U.S. patent application number 13/327951 was filed with the patent office on 2012-04-12 for s1p receptor modulating compounds and use thereof.
This patent application is currently assigned to EPIX PHARMACEUTICALS, INC.. Invention is credited to Roland Burli, Victor Cee, Brian Lanman, Susana C. Neira, Xiang Yu.
Application Number | 20120088749 13/327951 |
Document ID | / |
Family ID | 40468694 |
Filed Date | 2012-04-12 |
United States Patent
Application |
20120088749 |
Kind Code |
A1 |
Neira; Susana C. ; et
al. |
April 12, 2012 |
S1P Receptor Modulating Compounds and Use Thereof
Abstract
The present invention relates to amides that have activity as
S1P receptor modulating agents and the use of such compounds to
treat diseases associated with inappropriate S1P receptor activity.
The compounds may be used as immunomodulators, e.g., for treating
or preventing diseases such as autoimmune and related immune
disorders including systemic lupus erythematosus, inflammatory
bowel diseases such as Crohn's disease and ulcerative colitis, type
I diabetes, uveitis, psoriasis, myasthenia gravis, rheumatoid
arthritis, non-glomerular nephrosis, hepatitis, Behcet's disease,
glomerulonephritis, chronic thrombocytopenic purpura, hemolytic
anemia, hepatitis and Wegner's granuloma; and for treating other
conditions.
Inventors: |
Neira; Susana C.; (Thousand
Oaks, CA) ; Yu; Xiang; (Acton, MA) ; Burli;
Roland; (Bishop's Stortford, GB) ; Cee; Victor;
(Thousand Oaks, CA) ; Lanman; Brian; (Oak Park,
CA) |
Assignee: |
EPIX PHARMACEUTICALS, INC.
Lexington
MA
AMGEN, INC.
Thousand Oaks
CA
|
Family ID: |
40468694 |
Appl. No.: |
13/327951 |
Filed: |
December 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12234514 |
Sep 19, 2008 |
8080542 |
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13327951 |
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60994812 |
Sep 20, 2007 |
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Current U.S.
Class: |
514/210.18 ;
514/210.17; 546/268.1; 548/953 |
Current CPC
Class: |
A61P 15/00 20180101;
C07D 205/04 20130101; A61P 35/00 20180101; A61P 17/00 20180101;
A61P 1/16 20180101; A61P 11/06 20180101; A61P 7/04 20180101; A61P
17/02 20180101; A61P 25/00 20180101; A61P 7/06 20180101; A61P 1/18
20180101; A61P 11/00 20180101; A61P 19/02 20180101; A61P 43/00
20180101; A61P 13/12 20180101; A61P 17/06 20180101; A61P 27/02
20180101; A61P 3/10 20180101; A61P 21/04 20180101; A61P 13/08
20180101; A61P 1/00 20180101; A61P 19/04 20180101; A61P 1/04
20180101; A61P 5/00 20180101; A61P 35/02 20180101; A61P 29/00
20180101; C07D 401/10 20130101; A61P 37/06 20180101; A61P 9/00
20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/210.18 ;
548/953; 514/210.17; 546/268.1 |
International
Class: |
A61K 31/397 20060101
A61K031/397; C07D 401/10 20060101 C07D401/10; A61K 31/4427 20060101
A61K031/4427; A61P 37/06 20060101 A61P037/06; A61P 29/00 20060101
A61P029/00; A61P 35/02 20060101 A61P035/02; A61P 3/10 20060101
A61P003/10; A61P 25/00 20060101 A61P025/00; A61P 17/06 20060101
A61P017/06; A61P 1/04 20060101 A61P001/04; A61P 1/00 20060101
A61P001/00; C07D 205/04 20060101 C07D205/04; A61P 19/04 20060101
A61P019/04 |
Claims
1. A compound having the formula ##STR00030## or a
pharmaceutically-acceptable salt thereof, wherein: A is phenyl or a
six-membered heteroaryl containing 1 or 2 N atoms, the phenyl and
heteroaryl being substituted with 0, 1, 2, or 3 substituents
selected from F, Cl, Br, OC.sub.1-4alk, C.sub.1-4alk, and
C.sub.1-4haloalk; B is phenyl or a six-membered heteroaryl
containing 1 or 2 N atoms, the phenyl and heteroaryl being
substituted with 0, 1, 2, or 3 substituents selected from F, Cl,
Br, OC.sub.1-4alk, C.sub.1-4alk, and C.sub.1-4haloalk; wherein at
least one of A and B is a six-membered heteroaryl containing 1 or 2
N atoms and substituted with 0, 1, 2, or 3 substituents selected
from F, Cl, Br, OC.sub.1-4alk C.sub.1-4alk, and C.sub.1-4haloalk; L
is --C.ident.C--, --CH.sub.2CH.sub.2--, --N(R.sup.a)C(.dbd.O)--, or
--C(.dbd.O)N(R.sup.a)--; R.sup.a is, independently in each
instance, H or C.sub.1-6alk; R.sup.1 is selected from C.sub.1-6alk,
OC.sub.1-5alk, N(R.sup.a)C.sub.1-5alk, N(C.sub.1-5alk)C.sub.1-5alk,
aryl, and heteroaryl; X is selected from WC(.dbd.O)OR.sup.6a,
WP(.dbd.O)R.sup.6bR.sup.6c, WS(O).sub.2OH, WCONHSO.sub.3H, and
1H-tetrazol-5-yl; W is a direct bond, oxygen, or C.sub.1-4alk
having one or more substituents independently selected from
halogen, OH, cyano, NR.sup.aR.sup.a, arylamino, heteroarylamino,
OC.sub.1-4alk, and CO.sub.2H; R.sup.6a is hydrogen or C.sub.1-4alk;
R.sup.6b and R.sup.6c are independently hydrogen, OH, C.sub.1-4alk,
or C.sub.1-4haloalk; Y is residue of formula (a) wherein the left
and right asterisks indicate the point of attachment ##STR00031##
wherein the N, designated by the left asterisk, is attached to
Z.sup.1, and the carbon, designated by the right asterisk, is
attached to X, wherein Q is selected from a direct bond, C.dbd.O,
C.dbd.S, SO.sub.2, C.dbd.ONR.sup.a, and (CR.sup.10R.sup.11).sub.m;
m is 0, 1, 2, or 3; R.sup.7 and R.sup.8 are independently selected
from hydrogen, halogen, amino, C.sub.1-5alkamino, OH, cyano,
C.sub.1-6alk, C.sub.1-6alk(OH), SC.sub.1-5alk, OC.sub.1-5alk,
C.sub.1-6haloalk, and OC.sub.1-5alk; or R.sup.7 and R.sup.8 may be
joined together with the atoms to which they are attached atoll
form a 4- to 7-membered ring optionally containing an additional
heteroatom selected from N, O, and S; R.sup.9 is selected from
hydrogen, halogen, OH, cyano, C.sub.1-6alk, SC.sub.1-5alk,
OC.sub.1-5alk, C.sub.1-6haloalk, and OC.sub.1-5haloalk; R.sup.10
and R.sup.11 are independently selected from hydrogen, halogen, OH,
cyano, C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk,
C.sub.1-6haloalk, and OC.sub.1-5haloalk; Z.sup.1 and Z.sup.2 are
independently selected from O, NR.sup.3, S, S(.dbd.O),
S(.dbd.O).sub.2, S(.dbd.O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n,
C.dbd.O, C.dbd.S, C.dbd.N--R.sup.3, and a direct bond, wherein n is
0, 1, 2, or 3; R.sup.3 is selected from hydrogen, OH, SO.sub.2,
C.dbd.O, C.dbd.S, C.dbd.NH, C.sub.1-6alk, OC.sub.1-5alk,
SC.sub.1-5alk, C.sub.1-6haloalk, OC.sub.1-5haloalk, aryl, and
heteroaryl; or when Z.sup.2 is a direct bond, R.sup.3 is a
C.sub.3-C.sub.6 ring optionally containing a heteroatom; and
R.sup.4 and R.sup.5 are independently selected from hydrogen,
halogen, OH, cyano, C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk,
C.sub.1-6haloalk, OC.sub.1-5haloalk, aryl, and heteroaryl, or
R.sup.4 and R.sup.5 taken together with the carbon atom to which
they are attached form C.dbd.O.
2. A compound according to claim 1, wherein A is phenyl
additionally substituted by 0, 1, 2, or 3 substituents selected
from F, Cl, Br, OC.sub.1-4alk, C.sub.1-4alk, and
C.sub.1-4haloalk.
3. A compound according to claim 1, wherein B is phenyl
additionally substituted by 0, 1, 2, or 3 substituents selected
from F, Cl, Br, OC.sub.1-4alk, C.sub.1-4alk, and
C.sub.1-4haloalk.
4. (canceled)
5. A compound according to claim 1, wherein L is
--N(R.sup.a)C(.dbd.O)--.
6. A compound according to claim 1, wherein L is
--C(.dbd.O)N(R.sup.a)--.
7. A compound according to claim 1, wherein --Y--X is
##STR00032##
8. A compound having the formula ##STR00033## or a
pharmaceutically-acceptable salt thereof, wherein: A is phenyl or a
six-membered heteroaryl containing 1 or 2 N atoms, the phenyl and
heteroaryl being substituted with 0, 1, 2, or 3 substituents
selected from F, Cl, Br, OC.sub.1-4alk, C.sub.1-4alk, and
C.sub.1-4haloalk; B is phenyl or a six-membered heteroaryl
containing 1 or 2 N atoms, the phenyl and heteroaryl being
substituted with 0, 1, 2, or 3 substituents selected from F, Cl,
Br, OC.sub.1-4alk, C.sub.1-4alk, and C.sub.1-4haloalk; wherein at
least one of A and B is a six-membered heteroaryl containing 1 or 2
N atoms and substituted with 0, 1, 2, or 3 substituents selected
from F, Cl, Br, OC.sub.1-4alk C.sub.1-4alk, and C.sub.1-4haloalk; L
is --N(R.sup.a)C(.dbd.O)-- or --C(.dbd.O)N(R.sup.a)--; n is 0, 1,
2, or 3; R.sup.a is, independently in each instance, H or
C.sub.1-6alk; R.sup.1 is selected from C.sub.1-6alk, OC.sub.1-5alk,
N(R.sup.a)C.sub.1-5alk, N(C.sub.1-5alk)C.sub.1-5alk, aryl, and
heteroaryl; Z.sup.2 is selected from O, NR.sup.3, S, S(.dbd.O),
S(.dbd.O).sub.2, S(.dbd.O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n,
C.dbd.O, C.dbd.S, C.dbd.N--R.sup.3, and a direct bond; wherein
R.sup.3 is selected from hydrogen, OH, SO.sub.2, C.dbd.O, C.dbd.S,
C.dbd.NH, C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk,
C.sub.1-6haloalk, OC.sub.1-5haloalk, aryl, and heteroaryl; or when
Z.sup.2 is a direct bond, R.sup.3 is a C.sub.3-C.sub.6 ring
optionally containing a heteroatom; and R.sup.4 and R.sup.5 are
independently selected from hydrogen, halogen, OH, cyano,
C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk, C.sub.1-6haloalk,
OC.sub.1-5haloalk, aryl, and heteroaryl, or R.sup.4 and R.sup.5
taken together with the carbon atom to which they are attached form
C.dbd.O.
9. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable carrier or diluent.
10. A method of treating a condition selected from transplant
rejection related to solid organ transplant and islet cells;
transplant rejection related to tissue; rheumatoid arthritis;
lupus; insulin dependent diabetes; non-insulin dependent diabetes;
multiple sclerosis; psoriasis; ulcerative colitis; inflammatory
bowel disease; Crohn's disease; and acute and chronic lymphocytic
leukemias and lymphomas, wherein treating is lessening or or
reducing the symptoms of the condition, comprising administering to
a patient in need thereof a compound according to claim 1 in an
amount effective to treat the condition.
11. A compound according to claim 1 that is Methyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxy-
late;
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-ca-
rboxylic acid; or
1-(4-(2-(5-benzylpyridin-2-yl)ethyl)-3-fluorobenzyl)azetidine-3-carboxyli-
c acid.
Description
[0001] This application claims the benefit of U.S. provisional No.
60/994,812, filed Sep. 20, 2007, the content of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compounds that have
activity as S1P receptor modulating agents and the use of such
compounds to treat diseases associated with inappropriate S1P
receptor activity.
BACKGROUND OF THE INVENTION
[0003] Sphingosine-1-phosphate (S1P) has been demonstrated to
induce many cellular effects, including those that result in
platelet aggregation, cell proliferation, cell morphology, tumor
cell invasion, endothelial cell chemotaxis and endothelial cell in
vitro angiogenesis. S1P receptors are therefore good targets for
therapeutic applications such as wound healing and tumor growth
inhibition. S1P signals cells in part via a set of G
protein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5
(formerly called EDG-1, EDG-5, EDG-3, EDG-6, and EDG-8,
respectively). These receptors share 50-55% amino acid and cluster
identity with three other receptors (LPA1, LPA2, and LPA3 (formerly
EDG-2, EDG-4 and EDG-7)) for the structurally-related
lysophosphatidic acid (LPA).
[0004] A conformational shift is induced in the G-Protein Coupled
Receptor (GPCR) when the ligand binds to that receptor, causing GDP
to be replaced by GTP on the .alpha.-subunit of the associated
G-proteins and subsequent release of the G-proteins into the
cytoplasm. The .alpha.-subunit then dissociates from the
.beta..gamma.-subunit, and each subunit can then associate with
effector proteins, which activate second messengers leading to a
cellular response. Eventually the GTP on the G-proteins is
hydrolyzed to GDP, and the subunits of the G-proteins re-associate
with each other and then with the receptor. Amplification plays a
major role in the general GPCR pathway. The binding of one ligand
to one receptor leads to the activation of many G-proteins, each
capable of associating with many effector proteins, leading to an
amplified cellular response.
[0005] S1P receptors make good drug targets, because individual
receptors are both tissue- and response-specific. Tissue
specificity of the S1P receptors is important, because development
of an agonist or antagonist selective for one receptor localizes
the cellular response to tissues containing that receptor, limiting
unwanted side effects. Response specificity of the S1P receptors is
also important because it allows for development of agonists or
antagonists that initiate or to suppress certain cellular responses
without affecting other things. For example, the response
specificity of the S1P receptors could allow for an S1P mimetic
that initiates platelet aggregation without affecting cell
morphology.
[0006] S1P is formed as a metabolite of sphingosine in its reaction
with sphingosine kinase, and is abundantly stored in platelet
aggregates where high levels of sphingosine kinase exist and
sphingosine lyase is lacking. S1P is released during platelet
aggregation, accumulates in serum and is also found in malignant
ascites. S1P biodegradation most likely proceeds via hydrolysis by
ectophosphohydrolases, specifically the sphingosine 1-phosphate
phosphohydrolases.
SUMMARY OF THE INVENTION
[0007] The present invention relates to the use of new compositions
which include S1P modulators, e.g., agonists, partial agonists,
inverse agonists and antagonists, and their use in treating,
preventing or curing various S1P receptor-related conditions. The
invention features compounds which are S1P receptor modulators; in
an embodiment, such compounds include those having the formula
##STR00001##
[0008] and pharmaceutically acceptable salts thereof, wherein
R.sup.1, Z.sup.2, L, B, A, Z.sup.1, Z.sup.2, Y and X are defined
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0009] In one embodiment, the present invention relates to a
compound having the formula
##STR00002## [0010] or a pharmaceutically-acceptable salt thereof,
wherein: [0011] A is phenyl or a six-membered heteroaryl containing
1 or 2 N atoms, the phenyl and heteroaryl being substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk; [0012] B is phenyl or a
six-membered heteroaryl containing 1 or 2 N atoms, the phenyl and
heteroaryl being substituted by 0, 1, 2 or 3 substituents selected
from F, Cl, Br, OC.sub.1-4alk, C.sub.1-4alk, and C.sub.1-4haloalk;
[0013] L is --C.ident.C--, --CH.sub.2CH.sub.2--,
--N(R.sup.a)C(.dbd.O)-- or --C(.dbd.O)N(R.sup.a)--; [0014] n is 0,
1, 2 or 3; [0015] R.sup.a is, independently in each instance; H or
C.sub.1-6alk; [0016] R.sup.1 is selected from C.sub.1-6alk,
OC.sub.1-5alk, N(R.sup.a)C.sub.1-5alk, N(C.sub.1-5alk)C.sub.1-5alk,
aryl or heteroaryl. [0017] X is selected from WC(.dbd.O)OR.sup.6a,
WP(.dbd.O)R.sup.6bR.sup.6c, WS(.dbd.O).sub.2OH, WCONHSO.sub.3H or
1H-tetrazol-5-yl; wherein W is a direct bond, oxygen or
C.sub.1-4alk having one or more substituents independently selected
from halogen, OH, cyano, NR.sup.aR.sup.a, arylamino,
heteroarylamino, OC.sub.1-4alk and CO.sub.2H; R.sup.6a is hydrogen
or C.sub.1-4alk; and [0018] R.sup.6b and R.sup.6c are independently
hydrogen, OH, C.sub.1-4alk or C.sub.1-4haloalk; [0019] Y is residue
of formula (a) wherein the left and right asterisks indicate the
point of attachment
[0019] ##STR00003## [0020] wherein [0021] Q is selected from a
direct bond, C.dbd.O, C.dbd.S, SO.sub.2, C.dbd.ONR.sup.a or
(CR.sup.10R.sup.11).sub.m; and m is 0, 1, 2 or 3; [0022] R.sup.7
and R.sup.8 are independently selected from hydrogen, halogen,
amino, C.sub.1-5alkamino, OH, cyano, C.sub.1-6alk,
C.sub.1-6alk(OH), SC.sub.1-5alk, OC.sub.1-5alk, C.sub.1-6haloalk
and OC.sub.1-5alk; or R.sup.7 and R.sup.8 may be joined together
with the atoms to which they are attached to form a 4- to
7-membered ring, optionally having a heteroatom selected from N, O
and S; and [0023] R.sup.9 is selected from hydrogen, halogen, OH,
cyano, C.sub.1-6alk, SC.sub.1-5alk, OC.sub.1-5alk, C.sub.1-6haloalk
or OC.sub.1-5haloalk; [0024] R.sup.10 and R.sup.11 are
independently selected from hydrogen, halogen, OH, cyano,
C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk, C.sub.1-6haloalk or
OC.sub.1-5haloalk and [0025] Z.sup.1 and Z.sup.2 are independently
selected from O, NR.sup.3, S, S(.dbd.O), S(.dbd.O).sub.2,
S(.dbd.O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S,
C.dbd.N--R.sup.3, or a direct bond, wherein [0026] R.sup.3 is
selected from hydrogen, OH, SO.sub.2, C.dbd.O, C.dbd.S, C.dbd.NH,
C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk, C.sub.1-6haloalk and
OC.sub.1-5haloalk, aryl or heteroaryl; or when Z.sup.2 is a direct
bond, R.sub.3 is a C.sub.3-C.sub.6 ring optionally containing a
heteroatom; and [0027] R.sup.4 and R.sup.5 are independently
selected from hydrogen, halogen, OH, cyano, C.sub.1-6alk,
OC.sub.1-5alk, SC.sub.1-5alk, C.sub.1-6haloalk and
OC.sub.1-5haloalk, aryl and heteroaryl or together form
C.dbd.O.
[0028] In another embodiment, in conjunction with any above or
below embodiments, A is phenyl additionally substituted by 0, 1, 2
or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0029] In another embodiment, in conjunction with any above or
below embodiments, A is 1,4-phenyl additionally substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0030] In another embodiment, in conjunction with any above or
below embodiments, A is 1,3-phenyl additionally substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0031] In another embodiment, in conjunction with any above or
below embodiments, A is 1,2-phenyl additionally substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0032] In another embodiment, in conjunction with any above or
below embodiments, A is pyrdinyl additionally substituted by 0, 1,
2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0033] In another embodiment, in conjunction with any above or
below embodiments, A is pyrimidinyl additionally substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0034] In another embodiment, in conjunction with any above or
below embodiments, B is phenyl additionally substituted by 0, 1, 2
or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0035] In another embodiment, in conjunction with any above or
below embodiments, B is pyrdinyl additionally substituted by 0, 1,
2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0036] In another embodiment, in conjunction with any above or
below embodiments, B is pyrimidinyl additionally substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk.
[0037] In another embodiment, in conjunction with any above or
below embodiments, A is phenyl additionally substituted by 0, 1, 2
or 3 substituents selected from F, Cl, Br, OC.sub.1-4alk,
C.sub.1-4alk, and C.sub.1-4haloalk; and B is phenyl additionally
substituted by 0, 1, 2 or 3 substituents selected from F, Cl, Br,
OC.sub.1-4alk, C.sub.1-4alk, and C.sub.1-4haloalk.
[0038] In another embodiment, in conjunction with any above or
below embodiments, L is --N(R.sup.a)C(.dbd.O)--.
[0039] In another embodiment, in conjunction with any above or
below embodiments, L is --C(.dbd.O)N(R.sup.a)--.
[0040] In another embodiment, in conjunction with any above or
below embodiments, L is --C.ident.C--.
[0041] In another embodiment, in conjunction with any above or
below embodiments, L is --CH.sub.2CH.sub.2--.
[0042] In another embodiment, in conjunction with any above or
below embodiments, --Y--X is
##STR00004##
[0043] Another aspect of the invention relates to a compound having
the formula
##STR00005## [0044] or a pharmaceutically-acceptable salt thereof,
wherein: [0045] A is phenyl or a six-membered heteroaryl containing
1 or 2 N atoms, the phenyl and heteroaryl being substituted by 0,
1, 2 or 3 substituents selected from F, Cl, Br, C.sub.1-4alk, and
C.sub.1-4haloalk; [0046] B is phenyl or a six-membered heteroaryl
containing 1 or 2 N atoms, the phenyl and heteroaryl being
substituted by 0, 1, 2 or 3 substituents selected from F, Cl, Br,
C.sub.1-4alk, and C.sub.1-4haloalk; [0047] L is
--N(R.sup.a)C(.dbd.O)-- or --C(.dbd.O)N(R.sup.a)--; [0048] n is 0,
1, 2 or 3; [0049] R.sup.a is, independently in each instance; H or
C.sub.1-6alk; [0050] R.sup.1 is selected from C.sub.1-6alk,
OC.sub.1-5alk, N(R.sup.a)C.sub.1-5 alk,
N(C.sub.1-5alk)C.sub.1-5alk, aryl or heteroaryl. [0051] Z.sup.1 is
selected from O, NR.sup.3, S, S(.dbd.O), S(.dbd.O).sub.2,
S(.dbd.O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S,
C.dbd.N--R.sup.3, or a direct bond, wherein [0052] R.sup.3 is
selected from hydrogen, OH, SO.sub.2, C.dbd.O, C.dbd.S, C.dbd.NH,
C.sub.1-6alk, OC.sub.1-5alk, SC.sub.1-5alk, C.sub.1-6haloalk and
OC.sub.1-5haloalk, aryl or heteroaryl; or when Z.sup.2 is a direct
bond, R.sub.3 is a C.sub.3-C.sub.6 ring optionally containing a
heteroatom; and [0053] R.sup.4 and R.sup.5 are independently
selected from hydrogen, halogen, OH, cyano, C.sub.1-6alk,
OC.sub.1-5alk, SC.sub.1-5alk, C.sub.1-6haloalk and
OC.sub.1-5haloalk, aryl and heteroaryl or together form
C.dbd.O.
[0054] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is selected from phenyl
and heteroaryl; both of which are optionally substituted with
halogen.
[0055] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is phenyl optionally
substituted with halogen.
[0056] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is heteroaryl optionally
substituted with halogen.
[0057] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is phenyl.
[0058] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is heteroaryl.
[0059] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is 5- or 6-membered
unsaturated ring including one atom selected from N, O and S, and
0, 1, 2 or 3 additional N atoms.
[0060] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is selected from
pyridinyl, pyrimidine, thiazolyl, oxazolyl, furanyl and
thiophenyl.
[0061] In another embodiment of the invention, in conjunction with
the above and below embodiments, X is WC(O)OR.sup.6a,
WP(O)R.sup.6bR.sup.6c, WS(O).sub.2OH, WCONHSO.sub.3H or
1H-tetrazol-5-yl. W is a direct bond, oxygen or C.sub.1-4 alkyl
with substituents independently selected from halogen, hydroxyl,
cyano, amino, alkylamino, arylamino, heteroarylamino and C.sub.1-4
alkoxy; and R.sup.6a is hydrogen or C.sub.1-4alkyl; R.sup.6b and
R.sup.6c are independently selected from hydrogen, hydroxyl,
C.sub.1-4alkyl and halogen substituted C.sub.1-4alkyl.
[0062] In another embodiment of the invention, in conjunction with
the above and below embodiments, X is CO.sub.2H.
[0063] In another embodiment of the invention, in conjunction with
the above and below embodiments, Y is
##STR00006##
wherein R.sup.9 is selected from hydrogen, halogen, hydroxyl,
cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5 alkoxy,
halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy.
[0064] In another embodiment of the invention, in conjunction with
the above and below embodiments, Y is
##STR00007##
wherein R.sup.9 is selected from hydrogen, halogen and
hydroxyl.
[0065] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.9 is hydrogen.
[0066] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.9 is hydroxyl.
[0067] In another embodiment of the invention, in conjunction with
the above and below embodiments, Z.sup.1 is CR.sup.4R.sup.5;
wherein R.sup.4 and R.sup.5 are independently hydrogen, halogen,
hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5
alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy.
[0068] In another embodiment of the invention, in conjunction with
the above and below embodiments, Z.sup.1 is CR.sup.4R.sup.5;
wherein R.sup.4 and R.sup.5 are independently hydrogen, halogen,
C.sub.1-6 alkyl or halogen-substituted C.sub.1-6 alkyl.
[0069] In another embodiment of the invention, in conjunction with
the above and below embodiments, Z.sup.1 is CH.sub.2.
[0070] In another embodiment of the invention, in conjunction with
the above and below embodiments,
[0071] Z.sup.2 is selected from O, NR.sup.3, S, S(O), S(O).sub.2,
S(O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S, and
C.dbd.N--R.sup.3;
[0072] R.sup.3 is hydrogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy;
[0073] R.sup.4 and R.sup.5 are independently selected from
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy, or together form
"C.dbd.O"; and
[0074] n is 1, 2 or 3.
[0075] In another embodiment of the invention, in conjunction with
the above and below embodiments,
[0076] Z.sup.2 is selected from O, NR.sup.3, S, S(O), S(O).sub.2,
S(O).sub.2NR.sup.3, CR.sup.4R.sup.5, C.dbd.O, C.dbd.S, and
C.dbd.N--R.sup.3;
[0077] R.sup.3 is hydrogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy; and
[0078] R.sup.4 and R.sup.5 are independently selected from
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy.
[0079] In another embodiment of the invention, in conjunction with
the above and below embodiments Z.sup.2 is selected from O,
NR.sup.3, S, CR.sup.4R.sup.5, C.dbd.O, C.dbd.S, and
C.dbd.N--R.sup.3;
[0080] R.sup.3 is hydrogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy; and
[0081] R.sup.4 and R.sup.5 are independently selected from
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy.
[0082] In another embodiment of the invention, in conjunction with
the above and below embodiments Z.sup.2 is selected from O, S,
CH.sub.2, C.dbd.O, C.dbd.S and C.dbd.N--OH.
[0083] The specification and claims contain listing of species
using the language "selected from . . . and . . . " and "is . . .
or . . . " (sometimes referred to as Markush groups). When this
language is used in this application, unless otherwise stated it is
meant to include the group as a whole, or any single members
thereof, or any subgroups thereof. The use of this language is
merely for shorthand purposes and is not meant in any way to limit
the removal of individual elements or subgroups as needed.
[0084] In one aspect, the present invention provides methods for
modulating S1P-1 receptor mediated biological activity. The present
invention also provides methods for using S1P-1 modulators (i.e.,
agonists or antagonists) in treating or preventing diseases such as
ovarian cancer, peritoneal cancer, endometrial cancer, cervical
cancer, breast cancer, colorectal cancer, uterine cancer, stomach
cancer, small intestine cancer, thyroid cancer, lung cancer, kidney
cancer, pancreas cancer and prostrate cancer; acute lung diseases,
adult respiratory distress syndrome ("ARDS"), acute inflammatory
exacerbation of chronic lung diseases such as asthma, surface
epithelial cell injury such as transcorneal freezing or cutaneous
burns, and cardiovascular diseases such as ischemia in a subject in
need of such treatment or prevention.
[0085] In another aspect, the invention provides methods for using
S1P-1 modulators in treating or preventing disorders such as, but
not limited to, vasoconstriction in cerebral arteries, autoimmune
and related immune disorders including systemic lupus
erythematosus, inflammatory bowel diseases such as Crohn's disease
and ulcerative colitis, type I diabetes, uveitis, psoriasis,
myasthenia gravis, rheumatoid arthritis, non-glomerular nephrosis,
hepatitis, Behcet's disease, glomerulonephritis, chronic
thrombocytopenic purpura, hemolytic anemia, hepatitis and Wegner's
granuloma.
[0086] In still another aspect, the invention provides methods for
using S1P-1 modulators to treat or prevent a disease or disorder in
a subject, comprising administering to a subject in need of such
treatment or prevention a therapeutically effective amount of an
S1P-1 modulator, e.g., an agonist, that stimulates the immune
system. In certain embodiments, the subject is afflicted by an
infectious agent. In other embodiments, the subject is
immunocompromised.
[0087] In still another aspect, the present invention provides a
method of modulating an S1P-1 receptor-mediated biological activity
in a cell. A cell expressing the S1P-1 receptor is contacted with
an amount of an S1P-1 receptor modulator sufficient to modulate the
S1P-1 receptor mediated biological activity.
[0088] In yet another aspect, the present invention provides a
method for modulating an S1P-1 receptor mediated biological
activity in a subject. In such a method, an amount of a modulator
of the S1P-1 receptor effective to modulate an SIP-1
receptor-mediated biological activity is administered to the
subject.
[0089] In yet another aspect, the present invention provides a
method for treating, preventing or ameliorating an SIP-1 receptor
mediated condition in a subject. In such a method, an amount of a
modulator of the S1P-1 receptor effective to modulate an S1P-1
receptor-mediated biological activity is administered to the
subject. The S1P-1 receptor mediated condition may be, e.g.,
transplant rejection (solid organ transplant and islet cells);
transplant rejection (tissue); cancer; autoimmune/inflammatory
diseases; rheumatoid arthritis; lupus; insulin dependent diabetes
(Type I); non-insulin dependent diabetes (Type II); multiple
sclerosis; psoriasis; ulcerative colitis; inflammatory bowel
disease; Crohn's disease; acute and chronic lymphocytic leukemias
and lymphomas.
[0090] The features and other details of the invention will now be
more particularly described. It will be understood that particular
embodiments described herein are shown by way of illustration and
not as limitations of the invention. The principal features of this
invention can be employed in various embodiments without departing
from the scope of the invention. All parts and percentages are by
weight unless otherwise specified.
Definitions
[0091] For convenience, certain terms used in the specification and
examples are collected here.
[0092] "Treating", includes any effect, e.g., lessening, reducing,
modulating, or eliminating, that results in the improvement of the
condition, disease, disorder, etc.
[0093] "C.sub..alpha.-.beta.alk" means an alkyl group comprising a
minimum of .alpha. and a maximum of .beta. carbon atoms in a
branched, cyclical or linear relationship or any combination of the
three, wherein .alpha. and .beta. represent integers. The alkyl
groups described in this section may also contain one or two double
or triple bonds. A designation of C.sub.0alk indicates a direct
bond. Examples of C.sub.1-6alkyl include, but are not limited to
the following:
##STR00008##
[0094] "Halo" or "halogen" means a halogen atoms selected from F,
Cl, Br and I.
[0095] "C.sub.V-Whaloalk" means an alk group, as described above,
wherein any number--at least one--of the hydrogen atoms attached to
the alk chain are replaced by F, Cl, Br or I.
[0096] "Substituted alkyls" refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,
sulihydryl, alkylthio, thiocarboxylate, sulfates, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, or heterocyclyl.
[0097] "Aryl" includes groups with aromaticity, including 5- and
6-membered unconjugated (i.e., single-ring) aromatic groups that
may include from zero to four heteroatoms, as well as conjugated
(i.e., multicyclic) systems having at least one ring that is
aromatic. Examples of aryl groups include benzene, phenyl, tolyl
and the like. Multicyclic aryl groups include tricyclic and
bicyclic systems, e.g., naphthalene, benzoxazole, benzodioxazole,
benzothiazole, benzoimidazole, benzothiophene,
methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,
benzofuran, purine, benzofuran, deazapurine, indolizine, tetralin,
and methylenedioxyphenyl.
[0098] Aryl groups having heteroatoms in the ring structure may
also be referred to as "aryl heterocycles", "heterocycles,"
"heteroaryls" or "heteroaromatics"; e.g., pyrrole, furan,
thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine. The aromatic ring can be substituted at one or more
ring positions with, for example, halogen, hydroxyl, alkoxy,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,
aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,
arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, cyano, amino, acylamino, amidino, imino, sulfhydryl,
alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0099] An "alkylaryl" or an "aralkyl" moiety is an alkyl
substituted with an aryl group (e.g., phenylmethyl (benzyl)).
[0100] Unless the number of carbons is otherwise specified, "lower
alkyl" includes an alk group, as defined above, but having from one
to ten, more preferably from one to six, carbon atoms in its
backbone structure. "Lower alkenyl" and "lower alkynyl" have chain
lengths of, for example, 2-5 carbon atoms.
[0101] "Acyl" includes compounds and moieties which contain the
acyl radical (CH.sub.3CO--) or a carbonyl group. "Substituted acyl"
includes acyl groups where one or more of the hydrogen atoms are
replaced by for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0102] "Acylamino" includes moieties wherein an acyl moiety is
bonded to an amino group. For example, the term includes
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
"Alkylamino" includes moieties wherein an alkyl moiety is bonded to
an amino group; "dialkylamino", "arylamino", "diarylamino", and
"alkylarylamino" are analogously named. In some embodiments,
"amino" may include acylamino and/or alkylamino groups.
[0103] "Alkoxyalkyl", "alkylaminoalkyl" and "thioalkoxyalkyl"
include alkyl groups, as described above, which further include
oxygen, nitrogen or sulfur atoms replacing one or more hydrocarbon
backbone carbon atoms, e.g., oxygen, nitrogen or sulfur atoms.
[0104] "Alkoxy" includes alkyl, alkenyl, and alkynyl groups
covalently linked to an oxygen atom. Examples of alkoxy groups
include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy
groups. Examples of "substituted alkoxy" groups include halogenated
alkoxy groups. Substituted alkoxy groups can include alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, or heterocyclyl substituents.
Examples of halogen-substituted alkoxy groups include
fluoromethoxy, difluoromethoxy, trifluoromethoxy, chlorotnethoxy,
dichloromethoxy, and trichloromethoxy.
[0105] The terms "heterocyclyl" or "heterocyclic group" include
closed ring structures, e.g., 3- to 10-, or 4- to 7-membered rings
which include one or more heteroatoms. Heterocyclyl groups can be
saturated or unsaturated and include pyrrolidine, oxolane,
thiolane, piperidine, piperizine, morpholine, lactones, lactams
such as azetidinones and pyrrolidinones, sultams, sultones, and the
like.
[0106] Heterocyclic rings may be substituted at one or more
positions with such substituents as described above, as for
example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate, phosphonato, phosphinato, cyano, amino, acylamino,
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic or
heteroaromatic moiety.
[0107] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0108] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl" which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0109] The term "ester" includes compounds and moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl,
alkenyl, or alkynyl groups are as defined above.
[0110] The term "thioether" includes compounds and moieties which
contain a sulfur atom bonded to two different carbon or
heteroatoms. Examples of thioethers include, but are not limited to
alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term
"alkthioalkyls" include compounds with an alkyl, alkenyl, or
alkynyl group bonded to a sulfur atom which is bonded to an alkyl
group. Similarly, the term "alkthioalkenyls" and alkthioalkynyls"
refer to compounds or moieties wherein an alkyl, alkenyl, or
alkynyl group is bonded to a sulfur atom which is covalently bonded
to an alkynyl group.
[0111] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or
[0112] The term "halogen" includes fluorine, bromine, chlorine,
iodine, etc. The term "perhalogenated" generally refers to a moiety
wherein all hydrogens are replaced by halogen atoms.
[0113] "Heteroatom" includes atoms of any element other than carbon
or hydrogen. Examples of heteroatoms include nitrogen, oxygen,
sulfur and phosphorus.
[0114] "At least partially aromatic bicyclic ring system", means a
bicyclic ring system where either or both of the rings forming the
bicycle are aromatic.
[0115] It will be noted that the structure of some of the compounds
of the invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(e.g., all enantiomers and diastereomers) are included within the
scope of the invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by classical separation
techniques and by stereochemically controlled synthesis.
Furthermore, the structures and other compounds and moieties
discussed in this application also include all tautomers thereof.
Alkenes can include either the E- or Z-geometry, where
appropriate.
[0116] "Combination therapy" (or "co-therapy") includes the
administration of a S1P receptor modulator of the invention and at
least a second agent as part of a specific treatment regimen
intended to provide the beneficial effect from the co-action of
these therapeutic agents. The beneficial effect of the combination
includes, but is not limited to, pharmacokinetic or pharmacodynamic
co-action resulting from the combination of therapeutic agents.
Administration of these therapeutic agents in combination typically
is carried out over a defined time period (usually minutes, hours,
days or weeks depending upon the combination selected).
"Combination therapy" may, but generally is not, intended to
encompass the administration of two or more of these therapeutic
agents as part of separate monotherapy regimens that incidentally
and arbitrarily result in the combinations of the present
invention. "Combination therapy" is intended to embrace
administration of these therapeutic agents in a sequential manner,
that is, wherein each therapeutic agent is administered at a
different time, as well as administration of these therapeutic
agents, or at least two of the therapeutic agents, in a
substantially simultaneous manner. Substantially simultaneous
administration can be accomplished, for example, by administering
to the subject a single capsule having a fixed ratio of each
therapeutic agent or in multiple, single capsules for each of the
therapeutic agents. Sequential or substantially simultaneous
administration of each therapeutic agent can be effected by any
appropriate route including, but not limited to, oral routes,
intravenous routes, intramuscular routes, and direct absorption
through mucous membrane tissues. The therapeutic agents can be
administered by the same route or by different routes. For example,
a first therapeutic agent of the combination selected may be
administered by intravenous injection while the other therapeutic
agents of the combination may be administered orally.
Alternatively, for example, all therapeutic agents may be
administered orally or all therapeutic agents may be administered
by intravenous injection. The sequence in which the therapeutic
agents are administered is not narrowly critical. "Combination
therapy" also can embrace the administration of the therapeutic
agents as described above in further combination with other
biologically active ingredients and non-drug therapies (e.g.,
surgery or radiation treatment.) Where the combination therapy
further comprises a non-drug treatment, the non-drug treatment may
be conducted at any suitable time so long as a beneficial effect
from the co-action of the combination of the therapeutic agents and
non-drug treatment is achieved. For example, in appropriate cases,
the beneficial effect is still achieved when the non-drug treatment
is temporally removed from the administration of the therapeutic
agents, perhaps by days or even weeks.
[0117] An "anionic group," as used herein, refers to a group that
is negatively charged at physiological pH. Preferred anionic groups
include carboxylate, sulfate, sulfonate, sulfinate, sulfamate,
tetrazolyl, phosphate, phosphonate, phosphinate, or
phosphorothioate or functional equivalents thereof "Functional
equivalents" of anionic groups are intended to include
bioisosteres, e.g., bioisosteres of a carboxylate group.
Bioisosteres encompass both classical bioisosteric equivalents and
non-classical bioisosteric equivalents. Classical and non-classical
bioisosteres are known in the art (see, e.g., Silverman, R. B. The
Organic Chemistry of Drug Design and Drug Action, Academic Press,
Inc.: San Diego, Calif., 1992, pp. 19-23). A particularly preferred
anionic group is a carboxylate.
[0118] The term "heterocyclic group" is intended to include closed
ring structures in which one or more of the atoms in the ring is an
element other than carbon, for example, nitrogen, or oxygen or
sulfur. Heterocyclic groups can be saturated or unsaturated and
heterocyclic groups such as pyrrole and furan can have aromatic
character. They include fused ring structures such as quinoline and
isoquinoline. Other examples of heterocyclic groups include
pyridine and purine. Heterocyclic groups can also be substituted at
one or more constituent atoms with, for example, a halogen, a lower
alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower
alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, --CF.sub.3,
--CN, or the like.
[0119] An "S1P-modulating agent" includes compound or compositions
capable of inducing a detectable change in S1P receptor activity in
vivo or in vitro, e.g., at least 10% increase or decrease in S1P
activity as measured by a given assay such as the bioassay
described hereinbelow.
[0120] "EC.sub.50 of an agent" included that concentration of an
agent at which a given activity, including binding of sphingosine
or other ligand of an S1P receptor and/or a functional activity of
a S1P receptor (e.g., a signaling activity), is 50% maximal for
that S1P receptor. Stated differently, the EC.sub.50 is the
concentration of agent that gives 50% activation, when 100%
activation is set at the amount of activity of the S1P receptor
which does not increase with the addition of more ligand/agonist
and 0% activation is set at the amount of activity in the assay in
the absence of added ligand/agonist.
[0121] "Purified" and like terms relate to the isolation of a
molecule or compound in a form that is substantially free of
contaminants normally associated with the molecule or compound in a
native or natural environment.
[0122] An "effective amount" includes an amount sufficient to
produce a selected effect. For example, an effective amount of an
S1P receptor antagonist is an amount that decreases the cell
signaling activity of the S1P receptor.
[0123] "Immunomodulation" includes effects on the functioning of
the immune system, and includes both the enhancement of an immune
response as well as suppression of the immune response.
[0124] The compounds of the invention and the other
pharmacologically active agent may be administered to a patient
simultaneously, sequentially or in combination. It will be
appreciated that when using a combination of the invention, the
compound of the invention and the other pharmacologically active
agent may be in the same pharmaceutically acceptable carrier and
therefore administered simultaneously. They may be in separate
pharmaceutical carriers such as conventional oral dosage forms
which are taken simultaneously. The term "combination" further
refers to the case where the compounds are provided in separate
dosage forms and are administered sequentially.
[0125] The compounds of the invention may be administered to
patients (animals and humans) in need of such treatment in dosages
that will provide optimal pharmaceutical efficacy. It will be
appreciated that the dose required for use in any particular
application will vary from patient to patient, not only with the
particular compound or composition selected, but also with the
route of administration, the nature of the condition being treated,
the age and condition of the patient, concurrent medication or
special diets then being followed by the patient, and other factors
which those skilled in the art will recognize, with the appropriate
dosage ultimately being at the discretion of the attendant
physician.
[0126] An appropriate dosage level will generally be about 0.001 to
50 mg per kg patient body weight per day, which may be administered
in single or multiple doses. Preferably, the dosage level will be
about 0.01 to about 25 mg/kg per day; more preferably about 0.05 to
about 10 mg/kg per day. For example, in the treatment or prevention
of a disorder of the central nervous system, a suitable dosage
level is about 0.001 to 10 mg/kg per day, preferably about 0.005 to
5 mg/kg per day, and especially about 0.01 to 1 mg/kg per day. The
compounds may be administered on a regimen of 1 to 4 times per day,
preferably once or twice per day.
[0127] It will be appreciated that the amount of the compound of
the invention required for use in any treatment will vary not only
with the particular compounds or composition selected but also with
the route of administration, the nature of the condition being
treated, and the age and condition of the patient, and will
ultimately be at the discretion of the attendant physician.
[0128] The compositions and combination therapies of the invention
may be administered in combination with a variety of pharmaceutical
excipients, including stabilizing agents, carriers and/or
encapsulation formulations as described herein.
[0129] Aqueous compositions of the present invention comprise an
effective amount of the compounds of the invention, dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous
medium.
[0130] "Pharmaceutically or pharmacologically acceptable" include
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate. "Pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0131] For human administration, preparations should meet
sterility, pyrogenicity, general safety and purity standards as
required by FDA Office of Biologics standards.
[0132] The compositions and combination therapies of the invention
will then generally be formulated for parenteral administration,
e.g., formulated for injection via the intravenous, intramuscular,
subcutaneous, intralesional, or even intraperitoneal routes. The
preparation of an aqueous composition that contains a composition
of the invention or an active component or ingredient will be known
to those of skill in the art in light of the present disclosure.
Typically, such compositions can be prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a liquid
prior to injection can also be prepared; and the preparations can
also be emulsified.
[0133] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
[0134] Solutions of active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0135] Therapeutic or pharmacological compositions of the present
invention will generally comprise an effective amount of the
component(s) of the combination therapy, dissolved or dispersed in
a pharmaceutically acceptable medium. Pharmaceutically acceptable
media or carriers include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well known in the
art. Supplementary active ingredients can also be incorporated into
the therapeutic compositions of the present invention.
[0136] The preparation of pharmaceutical or pharmacological
compositions will be known to those of skill in the art in light of
the present disclosure. Typically, such compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection; as tablets or other solids for oral
administration; as time release capsules; or in any other form
currently used, including cremes, lotions, mouthwashes, inhalants
and the like.
[0137] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0138] The preparation of more, or highly, concentrated solutions
for intramuscular injection is also contemplated. In this regard,
the use of DMSO as solvent is preferred as this will result in
extremely rapid penetration, delivering high concentrations of the
active compound(s) or agent(s) to a small area.
[0139] The use of sterile formulations, such as saline-based
washes, by surgeons, physicians or health care workers to cleanse a
particular area in the operating field may also be particularly
useful. Therapeutic formulations in accordance with the present
invention may also be reconstituted in the form of mouthwashes, or
in conjunction with antifungal reagents. Inhalant forms are also
envisioned. The therapeutic formulations of the invention may also
be prepared in forms suitable for topical administration, such as
in cremes and lotions.
[0140] Suitable preservatives for use in such a solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium bisulfite,
sodium metabisulfite, sodium thiosulfite, thiourea and the like.
Suitable wetting and clarifying agents include polysorbate 80,
polysorbate 20, poloxamer 282 and tyloxapol. Suitable
viscosity-increasing agents include dextran 40, dextran 70,
gelatin, glycerin, hydroxyethylcellulose,
hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like.
[0141] Upon formulation, therapeutics will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed.
[0142] In this context, the quantity of active ingredient and
volume of composition to be administered depends on the host animal
to be treated. Precise amounts of active compound required for
administration depend on the judgment of the practitioner and are
peculiar to each individual.
[0143] A minimal volume of a composition required to disperse the
active compounds is typically utilized. Suitable regimes for
administration are also variable, but would be typified by
initially administering the compound and monitoring the results and
then giving further controlled doses at further intervals. For
example, for parenteral administration, a suitably buffered, and if
necessary, isotonic aqueous solution would be prepared and used for
intravenous, intramuscular, subcutaneous or even intraperitoneal
administration. One dosage could be dissolved in 1 mL of isotonic
NaCl solution and either added to 1000 mL of hypodermolysis fluid
or injected at the proposed site of infusion, (see for example,
Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038
and 1570-1580).
[0144] In certain embodiments, active compounds may be administered
orally. This is contemplated for agents which are generally
resistant, or have been rendered resistant, to proteolysis by
digestive enzymes. Such compounds are contemplated to include
chemically designed or modified agents; dextrorotatory to peptides;
and peptide and liposomal formulations in time release capsules to
avoid peptidase and lipase degradation.
[0145] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0146] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0147] The preparation of more, or highly, concentrated solutions
for direct injection is also contemplated, where the use of DMSO as
solvent is envisioned to result in extremely rapid penetration,
delivering high concentrations of the active agents to a small
area.
[0148] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0149] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure.
[0150] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations;
time-release capsules; and any other form currently used, including
cremes.
[0151] Additional formulations suitable for other modes of
administration include suppositories. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%.
[0152] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders.
[0153] In certain defined embodiments, oral pharmaceutical
compositions will comprise an inert diluent or assimilable edible
carrier, or they may be enclosed in hard or soft shell gelatin
capsule, or they may be compressed into tablets, or they may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0154] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring.
[0155] When the dosage unit form is a capsule, it may contain, in
addition to materials of the above type, a liquid carrier. Various
other materials may be present as coatings or to otherwise modify
the physical form of the dosage unit. For instance, tablets, pills,
or capsules may be coated with shellac, sugar or both. A syrup of
elixir may contain the active compounds sucrose as a sweetening
agent methyl and propylparabens as preservatives, a dye and
flavoring, such as cherry or orange flavor.
[0156] The pharmaceutical compositions of this invention may be
used in the form of a pharmaceutical preparation, for example, in
solid, semisolid or liquid form, which contains one or more of the
compound of the invention, as an active ingredient, in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral or parenteral applications. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, and any
other form suitable for use. The carriers which can be used are
water, glucose, lactose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form,
and in addition auxiliary, stabilizing, thickening and coloring
agents and perfumes may be used. The active object compound is
included in the pharmaceutical composition in an amount sufficient
to produce the desired effect upon the process or condition of the
disease.
[0157] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g., conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g., water, to form a solid preformulation composition containing
a homogeneous mixture of a compound of the invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the invention. The
tablets or pills of the novel composition can be coated or
otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0158] The liquid forms in which the compositions of the invention
may be incorporated for administration orally or by injection
include aqueous solution, suitably flavored syrups, aqueous or oil
suspensions, and emulsions with acceptable oils such as cottonseed
oil, sesame oil, coconut oil or peanut oil, or with a solubilizing
or emulsifying agent suitable for intravenous use, as well as
elixirs and similar pharmaceutical vehicles. Suitable dispersing or
suspending agents for aqueous suspensions include synthetic and
natural gums such as tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or
gelatin.
[0159] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. Preferably the compositions
are administered by the oral or nasal respiratory route for local
or systemic effect. Compositions in preferably sterile
pharmaceutically acceptable solvents may be nebulized by use of
inert gases. Nebulized solutions may be breathed directly from the
nebulizing device or the nebulizing device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0160] For treating clinical conditions and diseases noted above,
the compound of this invention may be administered orally,
topically, parenterally, by inhalation spray or rectally in dosage
unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques.
[0161] The compounds of the present invention are high affinity
agonists (or antagonists) at various S IP receptors. The compounds
of the invention are also expected to evoke lymphopenia when
introduced into rodents, non human primate or humans. Thus the
compounds of the invention can be used as immune modulators, and
are useful in treating or preventing pathologies mediated by
lymphocyte actions, including acute or chronic rejection of tissue
grafts such as organ transplants, and autoimmune diseases.
Autoimmune diseases that may be treated with compounds of the
invention include: systemic lupus erythematosus, multiple
sclerosis, Behcet's disease, glomerulonephritis, rheumatoid
arthritis, inflammatory bowel diseases such as Crohn's disease and
ulcerative colitis, type I diabetes, uveitis, psoriasis, myasthenia
gravis, Hashimoto's thyroiditis, autoimmune hemolytic anemia,
autoimmune thrombocytopenic purpura, hepatitis and Wegner's
granuloma.
[0162] The compounds of the invention are useful also in treating
inflammatory disorders, including atopic asthma, inflammatory
glomerular injury and ischemia-reperfusion injury.
[0163] Lysophospholipids, S1P and lysophosphatidic acid (LPA),
stimulate cellular proliferation and affect numerous cellular
functions by signaling through G protein-coupled endothelial
differentiation gene-encoded (S1P) receptors. Accordingly, the S1P
receptor modulators of the invention are anticipated to have
utility in immunomodulation, e.g., in anti-angiogenesis therapy,
such as in neoplastic disease treatment.
[0164] In one embodiment of the invention, a pharmaceutical
composition comprising one or more of the S IP receptor agonists of
the present invention is administered to a mammalian species,
including humans, to enhance wound repair, improve neuronal
function or enhance an immune response of that species. It has also
been reported that S1P inhibits fibrosis in various organs.
Accordingly, the S1P receptor agonists of the invention can be used
to prevent/treat diseases associated with organ fibrosis, such as
pulmonary fibrosis, interstitial pneumonia, chronic hepatitis,
hepatic cirrhosis, chronic renal insufficiency or kidney glomerular
sclerosis. In one embodiment, a composition comprising an S1P
receptor agonist of the present invention is used to treat wounds,
including burns, cuts, lacerations, surgical incisions, bed sores,
and slow-healing ulcers such as those seen in diabetics.
[0165] In addition, S1P modulating compounds of the invention are
believed to mobilize lymphocytes and increase their homing to
secondary lymphoid tissues. Thus the present compounds can be used
to direct lymphocytes away from transplanted organs, e.g.,
allografts, or healthy cells, e.g., pancreatic islets as in type I
diabetes, myelin sheathing (multiple sclerosis), or other tissues
that may be subjected to an undesirable immunoresponse, and thus
decrease damage to such tissues from the immune system.
[0166] In another embodiment, the S1P receptor-modulating compounds
of the invention are administered to a subject to treat or prevent
a disorder of abnormal cell growth and differentiation. These
disorders include Alzheimer's disease, aberrant corpus luteum
formation, osteoporosis, anovulation, Parkinson's disease, and
cancer. In one embodiment, an S1P antagonist is administered to a
patient to treat a disease associated with abnormal growth.
[0167] In one embodiment, the compounds of the invention are used
as immunomodulators to alter immune system activities and prevent
damage to healthy tissue that would otherwise occur in autoimmune
diseases and in organ transplantation. In particular, the compounds
can be administered to patients as part of the treatment associated
with organ transplantation, including pancreas, pancreatic islets,
kidney, heart and lung transplantations. The S1P modulators can be
administered alone or in combination with known immunosuppressants
such as cyclosporine, tacrolimus, rapamycin, azathioprine,
cyclophosphamide, methotrexate and corticosteroids such as
cortisone, des-oxymetasone, betametasone, desametasone,
flunisolide, prednisolone, prednisone, amcinomide, desonide,
methylprednisolone, triamcinolone, and alclometasone.
[0168] S1P also acts as a survival factor in many cell types. In
particular, compounds of the invention having S1P antagonistic
activity are anticipated to be useful in protecting cells and
tissues from hypoxic conditions. In accordance with one embodiment,
compounds of the invention are administered to a patient judged to
be or actually in need of treatment, to treat cells and tissues
exposed to hypoxic conditions, including injury sustained as a
result of ischemia. In accordance with one embodiment, compounds of
the invention that show S1P receptor antagonist activity can be
used to treat ischemia reperfusion type injury. Interference with
the supply of oxygenated blood to tissues is defined as ischemia.
The effects of ischemia are known to be progressive, so that over
time cellular vitality continues to deteriorate and tissues become
necrotic. Total persistent ischemia, with limited oxygen perfusion
of tissues, results in cell death and eventually in
coagulation-induced necrosis despite reperfusion with arterial
blood. Evidence indicates that a significant proportion of the
injury associated with ischemia is a consequence of the events
associated with reperfusion of ischemic tissues, hence the term
reperfusion injury.
[0169] Pharmaceutical compositions comprising the compounds of the
invention may be administered to an individual in need by any
number of routes, including topical, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means. The oral
route is typically employed for most conditions requiring the
compounds of the invention. Preference is given to intravenous
injection or infusion for the acute treatments. For maintenance
regimens the oral or parenteral, e.g., intramuscular or
subcutaneous, route is preferred. In accordance with one embodiment
a composition is provided that comprises a compound of invention
and albumin, e.g., a compound of the present invention, a
pharmaceutically acceptable carrier and 0.1-1.0% albumin. Albumin
functions as a buffer and improves the solubility of the
compounds.
[0170] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention. In
accordance with one embodiment, a kit is provided for treating a
patient in need of immunomodulation, including instructions for use
of the kit. In this embodiment the kit comprises one or more of the
S1P modulators of the invention, and may also include one or more
known immunosuppressants. These pharmaceuticals can be packaged in
a variety of containers, e.g., vials, tubes, microtiter well
plates, bottles, and the like. Other reagents can be included in
separate containers and provided with the kit; e.g., positive
control samples, negative control samples, buffers, cell culture
media, etc. Preferably, the kits will also include instructions for
use.
[0171] The activity of compounds of the invention may be determined
by using an assay for detecting S1P receptor activity (such as the
[.gamma.-35 S]GTP binding assay) and assaying for activity in the
presence of S1P and the test compound. More particularly, in the
method described by Traynor et al., 1995, Mol. Pharmaeol. 47:
848-854, incorporated herein by reference, G-protein coupling to
membranes can be evaluated by measuring the binding of labeled
GTP.
[0172] For example, samples comprising membranes isolated from
cells expressing an S1P polypeptide can be incubated in a buffer
promoting binding of the polypeptide to ligand (i.e. S IP), in the
presence of radiolabeled GTP and unlabeled GDP (e.g., in 20 mM
HEPES, pH 7.4, 100 mM NaCl, and 10 mM MgCl.sub.2, 80 .mu.M
.sup.35S-GTP.sub..gamma.5 and 3 .mu.M GDP), with and without a
candidate modulator. The assay mixture is incubated for a suitable
period of time to permit binding to and activation of the receptor
(e.g., 60 minutes at 30.degree. C.), after which time unbound
labeled GTP is removed (e.g., by filtration onto GF/B filters).
Bound, labeled GTP can be measured by liquid scintillation
counting. A decrease of 10% or more in labeled GTP binding as
measured by scintillation counting in a sample containing a
candidate modulator, relative to a sample without the modulator,
indicates that the candidate modulator is an inhibitor of S1P
receptor activity.
[0173] A similar GTP-binding assay can be performed without the
presence of the ligand (SIP) to identify agents that act as
agonists. In this case, ligand-stimulated GTP binding is used as a
standard. An agent is considered an agonist if it induces at least
50% of the level of GTP binding induced by S1P when the agent is
present at 10 .mu.m or less, and preferably will induce a level
which is the same as or higher than that induced by the ligand.
[0174] GTPase activity can be measured by incubating cell membrane
extracts containing an S1P receptor with .gamma..sup.32P-GTP.
Active GTPase will release the label as inorganic phosphate, which
can be detected by separation of free inorganic phosphate in a 5%
suspension of activated charcoal in 20 mM H.sub.3PO.sub.4, followed
by scintillation counting. Controls would include assays using
membrane extracts isolated from cells not expressing an S1P
receptor (e.g., mock-transfected cells), in order to exclude
possible non-specific effects of the candidate modulator. In order
to assay for the effect of a candidate modulator on S1P-regulated
GTPase activity, cell membrane samples can be incubated with the
ligand (S1P), with and without the modulator, and a GTPase assay
can be performed as described above. A change (increase or
decrease) of 10% or more in the level of GTP binding or GTPase
activity relative to samples without modulator is indicative of S1P
modulation by a candidate modulator.
[0175] S1P receptor (hS1P1, hS1P3, rS1P1, rS1P3 and parental cell)
Ca.sup.2+ Flux protocol
[0176] 1. Material [0177] a. FL1PR buffer: 1.times.HBSS; 10 mM
HEPES [0178] b. Cell growth media: [0179] i. human and rat S1P1 and
S1P3: F 12-Ham's media; 10% FBS (qualified); 1.times.Pen/Strep/Glu;
300 ug/mL Hygromycin; 400 ug/mL Geneticin [0180] ii. Parental cell:
human and rat S1P1 and S1P3: F12-Ham's media; 10% FBS (qualified);
1.times.Pen/Strep/Glu; 300 ug/mL Hygromycin; [0181] c. Cell seeding
media; F12-Ham's media; 10% FBS (Charcoal/dextran stripped);
1.times.Pen/Strep/Glu; [0182] d. Cell dissociation buffer: Versene
from Invitrogen [0183] e. Agonist (S1P) dissolving buffer: 0.4%
(w/v) fatty-acid free BSA (Sigma # A8806) in FLIPR buffer [0184] f.
FLIPR dye: BD PBX Calcium assay kit; Cat#641077 is composed of
Calcium indicator (Cat#850000) and 100.times.PBX signal enhancer
(cat#850001). The 100.times.PBX signal enhancer is diluted into
FLIPR buffer 1:100 and the calcium indicator is then added at
1:1000 ratio. [0185] g. Cell plates (96-well): Greiner Cat#655090
[0186] h. Compound plates (96-well): Costar #3365 [0187] i. S1P
stock solution preparation: S1P is purchased from CalBioChem
(Catalog #970471; 1 mg vial; custom prep using methanol, &
nitrogen gas drying inside glass vial; Storage @-20.degree. C.).
Dissolve 1 vial of S1P into 26.4 ml of agonist dissolving buffer in
a 50 ml centrifuge tube; Remove label from bottle of S1P, open and
drop entire bottle into tube. Sonicate at 37.degree. for 1/2 hour.
Clear solution of 100 .mu.M will result. This stock solution is
aliquoted and stored at -80 C
[0188] 2. Cell line maintenance [0189] a. V5 tags were added at the
N-terminus of hS1P1, hS1P3, rS1P1 and rS1P3. All four genes were
transfected into CHO K1 cells which stably express Gqi5. [0190] b.
hS1P1 and hS1P3 were established as stable clones and rS1P1 and
rS1P3 were sorted by anti V5 tag and established as stable pools
after sorting. [0191] c. Parental cell line CHO/K1 Gqi5 is used as
the control. [0192] d. All the cells are maintained in cell growth
media and splitted twice a week using Versene. [0193] e. All the
cell lines are used under passage 30.
[0194] 3. Assay protocol [0195] a. Cell seeding: Cells are lifted
from the flask by Versene and seeded in cell plates at 50K/well in
cell seeding media. Cells are grown overnight at 37 degree. [0196]
b. Cell loading: Cell seeding media is discarded. Cells are loaded
with 50 ul of FLIPR dye at RT for 90 min. Signal is stable for up
to 5 h after dye loading. [0197] c. Agonist (S1P) preparation:
Frozen stock of S1P is thawed out and sonicated at 37 c for 30
minutes every time before use. The stock is then diluted into FLIPR
buffer at proper concentration. [0198] d. Compound preparation:
Compounds are dissolved in DMSO. A 3.times., point dilution of the
compounds are carried out in DMSO. Then the compounds are diluted
into assay buffer133.times. so that the DMSO concentration is
0.75%. [0199] e. Activity measurement: The fluorescence signal
change of the cells upon compound addition is monitored in
FLIPRtetra. 25 ul of compound is transferred into the cell plates
(50 ul of FLIPR dye; DMSO concentration: 0.25%). Signal is recorded
for 90 sec after compound addition. Then 50 ul of 500 nM S1P is
added in the cell plate, and signal is recorded for 90 seconds upon
addition.
[0200] 4. Data analysis [0201] a. Peak value is calculated for each
compound/S1P addition [0202] b. The peak value of S1P at 200 nM is
used as high control (100%), and the peak value of buffer only is
used as low control (0%). [0203] c. Data is normalized against high
and low controls using the following equation:
[0203] POC_S=100*(RAW-LO)/(HI-LO) [0204] d. Peak value is plotted
against the concentration of compound. [0205] e. Curve is fitted
using the 4 parameter fit:
[0205] Y=(A+(B/(1+((x/C) D)))) [0206] where: [0207] Y is POC_S (or
POC) [0208] X is compound concentration [0209] A is the minimum
(EC50min or IC50min) B is the maxmum (EC50max or IC50max) [0210] C
is the inflection point (EC50IP or IC50IP) [0211] D is the hill
slop (EC50 slope or IC50 slope).
[0212] Identified S1P receptor agonists and antagonists can be used
to treat a variety of human diseases and disorders, including, but
not limited to the treatment of infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
ulcers; asthma; allergy; benign prostatic hypertrophy; migraine;
vomiting; psychotic and neurological disorders, including anxiety,
schizophrenia, manic depression, depression, delirium, dementia,
and severe mental retardation.
[0213] Pain is a complex subjective sensation reflecting real or
potential tissue damage and the affective response to it. Acute
pain is a physiological signal indicating a potential or actual
injury. Chronic pain can either be somatogenetic (organic) or
psychogenic. Chronic pain is frequently accompanied or followed by
vegetative signs, which often result in depression.
[0214] Somatogenetic pain may be of nociceptive origin,
inflammatory or neuropathic. Nociceptive pain is judged to be
commensurate with ongoing activation of somatic or visceral
pain-sensitive nerve fibers. Neuropathic pain results from
dysfunction in the nervous system; it is believed to be sustained
by aberrant somatosensory processes in the peripheral nervous
system, the CNS, or both. Chronic pain results in individual
suffering and social economic costs of tremendous extent. Existing
pharmacological pain therapies are widely unsatisfying both in
terms of efficacy and of safety.
[0215] In one embodiment, S1P modulators of the present invention
are used as immunomodulators to suppress the immune system and
prevent damage to healthy tissue that would otherwise occur in
autoimmune diseases and in organ transplantation. The compounds can
be administered to patients as part of the treatment associated
with organ transplantation, including pancreas, pancreatic islets,
kidney, heart and lung transplantations. The S1P modulators can be
administered alone or in combination with known immunosuppressants
such as cyclosporine, tacrolimus, azatioprine, desoxymetasone,
cyclophosphamide, cortisone, betametasone, FK 506 (a fungal
macrolide immunosuppressant), desametasone, flunisolide,
prednisolone, prednisone, amcinomide desonide, methylprednisolone,
triamcinolone, alclometasone and methotrexate.
[0216] The dosage to be used is, of course, dependent on the
specific disorder to be treated, as well as additional factors
including the age, weight, general state of health, severity of the
symptoms, frequency of the treatment and whether additional
pharmaceuticals accompany the treatment. The dosages are in general
administered several times per day and preferably one to three
times per day. The amounts of the individual active compounds are
easily determined by routine procedures known to those of ordinary
skill in the art S1P also acts as a survival factor in many cell
types. S1P receptor modulators are anticipated to have activity in
protecting cells and tissues from hypoxic conditions. In accordance
with one embodiment compounds of the invention are administered to
treat cells and tissues exposed to hypoxic conditions, including
injury sustained as a result of ischemia. In accordance with one
embodiment, the S1P modulators having antagonistic activity can be
used to treat ischemia reperfusion type injury. Interference with
the supply of oxygenated blood to tissues is defined as ischemia.
The effects of ischemia are known to be progressive, such that over
time cellular vitality continues to deteriorate and tissues become
necrotic. Total persistent ischemia, with limited oxygen perfusion
of tissues, results in cell death and eventually in
coagulation-induced necrosis despite reperfusion with arterial
blood.
[0217] The compounds of the invention and the other
pharmacologically active agent may be administered to a patient
simultaneously, sequentially or in combination. It will be
appreciated that when using a combination of the invention, the
compound of the invention and the other pharmacologically active
agent may be in the same pharmaceutically acceptable carrier and
therefore administered simultaneously. They may be in separate
pharmaceutical carriers such as conventional oral dosage forms
which are taken simultaneously. The term "combination" further
refers to the case where the compounds are provided in separate
dosage forms and are administered sequentially.
[0218] The compounds of the invention may be administered to
patients (animals and humans) in need of such treatment in dosages
that will provide optimal pharmaceutical efficacy. It will be
appreciated that the dose required for use in any particular
application will vary from patient to patient, not only with the
particular compound or composition selected, but also with the
route of administration, the nature of the condition being treated,
the age and condition of the patient, concurrent medication or
special diets then being followed by the patient, and other factors
which those skilled in the art will recognize, with the appropriate
dosage ultimately being at the discretion of the attendant
physician.
[0219] An appropriate dosage level will generally be about 0.001 to
50 mg per kg patient body weight per day, which may be administered
in single or multiple doses. Preferably, the dosage level will be
about 0.01 to about 25 mg/kg per day; more preferably about 0.05 to
about 10 mg/kg per day. For example, in the treatment or prevention
of a disorder of the central nervous system, a suitable dosage
level is about 0.001 to 10 mg/kg per day, preferably about 0.005 to
5 mg/kg per day, and especially about 0.01 to 1 mg/kg per day. The
compounds may be administered on a regimen of 1 to 4 times per day,
preferably once or twice per day.
[0220] It will be appreciated that the amount of the compound of
the invention required for use in any treatment will vary not only
with the particular compounds or composition selected but also with
the route of administration, the nature of the condition being
treated, and the age and condition of the patient, and will
ultimately be at the discretion of the attendant physician.
[0221] The compositions and combination therapies of the invention
may be administered in combination with a variety of pharmaceutical
excipients, including stabilizing agents, carriers and/or
encapsulation formulations as described herein.
[0222] Aqueous compositions of the present invention comprise an
effective amount of the compounds of the invention, dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous
medium.
[0223] "Pharmaceutically or pharmacologically acceptable" include
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate. "Pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0224] For human administration, preparations should meet
sterility, pyrogenicity, general safety and purity standards as
required by FDA Office of Biologics standards.
[0225] The compositions and combination therapies of the invention
will then generally be formulated for parenteral administration,
e.g., formulated for injection via the intravenous, intramuscular,
subcutaneous, intralesional, or even intraperitoneal routes. The
preparation of an aqueous composition that contains a composition
of the invention or an active component or ingredient will be known
to those of skill in the art in light of the present disclosure.
Typically, such compositions can be prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a liquid
prior to injection can also be prepared; and the preparations can
also be emulsified.
[0226] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
[0227] Solutions of active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0228] Therapeutic or pharmacological compositions of the present
invention will generally comprise an effective amount of the
component(s) of the combination therapy, dissolved or dispersed in
a pharmaceutically acceptable medium. Pharmaceutically acceptable
media or carriers include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for phannaceutical active substances is well known in the
art. Supplementary active ingredients can also be incorporated into
the therapeutic compositions of the present invention.
[0229] The preparation of pharmaceutical or pharmacological
compositions will be known to those of skill in the art in light of
the present disclosure. Typically, such compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection; as tablets or other solids for oral
administration; as time release capsules; or in any other form
currently used, including cremes, lotions, mouthwashes, inhalants
and the like.
[0230] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0231] The preparation of more, or highly, concentrated solutions
for intramuscular injection is also contemplated. In this regard,
the use of DMSO as solvent is preferred as this will result in
extremely rapid penetration, delivering high concentrations of the
active compound(s) or agent(s) to a small area.
[0232] The use of sterile formulations, such as saline-based
washes, by surgeons, physicians or health care workers to cleanse a
particular area in the operating field may also be particularly
useful. Therapeutic formulations in accordance with the present
invention may also be reconstituted in the form of mouthwashes, or
in conjunction with antifungal reagents. Inhalant forms are also
envisioned. The therapeutic formulations of the invention may also
be prepared in forms suitable for topical administration, such as
in cremes and lotions.
[0233] Suitable preservatives for use in such a solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium bisulfite,
sodium metabisulfite, sodium thiosulfite, thiourea and the like.
Suitable wetting and clarifying agents include polysorbate 80,
polysorbate 20, poloxamer 282 and tyloxapol. Suitable
viscosity-increasing agents include dextran 40, dextran 70,
gelatin, glycerin, hydroxyethylcellulose,
hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like.
[0234] Upon formulation, therapeutics will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed.
[0235] In this context, the quantity of active ingredient and
volume of composition to be administered depends on the host animal
to be treated. Precise amounts of active compound required for
administration depend on the judgment of the practitioner and are
peculiar to each individual.
[0236] A minimal volume of a composition required to disperse the
active compounds is typically utilized. Suitable regimes for
administration are also variable, but would be typified by
initially administering the compound and monitoring the results and
then giving further controlled doses at further intervals. For
example, for parenteral administration, a suitably buffered, and if
necessary, isotonic aqueous solution would be prepared and used for
intravenous, intramuscular, subcutaneous or even intraperitoneal
administration. One dosage could be dissolved in 1 mL of isotonic
NaCl solution and either added to 1000 mL of hypodermolysis fluid
or injected at the proposed site of infusion, (see for example,
Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038
and 1570-1580).
[0237] In certain embodiments, active compounds may be administered
orally. This is contemplated for agents which are generally
resistant, or have been rendered resistant, to proteolysis by
digestive enzymes. Such compounds are contemplated to include
chemically designed or modified agents; dextrorotatory peptides;
and peptide and liposomal formulations in time release capsules to
avoid peptidase and lipase degradation.
[0238] Pharmaceutically acceptable salts include acid addition
salts and which are formed with inorganic acids such as, for
example, hydrochloric, hydrobromic, boric, phosphoric, sulfuric
acids or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, maleic, fumaric, citric, succinic, mesylic, mandelic,
succinic, benzoic, ascorbic, methanesulphonic, a-keto glutaric,
a-glycerophosphoric, glucose-1-phosphoric acids and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, magnesium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Other examples of pharmaceutically acceptable salts include
quaternary derivatives, and internal salts such as N-oxides.
[0239] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0240] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0241] The preparation of more, or highly, concentrated solutions
for direct injection is also contemplated, where the use of DMSO as
solvent is envisioned to result in extremely rapid penetration,
delivering high concentrations of the active agents to a small
area.
[0242] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0243] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure.
[0244] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations;
time-release capsules; and any other form currently used, including
cremes.
[0245] Additional formulations suitable for other modes of
administration include suppositories. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%.
[0246] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders.
[0247] In certain defined embodiments, oral pharmaceutical
compositions will comprise an inert diluent or assimilable edible
carrier, or they may be enclosed in hard or soft shell gelatin
capsule, or they may be compressed into tablets, or they may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0248] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange
flavor.
[0249] The pharmaceutical compositions of this invention may be
used in the form of a pharmaceutical preparation, for example, in
solid, semisolid or liquid form, which contains one or more of the
compound of the invention, as an active ingredient, in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral or parenteral applications. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, and any
other form suitable for use. The carriers which can be used are
water, glucose, lactose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form,
and in addition auxiliary, stabilizing, thickening and coloring
agents and perfumes may be used. The active object compound is
included in the pharmaceutical composition in an amount sufficient
to produce the desired effect upon the process or condition of the
disease.
[0250] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g., conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g., water, to form a solid preformulation composition containing
a homogeneous mixture of a compound of the invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the invention. The
tablets or pills of the novel composition can be coated or
otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0251] The liquid forms in which the compositions of the invention
may be incorporated for administration orally or by injection
include aqueous solution, suitably flavored syrups, aqueous or oil
suspensions, and emulsions with acceptable oils such as cottonseed
oil, sesame oil, coconut oil or peanut oil, or with a solubilizing
or emulsifying agent suitable for intravenous use, as well as
elixirs and similar pharmaceutical vehicles. Suitable dispersing or
suspending agents for aqueous suspensions include synthetic and
natural gums such as tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or
gelatin.
[0252] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. Preferably the compositions
are administered by the oral or nasal respiratory route for local
or systemic effect. Compositions in preferably sterile
pharmaceutically acceptable solvents may be nebulized by use of
inert gases. Nebulized solutions may be breathed directly from the
nebulizing device or the nebulizing device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0253] For treating clinical conditions and diseases noted above,
the compound of this invention may be administered orally,
topically, parenterally, by inhalation spray or rectally in dosage
unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques.
[0254] The following examples are given for the purpose of
illustrating the invention, but not for limiting the scope or
spirit of the invention.
##STR00009##
General Procedure of Reductive Amination
[0255] A mixture of aldehyde (1.0 mmol), acetic acid (1.5 mmol) and
azetidine-3-carboxylic acid or piperidine-4-carboxylic acid
(1.2-1.5 mmol) in dichloromethane (DCM)/MeOH (1:1, 10 mL) was
stirred at room temperature for 1 h. Sodium cyanoborohydride (0.5
mmol) was added and the reaction mixture was stirred for 2-3 h at
room temperature. After concentration of solvent under reduced
pressure, the resulting residue was dissolved in DMSO, filtered and
purified by reverse phase preparative HPLC (Phenomenex reverse
phase Luna 5u C18(2) column, 60.times.21.2 mm ID, mobile phase:
A=0.05% TFA in water; B=0.05% trifluoroacetic acid (TFA) in
acetonitrile. The flow rate was 10-12 mL/minute) to yield the
desired final product with puritiy greater than 95%. All final
products were obtained as the TFA salts unless stated otherwise.
Alternatively, the crude mixture of reductive amination can be
purified by trituration with MeOH and water.
Example 1
1-(4-(4-Benzylbenzamido)-3-fluorobenzyl)azetidine-3-carboxylic
acid
Ethyl 4-(4-benzylbenzamido)-3-fluorobenzoate
##STR00010##
[0257] A solution of 4-benzylbenzoic acid (1.06 g, 5 mmole) in
dichloromethane (10 mL) was added oxalyl dichloride (1 mL) followed
by a few drops of DMF at 0.degree. C. The resulting mixture was
stirred room temperature for 3 hours. Removal of the solvents gave
the residue which was dissolved in dichloromethane (2 mL) and added
to the solution of triethylamine (1.4 mL, 10 mmol) and ethyl
4-amino-3-fluorobenzoate (916 mg, 5 mmole) in dichloromethane (50
mL) at 0.degree. C. After stirring at room temperature for 48
hours, the mixture was washed with saturated sodium bicarbonate and
1 N HCl. The combined organic extracts were washed with brine,
dried and concentrated under reduced pressure to give the residue
which was purification by silica gel chromatography on ISCO system.
The desired product was obtained as an off white solid: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.63 (t, J=8.8 Hz, 1H), 8.20 (s, 1H),
7.83 (m, 4H), 7.18-7.35 (m, 6H), 4.39 (q, J=7.2 Hz, 1H), 4.07 (s,
2H), 1.40 (t, J=7.2 Hz, 3H).
4-Benzyl-N-(2-fluoro-4-(hydroxymethyl)phenyl)benzamide
##STR00011##
[0259] A solution of methyl ethyl
4-(4-benzylbenzamido)-3-fluorobenzoate (100 mg, 0.265 mmol) in MeOH
(10 mL) at room temperature was added sodium borohydride (200 mg,
5.3 mmol). The mixture was stirred at room temperature for 3 days.
Removal of the solvent gave the residue which was diluted with
EtOAc (50 mL) and washed with 1 N NaOH and brine. The organic layer
was separated, dried and concentrated. The residue was purified on
ISCO system (1% methanol in dichloromethane) to give a pure product
as a white crystalline. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.44 (t, J=8.0 Hz, 1H), 8.02 (s, 1H), 7.80 (d, J=8.4 Hz, 2H),
7.15-7.34 (m, 8H), 4.69 (d, J=6.0 Hz, 1H), 4.06 (s, 2H).
4-Benzyl-N-(2-fluoro-4-formylphenyl)benzamide
##STR00012##
[0261] A mixture of
4-benzyl-N-(2-fluoro-4-(hydroxymethyl)phenyl)benzamide (200 mg,
0.60 mmol), 4-methylmorpholine N-oxide (140 mg, 1.2 mmol), and 4 A
molecular sieves (1 g) in 10 ml of CH.sub.3CN was treated with
tetrapropylammonium perruthnate (10 mg, 0.03 mmol) and the
resulting mixture was stirred at room temperature for 12 hours. The
solids were filtered and the filtrated was concentrated. The
residue was purified on ISCO system (80% hexane in dichloromethane)
to give a pure product as a white crystalline: .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 9.93 (s, 1H), 8.80 (t, J=8.0 Hz, 1H), 8.25
(s, 1H), 7.84 (d, J=7.2 Hz, 2H), 7.74 (d, J=8.4 Hz, 1H), 7.69 (d,
J=10.8 Hz, 1H), 7.19-7.37 (m, 6H), 4.07 (s, 2H).
1-(4-(4-Benzylbenzamido)-3-fluorobenzyl)azetidine-3-carboxylic
acid
##STR00013##
[0263] The title compound was prepared by the general method for
reductive amination to give the title compound: .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 7.86-7.94 (m, 4H), 7.17-7.39 (m, 8H), 4.42
(s, 2H), 4.33 (m, 4H), 4.06 (s, 2H), 3.71 (m, 1H). .sup.19F NMR
(376 MHz, CD.sub.3OD) .delta. -77.4 (TFA), -123.4. MS (ESI) m/z:
Calculated: 418.46; Observed: 419.0 (M.sup.++1).
##STR00014##
Example 2
1-(4-((4-benzylphenyl)carbamoyl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
1-Bromo-4-(diethoxymethyl)-2-fluorobenzene
##STR00015##
[0265] To a solution of 3-fluoro-4-bromobenzaldehyde (20.0 g, 98.5
mmol) in dry EtOH (120 mL) was added acetyl chloride (2.04 mL, 29.6
mmol) followed by the addition of triethyl orthoformate (6.55 mL,
39.4 mmol) and the contents were heated to 70.degree. C. for 3 h.
The contents were cooled to room temperature and shifted to a
rotary evaporator and subjected to reduced pressure (280 mm Hg)
with bath temperature 65.degree. C. for 45 min. The pressure was
further lowered to remove all the solvent. To this mixture, fresh
Ethanol (60 mL), acetyl chloride (1.5 mL), triethyl orthoformate
(5.0 mL) and heated to 70.degree. C. for 2 h. The solvent was
removed under the reduced pressure and diluted with EtOAc (200 mL),
washed with saturated sodium bicarbonate (3.times.100 mL), brine
and dried over anhydrous sodium sulfate. The solvent was evaporated
and the residue purified by silica gel column (basified with 5%
Et.sub.3N, eluent: EtOAc/hexanes, 1/20) to afford
1-bromo-4-(diethoxymethyl)-2-fluorobenzene as a colorless oil.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.03 (t, J=8.1 Hz,
1H), 7.36-7.33 (m, 2H), 5.54 (s, 1H), 3.63-3.52 (m, 4H), 1.25 (m,
6H).
2-Fluoro-4-formylbenzoic acid
##STR00016##
[0267] To a solution of 1-bromo-4-(diethoxymethyl)-2-fluorobenzene
(10.12 g, 36.53 mmol) in dry THF (90 mL) cooled to -78.degree. C.
was added n-butyllithium (2.5 M in hexanes, 16.5 mL, 43.83 mmol)
was added dropwise over a period of 10 min. The contents were
further stirred for 30 min and CO.sub.2 was bubbled through the
mixture for 0.5 h. (exothermic). The cooling bath was removed and
the contents warmed to room temperature. The mixture was treated
with aqueous NaOH (1N, 100 mL) and washed with EtOAc. The aqueous
layer was acidified to pH 2 with HCl (5N) and the free acid was
extracted with EtOAc (3.times.75 mL). The combined organic layers
were washed with water and brine, dried over MgSO.sub.4 and
evaporated. The residue was dissolved in ether (30 mL), TFA (1.5
mL) and water (2.0 mL) and stirred overnight. The volatiles were
removed under reduced pressure and co-evaporated with toluene. The
residue was then treated with diethyl ether (75 mL) and filtered.
The filter cake was dried under vacuum without further purification
to give 2-fluoro-4-formylbenzoic acid as a white solid. 1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 13.48 (s, 1H), 10.06 (s, 1H), 8.06
(t, J=7.4 Hz, 1H), 7.84-7.79 (m, 2H). MS (ESI) m/z: Calculated:
168.0; Observed: 167.0 (M.sup.--1).
N-(4-benzylphenyl)-2-fluoro-4-formylbenzamide
##STR00017##
[0269] To a solution of 4-benzylbenzenamine (0.100 g, 0.55 mmol),
2-fluoro-4-formylbenzoic acid (0.092 g, 0.55 mmol),
N,N-diethylpropan-2-amine (0.10 ml, 0.65 mmol) in DMF (2.00 ml) was
added HBTU. The reaction mixture was stirred at room temperature
for 1 h. The mixture was diluted with EtOAc, washed with water and
dried over MgSO.sub.4. The solvent was evaporated and the residue
was purified by flash chromatography in a EtOAc/Hexene system to
give the title compound. MS (ESI) m/z: Calculated; 333.3: Observed;
332.1. 1H NMR (300 MHz, CHLOROFORM-d) 8 ppm 10.05-10.10 (1H, m),
8.31-8.44 (2H, m), 7.80-7.85 (1H, m), 7.67-7.73 (1H, m), 7.56-7.62
(2H, m), 7.15-7.40 (6H, m), 3.99 (2H, s)
1-(4((4-benzylphenyl)carbamoyl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
##STR00018##
[0270] The compound was prepared by the general method for
reductive amination to give the title compound. MS (ESI) m/z:
Calculated; 418.46: Observed; 419.1. 1H NMR (300 MHz, DMSO-d.sub.6)
.delta. ppm 10.25-10.34 (1H, m), 7.51-7.67 (3H, m), 7.09-7.33 (8H,
m), 3.87-3.94 (2H, s), 3.56-3.63 (2H, s), 3.38 (2H, br. s.), 3.17
(3H, m.)
##STR00019##
##STR00020##
Example 3
Methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate
Methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
##STR00021##
[0271] Azetidine-3-carboxylic acid (43 g, 421 mmol),
4-bromo-3-fluorobenzaldehyde (81.4 g, 401 mmol), methyl
orthoformate (219 mL, 2005 mmol), and AcOH (34 mL, 601 mmol) were
added to DCM (700 mL) at rt under an N.sub.2 atmosphere. The
mixture was stirred for 15 min, at which point sodium
triacetoxyborohydride (127 g, 601 mmol) was added portion-wise
(exothermic). After 2 h, solvent swap with MeOH (257 g, 8019 mmol),
and sulfuric acid (79 g, 802 mmol) was added slowly (exothermic).
The mixture was heated at reflux for 18 h. Solvent was removed and
the mixture was extracted using DCM and water. The organic layer
was purified using a Biotage column (isopropanol/heptane),
affording methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
as a clear oil. MS (ESI) m/z: Calculated: 301.0; Observed: 302.0
(M.sup.++1).
Methyl
1-(3-fluoro-4-(2-(trimethylsilyl)ethynyl)benzyl)azetidine-3-carboxy-
late
##STR00022##
[0272] Methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
(25.00 g, 82.7 mmol), copper (I) iodide (3.14 g, 16.5 mmol),
(trimethylsilyl)acetylene (81.9 mL, 579 mmol),
bis(triphenylphosphine)palladium(II) chloride (5.81 g, 8.27 mmol),
and Hunig's base (115 mL, 662 mmol) were combined in a sealable
tube along with 100 mL THF. The tube was sealed and heated to 80
deg. C. under vigorous stirring for 24 h. The mixture was then
cooled to room temperature, filtered, and evaporated. The resulting
oil was purified by Biotage (75L, 0-50% EtOAc/hexanes), affording
methyl
1-(3-fluoro-4-(2-(trimethylsilyl)ethynyl)benzyl)azetidine-3-carboxylate
as a transparent brown oil. MS (ESI) m/z: Calculated: 319.1;
Observed: 320.1 (M.sup.++1).
Methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate
##STR00023##
[0273] Methyl
1-(3-fluoro-4-(2-(trimethylsilyl)ethynyl)benzyl)azetidine-3-carboxylate
(20.9 g, 65 mmol) and cesium fluoride (11 g, 72 mmol) were added to
DMF (50 mL). MeOH (100 mL) was added. After 2 h, MeOH was removed
and the mixture was extracted with DCM and water. The organic layer
was washed with brine and dried over magnesium sulfate. The solvent
was removed and the material was purified by Biotage (75L, 7-100%
EtOAc/hexanes), affording methyl
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate as light yellow
oil. MS (ESI) m/z: Calculated: 247.1; Observed: 248.0
(M.sup.++1).
1-(4-(2-(5-Benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxyl-
ic acid and
1-(4-(2-(5-Benzylpyridin-2-yl)ethyl)-3-fluorobenzyl)azetidine-3-carboxyli-
c acid
Methyl
1-(4-(2-(5-bromopyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-ca-
rboxylate
##STR00024##
[0275] A solution of 5-bromo-2-iodopyridine (800 mg, 2818 mop,
methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (704 mg,
2846 .mu.mol), and copper(I) iodide (21.5 mg, 113 mol) in
triethylamine (9.4 mL) was sparged with argon (30 sec), and
PdCl.sub.2(PPh.sub.3).sub.2 (79.1 mg, 113 .mu.mol) was then added
in one portion at 25.degree. C. The reaction mixture was cooled to
0.degree. C. and stirred for 40 min, and then allowed to stir at
25.degree. C. for 1.5 h. The mixture was subsequently diluted with
EtOAc (120 mL) and sequentially washed with water (4.times.30 mL)
and brine (30 mL). The organic layer was dried over sodium sulfate,
filtered, and concentrated in vacuo. Chromatographic purification
of the residue (silica gel, 0-100% EtOAc/Hexanes) furnished methyl
1-(4-(2-(5-bromopyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-c-
arboxylate as a light yellow oil. MS (ESI) m/z: Calculated:
402.0/404.0; Observed: 402.8/404.8 (M.sup.++1).
Methyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-c-
arboxylate
##STR00025##
[0276] Benzyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxy-
late
##STR00026##
[0278] Methyl
1-(4-(2-(5-bromopyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxyl-
ate (340.0 mg, 843 .mu.mol), PdCl.sub.2(P(t-Bu).sub.2Ph).sub.2 (37
mg, 59 .mu.tmol), and THF (4.0 mL) were combined under an argon
atmosphere. Benzylzinc(II) bromide (0.5M in THF; 1.92 mL, 961
.mu.mol) was added via syringe, and the resulting solution was
stirred at 25.degree. C. for 1 h. Additional benzylzinc(II) bromide
(0.5M in THF; 1.92 mL) was then added, and the resulting solution
was stirred for 1 h at 25.degree. C. A third portion of
benzylzinc(II) bromide (0.5M in THF; 1.92 mL) was then added, and
the resulting solution was stirred for 1 h at 25.degree. C. The
mixture was subsequently partitioned between half-saturated aqueous
NaHCO.sub.3 (50 mL) and EtOAc (100 mL). The organic layer was
separated, washed with brine (30 mL), dried over Na.sub.2SO.sub.4,
filtered, and concentrated in vacuo. Chromatographic purification
of the residue (silica gel, 0-100% EtOAc/Hexanes) separately
furnished benzyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxy-
late (MS (EST) m/z: Calculated: 490.2; Observed: 490.8 (M.sup.++1))
and methyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3--
carboxylate (MS (ESI) m/z: Calculated: 414.2; Observed: 414.8
(M.sup.++1)) as a yellow oils.
1-(4-(2-(5-Benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxyl-
ic acid
##STR00027##
[0280] A mixture of lithium hydroxide hydrate (28.4 mg, 677
.mu.mol) and methyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3--
carboxylate (77.9 mg, 188 .mu.mol) in THF (2.7 mL) and water (0.69
mL) was stirred at 25.degree. C. for 3 h. The reaction mixture was
then partially concentrated in vacuo (to remove THF), water (1.5
mL) was added, and the resulting solution was sonicated for 1 min.
The reaction solution was subsequently acidified with 1.0N HCl (670
.mu.L), brought to pH 6 with 1M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4
phosphate buffer (1.0 mL), and sonicated. The resulting precipitate
was collected by vacuum filtration, washed with water (3.0 mL), and
dried in vacuo to provide
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)azetidine-3-carboxy-
lic acid as a light yellow solid. MS (ESI) m/z: Calculated: 400.2;
Observed: 401.1 (M.sup.++1).
Example 4
1-(4-(2-(5-Benzylpyridin-2-yl)ethyl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
##STR00028##
[0282] A mixture of benzyl
1-(4-(2-(5-benzylpyridin-2-yl)ethynyl)-3-fluorobenzyl)-azetidine-3-carbox-
ylate (103.0 mg, 210 .mu.mol) and 10% palladium on carbon (22 mg,
21 .mu.mol) in MeOH (4.0 mL) was cycled under an H.sub.2 atmosphere
(alternately evacuating flask to 1 Ton, then refilling with H.sub.2
(1 atm); repeated 3.times.), then stirred under H.sub.2 (1 atm) at
25.degree. C. for 5 h. The reaction suspension was subsequently
filtered through Celite (washing with MeOH (10 mL)), and the
filtrate was concentrated in vacuo. rpHPLC purification of the
residue (C18, 0-100% CH.sub.3CN/H.sub.2O+0.1% TFA) furnished
1-(4-(2-(5-benzylpyridin-2-yl)ethyl)-3-fluorobenzyl)azetidine-3-carboxyli-
c acid, bis(trifluoroacetic acid) salt as a colorless oil. MS (ESI)
m/z: Calculated: 404.2; Observed: 405.1 (M.sup.++1).
Example 5
1-(4-(2-(5-Benzyl-2-(methylamino)phenyl)ethynyl)-3-fluorobenzyl)azetidine--
3-carboxylic acid
##STR00029##
[0284] The title compound can be made using methods similar to
those in Example 3. MS m/z: Calculated 428.19: Observed: 429.2.
Activity of Compounds of the Invention
[0285] The compounds of the invention made according to the
synthesis noted above can be assayed for their ability to modulate
the S1P-1 receptor. Compounds can be evaluated for the ability to
induce S1P1-specific receptor internalization using standard
in-vitro receptor internalization assays and their utility as
immunoregulatory agents can be demonstrated by their activity as
agonists of the S1P1 receptor measured in the receptor
internalization assay (>50% of S1P control at 10 nM or 300 nM).
The compounds accordingly are expected to be useful as S1P-1
receptor modulators, e.g., in the treatment of a variety of S1P-1
receptor-mediated clinical conditions. Such conditions include
transplant rejection (solid organ transplant and islet cells);
transplant rejection (tissue); cancer; autoimmune/inflammatory
diseases; rheumatoid arthritis; lupus; insulin dependent diabetes
(Type I); non-insulin dependent diabetes (Type II); multiple
sclerosis; psoriasis; ulcerative colitis; inflammatory bowel
disease; Crohn's disease; acute and chronic lymphocytic leukemias
and lymphomas.
[0286] To further demonstrate the suitability of compounds of the
invention as S1P-1 receptor modulators for treating conditions such
as transplant rejection; cancer; autoimmune/inflammatory diseases;
rheumatoid arthritis; lupus; diabetes; multiple sclerosis;
psoriasis; ulcerative colitis; inflammatory bowel disease; Crohn's
disease; acute and chronic lymphocytic leukemias and lymphomas
where immunosuppression is central (of which reduction of
lymphopenia is therefore a well-established indicator), compounds
of the invention can be evaluated in laboratory animals as
described below.
Protocol
Mice
[0287] C57BL/6J mice (B6, Jackson Laboratories, Bar Harbor, Me.)
can be maintained in a specific pathogen-free environment under a
microisolator containment system. Both adult male and female
age-matched mice can be used for all experiments, which can be
reviewed and approved by the Animal Care and Use Committee at the
University of Virginia. Whenever the protocol stated Mice were
anesthetized via intraperitoneal injections of ketamine
hydrochloride (125 mg/kg; Sanofi Winthrop Pharmaceuticals, New
York, N.Y.), xylazine (12.5 mg/kg TranquiVed; Phoenix Scientific,
St. Joseph, Mo.), and atropine sulfate (0.025 mg/kg; Fujisawa USA,
Deerfield, Ill.).
Flow Cytometry Preparation and Analysis
[0288] Blood can be harvested from at least six mice for each time
point of 0, 4, 8, 24, 48, 72 h following one day, 3 days or 7 days
daily dosing with the test compound. Following terminal bleeds
brain and certain other tissues can be harvested from all animals
undergoing treatment. Cell counts can be determined from whole
blood, yielding cell counts in thousands of cells per microliter
(K/1L).
[0289] To identify and quantify lymphocyte subsets, cell
suspensions were analyzed by flow cytometry. Following red blood
cell lysis, cells were stained with anti-mouse monoclonal
antibodies against CD3, CD4, CD8, CD19, and NK 1.1 (BD Biosciences,
San Jose, Calif.). Cells were analyzed via four-color flow
cytometry on a FACSCalibur (BD Biosciences) in the University of
Virginia Cancer Center Core Facility. Lymphocyte subsets, including
B cells, total T cells, CD4 T cells, CD8 T cells, double-positive
thymocytes, double-negative thymocytes, NK cells, and NK/T cells,
were analyzed. The size of each cell population was calculated as
the product of the total lymphocyte count recorded by the Hemavet
or hemocytometer and the percentage of positive lymphocytes
recorded by the flow cytometer. All data were analyzed with BD
Biosciences Cell Quest analysis software.
[0290] The compounds of the invention are expected to be useful
drugs for treating conditions such as transplant rejection; cancer;
autoimmune/inflammatory diseases; rheumatoid arthritis; lupus;
diabetes; multiple sclerosis; psoriasis; ulcerative colitis;
inflammatory bowel disease; Crohn's disease; acute and chronic
lymphocytic leukemias and lymphomas where immunosuppression is
central.
[0291] Rat Lymphopenia Study Protocol
[0292] Animals:
[0293] Female Lewis rats (150-175 gms, 6-8 wks) are received from
Charles River Laboratories and allowed to acclimatize for at least
one week before being placed on study.
[0294] Procedure:
[0295] 1) Rats (n=4/group) are administered compound or vehicle
(12.5% captisol in water) orally (PO, 10 mL/kg) at time 0.
[0296] 2) At various time points following dosing (1, 4, 8, or 24
firs), animals are sacrificed by CO.sub.2 inhalation.
[0297] 3) Using a 20 G needle and lcc syringe, blood is collected
by cardiac puncture.
[0298] 4) Approximately 500 .mu.L of blood is placed in a
microtainer tube containing EDTA (BD #365973), and the sample is
mixed thoroughly.
[0299] 5) Differential cell counts are perfomed using an Advia 120
hematology system by Bayer.
EQUIVALENTS
[0300] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of the invention.
Various substitutions, alterations, and modifications may be made
to the invention without departing from the spirit and scope of the
invention. Other aspects, advantages, and modifications are within
the scope of the invention. The contents of all references, issued
patents, and published patent applications cited throughout this
application are hereby incorporated by reference. The appropriate
components, processes, and methods of those patents, applications
and other documents may be selected for the invention and
embodiments thereof.
* * * * *