U.S. patent application number 12/189010 was filed with the patent office on 2009-02-12 for bicyclic sphingosine 1-phosphate analogs.
This patent application is currently assigned to UNIVERSITY OF VIRGINIA PATENT FOUNDATION. Invention is credited to Kevin R. LYNCH, Timothy L. Macdonald.
Application Number | 20090042955 12/189010 |
Document ID | / |
Family ID | 38180368 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090042955 |
Kind Code |
A1 |
LYNCH; Kevin R. ; et
al. |
February 12, 2009 |
BICYCLIC SPHINGOSINE 1-PHOSPHATE ANALOGS
Abstract
Compounds that have agonist activity at one or more of the S1P
receptors are provided. The compounds are sphingosine analogs that,
after phosphorylation, can behave as agonists at S1P receptors.
Inventors: |
LYNCH; Kevin R.;
(Charlottesville, VA) ; Macdonald; Timothy L.;
(Charlottesville, VA) |
Correspondence
Address: |
IPLM GROUP, P.A.
POST OFFICE BOX 18455
MINNEAPOLIS
MN
55418
US
|
Assignee: |
UNIVERSITY OF VIRGINIA PATENT
FOUNDATION
|
Family ID: |
38180368 |
Appl. No.: |
12/189010 |
Filed: |
August 8, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US07/03645 |
Feb 9, 2007 |
|
|
|
12189010 |
|
|
|
|
60771789 |
Feb 9, 2006 |
|
|
|
60855960 |
Nov 1, 2006 |
|
|
|
60860694 |
Nov 21, 2006 |
|
|
|
Current U.S.
Class: |
514/364 ;
514/533; 548/131; 560/54 |
Current CPC
Class: |
C07D 333/16 20130101;
A61P 27/02 20180101; A61P 37/06 20180101; C07F 9/091 20130101; A61P
25/00 20180101; A61P 3/00 20180101; A61P 29/00 20180101; A61P 43/00
20180101; C07D 419/04 20130101; C07C 217/64 20130101; A61P 19/00
20180101; A61P 3/10 20180101; A61P 1/00 20180101; C07F 9/093
20130101; A61P 25/28 20180101; C07D 271/06 20130101; A61P 37/00
20180101; A61P 19/02 20180101; A61P 1/04 20180101; C07C 215/28
20130101 |
Class at
Publication: |
514/364 ;
548/131; 560/54; 514/533 |
International
Class: |
A61K 31/4245 20060101
A61K031/4245; C07D 271/06 20060101 C07D271/06; A61K 31/225 20060101
A61K031/225; A61P 37/00 20060101 A61P037/00; C07C 69/75 20060101
C07C069/75 |
Goverment Interests
US GOVERNMENT RIGHTS
[0002] This invention was made with United States Government
support under Grant No. RO1 GM 067958 awarded by the National
Institutes of Health. The United States Government may have certain
rights in the invention.
Claims
1. A compound of formula I ##STR00039## wherein X.sup.1, Y.sup.1
and Z.sup.1 are independently O, CR.sup.a, CR.sup.aR.sup.b, N,
NR.sup.c, or S; R.sup.1 is hydrogen, halo (C.sub.1-C.sub.10)alkyl,
(C.sub.1-C.sub.10)haloalkyl, or (C.sub.1-C.sub.10)alkoxy; R.sup.2
is hydrogen, halo, (C.sub.1-C.sub.20)alkyl,
(C.sub.1-C.sub.20)alkoxy; (C.sub.2-C.sub.26)alkoxyalkyl;
(C.sub.2-C.sub.20)alkenyl, (C.sub.2-C.sub.20)alkynyl,
(C.sub.3-C.sub.12)cycloalkyl, (C.sub.6-C.sub.10)aryl,
(C.sub.7-C.sub.30)arylalkyl, (C.sub.2-C.sub.10)heterocyclic, and
(C.sub.5-C.sub.10)heteroaryl; or R.sup.2 can be a group having
formula II, III, IV, V, or VI; ##STR00040## where R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12R.sup.13, and R.sup.14 are
independently O, S, C, CR.sup.15CR.sup.16R.sup.17,C.dbd.O, N or
NR.sup.18 R.sup.15, R.sup.16 and R.sup.17 are independently
hydrogen, halo, (C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkyl
substituted with halo, hydroxy, (C.sub.1-C.sub.10)alkoxy, or cyano;
and where R.sup.18 can be hydrogen or (C.sub.1-C.sub.10)alkyl;
where at least one of R.sup.10, R.sup.11R.sup.12, R.sup.13, or
R.sup.14 is a heteroatom; where Z.sup.2 is (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, substituted alkyl,
(C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6)alkynyl, aryl, alkyl
substituted aryl, arylalkyl, or aryl substituted arylalkyl; wherein
the alkyl groups of Z.sup.2 are optionally substituted with 1, 2,
3, or 4 substituent groups where the substituent groups
independently are halo, (C.sub.1-C.sub.10)alkoxy or cyano;
##STR00041## indicates one or more optional double bonds; wherein
Y.sup.2 is O, C.dbd.O, or CH.sub.2; W.sup.1 is a bond or
--CH.sub.2--CH.sub.2--CH.sub.2--; W.sup.2 is a bond or --CH.sub.2--
and m is 1, 2, or 3, or (C.dbd.O)(CH.sub.2).sub.1-5 and m is 1; and
n is 0, 1, 2, or 3; each represents an optional double bond;
R.sup.3 is hydrogen, (C.sub.1-C.sub.10)alkyl, or
(C.sub.1-C.sub.10)alkoxy; R.sup.4 is hydroxyl (--OH), phosphate
(--OPO.sub.3H.sub.2), phosphonate (--CH.sub.2PO.sub.3H.sub.2), or
alpha-substituted phosphonate; R.sup.a, R.sup.b, and R.sup.c are
independently hydrogen, or (C.sub.1-C.sub.10)alkyl; wherein the
alkyl groups of R.sup.1 are optionally substituted with 1, 2, 3, or
4 substituent groups where the substituent groups independently are
halo, (C.sub.1-C.sub.10)alkoxy or cyano; wherein any of the alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, or heteroaryl
groups of R.sup.2 are optionally substituted with 1, 2, 3, or 4
substituent groups where the substituent groups independently are
oxo (.dbd.O), imino (.dbd.NR.sup.d), (C.sub.1-C.sub.10)alkyl,
(C.sub.1-C.sub.10)alkoxy, or C.sub.6-aryl; or wherein one or more
of the carbon atoms in the R.sup.2 alkyl groups can be
independently replaced with non-peroxide oxygen, sulfur or
NR.sup.c; the alkyl groups of R.sup.3 are optionally substituted
with 1, or 2 hydroxy groups; and R.sup.d is hydrogen, or
(C.sub.1-C.sub.10)alkyl; or a pharmaceutically acceptable salt or
ester thereof.
2. The compound of claim 1, wherein R.sup.1 is hydrogen, fluorine,
chlorine, bromine, trifluoromethyl, methoxy,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)haloalkyl,
(C.sub.1-C.sub.6)alkyl substituted with, alkoxy or cyano,
alkyl-substituted aryl, aryl-substituted alkyl, or aryl-substituted
arylalkyl.
3. The compound of claim 2, wherein R.sup.1 is hydrogen,
trifluoromethyl, or --CH.sub.2CF.sub.3.
4. The compound of claim 2, wherein R.sup.1 is benzyl, phenylethyl,
or methyl benzyl.
5. The compound of claim 1, wherein R.sup.2 comprises
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--.
6. The compound of claim 1, wherein R.sup.2 is ##STR00042##
7. The compound of claim 6, wherein R.sup.2 is: ##STR00043## where
Y.sup.3 is (CH.sub.3).sub.3C--,
CH.sub.3CH.sub.2(CH.sub.3).sub.2C--, CH.sub.3CH.sub.2CH.sub.2--,
CH.sub.3(CH.sub.2).sub.2CH.sub.2--,
CH.sub.3(CH.sub.2).sub.4--CH.sub.2--, (CH.sub.3).sub.2CHCH.sub.2--,
(CH.sub.3).sub.3CCH.sub.2--, CH.sub.3CH.sub.2O--,
(CH.sub.3).sub.2CHO--, or CF.sub.3CH.sub.2CH.sub.2-- or a group
having the formula: ##STR00044##
8. The compound of claim 7, wherein R.sup.2 is: ##STR00045##
9. The compound of claim 8, wherein R.sup.2 is: ##STR00046##
10. The compound of claim 6, wherein R.sup.2 is: ##STR00047##
11. The compound of claim 10, wherein R.sup.2 is ##STR00048##
12. The compound of claim 1, wherein R.sup.2 has formula IV
##STR00049##
13. The compound of claim 12, wherein R.sup.2 is ##STR00050##
14. The compound of claim 1, wherein R.sup.2 is
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.10)alkenyl and
(C.sub.2-C.sub.14)alkynyl, (C.sub.1-C.sub.10)alkoxy or
(C.sub.2-C.sub.16)alkoxyalkyl.
15. The compound of claim 14, wherein R.sup.2 is
(C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkoxy or
(C.sub.2-C.sub.12)alkoxyalkyl.
16. The compound of claim 15, wherein R.sup.2 is methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl, trifluoromethyl,
trifluoroethyl, trifluoromethoxy, trifluoroethoxy, methoxy, ethoxy,
propoxy, butoxy, pentoxy, heptoxy, or octoxy.
17. The compound of claim 1, wherein each of X.sup.1, Y.sup.1 and
Z.sup.1 is CH.sub.2.
18. The compound of claim 1, wherein R.sup.3 is hydrogen, methyl,
hydroxymethyl, ethyl, hydroxyethyl, propyl, or isopropyl.
19. The compound of claim 18, wherein R.sup.3 is hydrogen, methyl,
hydroxymethyl, ethyl, or hydroxyethyl.
20. The compound of claim 1, having the formula ##STR00051##
##STR00052##
21. The compound of claim 20, having the formula: ##STR00053##
22. A method for prevention or treatment of a pathological
condition or symptom in a mammal, wherein the activity of
sphingosine 1-phosphate receptors is implicated and agonism of such
activity is desired, comprising administering to said mammal an
effective amount of a compound of claim 1.
23. The method of claim 21, wherein the pathological condition is
an autoimmune disease.
24. The method of claim 22, wherein the autoimmune disease is
uveitis, type I diabetes, rheumatoid arthritis, inflammatory bowel
diseases, or multiple sclerosis.
25. The method of claim 24, wherein the autoimmune disease is
multiple sclerosis.
26. The method of claim 22, wherein the prevention or treatment of
a pathological condition is altering lymphocyte trafficking.
27. The method of claim 26, wherein altering lymphocyte trafficking
provides prolonged allograft survival.
28. The method of claim 27, wherein the allograft is for
transplantation.
29. A method for prevention or treatment of a pathological
condition or symptom in a mammal, wherein the activity S1P lyase
implicated and inhibition of the S1P lyase is desired, comprising
administering to said mammal an effective amount of a compound of
claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation under 35 U.S.C. 111(a) of
International Application No. PCT/US2007/003645, filed on Feb. 9,
2007, which claims priority to Provisional Application Ser. Nos.
60/771,789, filed Feb. 9, 2006, 60/855,960, filed Nov. 1, 2006 and
60/860,694, filed Nov. 21, 2006, the disclosures of which are
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator
that evokes a variety of cellular responses by stimulation of five
members of the endothelial cell differentiation gene (EDG) receptor
family. The EDG receptors are G-protein coupled receptors (GPCRs)
and on stimulation propagate second messenger signals via
activation of heterotrimeric G-protein alpha (G.sub..alpha.)
subunits and beta-gamma (G.sub..beta..gamma.) dimers. Ultimately,
this S1P-driven signaling results in cell survival, increased cell
migration and, often, mitogenesis. The recent development of
agonists targeting S1P receptors has provided insight regarding the
role of this signaling system in physiologic homeostasis. For
example, the immunomodulator, FTY720
(2-amino-2-[2-(4-octylphenyl)ethyl]propane 1,3-diol), that
following phosphorylation, is an agonist at 4 of 5 S1P receptors,
revealed that enhancing S1P tone influences lymphocyte trafficking.
Further, S1P type 1 receptor (S1P.sub.1) antagonists cause leakage
of the lung capillary endothelium, which suggests that S1P may be
involved in maintaining the integrity of the endothelial barrier in
some tissue beds.
[0004] Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator
that evokes a variety of cellular responses by stimulation of five
members of the endothelial cell differentiation gene (EDG) receptor
family.
[0005] Sphingosine-1-phosphate (S1P) has been demonstrated to
induce many cellular processes, including those that result in
platelet aggregation, cell proliferation, cell morphology,
tumor-cell invasion, endothelial cell chemotaxis and angiogenesis.
For these reasons, S1P receptors are good targets for therapeutic
applications such as wound healing and tumor growth inhibition.
[0006] Sphingosine-1-phosphate signals cells in part via a set of G
protein-coupled receptors named S1P.sub.1, S1P.sub.2, S1P.sub.3,
S1P.sub.4, and S1P.sub.5 (formerly EDG1, EDG5, EDG3, EDG6 and
EDG8). The EDG receptors are G-protein coupled receptors (GPCRs)
and on stimulation propagate second messenger signals via
activation of heterotrimeric G-protein alpha (G.sub..alpha.)
subunits and beta-gamma (G.sub..beta..gamma.) dimers. These
receptors share 50-55% amino acid sequence identity and cluster
with three other receptors (LPA.sub.1, LPA.sub.2, and LPA.sub.3
(formerly EDG2, EDG4 and EDG7) for the structurally related
lysophosphatidic acid (LPA).
[0007] 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 reassociate
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.
[0008] S1P receptors make good drug targets because individual
receptors are both tissue and response specific. Tissue specificity
of the S1P receptors is desirable 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 of
importance because it allows for the development of agonists or
antagonists that initiate or suppress certain cellular responses
without affecting other responses. For example, the response
specificity of the S1P receptors could allow for an S1P mimetic
that initiates platelet aggregation without affecting cell
morphology.
[0009] Sphingosine-1-phosphate is formed as a metabolite of
sphingosine in its reaction with sphingosine kinase and is stored
in abundance in the aggregates of platelets 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. Reversible biodegradation of S1P
most likely proceeds via hydrolysis by ectophosphohydrolases,
specifically the sphingosine 1-phosphate phosphohydrolases.
Irreversible degradation of S1P is catalyzed by S1P lyase yielding
ethanolamine phosphate and hexadecenal.
[0010] Currently, there is a need for novel, potent, and selective
agents that are agonists of the S1P receptor having enhanced
potency, selectivity, and oral bioavailability. In addition, there
is a need in the art for identification of, as well as the
synthesis and use of such compounds. The present invention
satisfies these needs.
SUMMARY
[0011] The present invention provides in one aspect compounds that
have agonist activity at one or more of the S1P receptors. The
compounds are sphingosine analogs that, after phosphorylation, can
behave as agonists at S1P receptors. Accordingly, there is provided
a compound of formula I:
##STR00001##
wherein X.sup.1, Y.sup.1 and Z.sup.1 are independently O, CR.sup.a,
CR.sup.aR.sup.b, N, NR.sup.c, or S; R.sup.1 is hydrogen, halo
(C.sub.1-C.sub.10)alkyl, or (C.sub.1-C.sub.10)alkoxy; R.sup.2 is
hydrogen, halo, (C.sub.1-C.sub.20)alkyl, (C.sub.1-C.sub.20)alkoxy;
(C.sub.2-C.sub.26)alkoxyalkyl; (C.sub.2-C.sub.20)alkenyl,
(C.sub.2-C.sub.20)alkynyl, (C.sub.3-C.sub.12)cycloalkyl,
(C.sub.6-C.sub.10)aryl, (C.sub.7-C.sub.30)arylalkyl,
(C.sub.2-C.sub.10)heterocyclic, (C.sub.4-C.sub.10)heteroaryl, or
R.sup.2 can be a group having formula II, III, IV, V, or VI:
##STR00002##
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13,
and R.sup.14 are independently O, S, C, CR.sup.15,
CR.sup.16R.sup.17, C.dbd.O, N or NR.sup.18; R.sup.15, R.sup.16 and
R.sup.17 are independently hydrogen, halo, (C.sub.1-C.sub.10)alkyl,
(C.sub.1-C.sub.10)alkyl substituted with halo, hydroxy,
(C.sub.1-C.sub.10)alkoxy, or cyano; and where R.sup.18 can be
hydrogen or (C.sub.1-C.sub.10)alkyl and at least one of R.sup.10,
R.sup.11, R.sup.12, R.sup.13, or R.sup.14 is a heteroatom (O, S or
N); Z.sup.2 is (C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl,
substituted alkyl, (C.sub.2-C.sub.6)alkenyl,
(C.sub.2-C.sub.6)alkynyl, (C.sub.6-C.sub.10)aryl, alkyl substituted
aryl, (C.sub.7-C.sub.16)arylalkyl, or aryl substituted arylalkyl;
wherein the alkyl groups of Z.sup.2 are optionally substituted with
1, 2, 3, or 4 groups independently are halo,
(C.sub.1-C.sub.10)alkoxy or cyano; indicates one or more optional
double bonds; Y.sup.2 is O, C.dbd.O, or CH.sub.2; W.sup.1 is a bond
or --CH.sub.2--CH.sub.2--CH.sub.2--; W.sup.2 is a bond or
--CH.sub.2-- and m is 1, 2, or 3, or (C.dbd.O)(CH.sub.2).sub.1-5
and m is 1. Each represents an optional double bond; n is 0, 1, 2,
or 3, and q is 0, 1, 2, or 3.
[0012] R.sup.3 is hydrogen, (C.sub.1-C.sub.10)alkyl, or
(C.sub.1-C.sub.10)alkoxy; and R.sup.4 is hydroxyl (--OH), phosphate
(--OPO.sub.3H.sub.2), phosphonate (--CH.sub.2PO.sub.3H.sub.2), or
alpha-substituted phosphonate; R.sup.c is hydrogen, or
(C.sub.1-C.sub.10)alkyl. R.sup.a, R.sup.b, and R.sup.c are
independently hydrogen, or (C.sub.1-C.sub.10)alkyl.
[0013] The alkyl groups of R.sup.1 can be optionally substituted
with 1, 2, 3, or 4 substituent groups, where the substituent groups
independently are halo, (C.sub.1-C.sub.10)alkoxy or cyano. Any of
the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, or
heteroaryl groups of R.sup.2 are optionally substituted with 1, 2,
3, or 4 substituent groups, where the substituent groups
independently are oxo (.dbd.O), imino (.dbd.NR.sup.d),
(C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkoxy, or C.sub.6-aryl,
or wherein one or more of the carbon atoms in the R.sup.2 alkyl
groups can be independently replaced with non-peroxide oxygen,
sulfur or NR.sup.c; the alkyl groups of R.sup.3 are optionally
substituted with 1, or 2 hydroxy groups; and R.sup.d is hydrogen,
or (C.sub.1-C.sub.10)alkyl. The invention includes pharmaceutically
acceptable salts or esters of the compounds of formula I.
[0014] In another aspect, the invention provides phosphate esters
having formula VII.
##STR00003##
[0015] In another aspect, the invention provides pro-drugs of the
compounds of formula I. In another aspect, the invention also
provides compounds of formula I or pharmaceutically acceptable
salts or esters thereof for use in medical therapy.
[0016] In another aspect, the present invention provides a method
for inhibiting angiogenesis in a tumor, comprising contacting the
cancerous cells with an effective amount of a compound of formula
I, or a pharmaceutically acceptable salt thereof.
[0017] In another aspect, the invention provides a method for
modulating the immune system by altering lymphocyte trafficking for
treatment of autoimmune diseases or prolongation of allograft
transplant survival, said method comprising administering an
effective amount of at least one compound of formula I to a subject
in need thereof.
[0018] In another aspect, the invention provides a method for to
preventing, inhibiting or treating neuropathic pain, wherein the
method comprises administering an effective amount of at least one
compound of formula I or a compound of formula I and a
pharmaceutically-acceptable carrier is administered to a subject in
need thereof. Pain can be nociceptive or neuropathic in nature.
Neuropathic pain is characterized by its chronic nature, an absence
of an obvious, direct cause (e.g., tissue damage), hyperalgesia or
allodynia. Hyperalgesia is an exaggerated response to a painful
stimulus. Allodynia is the perception of normal stimuli as painful
(examples include the touch of clothing, warm or cool air, etc.).
Neuropathic pain can be a sequel to nerve damage in an extremity
such as an arm, or more often a leg. Precipitating events can
include trauma, e.g., motor vehicle accidents or amputations (e.g.,
phantom limb pain). Neuropathic pain can occur due to an adverse
effect of drug therapies, e.g., vincristine or paclitaxel
(TAXOL.TM.) or can occur as a component of disease pathologies,
such as diabetes type 1 or type2, shingles, HIV-1 infections, etc.
Typically, neuropathic pain is not responsive to opiates or
non-steroidal anti-inflammatory drugs such as aspirin.
[0019] In another aspect, the invention provides a method for
repairing vascular injury following catheterization, comprising
contacting the lumen of the affected vessel with an effective
amount of the compound of formula I. In another aspect, the
invention includes coating indwelling stents with a compound of
formula I.
[0020] In another aspect, the present invention provides
compositions and methods for the use of S1P analogs to prevent and
inhibit vascular restenosis following vascular injury. For example,
the injury can be due to balloon angioplasty. In another aspect,
the present invention includes a method for treating subjects to
prevent vascular restenosis.
[0021] In another aspect, the present invention provides
compositions and methods for the use of sphingosine analogs
(including S1P pro-drugs) to prevent asthma attacks. In one aspect,
the asthma could be due to over production of cysteinyl
leukotrienes. In another aspect, the present invention includes a
method for treating subjects to treat asthma.
[0022] In another aspect, the present invention provides
compositions and methods for the use of sphingosine analogs of
formula I (including S1P pro-drugs) to treat obesity.
[0023] In another aspect, the present invention provides
compositions and methods for the use of sphingosine analogs
(including S1P pro-drugs) to normalize blood lipid composition. In
one aspect, blood low density lipoprotein (LDL or `bad
cholesterol`) levels could be lowered. In another aspect, blood
triglyceride levels could be lowered.
[0024] In another aspect, the present invention provides
compositions and methods for the use of S1P analogs and S1P
pro-drugs for the prevention and treatment of arteriosclerosis.
[0025] In another aspect, the present invention provides
compositions and methods for the use of S1P analogs and S1P
pro-drugs for the treatment of neoplastic disease. In one aspect,
this treatment is effected by application of S1P receptor
antagonists that are efficacious by virtue of their anti-angiogenic
properties. In another aspect, the treatment is effected by
administration of sphingosine analogs of formula I that inhibit the
multiple substrate lipid kinase.
[0026] In another aspect, the present invention provides
compositions and methods for the use of S1P analogs and S1P
pro-drugs for the treatment of neurodegenerative diseases. In one
aspect, the treatment is for senile dementia of the Alzheimers
type.
[0027] In another aspect, the invention provides a compound of
formula I, or a pharmaceutically acceptable salt thereof for use in
medical treatment (for example, treatment of neoplastic disease,
treatment of neuropathic pain, treatment of autoimmune disease,
prolongation of allograft survival).
[0028] In another aspect, the invention provides a method for the
use of a compound of formula I or a pharmaceutically acceptable
salt thereof to prepare a medicament for inhibiting tumor growth,
metastasis or tumor angiogenesis in a mammalian species (for
example, a human).
[0029] In another aspect, the invention provides for the use of a
compound of formula I or a pharmaceutically acceptable salt thereof
to prepare a medicament for treating an autoimmune disease or
prolonging allograft survival in a mammalian species (for example,
a human).
[0030] In another aspect, the invention provides for the use of a
compound of formula I or a pharmaceutically acceptable salt thereof
to prepare a medicament for treating neuropathic pain in a
mammalian species (for example, a human).
[0031] In another aspect, the invention provides a method for
assessing a compound of formula I (e.g., S1P receptor pro-drugs) as
a substrate for sphingosine kinase types 1 or 2, in vitro and in
vivo. In another aspect, the invention includes a method of
assessing a compound of formula I for binding to designated
receptor sites comprising in vivo or in vitro, with an amount of a
compound of formula I effective to bind said receptors. Tissue
comprising ligand bound designated S1P receptor sites can be used
to measure the selectivity of test compounds for specific receptor
subtypes, or can be used as a tool to identify potential
therapeutic agents for the treatment of diseases, by contacting
said agents with said ligand-receptor complexes, and measuring the
extent of displacement of the ligand or binding of the agent.
[0032] In another aspect, the invention provides novel
intermediates and processes disclosed herein that are useful for
preparing compounds of formula I, including the generic and
specific intermediates as well as the synthetic processes described
herein.
[0033] In another aspect, the present invention provides synthetic
schemes and methods of use of compounds having formula I and
analogs or derivatives thereof. In another aspect, the invention
provides synthetic and modification schemes for preparing analogs
and derivatives of the compounds of formula I, as well as
compositions and methods for the use of such analogs and
derivatives.
[0034] The above summary of the present invention is not intended
to describe each disclosed embodiment or every implementation of
the present invention. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive
list.
[0035] The details of one or more embodiments of the invention are
set forth in the accompanying description below. Other features,
objects, and advantages of the invention will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is an illustration of three S1P agonists FTY720,
AA151 compound XXIX and additional compounds of formula I.
[0037] FIGS. 2, 3 and 4 are schemes illustrating syntheses of
compounds of formula I.
[0038] FIGS. 5A, 5B and 6 illustrate the results of sphingosine
kinase type 2 (SPHK2) activity for S1P agonist compounds FTY720,
AA151, VIII and XVIII.
[0039] FIGS. 7 and 8 graphically illustrate the results of a broken
cell GTP.gamma..sup.35S binding assay for the human S1P.sub.1
receptor, testing S1P and compounds XXVI, XXVII and XXVIII.
[0040] FIG. 9 is graphically illustrates total lymphocyte counts
(k/.mu.l) 24 hours (left bar of each group) and 48 hours (right bar
of each group) after an oral dose of the test compounds are
administered to mice.
[0041] FIG. 10 is graphically illustrates total lymphocyte counts
(k/.mu.l) 18 hours after an oral dose of the test compounds are
administered to mice.
[0042] FIG. 11 is a graphical representation of the results of an
assay illustrating that compound VIII has no effect on the heart
rate of mice. In the assay the test compound was administered via
IV Dosing and the vehicle was 2% cyclodextrin.
DETAILED DESCRIPTION
[0043] The following abbreviations are used herein: S1P,
sphingosine-1-phosphate; S1P.sub.1-5 S1P receptor types; GPCR,
G-protein coupled receptor; SAR, structure-activity relationship;
EDG, endothelial cell differentiation gene; EAE, experimental
autoimmune encephalomyelitis; NOD non-obese diabetic; TNF.alpha.,
tumor necrosis factor alpha; HDL, high density lipoprotein; and
RT-PCR, reverse transcriptase polymerase chain reaction.
[0044] In describing and claiming the invention, unless otherwise
defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. Although any materials and
methods similar or equivalent to those described herein can be used
in the practice or testing of the present invention, the preferred
materials and methods are described herein. Each of the following
terms has meaning associated with it in this section. Specific and
preferred values listed below for radicals, substituents, and
ranges are for illustrations only; they do not exclude other
defined values or other values within defined ranges for the
radicals and substituents.
[0045] The terms "a," "an," "the," "at least one," and "one or
more" are used interchangeably. Thus, for example, a composition
that comprises "an" element means one element or more than one
element.
[0046] The term "receptor agonists" are compounds that mimic the
action of S1P at one or more of its receptors but may have
differing potency or efficacy. The term "receptor antagonists" are
compounds that 1) lack intrinsic agonist activity and 2) block
agonist (e.g., S1P) activation of the S1P receptor(s), often in a
manner that is both fully surmountable and reversible (`competitive
antagonist`).
[0047] The term "affected cell" refers to a cell of a subject
afflicted with a disease or disorder, which affected cell has an
altered phenotype relative to a subject not afflicted with a
disease or disorder.
[0048] Cells or tissue are "affected" by a disease or disorder if
the cells or tissue have an altered phenotype relative to the same
cells or tissue in a subject not afflicted with a disease or
disorder.
[0049] A disease or disorder is "alleviated" if the severity of a
symptom of the disease or disorder, the frequency with which such a
symptom is experienced by a patient, or both, is reduced.
[0050] An "analog" of a chemical compound is a compound that, by
way of example, resembles another in structure but is not
necessarily an isomer (e.g., 5-fluorouracil is an analog of
thymine).
[0051] The terms "cell," "cell line," and "cell culture" may be
used interchangeably.
[0052] A "control" cell, tissue, sample, or subject is a cell,
tissue, sample, or subject of the same type as a test cell, tissue,
sample, or subject. The control may, for example, be examined at
precisely or nearly the same time the test cell, tissue, sample, or
subject is examined. The control may also, for example, be examined
at a time distant from the time at which the test cell, tissue,
sample, or subject is examined, and the results of the examination
of the control may be recorded so that the recorded results may be
compared with results obtained by examination of a test cell,
tissue, sample, or subject. The control may also be obtained from
another source or similar source other than the test group or a
test subject, where the test sample is obtained from a subject
suspected of having a disease or disorder for which the test is
being performed.
[0053] A "test" cell, tissue, sample, or subject is one being
examined or treated.
[0054] A "pathoindicative" cell, tissue, or sample is one which,
when present, is an indication that the animal in which the cell,
tissue, or sample is located (or from which the tissue was
obtained) is afflicted with a disease or disorder. By way of
example, the presence of one or more breast cells in a lung tissue
of an animal is an indication that the animal is afflicted with
metastatic breast cancer.
[0055] A tissue "normally comprises" a cell if one or more of the
cell are present in the tissue in an animal not afflicted with a
disease or disorder.
[0056] The use of the word "detect" and its grammatical variants is
meant to refer to measurement of the species without
quantification, whereas use of the word "determine" or "measure"
with their grammatical variants are meant to refer to measurement
of the species with quantification. The terms "detect" and
"identify" are used interchangeably herein.
[0057] A "detectable marker" or a "reporter molecule" is an atom or
a molecule that permits the specific detection of a compound
comprising the marker in the presence of similar compounds without
a marker. Detectable markers or reporter molecules include, e.g.,
radioactive isotopes, antigenic determinants, enzymes, nucleic
acids available for hybridization, chromophores, fluorophores,
chemiluminescent molecules, electrochemically detectable molecules,
and molecules that provide for altered fluorescence-polarization or
altered light-scattering.
[0058] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0059] A "disorder" in an animal is a state of health in which the
animal is able to maintain homeostasis, but in which the animal's
state of health is less favorable than it would be in the absence
of the disorder. Left untreated, a disorder does not necessarily
cause a further decrease in the animal's state of health.
[0060] An "effective amount" means 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.
[0061] A "functional" molecule is a molecule in a form in which it
exhibits a property by which it is characterized. By way of
example, a functional enzyme is one which exhibits the
characteristic catalytic activity by which the enzyme is
characterized.
[0062] The term "inhibit" refers to the ability of a disclosed
compound to reduce or impede a described function. Preferably,
inhibition is by at least 10%, more preferably by at least 25%,
even more preferably by at least 50%, and most preferably, the
function is inhibited by at least 75%.
[0063] "Instructional material" includes a publication, a
recording, a diagram, or any other medium of expression which can
be used to communicate the usefulness of the disclosed compounds in
the kit for effecting alleviation of the various diseases or
disorders recited herein. Optionally, or alternately, the
instructional material may describe one or more methods of
alleviating the diseases or disorders in a cell or a tissue of a
mammal. The instructional material of the kit may, for example, be
affixed to a container which contains a disclosed compound or be
shipped together with a container which contains the identified
compound. Alternatively, the instructional material may be shipped
separately from the container with the intention that the
instructional material and the compound be used cooperatively by
the recipient.
[0064] The term "parenteral" means not through the alimentary canal
but by some other route such as subcutaneous, intramuscular,
intraspinal, or intravenous.
[0065] The term "pharmaceutically acceptable carrier" includes any
of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water and emulsions such as an oil/water
or water/oil emulsion, and various types of wetting agents. The
term also encompasses any of the agents approved by a regulatory
agency of the U.S. Federal government or listed in the U.S.
Pharmacopeia for use in animals, including humans.
[0066] The term "purified" and similar terms relate to the
isolation of a molecule or compound in a form that is substantially
free (at least 75% free, preferably 90% free, and most preferably
at least 95% free) from other components normally associated with
the molecule or compound in a native environment. The term
"purified" does not necessarily indicate that complete purity of
the particular molecules achieved during the process. A "very pure"
compound refers to a compound that is greater than 90% pure. A
"highly purified" compound refers to a compound that is greater
than 95% pure.
[0067] A "sample" refers preferably to a biological sample from a
subject, including, but not limited to, normal tissue samples,
diseased tissue samples, biopsies, blood, saliva, feces, semen,
tears, and urine. A sample can also be any other source of material
obtained from a subject, which contains cells, tissues, or fluid of
interest. A sample can also be obtained from cell or tissue
culture.
[0068] The term "standard," refers to something used for
comparison. For example, a standard can be a known standard agent
or compound which is administered or added to a control sample and
used for comparing results when measuring said compound in a test
sample. Standard can also refer to an "internal standard," such as
an agent or compound which is added at known amounts to a sample
and is useful in determining such things as purification or
recovery rates when a sample is processed or subjected to
purification or extraction procedures before a marker of interest
is measured.
[0069] A "subject" of analysis, diagnosis, or treatment is an
animal. Such animals include mammals, preferably a human.
[0070] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology for the purpose of
diminishing or eliminating those signs.
[0071] A "therapeutically effective amount" of a compound is that
amount of compound which is sufficient to provide a beneficial
effect to the subject to which the compound is administered.
[0072] The term "treating" includes prophylaxis of the specific
disorder or condition, or alleviation of the symptoms associated
with a specific disorder or condition or preventing or eliminating
said symptoms.
[0073] The disclosed compounds are generally named according to the
IUPAC or CAS nomenclature system. Abbreviations which are well
known to one of ordinary skill in the art may be used (e.g., "Ph"
for phenyl, "Me" for methyl, "Et" for ethyl, "h" for hour or hours,
"rt" for room temperature, and "rac" for racemic mixture).
[0074] The values listed below for radicals, substituents, and
ranges, are for illustration only; they do not exclude other
defined values or other values within defined ranges for the
radicals and substituents. The disclosed compounds include
compounds of formula I having any combination of the values,
specific values, more specific values, and preferred values
described herein.
[0075] The term "halogen" or "halo" includes bromo, chloro, fluoro,
and iodo. The term "haloalkyl", refers to an alkyl radical bearing
at least one halogen substituent, non-limiting examples include,
but are not limited to, chloromethyl, fluoroethyl or
trifluoromethyl and the like. The term "C.sub.1-C.sub.20 alkyl"
refers to a branched or linear alkyl group having from one to nine
carbons. Non-limiting examples include, but are not limited to,
methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl,
tert-butyl, pentyl, hexyl, heptyl, octyl and the like. The term
"C.sub.2-C.sub.20 alkenyl", refers to an olefinically unsaturated
branched or linear group having from two to nine carbon atoms and
at least one double bond. Typically, C.sub.2-C.sub.20 alkenyl
groups include, but are not limited to, 1-propenyl, 2-propenyl,
1,3-butadienyl, 1-butenyl, hexenyl, pentenyl, hexenyl, heptenyl,
octenyl and the like. The term (C.sub.2-C.sub.20)alkynyl can be
ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,
1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl,
2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl, and the like. The
term "(C.sub.1-C.sub.10)alkoxy or alkoxyl" refers to an alkyl group
attached through an oxygen atom. Examples of
(C.sub.1-C.sub.10)alkoxy can be methoxy, ethoxy, propoxy,
isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or
hexyloxy and the like. The term "C.sub.3-C.sub.12 cycloalkyl", can
be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl and the like.
[0076] The term "optionally substituted" refers to zero, one, two,
three or four substituents, wherein the substituents are each
independently selected. Each of the independently selected
substituents may be the same or different than other
substituents.
[0077] The term "(C.sub.6-C.sub.10)aryl" refers to a mono or
bicyclic carbocyclic ring system having one or two aromatic rings
including, but not limited to, phenyl, benzyl, naphthyl,
tetrahydronaphthyl, indanyl, indenyl, and the like.
[0078] The term "aryl(C.sub.1-C.sub.20)alkyl" or "aralkyl" refers
to an alkyl group substituted with a mono or bicyclic carbocyclic
ring system having one or two aromatic rings including, a group
such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and
the like. Non-limiting examples of arylalkyl include benzyl,
phenylethyl, and the like.
[0079] The term "optionally substituted aryl" includes aryl
compounds having zero, one, two, three or four substituents, and a
substituted aryl includes aryl compounds having one, two, three or
four substituents, wherein the substituents include groups such as,
for example, alkyl, halo, or amino substituents.
[0080] The "(C.sub.2-C.sub.10)heterocyclic group" refers to an
optionally substituted mono- or bicyclic carbocyclic ring system
containing one, two, or three heteroatoms (optionally in each ring)
wherein the heteroatoms are oxygen, sulfur, and nitrogen.
[0081] The term "(C.sub.4-C.sub.10)heteroaryl" refers to an
optionally substituted mono- or bicyclic carbocyclic ring system
containing one, two, or three heteroatoms (optionally in each ring)
wherein the heteroatoms are oxygen, sulfur, and nitrogen.
Non-limiting examples of heteroaryl groups include furyl, thienyl,
pyridyl, and the like.
[0082] The term "bicyclic" represents either an unsaturated or
saturated stable bridged or fused bicyclic carbon ring. The
bicyclic ring may be attached at any carbon atom which affords a
stable structure. Typically a bicyclic ring system can have from
about 7- to about 12 atoms in the ring system. The term includes,
but is not limited to, naphthyl, dicyclohexyl, dicyclohexenyl, and
the like.
[0083] The term "phosphate analog" and "phosphonate analog"
comprise analogs of phosphate and phosphonate wherein the
phosphorous atom is in the +5 oxidation state and one or more of
the oxygen atoms is replaced with a non-oxygen moiety, including
for example, the phosphate analogs phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate,
boronophosphates, and the like, including associated counterions,
e.g., H, NH.sub.4, Na, K, and the like if such counterions are
present.
[0084] A "derivative" of a compound refers to a chemical compound
that may be produced from another compound of similar structure in
one or more steps, such as replacement of hydrogen by an alkyl,
acyl, or amino group.
[0085] The term "pharmaceutically acceptable carrier" includes any
of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, hydroxypropyl beta-cyclodextrins
(HO-propyl beta cyclodextrins), water, emulsions such as an
oil/water or water/oil emulsion, and various types of wetting
agents. The term also encompasses any of the agents approved by a
regulatory agency of the U.S. Federal government or listed in the
U.S. Pharmacopeia for use in animals, including humans.
[0086] The term "pharmaceutically-acceptable salt" refers to salts
which retain the biological effectiveness and properties of the
disclosed compounds and which are not biologically or otherwise
undesirable. In many cases, the disclosed compounds are capable of
forming acid or base salts by virtue of the presence of amino or
carboxyl groups or groups similar thereto.
[0087] An "effective amount" means an amount sufficient to produce
a selected effect. For example, an effective amount of an S1P
receptor agonist is an amount that decreases the cell signaling
activity of the S1P receptor.
[0088] The disclosed compounds can contain one or more asymmetric
centers in the molecule. In accordance with the present disclosure
any structure that does not designate the stereochemistry is to be
understood as embracing all the various optical isomers, as well as
racemic mixtures thereof.
[0089] The disclosed compounds may exist in tautomeric forms and
the invention includes both mixtures and separate individual
tautomers. For example, the following structure:
##STR00004##
is understood to represent a mixture of the structures:
##STR00005##
as well as
##STR00006##
[0090] The terms 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers
to a branched or straight alkyl or alkenyl group, wherein the first
integer represents the total number of carbons in the group and the
second integer represent the number of double bonds in the
group.
[0091] An "S1P modulating agent" refers a compound or composition
that is 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 in the examples and known in the art. "S1P
receptor," refers to all of the S1P receptor subtypes (for example,
the S1P receptors S1P.sub.1, S1P.sub.2, S1P.sub.3, S1P.sub.4, and
S1P.sub.5), unless the specific subtype is indicated.
[0092] It will be appreciated by those skilled in the art that the
disclosed compounds having chiral centers may exist in and be
isolated in optically active and racemic forms. It is to be
understood that the disclosed compounds encompass any racemic,
optically active or stereoisomeric form, or mixtures thereof, of
the compound, which possess the useful properties described herein,
such as the S,R; S,S; R,R; or R,S diastereomers. It is well known
in the art how to prepare such optically active forms (for example,
by resolution of the racemic form by recrystallization techniques,
by synthesis from optically-active starting materials, by chiral
synthesis, or by chromatographic separation using a chiral
stationary phase) and how to determine S1P agonist activity using
the standard tests described herein, or using other similar tests
which are well known in the art. In addition, some compounds may
exhibit polymorphism.
[0093] Potential uses of an S1P receptor agonist pro-drugs
(S1P.sub.1 receptor type selective agonists preferred) include, but
are not limited to, altering lymphocyte trafficking as a method of
treatment for autoimmune pathologies such as uveitis, type I
diabetes, rheumatoid arthritis, inflammatory bowel diseases, and,
most particularly, multiple sclerosis. "Treatment" of multiple
sclerosis includes the various forms of the disease including
relapsing-remitting, chronic progressive, etc., and the S1P
receptor agonists can be used alone or in conjunction with other
agents to relieve signs and symptoms of the disease as well as
prophylactically.
[0094] In addition, the disclosed compounds can be used for
altering lymphocyte trafficking as a method for prolonging
allograft survival, for example solid organ transplants, treatment
of graft vs. host disease, bone marrow transplantation, and the
like.
[0095] In addition, the disclosed compounds can be used to inhibit
autotaxin. Autotaxin, a plasma phosphodiesterase, has been
demonstrated to undergo end product inhibition. Autotaxin
hydrolyzes several substrates to yield lysophosphatidic acid and
sphingosine 1-phosphate, and has been implicated in cancer
progression and angiogenesis. Therefore, S1P receptor agonist
pro-drugs of the disclosed compounds can be used to inhibit
autotaxin. This activity may be combined with agonism at S1P
receptors or may be independent of such activity.
[0096] In addition, disclosed compounds can be useful for
inhibition of S1P lyase. S1P lyase is an intracellular enzyme that
irreversibly degrades S1P. Inhibition of S1P lyase disrupts
lymphocyte trafficking with concomitant lymphopenia. Accordingly,
S1P lyase inhibitors can be useful in modulating immune system
function. Therefore, the disclosed compounds can be used to inhibit
S1P lyase. This inhibition could be in concert with S1P receptor
activity, or be independent of activity at any S1P receptor.
[0097] In addition, disclosed compounds can be useful as
antagonists of the cannabinoid CB.sub.1 receptor. CB.sub.1
antagonism is associated with a decrease in body weight and an
improvement in blood lipid profiles. The CB.sub.1 antagonism could
be in concert with S1P receptor activity, or be independent of
activity at any S1P receptor.
[0098] In addition, disclosed compounds can be useful for
inhibition of group IVA cytosolic PLA.sub.2 (cPLA.sub.2).
cPLA.sub.2 catalyzes the release of eicosanoic acids (e.g.,
arachidonic acid). The eicosanoic acids are transformed to
pro-inflammatory eicosanoids such as prostaglandins and
leukotrienes. Thus, disclosed compounds may be useful as
anti-inflammatory agents. This inhibition could be in concert with
S1P receptor activity, or be independent of activity at any S1P
receptor.
[0099] In addition, disclosed compounds may be useful for
inhibition of the multiple substrate lipid kinase (MuLK). MuLK is
highly expressed in many human tumor cells and thus its inhibition
might slow the growth or spread of tumors.
[0100] "Treatment" of multiple sclerosis includes the various forms
of the disease including relapsing-remitting, chronic progressive,
etc., and the S1P receptor agonists can be used alone or in
conjunction with other agents to relieve signs and symptoms of the
disease as well as prophylactically.
[0101] The present invention is also includes pharmaceutical
compositions comprising the compounds of formula I. More
particularly, such compounds can be formulated as pharmaceutical
compositions using standard pharmaceutically acceptable carriers,
fillers, solubilizing agents and stabilizers known to those skilled
in the art. For example, a pharmaceutical composition comprising a
compound of formula I, or analog, derivative, or modification
thereof, as described herein, is used to administer the appropriate
compound to a subject.
[0102] The compounds of formula I are useful for treating a disease
or disorder including administering to a subject in need thereof of
a therapeutically acceptable amount of a compound of formula I, or
a pharmaceutical composition comprising a therapeutically effective
amount of a compound of formula I, and a
pharmaceutically-acceptable carrier.
[0103] The disclosed compounds and method are directed to
sphingosine 1-phosphate (S1P) analogs that have activity as
receptor receptor agonists or antagonists at one or more S1P
receptors, specifically the S1P.sub.1, S1P.sub.4 and S1P.sub.5
receptor types. The disclosed compounds and method include both
compounds that have a phosphate moiety as well as compounds with
hydrolysis-resistant phosphate surrogates such as phosphonates,
alpha-substituted phosphonates particularly where the alpha
substitution is a halogen and phosphothionates.
[0104] The values listed below for radicals, substituents, and
ranges, are for illustration only; they do not exclude other
defined values or other values within defined ranges for the
radicals and substituents.
[0105] X.sup.1, Y.sup.1 and Z.sup.1 are independently O, CH,
CH.sub.2, CHCF.sub.3, N, NH, or S.
[0106] Another value for X.sup.1, Y.sup.1 and Z.sup.1 is
CH.sub.2.
[0107] R.sup.1 can be hydrogen, fluorine, chlorine, bromine,
trifluoromethyl, methoxy, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)haloalkyl, or (C.sub.1-C.sub.6)alkyl substituted
with, alkoxy or cyano.
[0108] Additional values for R.sup.1 are hydrogen, trifluoromethyl,
or --CH.sub.2CF.sub.3.
[0109] More additional values for R.sup.1 are alkyl-substituted
aryl, aryl-substituted alkyl, or aryl-substituted arylalkyl.
[0110] More additional values for R.sup.1 are benzyl, phenylethyl,
or methyl benzyl.
[0111] Compounds having formula I can have an R.sup.2 group that
includes a chain having the structure
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--.
[0112] Values for R.sup.2 include
##STR00007##
[0113] Additional values for R.sup.2 having formula II are
##STR00008##
where Y.sup.3 is (CH.sub.3).sub.3C--,
CH.sub.3CH.sub.2(CH.sub.3).sub.2C--, CH.sub.3CH.sub.2CH.sub.2--,
CH.sub.3(CH.sub.2).sub.2CH.sub.2--,
CH.sub.3(CH.sub.2).sub.4--CH.sub.2--(CH.sub.3).sub.2CHCH.sub.2--,
(CH.sub.3).sub.3CCH.sub.2--, CH.sub.3CH.sub.2O--,
(CH.sub.3).sub.2CHO--, or CF.sub.3CH.sub.2CH.sub.2-- or a group
having the formula:
##STR00009##
[0114] An additional value for R.sup.2 having formula II (para
substituted 3,5-diphenyl-(1,2,4)-oxadiazole) is;
##STR00010##
[0115] Another value for R.sup.2 having formula II is;
##STR00011##
[0116] Another value for R.sup.2 having formula II is;
##STR00012##
[0117] Additional values for R.sup.2 having formula III are;
##STR00013##
[0118] Another value for R.sup.2 having formula III is;
##STR00014##
[0119] Additional values for R.sup.2 include
(C.sub.1-C.sub.20)alkyl, (C.sub.1-C.sub.20)alkoxy, or
(C.sub.2-C.sub.26)alkoxyalkyl.
[0120] More additional values for R.sup.2 include
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.10)alkenyl and
(C.sub.2-C.sub.14)alkynyl or (C.sub.1-C.sub.10)alkoxy optionally
substituted with carbonyl (C.dbd.O) or oxime (C.dbd.NR.sup.d)
groups.
[0121] Additional values for R.sup.2 include methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, trifluoromethyl,
trifluoroethyl, trifluoromethoxy, trifluoroethoxy, methoxy, ethoxy,
propoxy, butoxy, pentoxy, heptoxy, or octoxy.
[0122] Another value for R.sup.3 is methyl, hydroxymethyl, ethyl,
hydroxyethyl, propyl, hydroxypropyl, or isopropyl.
[0123] Another value for R.sup.3 is methyl, hydroxymethyl, ethyl,
or hydroxyethyl.
[0124] A value for R.sup.4 is hydroxy, or phosphate
(--OPO.sub.3H.sub.2).
[0125] A specific compound has the formula
##STR00015##
[0126] Additional compounds have formulas
##STR00016## ##STR00017##
[0127] Additional compounds having formula I include compounds
above or in FIG. 1 where one of more of the hydrogen atoms from a
hydroxy group is replaced with a phosphate group
--OP(.dbd.O)(OH).sub.2.
[0128] Additional compounds of formula I are illustrated in table
1, below.
TABLE-US-00001 TABLE 1 ##STR00018## ##STR00019## Compound R.sup.e
XX ##STR00020## XXI ##STR00021## XXII ##STR00022## XXIII
##STR00023## XXIV ##STR00024## XXV ##STR00025##
[0129] In another aspect, the invention provides S1P receptor
pro-drug compounds having the general structure of formula I, is
provided by a compound with a mono-substituted tetralin ring system
that has the formula VIII. In some embodiments of formula I, the
structure (e.g., IX and X) has only a single chiral center and that
the amino carbon is pro-chiral, e.g., will become chiral following
enzyme-catalyzed phosphorylation.
[0130] Without wishing to be bound by any particular theory, it is
expected that the compounds described herein are pro-drugs, e.g.,
are activated by phosphorylation of the primary alcohol to form the
mono-phosphorylated analog. Additionally, the active drugs are
expected to be agonists at the S1P type 1 receptor.
[0131] In cases where compounds of formula I are sufficiently basic
or acidic to form stable nontoxic acid or base salts, preparation
and administration of the compounds as pharmaceutically acceptable
salts may be appropriate. Examples of pharmaceutically acceptable
salts are organic acid addition salts formed with acids which form
a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.alpha.-glycerophosphate. Inorganic salts may also be formed,
including hydrochloride, sulfate, nitrate, bicarbonate, and
carbonate salts.
[0132] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0133] Pharmaceutically-acceptable base addition salts can be
prepared from inorganic and organic bases. Salts from inorganic
bases, include but are not limited to, sodium, potassium, lithium,
ammonium, calcium and magnesium salts. Salts derived from organic
bases include, but are not limited to, salts of primary, secondary
and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl
amines, substituted alkyl amines, di(substituted alkyl) amines,
tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines,
trialkenyl amines, substituted alkenyl amines, di(substituted
alkenyl) amines, tri(substituted alkenyl) amines, cycloalkyl
amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted
cycloalkyl amines, disubstituted cycloalkyl amine, trisubstituted
cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl) amines,
tri(cycloalkenyl) amines, substituted cycloalkenyl amines,
disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl
amines, aryl amines, diaryl amines, triaryl amines, heteroaryl
amines, diheteroaryl amines, triheteroaryl amines, heterocyclic
amines, diheterocyclic amines, triheterocyclic amines, mixed di-
and tri-amines where at least two of the substituents on the amine
are different and are alkyl, substituted alkyl, alkenyl,
substituted alkenyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, or
heterocyclic and the like. Also included are amines where the two
or three substituents, together with the amino nitrogen, form a
heterocyclic or heteroaryl group. Mon-limiting examples of amines
include, isopropylamine, trimethyl amine, diethyl amine,
tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine,
2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine,
caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, N-alkylglucamines, theobromine, purines, piperazine,
piperidine, morpholine, N-ethylpiperidine, and the like. It should
also be understood that other carboxylic acid derivatives would be
useful, for example, carboxylic acid amides, including
carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and
the like.
[0134] The compounds of formula I can be formulated as
pharmaceutical compositions and administered to a mammalian host,
such as a human patient in a variety of forms adapted to the chosen
route of administration, e.g., orally or parenterally, by
intravenous, intramuscular, topical or subcutaneous routes.
[0135] Thus, the present compounds may be systemically
administered, e.g., orally, in combination with a pharmaceutically
acceptable vehicle such as an inert diluent or an assimilable
edible carrier. They may be enclosed in hard or soft shell gelatin
capsules, may be compressed into tablets, or may be incorporated
directly with the food of the patient's diet. For oral therapeutic
administration, the active compound may be combined with one or
more excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such compositions and preparations should contain at
least about 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 60% of the weight of a
given unit dosage form. The amount of active compound in such
therapeutically useful compositions is such that an effective
dosage level will be obtained.
[0136] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch 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, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0137] The active compound may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0138] Exemplary pharmaceutical dosage forms for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or 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, buffers 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.
[0139] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0140] For topical administration, the present compounds may be
applied in pure form, e.g., when they are liquids. However, it will
generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0141] Exemplary solid carriers include finely divided solids such
as talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0142] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0143] Examples of useful dermatological compositions which can be
used to deliver the compounds of formula I to the skin are known to
the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),
Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.
4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0144] Useful dosages of the compounds of formula I can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
[0145] Generally, the concentration of the compound(s) of formula I
in a liquid composition, such as a lotion, will be from about 0.1
to about 25 weight percent, preferably from about 0.5-10 weight
percent. The concentration in a semi-solid or solid composition
such as a gel or a powder will be about 0.1-5 wt-%, preferably
about 0.5-2.5 weight percent based on the total weight of the
composition.
[0146] The amount of the compound, or an active salt or derivative
thereof, required for use in treatment will vary not only with the
particular salt 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 be ultimately at the discretion of the
attendant physician or clinician. In general, however, a dose will
be in the range of from about 0.1 to about 10 mg/kg of body weight
per day.
[0147] The compound is conveniently administered in unit dosage
form; for example, containing 5 to 1000 mg, conveniently 10 to 750
mg, most conveniently, 50 to 500 mg of active ingredient per unit
dosage form.
[0148] Ideally, the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.5 to about 75 .mu.M, preferably, about 1 to 50 .mu.M, most
preferably, about 2 to about 30 .mu.M. This may be achieved, for
example, by the intravenous injection of a 0.05 to 5% solution of
the active ingredient, optionally in saline, or orally administered
as a bolus containing about 1-100 mg of the active ingredient.
Desirable blood levels may be maintained by continuous infusion to
provide about 0.01-5.0 mg/kg/hr or by intermittent infusions
containing about 0.4-15 mg/kg of the active ingredient(s).
[0149] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four, or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0150] The disclosed method includes a kit comprising an inhibitor
compound of formula I and instructional material which describes
administering the inhibitor compound or a composition comprising
the inhibitor compound to a cell or a subject. This should be
construed to include other embodiments of kits that are known to
those skilled in the art, such as a kit comprising a (preferably
sterile) solvent for dissolving or suspending the inhibitor
compound or composition prior to administering the compound or
composition to a cell or a subject. Preferably, the subject is a
human.
[0151] In accordance with the disclosed compounds and methods, as
described above or as discussed in the Examples below, there can be
employed conventional chemical, cellular, histochemical,
biochemical, molecular biology, microbiology, and in vivo
techniques which are known to those of skill in the art. Such
techniques are explained fully in the literature.
[0152] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the disclosed
compounds.
[0153] Processes for preparing compounds of formula I or for
preparing intermediates useful for preparing compounds of formula I
are provided as further embodiments. Intermediates useful for
preparing compounds of formula I are also provided as further
embodiments. The processes are provided as further embodiments and
are illustrated in the schemes herein wherein the meanings of the
generic radicals are as given above unless otherwise qualified.
[0154] An example of the synthesis of several disclosed compounds
is illustrated in Scheme 1 (FIG. 2). The reagents and conditions
are as follows: a) Tf.sub.2O, 2,6-lutidine, CH.sub.2Cl.sub.2,
0.degree. C., 2 h, 93%; b) 1-octene, 9-BBN, K.sub.3PO.sub.4, KBr,
H.sub.2O, Pd(PPh.sub.3).sub.4, 65.degree. C., 2 h, 82%; c)
CuBr.sub.2, EtOAc, CHCl.sub.3, reflux 6 h, 80%; d) NaH,
N-acetamido-diethylmalonate, DMF, 0.degree. C.-rt, overnight, 75%;
e) Et.sub.3SH, TiCl.sub.4,CH.sub.2Cl.sub.2, rt, 12 h, 65%; f)
LiBH.sub.4, rt, THF, 48 h, 33%; g) LiOH, H.sub.2O, MeOH, THF,
50.degree. C., 5 h, 75%; h) P.sub.2O.sub.5, H.sub.3PO.sub.4,
100.degree. C., 2 h, 37%; i) 12M HCL, MeOH, reflux, 2 h; j)
LiAlH.sub.4, THF, reflux, 12 h, 21%, two steps; k) P.sub.2O.sub.5,
H.sub.3PO.sub.4, 100.degree. C., 2 h, 50%.
[0155] The invention is now described with reference to the
following Examples and Embodiments. Without further description, it
is believed that one of ordinary skill in the art can, using the
preceding description and the following illustrative examples, make
and utilize the disclosed compounds. The following working examples
therefore, are provided for the purpose of illustration only and
specifically point out the preferred embodiments, and are not to be
construed as limiting in any way the remainder of the disclosure.
Therefore, the examples should be construed to encompass any and
all variations which become evident as a result of the teaching
provided herein.
EXAMPLES
Example 1
Trifluoromethanesulfonic acid
5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl ester (2)
##STR00026##
[0157] Trifluoromethanesulfonic anhydride (1.7 mL, 10 mmol) was
added slowly over 1 hour to a solution of 6-hydroxy-1-tetralone
(1.62 g, 10 mmol) and 2,6-lutidine (1.28 mL, 10 mmol) in dry
dichloromethane (10 mL) cooled to 0.degree. C. After 1 hour the
solution was diluted with dichloromethane (10 mL) and washed with 1
M hydrochloric acid (20 mL). The organic layer was re-extracted
with dichloromethane (50 mL) and the combined organics washed with
1 M hydrochloric acid (10 mL). The organics were dried over
magnesium sulfate and concentrated under vacuum. The residue was
purified by column chromatography (Silica gel, CH.sub.2Cl.sub.2) to
provide 2.7 g of compound 2 (93%).
[0158] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.13 (p, 2H,
J=6.22 Hz), 2.63 (t, 2H, J=6.95 Hz), 2.98 (t, 2H, J=6.22 Hz), 7.15
(m, 2H), 8.07 (m, 1H); .sup.13C NMR .delta. 23.08, 29.88, 38.92,
116.74, 119.81, 121.56, 130.14, 132.58, 147.38, 152.52, 196.53.
Example 2
6-Octyl-3,4-dihydro-2H-naphthalen-1-one (3)
##STR00027##
[0160] 9-BBN (0.5 M solution in THF, 20.2 mL, 10.1 mmol) was added
to 1-octene (1.6 mL, 10.1 mmol) at room temperature. The solution
was stirred, at room temperature, overnight. After this time,
K.sub.3PO.sub.4 (2.93 g, 13.8 mmol), Pd(Ph.sub.3P).sub.4 (191 mg,
0.17 mmol, 1.8 mol %), KBr (1.2 g, 10.1 mmol) and degassed H.sub.2O
(0.18 mL, 10 mmol) were added. This was followed by a solution of
compound 2 (2.7 g, 9.2 mmol) in dry degassed THF (10 mL). The
reaction mixture was heated at 65.degree. C. under argon for 2
hours. After cooling, the solution was acidified to pH 1 and
extracted into EtOAc (100 mL). The aqueous layer was re-extracted
with EtOAc (50 mL) and the combined organics washed with H.sub.2O
(20 mL) and brine (40 mL). The organic layer was dried over
magnesium sulfate and concentrated under vacuum. The residue was
purified by column chromatography (Silica gel, 5% EtOAc in hexanes)
to provide 1.93 g of compound 3 (82%).
[0161] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.85 (t, 3H,
J=6.95 Hz), 1.24 (bs, 10H), 1.58 (p, 2H, J=6.95 Hz), 2.06 (p, 2H,
J=5.85 Hz), 2.57 (t, 4H, J=6.95 Hz), 2.87 (t, 2H, J=6.22 Hz), 7.01
(s, 1H), 7.06 (d, 1H, J=8.05 Hz), 7.91 (d, 1H, J=8.06 Hz); .sup.13C
NMR .delta. 14.32, 22.88, 23.61, 29.44, 29.55, 29.66, 29.96, 31.32,
32.08, 36.31, 39.33, 127.12, 127.45, 128.73, 130.75, 144.70,
149.28, 198.09.
Example 3
2-Bromo-6-octyl-3,4-dihydro-2H-naphthalen-1-one (4)
##STR00028##
[0163] Cupric bromide (3.34 g, 15.0 mmol,) was heated at reflux in
ethyl acetate (10 mL) with stirring. To this was added compound 3
(1.93 g, 7.5 mmol) in chloroform (10 mL). The reaction was heated
at reflux for a further 6 hours and cooled. Copper bromide and
cupric bromide residues were filtered off and the filtrate was
decolorized with activated charcoal and filtered through a bed of
Celite and washed with ethyl acetate (4.times.50 mL). The solvent
was removed under reduced pressure and the residue was purified by
column chromatography (Silica gel, 2% EtOAc in hexanes) to provide
2.02 g of compound 4 (80%).
[0164] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.87 (t, 3H,
J=6.95 Hz), 1.26 (bs, 10H), 1.61 (p, 2H, J=6.96 Hz), 2.46 (m, 2H),
2.62 (t, 2H, J=7.69 Hz), 2.86 (dt, 1H, J=16.34 Hz, 4.39 Hz), 3.27
(dt, 1H, J=16.83 Hz, 4.39 Hz), 4.69 (t, 1H, J=4.02 Hz), 7.07 (s,
1H), 7.14 (d, 1H, J=8.05 Hz), 7.99 (d, 1H, J=8.05 Hz); .sup.13C NMR
.delta. 14.34, 22.88, 26.42, 29.44, 29.57, 29.64, 31.25, 32.08,
32.32, 36.39, 127.75, 128.00, 128.73, 129.00, 144.30, 150.39,
190.54.
Example 4
2-Acetylamino-2-(6-octyl-1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonic
acid diethyl ester (5)
##STR00029##
[0166] Sodium hydride (720 mg, 18.0 mmol) 60% in mineral oil was
suspended in dry DMF (10 mL) and a solution of diethyl
acetamidomalonate (3.26 g, 15 mmol) in dry DMF (10 mL) was added.
The solution was stirred at 0.degree. C. for 3 hours until the
anion had formed. A solution of 4 (2.02 g, 6.0 mmol) in dry DMF (10
mL) was added and the solution warmed to room temperature and
stirred overnight. The mixture was poured into distilled water (50
mL), in an ice-bath, acidified to pH 3 with 1M hydrochloric acid
and extracted with ethyl acetate (3.times.50 mL). The organic
phases were washed with brine (2.times.30 mL) and dried over
magnesium sulfate and concentrated under vacuum. The residue was
purified by column chromatography (Silica gel, 40% EtOAc in
hexanes) to provide 2.12 g of compound 5 (75%).
[0167] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.85 (t, 3H,
J=6.22 Hz), 1.24 (m, 16H), 1.58 (p, 2H, J=6.95 Hz), 1.97 (s, 3H),
2.59 (t, 2H, J=7.32 Hz), 2.83-3.21 (m, 4H), 3.88 (dd, 1H, J=14.00
Hz, 3.68 Hz), 4.14-4.32 (m, 4H), 6.86 (s, 1H), 7.03 (s, 1H), 7.07
(d, 1H, J=8.69 Hz), 7.84 (d, 1H, J=8.36 Hz); .sup.13C NMR .delta.
14.05, 14.16, 14.30, 22.85, 23.31, 26.98, 29.40, 29.49, 29.61,
29.98, 31.28, 32.05, 36.32, 56.16, 62.40, 63.13, 66.33, 127.16,
127.63, 128.78, 144.84, 150.07, 166.38, 168.70, 169.83, 197.63.
Example 5
2-Acetylamino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonic
acid diethyl ester (6)
##STR00030##
[0169] To a solution of triethylsilane (1.3 ml, 8.2 mmol) in 5 ml
of CH.sub.2Cl.sub.2 was added compound 5 (1 g, 2.1 mmol) in 5 ml of
CH.sub.2Cl.sub.2. The reaction mixture was stirred at room
temperature under Ar and TiCl.sub.4 (0.09 ml, 8.2 mmol) was added
dropwise. The resulting solution was stirred for 12 hours, cooled
to 0.degree. C. and quenched by slow addition of 10 ml of saturated
NaHCO.sub.3. The aqueous layer was extracted with CH.sub.2Cl.sub.2
(3.times.30 mL). The combined organic layers were washed with brine
(2.times.30 mL) and dried over magnesium sulfate and concentrated
under vacuum. The residue was purified by column chromatography
(Silica gel, 20% EtOAc in hexanes) to provide 630 mg of compound 6
(65%).
[0170] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.87 (t, 3H,
J=6.46 Hz), 1.26 (m, 16H), 1.58 (p, 2H, J=6.79 Hz), 2.03 (s, 3H),
2.28 (b, 1H), 2.49-2.68 (m, 4H), 2.82-2.92 (m, 2H), 4.20-4.34 (m,
4H), 6.69 (s, 1H), 6.89-7.05 (m, 3H); .sup.13C NMR .delta. 14.21,
14.25, 14.37, 22.92, 23.37, 25.48, 29.50, 29.63, 29.72, 29.76,
30.39, 31.93, 32.13, 35.78, 40.33, 62.46, 62.79, 68.80, 126.04,
128.81, 129.30, 132.85, 136.28, 140.66, 150.07, 167.63, 168.32,
169.42.
Example 6
N-[2-Hydroxy-1-hydroxymethyl-1-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl-
)-ethyl]-acetamide (7)
##STR00031##
[0172] Lithium borohydride (2M solution in THF, 0.88 ml, 1.76 mmol)
was added to compound 6 (200 mg, 0.44 mmol) in 5 ml THF at
0.degree. C. The reaction mixture was stirred at room temperature
for 48 hours and diluted with 40 ml ethyl acetate. The solution was
washed with brine (2.times.20 mL) and dried over magnesium sulfate
and concentrated under vacuum. The residue was purified by column
chromatography (Silica gel, 4% MeOH in CH.sub.2Cl.sub.2) to provide
55 of mg compound 7 (33%).
[0173] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.88 (t, 3H,
J=6.56 Hz), 1.29 (m, 10H), 1.57 (p, 2H, J=6.25 Hz), 1.94-1.98 (m,
2H), 2.05 (s, 3H), 2.33 (m, 1H), 2.51 (t, 2H, J=7.32), 2.60-2.85
(m, 4H), 3.69 (d, 2H, J=11.61), 3.89 (dd, 2H, J=11.61 Hz, 7.25 Hz),
6.22 (s, 1H), 6.88-6.99 (m, 3H); .sup.13C NMR .delta. 14.38, 22.92,
24.20, 24.35, 29.52, 29.66, 29.73, 29.95, 30.32, 31.94, 32.14,
35.78, 38.26, 63.55, 64.34, 64.46, 126.18, 128.85, 129.30, 133.06,
136.22, 140.75, 172.40.
Example 7
2-Amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-propane-1,3-diol
(VPC104061)
##STR00032##
[0175] A solution of compound 7 (53 mg, 0.14 mmol) and
LiOH.H.sub.2O (45 mg, 1.1 mmol) in MeOH (3 ml), THF (1.5 ml) and
water (3 ml) was stirred at 50.degree. C. for 5 hours and diluted
with ethyl acetate (20 ml). The solution was washed with brine
(2.times.10 mL) and dried over magnesium sulfate and concentrated
under vacuum. The residue was purified by column chromatography
(Silica gel, 50% MeOH in CH.sub.2Cl.sub.2) to provide 35 mg of
compound VPC104061 (75%).
[0176] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.88 (t, 3H,
J=6.17 Hz), 1.29 (m, 10H), 1.56 (p, 2H, J=6.17 Hz), 1.82-1.98 (m,
2H), 2.51 (t, 2H, J=6.95), 2.58-2.88 (m, 5H), 3.19 (b, 4H), 3.61
(d, 2H, J=10.98), 3.73 (d, 2H, J=10.61 Hz), 6.87-6.98 (m, 3H);
.sup.13C NMR .delta. 14.37, 22.93, 24.02, 29.32, 29.53, 29.70,
29.75, 29.85, 30.26, 31.94, 32.14, 35.08, 39.58, 57.74, 66.13,
66.19, 126.09, 128.81, 129.39, 133.28, 136.26, 140.64. MS (ESI) m/z
334.1 [M+H].sup.+.
Example 8
Phosphoric acid
mono-[2-amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-3-phosphonoo-
xypropyl]ester (VPC104081)
##STR00033##
[0178] Phosphorus pentoxide (2.0 g, 14 mmol) in phosphoric acid
(85% in water, 2 ml, 29 mmol) was added to VPC104061 (25 mg, 0.07
mmol). The mixture was stirred at 100.degree. C. for 2 hours and
cooled to 0.degree. C. The product was precipitated by adding water
(14 mg, 37%). MS (ESI) m/z 494.4 [M+H].sup.+.
Example 9
Amino-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-acetic acid
(8)
##STR00034##
[0180] Compound 6 (300 mg, 0.65 mmol) was added to 12 M HCl (10
ml). The mixture was heated to reflux and MeOH (5 ml) was added
until the mixture became homogenous. Reflux was continued for 2
hours until the starting material was consumed as determined by
thin layer chromatography (TLC). The reaction mixture was
concentrated under reduced pressure and co-evaporated with MeOH and
diethyl ether multiple times. The desired compound 8 was
recrystallized from diethyl ether and hexanes to provide a light
brown solid and used directly for the next reaction.
Example 10
2-Amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-ethanol
(VPC104059)
##STR00035##
[0182] The amino acid 8 prepared in Example 9 was added to a
refluxing solution of lithium aluminum hydride (62 mg, 1.63 mmol)
in THF (10 ml). The reaction mixture was heated at reflux for 12
hours, subsequently cooled to 0.degree. C. and 10 M NaOH was added
and stirred for 20 minutes. Ethyl acetate (20 ml) was added and the
mixture was filtered through Celite and magnesium sulfate. The
filtrate was concentrated under vacuum and purified by column
chromatography (Silica gel, 50% MeOH in CH.sub.2Cl.sub.2) to
provide 41 mg of the product, VPC104059 (21%, two steps).
[0183] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.88 (t, 3H,
J=6.39 Hz), 1.28 (m, 10H), 1.55 (p, 2H, J=7.16 Hz), 1.67-2.11 (m,
3H), 2.48 (t, 2H, J=7.69), 2.56-2.83 (m, 5H), 3.19 (b, 4H),
3.47-3.75 (m, 2H), 6.82-6.96 (m, 3H); .sup.13C NMR .delta. 13.34,
22.58, 26.45, 29.14, 29.21, 29.29, 29.47, 31.70 31.90, 32.26,
35.40, 47.56, 125.64, 128.36, 128.86, 128.93, 133.19, 139.93, MS
(ESI) m/z 303.9 [M+H].sup.+.
Example 11
Phosphoric acid
mono-[2-amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-ethyl]ester
(VPC104127)
##STR00036##
[0185] Phosphorus pentoxide (1.5 g, 10.5 mmol) in phosphoric acid
(85% in water, 1.5 ml, 22 mmol) was added to VPC104059 (25 mg, 0.08
mmol). The mixture was stirred at 100.degree. C. for 2 h and cooled
to 0.degree. C. The product was precipitated by adding water (10
mg, 50%). MS (ESI) m/z 384.2 [M+H].sup.+.
Example 12
Synthesis of Structure (X)
[0186] The synthesis of an ether containing compound having formula
IX is illustrated in Scheme 2 (FIG. 3). Keto-alcohol 1A, is
converted to the keto-ether 1B, using standard reagents and
techniques. The keto-ether is halogenated to provide halo-ether 1C
in a manner similar to Example 3. The halo-ether is alkylated to
provide diester-ether 1D, in a manner similar to the procedure
described in Example 4. The diester is converted to ether-triol 1E,
using standard reducing agents known in the art. The triol is
converted to a diol and deprotected using standard methods known in
the art, to provide compound IX.
Example 13
2-Acetylamino-2-(6-octyl-1-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-mal-
onic acid diethyl ester (A)
##STR00037##
[0188] To a solution of sodium borohydride (75 mg, 2.00 mmol) in 5
ml of ethanol at room temperature is added
2-acetylamino-2-(6-octyl-1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl)-maloni-
c acid diethyl ester (compound 5) (1.00 g, 2.1 mmol) in 5 ml of
ethanol. The reaction mixture is stirred at room temperature under
argon for an additional hour, quenched by the addition of water (20
mL) and methylene chloride (20 mL). The organic layer is removed
and the aqueous layer is extracted with methylene chloride
(2.times.20 mL). The combined organic layers were washed with brine
(2.times.20 mL) and dried over magnesium sulfate and concentrated
under vacuum. The residue is purified by column chromatography
(silica gel, 20% ethyl acetate in hexanes) to provide 755 mg of
compound A (75%).
Example 14
2-Acetylamino-2-(6-octyl-3,4-dihydro-naphthalen-2-yl)-malonic acid
diethyl ester (B)
##STR00038##
[0190]
2-Acetylamino-2-(6-octyl-1-hydroxy-1,2,3,4-tetrahydro-naphthalen-2--
yl)-malonic acid diethyl ester (compound A, 755 mg, 1.58 mmol)) is
dissolved in acetic anhydride (5 mL) followed at 0.degree. C. by a
catalytic amount of ferric chloride (66 mg, 0.4 mmol). The reaction
is stirred at 0.degree. C. for an additional 2 hours, and 20 mL of
diethyl ether is added. The reaction is carefully poured into 50 mL
of ice cold water and the organic layer quickly separated. The
organic layer is re-extracted with methylene chloride (2.times.20
mL) and the combined organic layers were washed once with brine (20
mL) and dried over magnesium sulfate. The organic layer is
concentrated under vacuum and the residue is purified by column
chromatography (silica gel, 10% ethyl acetate in hexanes) to
provide 458 mg of compound B (60%). The complete synthesis is
illustrated in FIG. 4.
Example 14
Sphingosine Kinase Assay
[0191] Recombinant sphingosine kinase type 2 (SPHK2) is prepared by
forcing the expression of the mouse or human recombinant enzyme by
transfecting the relevant plasmid DNA into HEK293T cells. After
about 60 hours, cells are harvested, broken and the non-microsomal
(e.g., soluble) fraction is retained. The broken cell supernatant
fluid containing the recombinant enzyme is mixed with test
compounds (FTY720, AA151, VIII and XVIII) (5-50 micromolar) and
.gamma.-32P-ATP and incubated for 0.5-2.0 hours at 37.degree. C.
The lipids in the reaction mixture are extracted into an organic
solvent and displayed by normal phase thin layer chromatography.
The radio-labeled bands are detected by autoradiography, scraped
from the plate and quantified by scintillation counting. In the
histogram shown, (FIGS. 5A, 5B and 6) sphingosine was present at 15
.mu.M, and the test compounds were at a concentration of 50 .mu.M,
incubation time was 20 minutes.
Example 15
GTP.gamma.S-35 Binding Assay
[0192] This assay illustrates agonist activation of G protein
coupled receptors (GPCRs) in isolation. The assay forces expression
concomitantly of a recombinant GPCR (e.g., the S1P1-5 receptor) and
each of the three subunits (typically, .alpha.-2, .beta.-1, or
.gamma.-2) of a heterotrimeric G protein in a HEK293T cell by
transfecting the cell with four plasmid DNAs encoding the
respective proteins. About 60 hours after transfection the cells
are harvested, opened, and the nucleus discarded. The crude
microsome is prepared from the remainder. Agonist (e.g., S1P)
stimulation of the receptor-G protein complex on the microsomes
results in the exchange of GTP for GDP on the .alpha.-subunit in a
dose-dependent manner. The GTP-bound .alpha.-subunit is detected
using a GTP analog (GTP.gamma.S-35), which is a radionuclide
(sulfur-35) labeled phosphothionate that is not hydrolyzed to GDP.
The microsomes with the adherent G proteins are collected by
filtration and the bound GTP.gamma.S-35 quantified in a liquid
scintillation counter. The assay yields relative potency (EC.sub.50
values) and maximum effect (efficacy, E.sub.max). Antagonist
activity is detected as rightward shifts in the agonist
dose-response curve in the presence of a fixed amount of
antagonist. If the antagonist behaves competitively, the affinity
of the receptor/antagonist pair (K.sub.i) can be determined.
[0193] The phosphorylated forms of compound VIII (a mixture of
mono, XXVI, and diphosphorylated, XXVII, isomers) and compound
XXVIII are low potency, partial agonists at the S1P.sub.3 receptor
(See FIG. 8.). Compounds XXVI, XXVII and XXVIII are more potent at
S1P.sub.1 and less potent at S1P.sub.3, relative to S1P. (See FIG.
7.) The assay was performed as described in Davis, M. D., J. J.
Clemens, T. L. Macdonald and K. R. Lynch (2005) "S1P Analogs as
Receptor Antagonists" Journal of Biological Chemistry, vol. 280,
pp. 9833-9841.
Example 16
Lymphopenia Assay
[0194] Compounds (e.g., primary alcohols such as compound VIII) are
dissolved in 2% hydroxypropyl beta-cyclodextrin and introduced into
groups of mice by oral gavage at doses from 0.01, 1.0 and 10 mg/kg
body weight. After 24 hours and 48 hours, the mice are lightly
anesthetized and ca. 0.1 ml of blood is drawn from the orbital
sinus. The number of lymphocytes (in thousands per microliter of
blood; normal is 4-11) is determined using a Hemavet blood
analyzer. In the histogram showing compound VIII, the 100% value
for the vehicle treated mouse was 7.5 at 24 hours; at 96 hours it
was 5. There were three mice/group, the strain was mixed
sv129.times.C57BL/6. Active compounds (e.g., compound VIII and
compound XXVIII) are dissolved in acidified DMSO at 20 mM, and
diluted 1:20 into 2% hydroxypropyl beta-cyclodextrin in water with
mixing. This solution is introduced into mice by intraperitoneal
(i.p.) injection at doses of 0.01, 1.0 and 10 mg/kg body
weight.
[0195] In FIG. 9 the results of an assay using compound VIII and
compound XXVIII administered via oral gavage are graphically
illustrated. The total lymphocyte counts (k/.mu.l) 24 hours after
an IV dose of compound VIII is reported.
[0196] Compounds were dissolved in 2% hydroxypropyl
.beta.-cyclodextrin and administered to sets of three, 12 week old
C57B1/6 female mice by oral gavage (PO). At 18 hours post dosing,
blood was drawn from the orbital sinus and lymphocyte numbers were
determined with a Hemavet blood analyzer. Doses were: compound XXIX
(1 mg/kg), compound VIII (10 mg/kg), and FTY720 (0.3 mg/kg).
Compound XXIX, 3,5-diphenyl-1,2,4-oxadiazole, is a S1P1 receptor
agonist described by Li et al. (Journal Medicinal Chemistry, vol.
48, p. 6169 (2005)) was used as a comparison. The results are
illustrated in FIG. 10.
Example 17
Heart Rate Assay
[0197] Mice were dosed with compound VIII, FTY720 (intravenous, 3
mg/kg) or vehicle (2% hydroxypropyl beta-cyclodextrin) and heart
rate measured at 1 hour post dosing. Heart rate was captured in
unrestrained, conscious animals using the ECGenie.TM. system. The
results are illustrated in FIG. 11.
[0198] The invention should not be construed to be limited solely
to the assays and methods described herein, but should be construed
to include other methods and assays as well. Other methods which
were used but not described herein are well known and within the
competence of one of ordinary skill in the art of chemistry,
biochemistry, molecular biology, and clinical medicine. One of
ordinary skill in the art will know that other assays and methods
are available to perform the procedures described herein.
[0199] The abbreviations used herein have their conventional
meaning within the clinical, chemical, and biological arts. In the
case of any inconsistencies, the present disclosure, including any
definitions therein will prevail.
[0200] The disclosures of each and every patent, patent
application, and publication cited herein are expressly
incorporated herein by reference in their entirety into this
disclosure. Illustrative embodiments of this disclosure are
discussed and reference has been made to possible variations within
the scope of this disclosure. These and other variations and
modifications in the disclosure will be apparent to those skilled
in the art without departing from the scope of the disclosure, and
it should be understood that this disclosure and the claims shown
below are not limited to the illustrative embodiments set forth
herein.
* * * * *