U.S. patent application number 11/069930 was filed with the patent office on 2005-09-22 for rxr activating molecules.
Invention is credited to Al-Shamma, Hussien A., Fanjul, Andrea N., Fine, Richard, Guo, Jianhua, Jakubowicz-Jaillardon, Karine, Pfahl, Magnus, Pleynet, David P.M., Spruce, Lyle W., Tachdjian, Catherine, Zapf, James W..
Application Number | 20050209286 11/069930 |
Document ID | / |
Family ID | 23047783 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050209286 |
Kind Code |
A1 |
Pfahl, Magnus ; et
al. |
September 22, 2005 |
RXR activating molecules
Abstract
Disclosed are molecules that activate an RXR receptor, wherein
the molecule comprises an RXR binding portion which binds the RXR
receptor and comprises a side pocket contacting residue which
contacts a side-pocket 1 of an RXR receptor.
Inventors: |
Pfahl, Magnus; (Solana
Beach, CA) ; Tachdjian, Catherine; (San Diego,
CA) ; Al-Shamma, Hussien A.; (Encinitas, CA) ;
Fanjul, Andrea N.; (San Diego, CA) ; Pleynet, David
P.M.; (San Diego, CA) ; Spruce, Lyle W.;
(Chula Vista, CA) ; Fine, Richard; (Ridgewood,
NJ) ; Zapf, James W.; (San Diego, CA) ; Guo,
Jianhua; (San Diego, CA) ; Jakubowicz-Jaillardon,
Karine; (San Diego, CA) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
23047783 |
Appl. No.: |
11/069930 |
Filed: |
March 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11069930 |
Mar 1, 2005 |
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10098184 |
Mar 8, 2002 |
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60274342 |
Mar 8, 2001 |
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Current U.S.
Class: |
514/341 ;
514/364; 546/269.1; 548/134; 702/19 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 9/10 20180101; A61P 1/00 20180101; C07D 277/20 20130101; A61P
35/00 20180101; A61P 7/12 20180101; A61P 35/02 20180101; A61P 37/02
20180101; C07D 417/10 20130101; A61P 3/06 20180101; C07D 271/07
20130101; A61P 3/10 20180101; A61P 29/00 20180101; A61P 19/02
20180101 |
Class at
Publication: |
514/341 ;
514/364; 546/269.1; 548/134; 702/019 |
International
Class: |
A61K 031/4439; A61K
031/4245; G06F 019/00; G01N 033/48; G01N 033/50; C07D 285/14 |
Claims
What is claimed is:
1. A molecule that activates an RXR receptor to at least 60% of the
activation of 9-cis retinoic acid, wherein the molecule comprises
an RXR binding portion which binds the RXR receptor and comprises a
side pocket contacting residue which has at least four contacts
with the region consisting of Leu 433, Gly 429, Ile 310, Asn 306,
and Trp 305 of side-pocket 1 of an RXR receptor, wherein the
contacts are less than or equal to 3.5 Angstroms, and wherein the
molecule does not have the structure: 19wherein: n and m are
independently 0 or 1; R.sub.1 and R.sub.2 are 1) independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, hydroxyl, acyl, amino, mono-substituted amino,
di-substituted amino, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide or haloalkoxy; or 2) R.sub.1 and R.sub.2
together with the aromatic ring bonded thereto form a cycloalkyl,
substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl
residue that may optionally comprise 1 or 2 heteroatoms selected
from O, S, NH or N-alkyl; R.sub.3 and R.sub.4 are independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano,
nitro, hydroxyl, acyloxy, amino, mono-substituted amino,
di-substituted amino, alkylsulfonamide, arylsulfonamide, alkylurea,
arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy,
substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide or substituted dialkylcarboxamide; A is
--CR.sub.6R.sub.7-- where R6 and R7 are independently or together
hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy or
haloalkoxy; or R.sub.6 and R.sub.7 together form a cycloalkyl
residue that may optionally comprise 1 or 2 heteroatoms selected
from O, S, NH and N-alkyl; Ar is Formula (II), (III), (IV) or (V):
20wherein R.sub.8, R.sub.9 and R.sub.10 are independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano,
nitro, hydroxyl, acyloxy, amino, mono-substituted amino,
di-substituted amino, alkylamide, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl,
thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide or substituted
dialkylcarboxamide; R.sub.5 is hydrogen, halogen, hydroxy, alkyl or
substituted alkyl; 21represents a bond present or absent; and W, X,
Y and Z are independently or together --C(O)--, --C(S)--, --S--,
--O-- or --NH-- residues that together form a
2,4-thiazolidinedione, 2-thioxo-4-thiazolidinedione,
isoxazolidinedione, 2,4-imidazolidinedione or
2-thioxo-4-imidazolidinedione residue; or a pharmaceutically
acceptable salt thereof.
2. A composition comprising an RXR receptor complexed with a
molecule that activates an RXR receptor to at least 60% of the
activation of 9-cis retinoic acid, wherein the molecule comprises
an RXR binding portion which binds the RXR receptor and comprises a
side pocket contacting residue which has at least four contacts
with the region consisting of Leu 433, Gly 429, Ile 310, Asn 306,
and Trp 305 of side-pocket 1 of an RXR receptor, wherein the
contacts are less than or equal to 3.5 Angstroms, wherein the
molecule does not have the structure 22wherein: n and m are
independently 0 or 1; R.sub.1 and R.sub.2 are 1) independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, hydroxyl, acyl, amino, mono-substituted amino,
di-substituted amino, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide or haloalkoxy; or 2) R.sub.1 and R.sub.2
together with the aromatic ring bonded thereto form a cycloalkyl,
substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl
residue that may optionally comprise 1 or 2 heteroatoms selected
from O, S, NH or N-alkyl; R.sub.3 and R.sub.4 are independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano,
nitro, hydroxyl, acyloxy, amino, mono-substituted amino,
di-substituted amino, alkylsulfonamide, arylsulfonamide, alkylurea,
arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy,
substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide or substituted dialkylcarboxamide; A is
--CR.sub.6R.sub.7-- where R6 and R7 are independently or together
hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy or
haloalkoxy; or R.sub.6 and R.sub.7 together form a cycloalkyl
residue that may optionally comprise 1 or 2 heteroatoms selected
from O, S, NH and N-alkyl; Ar is Formula (II), (III), (IV) or (V):
23wherein R.sub.8, R.sub.9 and R.sub.10 are independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano,
nitro, hydroxyl, acyloxy, amino, mono-substituted amino,
di-substituted amino, alkylamide, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl,
thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide or substituted
dialkylcarboxamide; R.sub.5 is hydrogen, halogen, hydroxy, alkyl or
substituted alkyl; 24represents a bond present or absent; and W, X,
Y and Z are independently or together --C(O)--, --C(S)--, --S--,
--O-- or --NH-- residues that together form a
2,4-thiazolidinedione, 2-thioxo-4-thiazolidinedione,
isoxazolidinedione, 2,4-imidazolidinedione or
2-thioxo-4-imidazolidinedione residue; or a pharmaceutically
acceptable salt thereof.
3. A molecule that activates an RXR at least one fifth of the
activity of compound 1 as measured in the adipocyte differentiation
assay, wherein the molecule comprises an RXR binding portion which
binds the RXR receptor and comprises a side pocket contacting
residue which has at least four contacts with the region consisting
of Leu 433, Gly 429, Ile 310, Asn 306, and Trp 305 of side-pocket 1
of an RXR receptor, wherein the contacts are less than or equal to
3.5 Angstroms, wherein the molecule does not have the structure:
25wherein: n and m are independently 0 or 1; R.sub.1 and R.sub.2
are 1) independently or together hydrogen, alkyl, substituted
alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, hydroxyl, acyl,
amino, mono-substituted amino, di-substituted amino, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide, substituted dialkylcarboxamide or haloalkoxy;
or 2) R.sub.1 and R.sub.2 together with the aromatic ring bonded
thereto form a cycloalkyl, substituted cycloalkyl, cycloalkenyl or
substituted cycloalkenyl residue that may optionally comprise 1 or
2 heteroatoms selected from O, S, NH or N-alkyl; R.sub.3 and R4 are
independently or together hydrogen, alkyl, substituted alkyl,
haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, halogen, cyano, nitro, hydroxyl, acyloxy, amino,
mono-substituted amino, di-substituted amino, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy,
thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide or substituted
dialkylcarboxamide; A is --CR.sub.6R.sub.7-- where R6 and R7 are
independently or together hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy or haloalkoxy; or R.sub.6 and R.sub.7
together form a cycloalkyl residue that may optionally comprise 1
or 2 heteroatoms selected from O, S, NH and N-alkyl; Ar is Formula
(II), (III), (IV) or (V): 26wherein R.sub.8, R.sub.9 and R.sub.10
are independently or together hydrogen, alkyl, substituted alkyl,
haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, halogen, cyano, nitro, hydroxyl, acyloxy, amino,
mono-substituted amino, di-substituted amino, alkylamide,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, alkoxy, substituted alkoxy,
haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide; R.sub.5 is hydrogen, halogen,
hydroxy, alkyl or substituted alkyl; 27represents a bond present or
absent; and W, X, Y and Z are independently or together --C(O)--,
--C(S)--, --S--, --O-- or --NH-- residues that together form a
2,4-thiazolidinedione, 2-thioxo-4-thiazolidinedione,
isoxazolidinedione, 2,4-imidazolidinedione or
2-thioxo-4-imidazolidinedione residue; or a pharmaceutically
acceptable salt thereof.
4. The molecule of claims 1, 2, or 3, wherein the molecule has at
least four contacts with the region consisting of the amino acids
Ile 310, Asn 306, and Trp 305 of SEQ ID NO:1.
5. The molecule of claims 1, 2, or 3, wherein the molecule
activates the RXR receptor at a concentration of less than or equal
to 1 uM.
6. The molecule of claim 4, wherein the molecule activates the RXR
receptor at a concentration is less than or equal to 100 nM.
7. The molecule of claim 1, 2, or 3, wherein the side-pocket
contacting residue comprises at least three connected atoms.
8. The molecule of claim 1, 2, or 3, wherein there are at least 14
contacts between the atoms of the side pocket contacting residue
and the region consisting of Leu 433, Gly 429, Ile 310, Asn 306,
and Trp 305 of side pocket 1.
9. A pharmaceutical composition comprising the molecules of claim
1, 2, 3 or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
10. The pharmaceutical composition of claim 9 that is effective to
reduce blood glucose levels and lower triglyceride levels in
KKA.sub.y mice.
11. The molecules of claim 1, 2, or 3, wherein the side pocket
contacting residue comprises between 3 and 12 connected atoms.
12. The molecules of claim 1, 2, or 3, wherein the side pocket
contacting residue comprises greater than 8 connected atoms.
13. The composition of claims 11, wherein the connected atoms
comprise carbon, hydrogen, nitrogen, phosphorus, oxygen, sulfur,
fluorine, chlorine, bromine, iodine, or mixtures thereof.
14. The composition of claims 1, 2, or 3, wherein the side pocket
contacting residue is an alkyl, a substituted alkyl, a haloalkyl,
an alkenyl, a substituted alkenyl, an alkynyl, a substituted
alkynyl, a halogen, cyano, nitro, hydroxyl, acyloxy, amino,
mono-substituted amino, di-substituted amino, alkylamide,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, alkoxy, substituted alkoxy,
haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide residue.
15. The composition of claims 1, 2, or 3, wherein the side pocket
contacting residue is a haloalkoxy residue comprising 1 to 5 carbon
atoms.
16. The composition of claims 1, 2, or 3, wherein the RXR binding
portion comprises a substituted or unsubstituted C.sub.2-C.sub.18
cyclic organic residue.
17. The composition of claims 16, wherein the C.sub.2-C.sub.18
cyclic organic residue is a substituted or unsubstituted
C.sub.6-C.sub.18 aromatic ring residue wherein all ring atoms are
carbon, a substituted or unsubstituted C.sub.2-C.sub.18
heteroaromatic ring residue having from one to three ring atoms
selected from O, S, N, NH, or a substituted or unsubstituted
C.sub.2-C.sub.18 heteroaromatic ring residue having from one to
three ring atoms selected from O, S, N, NH and N--R atoms or
residues, wherein R comprises an alkyl, a substituted alkyl, an
aryl, a substituted aryl, an acyl, a heteroaryl, or a substituted
heteroaryl group.
18. The composition of claim 16, wherein the combination of side
pocket contacting residue and the C.sub.2-C.sub.18 cyclic organic
residue is a residue of Formula (II), (III), (IV) or (V): 28wherein
R.sub.8, R.sub.9 and R.sub.10 are independently or together
hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro,
hydroxyl, acyloxy, amino, mono-substituted amino, di-substituted
amino, alkylamide, alkylsulfonamide, arylsulfonamide, alkylurea,
arylurea, alkylcarbamate, arylcarbamate, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide.
19. The composition of claim 16, wherein the C.sub.2-C.sub.18
cyclic organic residue is connected to a polar binding portion
comprising at least one functional group having at least one polar
carbon-heteroatom or heteroatom-hydrogen bond.
20. The composition of claim 19, wherein the polar binding portion
comprises a residue of the formula: 29wherein m is an integer 0 or
1; R.sub.5 is hydrogen, halogen, hydroxy, alkyl or substituted
alkyl; 30represents a bond present or absent; W, X, Y and Z are
independently or together --C(O)--, --C(S)--, --S--, --O-- or
--NH-- residues that together form a 2,4-thiazolidinedione,
2-thioxo-4-thiazolidinedione, isoxazolidinedione,
2,4-imidazolidinedione or 2-thioxo-4-imidazolidinedio- ne
residue.
21. The composition of claim 16, wherein the C.sub.2-C.sub.18
cyclic organic residue is connected to an organic binding portion
comprising a C.sub.4 to C.sub.25 substituted or unsubstituted
hydrocarbon residue.
22. The composition of claim 21, wherein the organic binding
portion comprises 31wherein: n and m are independently 0 or 1;
R.sub.1 and R.sub.2 are 1) independently or together hydrogen,
alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, hydroxyl,
acyl, amino, mono-substituted amino, di-substituted amino, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide, substituted dialkylcarboxamide or haloalkoxy;
or 2) R1 and R2 together with the aromatic ring bonded thereto form
a cycloalkyl, substituted cycloalkyl, cycloalkenyl or substituted
cycloalkenyl residue that may optionally comprise 1 or 2
heteroatoms selected from O, S, NH or N-alkyl; R.sub.3 and R.sub.4
are independently or together hydrogen, alkyl, substituted alkyl,
haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, halogen, cyano, nitro, hydroxyl, acyloxy, amino,
mono-substituted amino, di-substituted amino, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy,
thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide or substituted
dialkylcarboxamide; and A is --CR.sub.6R.sub.7-- where R.sub.6 and
R.sub.7 are independently or together hydrogen, alkyl, substituted
alkyl, alkoxy, substituted alkoxy or haloalkoxy; or R.sub.6 and
R.sub.7 together form a cycloalkyl residue that may optionally
comprise 1 or 2 heteroatoms selected from O, S, NH and N-alkyl.
23. A method of treating a disease comprising administering a
composition of claim 9 to a mammal.
24. A method of treating a disease comprising administering a
composition of claim 9 to a human.
25. The method of claim 24, wherein the disease is selected from
the group consisting of type 2 diabetes, hypercholesteremia, and
atherosclerosis.
26. A method for selecting compositions that are RXR ligands which
interact with a side-pocket 1 of an RXR receptor comprising 1)
interacting a library of molecules with an RXR receptor, 2)
removing molecules that do not interact with the RXR receptor at a
predetermined level, 3) collecting the molecules that interact with
side-pocket 1 of the RXR receptor.
27. A compounds of the formula: 32
28. A method for selecting molecules that activate an RXR receptor
having a side pocket 1 comprising: constructing a computer
representation of the 3 dimensional structure of an RXR receptor
and it's side pocket 1, constructing a computer representation of
the 3 dimensional structure of one or more candidate molecules,
employing a computer algorithm to calculate at least some of the
intermolecular interactions of the of the candidate molecule with
RXR receptor and the interactions of the candidate molecule with
the side pocket 1 of the RXR receptor, and selecting, based on the
calculated intermolecular interactions a molecule that binds to the
RXR receptor and contacts side pocket 1.
29. A method of selecting compounds for the treatment of type 1
diabetes, type 2 diabetes, hypercholestermia or atherosclerosis
comprising designing molecules to contact side pocket 1 of an RXR
receptor.
30. The method of claim 29, wherein the molecules are designed to
have at least 4 contacts with side pocket 1 of an RXR receptor.
31. The method of claim 29, wherein the molecules are designed to
maximize contacts with side pocket 1 of an RXR receptor.
32. The method of claim 29, wherein the molecules are designed to
comprise between 4 and 20 contacts with side pocket 1 of an RXR
receptor.
33. The methods of claim 30-32, wherein the molecule does not have
the structure 33wherein: n and m are independently 0 or 1; R.sub.1
and R.sub.2 are 1) independently or together hydrogen, alkyl,
substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, hydroxyl,
acyl, amino, mono-substituted amino, di-substituted amino, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide, substituted dialkylcarboxamide or haloalkoxy;
or 2) R.sub.1 and R.sub.2 together with the aromatic ring bonded
thereto form a cycloalkyl, substituted cycloalkyl, cycloalkenyl or
substituted cycloalkenyl residue that may optionally comprise 1 or
2 heteroatoms selected from O, S, NH or N-alkyl; R.sub.3 and
R.sub.4 are independently or together hydrogen, alkyl, substituted
alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, halogen, cyano, nitro, hydroxyl, acyloxy,
amino, mono-substituted amino, di-substituted amino,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide; A is --CR.sub.6R.sub.7-- where
R6 and R7 are independently or together hydrogen, alkyl,
substituted alkyl, alkoxy, substituted alkoxy or haloalkoxy; or
R.sub.6 and R.sub.7 together form a cycloalkyl residue that may
optionally comprise 1 or 2 heteroatoms selected from O, S, NH and
N-alkyl; Ar is Formula (II), (III), (IV) or (V): 34wherein R.sub.8,
R.sub.9 and R.sub.10 are independently or together hydrogen, alkyl,
substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl,
acyloxy, amino, mono-substituted amino, di-substituted amino,
alkylamide, alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, alkoxy, substituted alkoxy,
haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide; R.sub.5 is hydrogen, halogen,
hydroxy, alkyl or substituted alkyl; 35represents a bond present or
absent; and W, X, Y and Z are independently or together --C(O)--,
--C(S)--, --S--, --O-- or --NH-- residues that together form a
2,4-thiazolidinedione, 2-thioxo-4-thiazolidinedione,
isoxazolidinedione, 2,4-imidazolidinedione or
2-thioxo-4-imidazolidinedione residue; or a pharmaceutically
acceptable salt thereof.
34. The molecules of claims 1-3 wherein the molecule is a processed
molecule.
Description
I. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of and claims
the priority of U.S. patent application Ser. No. 10/098,184, filed
Mar. 08, 2002, which status is allowed, and also claims priority to
U.S. provisional application No. 60/274342, for RXR Activating
Molecules, filed on Mar. 8, 2001, both of which applications herein
incorporated by reference in their entirety. U.S. patent
application Ser. No. 09/652,810, now issued as U.S. Pat. No.
6,515,033, and U.S. patent application Ser. No. 09/655,460, both
filed Aug. 31, 2000, are also hereby incorporated by this reference
in their entireties.
II. BACKGROUND OF THE INVENTION
[0002] Type 2 diabetes, also referred to as non-insulin dependent
diabetes mellitus (NIDDM), is the major cause of diabetes in
developed countries. In the United States alone, approximately 17
million people, and more than 120 million worldwide, are affected.
Because this disorder is a late onset disease and occurs often in
overweight persons, it can be expected that the number of patients
suffering from this disease will increase further. Patients
suffering from type 2 diabetes usually still produce insulin, but
become increasingly resistant to their own insulin and insulin
therapy. A promising new class of drugs has been recently
introduced that resensitizes patients to their own insulin (insulin
sensitizers), thereby reducing blood glucose and triglyceride
levels, and thus abolishing, or at least reducing, the requirement
for exogenous insulin. Rosiglitazone (Avandia.TM.) and Pioglitazone
(Actos.TM.) bind to the nuclear receptor PPAR.quadrature. and are
the first representatives of this class of receptor ligands
approved for the treatment of type 2 diabetes in the United States
and several other countries. These compounds, however, have side
effects including rare but severe liver toxicities (i.e.,
troglitazone) and they can increase body weight and cause edema in
humans and may also lead to a worsening situation for patients with
certain heart conditions. Such side effects are of major concern
for patients who might require treatment for a decade or longer.
Therefore, new and better drugs for the treatment of type 2
diabetes and related disorders are needed. Ligands for the retinoid
X receptor (RXR) have been suggested and examined as alternative
choices for the development of a new class of insulin sensitizer
drugs, that could avoid side effects seen with the PPAR.quadrature.
insulin sensitizers. RXR ligands can also influence cholesterol
metabolism and transport and could therefore address additional
aspects such as hypercholesteremia and arteriosclerosis, often seen
with type 2 diabetic patients. In fact, a majority of type 2
diabetic patients appear to die of an arteriogenic event. However,
typical RXR ligands lead to increases in triglyceride levels in
animals and humans, which makes them undesirable for the treatment
of most type 2 diabetic patients that very often have already
elevated blood triglyceride levels. A subgroup of heterocyclic
derivatives that does not lead to such undesirable side effects and
which interacts with RXR in a highly specific manner is disclosed.
Such RXR ligands with these unexpected properties are useful for
the treatment of type 2 diabetes, hypercholesteremia,
arteriosclerosis and disorders related to these diseases. Disclosed
are molecules that interact with a side pocket of their specific
receptor ligand-binding domain, where the receptor is a retinoid X
receptor. Such molecules are useful for the treatment of type 2
diabetes, hypercholesteremia and related diseases, including
arteriosclerosis.
III. SUMMARY OF THE INVENTION
[0003] In accordance with the purpose(s) of this invention, as
embodied and broadly described herein, this invention, in one
aspect, relates to a molecule that activates an RXR receptor,
wherein the molecule comprises an RXR binding portion which binds
the RXR receptor and comprises a side pocket contacting residue
which contacts a side-pocket 1 of an RXR receptor.
[0004] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
exemplary embodiments of the invention and together with the
description, serve to explain the principles of the invention.
[0006] FIG. 1 shows the Connolly surface of the entire active site
of RXR, with one amino acid Arg A316 shown for reference. The
Connolly surface is generated as described in IUPAC-IUB Commission
on Biochemical Nomenclature (1970). Abbreviations and symbols for
the description of the conformation of polypeptide chains. J. Mol.
Biol., 52, 1-17.
[0007] FIG. 2 shows that portion of the RXR ligand pocket that
comprises side pocket 1, with reference to C19 of 9-cis-RA, a
natural ligand of RXR (numbering as in 1FBY). While 9-cis-RA
occupies a major portion of the RXR ligand pocket, side pocket 1
comprises unoccupied space in the crystal structure that is
adjacent to C19.
[0008] FIG. 3 shows the amino acids lining side pocket 1.
[0009] FIG. 4 shows the same view of FIG. 3 with the native ligand
9-cis-RA in the RXR ligand pocket site.
[0010] FIG. 5 shows the same view of FIG. 3 with compound 1 in the
RXR ligand pocket. Note that compound 1 has a side pocket 1
contacting residue.
[0011] FIG. 6 shows the labeling of specific atoms from the amino
acids shown in FIG. 3 that are close to the Connolly surface of the
side pocket 1. In this figure, wherever connected atoms are
selected, they are shown as lines connecting the atoms. Wherever
isolated atoms are selected, stars represent these atoms. Where
Association of the name and the atom requires, an arrow is provided
pointing from the amino acid name to the individual atoms (as in,
for example, Leu A436). The list of specific atoms are those of
C1L.
[0012] FIG. 7 shows the position of compound 1 relative to the
Connolly surface of the side pocket 1. Note that compound 1 has a
side pocket 1 contacting residue.
[0013] FIG. 8 shows the position of compound 2 relative to the
Connolly surface of the side pocket 1.
[0014] FIG. 9 shows the position of compound 4 relative to the
Connolly surface of the side pocket 1.
[0015] FIG. 10 shows adult diabetic KKA.sup.y mice were housed in a
fixed 12-12-hr artificial light-dark cycle, and maintained on a
standard rodent diet. Prior to commencing treatment, all of the
animals are bled from the tail vein and serum levels of glucose and
triglyceride are measured in duplicate. The animals are then sorted
into different treatment groups with equal average triglyceride
levels. The animals are treated with a single daily oral dose of
the test compound suspended in sesame oil (dose volume of 3ml/kg).
Type 2 diabetic KKA.sup.y mice treated with Compound 1 at 5 mg/kg
showed a significant decrease in serum glucose levels compared to
vehicle treated control mice (p.ltoreq.0.01: Fisher's LSD test). By
contrast, diabetic mice treated with Compound 2 at 5 and 15 mg/kg
showed no difference in serum glucose levels compared to vehicle
treated controls.
[0016] FIG. 11 shows type 2 diabetic KKA.sup.y mice were treated as
described in FIG. 10 legend. Animals treated with Compound 1 at 5
mg/kg showed a significant decrease in serum triglyceride levels
compared to vehicle treated control mice (p.ltoreq.0.05; Fisher's
LSD test). By contrast, diabetic mice treated with Compound 2 at 5
and 15 mg/kg showed an increase in serum triglyceride levels
compared to vehicle treated controls (statistically significant at
5 mg/kg, p.ltoreq.0.05; Fisher's LSD test).
[0017] FIG. 12 shows adult male BALB/c mice were housed in a fixed
12-12-hr artificial light-dark cycle, and maintained on a standard
rodent diet. The animals were treated with a single daily oral dose
of the test compound suspended in sesame oil (dose volume of 5
ml/kg). Treatment was for 14 days. Animas treated with 10 and 30
mg/kg of Compound 1, which are doses that are 30 and 100 times the
efficacious does in diabetic db/db mice, respectively, caused no
increase in serum triglyceride levels compared to vehicle treated
control animals.
[0018] FIG. 13 shows adult BALB/c mice were treated as described in
the FIG. 12 legend. Animals treated with 50 and 100 mg/kg of
Compound 2 showed an increase in serum triglyceride levels compared
to vehicle treated control animals (statistically significant at
100 mg/kg, p.ltoreq.0.01; Fisher's LSD test).
[0019] FIG. 14 shows chemical structures of various compounds.
[0020] FIG. 15 shows a 3T3-L1 differentiation Assay. Mouse 3T3-L1
cells were grown in 96-well tissue culture plates containing
growing medium (DME containing 10% calf serum (CS) plus
glutamine-pen-strep) at a density of 3,000 cells/well. Two days
after reaching confluence, cells are treated with the different
compounds in DM (Day 0). Compound 1 is included in all the
experiments, and its ability to differentiate 3T3-L1 cells at 0.1
.quadrature.M is taken as reference for 100% differentiation. After
7 days the lipid content of the cells was determined.
[0021] FIG. 16 shows glucose lowering activity for compound 7 as
disclosed herein.
[0022] FIG. 17 shows triglyceride lowering activity for compound 7
as disclosed herein.
V. DETAILED DESCRIPTION
[0023] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the Examples included therein and
to the Figures and their previous and following description.
[0024] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that this invention is not limited to specific synthetic
methods or to specific reagents, as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting.
[0025] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an aromatic compound" includes mixtures of aromatic
compounds, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like.
[0026] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0027] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0028] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not. For example, the phrase
"optionally substituted lower alkyl" means that the lower alkyl
group may or may not be substituted and that the description
includes both unsubstituted lower alkyl and lower alkyl where there
is substitution.
[0029] In the specification and Formulae described herein the
following terms are hereby defined.
[0030] The term "alkyl" denotes a radical containing 1 to 12
carbons, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,
sec-butyl, t-butyl, amyl, t-amyl, n-pentyl and the like.
[0031] The term "alkenyl" denotes a radical containing 1 to 12
carbons such as vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl,
3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl,
2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl and the
like. The term "alkenyl" includes dienes and trienes of straight
and branch chains.
[0032] The term "alkynyl" denotes a radical containing 1 to 12
carbons, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,
2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,
4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl
and the like. The term "alkynyl" includes di- and tri-ynes.
[0033] The term "substituted alkyl" denotes a radical containing 1
to 12 carbons of the above definitions that are substituted with
one or more groups, but preferably one, two or three groups,
selected from hydroxyl, cycloalkyl, amino, mono-substituted amino,
di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl,
thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy or haloalkoxy.
When more than one group is present then they may be the same or
different.
[0034] The term "substituted alkenyl" denotes a radical containing
1 to 12 carbons of the above definitions that are substituted with
one or more groups, but preferably one, two or three groups,
selected from halogen, hydroxyl, cycloalkyl, amino,
mono-substituted amino, di-substituted amino, acyloxy, nitro,
cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,
thiohaloalkyl, alkoxy, substituted alkoxy or haloalkoxy. When more
than one group is present then they may be the same or
different.
[0035] The term "substituted alkynyl" denotes a radical containing
1 to 8 carbons of the above definitions that are substituted with
one or more groups, but preferably one or two groups, selected from
halogen, hydroxyl, cycloalkyl, amino, mono-substituted amino,
di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl,
thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy or
haloalkoxy.
[0036] The term "cycloalkyl" denotes a radical containing 3 to 8
carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopenyl,
cyclohexyl, cycloheptyl and the like. The term "substituted
cycloalkyl" denotes a cycloalkyl as defined above that is further
substituted with one or more groups selected from halogen, alkyl,
hydroxyl, alkoxy, substituted alkoxy, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, amino, mono-substituted amino or
di-substituted amino. When the cycloalkyl is substituted with more
than one group, they may be the same or different.
[0037] The term "cycloalkenyl" denotes a radical containing 3 to 8
carbons, such as cyclopropenyl, 1-cyclobutenyl, 2-cyclobutenyl,
1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexyl,
2-cyclohexyl, 3-cyclohexyl and the like. The term "substituted
cycloalkenyl" denotes a cycloalkyl as defined above further
substituted with one or more groups selected from halogen, alkyl,
hydroxyl, alkoxy, substituted alkoxy, haloalkoxy, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide, substituted dialkylcarboxamide, amino,
mono-substituted amino or di-substituted amino. When the
cycloalkenyl is substituted with more than one group, they may be
the same or different.
[0038] The term "alkoxy" as used herein denotes a radical alkyl,
defined above, attached directly to an oxygen such as methoxy,
ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, iso-butoxy and
the like.
[0039] The term "substituted alkoxy" denotes a radical alkoxy of
the above definition that is substituted with one or more groups,
but preferably one or two groups, selected from hydroxyl,
cycloalkyl, amino, mono-substituted amino, di-substituted amino,
acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,
thiohaloalkyl, alkoxy, substituted alkoxy or haloalkoxy. When more
than one group is present then they may be the same or
different.
[0040] The term "mono-substituted amino" denotes an amino
substituted with one group selected from alkyl, substituted alkyl
or arylalkyl wherein the terms have the same definitions found
throughout.
[0041] The term "di-substituted amino" denotes an amino substituted
with two radicals that may be same or different selected from aryl,
substituted aryl, alkyl, substituted alkyl or arylalkyl wherein the
terms have the same definitions found throughout. Some examples
include dimethylamino, methylethylamino, diethylamino and the
like.
[0042] The term "haloalkyl" denotes a radical alkyl, defined above,
substituted with one or more halogens, preferably fluorine, such as
a trifluoromethyl, pentafluoroethyl and the like.
[0043] The term "haloalkoxy" denotes a haloalkyl, as defined above,
that is directly attached to an oxygen to form trifluoromethoxy,
pentafluoroethoxy and the like.
[0044] The term "acyl" denotes a radical containing 1 to 8 carbons
such as formyl, acetyl, propionyl, butanoyl, iso-butanoyl,
pentanoyl, hexanoyl, heptanoyl, benzoyl and the like.
[0045] The term "acyloxy" denotes a radical containing 1 to 8
carbons of an acyl group defined above directly attached to an
oxygen such as acetyloxy, propionyloxy, butanoyloxy,
iso-butanoyloxy, benzoyloxy and the like.
[0046] The term "aryl" denotes an aromatic ring radical containing
6 to 10 carbons that includes phenyl and naphthyl. The term
"substituted aryl" denotes an aromatic radical as defined above
that is substituted with one or more selected from hydroxyl,
cycloalkyl, aryl, substituted aryl, heteroaryl, heterocyclic ring,
substituted heterocyclic ring, amino, mono-substituted amino,
di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl,
alkylthio, alkoxy, substituted alkoxy or haloalkoxy, wherein the
terms are defined herein.
[0047] The term "halo" or "halogen" refers to a fluoro, chloro,
bromo or iodo group.
[0048] The term "thioalkyl" denotes a sulfide radical containing 1
to 8 carbons, linear or branched. Examples include methylsulfide,
ethyl sulfide, isopropylsulfide and the like.
[0049] The term "thiohaloalkyl" denotes a thioalkyl radical
substituted with one or more halogens. Examples include
trifluoromethylthio, 1,1-difluoroethylthio,
2,2,2-trifluoroethylthio and the like.
[0050] The term "carboalkoxy" refers to an alkyl ester of a
carboxylic acid, wherein alkyl has the same definition as found
above. Examples include carbomethoxy, carboethoxy, carboisopropoxy
and the like.
[0051] The term "alkylcarboxamide" denotes a single alkyl group
attached to the amine of an amide, wherein alkyl has the same
definition as found above. Examples include N-methylcarboxamide,
N-ethylcarboxamide, N-(iso-propyl)carboxamide and the like. The
term "substituted alkylcarboxamide" denotes a single "substituted
alkyl" group, as defined above, attached to the amine of an
amide.
[0052] The term "dialkylcarboxamide" denotes two alkyl or arylalkyl
groups that are the same or different attached to the amine of an
amide, wherein alkyl has the same definition as found above.
Examples of a dialkylcarboxamide include N,N-dimethylcarboxamide,
N-methyl-N-ethylcarboxamide and the like. The term "substituted
dialkylcarboxamide" denotes two alkyl groups attached to the amine
of an amide, where one or both groups is a "substituted alkyl", as
defined above. It is understood that these groups may be the same
or different. Examples include N,N-dibenzylcarboxamide,
N-benzyl-N-methylcarboxamide and the like.
[0053] The term "alkylamide" denotes an acyl radical attached to an
amine or monoalkylamine, wherein the term acyl has the same
definition as found above. Examples of "alkylamide" include
acetamido, propionamido and the like.
[0054] The term "arylalkyl" defines an alkylene, such as
--CH.sub.2-- for example, which is substituted with an aryl group
that may be substituted or unsubstituted as defined above. Examples
of an "arylalkyl" include benzyl, phenethylene and the like.
[0055] A residue of a chemical species, as used in the
specification and concluding claims, refers to the moiety that is
the resulting product of the chemical species in a particular
reaction scheme or subsequent formulation or chemical product,
regardless of whether the moiety is actually obtained from the
chemical species. Thus, an ethylene glycol residue in a polyester
refers to one or more --OCH2CH2O-- repeat units in the polyester,
regardless of whether ethylene glycol is used to prepare the
polyester. Similarly, a 2,4-thiazolidinedione residue in a chemical
compound refers to one or more -2,4-thiazolidinedione moieties of
the compound, regardless of whether the residue was obtained by
reacting 2,4-thiazolidinedione to obtain the compound.
[0056] The term the "RXR binding portion" as used herein refers a
part of the molecules of the invention that is bound to but
excludes the side pocket contacting residue. The RXR binding
portion is an organic residue of a suitable polarity, size, and
shape, so as to fit within and become bound to and/or complexed to
the ligand binding domain of the RXR receptor that binds 9-cis
retinoic acid as described by Egea et. al. The RXR binding portion
is an organic residue that can serve various functions, which
includes contributing at least a significant proportion of the
interactions with the RXR receptor which bind the molecules, and
additionally provides the function of supporting and geometrically
directing the side pocket contacting residue so as to come into
contact with side pocket 1.
[0057] The term "cyclic organic residue" as used herein refers to a
sub-portion of the RXR binding portion, comprising a cyclic organic
residue having one or more cyclic rings, and from 2 to 18 carbon
atoms, or preferably from about 4 to 12 carbon atoms. The cyclic
organic residue may contain suitable heteroatoms, which include but
are not limited to nitrogen, oxygen, and sulfur, so as to form
heterocyclic organic residues, which are a well known class of
compounds to those of skill in the art. The cyclic organic residue
may be saturated, unsaturated, or aromatic, and is preferably
aromatic. The cyclic organic residue is bonded to the side pocket
contacting residue, and can serve various functions, which includes
at least the function of supporting and geometrically directing the
side pocket contacting residue so as to come into contact with side
pocket 1. The cyclic organic residue may be unsubstituted, but
preferably is substituted with one or more organic or inorganic
substituent groups or residues, including but not limited to a
halide, a hydroxide, a thiol, an amino, an alkyl, an aryl, a
heteroaryl, an alkoxy, or other well known organic or inorganic
groups or residues as defined in the definitions section of this
application. Preferably, the cyclic organic residue is bonded to
one or both of the polar organic binding portions, and/or the
organic portion, as described herein below.
[0058] The term the "polar binding portion," as used herein refers
to a sub-portion of the RXR binding portion that is optionally
bound to the cyclic organic residue. The polar binding portion can
serve various functions, which include contributing at least a
significant proportion of the binding interactions with the RXR
receptor, especially by interacting with the relatively polar
portions of the RXR receptor (as described by Egea et. al.)that
normally binds the carboxyl group of 9-cis retinoic acid.
[0059] The term the "organic binding portion," as used herein
refers to a sub-portion of the RXR binding portion that is
optionally bound to the cyclic organic residue. The polar organic
binding portion can serve various functions, which include
contributing at least a significant proportion of the binding
interactions with the RXR receptor, especially by interacting with
the relatively non-polar portions of the RXR receptor (as described
by Egea et. al.)that normally binds the cyclohexenyl residue of
9-cis retinoic acid. Reference will now be made in detail to the
present preferred embodiment(s) of the invention, an example(s) of
which is [are] illustrated in the accompanying drawings. Wherever
possible, the same reference numbers are used throughout the
drawings to refer to the same or like parts.
[0060] A molecule can be considered to activate an RXR if the
molecule shows 60% of the activity of the agonist 9-cis retenoic
acid at 10.sup.-6 M which activity can be considered to activate
transcription in the RXR transcription assay discussed in Example
4.
[0061] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains. The references disclosed are also
individually and specifically incorporated by reference herein for
the material contained in them that is discussed in the sentence in
which the reference is relied upon.
[0062] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
A. Compositions
[0063] This invention provides a molecule that activates an RXR
receptor, wherein the molecule comprises an RXR binding portion
which binds the RXR receptor and comprises a side pocket contacting
residue which contacts side-pocket 1 of an RXR receptor. The side
pocket can comprise at least 4 or more contacts between the atoms
of the side pocket.
[0064] 1. RXR Receptors
[0065] a) Retinoid X Receptors (RXR)
[0066] (1) Function
[0067] RXRs belong to the large family of nuclear receptor
proteins. This family of receptors includes the estrogen, androgen,
thyroid hormone (TR), vitamin D3 (VDR) and other receptors that can
be activated by small molecule hormones, vitamins and other signal
molecules as has been reviewed, for instance by Mangelsdorf D J,
Umesono K, Evans R: The Retinoid Receptors. In The Retinoids
(Second Edition), Sporn M B, Roberts A B, Goodman D S (Eds.),
Academic Press, Inc., Orlando, Fla. (1994):319-349. RXRs are part
of the retinoid receptor subgroup which consists of two classes of
receptors: the retinoic acid receptors (RARs) and the retinoid X
receptors (RXRs). Each class contains three subtypes, .alpha.,
.beta. and .gamma. (encoded by separate genes), of which various
isoforms are expressed in a tissue and development specific
pattern. The receptors bind to small DNA segments called response
elements from which they direct transcription in the presence of a
ligand. RXRs also forms heterodimers with other receptors, such as
TR, VDR and the PPARs. Through their interactions with PPAR.gamma.,
RXRs can influence metabolic pathways that can be affected in type
2 diabetes patients (see Krey, et al., 1995, Molecular Endocrinol.
9:219-231 and Schulman, et al., 1998, Mol. Cell Biol. 18:3483-3494
and Mukherjee, et al. 1997, Nature. 386:407-410). However,
experience has shown that not all ligands for RXRs activate the
same biological responses.
[0068] (2) Different RXR Receptor Subtypes
[0069] In mammals, including humans, RXR receptors are usually
encoded by 3 genes: RXR.alpha., .beta. and .gamma.. While certain
portions, especially those of the amino terminal end of the RXRs
can differ, RXR.alpha., .beta. and .gamma. are essentially
identical in the amino acids that make up their ligand binding
domain. This also includes the amino acids that encode side pocket
1 in human RXR.alpha., .beta. and .gamma.. The sequences of human
RXR.alpha., .beta. and .gamma. are shown.
[0070] b) Side Pocket
[0071] As described in more detail in examples 1 and 2, the side
pocket 1 of human RXR.alpha. comprises amino acids Leu 436, Leu
433, Cys 432, Gly 429, Ile 310, Asn 306, and Trp 305. As discussed
by others, (see Egea, et al. 2000, EMBO J. 19: 2592-2601) the
various binding pockets are conserved between RXR subtypes. Thus,
this cluster of amino acids can fulfill essentially the same roles
in human RXR.beta. and .gamma.. The exact amino acid position
corresponding to the RXR.A-inverted. positions can readily be
determined by comparing SEQ ID Nos.
[0072] One way of defining the side-pocket 1 is at the level of the
amino acids that make up the side-pocket 1. In human RXR alpha,
side-pocket 1 can preferably be formed by the positioning of amino
acids Leu 436, Leu 433, Cys 432, Ile 310, Asn 306, Gly 429 and Trp
305 all of SEQ ID NO: 1. These seven amino acid residues, 2 Leu,
Cys, Ile, Asn, Gly and Trp, are highly conserved in other isoforms
of RXR, such as, for example, RXR.quadrature. and RXR.quadrature.,
and as such, are within the scope of the invention. The sequences
of RXR.alpha., RXR.beta. and RXR .gamma. are shown in SEQ ID Nos:
1, 2, and 3.
[0073] Side pocket 1 can be defined using the protein coordinates
of human RXR.alpha. protein that has been determined and which has
been deposited under 1FBY in the Protein Databank as further
described in Example 1. Specifically, that portion of the protein
coordinates of 1FBY that form side pocket 1, those atoms in amino
acids Leu 436, Cys 432, Leu 433, Ile 310, Asn 306, Gly 429 and Trp
305. Herein amino acids are specified using the three letter amino
acid code to specify the amino acid type, while the sequence number
(for example 305, 306 or 310) is as reported in the crystal
structure 1FBY from the Protein Databank. The prefix A specifies
that the A polypeptide chain from 1FBY from the Protein Databank.
The prefix A specifies that the A polypeptide chain from 1FBY is
used. From these coordinates a cavity list can be made of the atoms
of the amino acids making up the side pocket as disclosed herein.
For example, specific atoms lining the side pocket 1 can comprise a
"Cavity 1 List" (C1L). C1L can consist of TRP A305:CE3,CZ3,CH2; ASN
A306:CA,C,O,CB,CG,ND2; ILE A310:CG1,CD1; GLYA429:0,C,CA; and LEU
A433:N,CA,C,CD2, where individual atom names are defined in 1FBY.
The side pocket formed by these atoms is illustrated in the
Figures, which shows the amino acids comprising the pocket as line
drawings, as well as a Connolly surface in the side pocket 1 region
of the active site.
1 TABLE 1 Amino Acid Abbre- Amino Acid Abbreviations viations
alanine Ala A allosoleucine AIle arginine Arg R asparagine Asn N
aspartic acid Asp D cysteine Cys C glutamic acid Glu E glutamine
Gln K glycine Gly G histidine His H isolelucine Ile I leucine Leu L
lysine Lys K phenylalanine Phe F proline Pro P pyroglutamic acid
pGlu serine Ser S threonine Thr T tyrosine Tyr Y tryptophan Trp W
valine Val V
[0074] 2. Processed Molecules that Interact with RXR Receptors
[0075] The compositions that interact with the RXR receptors are
typically processed molecules. Processed molecules are not
naturally occurring molecules. For example, a molecule produced by
synthetic methods would be a processed molecule, but if the same
molecule were also synthesized naturally in a cell, the molecule
synthesized naturally in the cell, e.g. 9-cis RA, would not be a
processed molecule. Naturally occurring refers to pathways or
molecules that have not been purposefully manipulated. The
processed molecule can be further defined herein as comprising at
least two residues, these residues may be the same or different.
The first residues can be described as a "side-pocket 1 contacting"
region. This portion of the processed molecule contributes to the
contacts made with the side pocket 1, of the RXR.ident. receptor
defined by the amino acid residues Leu 436, Leu 433, Cys 432, Ile
310, Asn 306, Gly 429 and Trp 305 as present in SEQ ID NO:1 or a
subset of these amino acids: Leu 433, Ile 310, Asn 306, Gly 429 and
Trp 305. The second residue of the processed molecule, referred to
as the "RXR binding portion," is the remaining part of the
molecule, excluding the side-pocket 1 contacting residue, that
provides further contributing interactions with, for example, the
RXR.quadrature. receptor. The RXR binding portion can optionally be
divided into additional regions including a "cyclic organic
residue" a polar binding portion and an organic binding portion."
The cyclic organic residue can be directly attached to the
side-pocket 1 contacting residue and one or both of the polar
binding portion and/or the organic binding portion.
[0076] a) Interactions
[0077] The processed molecule contacts (interacts with) the RXR
receptor. These contacts can occur at a number of places between
the processed molecule and the RXR receptor, but must at least
occur between the processed molecule and the side-pocket 1 of the
RXR receptor. The sum of these contacts preferably are sufficient
to reduce blood glucose levels and lower triglyreride levels in
KKA.sub.y mice. One way of characterizing the interaction between
the processed molecule and the RXR receptor is to assess how
tightly the processed molecule and the RXR receptor bind. For
example, the processed molecule and the RXR receptor can interact
or bind each other with a k.sub.d of less than or equal to 1 .mu.M.
The binding can also preferably be defined by dissociation
constants of less than or equal to 100 nM. The binding can also
preferably be defined by dissociation constants of less than or
equal to 10 nM. The binding can also be defined by dissociation
constants of less than or equal to 1 nM. The binding can also
preferably be defined by dissociation constants of less than or
equal to 0.1 nM. It is understood that the dissociation constants
are not completely controlled by the specific contacts that are
made between the processed molecule and the side-pocket 1 of the
RXR receptor. The contacts with the side-pocket 1 of the RXR
receptor can contribute to the total affinity between the processed
molecule and the RXR receptor but do not necessarily increase the
affinity between the processed molecule and the RXR receptor.
[0078] b) Side Pocket Contacting Residue
[0079] The molecule has a side-pocket 1 contacting residue that is
capable of contacting with a precise region of the RXR receptors
defined as the side-pocket 1. This residue, present as one part of
the processed molecule, can optionally be an alkyl, a substituted
alkyl, a haloalkyl, an alkenyl, a substituted alkenyl, an alkynyl,
a substituted alkynyl, a halogen, cyano, nitro, hydroxyl, acyloxy,
amino, mono-substituted amino, di-substituted amino, alkylamide,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, alkoxy, substituted alkoxy,
haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, oxime, O-substitued
oxime, dialkylcarboxamide or substituted dialkylcarboxamide
residue.
[0080] c) Structure
[0081] One way of characterizing the side-pocket 1 contacting
residue is to quantify at the number of atoms that make up the
side-pocket 1 contacting residue. The atoms must be connected. The
side-pocket 1 contacting residue must have at least one connected
atom. Optionally there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, and 20 atoms or at least any one of these
numbers of atoms. Optionally, embodiments may either have between 3
and 15 atoms, or 4 and 14 atoms, or 5 and 13 atoms, 6 and 12 atoms,
7 and 12 atoms, 8 and 12 atoms, 9 and 12 atoms, 10 and 12 atoms, 11
and 12 atoms, 5 and 6 atoms, 5 and 7 atoms, 5 and 8 atoms, 5 and 9
atoms, 5 and 10 atoms, 5 and 11 atoms, and 5 and 12 atoms.
[0082] Other embodiments can optionally comprise of greater than 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and
20 connected atoms.
[0083] The side-pocket 1 contacting residue can consist of a number
of different types of atoms. The connected atoms of the side-pocket
1 contacting residues can optionally include carbon, hydrogen,
nitrogen, phosphorus, oxygen, sulfur, fluorine, chlorine, bromine,
iodine, or mixtures thereof.
[0084] The side pocket contacting residues can optionally be,
comprise, or contain an alkyl, a substituted alkyl, a haloalkyl, an
alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl,
a halogen, cyano, nitro, hydroxyl, acyloxy, amino, mono-substituted
amino, di-substituted amino, alkylamide, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl,
thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, oxime, O-substitued oxime, dialkylcarboxamide or
substituted dialkylcarboxamide residue.
[0085] The side-pocket 1 contacting residue can optionally be a
haloalkoxy residue comprising 1 to 5 carbon atoms.
[0086] The RXR binding portion comprises a cyclic residue C
comprising a substituted or unsubstituted C6-C 18 aromatic ring
residue wherein all ring atoms are carbon, a substituted or
unsubstituted C2-C18 heteroaromatic ring residue having from one to
three ring atoms selected from O, S, N, NH, or a substituted or
unsubstituted C2-C18 heteroaromatic ring residue having from one to
three ring atoms selected from O, S, N, NH and N--R atoms or
residues, wherein R comprises an alkyl, a substituted alkyl, an
aryl, a substituted aryl, an acyl, a heteroaryl, or a substituted
heteroaryl group.
[0087] The combination of side pocket contacting residue and the
cyclic residue C comprises a residue of Formula (II), (III), (IV)
or (V): 1
[0088] wherein R8, R9 and R10 are independently or together
hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro,
hydroxyl, acyloxy, amino, mono-substituted amino, di-substituted
amino, alkylamide, alkylsulfonamide, arylsulfonamide, alkylurea,
arylurea, alkylcarbamate, arylcarbamate, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, oxime, O-substitued
oxime, dialkylcarboxamide or substituted dialkylcarboxamide.
[0089] The cyclic residue C is connected to a polar binding residue
comprising at least one functional group having at least one polar
carbon-heteroatom or heteroatom-hydrogen bond.
[0090] In one embodiment the polar binding residue comprises a
residue of the formula: 2
[0091] wherein
[0092] a. m is an integer 0 or 1;
[0093] b. R5 is hydrogen, halogen, hydroxy, alkyl or substituted
alkyl;
[0094] c. - - - represents a bond present or absent;
[0095] d. V.dbd.C or N;
[0096] e. W, X, Y and Z are independently or together --C(O)--,
--C(S)--, --S--, --O-- or --NH-- residues. In one aspect of the
invention V, W, X, Y, and Z together form a 2,4-thiazolidinedione,
2-thioxo-4-thiazolidinedi- one, isoxazolidinedione,
2,4-imidazolidinedione 2-thioxo-4-imidazolidinedi- one or
[1,2,4]-oxadiazolidine-3,5-dione residue.
[0097] The cyclic residue C can be connected to residue D
comprising a C.sub.4 to C.sub.25 substituted or unsubstituted
hydrocarbon residue.
[0098] The cyclic residue C comprises the following formula: 3
[0099] wherein:
[0100] n is independently 0 or 1;
[0101] R.sub.1 and R.sub.2 are 1) independently or together
hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
hydroxyl, acyl, amino, mono-substituted amino, di-substituted
amino, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide or haloalkoxy; or 2) R.sub.1 and R.sub.2
together with the aromatic ring bonded thereto form a cycloalkyl,
substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl
residue that may optionally comprise 1 or 2 heteroatoms selected
from O, S, NH or N-alkyl.
[0102] R.sub.3 and R.sub.4 are independently or together hydrogen,
alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl,
acyloxy, amino, mono-substituted amino, di-substituted amino,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide; and
[0103] A is --CR.sub.6R.sub.7-- where R.sub.6 and R.sub.7 are
independently or together hydrogen, alkyl, substituted alkyl,
alkoxy, substituted alkoxy or haloalkoxy; or R.sub.6 and R.sub.7
together form a cycloalkyl residue that may optionally comprise 1
or 2 heteroatoms selected from O, S, NH and N-alkyl.
[0104] d) Contact
[0105] A contact between the side pocket contacting residue and the
side pocket can be defined by the distance or closeness of an atom
of the side pocket contacting residue and any atom of one of the
amino acid residues that make up the side pocket, as described in
herein.
[0106] The number of contacts between the atoms of the side pocket
containing residue and atoms of the side pocket 1 can vary for each
RXR ligand. A contact is defined by the distance where the distance
between the contacting atoms is less than 4 Angstrom (.ANG.), e.g.,
1, 1.5, 2, 2.5, 3, 3.5. Thus, when an atom of the side pocket
contacting residue is within a distance of 1 to 4 .ANG. of an atom
of side pocket 1, a contact is made and especially when the
distance is 3.5 .ANG..
[0107] The number of contacts between the atoms of side-pocket 1
contacting residues of the processed molecule and the atoms of
side-pocket 1 of the RXR receptor is one way to quantitate the
interaction. To constitute an effective interaction between the
side-pocket 1 contacting residue of a processed molecule with the
RXR receptor there must be at least four contacts. Optionally,
there are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
and 20 contacts or at least any one of these numbers of contacts.
Preferred embodiments either have 7, 14, 15, 16, or 17 contacts or
at least any number of these numbers of contacts.
[0108] Contacts can occur with any subset of the amino acids
comprising side pocket 1, such as any subset of one or two or three
or four or five or six of the amino acids in any combination.
[0109] For example, contacts preferably could be made with Leu 433,
Ile 310, Asn 306, Gly 429 and Trp 305.
[0110] Contacts could also be made with, for example, the subsets
Leu 436, Leu 433, Cys 432, Ile 310, Asn 306 or Gly 429 and Trp 305
Leu 436 or Leu 433, Cys 432, Ile 310, Asn 306, Gly 429 or Trp 305,
Leu 433, Ile 310.
[0111] It is understood that any combination of the amino acids
defining side pocket 1 can be used for contacts with the side
pocket contacting residue.
[0112] In one preferred embodiment, disclosed are molecules that
activate an RXR receptor to at least 60% of the activation of 9-cis
retinoic acid, wherein the molecule comprises an RXR binding
portion which binds the RXR receptor and comprises a side pocket
contacting residue which has at least four contacts with the region
consisting of Leu 433, Gly 429, Ile 310, Asn 306, and Trp 305 of
side-pocket 1 of an RXR receptor, wherein the contacts are less
than or equal to 3.5 Angstroms. Also provided are 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 contacts in this
five amino acid region.
[0113] In another preferred embodiment, disclosed are molecules
comprising an RXR receptor complexed with a molecule that activates
an RXR receptor, wherein the molecule comprises an RXR binding
portion which binds the RXR receptor and comprises a side pocket
contacting residue which has at least four contacts with the region
consisting of Leu 433, Gly 429, Ile 310, Asn 306, and Trp 305 of
side-pocket 1 of an RXR receptor, wherein the contacts are less
than or equal to 3.5 Angstroms. Also provided are 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 contacts in this
five amino acid region.
[0114] In another preferred embodiment, disclosed are molecules
that activate an RXR at least one fifth of the activity of compound
1 as measured in the adipocyte differentiation assay comprising an
RXR binding portion which binds the RXR receptor and comprises a
side pocket contacting residue which has at least four contacts
with the region consisting of Leu 433, Gly 429, Ile 310, Asn 306,
and Trp 305 of side-pocket 1 of an RXR receptor, wherein the
contacts are less than or equal to 3.5 Angstroms. Also provided are
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20
contacts in this five amino acid region.
[0115] As shown in the Examples, these seven amino acids that can
form side-pocket 1 of a human RXR are typically arranged and
positioned such that a small hydrophobic pocket or indentation is
presented. For RXR alpha amino acids such as Leu 436, Ile 310, Cys
432, Leu 433, Gly 429 and Trp 305 add to this hydrophobicity. Amino
acids such as Asn 306 can also add to the total hydrophobicity of
the side-pocket 1.
[0116] e) Activation of the RXR Receptor by the Molecules that
Interact with the Receptor
[0117] In some embodiments the compositions activate the RXR
receptor with an EC 50 concentration less than or equal to 1 uM.
Still other embodiments activate the RXR receptor with an EC 50
concentration less than or equal to 100 nM.
[0118] f) Molecules that are Produced by Selection Methods
[0119] Combinatorial chemistry includes but is not limited to all
methods for isolating molecules that have specific activity, such
as binding another molecule, and macromolecules that are capable of
binding either a small molecule or another macromolecule.
[0120] Using methodology well known to those of skill in the art,
in combination with various combinatorial libraries, one can
isolate and characterize those molecules that bind to or interact
with the desired target. The relative binding affinity of these
compounds can be compared and optimum compounds identified using
competitive binding studies that are well known to those of skill
in the art. For example, the following are examples of various
combinatorial compositions and methods which are herein
incorporated by reference: U.S. Pat. Nos. 6,168,913; 5,565,324;
6,087,103; 6,060,596 and S. Brenner et al., "Encoded Combinatorial
Chemistry", Proc. Natl. Acad. Sci. USA, vol. 89, (1992), pp.
5381-5383; R. W. Armstrong et al., "Microchip Encoded Combinatorial
Libraries: Generation of a Spatially Encoded Library from a Pool
Synthesis", Medicinal Chemistry, vol. 50, No. 6, (1996), pp.
258-260; J. J. Baldwin et al., "Synthesis of a Small Molecule
Combinatorial Library Encoded with Molecular Tags", J. Am. Chem.
Soc., vol. 117, (1995), pp. 5588-5589; H. P. Nestler et al., "A
General Method for Molecular Tagging of Encoded Combinatorial
Chemistry Libraires", J. Org. Chem., vol. 59, No. 17, (1994), pp.
4723-4724.
[0121] In some embodiments the processed molecules can also be
produced by combinatorial chemistry and selection methods which are
based on the disclosure that the claimed molecules all interact
with a unique portion of the RXR receptor, side-pocket 1. The
knowledge of this interaction means that molecules that interact
with this side-pocket 1 of the RXR receptor can be isolated. The
goal is to isolate those processed molecules that at least
partially fill the side-pocket 1 region of the RXR receptor. In
general this can be achieved by 1) interacting a library of
molecules with an RXR receptor, 2) removing molecules that do not
interact with the RXR receptor at a predetermined level, and 3)
collecting the molecules that interact with side-pocket 1 of the
RXR receptor.
[0122] Using methodology well known to those of skill in the art,
in combination with various combinatorial libraries, one can
isolate and characterize those compounds that bind to or interact
with the desired target. The relative binding affinity of these
compounds can be compared and optimum compounds identified using
competitive binding studies which are well known to those of skill
in the art.
[0123] g) Molecules that are Produced by Computer Selection Methods
and Computer Assisted Design
[0124] Another way to isolate molecules that bind a molecule of
choice is through rational design. This is achieved through
structural information and computer modeling. Computer modeling
technology allows visualization of the three-dimensional atomic
structure of a selected molecule and the rational design of new
compounds that will interact with the molecule. The
three-dimensional construct typically depends on data from x-ray
crystallographic analyses or NMR imaging of the selected molecule.
The molecular dynamics require force field data. The computer
graphics systems enable prediction of how a new compound will link
to the target molecule and allow experimental manipulation of the
structures of the compound and target molecule to perfect binding
specificity. Prediction of what the molecule-compound interaction
will be when small changes are made in one or both requires
molecular mechanics software and computationally intensive
computers, usually coupled with user-friendly, menu-driven
interfaces between the molecular design program and the user.
[0125] Examples of molecular modeling systems are the CHARMm and
QUANTA programs, Polygen Corporation, Waltham, MA. CHARMm performs
the energy minimization and molecular dynamics functions. QUANTA
performs the construction, graphic modelling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0126] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen, et al., 1988
Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57
(Jun. 16, 1988); McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol.
Toxiciol. 29, 111-122; Perry and Davies, QSAR: Quantitative
Structure-Activity Relationships in Drug Design pp. 189-193 (Alan
R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236,
125-140 and 141-162; and, with respect to a model enzyme for
nucleic acid components, Askew, et al., 1989 J. Am. Chem. Soc. 111,
1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada,
and Hypercube, Inc., Cambridge, Ontario. Although these are
primarily designed for application to drugs specific to particular
proteins, they can be adapted to design of drugs specific to
regions of DNA or RNA, once that region is identified.
[0127] Although described above with reference to design and
generation of compounds which could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds which alter substrate binding or enzymatic
activity.
[0128] 3. Compounds
[0129] Disclosed are compounds of Formula (IX): 4
[0130] wherein: 5
[0131] are independently or together present or absent;
[0132] D is CH or CH.sub.2;
[0133] K is CH or a quaternary carbon;
[0134] G, E and V are independently a carbon or nitrogen atom;
[0135] F is of Formula (X), (XI), (XII) or (XIII) 6
[0136] wherein R.sub.30, R.sub.31 or R.sub.32 are independently or
together hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, halogen, cyano,
nitro, hydroxyl, acyloxy, amino, mono-substituted amino,
di-substituted amino, alkylamide, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, alkoxy, substituted alkoxy, haloalkoxy, thioalkyl,
thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, thiohaloalkoxy, alkylsulfone, oxime,
O-substituted oxime, dialkylcarboxamide or substituted
dialkylcarboxamide;
[0137] R.sub.20 and R.sub.21 are independently or together
hydrogen, alkyl, substituted alkyl;
[0138] R.sub.22 is hydrogen, alkyl, substituted alkyl, haloalkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
halogen, cyano, nitro, hydroxyl, acyloxy, amino, mono-substituted
amino, di-substituted amino, alkylsulfonamide, arylsulfonamide,
alkylurea, arylurea, alkylcarbamate, arylcarbamate, heteroaryl,
alkoxy, substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl,
carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide or substituted
dialkylcarboxamide;
[0139] R.sub.23 is absent, hydrogen, alkyl, substituted alkyl,
haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, halogen, cyano, nitro, hydroxyl, acyloxy, amino,
mono-substituted amino, di-substituted amino, alkylsulfonamide,
arylsulfonamide, alkylurea, arylurea, alkylcarbamate,
arylcarbamate, heteroaryl, alkoxy, substituted alkoxy, haloalkoxy,
thioalkyl, thiohaloalkyl, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide or substituted
dialkylcarboxamide;
[0140] R.sub.22 and R.sub.23 together form a cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocycle or substituted heterocycle.
[0141] R.sub.24 and R.sub.25 independently or together are
hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, halogen, cyano, nitro,
hydroxyl, acyloxy, amino, mono-substituted amino, di-substituted
amino, alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxarnide
or substituted dialkylcarboxamide;
[0142] R.sub.26 is hydrogen, halogen, hydroxy, alkyl or substituted
alkyl; and
[0143] W, X, Y and Z are independently or together --C(O)--,
--C(S)--, --S--, --O--or
[0144] --NH-residues that together form a 2,4-thiazolidinedione,
2-thioxo-4-thiazolidinedione, isoxazolidinedione,
2,4-imidazolidinedione, [1,2,4]-oxadiazolidine-3,5-dione or
2-thioxo-4-imidazolidinedione residue; or
[0145] pharmaceutically acceptable salts;
[0146] and do not have the structure of the Formula: 7
[0147] wherein:
[0148] R.sub.1 and R.sub.2 are 1) independently or together
hydrogen, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
hydroxyl, acyl, amino, mono-substituted amino, di-substituted
amino, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide or haloalkoxy; or 2) R.sub.1 and R.sub.2
together with the aromatic ring bonded thereto form a cycloalkyl,
substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl
residue that may optionally comprise 1 or 2 heteroatoms selected
from O, S, NH or N-alkyl;
[0149] R.sub.3 and R.sub.4 are independently or together hydrogen,
alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, halogen, cyano, nitro, hydroxyl,
acyloxy, amino, mono-substituted amino, di-substituted amino,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide;
[0150] A is --CR.sub.6R.sub.7-- where R6 and R7 are independently
or together hydrogen, alkyl, substituted alkyl, alkoxy, substituted
alkoxy or haloalkoxy; or R.sub.6 and R.sub.7 together form a
cycloalkyl residue that may optionally comprise 1 or 2 heteroatoms
selected from O, S, NH and N-alkyl; 8
[0151] Ar is Formula (II), (III), (IV) or (V):
[0152] where R.sub.8, R.sub.9 and R.sub.10 are independently or
together hydrogen alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, halogen, cayno,
nitro, hydroxyl, acyloxy, amino, mono-substituted amino,
di-substituted amino, alkylamide, alkysulfonamide, arylsulfonamide,
alkylurea, arylurea, alkylcarbamate, arylcarbamate, alkoxy,
substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide or substituted dialkylcarboxamide;
[0153] R.sub.5 is hydrogen, halogen, hydroxy, alkyl or substituted
alkyl; 9
[0154] represents a bond present or absent; and
[0155] W, X, Y and Z are independently or together --C(O)--,
--C(S)--, --S--, --O-- or --NH-residues that together form a
2,4-thiazolidinedione, 2-thioxo-4-thiazolidinedione,
isoxazolidinedione, 2,4-imidazolidinedione or
2-thioxo-4-imidazolidinedione residue; or a pharmaceutically
acceptable salt.
[0156] Also disclosed are compounds wherein F is Formula (X);
R.sub.20, R.sub.21, and R.sub.24 are independently or together
alkyl or substituted alkyl; and R.sub.30 is alkyl, substituted
alkyl, haloalkoxy, amino, mono-substituted amino, di-substituted
amino, thiohaloalkoxy, alkylsulfone, oxime or O-substituted
oxime.
[0157] Also disclosed are compounds wherein R.sub.20, R.sub.21, and
R.sub.24 are independently or together alkyl or substituted alkyl;
the bond between D and E is a double bond; the bond between E and G
is a single bond; R.sub.22 is hydrogen, alkyl or substituted alkyl;
and R.sub.23 is hydrogen, alkyl or substituted alkyl.
[0158] Also disclosed are compounds wherein R.sub.20, R.sub.21, and
R.sub.24 are independently or together alkyl or substituted alkyl;
the bond between D and E is a double bond; the bond between E and G
is a single bond; and R.sub.22 and R.sub.23 together form a
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocycle or substituted heterocycle.
[0159] Also disclosed are compounds wherein R.sub.20, R.sub.21, and
R.sub.24 are independently or together alkyl or substituted alkyl;
the bond between D and E is a single bond; the bond between E and G
is a double bond; R.sub.22 is hydroxy or alkoxy; G is a nitrogen;
and R.sub.23 is absent.
[0160] Also disclosed are compounds wherein R.sub.20, R.sub.21, and
R.sub.24 are independently or together alkyl or substituted alkyl;
the bond between D and E is a single bond; the bond between E and G
is a double bond; R.sub.22 and R.sub.23 is together form a
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, heterocycle or substituted heterocycle.
[0161] Also disclosed are compounds wherein R.sub.20, R.sub.21, and
R.sub.24 are independently or together alkyl or substituted alkyl;
K is a quaternary carbon, the bond between K and V is a double
bond; the bond between E and G is a double bond; R.sub.22 is
hydrogen, alkyl or substituted alkyl; G is a nitrogen; and R.sub.23
is absent.
[0162] Also disclosed are compounds of Formula (100): 10
[0163] wherein: 11
[0164] represents a bond present or absent;
[0165] Ar is a substituted or unsubstituted benzene or pyridene
ring;
[0166] R.sub.20 and R.sub.21 are independently selected from
hydrogen or an alkyl or substituted alkyl residue comprising 1 to 8
carbon atoms;
[0167] R.sub.22 is hydrogen, halogen, cyano, nitro, amino, or
hydroxyl residue; or an alkyl, substituted alkyl, haloalkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
acyloxy, mono-substituted amino, di-substituted amino,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, heteroaryl, alkoxy, substituted
alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide
or substituted dialkylcarboxamide residue comprising 1 to 8 carbon
atoms, and R.sub.22 may be oriented either syn or anti with respect
to the compound; and
[0168] R.sub.24 and R.sub.25 are independently selected from
hydrogen, halogen, cyano, nitro, amino, or hydroxyl residue, or an
alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, acyloxy, mono-substituted amino,
di-substituted amino, alkylsulfonamide, arylsulfonamide, alkylurea,
arylurea, alkylcarbamate, arylcarbamate, heteroaryl, alkoxy,
substituted alkoxy, haloalkoxy, thioalkyl, thiohaloalkyl, carboxy,
carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide,
dialkylcarboxamide or substituted dialkylcarboxamide residue
comprising 1 to 8 carbon atoms;
[0169] R.sub.26 is a hydrogen, halogen, or hydroxyl residue, or an
alkyl or substituted alkyl comprising 1 to 4 carbon atoms;
[0170] V is a C or N atom, and W, X, Y and Z are independently
selected from --C(O)--, --C(S)--, --S--, --O-- or --NH-residues
that together form a 2,4-thiazolidinedione,
2-thioxo-4-thiazolidinedione, isoxazolidinedione,
2,4-imidazolidinedione, [1,2,4]-oxadiazolidine-3,5-di- one or
2-thioxo-4-imidazolidinedione residue; or
[0171] a pharmaceutically acceptable salt thereof.
[0172] The compound of claim 46 wherein Ar has the Formula (X),
(XI), (XII) or (XIII) 12
[0173] wherein R.sub.30, R.sub.31 or R.sub.32 are independently
selected from a hydrogen, halogen, cyano, nitro, amino, or hydroxyl
residue, or an alkyl, substituted alkyl, haloalkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, acyloxy,
mono-substituted amino, di-substituted amino, alkylamide,
alkylsulfonamide, arylsulfonamide, alkylurea, arylurea,
alkylcarbamate, arylcarbamate, alkoxy, substituted alkoxy,
haloalkoxy, thioalkyl, thiohaloalkyl, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, thiohaloalkoxy,
alkylsulfone, oxime, O-substituted oxime, dialkylcarboxamide or
substituted dialkylcarboxamide residue comprising 1 to 8 carbon
atoms.
[0174] Also disclosed are compounds wherein at least one of
R.sub.30, R.sub.31 or R.sub.32 is not hydrogen.
[0175] Also disclosed are compounds which are processed molecules
wherein Ar has the Formula 13
[0176] wherein R.sub.30 is selected from an alkyl, substituted
alkyl, haloalkoxy, amino, mono-substituted amino, di-substituted
amino, thiohaloalkoxy, or alkylsulfone.
[0177] Disclosed are compounds wherein R.sub.20, R.sub.21, and
R.sub.24 are alkyl or substituted alkyl; - - - represents a bond
present; and R.sub.22 is hydroxy or an alkoxy residue having 1 to 4
carbon atoms.
[0178] The disclosed compounds are suitable for pharmaceutical
compositions comprising one or more of the disclosed oxime
compounds for administration in mammals for modulating lipid
metabolism, carbohydrate metabolism, lipid and carbohydrate
metabolism, or adipocyte differentiation. The compounds also can
treat type 2 diabetes, polycystic ovary syndrome or syndrome X,
type 2 diabetes, uncontrolled cellular proliferation, or cancer,
such as, carcinoma, lymphoma, leukemia, or sarcoma, wherein the
cancer is Hodgkin's Disease, myeloid leukemia, polycystic kidney
disease, bladder cancer brain cancer, head and neck cancer, kidney
cancer, lung cancer myeloma, neurolastoma/glioblastoma, ovarian
cancer, pancreatic cancer, prostate cancer, skin cancer, liver
cancer, melanoma, colon cancer, cervical carcinoma, breast cancer
epithelial cancer, and leukemia. Disclosed are compounds for
administration in mammals that can also be used for treatment of an
inflammatory disease, such as wherein the inflammatory disease is
osteoarthritis, rheumatoid arthritis, Crohn's Disease, pulmonary
fibrosis, or Inflammatory Bowel Disease.
[0179] Disclosed are methods of modulating lipid metabolism,
carbohydrate metabolism, lipd and carbohydrate metabolism, or
adipocyte differentiation comprising administering to a mammal
diagnosed as needing such modulation pharmaceutical compositions
disclosed herein, wherein the mammal is human for example.
Disclosed are methods wherein the mammal is diagnosed as having
type 2 diabetes.
[0180] Also disclosed are methods of treatment for a disease of
uncontrolled cellular proliferation comprising administering to a
mammal diagnosed as having a disease of uncontrolled proliferation,
wherein for example, the mammal is human, and wherein for example,
the mammal is diagnosed as having an inflammatory disease.
[0181] 4. Pharmaceutical Compositions
[0182] Also provided are pharmaceutical compositions comprising the
molecules of the invention. Pharmaceutical compositions are well
known in the art. The pharmaceutical composition can treat a
disease selected from the consisting of type 1 diabetes, type 2
diabetes, hypercholestermia, atherosclerosis, and related
disorders. The composition of the invention can reduce blood
glucose levels and lower triglyceride levels in KKA.sub.y mice.
B. Methods
[0183] 1. Treating
[0184] The invention provides a method of treating a disease
comprising administering a molecule that activates an RXR receptor,
wherein the molecule comprises an RXR binding portion which binds
the RXR receptor and comprises a side pocket contacting residue
which contacts a side-pocket 1 of an RXR receptor. The disease can
be selected from the group consisting of type 1 diabetes, type 2
diabetes, hypercholesteremia, atherosclerosis, and related
disorders.
[0185] 2. Screening
[0186] The invention also provides a method for selecting
compositions that are RXR ligands which interact with side-pocket 1
of an RXR receptor comprising 1) interacting a library of molecules
with an RXR receptor, 2) removing molecules that do not interact
with the RXR receptor at a predetermined level, 3) collecting the
molecules that interact with side-pocket 1 of the RXR receptor.
[0187] Also provided is a method of selecting compounds for the
treatment of type 1 diabetes, type 2 diabetes, hypercholestermia or
atherosclerosis comprising designing molecules to contact side
pocket 1 of an RXR receptor. The molecules can be designed to have
four or more contacts, e.g. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and 20; with a side pocket 1 of an RXR.
[0188] A compound that shows a good molecular modeling profile and
has become a candidate for the subgroup of RXR ligands with
unexpected properties which may make them useful for the treatment
of type 2 diabetes and other diseases can be subsequently tested in
the following in vitro and in vivo biological screens and testing
models:
[0189] a) Adipocyte Differentiation Assay:
[0190] Prior to testing compounds in vivo, they can be run through
the in vitro adipocyte differentiation assay based on the protocol
of Zhang, et al. (1996) Negative Regulation of Peroxisome
Proliferator-Activated Receptor-.quadrature. Gene Expression
Contributes to the Antiadipogenic effects of Tumor Necrosis
Facto{dot over ({tilde over (r)})}{dot over ({tilde over
(.quadrature.)})} Mol. Endo. 10: 1457-1456, incorporated herein by
reference. In short, mouse 3T3-L1 preadipocytes are grown in
culture medium containing 10% calf serum supplemented with
antibiotics. Two days after reaching confluence, cells are treated
with the selected compound at different concentrations dissolved in
medium containing 10% fetal calf serum supplemented with
antibiotics and kept at 10% CO.sub.2. The test compounds are
replaced every 2-3 days. Differentiation is assessed after 7 days
of treatment by the lipid content in the cells, using the
Triglyceride (INFINITY) reagent (Sigma Chemical, St. Louis, Mo.).
If the test compound shows significant activity at 10.sup.-7 M and
its activity is equal or no less than 1/5 that of compound 1 at
10.sup.-7 M, it is deemed suitable for testing in vivo.
[0191] b) RXR Agonist/Activator
[0192] RXR agonists activate the RXR receptor when interacting with
RXR by binding to its binding pocket. This activation can be
independent of whether the agonist, while interacting with portions
of the ligand binding pocket, also interacts with side pocket 1.
Activation, also often referred to as transcriptional activation,
of RXR by agonists can be measured by a number of commonly used
assays where the effect of the agonist ligand interacting with the
RXR ligand binding pocket is to stimulate or induce transcription
of a gene or genes in a cell. In one particular embodiment,
transient transfection experiments (as described essentially by
Zhang, et al. (1996) Negative Regulation of Peroxisome
Proliferator-Activated Receptor-.quadrature. Gene Expression
Contributes to the Antiadipogenic effects of Tumor Necrosis
Facto{dot over ({tilde over (r)})}{dot over ({tilde over
(.quadrature.)})} Mol. Endo. 10: 1457-1456) can be used. In these
cases, fill length RXR proteins are expressed in living cells.
Alternatively, hybrid proteins that contain a portion of another
DNA binding domain and the ligand binding portion of the human
RXR.alpha. receptor can be expressed in living cells instead of the
full length RXR protein. Such hybrid proteins can bind to and
activate specific reporter genes introduced into the same living
cell and activation of such reporter genes is measured. The assays
described here are very commonly used today by people skilled in
the art of molecular biology experimenting. Transient transection
assays are often used instead of ligand binding assays and can
serve to measure or estimate an affinity of an RXR agonist ligand
for RXR protein. The effective dose fifty (ED.sub.50, also called
EC.sub.50) is the concentration of the ligand necessary to lead to
one half maximal activation of the reporter gene in a living cell.
Compounds that show 60% of the activity of the agonist 9-cis
retenoic acid at 10.sup.-6 M can be considered an RXR
activator/agonist. Compounds which show 70% 80% 90% of the activity
of the agonist 9-cis retinoic acid are also provided.
[0193] c) Efficacy Testing in KKA.sup.y
[0194] Antidiabetic activity of selected molecules can be
demonstrated in the KKA.sup.y mouse, an animal model of type 2
diabetes (described in detail in Iwatsuka, et al., 1970 General
Survey of Diabetic Features of Yellow KK Mice. Endocrinol. Japon.
17: 23-35, incorporated herein by reference). In short, adult
diabetic KKA.sup.y mice are housed in a fixed 12-12-hr artificial
light-dark cycle, and maintained on a standard rodent diet. Prior
to commencing treatment, all of the animals are bled from the tail
vein and serum levels of glucose and triglyceride are measured in
duplicate. The animals are then sorted into different treatment
groups with equal average triglyceride levels. The animals are
treated with a single daily oral dose ranging from 3-30 mg/kg of
the test compound suspended in sesame oil (dose volume of 3-5
ml/kg). Following one week of treatment the animals are bled from
the tail vein and Serum glucose and triglycerides are measured in
duplicate. Compounds showing efficacy in lowering glucose and which
do not increase triglycerides or preferably, compounds that lower
both glucose and triglyceride levels in this severely
hypertriglyceridemic model, are preferably tested in further in
vivo screens.
[0195] d) Toxicity in Wild Type Mice
[0196] Test compounds are tested in wild type mice at 10-100 times
the efficacious dose in diabetic mice. Typically, adult male BALB/c
mice are housed in a fixed 12-12-hr artificial light-dark cycle,
and maintained on a standard rodent diet. The animals are treated
with a single daily oral dose of the test compound suspended in
sesame oil (dose volume of 5 ml/kg) as also described in the
examples. Approximately two weeks later the animals are bled from
the tail vein, then necropsied following sacrifice by carbon
dioxide asphyxiation. Several serum biochemical parameters are
measured. Of special significance is the effect the selected
compounds have on serum triglyceride levels compared to vehicle
treated control animals. If the compounds do not increase
triglyceride levels even when used at multiples of the
concentration at which they reduce glucose levels in KKA.sup.y
mice, they can be preferably pursued in further drug development
following procedures and tests typically used for such drug
candidates in preclinical and clinical studies.
C. EXAMPLES
[0197] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
1. Example 1
Computer Modeling
[0198] A requirement for a compound to be considered a candidate
for the subclass of heterocyclic derivatives with unexpected
properties which are useful for the treatment of disease such as
type 2 diabetes, hypercholesteremia, osteosclerosis and other
diseases is that these compounds can interact with a specific
cavity, which is described herein as side pocket 1, in the ligand
binding pocket of human RXR.alpha., as described by Egea, et al in
EMBO J 2000 Jun. 1;19(11):2592-601, incorporated herein by
reference or as found in the PDB database
(http:.backslash..backslash.www.pdb.org). To determine whether the
compound interacts with side pocket 1, computer modeling is used to
dock putative ligands into three dimensional structure of the
protein RXR. This is accomplished by 1: Representing the three
dimensional coordinates of the protein as a set of x, y, and z
coordinates specifying the centers of the atoms comprising the
protein. 2. Representing a starting structure for the putative
ligand by generating a conformation for the ligand in three
dimensions that is consistent with bond lengths and atomic radii as
specified by the atomic formula for the putative ligand. 3.
Identifying torsional bonds within the ligand and the RXR protein
as comprised of single bonds that are not a part of ring
structures. 4. Moving and orienting the ligand with respect to the
protein while simultaneously rotating the torsional bonds within
the ligand, or simultaneously rotating the torsional bonds within
both the ligand and the RXR protein, where the CA atoms of the
protein are fixed. 5. Finding optimal fits of the ligand into the
enclosed pocket of RXR using a scoring function that contains
contributions from van der Waals and electrostatic interactions of
the ligand with the protein. 6. Once an optimal docked conformation
is obtained the ligand is evaluated to determine if its optimal
docked structure interacts with side pocket 1 using a contact
criterion outlined below in Example 2.
2. Example 2
Evaluation of Interaction of a Compound with Side Pocket 1 and
Candidacy to belong to a Selected Subclass of RXR Ligands Useful
for the Treatment of Diseases
[0199] Side pocket 1 is defined using the protein coordinates of
the crystal structure of human RXR.alpha. protein that has been
determined and which has been deposited under 1FBY in the Protein
Databank as further described in Example 1. Specifically, that
portion of the protein coordinates of 1FBY that form side pocket 1
and more specifically, those atoms of the amino acids Trp A305, Asn
A306, Ile A310, Cys A432, Gly A429, Leu A436, and Leu A433. Herein
amino acids are specified using the three letter amino acid code to
specify the amino acid type, while the amino acid sequence and
sequence numbering scheme is as reported in the crystal structure
1FBY from the Protein Databank. The prefix A specifies that the A
polypeptide chain from 1FBY is used. Specific atoms lining the side
pocket 1 comprise a "Cavity 1 List" (C1L). C1L consists of Trp
A305:CE3,CZ3,CH2; Asn A306:C,CA,CB,O; Ile A310:CD1; Cys A432:
C,CB,O; Leu A436:CD1; Gly A429:0,C,CA, and Leu A433:N,CA,CD2, where
individual atom names are defined as in 1FBY and are consistent
with IUPAC-IUB naming conventions for amino acids with capital
roman letters substituted for Greek letters. IUPAC-IUB Commission
on Biochemical Nomenclature (1970). Abbreviations and symbols for
the description of the conformation of polypeptide chains. J. Mol.
Biol., 52, 1-17. The side pocket 1 formed by these atoms is
illustrated in FIGS. 1-5. FIG. 1 shows the Connolly surface of the
entire active site of RXR, with one amino acid Arg A316 shown for
reference. The Connolly surface is generated as described in
IUPAC-IUB Commission on Biochemical Nomenclature (1970).
Abbreviations and symbols for the description of the conformation
of polypeptide chains. J. Mol. Biol., 52, 1-17. FIG. 2 shows that
portion of the RXR ligand pocket that comprises side pocket 1, with
reference to C19 of 9-cis-RA, a natural ligand of RXR (numbering as
in 1FBY). While 9-cis-RA occupies a major portion of the RXR ligand
pocket, side pocket 1 comprises unoccupied space in the crystal
structure that is adjacent to C19. FIG. 3 shows the amino acids
lining side pocket 1; FIG. 4 shows the same view with the native
ligand 9-cis-RA in the RXR ligand pocket site. FIG. 5 shows the
same view with the compound 1 in the RXR ligand pocket. Note that
compound 1 has a side pocket 1 contacting residue.
[0200] It is understood that the coordinates of the atoms making up
the side pocket 1 can be obtained by using the coordinates of any
other structures of an RXR, including structures of the side pocket
1.
[0201] FIG. 6 labels specific atoms from the amino acids shown in
FIG. 3 that are close to the Connolly surface of the side pocket 1.
In this figure, wherever connected atoms are selected, they are
shown as lines connecting the atoms. Wherever isolated atoms are
selected, these atoms are represented by stars. Where Association
of the name and the atom requires, an arrow is provided pointing
from the amino acid name to the individual atoms (as in, for
example, Leu A436). The list of specific atoms are those of
C1L.
[0202] As described in Example 1, when a candidate compound is
evaluated, it is first docked into the protein structure using
procedures described. Docked compounds are then selected as
candidates for a special RXR ligand subclass, based on whether or
not they enter the space defined as side pocket 1. This can be
determined by evaluating the number of contacts--defined as
NCCL--between ligand atoms and atoms in the C1L. NCCL is computed
as follows. For each docked ligand a "Side Pocket 1 Contact List"
(SPCL) is initialized to contain zero entries. For each
non-hydrogen atom of the docked ligand all atoms in the C1L are
examined. An atom from the C1L is added to the SPCL if the distance
between the center of an atom of the side pocket contacting residue
of the ligand and the center of an atom of C1L is lower than 4.0
Angstroms. When all atoms of the docked ligand have been examined
the number NCCL of atoms added to the SPCL is computed. The docked
ligand is considered a special RXR subclass candidate if NCCL of
atoms added to the CCL list is greater than or equal to 4, which
indicates that the docked ligand penetrates the side pocket 1 and
interacts with the amino acids comprising it. Compound 1 (see FIGS.
5 and 7) and 3 satisfy this requirement and belong to the special
RXR ligand subclass. Compound 2 (see FIG. 8) and compound 4 (see
FIG. 9) do not fulfill these requirements.
[0203] [1] W. Welch, J. Ruppert, A. Jain, Hammerhead: Fast, Fully
Automated Docking of Flexible Ligands to Protein Binding Sites,
Chemistry and Biology 3: 449-462 1996.
[0204] Scoring Non-Covalent Ligand-Protein Interactions: A
Continuous Differential Function Tuned to Compute Binding
Affinities. A. N. Jain; Journal of Computer Aided Molecular Design
10:5, 427-440, 1996].
[0205] M. L. Connolly, "Solvent-accessible surfaces of proteins and
nucleic acids," Science, 221, 709 (1983)
3. Example 3
Measuring Adipocyte Differentiation Activity
[0206] The ability of a compound to induce preadipocytes to
differentiate into adipocytes is an indicator of its antidiabetic
activity. This allows for screening many compounds in a simple in
vitro assay, thus avoiding the task of testing all compounds in
vivo. The adipocyte differentiation assay is based on the protocol
of Zhang, et al. (1996) Negative Regulation of Peroxisome
Proliferator-Activated Receptor-.quadrature. Gene Expression
Contributes to the Antiadipogenic effects of Tumor Necrosis
Facto{dot over ({tilde over (r)})}{dot over ({tilde over
(.quadrature.)})} Mol. Endo. 10:1457-1456, incorporated herein by
reference at least for material related to adipocyte
differentiation assays. Mouse 3T3-L1 preadipocytes are grown in
culture medium containing 10% calf serum supplemented with
antibiotics. Two days after reaching confluence, cells are treated
with the selected compound at different concentrations dissolved in
medium containing 10% fetal calf serum supplemented with
antibiotics and kept at 10% CO.sub.2. The test compounds are
replaced every 2-3 days. Differentiation is assessed after 7 days
of treatment by the lipid content in the cells, using the
Triglyceride (INFINITY) reagent (Sigma Chemical, St. Louis, Mo.)
(see FIG. 15).
4. Example 4
RXR Activation
[0207] To confirm that the computer modeling/design of a compound
does result in an RXR activator or RXR agonist, i.e. a compound
that leads to RXR activation or transcriptional activation,
commonly used assays can be applied, where the effect of the
agonist or activator, interacting with the RXR ligand binding
pocket is to stimulate or induce transcription of a gene or genes
in a cell. In one particular embodiment, transient transfection
experiments (as described essentially by Zhang, et al. (1996)
Negative Regulation of Peroxisome Proliferator-Activated
Receptor-.quadrature. Gene Expression Contributes to the
Antiadipogenic effects of Tumor Necrosis Facto{dot over ({tilde
over (r)})}{dot over ({tilde over (.quadrature.)})} Mol. Endo.
10:1457-1456) can be used. In these cases, full length RXR proteins
are expressed in living cells. Alternatively, hybrid proteins that
contain a portion of another DNA binding domain and the ligand
binding portion of the human RXR.alpha. receptor can be expressed
in living cells instead of the full length RXR protein. Such hybrid
proteins can bind to and activate specific reporter genes
introduced into the same living cell and activation of such
reporter genes is measured (Brand, A. H. & Perrimon, 1993,
Development, 118:401-415; Moya-Camarena, S. Y. et. al., 1999, J.
Lipid Res., 40:1426:1433; Yi, Y. W. et al., 2000, Biochem. Biophys.
Res. Commun., 272:193-198, each are herein incorporated by
reference at least for material related to reporter assays). These
assays not only measure binding of the ligand to its target, which
could also be measured by an in vitro binding assay, but also
measure the agonistic or activator activity of the compound. In
such an assay, new compounds are compared to a standard activator
like compound I or the natural agonist of RXR: 9-cis retinoic acid
(see Example 7). For instance, all compounds that show at a given
concentration at least 60% of the activity of 9-cis retinoic acid
can be considered useful activators.
5. Example 5
Measuring Antidiabetic Activity of a Selected Subclass of RXR
Ligands
[0208] The antidiabetic activity of a selected subclass of RXR
ligands can be demonstrated in the KKA.sup.y mouse, an animal model
of type 2 diabetes (described in detail in Iwatsuka, et al., 1970
General Survey of Diabetic Features of Yellow KK Mice. Endocrinol.
Japon. 17: 23-35, incorporated herein by reference). In this
example, compound 1, which belongs to the novel subclass of RXR
ligands is compared to a typical RXR ligand like compound 2 in the
treatment of KKA.sup.y mice.
[0209] For this study, adult diabetic KKA.sup.y mice were housed in
a fixed 12-12-hr artificial light-dark cycle, and maintained on a
standard rodent diet. Prior to commencing treatment, all of the
animals are bled from the tail vein and serum levels of glucose and
triglyceride are measured in duplicate. The animals are then sorted
into different treatment groups with equal average triglyceride
levels. The animals are treated with a single daily oral dose of
the test compound suspended in sesame oil (dose volume of 3
ml/kg).
[0210] Compound 1 shows a statistically significant decrease in
both glucose and triglyceride levels following one and two weeks of
treatment compared to vehicle treated control mice (See FIGS. 16
and 17 for data related to compound 7). By contrast, compound 2,
which is known to lower glucose and triglyceride levels in type 2
diabetic db/db mice at the concentrations used here, failed to
decrease glucose levels, and significantly increased triglyceride
levels in KKA.sup.y mice following two weeks of treatment compared
to vehicle treated controls (ANOVA, Fisher's LSD test; FIGS. 10,
11).
6. Example 6
Measuring Effect of a Selected Subclass 0f RXR Ligands on
Triglyceride Levels in Mice
[0211] Different classes of RXR compounds have different effects on
wild type mice. Relevant to this document, is the effect of these
molecules on serum triglyceride levels. In this example, compound
1, which belongs to the selected subclass of RXR ligands is
compared to a typical RXR ligand like compound 2 in the treatment
of wild type BALB/c mice.
[0212] For these studies, adult male BALB/c mice were housed in a
fixed 12-12-hr artificial light-dark cycle, and maintained on a
standard rodent diet. The animals were treated with a single daily
oral dose of the test compound suspended in sesame oil (dose volume
of 5 ml/kg).
[0213] Treatment with compound 1 at 10 and 30 mg/kg/day for 10 days
(which represents approximately 30 to 100 fold excess over the dose
that lowers glucose in diabetic db/db mice) does not change
triglyceride levels compared to vehicle treated controls (FIG. 12).
By contrast, treatment with Compound 2, when used at a similar
excess over its effective glucose lowering dose in db/db mice,
causes a statistically significant, dose dependent increase in
serum triglyceride levels compared to vehicle treated controls
(ANOVA, Fisher's LSD test **p.ltoreq.0.01; FIG. 13).
7. Example 7
Compounds Modeled as Disclosed Herein
[0214] The compound numbers refer to the compounds designated in
FIG. 14.
2TABLE 2 Number of RXR Differentiation Efficacious in Compound
Contacts.sup.1 Activation.sup.2 Activity.sup.3 KKAy Mice.sup.4 1 17
110% 100% Yes 2 1 >100% >100% No 3 7 120% 100% Yes 4 0
>100% >100% No 5 1 >100% >100% No 6 5 >100% >100%
No 7 16 100% .sup. 80% Yes 8 15 90% .sup. 95% Yes 9 14 125% .sup.
30% Yes .sup.1The number of contacts between the side pocket
contacting residue and side pocket 1, as described in Example 2.
.sup.2Activation of the RXR receptor by the test compounds when
used at 1.quadrature. M compared to 9-cis-RA at equal
concentration, as described in Example 4. .sup.3The ability of the
compound to induce the differentiation of pre-adipocytes to
adipocytes when administered at 0.1.quadrature. M compared to
Compound 1 when administered at an equal concentration, as
described in Example 3. .sup.4Only compounds that lower glucose
without increasing triglycerides are considered efficacious, as
described in Example 5.
8. Example 8
3-[(1-Oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene-7-yl)-O-methyloxim-
e]-4-trifluoromethoxy-benzylidene-2,4-thiazolidinedione
[0215] 14
[0216] A mixture of toluene (5 mL), piperidine (38 .quadrature.L),
acetic acid (38 .quadrature.L),
3-[(1-Oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaph-
thalene-7-yl)-O-methyloxime]-4-trifluoromethoxy-benzaldehyde (0.515
g, 1.271 mmol) and 2,4-thiazolidinedione (0.164 g, 1.398 mmol) was
heated at reflux overnight. The reaction mixture was cooled to room
temperature, and the resulting crystalline compound was filtered
then recrystallized from dichloromethane and hexane. The white
solid was dried under high vacuum to afford 0.307 g (48%) of
3-[(1-Oxo-4,4,6-trimethyl-1,2,3,4-tetra-
hydronaphthalene-7-yl)-O-methyloxime]-4-trifluoromethoxy-benzylidene-2,4-t-
hiazolidinedione, mp 224.degree.. .sup.1H NMR (300 MHz;
DMSO-d.sub.6): 1.29 (s, 6 H), 1.70 (t, J=5.6 Hz, 2 H), 2.11 (s, 3
H), 2.72 (t, J=5.6 Hz, 2 H), 3.86 (s, 3 H), 5.77 (s, 1 H), 7.40 (s,
1 H), 7.63 (s, 1 H), 7.66 (d, J=8.8 Hz, 1 H), 7.75 (d, J=8.8 Hz, 1
H), 7.87 (s, 1 H), 12.71 (brs, 1 H).
[0217] The intermediate
3-[(1-Oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphth-
alene-7-yl)-O-methyloxime]-4-trifluoromethoxy-benzaldehyde was
prepared as follows:
a)
3-[(1-Oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene-7-yl)-O-methylo-
xime]-4-trifluoromethoxy-benzaldehyde
[0218] A mixture of 3-formyl-6-trifluoromethoxy-1-phenyl boronic
acid (0.59 g, 2.52 mmol),
7-bromo-1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaph-
thalene-O-methyloxime (0.62 g, 2.10 mmol) and potassium carbonate
(0.581 g, 4.20 mmol) in toluene (6.5 mL), ethanol (1.3 mL) and
water (0.8 mL) was degassed with argon for 15 minutes.
Tetrakis(triphenylphosphine)palla- dium(0) (0.049 g, 0.04 mmol) was
added and the mixture heated at reflux under argon overnight. The
solution was cooled to room temperature, diluted with ethyl acetate
and washed successively with water and brine, dried over anhydrous
magnesium sulfate, filtered and evaporated. The residue was
purified on silica gel (eluent: 10% ethyl acetate in hexane) to
give 0.52 g of
3-[(1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene-
-7-yl)-O-methyloxime]-4-trifluoromethoxy-benzaldehyde (61%).
.sup.1H NMR (300 MHz; CDCl.sub.3): 1.32 (s, 6 H), 1.75 (t, J=6.6
Hz, 2 H), 2.12 (s, 3 H), 2.79 (t, J=6.6 Hz, 2 H), 3.94 (s, 3 H),
7.24 (s, 1 H), 7.49 (d, J=8.4 Hz, 1 H), 7.77 (s, 7.84 (d, J=2.1 Hz,
1 H), 7.93 (dd, J.sub.1=2.1 Hz, J.sub.2=8.7 Hz, 1 H), 10.02 (s, 1
H).
b) 3-Formyl-6-trifluoromethoxy-1-phenyl boronic acid
[0219] To a mixture of 2-(3-bromo-4-trifluoromethoxy)-1,3-dioxolane
( 7.20 g, 22.9 mmol) in THF (70 mL) cooled to -78.degree. C. under
an atmosphere of argon was added n-BuLi (13.8 mL, 2.5 M, 34.4 mmol)
dropwise. The resulting suspension was stirred for 5 minutes and
triisopropylborate (15.9 mL, 68.7 mmol) was added dropwise via
syringe. The mixture was stirred at -50.degree. C. for 2 hours then
warmed up to room temperature and stirred overnight at room
temperature. 1.0 N HCl (50 mL) was slowly added to the reaction
mixture. After 3 hours the mixture was diluted with ethyl acetate
and the layers separated, the aqueous layer was extracted once with
ethyl acetate and the two organic layers combined. The resulting
organic layer was washed with water, brine and dried
(Mg.sub.2SO.sub.4). The mixture was filtered, evaporated and the
residue stirred in hexane. The resulting white suspension was
filtered and the white solid dried under high vacuum to afford 3.00
g of 3-formyl-6-trifluoromethoxy-1-phenyl boronic acid (56%).
.sup.1H NMR (300 MHz; DMSO-d.sub.6+1 drop of D.sub.2O):
.quadrature. 7.55 (d, J=5.3 Hz, 1 H); 8.07 (dd, J.sub.1=1.1 Hz,
J.sub.2=5.3 Hz, 1 H); 10.07 (s, 1 H).
c) 2-(3-Bromo-4-trifluoromethoxy)-1,3-dioxolane
[0220] To a solution of 3-bromo-4-trifluoromethoxybenzaldehyde
(ref, 20 g, 74.0 mmol) in toluene (200 mL) was added ethylene
glycol (82.6 mL, 1.48 mol) and p-toluenesulfonic acid monohydrate
(0.84 g, 4.44 mmol). The reaction mixture was heated at reflux
overnight and the water was removed using a Dean Stark apparatus.
The solution was cooled to room temperature, poured into aqueous
potassium carbonate (10%) and extracted with ethyl acetate. The
organic layer was washed with water, brine and dried
(Mg.sub.2SO.sub.4). The residue was purified on silica gel (eluent:
10% ethyl acetate in hexane) to give 15.4 g of
2-(3-bromo-4-trifluorometh- oxy)-1,3-dioxolane (66%). .sup.1H NMR
(500 MHz; CDCl.sub.3): .quadrature. 4.05 (m, 2 H); 4.11 (m, 2 H);
5.79 (s, 1 H); 7.32 (d, 1 H); 7.43 (d, 1 H); 7.77 (d, J=1.1 Hz, 1
H).
d)
7-Bromo-1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene-O-methyloxi-
me
[0221] A solution of
7-bromo-1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronapht- halene (1.02
g, 3.82 mmol), methoxylamine hydrochloride (0.638 mg, 7.63 mmol)
and pyridine (0.93 mL, 11.46 mmol) in ethanol (30 mL) was refluxed
for 3 hours. The mixture was concentrated in vacuo then diluted
with water and extracted twice with ethyl acetate. The combined
organic layer was dried (Mg.sub.2SO.sub.4), filtered and evaporated
to give 1.13 g of
7-bromo-1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene-O-methyloxime
as a yellow oil (quantitative) and used without further
purification in the Suzuki coupling (step a). .sup.1H NMR (300 MHz;
CDCl.sub.3): 1.25 (s, 6 H), 1.75 (t, J=6.9 Hz, 2 H), 2.38 (s, 3 H),
2.73 (t, J=6.9 Hz, 2 H), 3.98 (s, 3 H), 7.17 (s, 1 H), 8.11 (s, 1
H).
e) 7-Bromo-1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene
[0222] A solution of
1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene (40.0 g, 212
mmol) in dichloromethane (80 mL) was added dropwise at room
temperature under argon with vigourous stiring to a suspension of
aluminum chloride (56 g, 424 mmol) in dichloromethane (80 mL).
Bromine (13 mL, 254 mmol) was then added slowly. The reaction
mixture was stirred for 1.5 hours then poured into concentrated
hydrochloride acid (6N, 300 mL) and extracted with ether. The
organic layer was washed with water, aq NaHCO.sub.3, water, brine
and dried (Mg.sub.2SO.sub.4). The residue was purified on silica
gel (eluent: 5 to 8% ethyl acetate in hexane) to give 32.2 g of
7-bromo-1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene (64%).
.sup.1H NMR (300 MHz; CDCl.sub.3): 1.32 (s, 6 H), 1.94 (t, J=6.9
Hz, 2 H), 2.38 (s, 3 H), 2.64 (t, J=6.6 Hz, 2 H), 7.23 (s, 1 H),
8.06 (s, 1 H).
f) 1-Oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene
[0223] A solution of chromium(VI)oxide (86 g, 0.861 mol) in acetic
acid (400 mL) and water (40 mL) was added dropwise to a stirred
solution of 1,1,7-trimethyl-1,2,3,4-tetrahydronaphthalene in acetic
acid (70 mL) and the reaction mixture stirred 2.5 hours at room
temperature. Isopropanol (5 mL) was added and the whole
concentrated in vacuo. The residue was dissolved in hexane and
filtered over celite. The organic was washed with water and brine,
dried (Mg.sub.2SO.sub.4), filtered and evaporated to give 40.3 g of
1-oxo-4,4,6-trimethyl-1,2,3,4-tetrahydronaphthalene and used
without further purification in the bromination (step e). .sup.1H
NMR (300 MHz; CDCl.sub.3): 1.36 (s, 6 H), 1.98 (t, J=6.9 Hz, 2 H),
2.38 (s, 3 H), 2.68 (t, J=6.9 Hz, 2 H), 7.01 (dd, J.sub.1=0.6 Hz,
J.sub.2=7.9 Hz, 1 H), 7.19 (d, J=0.6 Hz, 1 H), 7.90 (d, J=8.1 Hz, 1
H).
g) 1,1,7-Trimethyl-1,2,3,4-tetrahydronaphthalene
[0224] A solution of 2-methyl-5-(p-tolyl)-3-pentanol (74 g, 0.385
mol) in dichloromethane (100 mL) was mixed with-polyphosphoric acid
(570 g) and the reaction mixture was heated to 60.degree. C. and
stirred overnight. After cooling, ice/water was slowly added and
the aqueous extracted with dichloromethane. The organic layer was
successively washed with water, aq NaHCO.sub.4, water and brine,
dried (Mg.sub.2SO.sub.4), filtered and evaporated to give 67 g of
1,1,7-trimethyl-1,2,3,4-tetrahydronaphthalene and used without
further purification in the next step (step f). .sup.1H NMR (300
MHz; CDCl.sub.3): 1.34 (s, 6 H), 1.69 (m, 2 H), 1.83 (m, 2 H), 2.37
(s, 3 H), 2.78 (m, 2 H), 6.99 (m, 2 H), 7.19 (s, 1 H).
h) 2-Methyl-5-p-tolyl-3-pentanol
[0225] To a solution of trans-4-methyl-1-p-tolyl-1-penten-3-ol
(43.2 g, 0.227 mol) in methanol (35 mL) was added 2 micro-spoon of
palladium, 10% on activated carbon and the reaction mixture was
hydrogenated overnight at 40 psi. The solution was diluted with
ethyl acetate, filtered over celite and evaporated to give 40 g of
2-methyl-5-(p-tolyl)-3-pentanol as a colorless oil and used without
further purification in the next step (step g). .sup.1H NMR (300
MHz; CDCl.sub.3): 0.90 (d, J=6.9 Hz, 6 H), 1.70 (m, 4 H), 2.31 (s,
3 H), 2.62 (m, 1 H), 2.78 (m, 1 H), 3.39 (m, 1 H), 7.09 (s, 4
H).
(l) trans-4-Methyl-1(p-tolyl)-1-penten-3-ol
[0226] To a solution of 4-methyl-1-p-tolyl-pent-1-en-3-one (77.0 g,
0.41 mol) in methanol (400 mL) was added slowly under argon sodium
borohydride (31 g, 0.82 mol). The reaction was stirred at room
temperature overnight and methanol (200 mL) was evaporated. The
solution was neutralized with hydrochloric acid (2N), extracted
with ethyl acetate. The organic layer was successively washed with
water, aqueous NaHCO.sub.3, water and brine, dried
(Mg.sub.2SO.sub.4), filtered and evaporated to give 80.4 g of
trans-4-methyl-1(-tolyl)-1-penten-3-ol and used without further
purification in the next step (step h). .sup.1H NMR (300 MHz;
CDCl.sub.3): 0.94 (d, J=6.9 Hz, 3 H), 0.99 (d, J=6.6 Hz, 3 H), 1.83
(m, 1 H), 2.33 (s, 3 H), 4.01 (br, 1 H), 6.16 (dd, J.sub.1=7.0 Hz,
J.sub.2=16.0 Hz, 1 H), 6.53 (d, J=16.0 Hz, 1 H), 7.12 (d, J=7.8 Hz,
1 H), 7.28 (d, J=7.8 Hz, 1 H).
(2) trans-4-Methyl-1-p-tolyl-pent-1-en-3-one
[0227] A solution of p-tolualdehyde (1 mL, 8.48 mmol),
3-methyl-2-butanone (1.81 mL, 16.96 mmol) and barium hydroxide (0.2
g, 1.17 mmol) in ethanol (4 mL) were mixed and the reaction mixture
was heated at reflux for 1 hour. After cooling the solution was
extracted with ethyl acetate. The organic layer was washed with
water and brine, dried (Mg.sub.2SO.sub.4), filtered and evaporated
to give 1.43 g of trans-4-methyl-1-p-tolyl-pent-1- -en-3-one (90%)
and used without further purification in the next step (step i).
.sup.1H NMR (300 MHz; CDCl.sub.3): 1.16 (d, J=7.2 Hz, 6 H), 2.35
(s, 3 H), 2.92 (m, 1 H), 6.79 (dd, J=16.0 Hz, 1 H), 7.19 (d, J=7.5
Hz, 1 H), 7.43 (d, J=8.1 Hz, 1 H), 7.28 (d, J=16.2 Hz, 1 H).
9. Example 9
2-(3-[3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthyl-2-yl]-4-trifluorome-
thoxybenzyl)-[1,2,4]-oxadiazolidine-3,5-dione
[0228] 15
[0229] To a mixture of
3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthyl-
-2-yl)-4-trifluoromethoxybenzaldehyde (hydroxyoxime) (0.74 g, 1.8
mmol) in THF (5.0 mL) cooled to -10.degree. C. was added
N-(chlorocarbonyl)isocyan- ate (0.153 g, 1.45 mmol) dropwise. The
mixture was stirred at 0.degree. C. for 30 min and the reaction was
quenched with 1 N HCl. The mixture was extracted with EtOAc, dried
over MgSO.sub.4 and filtered. The solvents were evaporated and the
crude product was purified on silica gel (eluent: hexane:EtOAc,
20:1 to CH.sub.2Cl.sub.2:CH.sub.3CN, 20:1 to 10:1) to give 0.28 g
of 2-(3-[3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthyl-2-yl]-4--
trifluoromethoxybenzyl)-[1,2,4]-oxadiazolidine-3,5-dione as
colorless oil. .sup.1H NMR (300 MHz; DMSO-d.sub.6): .quadrature.
[1.20 (s), 1.27 (s), 12 H], 1.65 (s, 4 H), 2.00 (s, 3 H), 4.85 (s,
2 H), 7.04 (s, 1 H), 7.24 (s, 1 H), 7.33 (brs, 1 H), 7.45-7.52 (m,
2 H).
[0230] The intermediate
3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthy-
l-2-yl)-4-trifluoromethoxybenzaldehyde (hydroxyoxime) was prepared
as follows:
a)
3-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthyl-2-yl)-4-trifluorome-
thoxybenzaldehyde (hydroxyoxime)
[0231] To a solution of
3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthy-
l-2-yl)-4-trifluoromethoxybenzaldehyde (0.781 g, 2.00 mmol) and
trace amount of methyl orange in 10 mL of MeOH:THF (1:1) was added
an aqueous solution of hydroxyamine hydrochloride (0.174 g, 2.5
mmol) at room temperature. The mixture was adjusted to pH=9 with 6
N KOH and additional MeOH:THF (1:1) was added. To the resulting
homogeneous solution was added solid sodium cyanoborohydride (0.126
g, 2.00 mmol) followed by addition of 2.0 N HCl in aqueous MeOH
until the mixture turned ruby red. Additional acid was added to
maintain the color during the course of the reaction. After 22
hours, the organics were removed under reduced vacuum and the
resulting aqueous solution was adjusted to pH=12 with 2 N KOH. The
mixture was extracted with CH.sub.2Cl.sub.2, washed with brine and
dried over MgSO.sub.4. The mixture was filtered, evaporated and the
resulting white solid dried under high vacuum to give 0.74 g of
3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthyl-2-yl)-4-trifluorometh-
oxybenzaldehyde (hydroxyoxime). Used without further
purification.
b)
3-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthyl-2-yl)-4-trifluorome-
thoxybenzaldehyde
[0232] To a solution of 3-bromo-4-trifluoromethoxybenzaldehyde
(10.0 g, 37.2 mmol),
(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) boronic
acid (11 g, 44.68 mmol, 1.2 eq) in a mixture of toluene (100 mL),
ethanol (20 mL) and water (15 mL) was added potassium carbonate
(10.28 g, 74.4 mmol, 2 eq). The solution was degassed with argon
for 40 minutes. Tetrakis(triphenylphosphine)palladium(0) (0.86 g,
0.74 mmol, 0.02 eq) was added and the mixture heated at reflux
under argon for 22 hours. The solution was cooled to room
temperature, diluted with ethyl acetate and washed successively
with water and brine, dried over MgSO.sub.4, filtered and
evaporated. The residue was chromatographed on silica gel (silica:
70-230 mesh, 60 A, 400 g, eluant: ethyl acetate/ hexane, 5:95) to
give
4-trifluoromethoxy-3-(3,5,5,8,7-pentamethyl-5,6,7,8-tetrahydronaphthalen--
2-yl) benzaldehyde (11.1 g, 76%). .sup.1H NMR (300 MHz; CDCl.sub.3)
1.25 (s, 6H); 1.32 (s, 6H); 1.70 (s, 4H); 2.08 (s, 3H); 7.06 (s,
1H); 7.18 (s, 1H); 7.48 (dd, J.sub.1=8.4 Hz, J.sub.2=1.5 Hz, 1H);
7.84 (d, J=2.0 Hz, 1H); 7.88 (dd, J.sub.1=2.0 Hz , J.sub.2=8.5 Hz
1H), 9.91 (s, 1H).
c) 3-Bromo-4-trifluoromethoxybenzaldehyde
[0233] To a solution of 4-trifluoromethoxybenzaldehyde (215 g, 1.13
mol) in a mixture of TFA (300 mL), CH.sub.2Cl.sub.2 (300 mL) and
H.sub.2SO.sub.4 (150 mL) was added at room temperature
N-bromosuccinimide (402 g, 2.26 mol) in equal portion over 7 hours.
The reaction mixture was stirred for 4 days at room temperature,
poured into ice-water and extracted with CH.sub.2Cl.sub.2. The
organic layer was washed with water then treated with saturated
NaHCO.sub.3 (1.5 L) for 2 hrs. The layers were separated and the
organic layer further washed with water and brine, dried over
MgSO.sub.4, filtered and evaporated. The residue was triturated
with hexane and filtered. After evaporation of the solvent, the
residue was distilled to give
3-bromo-4-trifluoromethoxybenzaldehyde (190.2 g, 81.degree. C., 1.0
mm/Hg, 62%).
10. Example 10
5-[3-(1-Ethyl-4,4,6-trimethyl-2-oxo-1,2,3,4-tetrahydro-quinolin-7-yl)-4-tr-
ifluoromethoxy-benzylidene]-thiazolidine-2,4-dione, which may be
Referred to as "Compound 8 in Table 2
[0234] 16
[0235] Prepared in a similar manner to example 1 using
3-(1-Ethyl-4,4,6-trimethyl-2-oxo-1,2,3,4-tetrahydro-quinolin-7-yl)-4-trif-
luoromethoxy-benzaldehyde. 56% yield after column chromatography on
silica gel (40% ethyl acetate in hexane). mp 156-154.degree. C.
.sup.1H-NMR (300 MHz, DMSO-d-6): 1.06 (t, J=7.5 Hz, 3 H); 1.26 (s,
6 H), 2.08 (s, 3 H), 2.46 (s, 2 H), 3.95 (br d, 2 H), 6.97 (s, 1
H), 7.31 (s, 1 H), 7.65 (s, 1 H), 7.66 (dd, J=1.5 Hz and 9 Hz, 1
H), 7.75 (dd, J=2.4 Hz and 8.7 Hz, 1 H), 7.87 (s, 1H), 12.71 (br s,
1 H).
[0236] The intermediate
3-(1-Ethyl-4,4,6-trimethyl-2-oxo-1,2,3,4-tetrahydr-
o-quinolin-7-yl)-4-trifluoromethoxy-benzaldehyde was prepared as
follows:
a)
3-(1-Ethyl-4,4,6-trimethyl-2-oxo-1,2,3,4-tetrahydro-quinolin-7-yl)-4-tr-
ifluoromethoxy-benzaldehyde
[0237] A mixture of 3-formyl-6-trifluoromethoxy-1-phenyl boronic
acid (Example f below) (8.2 g, 34.84 mmol),
7-bromo-1-ethyl-4,4,6-trimethyl-3,- 4-dihydro-1H-quinoline-2-one
(8.6 g, 29.03 mmol, Example b) and potassium carbonate (8 g, 58.06
mmol) in toluene (80 mL), ethanol (16 mL) and water (12 mL) was
degassed with argon for 30 minutes. Tetrakis(triphenylphosphi-
ne)palladium(0) (1.34 g, 0.04 mmol) was added and the mixture
heated at reflux under argon for 48 hrs. The solution was cooled to
room temperature, diluted with ethyl acetate and washed
successively with water and brine, dried over anhydrous magnesium
sulfate, filtered and evaporated. The residue was purified on
silica gel (30% ethyl acetate in hexane) to give 6.66 g of
3-(1-ethyl-4,4,6-trimethyl-2-oxo-1,2,3,4-tetrah-
ydro-quinolin-7-yl)-4-trifluoromethoxy-benzaldehyde (57%). .sup.1H
NMR (300 MHz; CDCl.sub.3): 1.20 (t, J=7.2 Hz, 3 H), 1.33 (s, 6 H),
1.62 (s, 3 H), 2.10 (s, 3 H), 2.53 (s, 2 H), 4.00 (br d, 2 H), 6.81
(s, 1 H), 7.19 (s, 1 H), 7.55 (dd, J=1.8 and 8.4Hz, 1 H), 7.85 (d,
J=2.4 Hz, 1 H), 7.97 (dd, J=2.1 and 8.4 Hz, 1 H), 10.05 (s, 1
H).
(1)
7-bromo-1-ethyl-4,4,6-trimethyl-3,4-dihydro-1H-quinoline-2-one
[0238] A mixture of powdered potassium hydroxide (3.35 g, 59.67
mmol) in DMSO (40 mL) was stirred at 0.degree. C. for 10 min.
7-bromo-4,4,6-trimethyl-3,4-dihydro-1H-quinoline-2-one (Example 2c)
(8.0 g, 29.83 mmol) was added cautiously, followed immediately by
the addition of ethyl iodide (12 mL, 149.17 mmol). The reaction
mixture was kept at 0.degree. C. for 30 min then slowly warmed up
to room temperature and stirred overnight at room temperature. The
reaction mixture was poured into water and extracted with
dichloromethane washed with water and brine, dried
(Mg.sub.2SO.sub.4), filtered and evaporated to give 8.8 g of
7-bromo-1,4,4,6-tetramethyl-3,4-dihydro-1H-quinoline-2-one and used
without further purification in the Suzuki coupling (step a).
.sup.1H NMR (300 MHz; CDCl.sub.3): 1.24 (t, J=7.2 Hz, 1 H), 1.25
(s, 6 H), 2.37 (s, 3 H), 2.45 (s, 2 H), 3.98 (q, 2 H), 7.13 (s, 1
H), 7.18 (s, 1 H).
(2) 7-bromo-4,4,6-trimethyl-3,4-dihydro-1H-quinoline-2-one
[0239] To a solution of 3-methyl-but-2-enoic acid
(3-bromo-4-methyl-phenyl- )-amide (70.0 g, 261 mmol, Example d) at
90.degree. C. was added portion wise, under argon, with vigorous
stirring aluminum chloride (52.3 g, 391 mmol) over 1.5 hr. The
reaction mixture was stirred for 2 hours at 110-120.degree. C. The
reaction mixture was cooled to room temperature and ice-water was
carefully added. The solution was extracted with dichloromethane
and the organic washed with 2N HCl, water, saturated aqueous
NaHCO.sub.3, water and brine, dried (Mg.sub.2SO.sub.4), filtered
and evaporated. The residue was crystallized from
dichloromethane/hexane to give 46 g of
7-bromo-4,4,6-trimethyl-3,4-dihydro-1H-quinoline-2-one. The mother
liquor was further chromatographed on silica gel(20% ethyl acetate
in hexane) to give 6.2 g more of product. (75%). .sup.1H NMR (300
MHz; CDCl.sub.3): 1.30 (s, 6 H), 2.33 (s, 3 H), 2.46 (s, 2 H), 7.07
(s, 1 H), 7.10 (s, 1 H), 9.87 (br s, 1 H).
(3) 3-Methyl-but-2-enoic acid (3-bromo-4-methyl-phenyl)-amide
[0240] To a biphasic mixture of 3-bromo-4-methylaniline (50 g,
0.269 mol, Example e), 10% NaOH (270 mL) and dichloromethane (160
mL) was added dropwise over a period of 2 hours
3,3-dimethylacryloyl chloride (36 mL, 0.322 mol) in dichloromethane
(95 mL). The solution was stirred at room temperature for 48 hours
then diluted with water (100 mL). The aqueous layer was further
extracted with dichloromethane. The organic layers were combined
and washed with water and brine, dried (Mg.sub.2SO.sub.4), filtered
and evaporated. The white solid was triturated with hexane and
collected to give 70 g (97%) of 3-Methyl-but-2-enoic acid
(3-bromo-4-methyl-phenyl)-amide. .sup.1H NMR (300 MHz; CDCl.sub.3):
1.89 (s, 3 H), 2.21 (s, 3 H), 2.33 (s, 3 H), 5.68 (s, 1 H), 7.14
(d, J=8.0 Hz, 1 H), 7.17 (br s, 1 H), 7.33 (d, J=8.0 Hz, 1 H), 7.79
(s, 1 H).
(4) 3-bromo-4-methylaniline
[0241] To a solution of 2-bromo-4-nitrotoluene (50 g, 0.231 mol in
ethylacetate (330 mL) and Ethanol (150 mL) was added
Tin(II)chloride dihydrate (208 g, 0.924 mol) portionwise. The
reaction mixture was stirred at room temperature overnight. The
solution was then treated with potassium carbonate until pH=7 and
filtered over celite. The filtrate was washed with water, aqueous
NaHCO.sub.3, water and brine, dried (Mg.sub.2SO.sub.4), filtered
and evaporated to give 42.71 g (100%) of 3-bromo-4-methylaniline.
.sup.1H NMR (300 MHz; CDCl.sub.3): 2.27 (s, 3 H), 3.57 (br s, 2 H),
6.54 (dd, J=2.7 Hz and 8.1 Hz, 1 H), 6.90 (d, J=2.1 Hz, 1 H), 6.98
(d, J=8.1 Hz, 1 H).
(5) 3-formyl-6-trifluoromethoxy-1-phenyl boronic acid
[0242] To a mixture of
2-(3-bromo-4-trifluoromethoxy-1-phenyl)-1,3-dioxola- ne (7.20 g,
22.9 mmol, Example g below) in THF (70 mL) cooled to -78.degree. C.
under an atmosphere of argon was added n-BuLi (13.8 mL, 2.5 M, 34.4
mmol) dropwise. The resulting suspension was stirred for 5 minutes
and triisopropylborate (15.9 mL, 68.7 mmol) was added dropwise via
syringe. The mixture was stirred at -50.degree. C. for 2 hours then
warmed up to room temperature and stirred overnight at room
temperature. 1.0 N HCl (50 mL) was slowly added to the reaction
mixture. After 3 hours the mixture was diluted with ethyl acetate
and the layers separated, the aqueous layer was extracted once with
ethyl acetate and the two organic layers combined. The resulting
organic layer was washed with water, brine and dried
(Mg.sub.2SO.sub.4). The mixture was filtered, evaporated and the
residue stirred in hexane. The resulting white suspension was
filtered and the white solid dried under high vacuum to afford 3.00
g of 3-formyl-6-trifluoromethoxy-1-phenyl boronic acid (56%).
.sup.1H NMR (300 MHz; CDCL.sub.3): .quadrature. 17.42 (d, J=7.0 Hz,
1 H), 8.07 (dd, J.sub.1=2.1 Hz, J.sub.2=8.7 Hz, 1 H), 8.47 (d,
J=1.8 Hz, 1 H), 10.05 (s, 1 H).
(6) 2-(3-bromo-4-trifluoromethoxy-1-phenyl)-1,3-dioxolane
[0243] To a solution of 3-bromo-4-trifluoromethoxybenzaldehyde (20
g, 74.0 mmol) in toluene (200 mL) was added ethylene glycol (82.6
mL, 1.48 mol) and p-toluenesulfonic acid monohydrate (0.84 g, 4.44
mmol). The reaction mixture was heated at reflux overnight and the
water was removed using a Dean Stark apparatus. The solution was
cooled to room temperature, poured into aqueous potassium carbonate
(10%) and extracted with ethyl acetate. The organic layer was
washed with water, brine and dried (Mg.sub.2SO.sub.4). The residue
was purified on silica gel (eluent: 10% ethyl acetate in hexane) to
give 15.4 g of 2-(3-bromo-4-trifluoromethoxy)- -1,3-dioxolane
(66%). .sup.1H NMR (500 MHz; CDCl.sub.3): .quadrature. 4.05 (m, 2
H), 4.11 (m, 2 H), 5.79 (s, 1 H), 7.32 (d, 1 H), 7.43 (d, 1 H),
7.77 (d, J=1.1 Hz, 1 H).
11. Example 11
5-[4-Dimethylamino-3-(8-methoxyimino-3,5,5-trimethyl-5,6,7,8-tetrahydro-na-
phthalen-2-yl)-benzylidene]-thiazolidine-2,4-dione
[0244] 17
[0245] Prepared in a similar manner to example 1. mp
254-256.degree. C. (300 MHz, DMSO) 1.27 (2 s, 6 H), 1.92 (t, J=6.6
Hz, 2H), 2.11 (s, 3 H), 2.56 (s, 6 H), 2.70 (t, J=6.6 Hz, 2 H),
3.86 (s, 3 H), 7.10 (d, J=9 Hz, 1 H), 7.22 (d, J=1.8 Hz, 1 H), 7.34
(s, 1 H), 7.49 (dd, J.sub.1=1.8 Hz, J.sub.2=8.7 Hz, 1 H), 7.68 (s,
1 H), 7.73 (s, 1 H), 12.44 (br s, 1H).
12. Example 12
5-[4-Chloro-3-(8-methoxyimino-3,5,5-trimethyl-5,6,7,8-tetrahydro-naphthale-
n-2-yl)-benzylidene]-thiazolidine-2,4-dione
[0246] 18
[0247] Prepared in a similar manner to example 1. mp
252-254.degree. C. (300 MHz, DMSO) 1.29 (s, 6 H), 1.70 (t, 2 H),
2.08 (s, 3 H), 2.71 (t, 2 H), 3.86 (s, 3 H), 7.40 (s, 1 H), 7.54
(s, 1 H), 7.59 (s, 1 H), 7.62 (d, J=8.4 Hz, 1 H), 7.75 (d, J=8.4
Hz, 1 H), 7.83 (s, 1 H), 12.68 (br s, 1 H).
D. REFERENCES
[0248] Iwatsuka H., Shino A., and Suzuoki Z. (1970) General Survey
of Diabetic Features of Yellow KK Mice. Endocrinol. Japon. 17:
23-35.
E. Sequences
[0249] SEQ ID NO: 1 RXR alpha
[0250] SEQ ID NO:2 RXR Beta
[0251] SEQ ID NO:3 RXR gamma
Sequence CWU 1
1
3 1 462 PRT Artificial Sequence Description of Artificial Sequence;
Note = synthetic construct 1 Met Asp Thr Lys His Phe Leu Pro Leu
Asp Phe Ser Thr Gln Val Asn 1 5 10 15 Ser Ser Leu Thr Ser Pro Thr
Gly Arg Gly Ser Met Ala Ala Pro Ser 20 25 30 Leu His Pro Ser Leu
Gly Pro Gly Ile Gly Ser Pro Gly Gln Leu His 35 40 45 Ser Pro Ile
Ser Thr Leu Ser Ser Pro Ile Asn Gly Met Gly Pro Pro 50 55 60 Phe
Ser Val Ile Ser Ser Pro Met Gly Pro His Ser Met Ser Val Pro 65 70
75 80 Thr Thr Pro Thr Leu Gly Phe Ser Thr Gly Ser Pro Gln Leu Ser
Ser 85 90 95 Pro Met Asn Pro Val Ser Ser Ser Glu Asp Ile Lys Pro
Pro Leu Gly 100 105 110 Leu Asn Gly Val Leu Lys Val Pro Ala His Pro
Ser Gly Asn Met Ala 115 120 125 Ser Phe Thr Lys His Ile Cys Ala Ile
Cys Gly Asp Arg Ser Ser Gly 130 135 140 Lys His Tyr Gly Val Tyr Ser
Cys Glu Gly Cys Lys Gly Phe Phe Lys 145 150 155 160 Arg Thr Val Arg
Lys Asp Leu Thr Tyr Thr Cys Arg Asp Asn Lys Asp 165 170 175 Cys Leu
Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr 180 185 190
Gln Lys Cys Leu Ala Met Gly Met Lys Arg Glu Ala Val Gln Glu Glu 195
200 205 Arg Gln Arg Gly Lys Asp Arg Asn Glu Asn Glu Val Glu Ser Thr
Ser 210 215 220 Ser Ala Asn Glu Asp Met Pro Val Glu Arg Ile Leu Glu
Ala Glu Leu 225 230 235 240 Ala Val Glu Pro Lys Thr Glu Thr Tyr Val
Glu Ala Asn Met Gly Leu 245 250 255 Asn Pro Ser Ser Pro Asn Asp Pro
Val Thr Asn Ile Cys Gln Ala Ala 260 265 270 Asp Lys Gln Leu Phe Thr
Leu Val Glu Trp Ala Lys Arg Ile Pro His 275 280 285 Phe Ser Glu Leu
Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly 290 295 300 Trp Asn
Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val 305 310 315
320 Lys Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser
325 330 335 Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu
Thr Glu 340 345 350 Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys
Thr Glu Leu Gly 355 360 365 Cys Leu Arg Ala Ile Val Leu Phe Asn Pro
Asp Ser Lys Gly Leu Ser 370 375 380 Asn Pro Ala Glu Val Glu Ala Leu
Arg Glu Lys Val Tyr Ala Ser Leu 385 390 395 400 Glu Ala Tyr Cys Lys
His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala 405 410 415 Lys Leu Leu
Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys 420 425 430 Leu
Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp 435 440
445 Thr Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Met Thr 450 455
460 2 533 PRT Artificial Sequence Description of Artificial
Sequence; Note = synthetic construct 2 Met Ser Trp Ala Ala Arg Pro
Pro Phe Leu Pro Gln Arg His Ala Ala 1 5 10 15 Gly Gln Cys Gly Pro
Val Gly Val Arg Lys Glu Met His Cys Gly Val 20 25 30 Ala Ser Arg
Trp Arg Arg Arg Arg Pro Trp Leu Asp Pro Ala Ala Ala 35 40 45 Ala
Ala Ala Ala Val Ala Gly Gly Glu Gln Gln Thr Pro Glu Pro Glu 50 55
60 Pro Gly Glu Ala Gly Arg Asp Gly Met Gly Asp Ser Gly Arg Asp Ser
65 70 75 80 Arg Ser Pro Asp Ser Ser Ser Pro Asn Pro Leu Pro Gln Gly
Val Pro 85 90 95 Pro Pro Ser Pro Pro Gly Pro Pro Leu Pro Pro Ser
Thr Ala Pro Ser 100 105 110 Leu Gly Gly Ser Gly Ala Pro Pro Pro Pro
Pro Met Pro Pro Pro Pro 115 120 125 Leu Gly Ser Pro Phe Pro Val Ile
Ser Ser Ser Met Gly Ser Pro Gly 130 135 140 Leu Pro Pro Pro Ala Pro
Pro Gly Phe Ser Gly Pro Val Ser Ser Pro 145 150 155 160 Gln Ile Asn
Ser Thr Val Ser Leu Pro Gly Gly Gly Ser Gly Pro Pro 165 170 175 Glu
Asp Val Lys Pro Pro Val Leu Gly Val Arg Gly Leu His Cys Pro 180 185
190 Pro Pro Pro Gly Gly Pro Gly Ala Gly Lys Arg Leu Cys Ala Ile Cys
195 200 205 Gly Asp Arg Ser Ser Gly Lys His Tyr Gly Val Tyr Ser Cys
Glu Gly 210 215 220 Cys Lys Gly Phe Phe Lys Arg Thr Ile Arg Lys Asp
Leu Thr Tyr Ser 225 230 235 240 Cys Arg Asp Asn Lys Asp Cys Thr Val
Asp Lys Arg Gln Arg Asn Arg 245 250 255 Cys Gln Tyr Cys Arg Tyr Gln
Lys Cys Leu Ala Thr Gly Met Lys Arg 260 265 270 Glu Ala Val Gln Glu
Glu Arg Gln Arg Gly Lys Asp Lys Asp Gly Asp 275 280 285 Gly Glu Gly
Ala Gly Gly Ala Pro Glu Glu Met Pro Val Asp Arg Ile 290 295 300 Leu
Glu Ala Glu Leu Ala Val Glu Gln Lys Ser Asp Gln Gly Val Glu 305 310
315 320 Gly Pro Gly Gly Thr Gly Gly Ser Gly Ser Ser Pro Asn Asp Pro
Val 325 330 335 Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr
Leu Val Glu 340 345 350 Trp Ala Lys Arg Ile Pro His Phe Ser Ser Leu
Pro Leu Asp Asp Gln 355 360 365 Val Ile Leu Leu Arg Ala Gly Trp Asn
Glu Leu Leu Ile Ala Ser Phe 370 375 380 Ser His Arg Ser Ile Asp Val
Arg Asp Gly Ile Leu Leu Ala Thr Gly 385 390 395 400 Leu His Val His
Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile 405 410 415 Phe Asp
Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Arg 420 425 430
Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Ile Leu Phe Asn 435
440 445 Pro Asp Ala Lys Gly Leu Ser Asn Pro Ser Glu Val Glu Val Leu
Arg 450 455 460 Glu Lys Val Tyr Ala Ser Leu Glu Thr Tyr Cys Lys Gln
Lys Tyr Pro 465 470 475 480 Glu Gln Gln Gly Arg Phe Ala Lys Leu Leu
Leu Arg Leu Pro Ala Leu 485 490 495 Arg Ser Ile Gly Leu Lys Cys Leu
Glu His Leu Phe Phe Phe Lys Leu 500 505 510 Ile Gly Asp Thr Pro Ile
Asp Thr Phe Leu Met Glu Met Leu Glu Ala 515 520 525 Pro His Gln Leu
Ala 530 3 463 PRT Artificial Sequence Description of Artificial
Sequence; Note = synthetic construct 3 Met Tyr Gly Asn Tyr Ser His
Phe Met Lys Phe Pro Ala Gly Tyr Gly 1 5 10 15 Gly Ser Pro Gly His
Thr Gly Ser Thr Ser Met Ser Pro Ser Ala Ala 20 25 30 Leu Ser Thr
Gly Lys Pro Met Asp Ser His Pro Ser Tyr Thr Asp Thr 35 40 45 Pro
Val Ser Ala Pro Arg Thr Leu Ser Ala Val Gly Thr Pro Leu Asn 50 55
60 Ala Leu Gly Ser Pro Tyr Arg Val Ile Thr Ser Ala Met Gly Pro Pro
65 70 75 80 Ser Gly Ala Leu Ala Ala Pro Pro Gly Ile Asn Leu Val Ala
Pro Pro 85 90 95 Ser Ser Gln Leu Asn Val Val Asn Ser Val Ser Ser
Ser Glu Asp Ile 100 105 110 Lys Pro Leu Pro Gly Leu Pro Gly Ile Gly
Asn Met Asn Tyr Pro Ser 115 120 125 Thr Ser Pro Gly Ser Leu Val Lys
His Ile Cys Ala Ile Cys Gly Asp 130 135 140 Arg Ser Ser Gly Lys His
Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys 145 150 155 160 Gly Phe Phe
Lys Arg Thr Ile Arg Lys Asp Leu Ile Tyr Thr Cys Arg 165 170 175 Asp
Asn Lys Asp Cys Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln 180 185
190 Tyr Cys Arg Tyr Gln Lys Cys Leu Val Met Gly Met Lys Arg Glu Ala
195 200 205 Val Gln Glu Glu Arg Gln Arg Ser Arg Glu Arg Ala Glu Ser
Glu Ala 210 215 220 Glu Cys Ala Thr Ser Gly His Glu Asp Met Pro Val
Glu Arg Ile Leu 225 230 235 240 Glu Ala Glu Leu Ala Val Glu Pro Lys
Thr Glu Ser Tyr Gly Asp Met 245 250 255 Asn Met Glu Asn Ser Thr Asn
Asp Pro Val Thr Asn Ile Cys His Ala 260 265 270 Ala Asp Lys Gln Leu
Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro 275 280 285 His Phe Ser
Asp Leu Thr Leu Glu Asp Gln Val Ile Leu Leu Arg Ala 290 295 300 Gly
Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Val Ser 305 310
315 320 Val Gln Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg
Ser 325 330 335 Ser Ala His Ser Ala Gly Val Gly Ser Ile Phe Asp Arg
Val Leu Thr 340 345 350 Glu Leu Val Ser Lys Met Lys Asp Met Gln Met
Asp Lys Ser Glu Leu 355 360 365 Gly Cys Leu Arg Ala Ile Val Leu Phe
Asn Pro Asp Ala Lys Gly Leu 370 375 380 Ser Asn Pro Ser Glu Val Glu
Thr Leu Arg Glu Lys Val Tyr Ala Thr 385 390 395 400 Leu Glu Ala Tyr
Thr Lys Gln Lys Tyr Pro Glu Gln Pro Gly Arg Phe 405 410 415 Ala Lys
Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys 420 425 430
Cys Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile 435
440 445 Asp Thr Phe Leu Met Glu Met Leu Glu Thr Pro Leu Gln Ile Thr
450 455 460
* * * * *