U.S. patent application number 11/991971 was filed with the patent office on 2009-12-10 for alkoxy indolinone based protein kinase inhibitors.
This patent application is currently assigned to The Scripps Research Institute. Invention is credited to Yangbo Feng, Congxin Liang.
Application Number | 20090305382 11/991971 |
Document ID | / |
Family ID | 37900093 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090305382 |
Kind Code |
A1 |
Liang; Congxin ; et
al. |
December 10, 2009 |
ALKOXY INDOLINONE BASED PROTEIN KINASE INHIBITORS
Abstract
Alkoxy indolinone based acid and amide derivatives have enhanced
and unexpected drug properties as inhibitors of protein kinases and
are useful in treating disorders related to abnormal protein kinase
activities such as cancer.
Inventors: |
Liang; Congxin; (Jupiter,
FL) ; Feng; Yangbo; (Palm Beach Gardend, FL) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG & WOESSNER, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
The Scripps Research
Institute
La Jolla
CA
|
Family ID: |
37900093 |
Appl. No.: |
11/991971 |
Filed: |
September 21, 2006 |
PCT Filed: |
September 21, 2006 |
PCT NO: |
PCT/US2006/036946 |
371 Date: |
June 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60719474 |
Sep 22, 2005 |
|
|
|
Current U.S.
Class: |
435/184 |
Current CPC
Class: |
C07D 403/06 20130101;
C07D 403/14 20130101; C07D 413/14 20130101; A61P 35/00 20180101;
C07D 405/14 20130101; C07D 401/14 20130101 |
Class at
Publication: |
435/184 |
International
Class: |
C12N 9/99 20060101
C12N009/99 |
Claims
1-33. (canceled)
34. A method for the modulation of the catalytic activity of a
protein kinase, comprising contacting the kinase and an effective
amount of a compound represented by Formula (I): ##STR00044##
wherein: R.sup.1 is selected from the group consisting of hydrogen,
halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl,
hydroxy, (C1-C6) alkoxy, amino, (C1-C6) alkylamino, amide,
sulfonamide, cyano, substituted or unsubstituted (C6-C10) aryl;
R.sup.2 is selected from the group consisting of hydrogen, halo,
(C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy,
(C1-C6) alkoxy, (C2-C8) alkoxyalkyl, amino, (C1-C6) alkylamino,
(C6-C10) arylamino; R.sup.3 is selected from the group consisting
of hydrogen, (C1-C6) alkyl, (C6-C10) aryl, (C5-C10) heteroaryl, and
amide; R.sup.4, R.sup.5, R.sup.6 and R.sup.8 are independently
selected from the group consisting of hydrogen and (C1-C6) alkyl;
R.sup.7 is (C1-C6) alkyl; R.sup.9 is selected from the group
consisting of hydroxy, (C1-C6) O-alkyl, (C3-C8) O-cycloalkyl, and
NR.sup.10R.sup.11; where R.sup.10 and R.sup.11 are independently
selected from the group consisting of hydrogen, (C1-C6) alkyl,
(C1-C6) hydroxyalkyl, (C2-C6) dihydroxyalkyl, (C1-C6) alkoxy,
(C2-C6) alkyl carboxylic acid, (C1-C6) alkyl phosphonic acid,
(C1-C6) alkyl sulfonic acid, (C2-C6) hydroxyalkyl carboxylic acid,
(C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl,
(C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8) cycloalkyl carboxylic
acid, or R.sup.10 and R.sup.11 together with N forms a (C5-C8)
heterocyclic ring either unsubstituted or substituted with one or
more hydroxyls, ketones, ethers, and carboxylic acids; n is 1, 2,
or 3; and m is 0, 1, or 2; or, a pharmaceutically acceptable salt,
its tautomer, a pharmaceutically acceptable salt of its tautomer,
or a prodrug thereof.
35. The method of claim 34 wherein the compound is represented by
Formula (II): ##STR00045## wherein R.sup.12 is selected from the
group consisting of hydrogen, (C1-C6) alkyl, and (C3-C8)
cycloalkyl.
36. The method of claim 34 wherein the compound is represented by
Formula (III): ##STR00046##
37. The method of claim 34 wherein the compound is any of the
following: ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052##
38. The method of claim 34, wherein the compound is selected from
the set consisting of: ##STR00053## wherein R.sup.9 is any of the
following groups: ##STR00054## ##STR00055##
39. The method of claim 34, wherein the protein kinase is selected
from the group consisting of VEGF receptors and PDGF receptors.
Description
FIELD OF INVENTION
[0001] The invention relates to protein kinase inhibitors and to
their use in treating disorders related to abnormal protein kinase
activities such as cancer and inflammation. More particularly, the
invention relates to alkoxy indolinone based derivatives and their
pharmaceutically acceptable salts employable as protein kinase
inhibitors.
BACKGROUND
[0002] Protein kinases are enzymes that catalyze the
phosphorylation of hydroxyl groups of tyrosine, serine, and
threonine residues of proteins. Many aspects of cell life (for
example, cell growth, differentiation, proliferation, cell cycle
and survival) depend on protein kinase activities. Furthermore,
abnormal protein kinase activity has been related to a host of
disorders such as cancer and inflammation. Therefore, considerable
effort has been directed to identifying ways to modulate protein
kinase activities. In particular, many attempts have been made to
identify small molecules that act as protein kinase inhibitors.
[0003] Several pyrrolyl-indolinone derivatives have demonstrated
excellent activity as inhibitors of protein kinases (Larid et al.
FASEB J. 16, 681, 2002; Smolich et al. Blood, 97, 1413, 2001;
Mendel et al. Clinical Cancer Res. 9, 327, 2003; Sun et al. J. Med.
Chem. 46, 1116, 2003). The clinical utility of these compounds has
been promising, but has been partially compromised due to the
relatively poor aqueous solubility and/or other drug properties.
What is needed is a class of modified pyrrolyl-indolinone
derivatives having both inhibitory activity and enhanced drug
properties.
SUMMARY
[0004] The invention is directed to alkoxy indolinone based
derivatives and to their use as inhibitors of protein kinases. It
is disclosed herein that alkoxy indolinone based derivatives have
enhanced and unexpected drug properties that advantageously
distinguish this class of compounds over known pyrrolyl-indolinone
derivatives having protein kinase inhibition activity. It is also
disclosed herein that alkoxy indolinone based derivatives are
useful in treating disorders related to abnormal protein kinase
activities such as cancer.
[0005] One aspect of the invention is directed to a compound
represented by Formula (I):
##STR00001##
In Formula (I), R.sup.1 is selected from the group consisting of
hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6)
haloalkyl, hydroxy, (C1-C6) alkoxy, amino, (C1-C6) alkylamino,
amide, sulfonamide, cyano, substituted or unsubstituted (C6-C10)
aryl; R.sup.2 is selected from the group consisting of hydrogen,
halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl,
hydroxy, (C1-C6) alkoxy, (C2-C8) alkoxyalkyl, amino, (C1-C6)
alkylamino, (C6-C10) arylamino; R.sup.3 is selected from the group
consisting of hydrogen, (C1-C6) alkyl, (C6-C10) aryl, (C5-C10)
heteroaryl, and amide; R.sup.4, R.sup.5, R.sup.6 and R.sup.8 are
independently selected from the group consisting of hydrogen and
(C1-C6) alkyl; R.sup.7 is (C1-C6) alkyl; R.sup.9 is selected from
the group consisting of hydroxy, (C1-C6) O-alkyl, (C3-C8)
O-cycloalkyl, and NR.sup.10R.sup.11; where R.sup.10 and R.sup.11
are independently selected from the group consisting of hydrogen,
(C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C2-C6) dihydroxyalkyl,
(C1-C6) alkoxy, (C1-C6) alkyl carboxylic acid, (C1-C6) alkyl
phosphonic acid, (C1-C6) alkyl sulfonic acid, (C1-C6) hydroxyalkyl
carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8)
heterocycloalkyl, (C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8)
cycloalkyl carboxylic acid, or R.sup.10 and R.sup.11 together with
N forms a (C5-C8) heterocyclic ring either unsubstituted or
substituted with one or more hydroxyls, ketones, ethers, and
carboxylic acids; n is 1, 2, or 3; and m is 0, 1, or 2.
Alternatively, this aspect of the invention also is directed to a
pharmaceutically acceptable salt, its tautomer, a pharmaceutically
acceptable salt of its tautomer, or a prodrug of Formula (I).
[0006] A first preferred subgenus of this first aspect of the
invention is directed to the compound, salt, tautomer, or prodrug
represented by Formula (II):
##STR00002##
In Formula (II), R.sup.12 is selected from the group consisting of
hydrogen, (C1-C6) alkyl, and (C3-C8) cycloalkyl. Other groups are
as defined in Formula (I). In preferred embodiments, R.sup.1 and
R.sup.2 are independently selected from the group consisting of
hydrogen and fluoro; R.sup.3 and R.sup.4 are methyl; R.sup.5,
R.sup.6, R.sup.8, and R.sup.12 are hydrogen; R.sup.7 is (C1-C6)
alkyl; n is 1 or 2; and m is 0 or 1. Preferred species include the
following compounds:
##STR00003## ##STR00004##
[0007] A second preferred subgenus of this first aspect of the
invention is directed to a compound, salt, tautomer, or prodrug
represented by Formula (III):
##STR00005##
In Formula (III), the various R groups are the same as Formula (I).
In preferred embodiments, R.sup.1 and R.sup.2 are independently
selected from the group consisting of hydrogen, halo, cyano;
R.sup.3 is selected from the group consisting of hydrogen, (C1-C6)
alkyl, (C6-C10) aryl, (C5-C10) heteroaryl, and amide; R.sup.4,
R.sup.5, R.sup.6 and R.sup.8 are independently selected from the
group consisting of hydrogen and (C1-C6))alkyl; R.sup.7 is (C1-C6)
alkyl; n is 1 or 2; m is 0 or 1; and R.sup.10 and R.sup.11 are
selected from the group consisting of hydrogen, (C1-C6) alkyl,
(C1-C6) hydroxyalkyl, (C2-C6) dihydroxyalkyl, (C1-C6) alkoxy,
(C2-C6) alkyl carboxylic acid, (C1-C6) alkyl phosphonic acid,
(C1-C6) alkyl sulfonic acid, (C2-C6) hydroxyalkyl carboxylic acid,
(C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl,
(C6-C8) aryl, (C5-C8) heteroaryl, (C4-C8) cycloalkyl carboxylic
acid, or R.sup.10 and R.sup.11 together with N forms a (C5-C8)
heterocyclic ring either unsubstituted or substituted with one or
more hydroxyls, ketones, ethers, and carboxylic acids.
[0008] In a first subgroup of this second subgenus, m is 0.
Preferred species of this first subgroup are represented by the
following structures:
##STR00006## ##STR00007##
[0009] In a second subgroup of this second subgenus, m is 1.
Preferred species of this second subgroup are represented by the
following structures:
##STR00008##
[0010] Further species of the second aspect of the invention are
represented by the following structures:
##STR00009## ##STR00010##
wherein: R.sup.9 is selected from the group consisting of radicals
represented by the following structures:
##STR00011## ##STR00012##
[0011] Another aspect of the invention is directed to a method for
the modulation of the catalytic activity of a protein kinase with a
compound or salt of any one of the compounds of Formulas (I-III).
In a preferred mode, the protein kinase is selected from the group
consisting of VEGF receptors and PDGF receptors.
BRIEF DESCRIPTION OF FIGURES
[0012] FIG. 1 illustrates a scheme that is used for the synthesis
of the 3-alkoxy-4-acylaminoamide derivatives starting from methyl
3-hydroxy-4-aminobutanoate hydrochlorides and the activated
acylating agent 1-3.
[0013] FIG. 2 illustrates a scheme that is used for the synthesis
of the 2-alkoxy-3-acylaminoamide derivatives starting from methyl
2-hydroxy-3-aminopropionate hydrochlorides and the activated
acylating agent 1-3.
[0014] FIG. 3 illustrates a scheme that is used for the synthesis
of the (2S)-2-alkoxy-4-acylamino-amide derivatives starting from
methyl (2S)-2-hydroxy-4-aminobutanoate hydrochloride and the
activated acylating agent 1-3.
DETAILED DESCRIPTION
Examples 1-8
[0015] The synthesis of acids (1-4) and amides (1-5) is shown in
FIG. 1. Variations from this general synthetic procedure can be
understood and carried out by those skilled in the art. Thus, the
compounds of the present invention can be synthesized by those
skilled in the art.
Example 1
4-({5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1-
H-pyrrole-3-carbonyl}-amino)-3-methoxy-butyric acid
##STR00013##
[0017] To a suspension of methyl 4-amino-3-hydroxybutyrate (1.0
equiv, which was prepared by refluxing the free amino acid in dry
methanol with 1.2 equiv HCl) and DIEA (5 equiv) in DCM, Mmt-Cl (1.1
equiv) was added portion-wise at 25.degree. C. After stirring
overnight, the DCM was removed under reduced pressure. The residue
was suspended in ethyl acetate, washed with brine (3.times.), dried
over anhydrous Na.sub.2SO.sub.4. The ethyl acetate was then
removed, and the residue was dried overnight under high vacuum, and
subjected to flash chromatography to give compound 1-1. To a
solution of compound 1-1 in dry DMF, NaH (1.5 equiv) was added
under argon. After stirring at 25.degree. C. for 1 h, MeI (5 equiv)
was added to the solution, and the resulting suspension was gently
shaken at 25.degree. C. overnight. The DMF was removed under
vacuum; the residue was suspended in ethyl acetate, washed with
brine (3.times.), and dried over anhydrous Na.sub.2SO.sub.4. After
the ethyl acetate was removed via evaporation the resulting residue
was treated with 1% TFA in DCE/DCM for 30 min. The organic solvents
were then removed under reduced pressure, and the resulting residue
was triturated with hexane (3.times.) to obtain the free amino acid
1-2. This amino acid was used directly in the next step without any
purification and characterization. Thus, to a solution of 1-2 (2
equiv) and DIEA (5 equiv) in DMF, compound 1-3 (1 equiv) was added
at 25.degree. C. After stirring for 30 min (LC-MS show the complete
consumption of 1-3), KOH (5 equiv) in water was added, and the
solution was stirred for another 2 h (LC-MS demonstrated a complete
hydrolysis). The solvents were removed under reduced pressure, and
HCl (1N, excess) was added to give a precipitate. This precipitate
was collected and washed (by water) by filtration, dried under high
vacuum to give the title compound (95% based on compound 1-3).
LC-MS: single peak at 254 nm, MH.sup.+ calcd. for
C.sub.21H.sub.22FN.sub.3O.sub.5: 416, obtained: 416. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H), 12.18 (b, 1H),
10.90 (s, 1H), 7.75 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.71 (s, 1H),
7.64 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz,
1H), 3.73 (m, 1H), 3.43-3.31 (m, 2H), 3.22 (s, 3H), 2.52-2.35 (m,
2H), 2.43 (s, 3H), 2.41 (s, 3H).
Example 2
3-Ethoxy-4-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-d-
imethyl-1H-pyrrole-3-carbonyl}-amino)-butyric acid
##STR00014##
[0019] A similar route as that for the synthesis of Example 1 was
used to prepare the title compound. Iodoethane was used instead of
iodomethane to obtain the 3-ethoxy compound (9.7% based on compound
1-3). LC-MS: single peak at 254 nm, MH.sup.+ calcd. for
C.sub.22H.sub.24FN.sub.3O.sub.5: 430, obtained: 430.
Examples 3-8
[0020] The general procedure for the synthesis of amides (1-5): An
amine (2 equiv) was added to a solution of the acid (1-4), HATU
(1.05 mmol), and DIEA (5 equiv) in DMF (5 mL). After the solution
was stirred at 25.degree. C. for 2 h, aqueous HCl (2 mL, 1N) was
added. This solution was subjected to preparative HPLC to obtain
the pure amide product, which was subsequently characterized by
LC-MS and NMR spectroscopy.
Example 3
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(3-dimethylcarbamoyl-2-ethoxy-propyl)-amide
##STR00015##
[0022] Preparative HPLC gave 13 mg of the title compound (41%) from
30 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.24H.sub.29FN.sub.4O.sub.4: 457, obtained:
457. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, 9.2 Hz, 1H), 7.72 (s, 1H), 7.60
(t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, 8.4 Hz, 1H),
3.89 (m, 1H), 3.58-3.45 (m, 2H), 3.40-3.27 (m, 2H, buried in water
signals), 2.97 (s, 3H), 2.82 (s, 3H), 2.43 (s, 3H), 2.41 (s, 3H),
1.07 (t, J=7.2 Hz, 3H).
Example 4
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(3-dimethylcarbamoyl-2-methoxy-propyl)-amide
##STR00016##
[0024] Preparative HPLC gave 46 mg of the title compound (36%) from
120 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.23H.sub.27FN.sub.4O.sub.4: 443, obtained:
443. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, 9.2 Hz, 1H), 7.71 (s, 1H), 7.63
(t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, 8.8 Hz, 1H),
3.78 (m, 1H), 3.42-3.31 (m, 2H), 3.30 (s, 3H), 2.97 (s, 3H), 2.82
(s, 3H), 2.43 (s, 3H), 2.41 (s, 3H), 2.63-2.43 (m, 2H).
Example 5
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-methoxy-4-morpholin-4-yl-4-oxo-butyl)-amide
##STR00017##
[0026] Preparative HPLC gave 48 mg of the title compound (37%) from
110 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.25H.sub.29FN.sub.4O.sub.6: 485, obtained:
485. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, 9.2 Hz, 1H), 7.71 (s, 1H), 7.63
(t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, 8.4 Hz, 1H),
3.80 (m, 1H), 3.55 (m, 4H), 3.47 (m, 4H), 3.38 (m, 2H), 3.31 (s,
3H), 2.60 (m, 1H), 2.45 (m, 1H), 2.43 (s, 3H), 2.41 (s, 3H).
Example 6
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
[4-(4-hydroxy-piperidin-1-yl)-2-methoxy-4-oxo-butyl]-amide
##STR00018##
[0028] Preparative HPLC gave 20 mg of the title compound (33%) from
50 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.26H.sub.31FN.sub.4O.sub.5: 499, obtained:
499. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, 9.6 Hz, 1H), 7.72 (s, 1H), 7.63
(t, J=5.6 Hz, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.4 Hz, 8.4 Hz, 1H),
3.92 (m, 1H), 3.78 (m, 1H), 3.68 (b, 1H), 3.30 (s, 3H), 3.15 (m,
1H), 3.01 (m, 1H), 2.60 (m, 1H), 2.55 (m, 2H), 2.50 (m, 1H), 2.45
(m, 2H), 2.43 (s, 3H), 2.41 (s, 3H), 1.70 (m, 2H), 1.30 (m,
2H).
Example 7
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-methoxy-4-oxo-4-pyrrolidin-1-yl-butyl)-amide
##STR00019##
[0030] Preparative HPLC gave 40 mg of the title compound (32%) from
110 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.25H.sub.29FN.sub.4O.sub.4: 469, obtained:
469. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, 9.6 Hz, 1H), 7.71 (s, 1H), 7.63
(t, J=5.6 Hz, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, 8.8 Hz, 1H),
3.82 (m, 1H), 3.50-3.25 (m, 6H), 3.30 (s, 3H), 2.55-2.45 (m, 2H),
2.43 (s, 3H), 2.41 (s, 3H), 1.86 (m, 2H), 1.76 (m, 2H).
Example 8
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
[2-methoxy-3-(methoxy-methyl-carbamoyl)-propyl]-amide
##STR00020##
[0032] Preparative HPLC gave 15 mg of the title compound (15%) from
80 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.23H.sub.27FN.sub.4O.sub.5: 459, obtained:
459. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.90 (s, 1H), 7.76 (dd, J=2.4 Hz, 9.2 Hz, 1H), 7.72 (s, 1H), 7.68
(t, J=6.0 Hz, 1H), 6.93 (m, 1H), 6.84 (dd, J=4.4 Hz, 8.4 Hz, 1H),
3.79 (m, 1H), 3.66 (s, 3H), 3.50-3.35 (m, 2H), 3.31 (s, 3H), 3.13
(s, 3H), 2.55-2.45 (m, 2H), 2.43 (s, 3H), 2.41 (s, 3H).
Examples 9-15
[0033] The synthesis of acids (2-3) and amides (2-4) is shown in
FIG. 2. Variations from this general synthetic procedure can be
understood and carried out by those skilled in the art. Thus, the
compounds of the present invention can be synthesized by those
skilled in the art.
Example 9
3-({5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1-
H-pyrrole-3-carbonyl}-amino)-2-methoxy-propionic acid
##STR00021##
[0035] To a suspension of methyl 3-amino-2-hydroxypropionate (1.0
equiv, which was prepared by refluxing the free amino acid
isoserine in dry methanol with 1.2 equiv HCl) and DIEA (5 equiv) in
DCM, Mmt-Cl (1.1 equiv) was added portion-wise at 25.degree. C.
After stirring overnight, the DCM was removed under reduced
pressure. The residue was suspended in ethyl acetate, washed with
brine (3.times.), dried over anhydrous Na.sub.2SO.sub.4. The ethyl
acetate was then removed, and the residue was dried overnight under
high vacuum, and subjected to flash chromatography to give compound
2-1. To a solution of compound 2-1 in dry DMF, NaH (1.5 equiv) was
added under argon. After stirring at 25.degree. C. for 1 h, MeI (5
equiv) was added to the solution, and the resulting suspension was
gently stirred at 25.degree. C. overnight. The DMF was removed
under vacuum; the residue was suspended in ethyl acetate, washed
with brine (3.times.), and dried over anhydrous Na.sub.2SO.sub.4.
After the ethyl acetate was removed via evaporation the resulting
residue was treated with 1% TFA in DCE/DCM for 30 min. The organic
solvents were then removed under reduced pressure, and the
resulting residue was triturated with hexane (3.times.) to obtain
the free amino acid 2-2. This amino acid was used directly in the
next step without any purification and characterizations. Thus, to
a solution of 2-2 (2 equiv) and DIEA (5 equiv) in DMF, compound 1-3
(1 equiv) was added at 25.degree. C. After stirring for 30 min
(LC-MS show the complete consumption of 1-3), KOH (5 equiv) in
water was added, and the solution was stirred for another 2 h
(LC-MS demonstrated a complete hydrolysis). The solvents were
removed under reduced pressure, and HCl (1N, excess) was added to
give a precipitate. This precipitate was collected by filtration,
washed with water and dried under high vacuum to give the title
compound (99% based on compound 1-3). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.20H.sub.20FN.sub.3O.sub.5: 402, obtained:
402. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H),
12.83 (b, 1H), 10.90 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H),
7.71 (s, 1H), 7.69 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.82 (dd, J=4.8
Hz, J=8.4 Hz, 1H), 3.90 (m, 1H), 3.55 (m, 1H), 3.41 (m, 1H), 3.32
(s, 3H), 2.42 (s, 3H), 2.40 (s, 3H).
Example 10
2-Ethoxy-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-d-
imethyl-1H-pyrrole-3-carbonyl}-amino)-propionic acid
##STR00022##
[0037] A similar route as that for the synthesis of Example 9 was
used to prepare the title compound. Iodoethane was used instead of
iodomethane to obtain the 2-ethoxy compound (38% based on compound
1-3). LC-MS: single peak at 254 nm, MH.sup.+ calcd. for
C.sub.21H.sub.22FN.sub.4O.sub.5: 416, obtained: 416. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H), 12.80 (b, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.71 (s, 1H),
7.68 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz,
1H), 4.00 (dd, J=5.2 Hz, J=7.6 Hz, 1H), 3.58 (m, 2H), 3.41 (m, 2H),
2.43 (s, 3H), 2.41 (s, 3H), 1.14 (t, J=6.8 Hz, 3H).
Examples 11-15
[0038] The general procedure for the synthesis of amides (compounds
2-4): A corresponding amine (2 equiv) was added to a solution of
the acid (compound 2-3), HATU (1.05 mmol), and DIEA (5 equiv) in
DMF (5 mL). After the solution was stirred at 25.degree. C. for 2
h, aqueous HCl (2 mL, 1N) was added. This solution was subjected to
preparative HPLC to obtain the pure amide product, which was
subsequently characterized by LC-MS and NMR spectroscopy.
Example 11
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-dimethylcarbamoyl-2-ethoxy-ethyl)-amide
##STR00023##
[0040] Preparative HPLC gave 46 mg of the title compound (62%) from
70 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.23H.sub.27FN.sub.4O.sub.4: 443, obtained:
443.
Example 12
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-ethoxy-3-morpholin-4-yl-3-oxo-propyl)-amide
##STR00024##
[0042] Preparative HPLC gave 40 mg of the title compound (49%) from
70 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.25H.sub.29FN.sub.4O.sub.5: 485, obtained:
485. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.71 (s, 1H),
7.70 (m, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz, 1H), 4.40
(m, 1H), 3.73-3.35 (m, 12H), 2.43 (s, 3H), 2.41 (s, 3H), 1.12 (t,
J=7.2 Hz, 3H).
Example 13
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-dimethylcarbamoyl-2-methoxy-ethyl)-amide
##STR00025##
[0044] Preparative HPLC gave 93 mg of the title compound (76%) from
115 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.22H.sub.25FN.sub.4O.sub.4: 429, obtained:
429. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.90 (s, 1H), 7.75 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.72 (m, 1H),
7.71 (s, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.40
(dd, J=4.8 Hz, J=7.2 Hz, 1H), 3.50 (m, 1H), 3.32 (m, 1H), 3.24 (s,
3H), 3.10 (s, 3H), 2.86 (s, 3H), 2.43 (s, 3H), 2.41 (s, 3H).
Example 14
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-methoxy-3-morpholin-4-yl-3-oxo-propyl)-amide
##STR00026##
[0046] Preparative HPLC gave 98 mg of the title compound (73%) from
115 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.24H.sub.27FN.sub.4O.sub.5: 471, obtained:
471. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H),
10.89 (s, 1H), 7.75 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.71 (s, 1H),
7.70 (m, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.34
(dd, J=4.8 Hz, J=7.2 Hz, 1H), 3.85-3.30 (m, 10H), 3.26 (s, 3H),
2.44 (s, 3H), 2.42 (s, 3H).
Example 15
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
(2-methoxy-3-oxo-3-pyrrolidin-1-yl-propyl)-amide
##STR00027##
[0048] Preparative HPLC gave 86 mg of the title compound (66%) from
115 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.24H.sub.27FN.sub.4O.sub.4: 455, obtained:
455. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.70 (m, 1H),
7.71 (s, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.4 Hz, J=8.4 Hz, 1H), 4.20
(dd, J=5.2 Hz, J=7.2 Hz, 1H), 3.60-3.47 (m, 3H), 3.43-3.28 (m, 3H),
3.26 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H), 1.88 (m, 2H), 1.78 (m,
2H).
Examples 16-315
[0049] Still further amide examples are shown in the following
table:
TABLE-US-00001 ##STR00028## CORE I ##STR00029## CORE II
##STR00030## CORE III ##STR00031## CORE IV ##STR00032## CORE V and
##STR00033## CORE VI Ex # Core R 16 I a 17 I b 18 I c 19 I d 20 I e
21 I f 22 I g 23 I h 24 I i 25 I j 26 I k 27 I l 28 I m 29 I n 30 I
o 31 I p 32 I q 33 I r 34 I s 35 I t 36 I u 37 I v 38 I w 39 I x 40
I y 41 I z 42 I aa 43 I ab 44 I ac 45 I ad 46 I ae 47 I af 48 I ag
49 I ah 50 I ai 51 I aj 52 I ak 53 I al 54 I am 55 I an 56 I ao 57
I ap 58 I aq 59 I ar 60 I as 61 I at 62 I au 63 I av 64 I aw 65 I
ax 66 II a 67 II b 68 II c 69 II d 70 II e 71 II f 72 II g 73 II h
74 II i 75 II j 76 II k 77 II l 78 II m 79 II n 80 II o 81 II p 82
II q 83 II r 84 II s 85 II t 86 II u 87 II v 88 II w 89 II x 90 II
y 91 II z 92 II aa 93 II ab 94 II ac 95 II ad 96 II ae 97 II af 98
II ag 99 II ah 100 II ai 101 II aj 102 II ak 103 II al 104 II am
105 II an 106 II ao 107 II ap 108 II aq 109 II ar 110 II as 111 II
at 112 II au 113 II av 114 II aw 115 II ax 116 III a 117 III b 118
III c 119 III d 120 III e 121 III f 122 III g 123 III h 124 III i
125 III j 126 III k 127 III l 128 III m 129 III n 130 III o 131 III
p 132 III q 133 III r 134 III s 135 III t 136 III u 137 III v 138
III w 139 III x 140 III y 141 III z 142 III aa 143 III ab 144 III
ac 145 III ad 146 III ae 147 III af 148 III ag 149 III ah 150 III
ai 151 III aj 152 III ak 153 III al 154 III am 155 III an 156 III
ao 157 III ap 158 III aq 159 III ar 160 III as 161 III at 162 III
au 163 III av 164 III aw 165 III ax 166 IV a 167 IV b 168 IV c 169
IV d 170 IV e 171 IV f 172 IV g 173 IV h 174 IV i 175 IV j 176 IV k
177 IV l 178 IV m 179 IV n 180 IV o 181 IV p 182 IV q 183 IV r 184
IV s 185 IV t 186 IV u 187 IV v 188 IV w 189 IV x 190 IV y 191 IV z
192 IV aa 193 IV ab 194 IV ac 195 IV ad 196 IV ae 197 IV af 198 IV
ag 199 IV ah 200 IV ai 201 IV aj 202 IV ak 203 IV al 204 IV am 205
IV an 206 IV ao 207 IV ap 208 IV aq 209 IV ar 210 IV as 211 IV at
212 IV au 213 IV av 214 IV aw 215 IV ax 216 V a 217 V b 218 V c 219
V d 220 V e 221 V f 222 V g 223 V h 224 V i 225 V j 226 V k 227 V l
228 V m 229 V n 230 V o 231 V p 232 V q 233 V r 234 V s 235 V t 236
V u 237 V v 238 V w 239 V x 240 V y 241 V z 242 V aa
243 V ab 244 V ac 245 V ad 246 V ae 247 V af 248 V ag 249 V ah 250
V ai 251 V aj 252 V ak 253 V al 254 V am 255 V an 256 V ao 257 V ap
258 V aq 259 V ar 260 V as 261 V at 262 V au 263 V av 264 V aw 265
V ax 266 VI a 267 VI b 268 VI c 269 VI d 270 VI e 271 VI f 272 VI g
273 VI h 274 VI i 275 VI j 276 VI k 277 VI l 278 VI m 279 VI n 280
VI o 281 VI p 282 VI q 283 VI r 284 VI s 285 VI t 286 VI u 287 VI v
288 VI w 289 VI x 290 VI y 291 VI z 292 VI aa 293 VI ab 294 VI ac
295 VI ad 296 VI ae 297 VI af 298 VI ag 299 VI ah 300 VI ai 301 VI
aj 302 VI ak 303 VI al 304 VI am 305 VI an 306 VI ao 307 VI ap 308
VI aq 309 VI ar 310 VI as 311 VI at 312 VI au 313 VI av 314 VI aw
315 VI ax
[0050] In the above table, R.sup.9 is selected from the following
radicals:
##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038##
[0051] These amide examples 16-315 can be made by those skilled in
the art following the above procedure and/or known procedures.
Examples 316-320
[0052] The synthesis of acids (3-3) and amides (3-4) is shown in
FIG. 3. Variations from this general synthetic procedure can be
understood and carried out by those skilled in the art. Thus, the
compounds of the present invention can be synthesized by those
skilled in the art.
Example 316
(S)-4-({5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimeth-
yl-1H-pyrrole-3-carbonyl}-amino)-2-methoxy-butyric acid
##STR00039##
[0054] To a suspension of methyl 4-amino-2-hydroxybutyrate (1.0
equiv, which was prepared by refluxing the free amino acid in dry
methanol with 1.2 equiv HCl) and DIEA (5 equiv) in DCM, Mmt-Cl (1.1
equiv) was added portion-wise at 25.degree. C. After stirring
overnight, the DCM was removed under reduced pressure. The residue
was suspended in ethyl acetate, washed with brine (3.times.), dried
over anhydrous Na.sub.2SO.sub.4. The ethyl acetate was then
removed, and the residue was dried overnight under high vacuum, and
subjected to flash chromatography to give compound 3-1. To a
solution of compound 3-1 in dry DMF, NaH (1.5 equiv) was added
under argon. After stirring at 25.degree. C. for 1 h, MeI (5 equiv)
was added to the solution, and the resulting-suspension was gently
stirred at 25.degree. C. overnight. The DMF was removed under
vacuum; the residue was suspended in ethyl acetate, washed with
brine (3.times.), and dried over anhydrous Na.sub.2SO.sub.4. After
the ethyl acetate was removed via evaporation the resulting residue
was treated with 1% TFA in DCE/DCM for 30 min. The organic solvents
were then removed under reduced pressure, and the resulting residue
was triturated with hexane (3.times.) to obtain the free amino acid
3-2. This amino acid was used directly in the next step without any
purification and characterization. Thus, to a solution of 3-2 (2
equiv) and DIEA (5 equiv) in DMF, compound 1-3 (1 equiv) was added
at 25.degree. C. After stirring for 30 min (LC-MS show the complete
consumption of 1-3), KOH (5 equiv) in water was added, and the
solution was stirred for another 2 h (LC-MS demonstrated a complete
hydrolysis). The solvents were removed under reduced pressure, and
HCl (1N, excess) was added to give a precipitate. This precipitate
was collected and washed (by water) by filtration, dried under high
vacuum to give the title compound (97% based on compound 1-3).
LC-MS: single peak at 254 nm, MH.sup.+ calcd. for
C.sub.21H.sub.22FN.sub.3O.sub.5: 416, obtained: 416. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H), 12.80 (b, 1H),
10.90 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.71 (s, 1H),
7.65 (t, J=5.6 Hz, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz,
1H), 3.77 (dd, J=4.0 Hz, J=8.8 Hz, 1H), 3.40-3.30 (m, 2H), 3.30 (s,
3H), 2.43 (s, 3H), 2.41 (s, 3H), 1.92 (m, 1H), 1.78 (m, 1H).
Example 317
(S)-2-Ethoxy-4-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2-
,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-butyric acid
##STR00040##
[0056] A similar route as that for the synthesis of Example 316 was
used to prepare the title compound. Iodoethane was used instead of
iodomethane to obtain the 2-ethoxy compound (84% based on compound
1-3). LC-MS: single peak at 254 nm, MH.sup.+ calcd. for
C.sub.22H.sub.24FN.sub.3O.sub.5: 430, obtained: 430. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H), 12.70 (b, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.71 (s, 1H),
7.66 (t, J=5.6 Hz, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz,
1H), 3.85 (dd, J=4.0 Hz, J=8.4 Hz, 1H), 3.58 (m, 1H), 3.40-3.25 (m,
3H), 2.43 (s, 3H), 2.41 (s, 3H), 1.92 (m, 1H), 1.77 (m, 1H), 1.13
(t, J=7.2 Hz, 3H).
Example 318-320
[0057] The general procedure for the synthesis of amides (compounds
3-4): A corresponding amine (2 equiv) was added to a solution of
the acid (compound 3-3), HATU (1.05 mmol), and DIEA (5 equiv) in
DMF (5 mL). After the solution was stirred at 25.degree. C. for 2
h, aqueous HCl (2 mL, 1N) was added. This solution was subjected to
preparative HPLC to obtain the pure amide product, which was
subsequently characterized by LC-MS and NMR spectroscopy.
Example 318
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
((S)-3-dimethylcarbamoyl-3-methoxy-propyl)-amide
##STR00041##
[0059] Preparative HPLC gave 37 mg of the title compound (58%) from
60 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.23H.sub.27FN.sub.4O.sub.4: 443, obtained:
443. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.72 (s, 1H),
7.65 (t, J=5.6 Hz, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz,
1H), 4.20 (dd, J=4.0 Hz, J=8.0 Hz, 1H), 3.30 (m, 2H), 3.27 (s, 3H),
3.04 (s, 3H), 2.88 (s, 3H), 2.43 (s, 3H), 2.41 (s, 3H), 1.80 (m,
2H).
Example 319
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
((S)-3-methoxy-4-morpholin-4-yl-4-oxo-butyl)-amide
##STR00042##
[0061] Preparative HPLC gave 32 mg of the title compound (46%) from
60 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.25H.sub.29FN.sub.4O.sub.5: 485, obtained:
485. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.68 (s, 1H),
10.89 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.72 (s, 1H),
7.65 (t, J=5.6 Hz, 1H), 6.93 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.4 Hz,
1H), 4.19 (dd, J=4.8 Hz, J=8.0 Hz, 1H), 3.57 (m, 6H), 3.47 (m, 2H),
3.28 (m, 2H), 3.23 (s, 3H), 2.44 (s, 3H), 2.41 (s, 3H), 1.79 (m,
2H).
Example 320
5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-
rrole-3-carboxylic acid
((S)-3-dimethylcarbamoyl-3-ethoxy-propyl)-amide
##STR00043##
[0063] Preparative HPLC gave 67 mg of the title compound (57%) from
120 mg starting material (acid). LC-MS: single peak at 254 nm,
MH.sup.+ calcd. for C.sub.24H.sub.29FN.sub.4O.sub.4: 457, obtained:
457. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz), .delta. 13.67 (s, 1H),
10.88 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.71 (s, 1H),
7.56 (m, 1H), 6.91 (m, 1H), 6.83 (m, 1H), 4.25 (m, 1H), 3.45-3.25
(m, 4H), 3.03 (s, 3H), 2.83 (s, 3H), 2.43 (s, 3H), 2.41 (s, 3H),
1.80 (m, 2H).
[0064] The compounds described herein are presently representative
of preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. It will be readily
apparent to one skilled in the art that varying substitutions and
modifications may be made to the invention disclosed herein without
departing from the scope and spirit of the invention.
VEGFR Biochemical Assay
[0065] The compounds were assayed for biochemical activity by
Upstate Ltd at Dundee, United Kingdom, according to the following
procedure. In a final reaction volume of 25 .mu.l, KDR (h) (5-10
mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.33 mg/ml
myelin basic protein, 10 mM MgAcetate and [.gamma.-.sup.33P-ATP]
(specific activity approx. 500 cpm/pmol, concentration as
required). The reaction is initiated by the addition of the MgATP
mix. After incubation for 40 minutes at room temperature, the
reaction is stopped by the addition of 5 .mu.l of a 3% phosphoric
acid solution. 10 .mu.l of the reaction is then spotted onto a P30
filtermat and washed three times for 5 minutes in 75 mM phosphoric
acid and once in methanol prior to drying and scintillation
counting.
[0066] Compounds of the present invention were tested in this assay
and exhibited IC.sub.50 between 1-5,000 nM.
PDGFR Phosphorylation Assay
[0067] NIH3T3 cells are plated in a 96 well plate in DMEM+10% FBS.
Following cell attachment the cells are serum starved overnight
before adding the chemical test compounds to a final concentration
of 0.1% DMSO. Following a 1 hour incubation at 37.degree. C. cells
are removed from the incubator and allowed to cool to RT for 20 min
before stimulation with PDGF-BB for 15 min at RT. Cells are placed
on ice for 5 min, the media removed and the cells are lysed with
100 .mu.L/well lysis buffer for 1 hour at 4.degree. C. Plates are
spun at 2000 rpm for 30 min at 4.degree. C. and solubilized
phosphorylated PDGFR is quantitated by ELISA.
[0068] High binding microplates are incubated overnight at RT with
anti-mouse PDGFR-b capture-antibody in PBS, washed with PBS+0.05%
Tween20 and blocked for 4 h at RT with PBS+1% BSA and washed again.
100 .mu.L lysate/well is incubated overnight at 4.degree. C. Plates
are washed and wells are incubated with 100 .mu.L/well of mouse
anti-phosphotyrosine-HRP antibody for 2 h at 37.degree. C. Plates
are washed again and colorimetric detection is performed using TMB
as substrate.
[0069] Most of the compounds in this invention showed IC.sub.50 of
less than 1 .mu.M in this assay.
VEGFR Phosphorylation Assay
[0070] NIHT3T cells overexpressing mouse VEGFR-2 (FLK-1) are plated
in a 96 well plate in DMEM+10% FBS. Following cell attachment for 4
hours the cells are serum starved overnight before adding the
chemical test compounds to a final concentration of 0.1% DMSO.
Following a 1 hour incubation at 37.degree. C. cells are stimulated
for 15 min at 37.degree. C. with VEGF165. Cells are placed on ice
for 5 min, the media removed, washed once with ice cold PBS and the
cells are lysed with 50 .mu.L/well lysis buffer for 1 hour at
4.degree. C. Plates are spun for 10 min at 2000 rpm at 4.degree. C.
and solubilized phosphorylated VEGFR is quantitated by ELISA.
[0071] High binding microplates are incubated overnight at room
temperature with VEGFR antibody in 50 .mu.L PBS, washed with
PBS+0.05% Tween20 and blocked for 4 h at RT with PBS+1% BSA and
washed again. 50 .mu.L lysate/well is incubated overnight at
4.degree. C. Plates are washed and wells are incubated with 50
.mu.L/well of mouse anti-phosphotyrosine-HRP antibody for 2 h at
37.degree. C. Plates are washed again and colorimetric detection is
performed using TMB as substrate.
[0072] Most of the compounds in this invention showed IC.sub.50 of
less than 1 .mu.M in this assay.
Cellular Assay: HUVEC: VEGF Induced Proliferation
[0073] The compounds were assayed for cellular activity in the VEGF
induced proliferation of HUVEC cells. HUVEC cells (Cambrex,
CC-2517) were maintained in EGM (Cambrex, CC-3124) at 37.degree. C.
and 5% CO.sub.2. HUVEC cells were plated at a density 5000
cells/well (96 well plate) in EGM. Following cell attachment (1
hour) the EGM-medium was replaced by EBM (Cambrex, CC-3129)+0.1%
FBS (ATTC, 30-2020) and the cells were incubated for 20 hours at
37.degree. C. The medium was replaced by EBM+1% FBS, the compounds
were serial diluted in DMSO and added to the cells to a final
concentration of 0-5,000 nM and 1% DMSO. Following a 1 hour
pre-incubation at 37.degree. C. cells were stimulated with 10 ng/ml
VEGF (Sigma, V7259) and incubated for 45 hours at 37.degree. C.
Cell proliferation was measured by BrdU DNA incorporation for 4
hours and BrdU label was quantitated by ELISA (Roche kit, 16472229)
using 1M H.sub.2SO.sub.4 to stop the reaction. Absorbance was
measured at 450 nm using a reference wavelength at 690 nm.
DETAILED DESCRIPTION OF FIGURES
[0074] FIG. 1 shows a scheme that is used for the synthesis of the
3-alkoxy-4-acylaminoamide derivatives starting from methyl
3-hydroxy-4-aminobutanoate hydrochlorides and the activated
acylating agent 1-3. The amino ester hydrochloride starting
material was prepared by refluxing the free amino acid in anhydrous
methanol in the presence of 1.2 eq of HCl. The amino group was
protected as its monomethoxytrityl derivative in the presence of
the secondary hydroxyl group to give the neutral hydroxy ester 1-1.
The hydroxyl group was alkylated using methyl- or ethyl iodide to
form the protected amino alkoxy ester. The Mmt group was removed in
1% trifluoroacetic acid leaving the amino hydrochloride or
trifluoracetate compound 1-2. This compound was quickly acylated
with the preformed acylating agent 1-3 and the methyl ester was
hydrolyzed by potassium hydroxide in water/DMF to give 1-4. The
free acid was then exposed to HATU, amine and diisopropylethyl
amine in DMF to give the alkoxy amide 1-5.
[0075] FIG. 2 shows a scheme that is used for the synthesis of the
2-alkoxy-3-acylaminoamide derivatives starting from methyl
2-hydroxy-3-aminopropionate hydrochlorides and the activated
acylating agent 1-3. The amino ester hydrochloride starting
material was prepared by refluxing the free amino acid in anhydrous
methanol in the presence of 1.2 eq of HCl. The amino group was
protected as its monomethoxytrityl derivative in the presence of
the secondary hydroxyl group to give 2-1. The hydroxyl group was
alkylated using methyl- or ethyl iodide to form the protected amino
alkoxy ester. The Mmt group was removed in 1% trifluoroacetic acid
leaving the amino hydrochloride or trifluoracetate compound 2-2.
This compound was quickly acylated with the preformed acylating
agent 1-3 and the methyl ester was hydrolyzed by potassium
hydroxide in water/DMF to give 2-4. The free acid was then exposed
to HATU, amine and diisopropylethyl amine in DMF to give the alkoxy
amide 2-5.
[0076] FIG. 3 shows a scheme that is used for the synthesis of the
(2S)-2-alkoxy-4-acylamino-amide derivatives starting from methyl
(2S)-2-hydroxy-4-aminobutanoate hydrochloride and the activated
acylating agent 1-3. The amino ester hydrochloride starting
material was prepared by refluxing the free amino acid in anhydrous
methanol in the presence of 1.2 eq of HCl. The amino group was
protected as its monomethoxytrityl derivative in the presence of
the secondary hydroxyl group to give the neutral hydroxy ester 3-1.
The hydroxyl group was alkylated using methyl- or ethyl iodide to
form the protected amino alkoxy ester. The Mmt group was removed in
1% trifluoroacetic acid leaving the amino hydrochloride or
trifluoracetate compound 3-2. This compound was quickly acylated
with the preformed acylating agent 1-3 and the methyl ester was
hydrolyzed by potassium hydroxide in water/DMF to give 3-4. The
free acid was then exposed to HATU, amine and diisopropylethyl
amine in DMF to give the alkoxy amide 3-5.
* * * * *