H-pyrazol-4-yl, pyrazin-2-yl,
pyridazin-3-yl, pyridazin-4-yl, 1H-pyrazol-1-yl, 1H-pyrazol-3-yl,
1H-imidazol-1-yl, thiazol-4-yl, 1H-pyrrol-2-yl, thiazol-5-yl or
pyridin-3-yl of R.sub.1 is optionally substituted with 1 to 3
radicals independently selected from cyano, methyl,
methyl-sulfonyl, methoxy, halo, hydroxy, carboxyl, ethoxy-carbonyl,
methyl-amino-carbonyl and amino; with the proviso that R.sub.1 and
R.sub.3 are not both hydrogen.
[0055] In another embodiment, R.sub.2 is selected from
amino-sulfonyl, methyl-carbonyl-amino, methyl-sulfonyl-amino,
amino-sulfonyl-oxy, urea, phenyl, 1H-indol-2-yl, 1H-indol-3-yl,
benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl,
benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl,
furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
3-oxopiperazin-1-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl and 1H-imidazol-4-yl;
wherein said phenyl, 1H-indol-2-yl, 1H-indol-3-yl,
benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, benzofuran-2-yl,
benzofuran-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl,
furan-3-yl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
piperidin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl,
2-oxoimidazolidin-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl,
3-oxopiperazin-1-yl, 2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl,
1,2,3,4-tetrahydronaphthalen-2-yl, indolin-5-yl, 2-oxoindolin-5-yl,
1H-benzo[d]imidazol-5-yl, 1H-indazol-5-yl and 1H-imidazol-4-yl of
R.sub.2 is optionally substituted with hydroxy, methoxy, methyl,
halo, amino, amino-sulfonyl,
5-((3aS,4S,6aR)-2-oxohexahydro-H-thieno[3,4-d]imidazol-4-yl)pentanoyloxy,
2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentan-
amido)ethoxy)ethoxy and
2-(4-(4-hex-5-ynamidobenzoyl)phenylamino)-2-oxoethoxy.
[0056] In another embodiment, R.sub.3 is selected from hydrogen,
methyl, and biphenyl; and R.sub.4 is selected from isopropyl,
isobutyl, sec-butyl, 1-hydroxypropan-2-yl, cyclopropyl,
oxetan-3-yl, oxetan-2-yl, benzhydryl, piperidin-4-yl,
piperidin-3-yl, piperidin-2-yl, tetrahydro-2H-pyran-2-yl,
tetrahydro-2H-pyran-3-yl, tetrahydro-2H-pyran-4-yl, phenyl,
tetrahydrofuran-3-yl, tetrahydrofuran-2-yl, benzyl,
(4-pentylphenyl)(phenyl)methyl and
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl; wherein said cyclopropyl, oxetan-3-yl, oxetan-2-yl,
benzhydryl, piperidin-4-yl, piperidin-3-yl, piperidin-2-yl,
tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl,
tetrahydro-2H-pyran-4-yl, phenyl, tetrahydrofuran-3-yl,
tetrahydrofuran-2-yl, benzyl, (4-pentylphenyl)(phenyl)methyl or
1-(1-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)et-
hyl can be optionally substituted with 1 to 3 radicals
independently selected from methyl and trifluoromethyl.
[0057] In another embodiment are compounds selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydro-2H-pyran-3-yl)-9H-
-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-(trifluoromethyl)benzyl)-9H-purin-6--
ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isobutyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-methyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(4-methylbenzyl)-9H-purin-6-ylamino)e-
thyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-
-6-amine;
2-(benzo[b]thiophen-3-yl)-9-isopropyl-N-(2-(thiophen-3-yl)ethyl)-
-9H-purin-6-amine;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-isopropyl-9H-purin-6-
-amine;
N-(4-aminophenethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-puri-
n-6-amine;
4-(2-(9-isopropyl-2-(pyrimidin-5-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-phenyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(thiophen-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(furan-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
2-(benzo[b]thiophen-3-yl)-N-(4-fluorophenethyl)-9-phenyl-9H-purin-6-amine-
; N-benzyl-8-(biphenyl-4-yl)-9-isopropyl-9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-((4-pentylphenyl)(phenyl)methyl)--
9H-purin-6-ylamino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-
-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)e-
thyl)phenol;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-ol;
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pe-
ntanoate;
N-(2-(2-(3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6--
ylamino)ethyl)-1H-indol-5-yloxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahyd-
ro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide;
N-(4-(4-(2-(3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamin-
o)ethyl)-1H-indol-5-yloxy)acetamido)benzoyl)phenyl)hex-5-ynamide;
N-(2-(2-(2-(2-(4-(1-(2-(benzo[b]thiophen-3-yl)-6-(4-hydroxyphenethylamino-
)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)ac-
etamide;
4-(2-(9-isopropyl-2-(pyridin-4-yl)-9H-purin-6-ylamino)ethyl)pheno-
l; ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotina-
te; ethyl
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotin-
ate;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(9-isopropyl-2-(2-methoxypyridin-3-yl)-9H-purin-6-ylamino)et-
hyl)phenol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonitrile;
4-(2-(9-isopropyl-2-(pyrrolidin-1-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(1H-pyrazol-1-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(1H-imidazol-1-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridazin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridazin-4-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyrazin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(pyridin-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-ylamino)e-
thyl)phenol;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(2-chloropyridin-3-yl)-6-isopropyl-2,6-dihydroimidazo[4,5-c]pyr-
azol-3-ylamino)ethyl)phenol;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(4-methoxypyridin-3-yl)-9H-purin-6-ylamino)ethyl)ph-
enol;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(thiazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol-
;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(1H-pyrazol-3-yl)-9H-purin-6-ylamino)ethyl)phe-
nol;
4-(2-(9-isopropyl-2-(1H-pyrazol-4-yl)-9H-purin-6-ylamino)ethyl)phenol-
;
4-(2-(9-isopropyl-2-(thiophen-2-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)thiophene-2-carb-
oxylic acid;
4-(2-(2-(furan-2-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(4-methylthiophen-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-methoxy-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-met-
hoxyphenol;
N-[2-(6-methoxy-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H--
purin-6-amine;
N-[2-(5-methyl-1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-p-
urin-6-amine;
N-[2-(piperidin-4-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-a-
mine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pi-
peridin-4-ol; methyl
(2S)-3-(4-hydroxyphenyl)-2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6--
yl]amino}propanoate;
4-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)benzene-
-1-sulfonamide;
2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethane-1-
-sulfonamide;
4-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)benzene-
-1,2-diol;
N-[2-(1H-imidazol-4-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)--
9H-purin-6-amine;
1-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)imidazo-
lidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-y-
l)-9H-purin-6-amine;
N-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)pyridin-
-2-amine;
9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-2-(pyridin-3-yl)-9-
H-purin-6-amine;
N-[2-({[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}methyl)propy-
l]acetamide;
4-(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)piperaz-
in-2-one;
N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]ethyl}-9-(propan--
2-yl)-2-(pyridin-3-yl)-9H-purin-6-amine;
N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(propan-2-yl)-2-(pyridin-3-y-
l)-9H-purin-6-amine;
(2-{[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}ethyl)urea;
5-({[9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-yl]amino}methyl)-2,3-dih-
ydro-1H-1,3-benzodiazol-2-one;
2-(1-benzothiophen-3-yl)-N-[2-(1H-imidazol-4-yl)ethyl]-9-(propan-2-yl)-9H-
-purin-6-amine;
1-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)imidazolidin-2-one;
N-[2-(5-amino-1H-1,2,4-triazol-3-yl)ethyl]-2-(1-benzothiophen-3-yl)-9-(pr-
opan-2-yl)-9H-purin-6-amine;
N-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)pyridin-2-amine;
2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-N-[3-(1H-pyrazol-4-yl)propyl]-9H-
-purin-6-amine;
N-[2-({[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}meth-
yl)propyl]acetamide;
4-(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl-
)piperazin-2-one;
2-(1-benzothiophen-3-yl)-N-{2-[(3-methyl-1H-1,2,4-triazol-5-yl)sulfanyl]e-
thyl}-9-(propan-2-yl)-9H-purin-6-amine;
2-(1-benzothiophen-3-yl)-N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)propyl]-9-(pr-
opan-2-yl)-9H-purin-6-amine;
(2-{[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}ethyl)u-
rea;
5-({[2-(1-benzothiophen-3-yl)-9-(propan-2-yl)-9H-purin-6-yl]amino}met-
hyl)-2,3-dihydro-1H-1,3-benzodiazol-2-one;
N-[2-(1H-indol-3-yl)ethyl]-9-(propan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-am-
ine;
N-(4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl-
)methanesulfonamide;
4-(2-(2-(pyridin-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)propyl)pheno-
l;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)-N-methylnicotin-
amide;
6-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)-5,6,7,8-tetrahy-
dronaphthalen-2-ol;
N-(2-(1H-indazol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amin-
e;
4-(2-((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)(methyl)amino)ethyl)p-
henol;
4-(2-(9-isopropyl-8-methyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethy-
l)phenol;
1-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-1H--
benzo[d]imidazol-2(3H)-one;
4-(3-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)propyl)phenol;
4-((((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)methyl)(methyl)amino)met-
hyl)phenol;
4-(((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)methylamino)methyl)phenol-
;
4-(((9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)methoxy)methyl)phenol;
N-(2-(indolin-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-amine;
4-(2-(9-(1-methylpiperidin-4-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethy-
l)phenol;
4-(2-(9-(piperidin-4-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
N-(2-(1H-indazol-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-pu-
rin-6-amine;
N-(2-(1H-benzo[d]imidazol-5-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-pur-
in-6-amine;
5-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)indolin-2-one-
;
4-(2-(9-cyclopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenyl
sulfamate;
2-(4-hydroxyphenyl)-N-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-yl)acetami-
de;
4-(5-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)isoxazol-3-yl)ph-
enol;
4-(5-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)-1,3,4-oxadiaz-
ol-2-yl)phenol;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(1-methyl-1H-pyrrol-2-yl)-9H-purin-6-ylamino)ethyl)-
phenol; and
4-(2-(9-isopropyl-2-(thiazol-5-yl)-9H-purin-6-ylamino)ethyl)phenol.
[0058] In another embodiment is a compound of formula 1f:
##STR00004##
[0059] in which: R.sub.2 is selected from TH-indol-3-yl and phenyl
optionally substituted with hydroxy; and R.sub.4 is selected from
isopropyl, sec-butyl, benzhydryl, nonan-2-yl, oxetan-3-yl and
tetrahy drofuran-3-yl.
[0060] In a further embodiment are compounds selected from:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-benzhydryl-2-(benzo[b]thiophen-3-yl)-9H-purin-6-ylamino)eth-
yl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropy-
l-9H-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(nonan-2-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-
-purin-6-amine;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
(S)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-pu-
rin-6-ylamino)ethyl)phenol; and
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-y-
lamino)ethyl)phenol.
[0061] In another embodiment is a compound of formula 1g:
##STR00005##
[0062] in which: R.sub.2 is selected from:
H-pyrrolo[2,3-b]pyridin-3-yl; H-indol-3-yl optionally substituted
with 1 to 2 radicals independently selected from halo, methyl and
methoxy; and phenyl optionally substituted with 1 to 2 radicals
independently selected from methyl, halo and hydroxy; R.sub.4 is
selected from isopropyl, sec-butyl, 1-hydroxypropan-2-yl,
prop-1-en-2-yl, benzhydryl, nonan-2-yl, oxetan-3-yl and
tetrahydrofuran-3-yl; and Ra, Rb and Rc are independently selected
from hydrogen, cyano, methyl, halo, --SO.sub.2CH.sub.3 and
trifluoromethyl.
[0063] In a further embodiment are compounds selected from:
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(6-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(9-isopropyl-2-(4-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)phe-
nol;
5-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(9-isopropyl-2-(5-(methylsulfonyl)pyridin-3-yl)-9H-purin-6-yla-
mino)ethyl)phenol;
4-(2-(2-(4-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
4-(2-(2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phe-
nol;
9-isopropyl-N-(2-(6-methoxy-1H-indol-3-yl)ethyl)-2-(pyridin-3-yl)-9H--
purin-6-amine;
9-isopropyl-N-(2-(5-methyl-1H-indol-3-yl)ethyl)-2-(pyridin-3-yl)-9H-purin-
-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-(oxetan-3-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-
phenol;
4-(2-(9-(1-hydroxypropan-2-yl)-2-(pyridin-3-yl)-9H-purin-6-ylamino-
)ethyl)phenol;
4-(2-(2-(2-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l;
5-(9-sec-butyl-6-(4-hydroxy-3-methylphenethylamino)-9H-purin-2-yl)nicot-
inonitrile;
4-(2-(2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-purin-6-ylamino)ethyl)p-
henol;
4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol;
4-(2-(9-isopropyl-2-(5-(trifluoromethyl)pyridin-3-yl)-9H-purin-6--
ylamino)ethyl)phenol;
5-(6-(2-(1H-indol-3-yl)ethylamino)-9-sec-butyl-9H-purin-2-yl)nicotinonitr-
ile;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-pu-
rin-6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-p-
urin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin--
6-amine;
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3--
yl)-9H-purin-6-amine;
(S)--N-(2-(1H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-p-
urin-6-amine;
5-(6-(4-hydroxyphenethylamino)-9-(oxetan-3-yl)-9H-purin-2-yl)nicotinonitr-
ile;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidi-
n-4-ylamino)ethyl)phenol;
3-(6-(4-hydroxyphenethylamino)-9-isopropyl-9H-purin-2-yl)isonicotinonitri-
le;
4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin--
4-ylamino)ethyl)phenol;
4-(2-(9-isopropyl-2-(6-methylpyridin-3-yl)-9H-purin-6-ylamino)ethyl)pheno-
l;
2-chloro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)ph-
enol;
3-fluoro-4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl-
)phenol;
N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)--
9H-purin-6-amine;
N-(2-(5-chloro-1H-indol-3-yl)ethyl)-9-isopropyl-2-(pyridin-3-yl)-9H-purin-
-6-amine;
4-(2-(9-isopropyl-2-(pyridin-3-yl)-9H-purin-6-ylamino)ethyl)-2-m-
ethylphenol;
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propan-1-ol;
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol;
(R)--N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(tetrahydrofur-
an-3-yl)-9H-purin-6-amine;
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methoxy-TH-indol-3-y-
l)ethyl)-9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(5-methoxy-TH-indol-3-yl)ethyl)-
-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(prop-1-en-2-yl)-9H-
-purin-6-amine;
N-(2-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-iso-
propyl-9H-purin-6-amine;
4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-yla-
mino)ethyl)phenol;
N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin--
6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(7-methyl-1H-indol-3-yl-
)ethyl)-9H-purin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)-9-(oxetan-3-yl)-9H-pu-
rin-6-amine;
N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-TH-indol-3-yl)ethyl)--
9H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(6-methyl-TH-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(4-fluoro-H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9-
H-purin-6-amine;
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(4-methyl-TH-indol-3-yl)ethyl)--
9H-purin-6-amine;
N-(2-(7-fluoro-H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9-
H-purin-6-amine; and
4-(2-(2-(5-fluoropyridin-3-yl)-9-(1-hydroxypropan-2-yl)-9H-purin-6-ylamin-
o)ethyl)-2-methylphenol.
[0064] In another embodiment is a method of using a compound of
Formula I to stimulate the expansion of stem cells by increasing
the number of divisions; said method comprising contacting the stem
cells with a compound of Formula I.
[0065] In another embodiment is a method in which the expansion of
stem cells is in vivo, in vitro, or ex vivo.
[0066] In another embodiment is a method in which the stem cells
are human hematopoietic stem cells.
[0067] In another embodiment is a cell population with expanded
hematopoietic stem cells, as obtained or obtainable by the method
of the invention.
[0068] In a further embodiment is a composition comprising a cell
population with expanded HSCs derived from one or two cord blood
units, preferably one cord blood unit, wherein said composition
contains a total amount of cells of at least 10.sup.5 cells,
10.sup.7 cells, 10.sup.8 cells or 10.sup.9 cells, and wherein
between 20-100% of total cells are CD34+ cells, for example between
40-80% of total cells are CD34+.
[0069] In another embodiment is a method for treating a disease or
disorder for which stem cell therapy would result in the
prevention, treatment or eradication of said disorder.
[0070] It is anticipated that as stem cell use progresses the
diseases that can be treated by stem cell transplantation will
expand. A non-limiting list of examples follows, infra.
[0071] In another embodiment is the use of a compound of Formula I
as defined in the Summary of the Invention, or a salt thereof, in
the preparation of a composition for the treatment of an inherited
immunodeficient disease, an autoimmune disease and/or a
hematopoietic disorder.
[0072] In a further embodiment, the administration is an autologous
transplantation and the hematopoietic disorder is selected from
Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Acute
myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune
disorders and Amyloidosis.
[0073] In a further embodiment, the autoimmune disorders are
selected from Systemic lupus erythematosus (SLE) and systemic
sclerosis.
[0074] In a further embodiment, the administration is an allogeneic
transplantation and the hematopoietic disorder is selected from
Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic
myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative
disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin
lymphoma, Hodgkin disease, Aplastic anemia, Pure red cell aplasia,
Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia
major, Sickle cell anemia, Severe combined immunodeficiency (SCID),
Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH)
and inborn errors of metabolism.
[0075] In a further embodiment, the inborn errors of metabolism are
selected from mucopolysaccharidosis, Gaucher disease, metachromatic
leukodystrophies and adrenoleukodystrophies.
[0076] In another embodiment is a method for treating an inherited
immunodeficient disease, an autoimmune disease and/or a
hematopoietic disorder comprising administration to a patient in
need of such treatment hematopoietic stem cells expanded by a
compound as described in the Summary of the Invention.
[0077] In a further embodiment, the administration is an autologous
transplantation and the hematopoietic disorder is selected from
Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Acute
myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune
disorders and Amyloidosis.
[0078] In a further embodiment, the autoimmune disorders are
selected from Systemic lupus erythematosus (SLE) and systemic
sclerosis.
[0079] In a further embodiment, the administration is an allogeneic
transplantation and the hematopoietic disorder is selected from
Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic
myeloid leukemia, Chronic lymphocytic leukemia, Myeloproliferative
disorders, Myelodysplastic syndromes, Multiple myeloma, Non-Hodgkin
lymphoma, Hodgkin disease, Aplastic anemia, Pure red cell aplasia,
Paroxysmal nocturnal hemoglobinuria, Fanconi anemi, Thalassemia
major, Sickle cell anemia, Severe combined immunodeficiency (SCID),
Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH)
and inborn errors of metabolism.
[0080] In a further embodiment, the inborn errors of metabolism are
selected from mucopolysaccharidosis, Gaucher disease, metachromatic
leukodystrophies and adrenoleukodystrophies.
Utility
[0081] HSCs are primitive cells capable of regenerating all blood
cells. During development, hematopoiesis translocates from the
fetal liver to the bone marrow, which then remains the site of
hematopoiesis throughout adulthood. Once hematopoiesis has been
established in the bone marrow, the HSCs are not distributed
randomly throughout the bone cavity. Instead, they are found in
close proximity to the endosteal surfaces. The more mature stem
cells increase in number as the distance from the bone surface
increases. Finally, as the central longitudinal axis of the bone is
approached terminal differentiation of mature cells occurs.
[0082] Expanding the number of stem cells, whether from adult,
umbilical cord blood, fetal, or embryonic sources, would have a
huge impact on transplantation and other therapies for hematology
and oncology diseases and disorders, the least of which would be
increased safety and reduced costs. As described in the methods
herein, HSC numbers are increased ex vivo. A method of increasing
stem cell numbers is important as currently, approximately 25% of
autologous donor transplants are prohibited for lack of sufficient
stem cells. In addition, less than 25% of patients in need of
allogeneic transplant can find a histocompatible donor. Umbilical
cord blood banks currently exist and cover the broad racial make-up
of the general population, but these banks are currently restricted
to use in children due to inadequate stem cell numbers in the
specimens for adult recipients. A method to increase stem cell
numbers permits cord blood to be useful for adult patients, thereby
expanding the use of allogeneic transplantation. Compounds of the
invention can also be used to expand the progenitor cell numbers
which are clinically useful, for example, to speed engraftment and
decrease the duration of neutopenia.
[0083] Accordingly, a method for increasing the number of HSCs is
provided. As used herein, an increase in HSCs means that the
subject has at least one more HSC, a 10% increase, a 20% increase,
a 30% increase or greater. HSCs may consist of a subset of CD34+
cells, increase of HSCs can be measured indirectly by counting the
number of CD34+ cells in a cell population and, optionally, by
assessing the differentiation properties of the CD34+ cells by
analyzing the colony forming units (CFU) as described in the
experimental part below: An increase of the number of CD34+ cells
culture of a least 10%, preferably 20% increase or 30% increase or
greater as compared with a control without expansion is indicative
of HSC expansion. The expanded population of HSCs is harvested, for
example, from a bone marrow sample of a subject or from a culture.
Harvesting HSCs is defined as the dislodging or separation of
cells. This is accomplished using a number of methods, such as
enzymatic, non-enzymatic, centrifugal, electrical, or size-based
methods, or preferably, by flushing the cells using culture media
(e.g., media in which cells are incubated) or buffered solution.
The cells are optionally collected, separated, and further expanded
generating even larger populations of HSCs and differentiated
progeny.
[0084] A method for making an expanded population of HSCs comprises
contacting an agent capable of down-regulating the activity and/or
expression of AHR and/or a downstream effector of AHR, e.g., a
compound of the invention, with a starting cell population (i.e.,
an unexpanded population of cells) comprising a mixture of HSCs and
optionally HSC supporting cells. The administration step occurs ex
vivo, in vivo and/or in vitro. As described herein, the expanded
population of HSCs is optionally administered to a subject. For ex
vivo expansion, such agent for HSC expansion, e.g. a compound of
the invention, may be formulated in DMSO or some other suitable
carrier, "washed" from the cells and the cells may be transferred,
for example, into an infusion buffer. A DMSO formulation, for
example, can contain 0.3 mg/ml of a compound of the invention in
60% DMSO/40% water solution. Thus, provided are methods of
providing an expanded population of HSCs to a subject comprising
administering to the subject the expanded population of HSCs
described herein or made by the methods described herein. The
expanded population of HSCs is optionally used to make blood cells.
The blood cells are optionally administered to a subject in need.
Optionally, the subject is the same subject from which the
unexpanded population of HSCs or mixture of HSCs and HSC supporting
cells was derived.
[0085] As used herein, the term HSC supporting cell refers to cells
naturally found in the vicinity of one or more HSCs such that
factors released by HSC supporting cells reach the HSC by
diffusion, for example. HSC supporting cells include, but are not
limited to, lymphoreticular stromal cells. Lymphoreticular stromal
cells as used herein include, but are not limited to, all cell
types present in a lymphoid tissue which are not lymphocytes or
lymphocyte precursors or progenitors. Thus, lymphoreticular stromal
cells include osteoblasts, epithelial cells, endothelial cells,
mesothelial cells, dendritic cells, splenocytes and macrophages.
Lymphoreticular stromal cells also include cells that would not
ordinarily function as lymphoreticular stromal cells, such as
fibroblasts, which have been genetically altered to secrete or
express on their cell surface the factors necessary for the
maintenance, growth or differentiation of HSCs, including their
progeny. Lymphoreticular stromal cells are optionally derived from
the disaggregation of a piece of lymphoid tissue. Such cells are
capable of supporting in vitro or in vivo the maintenance, growth
or differentiation of HSCs, including their progeny. By lymphoid
tissue it is meant to include bone marrow, peripheral blood
(including mobilized peripheral blood), umbilical cord blood,
placental blood, fetal liver, embryonic cells (including embryonic
stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid
soft tissue. Lymphoid soft tissue as used herein includes, but is
not limited to, tissues such as thymus, spleen, liver, lymph node,
skin, tonsil, adenoids and Peyer's patch, and combinations
thereof.
[0086] Lymphoreticular stromal cells provide the supporting
microenvironment in the intact lymphoid tissue for the maintenance,
growth or differentiation of HSCs, including their progeny. The
microenvironment includes soluble and cell surface factors
expressed by the various cell types which comprise the
lymphoreticular stroma. Generally, the support which the
lymphoreticular stromal cells provide is characterized as both
contact-dependent and non-contact-dependent.
[0087] Lymphoreticular stromal cells, for example, are autologous
(self) or non-autologous (non-self, e.g., heterologous, allogeneic,
syngeneic or xenogeneic) with respect to HSCs. Autologous, as used
herein, refers to cells from the same subject. Allogeneic, as used
herein, refers to cells of the same species that differ
genetically. Syngeneic, as used herein, refers to cells of a
different subject that are genetically identical to the cell in
comparison. Xenogeneic, as used herein, refers to cells of a
different species. Lymphoreticular stroma cells are obtained, for
example, from the lymphoid tissue of a human or a non-human subject
at any time after the organ/tissue has developed to a stage (i.e.,
the maturation stage) at which it can support the maintenance,
growth or differentiation of HSCs. The lymphoid tissue from which
lymphoreticular stromal cells are derived usually determines the
lineage-commitment HSCs undertake, resulting in the
lineage-specificity of the differentiated progeny.
[0088] The co-culture of HSCs (and progeny thereof) with
lymphoreticular stromal cells, usually occurs under conditions
known in the art (e.g., temperature, CO.sub.2 and O.sub.2 content,
nutritive media, duration, etc.). The time sufficient to increase
the number of cells is a time that can be easily determined by a
person skilled in the art, and varies depending upon the original
number of cells seeded. The amounts of HSCs and lymphoreticular
stromal cells initially introduced (and subsequently seeded) varies
according to the needs of the experiment. The ideal amounts are
easily determined by a person skilled in the art in accordance with
needs.
[0089] As used throughout, by a subject is meant an individual.
Thus, subjects include, for example, domesticated animals, such as
cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and
goats), laboratory animals (e.g., mice, rabbits, rats, and guinea
pigs), mammals, non-human mammals, primates, non-human primates,
rodents, birds, reptiles, amphibians, fish, and any other animal.
The subject is optionally a mammal such as a primate or a
human.
Methods For Expanding Hematopoietic Stem Cells
[0090] The invention therefore relates to a method for expanding
hematopoietic stem cells, comprising (a) providing a starting cell
population comprising hematopoietic stem cells and (b) culturing
said starting cell population ex vivo in presence of an agent
capable of down-regulating the activity and/or expression of aryl
hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway, under suitable conditions for
expanding hematopoietic stem cells.
[0091] The aryl hydrocarbon (dioxin) receptor (AHR) is a cytosolic
ligand-activated transcription factor known to mediate a large
number of toxic and carcinogenic effects in animals and possible in
human (Safe S 2001 Toxicol Lett 120: 1-7). As a consequence of AHR
activation by its ligands, many detoxification genes are
transcriptionally induced, including those coding for phase I
xenobiotic-metabolizing enzymes, such as the cytochromes P450
CYP1A1, CYP1A2, CYP1B1 and CYP2S1, and the phase II enzymes
UDP-glucuronosyltransferase UGT1A6, NAD(P)H-dependent quinone
oxidoreductase-1 (NQO1), the aldehyde dehydrogenase ALDH3A1, and
several glutathione-S-transferase.
[0092] In one embodiment, an agent capable of down-regulating the
activity and/or expression of aryl hydrocarbon receptor and/or a
down-stream effector of aryl hydrocarbon receptor pathway is
selected among the group consisting of: (i) an organic compound;
(ii) a small interference RNA (siRNA) molecule capable of
down-regulating the expression of AHR; and (iii) antisense
oligonucleotide capable of down-regulating the expression of
AHR.
[0093] In one specific embodiment, said method for expanding
hematopoietic stem cells, comprises (a) providing a starting cell
population comprising hematopoietic stem cells and (b) culturing
said starting cell population ex vivo in the presence of an agent
capable of down-regulating the activity and/or expression of aryl
hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway, under suitable conditions for
expanding hematopoietic stem cells, wherein said agent capable of
down-regulating the activity and/or expression of aryl hydrocarbon
receptor and/or a down-stream effector of aryl hydrocarbon receptor
pathway is not alpha-napthoflavone or
3'-methoxy-4'-nitroflavone.
[0094] Organic compound that inhibits AHR activity (also referred
herein as AHR antagonist) have been described in the art, for
example 2-methyl-2H-pyrazole-3-carboxylic acid
(2-methyl-4-o-tolylazophenyl)amide (CH.sub.223191), alpha
napthoflavone, resveratrol (Nutr. Metab. Cardiovasc. Dis., 2003
April; 13(2):104-13), 3'-methoxy-4'-nitroflavone (Biochem.
Pharmacol., 2007 May 15; 73(10):1622-34, Epub 2007 Jan. 30), and
6-methyl-1,3,8-trichlorodibenzofuran (Cancer Res., 2004, Apr. 15;
64(8):2889-97). An inhibitor of AHR activity refers to a compound
which decreases AHR activity to at least 10%, 20%, 30%, 50%, 60%,
70%, 80% or at least 90% the transcriptional activity of AHR as
observed under activated conditions. An assay to measure AHR
inhibitory activity is for example the dioxin-induced AHR dependent
luciferase reporter gene assay as described in the Examples. In one
embodiment, an inhibitor of AHR activity is a compound that has an
EC50 of less than 10 .mu.M, preferably less than 5 .mu.M as
measured in the dioxin-induced AHR dependent luciferase reporter
gene assay.
[0095] AHR is a transcriptional factor regulating the transcription
of various genes in human. In one embodiment, a downstream effector
of AHR pathway is a gene which is directly regulated at the
transcriptional level by AHR. Examples of such genes are selected
from Cyp1B1, Cyp1A1, and AHRR. AHR also functions in pathways
outside of its well-characterized role in xenobiotic enzyme
induction. Xenobiotic ligands of AHR have been shown to regulate
beta catenin, STAT5, STAT1, HES-1, c-Myc, C/EBP, PU.1,
.beta.-catenin, p21, P27, pRb, deoxynucleotidyl transferase, CXCR4,
and its chemokine ligand CXCL12 (SDF-1).
[0096] In one specific embodiment, an agent capable of
down-regulating the activity and/or expression of aryl hydrocarbon
receptor is a compound as defined in the Summary of the
Invention.
[0097] In another embodiment, an agent capable of down-regulating
the activity and/or expression of aryl hydrocarbon receptor is an
antisense oligonucleotide or a small interfering RNA molecule
(siRNA), capable of down-regulating AHR protein expression or the
protein expression of one more down-stream effectors of AHR.
[0098] Design of antisense oligonucleotides which can be used to
efficiently inhibit the AHR protein expression must be effected in
a way that such oligonucleotides specifically binds the designated
mRNA within cells in a way which inhibits translation thereof.
Sequence suitable for use in design and synthesis of antisense
oligonucleotides which specifically bind to AHR mRNA, genomic DNA
and/or its promoter or other control sequences are available in
published sequence of AHR, in particular human AHR. In addition,
algorithms for identifying sequences with the highest predicted
binding affinity for their target mRNA based on thermodynamic cycle
that accounts for the energetics of structural alterations in both
the target mRNA and the oligonucleotides are also available.
[0099] Synthesis of RNAi molecules suitable for use with the
present invention can be affected as follows: First, the AHR mRNA
sequence (or one or more of its down-stream effectors) is scanned
downstream of the AUG start codon for AA-dinucleotide sequences.
Occurrence of each AA and the 19 3'-adjacent is recorded as a
potential siRNA target site. Then, potential target sites are
compared to an appropriate genomic database (e.g, human, mouse,
rat, etc.) using any sequence alignment software. Putative target
site that exhibit significant homology to other coding sequences
are filtered out. Preferred sequences are then those including low
G/C content, in particular sequences with G/C content lower than
55%. Several target sites are then selected along the length of the
target gene. Methods or algorithms to identify putative target site
of siRNA are described for example in (Tilesi, et al., Curr. Opin.
Mol. Ther. 11:156, 2009). Examples of siRNA molecules which are
capable of down-regulating the expression of AHR are:
TABLE-US-00001 AHR 111S, (SEQ ID NO.: 1) 5'GCG GCA TAG AGA CCG ACT
TAA TTT CAA GAG AAT TAA GTC GGT CTC TAT GCC GCT TTT TTG G 3'; AHR
111AS, (SEQ ID NO.: 2) 5'CGC GCC AAA AAA GCG GCA TAG AGA CCG ACT
TAA TTC TCT TGA AAT TAA GTC GGT CTC TAT GCC GC 3'; AHR 242S, (SEQ
ID NO.: 3) 5'GGC TTC TTT GAT GTT GCA TTA ATT CAA GAG ATT AAT GCA
ACA TCA AAG AAG CCT TTT TTG G 3'; AHR 242A5, (SEQ ID NO.: 4) 5'CGC
GCC AAA AAA GGC TTC TTT GAT GTT GCA TTA ATC TCT TGA ATT AAT GCA ACA
TCA AAG AAG CC 3'.
[0100] The starting cell population comprising hematopoietic stem
cells will be selected by the person skilled in the art depending
on the envisaged use. Various sources of cells comprising
hematopoietic stem cells have been described in the art, including
bone marrow, peripheral blood, neonatal umbilical cord blood,
placenta or other sources such as liver, particularly fetal
liver.
[0101] The cell population may first be subjected to enrichment or
purification steps, including negative and/or positive selection of
cells based on specific cellular markers in order to provide the
starting cell population. Methods for isolating said starting cell
population based on specific cellular markers may use fluorescent
activated cell sorting (FACS) technology also called flow cytometry
or solid or insoluble substrate to which is bound antibodies or
ligands that interact with specific cell surface markers. For
example, cells may be contacted with a solid substrate (e.g.,
column of beads, flasks, magnetic particles) containing the
antibodies and any unbound cells are removed. When a solid
substrate comprising magnetic or paramagnetic beads is used, cells
bound to the beads can be readily isolated by a magnetic
separator.
[0102] In one embodiment, said starting cell population is enriched
in a desirable cell marker phenotype (e.g., CD34+, CD133+, CD90+)
or based on efflux of dyes such as rhodamine, Hoechst or aldehyde
dehydrogenase activity. In one specific embodiment, said starting
cell population is enriched in CD34+ cells. Methods for enriching
blood cell population in CD34+ cells include kits commercialized by
Miltenyi Biotec (CD34+ direct isolation kit, Miltenyi Biotec,
Bergisch, Gladbach, Germany) or by Baxter (Isolex 3000).
[0103] The amount of cord blood from a single birth is often
inadequate to treat an adult or an older child. One advantage of
the expansion methods using the compounds of the invention, or an
agent capable of down-regulating the activity and/or expression of
aryl hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway, is that it enables the production of
a sufficient amount of hematopoietic stem cells from only one cord
blood unit.
[0104] Accordingly, in one embodiment, the starting cell population
is derived from neonatal umbilical cord blood cells which have been
enriched in CD34+ cells. In one related embodiment, said starting
cell population is derived from one or two umbilical cord blood
units.
[0105] In another embodiment, the starting cell population is
derived from human mobilized peripheral blood cells which have been
enriched in CD34+ cells. In one related embodiment, said starting
cell population is derived from human mobilized peripheral blood
cells isolated from only one patient.
[0106] Said starting cell population may preferably contain at
least 50% CD34+ cells, in some embodiments, more than 90% of CD34+
cells, and may comprise between 10.sup.5 and 10.sup.9 nucleated
cells.
[0107] The starting cell population may be used directly for
expansion or frozen and stored for use at a later date.
[0108] Conditions for culturing the starting cell population for
hematopoietic stem cell expansion will vary depending, inter alia,
on the starting cell population, the desired final number of cells,
and desired final proportion of HSCs.
[0109] In one specific embodiment, in particular, using a starting
cell population from umbilical cord blood cells enriched in CD34+
cells, the culturing conditions comprises the use of other
cytokines and growth factors, generally known in the art for
hematopoietic stem cell expansion. Such cytokines and growth
factors include without limitation IL-1, IL-3, IL-6, IL-11, G-CSF,
GM-CSF, SCF, FlT3-L, thrombopoietin (TPO), erythropoeitin, and
analogs thereof. As used herein, "analogs" include any structural
variants of the cytokines and growth factors having the biological
activity as the naturally occurring forms, including without
limitation, variants with enhanced or decreased biological activity
when compared to the naturally occurring forms or cytokine receptor
agonists such as an agonist antibody against the TPO receptor (for
example, VB22B sc(Fv)2 as detailed in patent publication WO
2007/145227, and the like). Cytokine and growth factor combinations
are chosen to expand HSC and progenitor cells while limiting the
production of terminally differentiated cells. In one specific
embodiment, one or more cytokines and growth factors are selected
from the group consisting of SCF, Flt3-L and TPO. In one specific
embodiment, at least TPO is used in a serum-free medium under
suitable conditions for HSC expansion. In one related embodiment, a
mixture of IL6, SCF, Flt3-L and TPO is used in the method for
expanding HSCs in combination with the compound of the invention or
an agent capable of down-regulating the activity and/or expression
of aryl hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway.
[0110] Human IL6 or interleukin-6, also known as B-cell stimulatory
factor 2 has been described by (Kishimoto, Ann. review of Imm.
23:12005) and is commercially available. Human SCF or stem cell
factor, also known as c-kit ligand, mast cell growth factor or
Steel factor has been described (Smith, M A et al., ACTA
Haematologica, 105, 3:143, 2001) and is commercially available.
Flt3-L or FLT-3 Ligand, also referred as FL is a factor that binds
to flt3-receptor. It has been described (Hannum C, Nature 368
(6472): 643-8) and is commercially available. TPO or
thrombopoietin, also known as megakarayocyte growth factor (MGDF)
or c-Mpl ligand has been described (Kaushansky K (2006). N. Engl.
J. Med. 354 (19): 2034-45) and is commercially available.
[0111] The expansion of HSC may be carried out in a basal medium,
which is supplemented with the mixtures of cytokines and growth
factors described above. A basal medium typically comprises amino
acids, carbon sources, vitamins, serum proteins (e.g. albumin),
inorganic salts, divalent cations, buffers and any other element
suitable for use in expansion of HSC. Examples of such basal medium
appropriate for a method of expanding HSC include, without
limitation, StemSpan.COPYRGT. SFEM--Serum-Free Expansion Medium
(StemCell Technologies, Vancouver, Canada), StemSpan.RTM.
H3000--Defined Medium (StemCell Technologies, Vancouver, Canada),
CellGro.RTM. SCGM (CellGenix, Freiburg Germany), StemPro.RTM.-34
SFM (Invitrogen).
[0112] In one embodiment, the compound of the invention or the
agent capable of down-regulating the activity and/or expression of
aryl hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway, is administered during the expansion
method of said starting cell population under a concentration
appropriate for HSC expansion. In one specific embodiment, said
compound or AhR modulating agent is administered at a concentration
comprised between 1 pM and 100 .mu.M, for example between 10 pM and
10 .mu.M, or between 100 pM and 1 .mu.M.
[0113] In one specific embodiment where starting cell population
essentially consists of CD34+ enriched cells from one or two cord
blood units, the cells are grown under conditions for HSC expansion
from about 3 days to about 90 days, for example between 7 and 2
days and/or until the indicated fold expansion and the
characteristic cell populations are obtained. In one specific
embodiment, the cells are grown under conditions for HSC expansion
not more than 21 days, 14 days or 7 days.
[0114] In one embodiment, the starting cell population is cultured
during a time sufficient to reach an absolute number of CD34+ cells
of at least 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8 or 10.sup.9
cells. In another embodiment, said starting cell population is
cultured during a time sufficient for a 10 to 50000 fold expansion
of CD34+ cells, for example between 100 and 10000 fold
expansion.
[0115] The cell population obtained after the expansion method may
be used without further purification or may be subject to further
purification or selection steps.
[0116] The cell population may then be washed to remove the
compound of invention or any other agent capable of down-regulating
the activity and/or expression of aryl hydrocarbon receptor and/or
a down-stream effector of aryl hydrocarbon receptor pathway and/or
any other components of the cell culture and resuspended in an
appropriate cell suspension medium for short term use or in a
long-term storage medium, for example a medium suitable for
cryopreservation.
Cell Population with Expanded HSCs as Obtained by the Expansion
Method and Therapeutic Compositions
[0117] The invention further provides a cell population with
expanded HSCs, obtainable or obtained by the expansion method
described above. In one specific embodiment, such cell population
is resuspended in a pharmaceutically acceptable medium suitable for
administration to a mammalian host, thereby providing a therapeutic
composition.
[0118] The compound as defined in the Summary of the Invention or
an agent capable of down-regulating the activity and/or expression
of aryl hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway enables the expansion of HSCs, for
example from only one or two cord blood units, to provide a cell
population quantitatively and qualitatively appropriate for
efficient short and long term engraftment in human patient in need
thereof. In particular, the invention relates to a composition
comprising a cell population with expanded HSCs derived from not
more than one or two cord blood units, wherein said therapeutic
composition contains a total amount of cells of at least 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8 or 10.sup.9 cells, with between
20-100%, for example between 40-80% of total cells being CD34+
cells. In one related embodiment, said composition contains between
0.1-40%, for example between 0.1-10% of total cells being CD34+
Thy1+ and 20-80% of cells being CD34+ CD45RA+. In some specific
embodiments, said composition contains between 10-95% of cells
being CD38+ and between 5-70% of cells being CD133+.
Use of Therapeutic Compositions
[0119] The invention further provides the cell population with
expanded HSCs or its composition for use in allogeneic or
autologous stem cell transplantation in a mammalian subject.
[0120] The subject referred to herein is, for example, a bone
marrow donor or an individual with or at risk for depleted or
limited blood cell levels. Optionally, the subject is a bone marrow
donor prior to bone marrow harvesting or a bone marrow donor after
bone marrow harvesting. The subject is optionally a recipient of a
bone marrow transplant. The methods described herein are
particularly useful in subjects that have limited bone marrow
reserve such as elderly subjects or subjects previously exposed to
an immune depleting treatment or myeloablative treatment such as
chemotherapy, e.g., for treating leukemia or lymphomas. The
subject, optionally, has a decreased blood cell level or is at risk
for developing a decreased blood cell level as compared to a
control blood cell level. As used herein the term control blood
cell level refers to an average level of blood cells in a subject
prior to or in the substantial absence of an event that changes
blood cell levels in the subject. An event that changes blood cell
levels in a subject includes, for example, anemia, trauma,
chemotherapy, bone marrow transplant and radiation therapy. For
example, the subject has anemia or blood loss due to, for example,
trauma.
[0121] The expanded HSC population or the composition comprising
the cell population with expanded HSCs is administered to the
subject, for example, before, at the same time, or after
chemotherapy, radiation therapy or a bone marrow transplant. The
subject optionally has depleted bone marrow related to, for
example, congenital, genetic or acquired syndrome characterized by
bone marrow loss or depleted bone marrow. Thus, the subject is
optionally a subject in need of hematopoiesis. Optionally, the
subject is a bone marrow donor or is a subject with or at risk for
depleted bone marrow.
[0122] Hematopoietic stem cell manipulation is useful as a
supplemental treatment to chemotherapy or radiation therapy. For
example, HSCs are localized into the peripheral blood and then
isolated from a subject that will undergo chemotherapy, and after
the therapy the cells are returned. Thus, the subject is a subject
undergoing or expected to undergo an immune cell depleting
treatment such as chemotherapy, radiation therapy or serving as a
donor for a bone marrow transplant. Bone marrow is one of the most
prolific tissues in the body and is therefore often the organ that
is initially damaged by chemotherapy drugs and radiation. The
result is that blood cell production is rapidly destroyed during
chemotherapy or radiation treatment, and chemotherapy or radiation
must be terminated to allow the hematopoietic system to replenish
the blood cell supplies before a patient is re-treated with
chemotherapy. Therefore, as described herein, HSCs or blood cells
made by the methods described herein are optionally administered to
such subjects in need of additional blood cells.
[0123] Provided are HSCs expanded by a compound of the invention or
an agent capable of down-regulating the activity and/or expression
of aryl hydrocarbon receptor and/or a down-stream effector of aryl
hydrocarbon receptor pathway or the compositions with expanded HSCs
as described above in combination with a therapeutic capable of
enhancing the proliferation of HSCs in vivo, in vitro, or ex vivo
(for example, a small molecule, an antibody, or the like) and
optionally at least one pharmaceutically acceptable excipient or
carrier. By a therapeutic capable of enhancing HSC proliferation is
meant: an agonist antibody against the TPO receptor (for example,
VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and
the like); a cytokine such as SCF, IL-6, Flt-3 ligand, TPO or a TPO
mimetic (for example, such as described in WO/2007/022269;
WO/2007/009120; WO/2004/054515; WO/2003/103686; WO/2002/085343;
WO/2002/049413; WO/2001/089457; WO/2001/039773; WO/2001/034585;
WO/2001/021180; WO/2001/021180; WO/2001/017349; WO/2000/066112;
WO/2000/035446; WO/2000/028987; WO/2008/028645; and the like);
granulocyte colony stimulating factor (G-CSF); granulyte macrophage
colony stimulating factor (GM-CSF); a prostaglandin or a
prostaglandin receptor agonist (for example, prostaglandin E2
receptor-1 (EP-I) agonist, prostaglandin E2 receptor-2 (EP-2)
agonist, prostaglandin E2 receptor-3 (EP-3) agonist and
prostaglandin E2 receptor-4 (EP-4) agonists, as detailed in patent
publication WO/2008/073748); tetraethylenepentamine (TEPA);
Notch-ligands (Delta-1); and/or a WNT agonist. In addition,
culturing stem cells with mesenchymal stem cells (MSCs) prevents
graft-versus-host disease (GVHD) and may help stem cell expansion.
MSCs and stem cells can be transplanted as a whole culture.
[0124] By pharmaceutically acceptable is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to a subject or cell, without causing undesirable
biological effects or interacting in a deleterious manner with the
other components of the pharmaceutical composition in which it is
contained. The carrier or excipient is selected to minimize
degradation of the active ingredient and to minimize adverse side
effects in the subject or cell.
[0125] The compositions are formulated in any conventional manner
for use in the methods described herein. Administration is via any
route known to be effective by one of ordinary skill.
[0126] For example, the compositions is administered orally,
parenterally (e.g., intravenously), by intramuscular injection, by
intraperitoneal injection, transdermally, extracorporeally,
intranasally or topically.
[0127] The preferred method of administration is intravenous
infusion. The number of cells transfused will take into
consideration factors such as sex, age, weight, the types of
disease or disorder, stage of the disorder, the percentage of the
desired cells in the cell population and the amount of cells needed
to produce a therapeutic benefit. In one particular embodiment, the
composition is administered by intravenous infusion and comprises
at least 10.sup.4 cells/kg, from 10.sup.5 to 5.10.sup.7 cells/kg or
more if necessary. In one specific embodiment, the infused cells
are all deriving from expanded cord blood cells from a single
birth.
[0128] A pharmaceutically acceptable carrier for infusion of a
composition comprising cells into a patient typically comprise
buffered saline with 5% HSA or unsupplemented basal medium or
medium as known in the art.
[0129] For oral administration, the compositions take the form of,
for example, tablets or capsules prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets are
coated by methods well known in the art. Liquid preparations for
oral administration take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations are prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations
optionally contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0130] The compositions are formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection are presented in unit dosage
form, e.g., in ampules or in multi-dose containers, with or without
an added preservative. The compositions take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0131] Alternatively, the active ingredient is in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use. In general, water, suitable oil, saline, aqueous
dextrose (glucose), and related sugar solutions and glycols such as
propylene glycol or polyethylene glycols are suitable carriers for
parenteral solutions. Solutions for parenteral administration
contain, for example, a water soluble salt of the active
ingredient, suitable stabilizing agents and, if necessary, buffer
substances. Antioxidizing agents such as sodium bisulfate, sodium
sulfite or ascorbic acid, either alone or combined, are suitable
stabilizing agents. Also citric acid and its salts and sodium
ethylenediaminetetraacetic acid (EDTA) are optionally used. In
addition, parenteral solutions optionally contain preservatives
such as benzalkonium chloride, methyl- or propyl-paraben and
chlorobutanol. Suitable pharmaceutical carriers are described in
Remington: The Science and Practice of Pharmacy, 21st Edition,
David B. Troy, ed., Lippicott Williams & Wilkins (2005), which
is incorporated by reference in its entirety at least for the
material related to pharmaceutical carriers and compositions.
[0132] The compositions are optionally formulated as a depot
preparation. Such long acting formulations are optionally
administered by implantation. Thus, for example, the compositions
are formulated with suitable polymeric or hydrophobic materials
(for example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt. The compositions are applied to or embedded
with implants concurrent with or after surgical implant.
[0133] Additionally, standard pharmaceutical methods are employed
to control the duration of action. These include control release
preparations and appropriate macromolecules, for example, polymers,
polyesters, polyamino acids, polyvinyl, pyrolidone,
ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or
protamine sulfate. The concentration of macromolecules as well as
the methods of incorporation are adjusted in order to control
release. Optionally, the agent is incorporated into particles of
polymeric materials such as polyesters, polyamino acids, hydrogels,
poly (lactic acid) or ethylenevinylacetate copolymers. In addition
to being incorporated, these agents are optionally used to trap the
compound in microcapsules.
[0134] A composition for use in the methods described herein is
optionally formulated as a sustained and/or timed release
formulation. Such sustained and/or timed release formulations are
made by sustained release means or delivery devices that are well
known to those of ordinary skill in the art. The compositions are
used to provide slow or sustained release of one or more of the
active ingredients using, for example, hydropropylmethyl cellulose,
other polymer matrices, gels, permeable membranes, osmotic systems,
multilayer coatings, microparticles, liposomes, microspheres or a
combination thereof to provide the desired release profile in
varying proportions. Suitable sustained release formulations are
selected for use with the compositions described herein. Thus,
single unit dosage forms suitable for oral administration, such as,
but not limited to, tablets, capsules, gelcaps, caplets, powders
that are adapted for sustained release are used.
[0135] The compositions are optionally delivered by a
controlled-release system. For example, the composition is
administered using intravenous infusion, an implantable osmotic
pump, liposomes, or other modes of administration. A controlled
release system is placed in proximity to the target.
[0136] Optionally, it is desirable to administer the composition
locally, i.e., to the area in need of treatment. For example, the
composition is administered by injection into the bone marrow of a
long bone, for example. Local administration is achieved, for
example, by local infusion during surgery, topical application
(e.g., in conjunction with a wound dressing after surgery),
injection, catheter, suppository, or implant. An implant is of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers.
[0137] The pharmaceutical compositions described herein are
administered by any conventional means available for use in
conjunction with pharmaceuticals, either as individual therapeutic
active ingredients or in a combination of therapeutic active
ingredients. They are optionally administered alone, but are
generally administered with a pharmaceutical carrier selected on
the basis of the chosen route of administration and standard
pharmaceutical practice.
[0138] The compounds described herein are provided in a
pharmaceutically acceptable form including pharmaceutically
acceptable salts and derivatives thereof. The term pharmaceutically
acceptable form refers to compositions including the compounds
described herein that are generally safe, relatively non-toxic and
neither biologically nor otherwise undesirable. These compositions
optionally include pharmaceutically acceptable carriers or
stabilizers that are nontoxic to the cell or subject being exposed
thereto at the dosages and concentrations employed.
[0139] Examples of physiologically acceptable carriers include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptide; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM. (Unigema, United Kingdom),
polyethylene glycol (PEG), and PLURONICS.TM. (BASF, Germany).
[0140] The term pharmaceutically acceptable acid salts and
derivatives refers to salts and derivatives of the compounds of
Formula I described herein that retain the biological effectiveness
and properties as described, and that are not biologically or
otherwise undesirable.
[0141] Pharmaceutically acceptable salts are formed, for example,
with inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, propionic acid, glycolic acid,
pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, p-toluenesulfonic acid, salicylic acid, and the like.
[0142] The chemical stability of a composition comprising a
compound of Formula I or a pharmaceutically acceptable salt or
ester thereof is enhanced by methods known to those of skill in the
art. For example, an alkanoic acid ester of a polyethoxylated
sorbitol (a polysorbate) is added to a composition containing a
compound of Formula I in an amount effective to enhance the
chemical stability of the compound.
[0143] The data obtained from the cell culture assays and animal
studies are optionally used in formulating a range of dosage for
use in humans. The dosage of such compounds lies preferably within
a range of circulating concentrations that include little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed and the route of administration utilized. For
any compound used in the provided methods, the therapeutically
effective dose is estimated initially from cell culture assays.
[0144] Also provided herein is a pack or kit comprising one or more
containers filled with one or more of the ingredients described
herein. Such kits optionally comprise solutions and buffers as
needed or desired. The kit optionally includes an expanded
population of stem cells made by the methods described above or can
contain containers or compositions for making an expanded
population of HSCs. In particular, the invention provides a kit for
expanding ex vivo hematopoietic stem cells, comprising a compound
as defined in the Summary of Invention and instructions for use of
such compound in a method for HSC expansion and, optionally, one
ore more cytokines or growth factors, or media for cell growth, in
particular media for hematopoietic stem cell growth as described
above. The kit may further comprise antibodies for monitoring
production of the cells, such as anti-CD34, anti-CD133, anti-CD38,
anti-CD45RA and/or anti-Thy1 antibodies. In one specific
embodiment, such kit further include one or more cytokines or
growth factors selected from the group consisting of IL6, FLT3-L,
SCF and TPO. Optionally associated with such pack(s) or kit(s) are
instructions for use.
[0145] Also provided is a kit for providing an effective amount of
a compound of the invention to increase HSCs in a subject
comprising one or more doses of the compound for use over a period
of time, wherein the total number of doses of the compound of the
invention in the kit equals the effective amount sufficient to
increase HSCs in a subject. The period of time is from about one to
several days or weeks or months. Thus, the period of time is from
at least about 5, 6, 7, 8, 10, 12, 14, 20, 21, 30 or 60 days or
more or any number of days between one and 90.
Processes for Making Compounds of the Invention
[0146] The present invention also includes processes for the
preparation of compounds of the invention. In the reactions
described, it can be necessary to protect reactive functional
groups, for example hydroxy, amino, imino, thio or carboxy groups,
where these are desired in the final product, to avoid their
unwanted participation in the reactions. Conventional protecting
groups can be used in accordance with standard practice, for
example, see T. W. Greene and P. G. M. Wuts in "Protective Groups
in Organic Chemistry", John Wiley and Sons, 1991.
[0147] The following reaction schemes 1-5 detail the preparation of
compounds of the invention. It will be appreciated by one skilled
in the art that, following introduction by the methods detailed
below, any of the groups R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
L.sub.1 may optionally be further elaborated by known
transformations to arrive at the desired final compounds of Formula
I.
[0148] Compounds of Formula I can be prepared according the
following Reaction Scheme 1:
##STR00006##
[0149] in which G.sub.1, G.sub.2, G.sub.3, G.sub.4, R.sub.1,
R.sub.2 and R.sub.4 are as defined for Formula I in the Summary of
the Invention and L of Formula I is defined in the reaction scheme
as --NH-L.sub.1-- which is equivalent to, for example,
--NR.sub.5a(CH.sub.2).sub.0-3-- where R.sub.5a is hydrogen and
--(CH.sub.2).sub.0-3-- is L.sub.1.
[0150] Compounds of Formula I can be prepared by reacting a
compound of Formula 2 with a compound of Formula 3 in the presence
of a suitable catalyst (e.g., Pd.sub.2(dba).sub.3, or the like) in
the presence of an appropriate ligand (e.g.,
1,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable
base (e.g., Cs.sub.2CO.sub.3, or the like) and an appropriate
solvent (e.g., 1,4-dioxane) at a temperature of about 80 to
100.degree. C. for 2 to about 48 hours. Compounds of Formula 2 in
turn can be prepared by reacting a compound of Formula 4 with a
slight excess of an amine compound of Formula 5 in an appropriate
solvent (e.g. isopropanol) at a temperature of about room
temperature to about 80.degree. C. Compounds of Formula 4 can be
prepared by alkylation of a compound of Formula 6 with a suitable
alkylating agent 7, in which Xi is chlorine, bromine, iodine, or a
sulfonate ester, in the presence of a suitable base (e.g. sodium
hydride or potassium carbonate), in a suitable solvent (e.g. DMF),
at a temperature of about 0.degree. C. to about 80.degree. C.
Alternatively, the reaction can be performed under Mitsunobu
conditions using a suitable alcohol R.sub.4--OH in the presence of
a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate
(e.g. diethylazodicarboxylate), in an inert solvent such as THF or
toluene, at a temperature from about 0.degree. C. to about room
temperature.
[0151] Compounds of Formula Ia, in which G.sub.1 is CR.sub.3 and in
which all other G groups are N, can also be prepared by proceeding
as in the following Reaction Scheme 2:
##STR00007##
[0152] in which R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as
defined for Formula I in the Summary of the Invention and L of
Formula I is defined in the reaction scheme as --NH-L.sub.1- which
is equivalent to, for example, --NR.sub.5a(CH.sub.2).sub.0-3--
where R.sub.5a is hydrogen and --(CH.sub.2).sub.0-3-- is
L.sub.1.
[0153] Compounds of Formula I can be prepared by reacting a
compound of Formula 8 with an amine compound of Formula 5 in an
appropriate solvent (e.g. isopropanol) at a temperature of about
room temperature to about 100.degree. C. Compounds of Formula 8 can
in turn be prepared by reacting a compound of Formula 9 with a
compound of Formula 3 in the presence of a suitable catalyst (e.g.,
Pd(Ph.sub.3P).sub.4, Pd.sub.2(dba).sub.3, or the like), optionally
in the presence of an appropriate ligand (e.g.,
1,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable
base (e.g., Cs.sub.2CO.sub.3, or the like) and an appropriate
solvent (e.g., 1,4-dioxane) at a temperature of about 80 to
100.degree. C. for 2 to about 48 hours. Compounds of Formula 9 in
turn can be prepared by reacting a compound of Formula 10 with a
mixture of di-iodomethane, copper(I) iodide, and an alkyl nitrite
(e.g. isoamylnitrite), optionally in the presence of an inert
solvent, at a temperature of about 50 to 100.degree. C. Compounds
of Formula 10 can be prepared by alkylation of a compound of
Formula 11 with a suitable alkylating agent 7, in which Xi is
chlorine, bromine, iodine, or a sulfonate ester, in the presence of
a suitable base (e.g. sodium hydride or potassium carbonate), in a
suitable solvent (e.g. DMF), at a temperature of about 0.degree. C.
to about 80.degree. C. Alternatively, the reaction can be performed
under Mitsunobu conditions using a suitable alcohol R.sub.4--OH in
the presence of a suitable phosphine (e.g. triphenylphosphine) and
azodicarboxylate (e.g. diethylazodicarboxylate), in an inert
solvent such as THF or toluene, at a temperature from about
0.degree. C. to about room temperature.
[0154] Compounds of Formula II, which are a subset of compounds of
Formula I in which R.sub.1 is N-linked heterocyclyl or N-linked
heteroaryl, can be prepared as detailed in the following Reaction
Scheme 3:
##STR00008##
[0155] G.sub.1, G.sub.2, G.sub.3, G.sub.4, R.sub.1, R.sub.2 and
R.sub.4 are as defined for Formula I in the Summary of the
Invention and L of Formula I is defined in the reaction scheme as
--NH-L.sub.1- which is equivalent to, for example,
--NR.sub.5a(CH.sub.2).sub.0-3-- where R.sub.5a is hydrogen and
--(CH.sub.2).sub.0-3-- is L.sub.1. Compounds of Formula II can be
prepared by reacting a compound of Formula 2 with a compound of
Formula in the presence of an excess of cyclic amine or NH-bearing
heterocycle (for example, substituted pyrazole, substituted
imidazole, and the like), at a temperature of about 50.degree. C.
to about 250.degree. C., for about 1 to about 24 hours, optionally
in the presence of a base such as sodium hydride or DBU.
[0156] Compounds of Formula 10 in which G.sub.1 is CR.sub.3, and in
which all other G groups are N, can also be prepared by proceeding
as in the following Reaction Scheme 4:
##STR00009##
[0157] in which R.sub.3 and R.sub.4 are as defined for Formula I in
the Summary of the Invention. Compounds of Formula 10 can be
prepared according to procedures described in J. Med. Chem, 1972,
456, and J. Med. Chem., 1992, 4180. An orthoester compound of
Formula 21 is reacted with a compound of Formula 22, optionally in
the presence of an acid such as acetic acid, at a temperature of
about room temperature to about 150.degree. C., for about 1 to
about 24 hr. A compound of Formula 22 can in turn be prepared by
reacting a compound of Formula 23 with a primary amine compound of
Formula 24, optionally in the presence of an acid such as pTSA, or
a base such as triethylamine or DBU, at a temperature of about 50
to about 200.degree. C.
[0158] Compounds of Formula IV can be prepared as detailed in the
following Reaction Scheme 5:
##STR00010##
[0159] in which G.sub.1, G.sub.2, G.sub.3, G.sub.4, R.sub.1 and
R.sub.2 are as defined for Formula I in the Summary of the
Invention and L of Formula I is defined in the reaction scheme as
--NH-L.sub.1- which is equivalent to, for example,
--NR.sub.5a(CH.sub.2).sub.0-3-- where R.sub.5a is hydrogen and
--(CH.sub.2).sub.0-3-- is L.sub.1. R.sub.20 and R.sub.21 are
independently selected from hydrogen and C.sub.1-4alkyl. A compound
of Formula IV, in which R.sub.21 is hydrogen, can be prepared from
a compound of Formula III by treatment with a suitable reducing
agent such as lithium aluminum hydride or di-isobutyl aluminum
hydride, in a suitable solvent such as THF or toluene, at a
temperature of about -78.degree. C. to about 50.degree. C. The
reaction takes about 0.5 to about 16 hr to complete. A compound of
Formula IV, in which R.sub.21 is lower alkyl, can be prepared by
treatment of a compound of Formula III with an alkyl lithium or
Grignard reagent, in a suitable solvent such as ether or
tetrahydrofuran, at a temperature of about -78.degree. C. to about
50.degree. C. The reaction takes about 0.5 to about 16 hr to
complete.
[0160] Detailed examples of the synthesis of a compound of Formula
I can be found in the Examples, infra.
Additional Processes for Making Compounds of the Invention
[0161] A compound of the invention can be prepared as a
pharmaceutically acceptable acid addition salt by reacting the free
base form of the compound with a pharmaceutically acceptable
inorganic or organic acid. Alternatively, a pharmaceutically
acceptable base addition salt of a compound of the invention can be
prepared by reacting the free acid form of the compound with a
pharmaceutically acceptable inorganic or organic base.
Alternatively, the salt forms of the compounds of the invention can
be prepared using salts of the starting materials or
intermediates.
[0162] For example, salt forms of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (Example 1, infra) were synthesized as follows:
[0163] Mesylate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 12 ml acetone
at 50.degree. C. Methanesulfonic acid (0.137 g; 1.40 mmoles) is
added drop wise. The crystallization takes place rapidly. The white
suspension is allowed to cool over about 30 minutes with cooling to
room temperature. The slurry is stirred for 18 hours at room
temperature and filtered. The solid is washed with acetone (6 ml)
in three portions and dried first for about 3 hours at 50.degree.
C./ca. 10 mbar and then for about 16 hours at 80.degree. C./ca.10
mbar. The material has a melting point at about 233.degree. C. with
a melting enthalpy of 98 g/J. The material produced exhibited a
loss on drying of 0.2%. The water uptake was estimated by
thermogravimetry after exposure to relative humidity (80% rh)
during 24 hours. A water uptake of 0.4% was observed.
[0164] In another embodiment, the invention provides a mesylate
salt of the compound of Example 1. In a further embodiment, the
invention provides the mesylate salt of the compound of Example 1
comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 6.4, 6.7, 18.3, 18.6, 26.9; and which in an
additional embodiment comprises the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 6.4, 6.7, 10.3, 12.9,
16.4, 18.3, 25.8, 26.5, 26.9.
[0165] In a yet further embodiment, the invention provides the
mesylate salt of the compound of Example 1 having the powder X-ray
diffraction pattern shown in FIG. 3 herein.
[0166] Tosylate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 12 ml at
50.degree. C. A solution of para-toluenesulfonic acid mono-hydrate
(0.271 g; 1.40 mmoles) in acetone (1.2 ml) is added drop wise. The
solution is seeded at 50.degree. C. and crystallization takes place
quickly. The suspension is allowed to cool over about 30 minutes to
room temperature and stirred for about 18 hours. After filtration
the solid is washed with acetone (6 ml) in three portions and dried
first for about 3 hours at 50.degree. C./ca. 10 mbar and then for
about 16 hours at 80.degree. C./ca.10 mbar. The material has a
melting point at about 233.degree. C. with a melting enthalpy of 88
g/J. The material produced exhibited a loss on drying of 0.2%. The
water uptake was estimated by Thermogravimetry after exposure to
relative humidity (80% rh) during 24 hours. A water uptake of 0.4%
was observed.
[0167] In another embodiment, the invention provides a tosylate
salt of the compound of Example 1. In a further embodiment, the
invention provides the tosylate salt of the compound of Example 1
comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.):6.2, 13.3, 16.7, 19.5, 25.4; and which in an
additional embodiment comprises the following powder X-ray
diffraction peaks: 6.2, 7.6, 12.4, 13.3, 15.1, 16.7, 17.7, 19.5,
20.2, 24.6, 24.9, 25.4, 25.6.
[0168] In a yet further embodiment, the invention provides the
tosylate salt of the compound of Example 1 having the powder X-ray
diffraction pattern shown in FIG. 4 herein.
[0169] Sulfate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 10 ml acetone
and 1 ml water at about 55.degree. C. A solution of sulfuric acid
(0.280 g; 2.79 mmoles) in 1 ml water is added drop wise. The
crystallization takes place rapidly. The suspension is allowed to
cool over about 30 minutes with cooling to room temperature,
stirred for about 18 hours and filtered. The filter cake is washed
with 6 ml acetone in three portions and dried first for about 3
hours at 50.degree. C./ca. 10 mbar and then for about 16 hours at
80.degree. C./ca.10 mbar. The material has a melting point at about
224.degree. C. with a melting enthalpy of 91 g/J. The material
produced exhibited a loss on drying below 0.05%. The water uptake
was estimated by Thermogravimetry after exposure to relative
humidity (80% rh) during 24 hours. A water uptake of 0.2% was
observed.
[0170] In another embodiment, the invention provides a sulfate salt
of the compound of Example 1. In a further embodiment, the
invention provides the sulfate salt of the compound of Example 1
comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 6.5, 6.8, 10.7, 13.5, 26.4, 27.6; and which in
an additional embodiment comprises the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 6.5, 6.8, 10.7, 13.1,
13.5, 18.6, 18.8, 20.8, 26.4, 27.1, 27.6.
[0171] In a yet further embodiment, the invention provides the
sulfate salt of the compound of Example 1 having the powder X-ray
diffraction pattern shown in FIG. 6 herein.
[0172] Esylate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 12 ml acetone
at 50.degree. C.
[0173] Ethanesulfonic acid (0.155 g; 1.40 mmoles) is added drop
wise. The crystallization takes place quickly. The resulting white
suspension is allowed to cool over about 30 minutes to room
temperature. The suspension is stirred for about 18 hours at room
temperature and filtered. The solid is washed with 6 ml acetone in
three portions and dried first for about 3 hours at 50.degree.
C./ca. 10 mbar and then for about 16 hours at 80.degree. C./ca.10
mbar. The material has a melting point at about 231.degree. C. with
a melting enthalpy of 76 g/J. The material produced exhibited a
loss on drying of 0.6%. The water uptake was estimated by
Thermogravimetry after exposure to relative humidity (80% rh)
during 24 hours. A water uptake of 0.05% was observed.
[0174] In another embodiment, the invention provides a esylate salt
of the compound of Example 1. In a further embodiment, the
invention provides the esylate salt of the compound of Example 1
comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 6.3, 9.9, 18.4, 25.3, 26.1; and which in an
additional embodiment comprises the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 6.3, 9.9, 17.1, 17.9,
18.4, 19.0, 22.0, 25.3, 26.1, 27.1.
[0175] In a yet further embodiment, the invention provides the
esylate salt of the compound of Example 1 having the powder X-ray
diffraction pattern shown in FIG. 8 herein.
[0176] Hydrobromide salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 6 ml DMF at
65.degree. C. Hydrobromic acid 48% (0.235 g; 1.40 mmoles) is added
drop wise. The solution is allowed to cool over about 30 minutes to
room temperature. Seeds are added at 55.degree. C. and
crystallization takes place slowly. The suspension is stirred for
about 18 hours at room temperature and filtered. The solid is
washed with 4 ml DMF/water 1:1 and 6 ml water. The salt is dried
first for about 3 hours at 50.degree. C./ca. 10 mbar and then for
about 16 hours at 80.degree. C./ca.10 mbar. The material has a
melting point at about 285.degree. C. with a melting enthalpy of
119 g/J. The material produced exhibited a loss on drying of 1.0%.
The water uptake was estimated by Thermogravimetry after exposure
to relative humidity (80% rh) during 24 hours. No water uptake was
observed.
[0177] In another embodiment, the invention provides a hydrobromide
salt of the compound of Example 1. In a further embodiment, the
invention provides the hydrobromide salt of the compound of Example
1 comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 7.0, 25.9, 26.8, 27.9; and which in an
additional embodiment comprises the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 7.0, 11.4, 13.3, 21.4,
23.4, 25.9, 26.4, 26.8, 27.9.
[0178] Ina yet further embodiment, the invention provides the
hydrobromide salt of the compound of Example 1 having the powder
X-ray diffraction pattern shown in FIG. 9 herein.
[0179] Orotate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) and orotic acid (0.222 g; 1.40
mmoles) are dissolved in 7.8 ml NMP (1-Methyl-2-pyrrolidone) at
85.degree. C. The solution is cooled to 60.degree. C. and 6 ml
water is added drop wise over about 5 minutes. The resulting white
suspension is allowed to cool over about 30 minutes to room
temperature and stirred for 18 hours. After filtration the filter
cake is washed with 4 ml NMP/water 1:1 in two portions and 6 ml
water in three portions. The solid is dried first for about 3 hours
at 50.degree. C./ca. 10 mbar and then for about 16 hours at
80.degree. C./ca.10 mbar. The material has a melting point at about
240.degree. C. with a melting enthalpy of 130 g/J. The material
produced exhibited a loss on drying below 0.05%. The water uptake
was estimated by Thermogravimetry after exposure to relative
humidity (80% rh) during 24 hours. A water uptake of 1.7% was
observed.
[0180] In another embodiment, the invention provides an orotate
salt of the compound of Example 1. In a further embodiment, the
invention provides the orotate salt of the compound of Example 1
comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 7.1, 16.3, 19.2, 23.5, 25.6, 26.9; and which in
an additional embodiment comprises the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 7.1, 14.4, 16.3, 18.6,
19.2, 21.7, 23.0, 23.5, 25.6, 26.9, 28.7.
[0181] Ina yet further embodiment, the invention provides the
orotate salt of the compound of Example 1 having the powder X-ray
diffraction pattern shown in FIG. 10 herein.
[0182] Hemi-fumarate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 18 ml methanol
at 65.degree. C. Fumaric acid (0.164 g; 1.40 mmoles) and 6 ml
methanol are added. The solution is allowed to cool over about 30
minutes to room temperature. Some seed crystals are added at
60.degree. C. and crystallization takes place slowly. The
suspension is stirred for 18 hours at room temperature and
filtered. The solid is washed with 6 ml methanol in three portions
and dried first for about 3 hours at 50.degree. C./ca. 10 mbar and
then for about 16 hours at 80.degree. C./ca.10 mbar. The material
has a melting point at about 232.degree. C. with a melting enthalpy
of 83 g/J. The material produced exhibited a loss on drying below
0.05%. The water uptake was estimated by Thermogravimetry after
exposure to relative humidity (80% rh) during 24 hours. A water
uptake of 0.3% was observed.
[0183] In another embodiment, the invention provides a
hemi-fumarate salt of the compound of Example 1. In a further
embodiment, the invention provides the hemi-fumarate salt of the
compound of Example 1 comprising the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 7.2, 8.7, 14.4, 15.8,
17.4, 19.0, 23.7; and which in an additional embodiment comprises
the following powder X-ray diffraction peaks (Angle 2-Theta
.degree.): 7.2, 8.7, 10.8, 14.4, 15.8, 17.4, 17.8, 19.0, 20.1,
23.7, 27.5.
[0184] In a yet further embodiment, the invention provides the
hemi-fumarate salt of the compound of Example 1 having the powder
X-ray diffraction pattern shown in FIG. 11 herein.
[0185] Besylate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 12 ml acetone
at 50.degree. C. A solution of benzenesulfonic acid (0.225 g; 2.79
mmoles) in 1.2 ml acetone is added drop wise. Seed crystals are
added at 48.degree. C. and the crystallization takes place slowly.
The suspension is allowed to cool over about 30 minutes to room
temperature. The slurry is stirred for about 18 hours at room
temperature and filtered. The salt is washed with 6 ml acetone in
three portions and dried first for about 3 hours at 50.degree.
C./ca. 10 mbar and then for about 16 hours at 80.degree. C./ca.10
mbar. The material has a melting point at about 219.degree. C. with
a melting enthalpy of 92 g/J. The material produced exhibited a
loss on drying of 0.3%. The water uptake was estimated by
Thermogravimetry after exposure to relative humidity (80% rh)
during 24 hours. A water uptake of about 0.05% was observed.
[0186] In another embodiment, the invention provides a besylate
salt of the compound of Example 1. In a further embodiment, the
invention provides the besylate salt of the compound of Example 1
comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 6.2, 7.7, 17.7, 25.5; and which in an additional
embodiment comprises the following powder X-ray diffraction peaks
(Angle 2-Theta .degree.): 6.2, 7.7, 15.2, 16.7, 17.1, 17.7, 19.8,
20.2, 24.9, 25.2, 25.5.
[0187] In a yet further embodiment, the invention provides the
besylate salt of the compound of Example 1 having the powder X-ray
diffraction pattern shown in FIG. 7 herein.
[0188] Napadisylate salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) and 0.259 g
1,5-naphthalenedisulfonic acid (0.70 mmoles) are dissolved in 9 ml
DMF at 87.degree. C. The clear solution is allowed to cool over
about 30 minutes to room temperature. Seeds are added at 65.degree.
C. and crystallization takes place slowly. The suspension is
stirred for about 18 hours at room temperature and filtered. The
solid is washed with 4 ml DMF/water 1:1 in two portions and 6 ml
water in three portions. The salt is dried first for about 3 hours
at 50.degree. C./ca. 10 mbar and then for about 16 hours at
80.degree. C./ca.10 mbar. The material has a melting point at about
304.degree. C. with a melting enthalpy of 83 g/J. A broad
endothermic phenomenon is observed at 107.degree. C. that might be
attributed to the loss of water. The material produced exhibited a
loss on drying of 6.1%. The water uptake was estimated by
Thermogravimetry after exposure to relative humidity (80% rh)
during 24 hours. A water uptake less than 0.05% was observed.
[0189] In another embodiment, the invention provides a napadysilate
salt of the compound of Example 1. In a further embodiment, the
invention provides the napadysilate salt of the compound of Example
1 comprising the following powder X-ray diffraction peaks (Angle
2-Theta .degree.): 6.4, 9.6, 13.1, 15.7, 16.1, 26.0; and which in
an additional embodiment comprises the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 9.6, 13.1, 15.7, 16.1,
16.4, 20.4, 20.9, 23.7, 26.0, 26.9.
[0190] In a yet further embodiment, the invention provides the
napadysilate salt of the compound of Example 1 having the powder
X-ray diffraction pattern shown in FIG. 12 herein.
[0191] Hydrochloride salt:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol free base (0.60 g; 1.40 mmoles) are dissolved in 12 ml acetone
at 55.degree. C. Hydrochloric acid 37% (0.138 g; 1.40 mmoles) is
added drop wise. The crystallization takes place quickly. The white
suspension is allowed to cool over about 30 minutes to room
temperature and stirred for 18 hours. After filtration the solid is
washed with 6 ml acetone in three portions and dried first for
about 3 hours at 50.degree. C./ca. 10 mbar and then for about 16
hours at 80.degree. C./ca.10 mbar. The material is exhibiting an
exothermic event at about 162.degree. C. with an enthalpy of -13.8
J/g. This phenomenon might be attributed to a solid transformation
into a more stable modification. An endothermic event is then seen
at about 259.degree. C. with an enthalpy of 99.7 J/g. The material
produced exhibited a loss on drying of 0.6%. The water uptake was
estimated by Thermogravimetry after exposure to relative humidity
(80% rh) during 24 hours. A water uptake of 0.3% was observed.
[0192] In another embodiment, the invention provides a
hydrochloride salt of the compound of Example 1. In a further
embodiment, the invention provides the hydrochloride salt of the
compound of Example 1 comprising the following powder X-ray
diffraction peaks (Angle 2-Theta .degree.): 6.1, 7.0, 19.8, 26.1;
and which in an additional embodiment comprises the following
powder X-ray diffraction peaks (Angle 2-Theta .degree.): 6.1, 7.0,
18.1, 19.8, 24.7, 26.1, 27.0, 27.7.
[0193] In a yet further embodiment, the invention provides the
hydrochloride salt of the compound of Example 1 having the powder
X-ray diffraction pattern shown in FIG. 5 herein.
[0194] The free acid or free base forms of the compounds of the
invention can be prepared from the corresponding base addition salt
or acid addition salt form, respectively. For example a compound of
the invention in an acid addition salt form can be converted to the
corresponding free base by treating with a suitable base (e.g.,
ammonium hydroxide solution, sodium hydroxide, and the like). A
compound of the invention in a base addition salt form can be
converted to the corresponding free acid by treating with a
suitable acid (e.g., hydrochloric acid, etc.). The nitrate salt of
the compound of example 1 can be made using methods known to the
skilled person. The powder X-ray diffraction pattern is disclosed
in FIG. 2 herein.
[0195] Compounds of the invention in unoxidized form can be
prepared from N-oxides of compounds of the invention by treating
with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl
phosphine, lithium borohydride, sodium borohydride, phosphorus
trichloride, tribromide, or the like) in a suitable inert organic
solvent (e.g. acetonitrile, ethanol, aqueous dioxane, or the like)
at 0 to 80.degree. C.
[0196] Prodrug derivatives of the compounds of the invention can be
prepared by methods known to those of ordinary skill in the art
(e.g., for further details see Saulnier et al., (1994), Bioorganic
and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example,
appropriate prodrugs can be prepared by reacting a non-derivatized
compound of the invention with a suitable carbamylating agent
(e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl
carbonate, or the like).
[0197] Protected derivatives of the compounds of the invention can
be made by means known to those of ordinary skill in the art. A
detailed description of techniques applicable to the creation of
protecting groups and their removal can be found in T. W. Greene,
"Protecting Groups in Organic Chemistry", 3.sup.rd edition, John
Wiley and Sons, Inc., 1999.
[0198] Compounds of the present invention can be conveniently
prepared, or formed during the process of the invention, as
solvates (e.g., hydrates). Hydrates of compounds of the present
invention can be conveniently prepared by recrystallization from an
aqueous/organic solvent mixture, using organic solvents such as
dioxin, tetrahydrofuran or methanol.
[0199] Compounds of the invention can be prepared as their
individual stereoisomers by reacting a racemic mixture of the
compound with an optically active resolving agent to form a pair of
diastereoisomeric compounds, separating the diastereomers and
recovering the optically pure enantiomers. While resolution of
enantiomers can be carried out using covalent diastereomeric
derivatives of the compounds of the invention, dissociable
complexes are preferred (e.g., crystalline diastereomeric salts).
Diastereomers have distinct physical properties (e.g., melting
points, boiling points, solubilities, reactivity, etc.) and can be
readily separated by taking advantage of these dissimilarities. The
diastereomers can be separated by chromatography, or preferably, by
separation/resolution techniques based upon differences in
solubility. The optically pure enantiomer is then recovered, along
with the resolving agent, by any practical means that would not
result in racemization. A more detailed description of the
techniques applicable to the resolution of stereoisomers of
compounds from their racemic mixture can be found in Jean Jacques,
Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and
Resolutions", John Wiley And Sons, Inc., 1981. Compounds of the
invention can also be prepared as their individual stereoisomers by
using chiral chromatography techniques, in particular, by use of
HPLC or SFC chromatography using a chiral stationary phase.
[0200] Powder X-ray diffraction spectra as enclosed herein were
obtained using the instrument Bruker D8 Vario in transmission
geometry, irradiation CuK.alpha. (30 kV, 40 mA), scan range
2.degree.-40.degree. (2 theta value), step time 90.3 s.
Differential scanning calorimetry (DSC) of Example 1 amorphous
material was carried out using the instrument Perkin Elmer DSC7 at
a heating rate of 40.degree. C./min.
[0201] In summary, the compounds of Formula I can be made by a
process, which involves:
[0202] (a) those of reaction schemes 1-5; and
[0203] (b) optionally converting a compound of the invention into a
pharmaceutically acceptable salt;
[0204] (c) optionally converting a salt form of a compound of the
invention to a non-salt form;
[0205] (d) optionally converting an unoxidized form of a compound
of the invention into a pharmaceutically acceptable N-oxide;
[0206] (e) optionally converting an N-oxide form of a compound of
the invention to its unoxidized form;
[0207] (f) optionally resolving an individual isomer of a compound
of the invention from a mixture of isomers;
[0208] (g) optionally converting a non-derivatized compound of the
invention into a pharmaceutically acceptable prodrug derivative;
and
[0209] (h) optionally converting a prodrug derivative of a compound
of the invention to its non-derivatized form.
[0210] Insofar as the production of the starting materials is not
particularly described, the compounds are known or can be prepared
analogously to methods known in the art or as disclosed in the
Examples hereinafter.
[0211] One of skill in the art will appreciate that the above
transformations are only representative of methods for preparation
of the compounds of the present invention, and that other well
known methods can similarly be used.
EXAMPLES
[0212] The present invention is further exemplified, but not
limited, by the following examples that illustrate the preparation
of compounds of Formula I (Examples) according to the
invention.
Example 1
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l
##STR00011##
[0214] Synthesis of 2,6-dichloro-9-isopropyl-9H-purine (b): To
2,6-dichloro-9H-purine (a) (6.0 mmol) dissolved in anhydrous DMF
(5.0 mL) was slowly added sodium hydride (7.8 mmol) with stirring
at rt over 2 hr. 2-iodopropane was added and the mixture was
stirred for 16 hr. The mixture was concentrated. The residue was
purified by column chromatography on silica gel, eluting with
hexane/EtOAc (20:1 to 3:1) to afford the title compound as a white
solid. .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 8.15 (s, 1H),
4.91 (m, 1H), 1.63 (d, 6H).
[0215] Synthesis of
4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (c):
2,6-dichloro-9-isopropyl-9H-purine (1.1 mmol) was mixed with
tyramine (1.16 mmol) dissolved in i-PrOH (6 ml) and the mixture was
stirred overnight. The reaction mixture was concentrated, and the
residue purified by column chromatography on silica gel, eluting
with hexane/EtOAc (5:1 to 1:2) to afford the title compound as a
white solid. .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 9.21 (br,
1H), 8.49 (s, 1H), 7.80 (s, 1H), 7.10 (d, 2H), 6.73 (d, 2H), 4.87
(m, 1H), 4.03 (t, 2H), 3.01 (t, 2H), 1.68 (d, 6H); HRMS (EI) calcd
for C.sub.16H.sub.18ClN.sub.5O (M+H.sup.+) 332.1273, found
332.1278.
[0216] Synthesis of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (d): A flame-dried schlenk flask was charged with
4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (0.62
mmol), thianaphthene-3-boronic acid (0.94 mmol),
pd.sub.2(dba).sub.3 (0.062 mmol), Cs.sub.2CO.sub.3 (1.25 mmol) and
1,3-bis(2,4,6-trimethylphenyl) imidazolium chloride (0.125 mmol).
The flask was evacuated and backfilled with N.sub.2 and anhydrous
1,4-dioxane (2 mL) was added. The flask was sealed and the reaction
mixture was stirred at 80.degree. C. for 24 hours. The reaction
mixture was concentrated and purified directly by column
chromatography on silica gel, eluting with hexane/EtOAc (20:1 to
1:4) to afford the title compound as a yellowish solid.
[0217] Alternatively, the synthesis of Example 1 can be carried out
as follows:
[0218] Synthesis of
2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine (b): A
round-bottom flask was charged with
6-chloro-2-iodo-9-isopropyl-9H-purine (prepared in Example 15c,
3.31 g, 0.0103 mol), benzo[b]thiophen-3-ylboronic acid (2.74 g,
0.0154 mol), and tetrakis(triphenylphosphine)palladium(0)(1.19 g,
0.0103 mol). To this mixture was added toluene (80 ml), ethanol (25
ml) and aqueous sodium carbonate solution (2M, 21 ml). The flask
was sealed and the reaction mixture was stirred at 90.degree. C.
for 1 h. Water was added to the cooled mixture, which was extracted
with ethyl acetate. The organic fractions were combined, dried over
sodium sulfate, and concentrated. The residue was purified by
column chromatography on silica gel, eluting with 20 to 50% EtOAc
in hexane to afford the title compound as a solid, which was
recrystallized from 1:1 methanol/water. .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta.=9.15 (d, 1H), 8.85 (s, 2H), 8.17 (d, 1H),
7.62 (t, 1H), 7.53 (t, 1H), 5.06 (m, 1H), 1.71 (d, 6H); MS m/z
329.0 (M+1).
[0219] Synthesis of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (c): 2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine
(2.2 g, 0.0067 mol) was suspended in anhydrous 2-propanol (70 mL)
in a pressure tube. Tyramine (1.01 g, 0.0074 mol) was added. The
tube was sealed and heated at 85.degree. C. for 16 hr. Additional
tyramine (0.50 g, 0.0037 mol) was added and the mixture was heted
at 85.degree. C. for 48 hr. The reaction was concentrated. Aqueous
sodium bicarbonate solution was added to the residue, which was
extracted with EtOAc. The combined organic extracts were dried over
sodium sulfate, filtered, and concentrated. The residue was
purified by column chromatography on silica gel, eluting with 50 to
85% EtOAc in hexane to afford a solid. The solid was triturated
with methanol to provide the title compound as an off-white
solid.
[0220] Alternatively, the synthesis of Example 1 can be carried out
as follows:
[0221] Synthesis of 2,6-dichloro-9-isopropyl-9H-purine (b): To
2,6-dichloro-9H-purine (a) (998 g, 5.28 mol) dissolved in anhydrous
DMF (5.0 L) was added sodium hydride (60% dispersion, 254 g, 6.35
mol) with stirring at 10.degree. C. over 1 hr. 2-iodopropane (1595
g) was added and the mixture was stirred at rt for 24 hr. Water
(5.0 L) was added, and the resulting solid precipitate was
collected and washed with water (500 ml) and heptane (2.times.2.5
L). The crude solid was crystallized from isopropyl acetate (2.1 L)
to provide the title compound as a solid.
[0222] Synthesis of
4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (c):
2,6-dichloro-9-isopropyl-9H-purine (500 g) was added in portions to
a stirred mixture of tyramine (593 g), triethylamine (262 g), and
i-PrOH (5.0 L) at 50.degree. C. The mixture was stirred at that
temperature for 4 hr, then the reaction mixture was concentrated.
The residue was taken up in isopropyl acetate (6.0 L) and was
washed with 20% citric acid solution (2.0 L) and water (2.0 L). The
organic layer was concentrated to dryness, then was taken up in
ethanol (2.0 L) and again concentrated to dryness. The crude solid
was crystallized from ethanol (3.2 L) to provide the title compound
as a solid.
[0223] Synthesis of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (d): A mixture of
4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (950 g),
thianaphthene-3-boronic acid (561 g),
dichlorobis(triphenylphosphine)palladium(II)(10.1 g), potassium
carbonate (791 g), water (3.25 L), and DMA (3.25 L) was stirred
under a nitrogen atmosphere for 10 min. The stirred mixture was
then heated at 70.degree. C. for 14 h. Ethyl acetate (6.5 L) and
water (3.25 L) were added, and the mixture was filtered through
Celite (125 g) at 50.degree. C., rinsing with ethyl acetate (1.0
L). The layers were separated, and the aqueous layer was extracted
at 50.degree. C. with additional ethyl acetate (7.5 L). The
combined organic layers were washed with water (3.times.2.5 L),
then distilled to remove about 2.5 L of solvent. Tetrahydrofuran
(2.5 L) and silica bond thio silica gel (200 g) were added. The
mixture was stirred at 70.degree. C. for 16 hr, then was filtered,
washing the pad with ethyl acetate (1.0 L). The combined filtrates
were concentrated at atmospheric pressure to a volume of about 5 L,
then the mixture was allowed to cool. The resulting solid was
collected and washed with ethyl acetate (2.times.1.0 L) to provide
the title compound.
[0224] The compound of Example 1 can be recrystallised using a
toluene/ethanol mixture and washed at room temperature with
NaHCO.sub.3 aqueous solution.
[0225] In another embodiment, the invention provides a compound of
Example 1 in crystal form modification A, wherein modification A
comprises the following powder X-ray diffraction peaks (angle
2-Theta .degree.): 12.1, 16.9, 18.9, 21.3; and which in an
additional embodiment comprises the following powder X-ray
diffraction peaks (angle 2-Theta .degree.): 12.1, 15.9, 16.9, 17.3,
18.9, 21.3, 22.1, 23.6, 24.4, 27.3.
[0226] In a yet further embodiment, the invention provides the
compound of Example 1 as solid form modification A comprising the
following powder X-ray diffraction peaks (angle 2-Theta .degree.):
9.0, 12.1, 13.0, 13.1, 13.6, 14.4, 14.7, 15.1, 15.9, 16.9, 17.3,
17.7, 18.0, 18.9, 19.0, 20.1, 21.3, 22.1, 22.3, 22.6, 22.8, 23.4,
23.6, 24.4, 25.3, 26.3, 26.5, 27.3, 27.8, 28.2, 29.5, 29.7, 30.4,
30.7, 31.0, 31.4, 32.2, 32.8, 33.3, 34.3, 35.5, 36.4, 37.5, 38.4,
39.0, 39.4.
[0227] The powder X-ray diffraction pattern of the compound of
Example 1, modification A, is shown in FIG. 1 herein. Amorphous
material of the compound of Example 1 was produced in situ in a DSC
(differential scanning calorimetry) crucible by heating the
compound until melting and annealing/cooling. Upon the cooling
cycle the glass transition could be observed but upon the reheating
cycle is much more characterized at about 70-75.degree. C. The DSC
pattern is shown in FIG. 13 herein.
Example 15
4-(2-(Pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
##STR00012##
[0229] Synthesis of 2-Amino-6-chloro-9-isopropyl-9H-purine (b):
Sodium hydride (1.5 g of 60% dispersion in mineral oil, 38 mmol)
was added in portions over 10 min to a stirred suspension of
2-amino-6-chloro-9H-purine (5.34 g, 31.5 mmol) in anhydrous DMF (50
mL) at rt. After 45 min, the mixture was cooled in an ice bath,
then 2-iodopropane was added. The cooling bath was removed and the
stirred mixture was allowed to warm to rt over 16 h. The mixture
was cooled in ice, then water was added. The mixture was
concentrated, and the residue was treated with hot ethyl acetate.
The cooled mixture was filtered, and the filtrate was concentrated.
The residue was purified by column chromatography on silica gel,
eluting with 0 to 50% EtOAc in hexane to afford the title compound
as a solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.83 (s, 1H),
5.17 (s, 2H), 4.71-4.66 (m, 1H), 1.57 (d, 6H). MS m/z 212.1
(M+1).
[0230] Synthesis of 6-Chloro-2-iodo-9-isopropyl-9H-purine (c):
6-chloro-9-isopropyl-9H-purin-2-amine (2.68 g, 12.7 mmol) was
dissolved in THF (64 mL) at rt. Iodine (1.61 g, 6.25 mmol),
CH.sub.2I.sub.2 (10.6 mL) and CuI (1.27 g, 6.66 mmol) were added.
The mixture was stirred for 5 min at room temperature. Isopentyl
nitrite (5.33 mL) was added. The reaction mixture was refluxed for
45 min, and was then cooled to room temperature. Saturated aqueous
sodium bicarbonate solution was added, and the mixture was
extracted with EtOAc three times. The combined organic phase was
washed with brine, dried with MgSO.sub.4 and concentrated. The
residue was purified by column chromatography on silica gel,
eluting with 0 to 30% ethyl acetate in hexane to afford the title
compound as a solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.09
(s, 1H), 4.95-4.88 (m, 1H), 1.65 (d, 6H). MS m/z 323.0 (M+1).
[0231] Synthesis of 6-Chloro-2-(pyridin-3-yl)-9-isopropyl-9H-purine
(d): A round-bottom flask was charged with
6-chloro-2-iodo-9-isopropyl-9H-purine (1.2 g, 3.7 mmol),
pyridine-3-boronic acid 1,3-propanediol cyclic ester (0.91 g, 5.6
mmol), and tetrakis(triphenylphosphine)palladium(0)(430 mg, 0.37
mmol). To this mixture was added toluene (60 ml), ethanol (6 ml)
and aqueous sodium carbonate solution (2M, 15 ml). The flask was
sealed and the reaction mixture was stirred at 80.degree. C. for 4
h. Water was added to the cooled mixture, which was extracted with
ethyl acetate (50 ml.times.3). The organic fractions were combined,
dried over sodium sulfate, and concentrated. The residue was
purified by column chromatography on silica gel, eluting with 30 to
70% EtOAc in hexane to afford the title compound as a solid.
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 9.60 (d, 1H), 8.90-8.87
(m, 1H), 8.68 (s, 1H), 8.67 (d, 1H), 7.63-7.60 (m, 1H), 5.12-5.05
(m, 1H), 1.74 (d, 6H). MS m/z 274.1 (M+1).
[0232] Synthesis of
4-(2-(pyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
(e): 6-chloro-9-isopropyl-2-(pyridin-3-yl)-9H-purine (300 mg, 1.1
mmol) was suspended in anhydrous 2-propanol (40 mL) in a pressure
tube. Tyramine (300 mg, 2.2 mmol) was added. The tube was sealed
and heated to 85.degree. C. for 16 hr. The reaction was
concentrated and the residue was purified by column chromatography
on silica gel, eluting with 0 to 70% EtOAc in hexane to afford the
title compound as a solid.
Example 123
4-(2-(9-Isopropyl-2-(2-methyl-1H-imidazol-1-yl)-9H-purin-6-ylamino)ethyl)p-
henol
##STR00013##
[0234] A microwave reaction tube was charged with
4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (30 mg,
0.091 mmol), 2-methyl-1H-imidazole (59 mg, 0.73 mmol) and 0.5 ml of
NMP. The sealed tube was heated under microwave irradiation at
240.degree. C. for 2 hr. The reaction mixture was purified by
reverse-phase HPLC (Cis column, eluting with ACN-H.sub.2O 0.05%
TFA) to afford the title compound as an off-white solid.
Example 128
4-(2-(2-(5-Chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
##STR00014##
[0236] Synthesis of
4-(2-(2-iodo-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (b): A
mixture of 6-chloro-2-iodo-9-isopropyl-9H-purine (a) (1.0 g, 3.1
mmol), tyramine (0.64 g, 4.65 mmol), triethylamine (0.63 g, 6.2
mmol) and 2-propanol (30 mL) was heated at 85.degree. C. for 2 hr.
The reaction mixture was concentrated and saturated aqueous sodium
bicarbonate solution was added. The mixture was extracted with
ethyl acetate (50 ml.times.3). The combined organic layers were
dried over sodium sulfate, filtered, and concentrated. The residue
was purified by column chromatography on silica gel (25 to 75%
ethyl acetate in hexane eluant) to afford the title compound as a
solid. MS m/z 424.1 (M+1).
[0237] Synthesis of
4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)pheno-
l (c): Following the procedure of Example 15d,
4-(2-(2-iodo-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (b) was
reacted with 5-chloropyridin-3-ylboronic acid. The crude product
was purified by reverse-phase HPLC (Cis column, eluting with
ACN-H.sub.2O 0.05% TFA) to afford the title compound as an
off-white solid.
Example 134
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-y-
lamino)ethyl)phenol
##STR00015##
[0239] Synthesis of
4-(2-(6-chloro-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)ethyl)ph-
enol (b): Following the procedure of Example 128b,
4,6-dichloro-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidine (U.S. Pat.
No. 3,399,196) (a) (0.184 g, 0.795 mmol) was reacted with tyramine.
The crude residue was purified by silica gel chromatography (25 to
75% ethyl acetate in hexane eluant) to afford the title compound as
a solid. MS m/z 332.1 (M+1).
[0240] Synthesis of
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4--
ylamino)ethyl)phenol (c): Following the procedure of Example 15d,
4-(2-(6-chloro-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)ethyl)ph-
enol (b) was reacted with 5-fluoropyridin-3-ylboronic acid. The
crude residue was purified by reverse-phase HPLC (Cis column,
eluting with ACN-H.sub.2O 0.05% TFA) to afford the title compound
as an off-white solid.
Example 141
4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl-
amino)ethyl)phenol
##STR00016##
[0242] Synthesis of
2,4-dichloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine (b): Following
the procedure of Example 15b,
2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 2.67 mmol) was
reacted with 2-iodopropane. The crude residue was purified by
silica gel chromatography (15 to 25% ethyl acetate in hexane
eluant) to afford the title compound as a solid. MS m/z 230.2
(M+1).
[0243] Synthesis of
4-(2-(2-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phe-
nol (c): Following the procedure of Example 128b,
2,4-dichloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine (b) (0.278 g,
1.21 mmol) was reacted with tyramine. The crude residue was
purified by silica gel chromatography (25 to 75% ethyl acetate in
hexane eluant) to afford the title compound as a solid. MS m/z
331.1 (M+1).
[0244] Synthesis of
4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-y-
lamino)ethyl)phenol (d): Following the procedure of Example 15d,
4-(2-(2-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phe-
nol (20 mg, 0.06 mmol) was reacted with 5-fluoropyridin-3-ylboronic
acid. The crude residue was purified by reverse-phase HPLC
(C.sub.18 column, eluting with ACN-H.sub.2O 0.05% TFA) to afford
the title compound as an off-white solid.
Example 153
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-yl-
amino)ethyl)phenol
##STR00017##
[0246] Synthesis of
(R)-2,6-dichloro-9-(tetrahydrofuran-3-yl)-9H-purine (b): A solution
of 5,7-2,6-dichloro-9H-purine (400 mg, 2.12 mmol),
(S)-tetrahydrofuran-3-ol (88 mg, 2.5 mmol) and triphenylphosphine
(1.0 g, 3.8 mmol) in anhydrous THF (30 mL) was treated at
-78.degree. C. with diisopropyl azodicarboxylate (856 mg, 4.23
mmol). The reaction was allowed to warmed to rt and was stirred for
16 hr. Saturated aqueous sodium bicarbonate solution was added and
the mixture was extracted with ethyl acetate. The organic layers
were combined, dried over sodium sulfate, and concentrated. The
residue was purified by silica gel chromatography (10 to 80% ethyl
acetate in hexane eluant) to afford a white solid which consisted
of the title compound contaminated with triphenylphosphoxide. MS
m/z 258.0 (M+1).
[0247] Synthesis of
(R)-4-(2-(2-chloro-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol (c): Following the procedure of Example 128b,
(R)-2,6-dichloro-9-(tetrahydrofuran-3-yl)-9H-purine (b) was reacted
with tyramine. The crude reaction mixture was purified by
reverse-phase preparative HPLC. MS m/z 360.1 (M+1).
[0248] Synthesis of
(R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6-y-
lamino)ethyl)phenol: Following the procedure of Example 15d,
(R)-4-(2-(2-chloro-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phen-
ol (c) was reacted with benzo[b]thiophen-3-ylboronic acid (22.3 mg,
0.125 mmol). The crude residue was purified by reverse-phase
preparative HPLC to afford the title compound as an off-white
solid.
Example 157
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)-
propan-1-ol
##STR00018##
[0250] Synthesis of methyl 2-(2,6-dichloro-9H-purin-9-yl)propanoate
(b): A mixture of 2,6-dichloro-9H-purine (5.0 g, 26.5 mmol), methyl
2-bromopropanoate (5.3 g, 31.7 mmol) and potassium carbonate (11.0
g, 79.4 mmol) in anhydrous DMF (100 mL) was heated at 100.degree.
C. for 15 h. Sat. aqueous sodium bicarbonate solution was added and
reaction was extracted with ethyl acetate (150 ml.times.3). The
organic layers were combined, washed with brine, dried over sodium
sulfate, and concentrated. The residue was purified by silica gel
chromatography (10 to 80% ethyl acetate in hexane eluant) to afford
the title compound as a white solid. MS m/z 275.0 (M+1).
[0251] Synthesis of methyl
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-chloro-9H-purin-9-yl)propanoate
(c): A mixture of methyl 2-(2,6-dichloro-9H-purin-9-yl)propanoate
(b) (600 mg, 2.2 mmol), tryptamine (420 mg, 2.6 mmol) and
2-propanol (30 mL) was heated at 85.degree. C. in a sealed tube for
16 h. The reaction mixture was cooled to room temperature and
concentrated. The residue was purified by silica gel chromatography
(10 to 80% ethyl acetate in hexane eluant) to afford the title
compound as a white solid. MS m/z 360.1 (M+1).
[0252] Synthesis of methyl
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propanoate (d): A 150 ml pressure tube was charged with methyl
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-chloro-9H-purin-9-yl)propanoate
(c) (300 mg, 0.75 mmol), 5-fluoropyridin-3-ylboronic acid (159 mg,
1.1 mmol), tetrakis(triphenylphosphine)-palladium(0)(87 mg, 0.075
mmol), K.sub.3PO.sub.4 (638 mg, 3.0 mmol), and anhydrous dioxane
(15 mL). The pressure tube was sparged with nitrogen and was
sealed, then the reaction mixture was heated at 130.degree. C. for
6 h with stirring. Water was added to the cooled mixture, and the
mixture was extracted with ethyl acetate (50 ml.times.3). The
combined organic layers were washed with brine, dried over sodium
sulfate, and concentrated. The residue was purified by silica gel
chromatography (10 to 80% ethyl acetate in hexane eluant) to afford
the title compound contaminated with a small amount of
triphenylphosphine oxide. MS m/z 460.1 (M+1).
[0253] Synthesis of
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propan-1-ol: Lithium aluminum hydride (230 mg, 6.1 mmol) was added
in portions to a 0.degree. C. solution of methyl
2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl-
)propanoate (282 mg, 0.61 mmol) in anhydrous THF (15 mL). The
stirred reaction mixture was allowed to warm to rt over 2 h, then
water was added carefully. The mixture was extracted with EtOAc (50
ml.times.3). The organic fractions were combined, washed with
brine, dried over sodium sulfate, and concentrated. The residue was
purified by silica gel column chromatography (0 to 5% solvent B in
dichloromethane; solvent B=2M ammonia in methanol) to afford the
partially purified title product. This was further purified by
preparative TLC (5% solvent B in dichloromethane) to provide the
title compound as a white solid.
Examples 157R & 157S
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-
-yl)propan-1-ol &
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol
##STR00019##
[0255]
(R/S)-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H--
purin-9-yl)propan-1-ol was separated into the individual
enantiomers using preparative chiral HPLC on a 21.times.250 mm
Lux-Cellulose-2 (Phenomenex) chiral column. A 3 mg/ml solution of
the racemate in methanol was prepared and loaded onto the column
with 0.5 ml solution per injection. The column was eluted with
85/7.5/7.5 hexane/ethanol/methanol at a flow rate of 20 mL/min for
25 min. Peaks 1 and 2 were eluted at 20 min and 22.5 min,
respectively. Analytical chromatography was performed on a
4.6.times.100 mm Lux_Cellulose-2 (Phenomenex) chiral column,
eluting with 90/5/5 hexane/ethanol/methanol at 1 mL/min for 20 min.
Peaks 1 and 2 were eluted at 17.45 and 18.14 min, respectively.
Example 157R
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-
-yl)propan-1-ol
##STR00020##
[0257] Synthesis of
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-(1-(benzyloxy)propan-2-yl)-2-(5-fluorop-
yridin-3-yl)-9H-purin-6-amine (b): Following, in succession, the
procedures of Example 153b (using 2,6-dichloro-9H-purine and
(S)-1-(benzyloxy)propan-2-ol as reactants), Example 153c (using
tryptamine as reactant), and Example 153d (using
5-fluoropyridin-3-ylboronic acid as reactant), the title compound
was obtained.
[0258] Synthesis of
(R)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin--
9-yl)propan-1-ol (c): A solution of
(R)--N-(2-(1H-indol-3-yl)ethyl)-9-(1-(benzyloxy)propan-2-yl)-2-(5-fluorop-
yridin-3-yl)-9H-purin-6-amine (b) (0.15 g, 0.29 mmol) in DCM (10
ml) was treated with BCl.sub.3 (1M, 2.9 ml, 2.9 mmol) in DCM (10
ml) at -78.degree. C. for 2 hr. 1N aqueous sodium hydroxide
solution was added, and the mixture was extracted with DCM. The
combined organic extracts were dried over sodium sulfate, filtered,
and concentrated and the residue was purified by silica gel column
chromatography (5% MeOH in DCM eluant) to provide the title
compound. MS m/z 432.2 (M+1).
Example 157S
(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-
-yl)propan-1-ol
##STR00021##
[0260] Following the procedure of Example 157R, but employing
(R)-1-(benzyloxy)propan-2-ol in place of
(S)-1-(benzyloxy)propan-2-ol, the title compound was prepared. MS
m/z 432.2 (M+1).
Example 161
4-(2-(6-(5-Fluoropyridin-3-yl)-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-ylam-
ino)ethyl)phenol
##STR00022##
[0262] Synthesis of
4,6-dichloro-1-isopropyl-1H-imidazo[4,5-c]pyridine (b): Following
the procedure of Example 15b,
4,6-dichloro-1H-imidazo[4,5-c]pyridine (J. Het. Chem. 1965,
196-201) (0.19 g, 1.0 mmol) was reacted with 2-iodopropane. The
residue was purified by silica gel chromatography (25 to 35% ethyl
acetate in hexane eluant) to afford the title compound as a solid.
MS m/z 230.2 (M+1).
[0263] Synthesis of
4-(2-(6-chloro-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-ylamino)ethyl)pheno-
l (c): A mixture of
4,6-dichloro-1-isopropyl-H-imidazo[4,5-c]pyridine (b) (40 mg, 0.17
mmol), tyramine (120 mg, 0.86 mmol), and 2-butanol (2 mL) was
heated under microwave irradiation at 140.degree. C. for 8 hr. The
mixture was concentrated and the residue was purified by
reverse-phase HPLC (Cis column, eluting with ACN-H.sub.2O 0.05%
TFA) to afford the title compound as an off-white solid. MS m/z
331.1 (M+1).
[0264] Synthesis of
4-(2-(6-(5-fluoropyridin-3-yl)-1-isopropyl-H-imidazo[4,5-c]pyridin-4-ylam-
ino)ethyl)phenol (d): A 5 ml microwave reaction vial was charged
with
4-(2-(6-chloro-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-ylamino)ethyl)pheno-
l (c): (17 mg, 0.051 mmol), 5-fluoropyridin-3-ylboronic acid (72
mg, 0.51 mmol), and tetrakis(triphenylphosphine)palladium(0) (36
mg, 0.031 mmol). To this mixture was added toluene (1 ml), ethanol
(0.5 ml) and aqueous sodium carbonate solution (2M, 0.5 ml). The
vial was sealed and the reaction mixture was stirred at 140.degree.
C. under microwave irradiation for 2 hours. Water was added to the
cooled mixture, which was extracted with ethyl acetate (5
ml.times.3). The organic fractions were combined, dried over sodium
sulfate, and concentrated. The residue was purified by
reverse-phase HPLC (Cis column, eluting with ACN-H.sub.2O 0.05%
TFA) to afford the title compound as an off-white solid.
Example 177
4-(2-(5-(5-Fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylam-
ino)ethyl)phenol
##STR00023##
[0266] Synthesis of
5,7-dichloro-3-isopropyl-3H-imidazo[4,5-b]pyridine (b): Following
the procedure of Example 15b,
5,7-dichloro-3H-imidazo[4,5-b]pyridine (J. Med. Chem., 2007, 50,
828-834) (0.118 g, 0.624 mmol) was reacted with 2-iodopropane. The
crude product mixture was purified by silica gel chromatography (25
to 35% ethyl acetate in hexane eluant) to afford a mixture of the
title compound (major) and an isomeric product as a solid. MS m/z
230.2 (M+1).
[0267] Synthesis of
4-(2-(5-chloro-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)pheno-
l (c): The product mixture containing
5,7-dichloro-3-isopropyl-3H-imidazo[4,5-b]pyridine (b) (40 mg, 0.17
mmol), tyramine (120 mg, 0.87 mmol), and 2-propanol (2 mL) was
heated in a sealed vial at 140.degree. C. for 72 hr. The mixture
was concentrated, and the residue was purified by preparative TLC
(1:2 hexanes/ethyl acetate eluant) to afford the title compound as
an off-white solid. MS m/z 331.1 (M+1).
[0268] Synthesis of
4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-yla-
mino)ethyl)phenol (d): Following the procedure of Example 161d,
4-(2-(5-chloro-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)pheno-
l (c) (15 mg, 0.047 mmol) was reacted with
5-fluoropyridin-3-ylboronic acid. The crude residue was purified
preparative TLC (1:1 hexanes/ethyl acetate eluant) to afford the
title compound as an off-white solid.
[0269] By repeating the procedures described in the above examples,
using appropriate starting materials, the following compounds of
Formula I, as identified in Table 1, are obtained.
TABLE-US-00002 TABLE 1 EC.sub.50 Example Physical Data (% CD34+)
Number Structure .sup.1H NMR and/or MS .mu.M 1 ##STR00024## .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. = 9.20 (d, 1H), 8.58 (s, 1H),
8.00-7.80 (m, 2H), 7.55-7.38 (m, 3H), 7.11 (d, 2H), 6.72 (d, 2H),
6.18 (br, 1H), 5.01-4.68 (m, 1H), 4.02 (br, 2H), 3.00 (t, 2H), 1.68
(d, 6H); HRMS (EI) m/z 430.1698 (M + 1) 0.12 2 ##STR00025## .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. = 9.22 (d, 1H), 8.53 (s, 1H),
7.92 (d, 1H), 7.80 (s, 1H), 7.52-7.33 (m, 3H), 7.13 (d, 2H), 6.74
(d, 2H), 6.08 (br, 1H), 4.80- 4.62 (m, 1H), 4.02 (br, 2H), 3.01 (t,
2H), 2.20-1.90 (m, 2H), 1.77 (d, 3H), 0.92 (t, 3H); HRMS (EI) m/z
444.1857 (M + 1) 0.03 3 ##STR00026## HRMS (EI) m/z 554.2005 (M + 1)
0.15 4 ##STR00027## HRMS (EI) m/z 472.1807 (M + 1) 1.49 5
##STR00028## HRMS (EI) m/z 546.1571 (M + 1) 2.08 6 ##STR00029##
HRMS (EI) m/z 444.1857 (M + 1) 2.53 7 ##STR00030## HRMS (EI) m/z
402.1385 (M + 1) 7.2 8 ##STR00031## HRMS (EI) m/z 492.1856 (M + 1)
6.03 9 ##STR00032## .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. =
9.21 (d, 1H), 8.48 (s, 1H), 8.02 (br, 1H), 7.89 (d, 1H), 7.79 (s,
1H), 7.70 (d, 1H), 7.50-7.07 (m, 6H), 5.82 (br, 1H), 5.00- 4.88 (m,
1H), 4.13 (br, 2H), 3.22 (t, 2H), 1.69 (d, 6H); HRMS (EI) m/z
453.1857 (M + 1) 0.02 10 ##STR00033## HRMS (EI) m/z 420.1315 (M +
1) 1.38 11 ##STR00034## HRMS (EI) m/z 430.1697 (M + 1) 1.45 12
##STR00035## HRMS (EI) m/z 432.1655 (M + 1) 1.76 13 ##STR00036##
HRMS (EI) m/z 429.1853 (M + 1) 5.75 14 ##STR00037## HRMS (EI) m/z
376.1881 (M + 1) 0.17 15 ##STR00038## .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 9.57 (d, 1H), 8.85-8.83 (m, 1H), 8.59 (q, 1H),
8.16 (s, 1H), 7.57 (q, 1H), 7.13 (d, 2H), 6.72 (d, 2H), 4.98-4.91
(m, 1H), 3.91 (bs, H), 2.98 (t, 2H), 1.68 (d, 6H); HRMS (EI) m/z
375.1928 (M + 1) 0.19 16 ##STR00039## HRMS (EI) m/z 374.1976 (M +
1) 0.46 17 ##STR00040## HRMS (EI) m/z 380.1544 (M + 1) 0.97 18
##STR00041## HRMS (EI) m/z 364.1769 (M + 1) 3.9 19 ##STR00042##
HRMS (EI) m/z 466.1493 (M + 1) 1.1 20 ##STR00043## HRMS (EI) m/z
420.2184 (M + 1) 7.8 21 ##STR00044## HRMS (EI) m/z 514.2638 (M + 1)
0.13 23 ##STR00045## HRMS (EI) m/z 467.2013 (M + 1) 0.019 31
##STR00046## MS m/z 375.2 (M + 1) 0.66 32 ##STR00047## MS m/z 447.2
(M + 1) 5.6 33 ##STR00048## MS m/z 405.2 (M + 1) 0.27 34
##STR00049## MS m/z 393.2 (M + 1) 0.16 35 ##STR00050## MS m/z 389.2
(M + 1) 0.34 37 ##STR00051## MS m/z 400.2 (M + 1) 0.024 38
##STR00052## MS m/z 367.2 (M + 1) 1.6 40 ##STR00053## MS m/z 364.2
(M + 1) 0.26 42 ##STR00054## MS m/z 376.2 (M + 1) 0.64 43
##STR00055## MS m/z 376.2 (M + 1) 2.4 44 ##STR00056## MS m/z 375.2
(M + 1) 1.7 45 ##STR00057## MS m/z 389.2 (M + 1) 0.063 46
##STR00058## MS m/z 453.2 (M + 1) 0.65 48 ##STR00059## MS m/z 409.2
(M + 1) 0.51 50 ##STR00060## MS m/z 393.2 (M + 1) 0.034 52
##STR00061## MS m/z 378.2 (M + 1) 55 ##STR00062## MS m/z 380.2 (M +
1) 1.3 58 ##STR00063## MS m/z 394.2 (M + 1) 0.24 60 ##STR00064## MS
m/z 405.1 (M + 1) 3.2 61 ##STR00065## MS m/z 428.1 (M + 1) 0.13 62
##STR00066## MS m/z 412.1 (M + 1) 0.72 70 ##STR00067## MS m/z 367.2
(M + 1) 2.7 72 ##STR00068## MS m/z 375.2 (M + 1) 6.3 73
##STR00069## MS m/z 363.2 (M + 1) 8.2 76 ##STR00070## MS m/z 396.2
(M + 1) 6.0 81 ##STR00071## MS m/z 422.1 (M + 1) 2.7 82
##STR00072## MS m/z 420.1 (M + 1) 7.9 83 ##STR00073## MS m/z 430.1
(M + 1) 7.1 84 ##STR00074## MS m/z 418.1 (M + 1) 5.4 88
##STR00075## MS m/z 446.10 (M + 1) 2.6 89 ##STR00076## MS m/z
396.10 (M + 1) 1.4 90 ##STR00077## MS m/z 456.2 (M + 1) 3.3 91
##STR00078## MS m/z 398.1 (M + 1) 0.029 92 ##STR00079## MS m/z
452.2 (M + 1) 7.1 93S ##STR00080## MS m/z 403.1 (M + 1) 1.1 93R
##STR00081## MS m/z 403.1 (M + 1) 0.52 94 ##STR00082## MS m/z 389.1
(M + 1) 0.97 95 ##STR00083## MS m/z 389.1 (M + 1) 2.3 98
##STR00084## MS m/z 399.2 (M + 1) 8.2 99 ##STR00085## MS m/z 389.2
(M + 1) 7.5 113 ##STR00086## MS m/z 391.2 (M + 1) 0.54 114
##STR00087## MS m/z 454.1 (M + 1) 1.1 118 ##STR00088## MS m/z 393.2
(M + 1) 0.45 119 ##STR00089## MS m/z 377.2 (M + 1) 1.4 120
##STR00090## MS m/z 381.2 (M + 1) 1.4 121 ##STR00091## MS m/z 414.2
(M + 1) 0.086 122 ##STR00092## MS m/z 414.2 (M + 1) 0.42 123
##STR00093## .sup.1H NMR (400 MHz, DMSO): .delta. = 9.21 (br, 1H),
8.57 (t, 1H), 8.36 (s, 1H), 8.23 (d, 1H), 7.70 (d, 1H), 7.04 (d,
2H), 6.66 (d, 2H), 4.84-4.72 (m, 1H), 3.67 (q, 2H), 2.99 (s, 3H),
2.83 (t, 2H), 1.56 (d, 6H); MS m/z 378.2 (M + 1) 0.066 124
##STR00094## MS m/z 428.2 (M + 1) 0.003 125 ##STR00095## MS m/z
399.2 (M + 1) 126 ##STR00096## MS m/z 363.2 (M + 1) 5.0 127
##STR00097## MS m/z 407.3 (M + 1) 0.47 128 ##STR00098## .sup.1H NMR
(400 MHz, DMSO): .delta. = 9.47 (s, 1H), 8.71 (s, 1H), 8.67 (s,
1H), 8.32 (s, 1H), 8.04 (t, 1H), 7.10 (d, 2H), 6.69 (d, 2H),
4.91-4.81 (m, 1H), 3.80- 3.70 (m, 2H), 2.86 (t, 2H), 1.58 (d, 6H);
MS m/z 409.2 (M + 1) 0.019 129 ##STR00099## MS m/z 443.2 (M + 1)
0.12 130 ##STR00100## .sup.1H NMR (400 MHz, DMSO): .delta. = 10.82
(s, 1H), 9.74 (s, 1H), 9.10 (s, 1H), 8.99 (s, 1H), 8.32 (s, 1H),
8.13 (t, 1H), 7.65 (d, 1H), 7.32 (d, 1H), 7.22 (s, 1H), 7.06 (t,
1H), 6.99 (t, 1H), 4.72-4.60 (m, 1H), 3.96- 3.85 (m, 2H), 3.08 (t,
2H), 2.08-1.88 (m, 2H), 1.58 (d, 3H), 0.77 (t, 3H); MS m/z 437.2 (M
+ 1) 0.001 131 ##STR00101## .sup.1H NMR (400 MHz, DMSO): .delta. =
10.83 (s, 1H), 9.40 (s, 1H), 8.97 (s, 1H), 8.76 (s, 1H), 8.35 (s,
1H), 8.18 (t, 1H), 7.62 (d, 1H), 7.33 (d, 1H), 7.23 (s, 1H), 7.06
(t, 1H), 6.97 (t, 1H), 4.72-4.60 (m, 1H), 3.96- 3.82 (m, 2H), 3.10
(t, 2H), 2.53 (s, 3H), 2.09-1.89 (m, 2H), 1.58 (d, 3H), 0.77 (t,
3H); MS m/z 426.2 (M + 1) 0.004 131R ##STR00102## MS m/z 430.2 (M +
1) 0.001 131S ##STR00103## MS m/z 430.2 (M + 1) 0.002 132
##STR00104## .sup.1H NMR (400 MHz, DMSO): .delta. = 10.83 (s, 1H),
9.42 (s, 1H), 8.66 (s, 1H), 8.41 (d, 1H), 8.31 (s, 1H), 8.09 (t,
1H), 7.64 (d, 1H), 7.34 (d, 1H), 7.22 (s, 1H), 7.07 (t, 1H), 6.97
(t, 1H), 4.68-4.60 (m, 1H), 3.92- 3.84 (m, 2H), 3.08 (t, 2H),
2.08-1.90 (m, 2H), 1.58 (d, 3H), 0.77 (t, 3H); MS m/z 430.2 (M + 1)
0.003 132R ##STR00105## MS m/z 426.2 (M + 1) 0.003 132S
##STR00106## MS m/z 426.2 (M + 1) 0.003 133 ##STR00107## MS m/z
414.2 (M + 1) 0.18 134 ##STR00108## .sup.1H NMR (400 MHz, DMSO):
.delta. = 9.44 (s, 1H), 9.21 (s, 1H), 8.69 (d, 1H), 8.56 (t, 1H),
8.47 (d, 1H), 8.14 (s, 1H), 7.09 (d, 2H), 6.69 (d, 2H), 5.17-5.09
(m, 1H), 3.80-3.75 (m, 2H), 2.87 (t, 2H), 1.48 (d, 6H); MS m/z
393.2 (M + 1) 0.20 135 ##STR00109## MS m/z 430.2 (M + 1) 0.38 137
##STR00110## MS m/z 421.1 (M + 1) 138 ##STR00111## MS m/z 389.2 (M
+ 1) 0.40 139 ##STR00112## MS m/z 400.2 (M + 1) 1.3 140
##STR00113## MS m/z 400.2 (M + 1) 0.091 141 ##STR00114## .sup.1H
NMR (400 MHz, DMSO): .delta. = 9.42 (s, 1H), 8.63 (d, 1H), 8.42 (d,
1H), 7.79 (t, 1H), 7.35 (d, 1H), 7.09 (d, 2H), 6.70 (d, 2H), 6.61
(d, 1H), 5.08-5.00 (m, 1H), 3.76-3.70 (m, 2H), 2.87 (t, 2H), 1.47
(d, 6H); MS m/z 392.2 (M + 1) 0.16 143 ##STR00115## MS m/z 400.2 (M
+ 1) 4.3 144 ##STR00116## MS m/z 389.2 (M + 1) 0.16 145
##STR00117## MS m/z 425.2 (M + 1) 5.4 146 ##STR00118## MS m/z 409.1
(M + 1) 0.24 147 ##STR00119## MS m/z 393.2 (M + 1) 0.092 148
##STR00120## MS m/z 432.2 (M + 1) 0.75 149 ##STR00121## MS m/z
416.2 (M + 1) 0.52 150 ##STR00122## MS m/z 389.2 (M + 1) 0.057 151
##STR00123## MS m/z 444.1 (M + 1) 0.17 152 ##STR00124## MS m/z
458.2 (M + 1) 0.35 153 ##STR00125## .sup.1H NMR (400 MHz, CD3OD):
.delta. = 9.14 (s, 1H), 8.55 (s, 1H), 8.33 (s, 1H), 7.96 (d, 1H),
7.44 (t, 1H), 7.15 (d, 2H), 6.73 (d, 2H), 5.46-5.43 (m, 1H), 4.27-
3.94 (m, 6H), 2.98 (t, 2H),
2.73-2.64 (m, 1H), 2.46- 2.39 (m, 1H); MS m/z 458.2 (M + 1) 0.22
157 ##STR00126## .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. = 9.40
(s, 1H), 8.53-8.48 (m, 2H), 8.23 (s, 1H), 7.65 (d, 1H), 7.31 (d,
1H), 7.11 (s, 1H), 7.08-7.04 (m, 1H), 7.01-6.97 (m, 1H), 4.08-4.03
(m, 3H), 3.94 (dd, 1H), 3.35-3.30 (m, 1H), 3.19 (t, 2H), 1.68 (d,
3H); MS m/z 432.2 (M + 1) 0.005 157R ##STR00127## MS m/z 432.2 (M +
1). 0.008 157S ##STR00128## MS m/z 432.2 (M + 1) 0.003 158
##STR00129## MS m/z 444.2 (M + 1) 0.012 159 ##STR00130## MS m/z
415.2 (M + 1) 0.59 160 ##STR00131## MS m/z 415.2 (M + 1) 1.9 161
##STR00132## .sup.1H NMR (400 MHz, DMSO): .delta. = 9.11 (s, 1H),
8.64 (s, 1H), 8.51 (s, 1H), 8.30 (d, 1H), 7.74 (s, 1H), 7.09 (d,
2H), 6.69 (d, 2H), 4.88-4.76 (m, 1H), 3.88- 3.78 (m, 2H), 2.88 (t,
2H), 1.56 (d, 6H); MS m/z 392.2 (M + 1) 0.17 162 ##STR00133## MS
m/z 392.2 (M + 1) 0.14 166 ##STR00134## MS m/z 378.1 (M + 1) 7.5
167 ##STR00135## MS m/z 409.2 (M + 1) 0.29 169 ##STR00136## MS m/z
446.2 (M + 1) 0.044 170 ##STR00137## MS m/z 416.2 (M + 1) 0.006 172
##STR00138## MS m/z 446.2 (M + 1) 0.42 173 ##STR00139## MS m/z
414.1 (M + 1) 0.012 174 ##STR00140## MS m/z 394.2 (M + 1) 2.2 175
##STR00141## MS m/z 417.2 (M + 1) 0.42 176 ##STR00142## MS m/z
531.3 (M + 1) 1.1 177 ##STR00143## .sup.1H NMR (400 MHz, DMSO):
.delta. = 9.18 (s, 1H), 9.15 (s, 1H), 8.57 (d, 1H), 8.29 (d, 1H),
8.26 (s, 1H), 7.11 (d, 2H), 7.01 (s, 1H), 6.79 (t, 1H), 6.95 (d,
2H), 4.92-4.84 (m, 1H), 3.72- 3.62 (m, 2H), 2.83 (t, 2H), 1.56 (d,
6H); MS m/z 392.2 (M + 1) 0.14 178 ##STR00144## .sup.1H NMR (400
MHz, DMSO): .delta. = 10.83 (s, 1H), 8.67 (t, 1H), 8.37 (s, 1H),
8.15 (d, 1H), 7.71 (d, 1H), 7.57 (d, 1H), 7.33 (d, 1H), 7.20 (s,
1H), 7.06 (t, 1H), 6.96 (t, 1H), 4.60-4.48 (m, 1H), 3.86-3.76 (m,
2H), 3.06 (t, 2H), 2.96 (s, 3H), 2.05-1.85 (m, 2H), 1.56 (d, 3H),
0.76 (t, 3H); MS m/z 415.2 (M + 1) 0.003 180 ##STR00145## MS m/z
392.2 (M + 1) 0.13 181 ##STR00146## MS m/z 406.2 (M + 1) 2.5 182
##STR00147## MS m/z 432.2 (M + 1) 5.1 183 ##STR00148## .sup.1H NMR
(400 MHz, DMSO): .delta. = 10.84 (s, 1H), 9.37 (s, 1H), 8.52 (s,
1H), 8.50 (s, 1H), 8.29 (s, 1H), 8.01 (t, 1H), 7.66 (d, 1H), 7.34
(d, 1H), 7.23 (m, 1H), 7.07 (t, 1H), 6.98 (t, 1H), 4.89-4.83 (m,
1H), 3.95- 3.85 (m, 2H), 3.09 (t, 2H), 2.41 (s, 3H), 1.58 (d, 6H);
MS m/z 412.2 (M + 1) 0.01 184 ##STR00149## MS m/z 401.2 (M + 1)
0.008 185 ##STR00150## MS m/z 430.2 (M + 1) 0.024 186 ##STR00151##
MS m/z 430.2 (M + 1) 0.007 187 ##STR00152## .sup.1H NMR (400 MHz,
DMSO): .delta. = 10.84 (s, 1H), 9.38 (s, 1H), 8.49 (m, 1H), 8.47
(s, 1H), 8.10 (t, 1H), 7.67 (d, 1H), 7.35 (d, 1H), 7.22 (m, 1H),
7.07 (t, 1H), 6.98 (t, 1H), 5.85-5.78 (m, 1H), 5.17 (t, 2H), 5.03
(t, 2H), 3.84-3.84 (m, 2H), 3.09 (t, 2H), 2.40 (s, 3H); MS m/z
426.2 (M + 1) 0.034 188 ##STR00153## MS m/z 434.2 (M + 1) 0.005 189
##STR00154## .sup.1H NMR (400 MHz, DMSO): .delta. = 10.65 (s, 1H),
9.42 (s, 1H), 8.68 (m, 1H), 8.41 (d, 1H), 8.34 (s, 1H), 8.08 (t,
1H), 7.53 (d, 1H), 7.12 (m, 2H), 6.81 (d, 1H), 4.90-4.81 (m, 1H),
3.93- 3.80 (m, 2H), 3.05 (t, 2H), 2.38 (s, 3H), 1.58 (d, 6H); MS
m/z 432.0 (M + 1) 0.026 190 ##STR00155## .sup.1H NMR (400 MHz,
DMSO): .delta. = 10.71 (s, 1H), 9.42 (s, H), 8.67 (d, 1H), 8.38
(dd, 1H), 8.32 (s, 1H), 8.05 (t, 1H), 7.55 (d, 1H), 7.21 (d, 1H),
6.98 (t, 1H), 6.93 (t, 1H), 4.92-4.83 (m, 1H), 3.78-3.71 (m, 2H),
2.99 (t, 2H), 2.33 (s, 3H), 1.59 (d, 6H); MS m/z 430.2 (M + 1)
0.005 195 ##STR00156## HPLC-MS calculated MS m/z 434.2 (M + 1)
0.003 196 ##STR00157## MS m/z 434.2 (M + 1) 0.002 197 ##STR00158##
.sup.1H NMR (400 MHz, DMSO): .delta. = 10.79 (s, 1H), 9.37 (s, 1H),
8.64 (d, 1H), 8.39 (d, 1H), 8.31 (s, 1H), 8.06 (t, 1H), 7.15 (s,
1H), 7.13 (d, 1H), 6.90 (t, 1H), 6.69 (d, 1H), 4.90-4.83 (m, 1H),
3.83-3.87 (m, 2H), 3.24 (t, 2H), 2.65 (s, 3H), 1.57 (d, 6H); MS m/z
430.2 (M + 1) 0.011 198 ##STR00159## MS m/z 429.1 (M + 1) 1.1 199
##STR00160## MS m/z 399.2 (M + 1) 1.6 200 ##STR00161## MS m/z 423.2
(M + 1) 0.001 201 ##STR00162## 202 ##STR00163## 203
##STR00164##
[0270] Affinity probe compounds related to the compounds of the
invention can also be prepared, as described in the following
examples.
Example 210
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-5-yl
6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
pentanamido)hexanoate
##STR00165##
[0272] Synthesis of
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl 6-aminohexanoate (e): To a solution of
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl 6-(tert-butoxycarbonylamino)hexanoate (a) (80 mg, 0.117
mmol) in DCM (20 ml) was added TFA (5 ml). The reaction was stirred
at rt for 3 hr. It was concentrated. Aqueous sodium carbonate
solution was added and the mixture was extracted with DCM. The
organic fractions were combined, dried over sodium sulfate, and
concentrated to afford the product as an oil. MS m/z 582.2
(M+1).
[0273] Synthesis of
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl
6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl-
)pentanamido)hexanoate: To a solution of (+)-biotin (35 mg, 0.14
mmol) and Et.sub.3N (36 mg, 0.35 mmol) in DMF (1 ml) was added HATU
(90 mg, 0.24 mmol). The mixture was stirred for 10 min, and then
was added to a solution of
(3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-
-indol-5-yl 6-aminohexanoate (b) (68 mg, 0.12 mmol) in DMF (1 ml).
The reaction mixture was stirred for 16 hr at rt and then was
concentrated. The residue was purified by reverse-phase HPLC (Cis
column, eluting with MeOH--H.sub.2O 0.05% TFA) to afford the title
compound as an off-white solid. Example 210 showed an EC.sub.50
value in the % CD34+ assay of 2.1 .mu.M.
Example 211
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H-i-
ndol-5-yl 6-(tert-butoxycarbonylamino)hexanoate
##STR00166##
[0275] Synthesis of
2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine (b):
Following the procedure of Example 15d,
6-chloro-2-iodo-9-isopropyl-9H-purine (3.31 g, 0.0103 mol) was
reacted with benzo[b]thiophen-3-ylboronic acid. The crude product
was purified by silica gel chromatography (20 to 50% ethyl acetate
in hexane) to afford the title compound as a solid. MS m/z 329.0
(M+1). .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=9.15 (d, 1H),
8.85 (s, 2H), 8.17 (d, 1H), 7.62 (t, 1H), 7.53 (t, 1H), 5.06 (m,
1H), 1.71 (d, 6H).
[0276] Synthesis of
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-ol (c): Following the procedure of Example 15e,
2-(benzo[b]thiophen-3-yl)-6-chloro-9-isopropyl-9H-purine (b) (80
mg, 0.243 mmol) was reacted with serotonin. The reaction mixture
was concentrated, then aqueous sodium bicarbonate solution was
added. The mixture was extracted with ethyl acetate. The organic
fractions were combined, dried over sodium sulfate, and
concentrated. The residue was purified by silica gel chromatography
(0 to 5% MeOH in DCM eluant) to afford the title compound as an
off-white solid. MS m/z 469.2 (M+1).
[0277] Synthesis of
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-yl 6-(tert-butoxycarbonylamino)hexanoate (d): To a solution
of
3-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)-1H--
indol-5-ol (55.5 mg, 0.119 mmol) and
6-(tert-butoxycarbonylamino)hexanoic acid (30 mg, 0.113 mmol) in
DMF (3 ml) was added Et.sub.3N (24 mg, 0.237 mmol) and HATU (90 mg,
0.237 mmol). The mixture was stirred at rt for 16 hr, and then was
concentrated. Water was added and the reaction mixture was
extracted with ethyl acetate. The organic fractions were combined,
dried over sodium sulfate, and concentrated. The residue was
purified by silica gel chromatography (0 to 5% MeOH in DCM eluant)
to afford the title compound as an off-white solid.
[0278] By repeating the procedures described in the above examples,
using appropriate starting materials, the following affinity
probes, as identified in Table 2, are obtained:
TABLE-US-00003 TABLE 2 Example Physical Data Number Structure
.sup.1H NMR and/or MS 26 ##STR00167## HRMS (EI) m/z 695.2585 (M +
1) 29 ##STR00168## HRMS (EI) m/z 700.3027 (M + 1) 209 ##STR00169##
MS m/z 760.4 (M + 1) 210 ##STR00170## 1H NMR (400 MHz, DMSO):
.delta. = 10.95 (s, 1H), 9.17 (d, 1H), 8.54 (s, 1H), 8.34 (s, 1H),
8.06 (d, 1H), 7.94 (bs, 1H), 7.77 (t, 1H), 7.39-7.48 (m, 2H),
7.28-7.34 (m, 3H), 6.79 (dd, 1H), 6.42 (bs, 1H), 4.85-4.93 (m, 1H),
4.24-4.28 (m, 1H), 4.07-4.10 (m, 1H), 3.88-3.92 (m, 2H), 3.01-3.11
(m, 5H), 2.77 (dd, 1H), 2.53-2.57 (m, 1H), 2.00 (t, 2H), 1.63 (d,
6H), 1.23-1.60 (m, 12H); MS m/z 808.3 (M + 1) 211 ##STR00171## MS
m/z 682.2 (M + 1). 212 ##STR00172## MS m/z 809.4 (M + 1) 213
##STR00173## MS m/z 758.4 (M + 1) 214 ##STR00174## MS m/z 807.4 (M
+ 1)
Assays
[0279] The following assays are used to assess the activity of the
compounds of the invention to facilitate hematopoietic stem cell
(HSC) expansion.
[0280] Primary adult CD34.sup.+ human hematopoietic stem cells
(HSCs) are cultured and screened to identify compounds of the
invention that facilitate HSC expansion. The cells are analyzed for
the presence of the desired phenotype (CD34 expression). Compounds
of the invention promote HSC expansion in a dose dependent
manner.
[0281] Culture Medium: StemSpan SFEM medium is serum-free medium
(StemCell Technologies, Vancouver, BC) supplemented with the
following human recombinant cytokines: thrombopoietin,
interleukin-6, Flt-3 ligand, and stem cell factor (all from R&D
Systems, Minneapolis, Minn.), each at a final concentration of 50
ng/mL, with vehicle (DMSO) or a compound of the invention.
[0282] Human Cell Culture: Fresh human leukophoresed G-CSF
mobilized peripheral blood from normal donors, CD34+ cells from
adult bone marrow and cryopreserved human cord blood CD34.sup.+
cells are purchased from AllCells (Berkeley, Calif.). Human
CD34.sup.+ cells are enriched from leukophoresed G-CSF mobilized
peripheral blood using magnetic cell sorting (MACS, Direct CD34
Progenitor Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach,
Germany) and cryopreserved. CD34.sup.+ cell purity, checked by flow
Cytometry, is higher than 90%. After thawing, the cell viability
tested by trypan blue exclusion is higher than 70%. The thawed
cells are centrifuged and resuspended with StemSpan medium before
being aliquoted for immediate culture. Cells are plated at 10.sup.4
cells/mL in a 384 well plate (Greiner Bio-One, Monroe, N.C.) with
50 .mu.L of medium per well for 7 days. Every 7 days the cells are
transferred to larger well plates and fresh medium is added to keep
the cell density between 104 and 5.times.10.sup.5 cells/mL. Cells
were cultured at 37.degree. C. in 5% CO.sub.2. For transplantation,
cells were cultured in 75 cm.sup.2 flasks before the cells were
transplanted into mice. At a concentration of 1 micromolar,
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1),
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 2, table 1), and
N-(2-(1H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-
-6-amine (compound 9, table 1) each gives rise to a greater than
10-fold increase in the number of CD34.sup.+CD45RA.sup.- cells
derived from 1000 mPB CD34.sup.+ HSCs after 21 days compared to
vehicle. Compounds of the invention were assayed in a dose response
format (1 nM to 10 .mu.M) to determine the effective concentration
that produced the desired effect in 50% of the cells (EC.sub.50).
Compounds of the invention increased the total number and/or
percent of CD34+ cells with an EC50 of less than 10 .mu.M. The
results are shown in Table 1 and examples, supra.
[0283] Colony-Forming Units in Culture (CFU-C) Assay: Mononuclear
cells at 1000 per mL for cord blood 5 week and mPB 3 week culture
and 100 cells per mL for CB 3 week and mPB 1 week culture were
added to MethoCult SF H4436, serum-free methylcellulose medium
containing methylcellulose in Iscove's MDM, bovine serum albumin,
2-mercaptoethanol, L-glutamine, human transferring (iron
saturated), recombinant human insulin, and recombinant human
cytokines: stem cell factor, GM-CSF, IL-3, IL-6, G-CSF, and
erythropoietin (StemCell Technologies). The MethoCult is
supplemented with the following human recombinant cytokines:
thrombopoietin, and Flt-3 ligand (R&D Systems), each at a final
concentration of 50 ng/mL. After stirring, the mixture is divided
into three 35-mm dishes. The dishes are incubated for 14 days at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in air. At
the end of the incubation period, myeloid and erythroid colonies
are counted under an inverted microscope at 40.times.
magnification. CFU-C content of the expansion culture is calculated
as follows: number of scored colonies per three dishes.times.total
mononuclear cell number/input cell number. Up to one week, total
mononuclear cells are determined by multiplying the number of cells
per milliliter by the culture volume. From week 1 and on, the
number of passages is also taken into account. Cultures treated
with
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) at a concentration of 1
micromolar, generated a greater than 10-fold increase in the number
of colony forming cells after 21 days of culture of mPB CD34+ cells
compared to vehicle. Using 1.times.10.sup.3 CB CD34.sup.+ cells
treated with
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) at a concentration of 1
micromolar taken from the 5 week culture showed a >10-fold
increase in colony forming units compared to control. Cells treated
with a
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) generated more mixed colonies
associated with a >10-fold increase in erythrocyte colonies, a
>10-fold increase in granulocyte/macrophage colonies, and a
>10-fold increase in macrophage colonies. Cells treated with
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) also give rise mixed
granulocyte/erythrocyte/monocyte/macrophage colonies, which are not
observed in colonies derived from untreated cultures.
[0284] Cobblestone area-forming cell (CAFC) assays: The FBMD-1
stromal cells are maintained in 25-cm.sup.2 flasks and are
trypsinized after 1/3 confluence. Since this non-transformed line
ages, and therefore gradually loses its potential to support CAFC
growth at late stages, all feeders are used below passage 20. For
supporting CAFC growth in 96-well plates, 1.times.10.sup.3 stromal
cells are seeded per well. The cultures are maintained in Iscove's
medium supplemented with 10% fetal calf serum (FCS), 2.5% horse
serum (HS), 1% L-glutamine, 1% penicillin-streptomycin, and
1.times.10.sup.-5 M hydrocortisone at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air. After stromal layers reach
confluency, they are inoculated with CD34.sup.+ HSCs that have been
cultured for 5 days with vehicle or a compound of the invention.
MNCs are added at 8 serial 1:3 dilutions (starting at 25,000
cells/well), with 10 wells for each cell dose. The dilutions with
wells with at least one phase-dark hematopoietic clone (cobblestone
area) of at least five cells beneath the stromal layer are
determined at week 4. At a test concentration of 1 micromilar,
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1), stimulates a greater than
2-fold increase in the number of cobblestone area forming cells
derived from mPB CD34.sup.+ HSCs after 5 days of culture, compared
with control cultures that are treated with DMSO alone.
[0285] Surface Antigen Analysis: The cells are washed with staining
media (Hanks balanced salt solution containing FBS (2%) and EDTA (2
mM)) and stained (at 4.degree. C. for 30 minutes) with indicated
primary conjugated antibodies. The cells are washed in the
previously described buffer and analyzed using a BD LSR II flow
cytometer (Becton Dickinson, San Jose, Calif.). The cells are
passed at a rate of up to 1000 cells/second using 488-nm argon and
633-nm HeNe laser beams as the light source for excitation.
Emission of 10.sup.4 cells is measured using logarithmic
amplification and analyzed using FlowJo software (TreeStar Inc.
Ashland, Oreg.). Cells stained with primary conjugated isotype
control antibodies are used to determine background
fluorescence.
[0286] Determination of CD34.sup.+ cell subsets: The percentages of
CD34.sup.+ cell subsets are determined from aliquots of the cell
culture. Cells were stained with APC anti-Thy1.1, PerCP anti-CD34,
PECy7 anti CD45RA, FITC anti CD38, and PE anti-CD133 for
determination of CD34.sup.+Thy1.1.sup.+, CD34.sup.+CD45RA.sup.-,
CD34.sup.+CD38.sup.-, CD133.sup.+CD38.sup.- and
CD34.sup.+CD133.sup.+ cells. Antibodies to CD34, CD38, Thy1.1 and
CD45RA were purchased from Becton Dickinson and antibodies to CD133
were purchased Miltenyi Biotec. FACS analysis results of these
subsets are given as percentage of the total population. The
absolute number of each population of cells in the culture is
calculated from the total number of cells multiplied by the
percentage of each population. Starting with CB CD34+ cell, after
five weeks the total cell number in the cultures increased on
average greater than 2-fold in the
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) 1 micromolar treated cells
compared to control cultures. More importantly, >50% of
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) cultured cells were CD34.sup.+
compared to <10% of vehicle cultured cells resulting in a
greater than 10-fold expansion of CD34+ cells compared to control
and a greater than 10,000-fold expansion compared to input cells.
In addition, the presence of 1 micromolar
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol (compound 1, table 1; example 1) increased both the percentage
and total numbers of the CD34.sup.+ subpopulations,
CD34.sup.+CD45RA.sup.-, CD34.sup.+CD38.sup.-,
CD133.sup.+CD38.sup.-, and CD34.sup.+CD133.sup.+ resulting in a net
expansion of greater than 30-fold for each subset.
[0287] Transplantation of human CD34.sup.+ cells into
NOD.CB17-Prkdc.sup.scid mice (NOD/SCID): To assess the in vivo
repopulating capacity of CD34.sup.+ cells and their cultured
progeny, uncultured CD34.sup.+ or the progenies of cultured
CD34.sup.+ cells after 4 days (mPB) or 21 days (CB) with vehicle or
a test compound were injected intravenously via the retro-orbital
route into sub-lethally irradiated (3.0 Gy) 8- to 10-week-old
NOD/SCID (for mPB HSC experiments) or NOD/SCIDgc-/- (for CB HSC
experiments) mice. To monitor engraftment blood was drawn weekly
via the retro-orbital and treated with erythrocyte lysis solution
(Qiagen, Valencia, Calif.) to remove red blood cells, washed with
staining media, and analyzed by flow Cytometry. Engraftment was
measured by detection of anti-human CD45.sup.+ cells in the blood.
The mice are sacrificed at 10 weeks post-transplantation; BM is
collected from both femurs and tibiae. BM cells are washed in
staining media and stained with anti-human antibodies. Following
incubation, the suspension is treated with erythrocyte lysis
solution (Qiagen, Valencia, Calif.) to remove red blood cells,
washed with staining media, and analyzed by flow Cytometry, as
described earlier. Both mPB and CB derived HSCs cultured with
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl-
)phenol (compound 1, table 1; example 1) at a concentration of 1
micromolar give rise to a statistically significant increase in the
percentage of human cells 10 weeks after engraftment.
Target Identification
[0288] To identify the mechanism whereby a compound of the
invention expands HSCs in an undifferentiated state, a genome-wide
transcriptional profiling of mPB-derived CD34.sup.+ cells treated
for 24 hours with example 1 and a less active analog
(.about.20-fold) of example 1
(2-(benzo[b]thiophen-3-yl)-N-(3-(3,5-dimethyl-TH-pyrazol-4-yl)propyl)-9-i-
sopropyl-9H-purin-6-amine). Of the >50,000 probe sets analyzed,
only 5 genes were up-regulated greater than 3-fold upon treatment
with Example 1 and most were also induced to some degree by the
inactive analog. In addition, 5 genes were down-regulated by
>70% upon treatment with 1 pM of Example 1. All were
down-regulated in a dose dependent fashion and none were
significantly affected by the inactive analog. The two genes that
were the most highly repressed by treatment with Example 1
(cytochrome P450 1B1 [CYP1B1] and the aryl hydrocarbon receptor
repressor [AHRR]) are transcriptionally regulated by the aryl
hydrocarbon receptor (AHR). Therefore, compounds of the invention
could be acting as an antagonist of AHR signaling.
[0289] Further, the ability of Example 1 to block
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin)-mediated CYP1B1
mRNA expression by qPCR in mPB-derived CD34+ cells was determined.
Treatment with TCDD (3 nM) caused a 4.5-fold increase in the level
of CYP1B1 mRNA compared with the vehicle control (0.01% toluene).
This increase was inhibited by Example 1 in a dose-dependent manner
indicating that compounds of the invention can antagonize AHR
signaling. To determine the effects of Example 1 in AHR
transcription the ability of Example 1 to inhibit a dioxin-induced
AHR dependent luciferase reporter gene assay was tested. Inclusion
of Example 1 (1 .mu.M) completely abolished dioxin-induced AHR
dependent transcription when used on cells expressing human AHR.
Titration of Example 1 revealed an EC.sub.50 of 127 nM,
demonstrating that Example 1 is a potent AHR antagonist.
Interestingly, Example 1 only weakly inhibited dioxin induced
transcription in murine cells and had no activity on rat cells,
suggesting that Example 1 preferentially inhibits human AHR. This
correlates with a lack of activity of Example 1 on murine HSC, and
can explain the species selectivity of Example 1. Finally, Example
1 had only weak agonist activity on murine or rat cells, and failed
to induce AHR dependent transcription in human cells.
[0290] To further explore the role of AHR signaling in HSCs two
other AHR antagonists (alpha-naphthoflavone and CH-223191) were
tested. Both compounds lead to dose dependent increases in the
number of CD34.sup.+ cells when cultured with mPB-derived
CD34.sup.+ cell for 7 days: inclusion of 1 .mu.M CH223191 afforded
a 2.2-fold expansion of CD34.sup.+ cells; 0.75 .mu.M
.alpha.-naphthoflavone afforded a 1.9-fold expansion of CD34.sup.+
cells while 0.75 .mu.M Example 1 afforded a 3.4-fold expansion of
total CD34.sup.+ cells. To show a direct role for the AHR in
Example 1 induced HSC expansion, human CB-derived CD34.sup.+ HSCs
were treated with lentiviral particles containing a shRNA-targeting
AHR that co-expressed GFP or control virus. Forty-eight hours
following transduction, CD34.sup.+GFP.sup.+ cells were purified by
cell sorting and the levels of AHR were determined by qPCR. Both
AHR targeting shRNAs led to decreases in AHR expression following
transduction (81% with sh111 and 51% with sh242). These decreases
were not seen in cells lacking GFP or in cells transduced with
control virus. CB-derived CD34+ cells with decreased AHR expression
displayed a phenotype similar to Example 1 treated cells with
sustained expression of CD34+. These data show that inhibition of
AHR activity by a compound of the invention is sufficient to
promote ex-vivo expansion of HSC.
Method of Expanding HSCs from Human Neonatal Umbilical Cord
Blood
[0291] The culture medium used is StemSpan SFEM (StemCell
Technologies, Cat. #09650) supplemented with the following
recombinant human cytokines: TPO, IL6, Flt3 ligand, and SCF each at
a final concentration of 50 ng/mL. The culture media is prepared
fresh the day of use.
[0292] Compound dilution into media: a 10,000.times. concentrate of
a compound of the invention is used for the dilutions. The addition
of compound into the culture media occurs in two steps. The first
step is a 1:100 dilution (10 .mu.L of 10,000.times. concentrate
into 990 .mu.L of complete culture media (containing cytokines) in
a 1.5 mL effendorf tube [USA Scientific, Cat #1615-5500]) to
generate a 100.times. solution of compound in the culture media.
The second step is a 1:100 dilution into the culture media that
will be used to initiate the cell culture. The volume of the
culture is variable depending on the input number of cord blood
(CB) CD34.sup.+ cells. For example, 1.times.10.sup.6 CB CD34.sup.+
cells are seeded into 20 mL of media (5.times.10.sup.4 cells/mL).
In this case, 200 .mu.L of the 100.times. Example 1 solution is
added to the 20 mL of media in a 50 mL conical tube (Becton
Dickinson, Cat #352098) to reach the final concentration (see Table
3).
[0293] Cell culture initiation: purified human CB CD34.sup.+ cells
are used for the ex vivo expansion experiments. After thawing, the
cell viability, tested by trypan blue exclusion, is higher than
50%. The thawed cells are diluted 5-fold with culture media (no
cytokines or compounds of the invention such as Example 1) and
centrifuged at 300 g at 25.degree. C. for 8 minutes. After
aspirating the supernatant, the pellet is resuspended with the
appropriate volume of culture medium (5.times.10.sup.4 cells/mL,
Table 3) before being injected (22 gauge needle, Air-Tite products;
20 mL syringe, BD cat #309661) into AFC bags (Table 5) for
immediate culture. Cells are cultured at 37.degree. C. in 5%
CO.sub.2.
[0294] Addition of media to the cell culture: for media volumes up
to 80 mL the procedure above (compound dilution into media) is
used. For media volumes larger than 80 mL the first 1:100 dilution
is carried out in 10 mL conical tubes (Corning, Cat #430052, Table
4). The second step is a 1:100 dilution into the culture media, in
sterile containers, (BD Falcon, Cat #354015) that is added to the
AFC bag (22 gauge needle, Air-Tite products; 60 mLsyringe, BD cat
#309653).
TABLE-US-00004 TABLE 3 Example 1 dilutions for starting cord blood
derived CD34+ cell expansion Number of Starting volume of volume of
cord blood culture 100x volume of 100x 10,000x derived CD34+ volume
Example 1 Example 1 to Example 1 cells (.times.10.sup.6) (mL)
(.mu.L) needed prepare (mL) needed (.mu.L) 0.25 5 50 1 10 0.50 10
100 1 10 0.75 15 150 1 10 1.00 20 200 1 10 1.25 25 250 1 10 1.50 30
300 1 10 1.75 35 350 1 10 2.00 40 400 1 10 2.50 50 500 1 10 3.00 60
600 1 10 4.00 80 800 1 10
TABLE-US-00005 TABLE 4 Example 1 dilutions for adding media to the
cord blood derived CD34+ cell expansion Volume of volume of 100x
volume of 100x volume of 10,000x media to add Example 1 (.mu.l)
Example 1 to Example 1 needed (mL) needed prepare (mL) (.mu.L)
10.00 100 1 10 20.00 200 1 10 30.00 300 1 10 40.00 400 1 10 50.00
500 1 10 60.00 600 1 10 70.00 700 1 10 80.00 800 1 10 100.00 1,000
5 50 120.00 1,200 5 50 160.00 1,600 5 50 250.00 2,500 5 50 500.00
5,000 10 100 750.00 7,500 10 100 1,000.00 10,000 10 100
TABLE-US-00006 TABLE 5 Volume restrictions for American Fluoroseal
Corporation bags. AFC bag catalog Volume for optimal Maximum volume
of the number expansion (mL) bag (mL) 1PF-0007 7 7 2PF-0032 32 32
2PF-0072 71 130 2PF-0118 118 245 2PF-0197 179 580 2PF-0225 225 665
2PF-0270 270 960 2PF-750C 750
[0295] The same protocol can be used starting from mobilized
peripheral blood cells from a patient for autologous graft
transplantation.
[0296] A composition comprising a population of cells with expanded
HSCs appropriate for intravenous administration as an infusion can
also be prepared. To prepare cells for infusion, cultured cells are
pelletted by centrifugation for 10 minutes at 300 g and resuspended
in infusion buffer consisting of 5% HSA (Baxter) at a concentration
of between 10.sup.6 to 10.sup.8 cells/ml.
[0297] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
Sequence CWU 1
1
4161DNAArtificial SequencesiRNA targeting AHR 111AS 1gcggcataga
gaccgactta atttcaagag aattaagtcg gtctctatgc cgcttttttg 60g
61265DNAArtificial SequencesiRNA targeting AHR 111AS 2cgcgccaaaa
aagcggcata gagaccgact taattctctt gaaattaagt cggtctctat 60gccgc
65361DNAArtificial SequencesiRNA targeting AHR 242S 3ggcttctttg
atgttgcatt aattcaagag attaatgcaa catcaaagaa gccttttttg 60g
61465DNAArtificial SequencesiRNA targeting AHR 242AS 4cgcgccaaaa
aaggcttctt tgatgttgca ttaatctctt gaattaatgc aacatcaaag 60aagcc
65
* * * * *