U.S. patent application number 13/740669 was filed with the patent office on 2013-08-01 for sphingosine 1 phosphate receptor modulators and methods of chiral synthesis.
This patent application is currently assigned to RECEPTOS, INC.. The applicant listed for this patent is RECEPTOS, INC.. Invention is credited to Marcus F. Boehm, Enugurthi Brahmachary, Liming Huang, Esther Martinborough, Manisha Moorjani, Junko Tamiya, Adam Richard Yeager.
Application Number | 20130196966 13/740669 |
Document ID | / |
Family ID | 43992104 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130196966 |
Kind Code |
A1 |
Martinborough; Esther ; et
al. |
August 1, 2013 |
SPHINGOSINE 1 PHOSPHATE RECEPTOR MODULATORS AND METHODS OF CHIRAL
SYNTHESIS
Abstract
Compounds that selectively modulate the sphingosine 1 phosphate
receptor are provided including compounds which modulate subtype 1
of the S1P receptor. Methods of chiral synthesis of such compounds
are provided. Uses, methods of treatment or prevention and methods
of preparing inventive compositions including inventive compounds
are provided in connection with the treatment or prevention of
diseases, malconditions, and disorders for which modulation of the
sphingosine 1 phosphate receptor is medically indicated.
Inventors: |
Martinborough; Esther; (San
Diego, CA) ; Boehm; Marcus F.; (San Diego, CA)
; Yeager; Adam Richard; (La Mesa, CA) ; Tamiya;
Junko; (Carlsbad, CA) ; Huang; Liming; (San
Diego, CA) ; Brahmachary; Enugurthi; (San Diego,
CA) ; Moorjani; Manisha; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RECEPTOS, INC.; |
San Diego |
CA |
US |
|
|
Assignee: |
RECEPTOS, INC.
San Diego
CA
|
Family ID: |
43992104 |
Appl. No.: |
13/740669 |
Filed: |
January 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12946800 |
Nov 15, 2010 |
8357706 |
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13740669 |
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61261282 |
Nov 13, 2009 |
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61262474 |
Nov 18, 2009 |
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Current U.S.
Class: |
514/210.18 ;
514/236.2; 514/326; 514/364; 544/138; 546/209; 548/131; 558/423;
564/225 |
Current CPC
Class: |
A61P 37/06 20180101;
C07D 413/04 20130101; A61P 25/28 20180101; A61K 45/06 20130101;
A61P 31/16 20180101; C07D 413/10 20130101; A61K 31/4245 20130101;
C07D 263/32 20130101; C07D 271/06 20130101; A61P 43/00 20180101;
A61K 31/454 20130101; A61P 25/00 20180101; A61P 11/00 20180101;
A61P 29/00 20180101; C07D 413/12 20130101; A61K 31/5377 20130101;
A61P 1/00 20180101; A61P 1/04 20180101 |
Class at
Publication: |
514/210.18 ;
548/131; 544/138; 546/209; 514/364; 514/236.2; 514/326; 564/225;
558/423 |
International
Class: |
C07D 271/06 20060101
C07D271/06; A61K 45/06 20060101 A61K045/06; A61K 31/454 20060101
A61K031/454; A61K 31/5377 20060101 A61K031/5377; C07D 413/12
20060101 C07D413/12; A61K 31/4245 20060101 A61K031/4245 |
Claims
1. A compound having the structure of Formula I-R or I-S or a
pharmaceutically acceptable salt, ester, prodrug, homolog, hydrate
or solvate thereof: ##STR00197## wherein X is --NR'R'' or --OR''';
Y is --CN, --Cl, or --CF.sub.3; R' is H, C.sub.1-4 alkyl, n-hydroxy
C.sub.1-4 alkyl, --SO.sub.2--R.sup.1, or --CO--R.sup.1; R'' is H,
--SO.sub.2--R.sup.3, C.sub.1-4 alkyl optionally substituted with 1
or more R.sup.2, or a ring moiety optionally substituted with
R.sup.4 wherein such ring moiety is piperidinyl, cyclohexyl,
morpholinyl, thiazolyl, pyrazolyl, pyrrolidinyl, imidazolyl, or
phenyl; or R' and R'' taken together with the nitrogen atom to
which they are bound form a 4, 5, or 6 membered saturated
heterocyclic ring containing 0 or 1 additional heteroatoms where
such additional heteroatom is O or N wherein such heterocycle is
optionally singly or multiply substituted with substituents
independently selected from the group consisting of --OH, oxo,
--NH.sub.2, n-hydroxy-C.sub.1-4 alkyl, --COOH,
--(CH.sub.2).sub.m--COOH, --(CH.sub.2).sub.m--COOR.sup.1,
--N(R.sup.1R.sup.1), and --(CH.sub.2).sub.m--CO--N(R.sup.5R.sup.5);
R''' is H, C.sub.1-4 alkyl, or --CO--R.sup.1; each R.sup.1 is
independently C.sub.1-4 alkyl or H; each R.sup.2 is independently
H, halo, OH, oxo, .dbd.NH, NH.sub.2, --COOH, F, --NHR.sup.1,
--N(R.sup.5R.sup.5),--SO.sub.2--R.sup.1,
--SO.sub.2--N(R.sup.5R.sup.5), --N(R.sup.1)--SO.sub.2--R.sup.1,
--COOR.sup.1, --OCO--R.sup.1, --CO--N(R.sup.5R.sup.5),
--N(R.sup.1)--COR.sup.1, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, and a
ring moiety optionally substituted with R.sup.4 wherein such ring
moiety is piperazinyl, piperidinyl, morpholinyl, pyrrolidinyl,
pyrazolyl, imidazolyl, benzimidazolyl, azetidinyl, cyclobutinyl, or
phenyl; each R.sup.3 is independently R.sup.2, C.sub.1-4 alkyl,
C.sub.3-6 cycloalkyl, or C.sub.1-4 alkyl optionally substituted
with 1 or more R.sup.2; each R.sup.4 is independently halo, OH,
--NH.sub.2, --NHR.sup.1, --N(R.sup.1R.sup.1), --COOH, --COOR.sup.1,
--NHCO--R.sup.1; each R.sup.5 is independently C.sub.1-4 alkyl or
H, or two R.sup.5 taken together with the nitrogen atom to which
they are bound form a 4, 5, or 6 membered saturated heterocyclic
ring containing 0 or 1 additional heteroatoms where such additional
heteroatom is O or N wherein such heterocycle is optionally
substituted with --OH, --NH.sub.2, --N(R.sup.1R.sup.1), n-hydroxy
C.sub.1-4 alkyl, --(CH.sub.2).sub.m--COOH,
--(CH.sub.2).sub.m--COOR.sup.1; and each m is independently 0, 1,
2, or 3.
2. The compound of claim 1 wherein the compound has the structure
of Formula I-R or a pharmaceutically acceptable salt, ester,
prodrug, homolog, hydrate or solvate thereof.
3. The compound of claim 1 wherein the compound has the structure
of Formula I-S or a pharmaceutically acceptable salt, ester,
prodrug, homolog, hydrate or solvate thereof.
4. The compound of claim 1 wherein the compound is substantially
enantiomerically pure.
5-9. (canceled)
10. The compound of claim 1 wherein Y is Cl.
11. The compound of claim 1 wherein Y is CF.sub.3.
12. The compound of claim 1 wherein Y is CN.
13. The compound of claim 1 wherein X is --NR'R''.
14. The compound of claim 1 wherein X is --OR'''.
15. The compound of claim 14 wherein X is --OH.
16. The compound of claim 14 wherein X is --OCO--R.sup.1.
17. The compound of claim 16 wherein R.sup.1 is C.sub.1-3
alkyl.
18. The compound of claim 13 wherein R' is H.
19. The compound of claim 13 wherein R' is --COR.sup.1.
20. The compound of claim 13 wherein R' is --SO.sub.2--R'.
21. The compound of claim 13 wherein R'' is H.
22. The compound of claim 13 wherein R'' is
--SO.sub.2--R.sup.3.
23. The compound of claim 13 wherein R'' is C.sub.1-4 alkyl
optionally substituted with 1 or more R.sup.2.
24. The compound of claim 13 wherein R'' is
--(CR.sup.aR.sup.b).sub.n--R.sup.2; each R.sup.a and each R.sup.b
is independently selected from the group consisting of H, hydroxyl
and methyl or R.sup.a and R.sup.b bound to the same carbon taken
together are oxo; and n is 0, 1, 2, or 3.
25. The compound of claim 24 wherein n is 2.
26. The compound of claim 25 wherein R.sup.2 is --OH, --NH.sub.2,
--NHR.sup.1, --N(R.sup.5R.sup.5), or --COOH.
27. The compound of claim 22 wherein R.sup.3 is C.sub.1-4 alkyl
optionally substituted with 1 or more R.sup.2.
28. The compound of claim 22 wherein Y is CN.
29. The compound of claim 27 wherein R.sup.3 is
--C.sub.2H.sub.5--N((R.sup.5R.sup.5) or
--CH.sub.2--CO--N(R.sup.5R.sup.5).
30. The compound of claim 28 wherein R.sup.3 is
C.sub.2H.sub.5--O--R.sup.1.
31. The compound of claim 12 wherein X is
--NH--CO--N(R.sup.5R.sup.5).
32. The compound of claim 1 wherein the compound is selected from
the group consisting of: ##STR00198## ##STR00199## ##STR00200##
##STR00201## ##STR00202## ##STR00203## ##STR00204## ##STR00205##
##STR00206## ##STR00207## ##STR00208## ##STR00209## or a
pharmaceutically acceptable salt, ester, prodrug, homolog, hydrate
or solvate thereof.
33. The compound of claim 32 selected from the group consisting of:
##STR00210## or a pharmaceutically acceptable salt, ester, prodrug,
homolog, hydrate or solvate thereof.
34. A pharmaceutical composition comprising a compound of claim 1
and a suitable excipient.
35. A pharmaceutical combination comprising a compound of claim 1
and a second medicament.
36. The combination of claim 35 wherein the second medicament is
medically indicated for the treatment of multiple sclerosis,
transplant rejection, or acute respiratory distress syndrome.
37. (canceled)
38. A method of activation or agonism of a sphingosine-1-phosphate
receptor subtype 1 comprising contacting the receptor subtype 1
with an effective amount of the compound of claim 1 or the
composition of claim 34.
39. The method of claim 38 wherein the compound activates or
agonizes the sphingosine-1-phosphate receptor subtype 1 to a
greater extent than the compound activates or agonizes a
sphingosine-1-phosphate receptor subtype 3.
40. The method of claim 38 wherein the sphingosine-1-phosphate
receptor subtype 1 is disposed within a living mammal.
41. A method of treatment of a malcondition in a patient for which
activation or agonism of an sphingosine-1-phosphate receptor
subtype 1 is medically indicated, comprising administering an
effective amount of the compound of claim 1 to the patient at a
frequency and for a duration of time sufficient to provide a
beneficial effect to the patient.
42. The method of claim 41 wherein selective activation or agonism
of an S1P subtype 1 receptor with respect to other subtypes of S1P
receptor is medically indicated.
43. The method of claim 41 wherein the malcondition comprises
multiple sclerosis, transplant rejection, acute respiratory
distress syndrome, ulcerative colitis, influenza, Crohn's disease
or adult respiratory distress syndrome.
44-60. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/946,800, filed Nov. 15, 2010 (allowed), which claims
priority to U.S. Provisional Application No. 61/261,282, filed Nov.
13, 2009 and U.S. Provisional Application No. 61/262,474, filed
Nov. 18, 2009, the disclosures of which are incorporated herein in
their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to compounds which are agonists of the
sphingosine 1-phosphate receptor subtype 1, methods of their
synthesis and methods of their therapeutic and/or prophylactic
use.
BACKGROUND
[0003] The S1P.sub.1/EDG.sub.1 receptor is a G-protein coupled
receptor (GPCR) and is a member of the endothelial cell
differentiation gene (EDG) receptor family. Endogenous ligands for
EDG receptors include lysophospholipids, such as
sphingosine-1-phosphate (S1P). Like all GPCRs, ligation of the
receptor propagates second messenger signals via activation of
G-proteins (alpha, beta and gamma).
[0004] Development of small molecule S1P.sub.1 agonists and
antagonists has provided insight into some physiological roles of
the S1P.sub.1/S1P-receptor signaling system. Agonism of the
S1P.sub.1 receptor perturbs lymphocyte trafficking, sequestering
them in lymph nodes and other secondary lymphoid tissue. This leads
to rapid and reversible lymphopenia, and is probably due to
receptor ligation on both lymphatic endothelial cells and
lymphocytes themselves (Rosen et al, Immunol. Rev., 195:160-177,
2003). A clinically valuable consequence of lymphocyte
sequestration is exclusion of them from sights of inflammation
and/or auto-immune reactivity in peripheral tissues.
[0005] Agonism of S1P.sub.1 has also been reported to promote
survival of oligodendrocyte progenitors (Miron et al, Ann. Neurol.,
63:61-71, 2008). This activity, in conjunction with lymphocyte
sequestration would be useful in treating inflammatory and
autoimmune conditions of the central nervous system.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to heterocyclic compounds
adapted to act as agonists of S1P receptor subtype 1, S1P.sub.1;
methods of preparation and methods of use, such as in treatment of
a malcondition mediated by S1P.sub.1 activation, or when activation
of S1P.sub.1 is medically indicated.
[0007] Certain embodiments of the present invention comprise a
compound having the structure of Formula I-R or I-S or a
pharmaceutically acceptable salt, ester, prodrug, homolog, hydrate
or solvate thereof:
##STR00001##
X can be --NR'R'' or --OR''' and Y can be --CN, --Cl, or
--CF.sub.3.
[0008] R' can be H, C.sub.1-4 alkyl, n-hydroxy C.sub.1-4 alkyl,
--SO.sub.2--R.sup.1, or --CO--R.sup.1. R'' can be H,
--SO.sub.2--R.sup.3, C.sub.1-4 alkyl optionally substituted with 1
or more R.sup.2, or a ring moiety optionally substituted with
R.sup.4 wherein such ring moiety is piperidinyl, cyclohexyl,
morpholinyl, pyrrolidinyl, imidazolyl, or phenyl. R''' can be H,
C.sub.1-4 alkyl, or --CO--R.sup.1. Alternatively, R' and R'' taken
together with the nitrogen atom to which they are bound form a 4,
5, or 6 membered saturated heterocyclic ring containing 0 or 1
additional heteroatoms where such additional heteroatom is O or N
wherein such heterocycle is optionally singly or multiply
substituted with substituents independently selected from --OH,
oxo, --NH.sub.2, n-hydroxy-C.sub.1-4 alkyl, --COOH,
--(CH.sub.2).sub.m--COOH, --(CH.sub.2).sub.m--COOR.sup.1,
--N(R.sup.1R.sup.1), and --(CH.sub.2).sub.m--CO--N(R.sup.5R.sup.5).
Each R.sup.1 can be independently C.sub.1-4 alkyl or H and each
R.sup.2 can be independently H, halo, OH, oxo, .dbd.NH, NH.sub.2,
--COOH, F, --NHR.sup.1, --N(R.sup.5R.sup.5), --SO.sub.2--R.sup.1,
--SO.sub.2--N(R.sup.5R.sup.5), --N(R.sup.1)--SO.sub.2--R.sup.1,
--COOR.sup.1, --OCO--R.sup.1, --CO--N(R.sup.5R.sup.5),
--N(R.sup.1)--COR.sup.1, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, and a
ring moiety optionally substituted with R.sup.4 wherein such ring
moiety is piperazinyl, piperidinyl, morpholinyl, pyrrolidinyl,
pyrazolyl, imidazolyl, benzimidazolyl, azetidinyl, cyclobutinyl, or
phenyl. Each R.sup.3 can be independently R.sup.2, C.sub.1-4 alkyl,
C.sub.3-6 cycloalkyl, or C.sub.1-4 alkyl optionally substituted
with 1 or more R.sup.2; and each R.sup.4 can be independently halo,
OH, --NH.sub.2, --NHR.sup.1, --N(R.sup.1R.sup.1), --COOH,
--COOR.sup.1, --NHCO--R.sup.1. Each R.sup.5 can be independently
C.sub.1-4 alkyl or H, or alternatively two R.sup.5 taken together
with the nitrogen atom to which they are bound can form a 4, 5, or
6 membered saturated heterocyclic ring containing 0 or 1 additional
heteroatoms where such additional heteroatom is O or N wherein such
heterocycle is optionally substituted with --OH, --NH.sub.2,
--N(R.sup.1R.sup.1), n-hydroxy C.sub.1-4 alkyl,
--(CH.sub.2).sub.m--COOH, --(CH.sub.2).sub.m--COOR.sup.1. Each m is
independently 0, 1, 2, or 3.
[0009] In certain embodiments, a pharmaceutical composition
comprising a compound of the invention and a suitable excipient is
provided.
[0010] In certain embodiments, a method of use of an inventive
compound comprising preparation of a medicament is provided.
[0011] In certain combinations, a pharmaceutical combination
comprising a compound of the invention and a second medicament is
provided. In various embodiments the second medicament is medically
indicated for the treatment of multiple sclerosis, transplant
rejection, acute respiratory distress syndrome or adult respiratory
distress syndrome.
[0012] In certain embodiments, a method of activation or agonism of
a sphingosine-1-phosphate receptor subtype 1 comprising contacting
the receptor subtype 1 with a compound of claim 1 is provided. In
various embodiments, the compound of claim 1 activates or agonizes
the sphingosine-1-phosphate receptor subtype 1 to a greater degree
than the compound activates or agonizes a sphingosin-1-phosphate
receptor subtype 3.
[0013] In certain embodiments a method of treatment of a
malcondition in a patient for which activation or agonism of an
S1P.sub.1 receptor is medically indicated, is provided. In various
embodiment, selective activation or agonism of an S1P.sub.1
receptor, such as with respect to an S1P.sub.3 receptor, is
medically indicated. In various embodiments, the malcondition
comprises multiple sclerosis, transplant rejection, or acute
respiratory distress syndrome.
[0014] In certain embodiments, a method is provided for chiral
synthesis of certain compounds including compounds of the
invention. In certain other embodiments the invention provides
certain intermediate compounds associated with such methods of
chiral synthesis.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Certain embodiments of the present invention comprise a
compound having the structure of Formula I-R or I-S or a
pharmaceutically acceptable salt, ester, prodrug, homolog, hydrate
or solvate thereof:
##STR00002##
X can be --NR'R'' or --OR''' and Y can be --CN, --Cl, or
--CF.sub.3. R' can be H, C.sub.1-4 alkyl, n-hydroxy C.sub.1-4
alkyl, --SO.sub.2--R.sup.1, or --CO--R.sup.1. R'' can be H,
--SO.sub.2--R.sup.3, C.sub.1-4 alkyl optionally substituted with 1
or more R.sup.2, or a ring moiety optionally substituted with
R.sup.4 wherein such ring moiety is piperidinyl, cyclohexyl,
morpholinyl, pyrrolidinyl, imidazolyl, or phenyl. R''' can be H,
C.sub.1-4 alkyl, or --CO--R.sup.1. Alternatively, R' and R'' taken
together with the nitrogen atom to which they are bound form a 4,
5, or 6 membered saturated heterocyclic ring containing 0 or 1
additional heteroatoms where such additional heteroatom is O or N
wherein such heterocycle is optionally singly or multiply
substituted with substituents independently selected from --OH,
oxo, --NH.sub.2, n-hydroxy-C.sub.1-4 alkyl, --COOH,
--(CH.sub.2).sub.m--COOH, --(CH.sub.2).sub.m--COOR.sup.1,
--N(R.sup.1R.sup.1), and
--(CH.sub.2).sub.m--CO--N(R.sup.5R.sup.5).
[0016] Each R.sup.1 can be independently C.sub.1-4 alkyl or H and
each R.sup.2 can be independently H, halo, OH, oxo, .dbd.NH,
NH.sub.2, --COOH, F, --NHR.sup.1, --N(R.sup.5R.sup.5),
--SO.sub.2--R.sup.1, --SO.sub.2--N(R.sup.5R.sup.5),
--N(R.sup.1)--SO.sub.2--R.sup.1, --COOR.sup.1, --OCO--R.sup.1,
--CO--N(R.sup.5R.sup.5), --N(R.sup.1)--COR.sup.1, C.sub.1-3 alkyl,
C.sub.1-3 alkoxy, and a ring moiety optionally substituted with
R.sup.4 wherein such ring moiety is piperazinyl, piperidinyl,
morpholinyl, pyrrolidinyl, pyrazolyl, thiazolyl, imidazolyl,
benzimidazolyl, azetidinyl, cyclobutinyl, or phenyl.
[0017] Each R.sup.3 can be independently R.sup.2, C.sub.1-4 alkyl,
C.sub.3-6 cycloalkyl, or C.sub.1-4 alkyl optionally substituted
with 1 or more R.sup.2; and each R.sup.4 can be independently halo,
OH, --NH.sub.2, --NHR.sup.1, --N(R.sup.1R.sup.1), --COOH,
--COOR.sup.1, --NHCO--R.sup.1. Each R.sup.5 can be independently
C.sub.1-4 alkyl or H, or alternatively two R.sup.5 taken together
with the nitrogen atom to which they are bound can form a 4, 5, or
6 membered saturated heterocyclic ring containing 0 or 1 additional
heteroatoms where such additional heteroatom is O or N wherein such
heterocycle is optionally substituted with --OH, --NH.sub.2,
--N(R.sup.1R.sup.1), n-hydroxy C.sub.1-4 alkyl,
--(CH.sub.2).sub.m--COOH, --(CH.sub.2).sub.m--COOR.sup.1. Each m is
independently 0, 1, 2, or 3.
[0018] In certain embodiments, the compounds of the invention have
the structure of Formula I-R or a pharmaceutically acceptable salt,
ester, prodrug, homolog, hydrate or solvate thereof. In other
embodiments, the compounds of the invention have the structure of
Formula I-S or a pharmaceutically acceptable salt, ester, prodrug,
homolog, hydrate or solvate thereof.
[0019] In certain embodiments the invention provides compounds
which are substantially enantiomerically pure.
[0020] In certain embodiments the invention provides compounds
which have an EC.sub.50 as an agonist of the wild type S1P receptor
subtype 1 which is at least ten times smaller than the EC.sub.50 of
such compound as an agonist of a mutant S1P receptor subtype 1
having a single mutation with respect to wild type S1P receptor
subtype 1 such that the 101.sup.st amino acid residue is changed
from asparagine to alanine.
[0021] In certain embodiments the invention provides compounds
which have an EC.sub.50 as an agonist of the wild type S1P receptor
subtype 1 which is at least twenty times smaller than the EC.sub.50
of such compound as an agonist of a mutant S1P receptor subtype 1
having a single mutation with respect to wild type S1P receptor
subtype 1 such that the 101.sup.st amino acid residue is changed
from asparagine to alanine.
[0022] In certain embodiments the invention provides compounds
which have a therapeutic index of at least 5 as measured in rats
following 5 or 14 days of dosing of rats with the compound where
the therapeutic index is calculated as a ratio of (i) the highest
dose of such compound which achieves less than or equal to a ten
percent increase in the ratio of lung to terminal body weight at
the conclusion of such 5 or 14 days of dosing, to (ii) the dose of
such compound achieving 50% lymphopenia in rats. In certain
embodiments, such therapeutic index is at least 10 and in certain
embodiments the therapeutic index is at least 20. In certain
embodiments, the therapeutic index for a compound is at least five
times greater than the therapeutic index for the enantiomer of such
compound.
[0023] In certain embodiments the invention provides compounds
which have a therapeutic index of at least 5 as measured in rats
following 5 or 14 days of dosing of rats with the compound where
the therapeutic index is calculated as a ratio of (i) the highest
dose of such compound which achieves less than or equal to a ten
percent increase in the ratio of lung to terminal body weight at
the conclusion of such 5 or 14 days of dosing, to (ii) the dose of
such compound achieving 50% lymphopenia in rats. In certain
embodiments, such therapeutic index is at least 10 and in certain
embodiments the therapeutic index is at least 20. In certain
embodiments, the therapeutic index for a compound is greater than
the therapeutic index for the enantiomer of such compound. In
certain embodiments, the therapeutic index for a compound is at
least 150% of the therapeutic index for the enantiomer of such
compound.
[0024] In certain embodiments the invention provides compounds
where Y is Cl, in other embodiments the invention provides
compounds where Y is CF.sub.3 and in other embodiments the
invention provides compounds where Y is CN.
[0025] In certain embodiments the invention provides compounds
where X is --NR'R'', in other embodiments the invention provides
compounds where X is --OR'''. In certain embodiments the invention
provides compounds where X is --OR'''. In certain embodiments the
invention provides compounds where X is --OH and in other
embodiments the invention provides compounds where X is
--OCO--R.sup.1.
[0026] In certain embodiments the invention provides compounds
where R.sub.1 is C.sub.1-3 alkyl; in other embodiments the
invention provides compounds where R' is H.
[0027] In certain embodiments the invention provides compounds
where R' is --COR.sup.1; in other embodiments the invention
provides compounds where R' is SO.sub.2--R.sup.1. In certain
embodiments the invention provides compounds where R'' is H.
[0028] In certain embodiments the invention provides compounds
where R'' is --SO.sub.2--R.sup.3; in other embodiments the
invention provides compounds where R'' is C.sub.1-4 alkyl where the
C.sub.1-4 alkyl is optionally substituted with 1 or more
substituents defined by R.sup.2. In certain embodiments the
invention provides compounds where R'' is
--(CR.sup.aR.sup.b).sub.n--R.sup.2 and each R.sup.a and each
R.sup.b can be independently any of H, hydroxyl and methyl or where
R.sup.a and R.sup.b are bound to the same carbon they can be taken
together to form oxo (i.e. with the carbon to which they are bound
forming a carbonyl moiety). In certain such embodiments n can be 0,
1, 2, or 3 and in certain embodiments n is 2. In certain such
embodiments R.sub.2 can be --OH, --NH.sub.2, --NHR.sup.1,
--N(R.sup.5R.sup.5), or --COOH.
[0029] In certain embodiments the invention provides compounds
where R.sup.3 is C.sub.1-4 alkyl optionally substituted with 1 or
more R.sup.2. In certain embodiments the invention provides
compounds where R.sup.2 is OH; in other embodiments the invention
provides compounds where R.sup.2 is C.sub.1-3 alkoxy. In certain
embodiments the invention provides compounds where R.sup.3 is
(CH.sub.2).sub.2--OR.sup.1.
[0030] In certain embodiments the invention provides compounds
where Y is CN and X is --NH--SO.sub.2--R.sup.3. In certain
embodiments the invention provides compounds where R.sup.3 is
--C.sub.2H.sub.5--N((R.sup.5R.sup.5) or
--CH.sub.2--CO--N(R.sup.5R.sup.5). In certain embodiments the
invention provides compounds where Y is CN and X is
--NH--CO--N(R.sup.5R.sup.5).
[0031] In certain embodiments X is --NH.sub.2 and in certain of
such embodiments Y is CN.
[0032] In certain embodiments the invention provides one or more of
compounds 1-55:
##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014##
or any pharmaceutically acceptable salt, tautomer, stereoisomer,
solvate, hydrate, or prodrug thereof. In certain of such
embodiments, the invention provides a compound selected from
compounds 8, 13, 29, 33, 37, and 49 or any pharmaceutically
acceptable salt, ester, tautomer, stereoisomer, solvate, hydrate,
homolog, or prodrug thereof.
[0033] In certain embodiments, an invention compound of Formula I
is provided wherein the compound has at least one chiral center and
is substantially enantiomerically pure.
[0034] In other embodiments, a pharmaceutical composition
comprising an invention compound of Formula I and a suitable
excipient is provided.
[0035] In other embodiments, a pharmaceutical combination
comprising an invention compound and a second medicament is
provided. In still other embodiments, a pharmaceutical combination
comprising an invention compound and a second medicament is
provided wherein the second medicament is medically indicated for
the treatment of multiple sclerosis, transplant rejection, or adult
respiratory distress syndrome.
[0036] In certain embodiments, a method of use of an invention
compound for preparation of a medicament is provided.
[0037] In certain embodiments a method of activation or agonism of
a sphingosine-1-phosphate receptor subtype 1 by contacting the
receptor subtype 1 with an effective amount of an invention
compound. In further embodiments, a method of activation or agonism
of a sphingosine-1-phosphate receptor subtype 1 by contacting the
receptor subtype 1 with an effective amount of an invention
compound is provided, wherein the compound activates or agonizes
the sphingosine-1-phosphate receptor subtype 1 to a greater extent
than the compound activates or agonizes a sphingosine-1-phosphate
receptor subtype 3. In further embodiments, a method of activation
or agonism of a sphingosine-1-phosphate receptor subtype 1 by
contacting the receptor subtype 1 with an effective amount of an
invention compound is provided, wherein the sphingosine-1-phosphate
receptor subtype 1 is disposed within a living mammal.
[0038] In certain embodiments, a method is provided for treatment
of a malcondition in a patient for which activation or agonism of a
sphingosine-1-phosphate receptor subtype 1 is medically indicated,
by administering an effective amount of an invention compound to
the patient at a frequency and for a duration of time sufficient to
provide a beneficial effect to the patient. In further embodiments,
a method is provided for treatment of a malcondition in a patient
for which activation or agonism of an sphingosine-1-phosphate
receptor subtype 1 is medically indicated, by administering an
effective amount of an invention compound to the patient at a
frequency and for a duration of time sufficient to provide a
beneficial effect to the patient, wherein selective activation or
agonism of an S1P subtype 1 receptor with respect to other subtypes
of S1P receptor is medically indicated. In yet further embodiments,
a method is provided for treatment of a malcondition in a patient
for which activation or agonism of an sphingosine-1-phosphate
receptor subtype 1 is medically indicated, by administering an
effective amount of an invention compound to the patient at a
frequency and for a duration of time sufficient to provide a
beneficial effect to the patient, wherein the malcondition
comprises rejection of transplanted organs or tissue;
graft-versus-host diseases brought about by transplantation;
autoimmune syndromes including rheumatoid arthritis; acute
respiratory distress syndrome; adult respiratory distress syndrome;
influenza; cancer; systemic erythematosus; Hashimoto's thyroiditis;
lymphocytic thyroiditis; multiple sclerosis; myasthenia gravis;
type I and II diabetes; uveitis; posterior uveitis; uveitis
associated with Behcet's disease; uveomeningitis syndrome; allergic
encephalomyelitis; chronic allograft vasculopathy; post-infectious
autoimmune diseases including rheumatic fever and post-infectious
glomerulonephritis; inflammatory and hyperproliferative skin
diseases; cutaneous manifestations of immunologically-mediated
disorders; psoriasis; atopic dermatitis; osteomyelitis; contact
dermatitis; eczematous dermatitis; seborrhoeic dermatitis; lichen
planus; pemphigus; bullous pemphigoid; epidermolysis bullosa;
urticaria; angioedema; vasculitis; erythema; cutaneous
eosinophilia; acne; alopecia greata; keratoconjunctivitis; vernal
conjunctivitis; keratitis; herpetic keratitis; dystrophia
epithelialis corneae; corneal leukoma; ocular pemphigus; Mooren's
ulcer; ulcerative keratitis; scleritis; Graves' ophthalmopathy;
Vogt-Koyanagi-Harada syndrome; sarcoidosis; pollen allergies;
reversible obstructive airway disease; bronchial asthma; allergic
asthma; intrinsic asthma; extrinsic asthma; dust asthma; chronic or
inveterate asthma; late asthma and airway hyper-responsiveness;
bronchitis; gastric ulcers; ischemic bowel diseases; inflammatory
bowel diseases; necrotizing enterocolitis; intestinal lesions
associated with thermal burns; celiac diseases; proctitis;
eosinophilic gastroenteritis; mastocytosis; Crohn's disease;
ulcerative colitis; vascular damage caused by ischemic diseases and
thrombosis; atherosclerosis; fatty heart; myocarditis; cardiac
infarction; arteriosclerosis; aortitis syndrome; cachexia due to
viral disease; vascular thrombosis; migraine; rhinitis; eczema;
interstitial nephritis; IgA-induced nephropathy; Goodpasture's
syndrome; hemolytic-uremic syndrome; diabetic nephropathy;
glomerulosclerosis; glomerulonephritis; multiple myositis;
Guillain-Barre syndrome; Meniere's disease; polyneuritis; multiple
neuritis; mononeuritis; radiculopathy; hyperthyroidism; Basedow's
disease; thyrotoxicosis; pure red cell aplasia; aplastic anemia;
hypoplastic anemia; idiopathic thrombocytopenic purpura; autoimmune
hemolytic anemia; agranulocytosis; pernicious anemia; megaloblastic
anemia; anerythroplasia; osteoporosis; sarcoidosis; fibroid lung;
idiopathic interstitial pneumonia; dermatomyositis; leukoderma
vulgaris; ichthyosis vulgaris; photoallergic sensitivity; cutaneous
T cell lymphoma; polyarteritis nodosa; Huntington's chorea;
Sydenham's chorea; myocardosis; scleroderma; Wegener's granuloma;
Sjogren's syndrome; adiposis; eosinophilic fascitis; lesions of
gingiva, periodontium, alveolar bone, substantia ossea dentis; male
pattern alopecia or alopecia senilis; muscular dystrophy; pyoderma;
Sezary's syndrome; chronic adrenal insufficiency; Addison's
disease; ischemia-reperfusion injury of organs which occurs upon
preservation; endotoxin shock; pseudomembranous colitis; colitis
caused by drug or radiation; ischemic acute renal insufficiency;
chronic renal insufficiency; lung cancer; malignancy of lymphoid
origin; acute or chronic lymphocytic; leukemias; lymphoma;
psoriasis; inflammatory lung injury, pulmonary emphysema;
cataracta; siderosis; retinitis pigmentosa; senile macular
degeneration; vitreal scarring; inflammatory eye disease; corneal
alkali burn; dermatitis erythema; ballous dermatitis; cement
dermatitis; gingivitis; periodontitis; sepsis; pancreatitis;
carcinogenesis; metastasis of carcinoma; hypobaropathy; autoimmune
hepatitis; primary biliary cirrhosis; sclerosing cholangitis;
partial liver resection; acute liver necrosis; cirrhosis; alcoholic
cirrhosis; hepatic failure; fulminant hepatic failure; late-onset
hepatic failure; "acute-on-chronic" liver failure. In yet further
embodiments, the malcondition is one or more of rejection of
transplanted organs or tissue; graft-versus-host diseases brought
about by transplantation; autoimmune syndromes including rheumatoid
arthritis, multiple sclerosis, myasthenia gravis; pollen allergies;
type I diabetes; prevention of psoriasis; Crohn's disease;
ulcerative colitis, acute respiratory distress syndrome; adult
respiratory distress syndrome; influenza; post-infectious
autoimmune diseases including rheumatic fever and post-infectious
glomerulonephritis; and metastasis of carcinoma. In yet further
empbodiments the malcondition is one of influenza, ulcerative
colitis, multiple sclerosis, transplant rejection, acute
respiratory distress syndrome or adult respiratory distress
syndrome.
[0039] In certain embodiments, methods are provided for use of an
invention compound for preparation of a medicament adapted for
treatment of a disorder or a malcondition wherein activation or
inhibition of a sphingosine-1-phosphate receptor subtype 1 is
medically indicated.
[0040] In certain embodiments the invention provides a method for
the chiral synthesis of a compound comprising a
tetrahydronaphthalene moiety having a chiral carbon in the
six-membered saturated ring of the tetrahydronaphthalene moiety
where the compound is enantiomerically enriched with respect to the
chiral carbon. In such embodiments, the method of the invention
provides the steps of (i) providing a compound comprising a
tetrahydronaphthalene moiety where the ring carbon of the
six-membered saturated ring of the tetrahydronaphthalene moiety
where chiral substitution is desired is oxo substituted at such
carbon; and (ii) reacting such compound with a chiral reagent to
form a chiral center at the tetrahydronaphthalene moiety carbon
previously bound to the oxo group. In certain of such embodiments,
the chiral reagent is RuCl(p-cymene)[(R,R)-Ts-DPEN] or
RuCl(p-cymene)[(S,S)-Ts-DPEN].
[0041] In certain of such embodiments the compound comprising a
tetrahydronaphthalene moiety provided in step (i) is contacted with
the chiral reagent to form in step (ii) an intermediate of Formula
VI-R or VI-S:
##STR00015##
wherein Z is --CN, --Cl, or --CF.sub.3. In certain of such
embodiments Z is --CN.
[0042] In certain embodiments the invention provides the method
comprising the step of reversing the chiral configuration of the
chiral carbon in the six-membered saturated ring of the
tetrahydronaphthalene moiety that was previously bound to the oxo
group by treating the intermediate of Formula VI-R or VI-S with
diphenylphosphoryl azide (DPPA) to form an azido
tetrahydronaphthalene of Formula VII-S or VII-R:
##STR00016##
where the azido substituent in the six-membered saturated ring of
the tetrahydronaphthalene moiety replaces the hydroxy substituent
of Formula VI-R or VI-S and the resulting chiral carbon that is
bound to the azido substituent has a reverse chiral configuration
of the chiral carbon when it was previously bound to the hydroxy
substituent.
[0043] In certain embodiments the invention provides the method
where Z is --CN and the method further comprises the additional
steps of (a) forming a substituted 1,2,4-oxadiazole on the
tetrahydronaphthalene moiety by (a) reacting the intermediate of
VII-R or VII-S with a protecting agent and then reacting the
resulting protected form of the intermediate of VII-R or VII-S with
a hydroxylamine or a hydroxylamine hydrochloride to form a
hydroxyamidine at the phenyl carbon to which Z had been attached,
the resulting compound of such reaction having the Formula VIII-R
or VIII-S:
##STR00017##
and (b) contacting the intermediate of Formula VIII-R or VIII-S
with substituted benzoic acid and a coupling reagent to form a
compound of Formula IX-R or XI-S:
##STR00018##
where X is as defined above or in certain embodiments OH, N.sub.3,
NH-PG, NH.sub.2 or NR'R''; PG can be a protecting group; R' can be
H, C.sub.1-4 alkyl, n-hydroxy C.sub.1-4 alkyl, --SO.sub.2--R.sup.1,
or --CO--R.sup.1; R'' can be H, --SO.sub.2--R.sup.3, C.sub.1-4
alkyl optionally substituted with 1 or more R.sup.2, or a ring
moiety optionally substituted with R.sup.4 wherein such ring moiety
is piperidinyl, cyclohexyl, morpholinyl, thiazolyl, pyrazolyl,
pyrrolidinyl, imidazolyl, or phenyl; R.sup.a is lower alkyl and
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are as defined above. In
certain of such embodiments the compounds of Formula IX-R or IX-S
have the structures below:
##STR00019##
[0044] In certain of such embodiments, the coupling reagent can be
a mixture comprising hydroxybenzotriazole (HOBt) and
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC).
[0045] Protecting groups can render chemical functionality inert to
specific reaction conditions and can be appended to and removed
from such functionality in a molecule without substantially
damaging the remainder of the molecule. Practitioners in the art
would be familiar with suitable protecting groups for use in the
synthetic methods of the invention. See, e.g., Greene and Wuts,
Protective Groups in Organic Synthesis, 2.sup.nd ed., John Wiley
& Sons, New York, 1991.
[0046] In certain embodiments the invention provides the method
where the compound provided in step (i) is
##STR00020##
[0047] In certain embodiments the invention provides the method
where the resulting compound comprising a tetrahydronaphthalene
moiety having a chiral carbon in the six-membered saturated ring of
the tetrahydronaphthalene moiety is enantiomerically enriched at
least 90%. In certain such embodiments the resulting compound is
enantiomerically enriched at least 95%. In certain such embodiments
the resulting compound is enantiomerically enriched at least 98%.
In certain such embodiments the resulting compound is
enantiomerically enriched at least 99%.
[0048] In certain of such embodiments, the invention provides a
method for chiral synthesis of a chiral compound comprising a
tetrahydronaphthalene moiety having a chiral carbon in the
six-membered saturated ring of the tetrahydronaphthalene moiety or
a chiral compound comprising an oxadiazole-tetrahydronaphthalene
moiety having a chiral carbon in the six-membered saturated ring of
the tetrahydronaphthalene moiety where the chiral compound has an
enantiomeric enrichment of at least 75%, 85%, 90%, 95%, 98%, or
99%.
[0049] In certain of such embodiments, the invention provides a
method for synthesis of a chiral compound of the invention having
an enantiomeric enrichment of at least 75%, 85%, 90%, 95%, 98%, or
99%.
[0050] In certain embodiments, the invention provides compounds
which can be intermediates in the herein described methods for
chiral syntheses. In certain such embodiments, the invention
provides one or more of the following intermediate compounds:
##STR00021## ##STR00022##
[0051] In certain of such embodiments, the invention provides a
method for the synthesis of a compound comprising a
tetrahydronaphthalene moiety having a chiral carbon in the
six-membered saturated ring of the tetrahydronaphthalene moiety
where the compound is enantiomerically enriched with respect to
such chiral carbon, with the method comprising a step of providing
one of such intermediate compounds.
[0052] In certain embodiments, a method for the synthesis of a
compound comprising a tetrahydronaphthalene moiety having a chiral
carbon in the six-membered saturated ring of the
tetrahydronaphthalene moiety where the compound is enantiomerically
enriched with respect to the chiral carbon is provided. In certain
embodiments, a method comprising a step of providing a compound of
the structures described herein is provided.
[0053] In certain embodiments, the invention provides a method for
the synthesis of a compound of the Formula IX-R or XI-S:
##STR00023##
wherein X is as defined herein, with the method comprising a step
of providing one of the intermediate compounds described above. In
certain of such embodiments the invention provides a method for the
synthesis of a compound of the invention.
[0054] In certain embodiments the invention provides a method for
the chiral synthesis of the structure of Formula IX-R or IX-S or a
pharmaceutically acceptable salt, ester, prodrug, homolog, hydrate
or solvate thereof:
##STR00024##
[0055] where X is defined as above and where the compound is
enantiomerically enriched with respect to the chiral carbon. In
such embodiments, the method of the invention provides the steps of
[0056] (i) providing the compound
##STR00025##
[0056] and [0057] (ii) reacting such compound with a chiral reagent
RuCl(p-cymene)[(R,R)-Ts-DPEN] or RuCl(p-cymene)[(S,S)-Ts-DPEN]; and
[0058] (iii) forming a chiral center at the tetrahydronaphthalene
moiety carbon previously bound to the oxo group.
[0059] Additional steps for the preparation of such compounds can
be adapted from the synthetic methods disclosed herein including
recrystallization and other processes for purification.
[0060] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise.
[0061] As used herein, "individual" (as in the subject of the
treatment) means both mammals and non-mammals. Mammals include, for
example, humans; non-human primates, e.g., apes and monkeys;
cattle; horses; sheep; and goats. Non-mammals include, for example,
fish and birds.
[0062] The term "S1P.sub.1" as used herein refers to subtype 1 of a
sphingosine-1-phosphate receptor, while other
sphingosine-1-phosphate receptor subtypes are referred to in a
corresponding manner, for example, sphingosine-1-phosphate receptor
subtype 3 is referred to as "S1P.sub.3".
[0063] A "receptor", as is well known in the art, is a biomolecular
entity usually comprising a protein that specifically binds a
structural class of ligands or a single native ligand in a living
organism, the binding of which causes the receptor to transduce the
binding signal into another kind of biological action, such as
signaling a cell that a binding event has occurred, which causes
the cell to alter its function in some manner. An example of
transduction is receptor binding of a ligand causing alteration of
the activity of a "G-protein" in the cytoplasm of a living cell.
Any molecule, naturally occurring or not, that binds to a receptor
and activates it for signal transduction, is referred to as an
"agonist" or "activator." Any molecule, naturally occurring or not,
that binds to a receptor, but does not cause signal transduction to
occur, and which can block the binding of an agonist and its
consequent signal transduction, is referred to as an
"antagonist."
[0064] An "S1P.sub.1 compound" or "S1P.sub.1 agonist" or "S1P.sub.1
activator" or "S1P.sub.1 inhibitor" or "S1P.sub.1 antagonist" as
the terms are used herein refer to compounds that interact in some
way with the S1P receptor subtype 1. They can be agonist or
activators, or they can be antagonists or inhibitors. An "S1P.sub.1
compound" of the invention can be selective for action on subtype 1
of the S1P receptor family; for example a compound of the invention
can act at a lower concentration on subtype 1 of the S1P receptor
family than on other subtypes of the S1P receptor family; more
specifically, an "S1P.sub.1 compound" of the invention can
selectively act on subtype 1 receptors compared to its action on
subtype 3, or "S1P.sub.3" receptors.
[0065] In certain embodiments, compounds of the invention are
orthostatic agonists. In certain other embodiments, compounds of
the invention are allosteric agonists. Receptor agonists may be
classified as either orthosteric or allosteric. An orthosteric
agonist binds to a site in the receptor that significantly overlaps
with the binding of the natural ligand and replicates the key
interactions of the natural ligand with the receptor. An
orthosteric agonist will activate the receptor by a molecular
mechanism similar to that of the natural ligand, will be
competitive for the natural ligand, and will be competitively
antagonized by pharmacological agents that are competitive
antagonists for the natural ligand. An allosteric agonist binds to
a site in the receptor that makes some significant interactions
that are partly or wholly non-overlapping with the natural ligand.
Allosteric agonists are true agonists and not allosteric
potentiators. Consequently, they activate receptor signaling alone
and without a requirement for a sub-maximal concentration of the
natural ligand. Allosteric agonists may be identified when an
antagonist known to be competitive for the orthosteric ligand shows
non-competitive antagonism. The allosteric agonist site can also be
mapped by receptor mutagenesis. The introduction of single point
mutations in receptors that retain receptor activation by
allosteric agonist, while diminishing or abolishing signaling
induced by orthosteric agonist or vice versa provide formal
evidence for differences in binding interactions. Orthosteric
agonists may destabilize GPCR structure and conformation, while
allosteric agonists may either stabilize or destabilize GPCR
structure and conformation. Allosteric agonists, by virtue of their
different interactions with receptor, may be pharmaceutically
useful because the allosteric site may confer additional
opportunities for agonist potency and selectivity within a related
family of receptor subtypes that share a similar orthosteric
ligand. In addition, the allosteric site may require very different
physical and chemical properties of an agonist compared to the
orthosteric ligand. These chemico-physical properties, which
include hydrophobicity, aromaticity, charge distribution and
solubility, may also provide advantages in generating agonists of
varying pharmacokinetic, oral bioavailability, distributional and
metabolism profiles that facilitate the development of effective
pharmaceutical substances.
[0066] "Substantially" as the term is used herein means completely
or almost completely; for example, a composition that is
"substantially free" of a component either has none of the
component or contains such a trace amount that any relevant
functional property of the composition is unaffected by the
presence of the trace amount, or a compound is "substantially pure"
is there are only negligible traces of impurities present.
[0067] Substantially enantiomerically pure means a level of
enantiomeric enrichment of one enantiomer with respect to the other
enantiomer of at least 90%, 95%, 98%, 99%, 99.5% or 99.9%.
[0068] "Treating" or "treatment" within the meaning herein refers
to an alleviation of symptoms associated with a disorder or
disease, or inhibition of further progression or worsening of those
symptoms, or prevention or prophylaxis of the disease or
disorder.
[0069] The expression "effective amount", when used to describe use
of a compound of the invention in providing therapy to a patient
suffering from a disorder or malcondition mediated by a
sphingosine-1-phospate receptor of subtype 1 refers to the amount
of a compound of the invention that is effective to bind to as an
agonist or as an antagonist a S1P.sub.1 receptor in the
individual's tissues, wherein the S1P.sub.1 is implicated in the
disorder, wherein such binding occurs to an extent sufficient to
produce a beneficial therapeutic effect on the patient. Similarly,
as used herein, an "effective amount" or a "therapeutically
effective amount" of a compound of the invention refers to an
amount of the compound that alleviates, in whole or in part,
symptoms associated with the disorder or condition, or halts or
slows further progression or worsening of those symptoms, or
prevents or provides prophylaxis for the disorder or condition. In
particular, a "therapeutically effective amount" refers to an
amount effective, at dosages and for periods of time necessary, to
achieve the desired therapeutic result by acting as an agonist of
sphingosine-1-phosphate receptor subtype 1 (S1P.sub.1) activity. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of compounds of the invention are outweighed by
the therapeutically beneficial effects. For example, in the context
of treating a malcondition mediated by activation of S1P.sub.1, a
therapeutically effective amount of an S1P.sub.1 agonist of the
invention is an amount sufficient to control the malcondition, to
mitigate the progress of the malcondition, or to relieve the
symptoms of the malcondition. Examples of malconditions that can be
so treated include multiple sclerosis, transplant rejection, adult
respiratory distress syndrome.
[0070] Diseases, disorders and conditions which may be treated by
compounds of the invention include rejection of transplanted organs
or tissue; graft-versus-host diseases brought about by
transplantation; autoimmune syndromes including rheumatoid
arthritis; acute respiratory distress syndrome; adult respiratory
distress syndrome; influenza; cancer; systemic erythematosus;
Hashimoto's thyroiditis; lymphocytic thyroiditis; multiple
sclerosis; myasthenia gravis; type I and II diabetes; uveitis;
posterior uveitis; uveitis associated with Behcet's disease;
uveomeningitis syndrome; allergic encephalomyelitis; chronic
allograft vasculopathy; post-infectious autoimmune diseases
including rheumatic fever and post-infectious glomerulonephritis;
inflammatory and hyperproliferative skin diseases; cutaneous
manifestations of immunologically-mediated disorders; psoriasis;
atopic dermatitis; osteomyelitis; contact dermatitis; eczematous
dermatitis; seborrhoeic dermatitis; lichen planus; pemphigus;
bullous pemphigoid; epidermolysis bullosa; urticaria; angioedema;
vasculitis; erythema; cutaneous eosinophilia; acne; alopecia
greata; keratoconjunctivitis; vernal conjunctivitis; keratitis;
herpetic keratitis; dystrophia epithelialis corneae; corneal
leukoma; ocular pemphigus; Mooren's ulcer; ulcerative keratitis;
scleritis; Graves' ophthalmopathy; Vogt-Koyanagi-Harada syndrome;
sarcoidosis; pollen allergies; reversible obstructive airway
disease; bronchial asthma; allergic asthma; intrinsic asthma;
extrinsic asthma; dust asthma; chronic or inveterate asthma; late
asthma and airway hyper-responsiveness; bronchitis; gastric ulcers;
ischemic bowel diseases; inflammatory bowel diseases; necrotizing
enterocolitis; intestinal lesions associated with thermal burns;
celiac diseases; proctitis; eosinophilic gastroenteritis;
mastocytosis; Crohn's disease; ulcerative colitis; vascular damage
caused by ischemic diseases and thrombosis; atherosclerosis; fatty
heart; myocarditis; cardiac infarction; arteriosclerosis; aortitis
syndrome; cachexia due to viral disease; vascular thrombosis;
migraine; rhinitis; eczema; interstitial nephritis; IgA-induced
nephropathy; Goodpasture's syndrome; hemolytic-uremic syndrome;
diabetic nephropathy; glomerulosclerosis; glomerulonephritis;
multiple myositis; Guillain-Barre syndrome; Meniere's disease;
polyneuritis; multiple neuritis; mononeuritis; radiculopathy;
hyperthyroidism; Basedow's disease; thyrotoxicosis; pure red cell
aplasia; aplastic anemia; hypoplastic anemia; idiopathic
thrombocytopenic purpura; autoimmune hemolytic anemia;
agranulocytosis; pernicious anemia; megaloblastic anemia;
anerythroplasia; osteoporosis; sarcoidosis; fibroid lung;
idiopathic interstitial pneumonia; dermatomyositis; leukoderma
vulgaris; ichthyosis vulgaris; photoallergic sensitivity; cutaneous
T cell lymphoma; polyarteritis nodosa; Huntington's chorea;
Sydenham's chorea; myocardosis; scleroderma; Wegener's granuloma;
Sjogren's syndrome; adiposis; eosinophilic fascitis; lesions of
gingiva, periodontium, alveolar bone, substantia ossea dentis; male
pattern alopecia or alopecia senilis; muscular dystrophy; pyoderma;
Sezary's syndrome; chronic adrenal insufficiency; Addison's
disease; ischemia-reperfusion injury of organs which occurs upon
preservation; endotoxin shock; pseudomembranous colitis; colitis
caused by drug or radiation; ischemic acute renal insufficiency;
chronic renal insufficiency; lung cancer; malignancy of lymphoid
origin; acute or chronic lymphocytic; leukemias; lymphoma;
psoriasis; inflammatory lung injury, pulmonary emphysema;
cataracta; siderosis; retinitis pigmentosa; senile macular
degeneration; vitreal scarring; inflammatory eye disease; corneal
alkali burn; dermatitis erythema; ballous dermatitis; cement
dermatitis; gingivitis; periodontitis; sepsis; pancreatitis;
carcinogenesis; metastasis of carcinoma; hypobaropathy; autoimmune
hepatitis; primary biliary cirrhosis; sclerosing cholangitis;
partial liver resection; acute liver necrosis; cirrhosis; alcoholic
cirrhosis; hepatic failure; fulminant hepatic failure; late-onset
hepatic failure; "acute-on-chronic" liver failure. Particularly
preferred diseases and conditions which may be treated with
compounds of the invention comprise the group consisting of
rejection of transplanted organs or tissue; graft-versus-host
diseases brought about by transplantation; autoimmune syndromes
including rheumatoid arthritis, multiple sclerosis, myasthenia
gravis; pollen allergies; type I diabetes; prevention of psoriasis;
Crohn's disease; ulcerative colitis, acute respiratory distress
syndrome; adult respiratory distress syndrome; influenza;
post-infectious autoimmune diseases including rheumatic fever and
post-infectious glomerulonephritis; and metastasis of
carcinoma.
[0071] Furthermore, compounds of Formula I-R or I-S are also
useful, in combination with one or several immunosuppressant
agents, for the treatment of diseases, disorders and conditions
associated with an activated immune system and selected from the
list as above-mentioned. According to a preferred embodiment of the
invention, said immunosuppressant agent is selected from the group
comprising or consisting of cyclosporin, daclizumab, basiliximab,
everolimus, tacrolimus (FK506), azathiopirene, leflunomide,
15-deoxyspergualin, or other immunosuppressant drugs
[0072] All chiral, diastereomeric, racemic forms of a structure are
intended, unless a particular stereochemistry or isomeric form is
specifically indicated. Compounds used in the present invention can
include enriched or resolved optical isomers at any or all
asymmetric atoms as are apparent from the depictions, at any degree
of enrichment. Both racemic and diastereomeric mixtures, as well as
the individual optical isomers can be synthesized so as to be
substantially free of their enantiomeric or diastereomeric
partners, and these are all within the scope of certain embodiments
of the invention.
[0073] The isomers resulting from the presence of a chiral center
comprise a pair of non-superimposable isomers that are called
"enantiomers." Single enantiomers of a pure compound are optically
active, i.e., they are capable of rotating the plane of plane
polarized light. Single enantiomers are designated according to the
Cahn-Ingold-Prelog system. Once the priority ranking of the four
groups is determined, the molecule is oriented so that the lowest
ranking group is pointed away from the viewer. Then, if the
descending rank order of the other groups proceeds clockwise, the
molecule is designated (R) and if the descending rank of the other
groups proceeds counterclockwise, the molecule is designated (S).
In the examples, the Cahn-Ingold-Prelog ranking is
A>B>C>D. The lowest ranking atom, D is oriented away from
the viewer.
##STR00026##
[0074] "Isolated optical isomer" means a compound which has been
substantially purified from the corresponding optical isomer(s) of
the same formula. Preferably, the isolated isomer is at least about
80%, more preferably at least 90% pure, even more preferably at
least 98% pure, most preferably at least about 99% pure, by
weight.
Rotational Isomerism
[0075] It is understood that due to chemical properties (i.e.,
resonance lending some double bond character to the C--N bond) of
restricted rotation about the amide bond linkage (as illustrated
below) it is possible to observe separate rotamer species and even,
under some circumstances, to isolate such species, example shown
below. It is further understood that certain structural elements,
including steric bulk or substituents on the amide nitrogen, may
enhance the stability of a rotamer to the extent that a compound
may be isolated as, and exist indefinitely, as a single stable
rotamer. The present invention therefore includes any possible
stable rotamers of compounds of the invention which are
biologically active in the treatment of a disease, disorder or
condition for which a compound of the invention may be effective as
described herein.
##STR00027##
Regioisomerism
[0076] The preferred compounds of the present invention have a
particular spatial arrangement of substituents on the aromatic
rings, which is related to the structure activity relationship
demonstrated by the compound class. Often such substitution
arrangement is denoted by a numbering system; however, numbering
systems are often not consistent between different ring systems. In
six-membered aromatic systems, the spatial arrangements are
specified by the common nomenclature "para" for 1,4-substitution,
"meta" for 1,3-substitution and "ortho" for 1,2-substitution as
shown below.
##STR00028##
[0077] All structures encompassed within a claim are "chemically
feasible", by which is meant that the structure depicted by any
combination or subcombination of optional substituents meant to be
recited by the claim is physically capable of existence with at
least some stability as can be determined by the laws of structural
chemistry and by experimentation. Structures that are not
chemically feasible are not within a claimed set of compounds.
[0078] In general, "substituted" refers to an organic group as
defined herein in which one or more bonds to a hydrogen atom
contained therein are replaced by one or more bonds to a
non-hydrogen atom such as, but not limited to, a halogen (i.e., F,
Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups,
alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl)
groups, carboxyl groups including carboxylic acids, carboxylates,
and carboyxlate esters; a sulfur atom in groups such as thiol
groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone
groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in
groups such as amines, hydroxylamines, nitriles, nitro groups,
N-oxides, hydrazides, azides, and enamines; and other heteroatoms
in various other groups. Non-limiting examples of substituents that
can be bonded to a substituted carbon (or other) atom include F,
Cl, Br, I, OR', OC(O)N(R').sub.2, CN, CF.sub.3, OCF.sub.3, R', O,
S, C(O), S(O), methylenedioxy, ethylenedioxy, N(R').sub.2, SR',
SOR', SO.sub.2R', SO.sub.2N(R').sub.2, SO.sub.3R', C(O)R',
C(O)C(O)R', C(O)CH.sub.2C(O)R', C(S)R', C(O)OR', OC(O)R',
C(O)N(R').sub.2, OC(O)N(R').sub.2, C(S)N(R').sub.2,
(CH.sub.2).sub.0-2NHC(O)R', (CH.sub.2).sub.0-2N(R')N(R).sub.2,
N(R')N(R')C(O)R', N(R')N(R')C(O)OR', N(R')N(R')CON(R').sub.2,
N(R')SO.sub.2R', N(R')SO.sub.2N(R').sub.2, N(R)C(O)OR',
N(R')C(O)R', N(R')C(S)R', N(R')C(O)N(R').sub.2,
N(R')C(S)N(R').sub.2, N(COR')COR', N(OR')R', C(.dbd.NH)N(R').sub.2,
C(O)N(OR')R', or C(.dbd.NOR')R' wherein R' can be hydrogen or a
carbon-based moiety, and wherein the carbon-based moiety can itself
be further substituted.
[0079] Substituted alkyl, alkenyl, alkynyl, cycloalkyl, and
cycloalkenyl groups as well as other substituted groups also
include groups in which one or more bonds to a hydrogen atom are
replaced by one or more bonds, including double or triple bonds, to
a carbon atom, or to a heteroatom such as, but not limited to,
oxygen in carbonyl (oxo), carboxyl, ester, amide, imide, urethane,
and urea groups; and nitrogen in imines, hydroxyimines, oximes,
hydrazones, amidines, guanidines, and nitriles. The substituents of
the substituted groups can further be substituted with alkyl,
alkenyl, cycloalkyl, aryl, heteroaryl, and alkynyl groups as
defined herein, which can themselves be further substituted. For
example, a C.sub.1-4 alkyl group can be substituted with an amide,
and the amide can further be substituted with another C.sub.1-4
alkyl, which can further be substituted.
[0080] Substituted ring groups such as substituted aryl,
heterocyclyl and heteroaryl groups also include rings and fused
ring systems in which a bond to a hydrogen atom is replaced with a
bond to a carbon atom. Therefore, substituted aryl, heterocyclyl
and heteroaryl groups can also be substituted with alkyl, alkenyl,
cycloalkyl, aryl, heteroaryl, and alkynyl groups as defined herein,
which can themselves be further substituted.
[0081] The term "heteroatoms" as used herein refers to non-carbon
and non-hydrogen atoms, capable of forming covalent bonds with
carbon, and is not otherwise limited. Typical heteroatoms are N, O,
and S. When sulfur (S) is referred to, it is understood that the
sulfur can be in any of the oxidation states in which it is found,
thus including sulfoxides (R--S(O)--R') and sulfones
(R--S(O).sub.2--R'), unless the oxidation state is specified; thus,
the term "sulfone" encompasses only the sulfone form of sulfur; the
term "sulfide" encompasses only the sulfide (R--S--R') form of
sulfur. When the phrases such as "heteroatoms selected from the
group consisting of O, NH, NR' and S," or "[variable] is O, S . . .
" are used, they are understood to encompass all of the sulfide,
sulfoxide and sulfone oxidation states of sulfur.
[0082] Alkyl groups include straight chain and branched alkyl
groups and cycloalkyl groups having from 1 to about 20 carbon atoms
(C.sub.1-20 alkyl), and typically from 1 to 12 carbons (C.sub.1-12
alkyl) or, in some embodiments, from 1 to 8 carbon atoms (C.sub.1-8
alkyl) or, in some embodiments, from 1 to 4 carbon atoms (C.sub.1-4
alkyl) or, in some embodiments, from 1 to 3 carbon atoms (C.sub.1-3
alkyl). Examples of straight chain alkyl groups include, but are
not limited to methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,
n-heptyl, and n-octyl groups. Examples of branched alkyl groups
include, but are not limited to, isopropyl, iso-butyl, sec-butyl,
t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
Representative substituted alkyl groups can be substituted one or
more times with any of the groups listed above, for example, amino,
hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
The group "n-hydroxy C.sub.1-4 alkyl" represents a C.sub.1-4 alkyl
substituted with a terminal hydroxy group.
[0083] Cycloalkyl groups are alkyl groups forming a ring structure,
which can be substituted or unsubstituted. Examples of cycloalkyl
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In
some embodiments, the cycloalkyl group has 3 to 8 ring members,
whereas in other embodiments the number of ring carbon atoms range
from 3 to 5, 3 to 6, or 3 to 7. Cycloalkyl groups further include
polycyclic cycloalkyl groups such as, but not limited to,
norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl
groups, and fused rings such as, but not limited to, decalinyl, and
the like. Cycloalkyl groups also include rings that are substituted
with straight or branched chain alkyl groups as defined above.
Representative substituted cycloalkyl groups can be
mono-substituted or substituted more than once, such as, but not
limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstituted cyclohexyl
groups or mono-, di- or tri-substituted norbornyl or cycloheptyl
groups, which can be substituted with, for example, amino, hydroxy,
cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
[0084] The terms "carbocyclic" and "carbocycle" denote a ring
structure wherein the atoms of the ring are carbon. In some
embodiments, the carbocycle has 3 to 8 ring members, whereas in
other embodiments the number of ring carbon atoms is 4, 5, 6, or 7.
Unless specifically indicated to the contrary, the carbocyclic ring
can be substituted with as many as N substituents wherein N is the
size of the carbocyclic ring with for example, amino, hydroxy,
cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
[0085] (Cycloalkyl)alkyl groups, also denoted cycloalkylalkyl, are
alkyl groups as defined above in which a hydrogen or carbon bond of
the alkyl group is replaced with a bond to a cycloalkyl group as
defined above.
[0086] Alkenyl groups include straight and branched chain and
cyclic alkyl groups as defined above, except that at least one
double bond exists between two carbon atoms. Thus, alkenyl groups
have from 2 to about 20 carbon atoms, and typically from 2 to 12
carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples
include, but are not limited to --CH.dbd.CH(CH.sub.3),
--CH.dbd.C(CH.sub.3).sub.2, --C(CH.sub.3).dbd.CH.sub.2,
--C(CH.sub.3).dbd.CH(CH.sub.3), --C(CH.sub.2CH.sub.3).dbd.CH.sub.2,
vinyl, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl,
pentadienyl, and hexadienyl among others.
[0087] The term "cycloalkenyl" alone or in combination denotes a
cyclic alkenyl group wherein at least one double bond is present in
the ring structure. Cycloalkenyl groups include cycloalkyl groups
having at least one double bond between two adjacent carbon atoms.
Thus for example, cycloalkenyl groups include but are not limited
to cyclohexenyl, cyclopentenyl, and cyclohexadienyl groups.
[0088] (Cycloalkenyl)alkyl groups are alkyl groups as defined above
in which a hydrogen or carbon bond of the alkyl group is replaced
with a bond to a cycloalkenyl group as defined above.
[0089] Alkynyl groups include straight and branched chain alkyl
groups, except that at least one triple bond exists between two
carbon atoms. Thus, alkynyl groups have from 2 to about 20 carbon
atoms, and typically from 2 to 12 carbons or, in some embodiments,
from 2 to 8 carbon atoms. Examples include, but are not limited to
--C.ident.CH, --C.ident.C(CH.sub.3), --C.ident.C(CH.sub.2CH.sub.3),
--CH.sub.2C.ident.CH, --CH.sub.2C.ident.C(CH.sub.3), and
--CH.sub.2C.ident.C(CH.sub.2CH.sub.3), among others.
[0090] Aryl groups are cyclic aromatic hydrocarbons that do not
contain heteroatoms. Thus aryl groups include, but are not limited
to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl,
phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl,
biphenylenyl, anthracenyl, and naphthyl groups. In some
embodiments, aryl groups contain 6-14 carbons in the ring portions
of the groups. The phrase "aryl groups" includes groups containing
fused rings, such as fused aromatic-aliphatic ring systems (e.g.,
indanyl, tetrahydronaphthyl, and the like), and also includes
substituted aryl groups that have other groups, including but not
limited to alkyl, halo, amino, hydroxy, cyano, carboxy, nitro,
thio, or alkoxy groups, bonded to one of the ring atoms.
Representative substituted aryl groups can be mono-substituted or
substituted more than once, such as, but not limited to, 2-, 3-,
4-, 5-, or 6-substituted phenyl or naphthyl groups, which can be
substituted with groups including but not limited to those listed
above.
[0091] Aralkyl groups are alkyl groups as defined above in which a
hydrogen or carbon bond of an alkyl group is replaced with a bond
to an aryl group as defined above. Representative aralkyl groups
include benzyl and phenylethyl groups and fused
(cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. The aryl
moiety or the alkyl moiety or both are optionally substituted with
other groups, including but not limited to alkyl, halo, amino,
hydroxy, cyano, carboxy, nitro, thio, or alkoxy groups. Aralkenyl
group are alkenyl groups as defined above in which a hydrogen or
carbon bond of an alkyl group is replaced with a bond to an aryl
group as defined above.
[0092] Heterocyclyl groups include aromatic and non-aromatic ring
compounds (heterocyclic rings) containing 3 or more ring members,
of which one or more is a heteroatom such as, but not limited to,
N, O, S, or P. In some embodiments, heterocyclyl groups include 3
to 20 ring members, whereas other such groups have 3 to 15 ring
members. At least one ring contains a heteroatom, but every ring in
a polycyclic system need not contain a heteroatom. For example, a
dioxolanyl ring and a benzdioxolanyl ring system
(methylenedioxyphenyl ring system) are both heterocyclyl groups
within the meaning herein. A heterocyclyl group designated as a
C.sub.2-heterocyclyl can be a 5-membered ring with two carbon atoms
and three heteroatoms, a 6-membered ring with two carbon atoms and
four heteroatoms and so forth. Likewise a C.sub.4-heterocyclyl can
be a 5-membered ring with one heteroatom, a 6-membered ring with
two heteroatoms, and so forth. The number of carbon atoms plus the
number of heteroatoms sums up to equal the total number of ring
atoms. A saturated heterocyclic ring refers to a heterocyclic ring
containing no unsaturated carbon atoms.
[0093] The phrase "heterocyclyl group" includes fused ring species
including those having fused aromatic and non-aromatic groups. The
phrase also includes polycyclic ring systems containing a
heteroatom such as, but not limited to, quinuclidyl and also
includes heterocyclyl groups that have substituents, including but
not limited to alkyl, halo, amino, hydroxy, cyano, carboxy, nitro,
thio, or alkoxy groups, bonded to one of the ring members. A
heterocyclyl group as defined herein can be a heteroaryl group or a
partially or completely saturated cyclic group including at least
one ring heteroatom. Heterocyclyl groups include, but are not
limited to, pyrrolidinyl, furanyl, tetrahydrofuranyl, dioxolanyl,
piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl,
triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl,
thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl,
indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl,
azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,
imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl,
xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl,
tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.
Heterocyclyl groups can be substituted. Representative substituted
heterocyclyl groups can be mono-substituted or substituted more
than once, including but not limited to, rings containing at least
one heteroatom which are mono, di, tri, tetra, penta, hexa, or
higher-substituted with substituents such as those listed above,
including but not limited to alkyl, halo, amino, hydroxy, cyano,
carboxy, nitro, thio, and alkoxy groups.
[0094] Heteroaryl groups are aromatic ring compounds containing 5
or more ring members, of which, one or more is a heteroatom such
as, but not limited to, N, O, and S. A heteroaryl group designated
as a C.sub.2-heteroaryl can be a 5-membered ring with two carbon
atoms and three heteroatoms, a 6-membered ring with two carbon
atoms and four heteroatoms and so forth. Likewise a
C.sub.4-heteroaryl can be a 5-membered ring with one heteroatom, a
6-membered ring with two heteroatoms, and so forth. The number of
carbon atoms plus the number of heteroatoms sums up to equal the
total number of ring atoms. Heteroaryl groups include, but are not
limited to, groups such as pyrrolyl, pyrazolyl, triazolyl,
tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl,
benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl,
benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl,
benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl,
thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl,
quinolinyl, isoquinolinyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, quinoxalinyl, and quinazolinyl groups. The
terms "heteroaryl" and "heteroaryl groups" include fused ring
compounds such as wherein at least one ring, but not necessarily
all rings, are aromatic, including tetrahydroquinolinyl,
tetrahydroisoquinolinyl, indolyl and 2,3-dihydro indolyl. The term
also includes heteroaryl groups that have other groups bonded to
one of the ring members, including but not limited to alkyl, halo,
amino, hydroxy, cyano, carboxy, nitro, thio, or alkoxy groups.
Representative substituted heteroaryl groups can be substituted one
or more times with groups such as those listed above.
[0095] Additional examples of aryl and heteroaryl groups include
but are not limited to phenyl, biphenyl, indenyl, naphthyl
(1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl,
N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl,
3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl,
3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl,
fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl,
pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl
(1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl
(1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-yl,
1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl),
thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl
(2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl,
4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl,
pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl
(2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl,
7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl,
4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl,
8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl,
3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl,
6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl
(2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl),
4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),
6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl),
benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl,
4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl,
7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl,
(2-(2,3-dihydro-benzo[b]thiophenyl),
3-(2,3-dihydro-benzo[b]thiophenyl),
4-(2,3-dihydro-benzo[b]thiophenyl),
5-(2,3-dihydro-benzo[b]thiophenyl),
6-(2,3-dihydro-benzo[b]thiophenyl),
7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl,
3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole
(1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl,
7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl,
4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl,
7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl,
2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl,
2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl,
6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl,
2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepineo
(5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl,
5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl,
5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine
(10,11-dihydro-5H-dibenz[b,f]azepine-1-yl,
10,11-dihydro-5H-dibenz[b,f]azepine-2-yl,
10,11-dihydro-5H-dibenz[b,f]azepine-3-yl,
10,11-dihydro-5H-dibenz[b,f]azepine-4-yl,
10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.
[0096] Heterocyclylalkyl groups are alkyl groups as defined above
in which a hydrogen or carbon bond of an alkyl group is replaced
with a bond to a heterocyclyl group as defined above.
Representative heterocyclyl alkyl groups include, but are not
limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-2-yl
methyl (.alpha.-picolyl), pyridine-3-yl methyl (.beta.-picolyl),
pyridine-4-yl methyl (.gamma.-picolyl), tetrahydrofuran-2-yl ethyl,
and indol-2-yl propyl. Heterocyclylalkyl groups can be substituted
on the heterocyclyl moiety, the alkyl moiety, or both.
[0097] Heteroarylalkyl groups are alkyl groups as defined above in
which a hydrogen or carbon bond of an alkyl group is replaced with
a bond to a heteroaryl group as defined above. Heteroarylalkyl
groups can be substituted on the heteroaryl moiety, the alkyl
moiety, or both.
[0098] By a "ring system" as the term is used herein is meant a
moiety comprising one, two, three or more rings, which can be
substituted with non-ring groups or with other ring systems, or
both, which can be fully saturated, partially unsaturated, fully
unsaturated, or aromatic, and when the ring system includes more
than a single ring, the rings can be fused, bridging, or
spirocyclic. By "spirocyclic" is meant the class of structures
wherein two rings are fused at a single tetrahedral carbon atom, as
is well known in the art.
[0099] A "monocyclic, bicyclic or polycyclic, aromatic or partially
aromatic ring" as the term is used herein refers to a ring system
including an unsaturated ring possessing 4n+2 pi electrons, or a
partially reduced (hydrogenated) form thereof. The aromatic or
partially aromatic ring can include additional fused, bridged, or
spiro rings that are not themselves aromatic or partially aromatic.
For example, naphthalene and tetrahydronaphthalene are both a
"monocyclic, bicyclic or polycyclic, aromatic or partially aromatic
ring" within the meaning herein. Also, for example, a
benzo-[2.2.2]-bicyclooctane is also a "monocyclic, bicyclic or
polycyclic, aromatic or partially aromatic ring" within the meaning
herein, containing a phenyl ring fused to a bridged bicyclic
system. A fully saturated ring has no double bonds therein, and is
carbocyclic or heterocyclic depending on the presence of
heteroatoms within the meaning herein.
[0100] The term "alkoxy" refers to an oxygen atom connected to an
alkyl group, including a cycloalkyl group, as are defined above.
Examples of linear alkoxy groups include but are not limited to
methoxy, ethoxy, n-propoxy, n-butoxy, n-pentyloxy, n-hexyloxy, and
the like. Examples of branched alkoxy include but are not limited
to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy,
and the like. Examples of cyclic alkoxy include but are not limited
to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy,
and the like.
[0101] The terms "aryloxy" and "arylalkoxy" refer to, respectively,
an aryl group bonded to an oxygen atom and an aralkyl group bonded
to the oxygen atom at the alkyl moiety. Examples include but are
not limited to phenoxy, naphthyloxy, and benzyloxy.
[0102] An "acyl" group as the term is used herein refers to a group
containing a carbonyl moiety wherein the group is bonded via the
carbonyl carbon atom. The carbonyl carbon atom is also bonded to
another carbon atom, which can be part of an alkyl, aryl, aralkyl
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl group or the like. In the special case
wherein the carbonyl carbon atom is bonded to a hydrogen, the group
is a "formyl" group, an acyl group as the term is defined herein.
An acyl group can include 0 to about 12-20 additional carbon atoms
bonded to the carbonyl group. An acyl group can include double or
triple bonds within the meaning herein. An acryloyl group is an
example of an acyl group. An acyl group can also include
heteroatoms within the meaning here. A nicotinoyl group
(pyridyl-3-carbonyl) group is an example of an acyl group within
the meaning herein. Other examples include acetyl, benzoyl,
phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the
like. When the group containing the carbon atom that is bonded to
the carbonyl carbon atom contains a halogen, the group is termed a
"haloacyl" group. An example is a trifluoroacetyl group.
[0103] The term "amine" includes primary, secondary, and tertiary
amines having, e.g., the formula N(group).sub.3 wherein each group
can independently be H or non-H, such as alkyl, aryl, and the like.
Amines include but are not limited to RNH.sub.2, for example,
alkylamines, arylamines, alkylarylamines; R.sub.2NH wherein each R
is independently selected, such as dialkylamines, diarylamines,
aralkylamines, heterocyclylamines and the like; and RN wherein each
R is independently selected, such as trialkylamines,
dialkylarylamines, alkyldiarylamines, triarylamines, and the like.
The term "amine" also includes ammonium ions as used herein.
[0104] An "amino" group is a substituent of the form --NH.sub.2,
--NHR, --NR.sub.2, --NR.sub.3.sup.+, wherein each R is
independently selected, and protonated forms of each. Accordingly,
any compound substituted with an amino group can be viewed as an
amine.
[0105] An "ammonium" ion includes the unsubstituted ammonium ion
NH.sub.4.sup.+, but unless otherwise specified, it also includes
any protonated or quaternarized forms of amines. Thus,
trimethylammonium hydrochloride and tetramethylammonium chloride
are both ammonium ions, and amines, within the meaning herein.
[0106] The term "amide" (or "amido") includes C- and N-amide
groups, i.e., --C(O)NR'R'', and --NR'C(O)R'' groups, respectively.
The R' and R'' of the C-amide may join together to form a
heterocyclic ring with the nitrogen atom. Amide groups therefore
include but are not limited to carbamoyl groups (--C(O)NH.sub.2)
and formamide groups (--NHC(O)H). A "carboxamido" group is a group
of the formula C(O)NR.sub.2, wherein R can be H, alkyl, aryl,
etc.
[0107] The term "urethane" (or "carbamyl") includes N- and
O-urethane groups, i.e., --NRC(O)OR and --OC(O)NR.sub.2 groups,
respectively.
[0108] The term "sulfonamide" (or "sulfonamido") includes S- and
N-sulfonamide groups, i.e., --SO.sub.2NR.sub.2 and --NRSO.sub.2R
groups, respectively. Sulfonamide groups therefore include but are
not limited to sulfamoyl groups (--SO.sub.2NH.sub.2).
[0109] The term "amidine" or "amidino" includes groups of the
formula --C(NR)NR.sub.2. Typically, an amidino group is
--C(NH)NH.sub.2.
[0110] The term "guanidine" or "guanidino" includes groups of the
formula --NRC(NR)NR.sub.2. Typically, a guanidino group is
--NHC(NH)NH.sub.2.
[0111] "Halo," "halogen," and "halide" include fluorine, chlorine,
bromine and iodine.
[0112] The terms "comprising," "including," "having," "composed
of," are open-ended terms as used herein, and do not preclude the
existence of additional elements or components. In a claim element,
use of the forms "comprising," "including," "having," or "composed
of" means that whatever element is comprised, had, included, or
composes is not necessarily the only element encompassed by the
subject of the clause that contains that word.
[0113] A "salt" as is well known in the art includes an organic
compound such as a carboxylic acid, a sulfonic acid, or an amine,
in ionic form, in combination with a counterion. For example, acids
in their anionic form can form salts with cations such as metal
cations, for example sodium, potassium, and the like; with ammonium
salts such as NH.sub.4.sup.+ or the cations of various amines,
including tetraalkyl ammonium salts such as tetramethylammonium and
alkyl ammonium salts such as tromethamine salts, or other cations
such as trimethylsulfonium, and the like. A "pharmaceutically
acceptable" or "pharmacologically acceptable" salt is a salt formed
from an ion that has been approved for human consumption and is
generally non-toxic, such as a chloride salt or a sodium salt. A
"zwitterion" is an internal salt such as can be formed in a
molecule that has at least two ionizable groups, one forming an
anion and the other a cation, which serve to balance each other.
For example, amino acids such as glycine can exist in a
zwitterionic form. A "zwitterion" is a salt within the meaning
herein. The compounds of the present invention may take the form of
salts. The term "salts" embraces addition salts of free acids or
free bases which are compounds of the invention. Salts can be
"pharmaceutically-acceptable salts." The term
"pharmaceutically-acceptable salt" refers to salts which possess
toxicity profiles within a range that affords utility in
pharmaceutical applications. Pharmaceutically unacceptable salts
may nonetheless possess properties such as high crystallinity,
which have utility in the practice of the present invention, such
as for example utility in process of synthesis, purification or
formulation of compounds of the invention.
[0114] Suitable pharmaceutically-acceptable acid addition salts may
be prepared from an inorganic acid or from an organic acid.
Examples of inorganic acids include hydrochloric, hydrobromic,
hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
Appropriate organic acids may be selected from aliphatic,
cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and
sulfonic classes of organic acids, examples of which include
formic, acetic, propionic, succinic, glycolic, gluconic, lactic,
malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric,
pyruvic, aspartic, glutamic, benzoic, anthranilic,
4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),
methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,
trifluoromethanesulfonic, 2-hydroxyethanesulfonic,
p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic,
alginic, .beta.-hydroxybutyric, salicylic, galactaric and
galacturonic acid. Examples of pharmaceutically unacceptable acid
addition salts include, for example, perchlorates and
tetrafluoroborates.
[0115] Suitable pharmaceutically acceptable base addition salts of
compounds of the invention include, for example, metallic salts
including alkali metal, alkaline earth metal and transition metal
salts such as, for example, calcium, magnesium, potassium, sodium
and zinc salts. Pharmaceutically acceptable base addition salts
also include organic salts made from basic amines such as, for
example, N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine. Examples of pharmaceutically unacceptable base addition
salts include lithium salts and cyanate salts. Although
pharmaceutically unacceptable salts are not generally useful as
medicaments, such salts may be useful, for example as intermediates
in the synthesis of compounds, for example in their purification by
recrystallization. All of these salts may be prepared by
conventional means from the corresponding compound by reacting, for
example, the appropriate acid or base with the compound. The term
"pharmaceutically acceptable salts" refers to nontoxic inorganic or
organic acid and/or base addition salts, see, for example, Lit et
al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33,
201-217, incorporated by reference herein
[0116] Nonlimiting examples of potential salts of this invention
include but are not limited to hydrochloride, citrate, glycolate,
fumarate, malate, tartrate, mesylate, esylate, cinnamate,
isethionate, sulfate, phosphate, diphosphate, nitrate,
hydrobromide, hydroiodide, succinate, formate, acetate,
dichloroacetate, lactate, p-toluenesulfonate, pamitate, pidolate,
pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamido
benzoate, glutamate, aspartate, glycolate, adipate, alginate,
ascorbate, besylate, camphorate, camphorsulfonate, camsylate,
caprate, caproate, cyclamate, laurylsulfate, edisylate, gentisate,
galactarate, gluceptate, gluconate, glucuronate, oxoglutarate,
hippurate, lactobionate, malonate, maleate, mandalate, napsylate,
napadisylate, oxalate, oleate, sebacate, stearate, succinate,
thiocyanate, undecylenate, and xinafoate.
[0117] A "hydrate" is a compound that exists in a composition with
water molecules. The composition can include water in stoichiometic
quantities, such as a monohydrate or a dihydrate, or can include
water in random amounts. As the term is used herein a "hydrate"
refers to a solid form, i.e., a compound in water solution, while
it may be hydrated, is not a hydrate as the term is used
herein.
[0118] A "homolog" of a compound of the invention is a compound
having one or more atoms of the compound replaced by an isotope of
such atom. For example, homologs include compounds with deuterium
in place of some hydrogen atoms of the compound such as compounds
of the invention in which the methyl groups of the isopropoxy
moiety of Formulas I-R and I-S are fully or partially deuterated
(e.g., (D.sub.3C).sub.2C--O--). Isotopic substitutions which may be
made in the formation of homologs of the invention include
non-radioactive (stable) atoms such as deuterium and carbon 13, as
well as radioactive (unstable) atoms such as tritium, carbon 14,
iodine 123, iodine 125, etc.
[0119] A "solvate" is a similar composition except that a solvent
other that water replaces the water. For example, methanol or
ethanol can form an "alcoholate", which can again be stoichiometic
or non-stoichiometric. As the term is used herein a "solvate"
refers to a solid form, i.e., a compound in solution in a solvent,
while it may be solvated, is not a solvate as the term is used
herein.
[0120] A "prodrug" as is well known in the art is a substance that
can be administered to a patient where the substance is converted
in vivo by the action of biochemicals within the patient's body,
such as enzymes, to the active pharmaceutical ingredient. Examples
of prodrugs include esters of carboxylic acid groups, which can be
hydrolyzed by endogenous esterases as are found in the bloodstream
of humans and other mammals.
[0121] Any compound which can be converted in vivo to the active
drug by chemical or biochemical transformations functions as a
prodrug. Prodrugs of claimed compounds are covered under this
invention.
[0122] Some examples of prodrugs within the scope of this invention
include: [0123] i. If the compound contains a hydroxyl group, the
hydroxyl group may be modified to form an ester, carbonate, or
carbamate. Examples include acetate, pivalate, methyl and ethyl
carbonates, and dimethylcarbamate. The ester may also be derived
from amino acids such as glycine, serine, or lysine. [0124] ii. If
the compound contains an amine group, the amine group may be
modified to form an amide. Examples include acetamide or
derivatization with amino acids such as glycine, serine, or
lysine.
[0125] Certain compounds of the invention and their salts may exist
in more than one crystal form and the present invention includes
each crystal form and mixtures thereof. In addition, the compounds
of the present invention can exist in unsolvated as well as
solvated forms with pharmaceutically acceptable solvents such as
water to form hydrates or adducts with alcohols such as
C.sub.1-4-alkanols, and the like. Furthermore, compounds of this
invention can be isolated in association with solvent molecules by
crystallization from evaporation of an appropriate solvent. Such
solvents include but are not limited to toluene, tetrahydrofuran,
dioxane, dimethylformamide, acetonitrile, acetates such as methyl
acetate, ethyl acetate, butyl acetate, isobutyl acetate, propyl-
and isopropyl acetate, ethers such as diethyl ether and ethyl
ether, alcohols such as methanol, ethanol, 1- or 2-butanol, 1- or
2-propanol, pentanol, and dimethylsulfoxide. In general, a
depiction for the compound by structure or name is considered to
embrace the compound in any form (e.g., by itself, as a hydrate,
solvate, or otherwise in a mixture).
[0126] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
For example, if X is described as selected from the group
consisting of bromine, chlorine, and iodine, claims for X being
bromine and claims for X being bromine and chlorine are fully
described. Moreover, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any combination of individual members or subgroups of members of
Markush groups. Thus, for example, if X is described as selected
from the group consisting of bromine, chlorine, and iodine, and Y
is described as selected from the group consisting of methyl,
ethyl, and propyl, claims for X being bromine and Y being methyl
are fully described.
Compositions and Combination Treatments
[0127] The S1P.sub.1 compounds, their pharmaceutically acceptable
salts or hydrolyzable esters of the present invention may be
combined with a pharmaceutically acceptable carrier to provide
pharmaceutical compositions useful for treating the biological
conditions or disorders noted herein in mammalian species, and more
preferably, in humans. The particular carrier employed in these
pharmaceutical compositions may vary depending upon the type of
administration desired (e.g. intravenous, oral, topical,
suppository, or parenteral).
[0128] In preparing the compositions in oral liquid dosage forms
(e.g., suspensions, elixirs and solutions), typical pharmaceutical
media, such as water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like can be employed.
Similarly, when preparing oral solid dosage forms (e.g., powders,
tablets and capsules), carriers such as starches, sugars, diluents,
granulating agents, lubricants, binders, disintegrating agents and
the like can be employed.
[0129] Another aspect of an embodiment of the invention provides
compositions of the compounds of the invention, alone or in
combination with another S1P.sub.1 inhibitor or another type of
therapeutic agent, or both. As set forth herein, compounds of the
invention include stereoisomers, tautomers, solvates, hydrates,
salts including pharmaceutically acceptable salts, and mixtures
thereof. Compositions containing a compound of the invention can be
prepared by conventional techniques, e.g. as described in
Remington: The Science and Practice of Pharmacy, 19th Ed., 1995,
incorporated by reference herein. The compositions can appear in
conventional forms, for example capsules, tablets, aerosols,
solutions, suspensions or topical applications.
[0130] Typical compositions include a compound of the invention and
a pharmaceutically acceptable excipient which can be a carrier or a
diluent. For example, the active compound will usually be mixed
with a carrier, or diluted by a carrier, or enclosed within a
carrier which can be in the form of an ampoule, capsule, sachet,
paper, or other container. When the active compound is mixed with a
carrier, or when the carrier serves as a diluent, it can be solid,
semi-solid, or liquid material that acts as a vehicle, excipient,
or medium for the active compound. The active compound can be
adsorbed on a granular solid carrier, for example contained in a
sachet. Some examples of suitable carriers are water, salt
solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated
castor oil, peanut oil, olive oil, gelatin, lactose, terra alba,
sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin,
amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia,
stearic acid or lower alkyl ethers of cellulose, silicic acid,
fatty acids, fatty acid amines, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, polyoxyethylene,
hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the
carrier or diluent can include any sustained release material known
in the art, such as glyceryl monostearate or glyceryl distearate,
alone or mixed with a wax.
[0131] The formulations can be mixed with auxiliary agents which do
not deleteriously react with the active compounds. Such additives
can include wetting agents, emulsifying and suspending agents, salt
for influencing osmotic pressure, buffers and/or coloring
substances preserving agents, sweetening agents or flavoring
agents. The compositions can also be sterilized if desired.
[0132] The route of administration can be any route which
effectively transports the active compound of the invention which
inhibits the enzymatic activity of the focal adhesion kinase to the
appropriate or desired site of action, such as oral, nasal,
pulmonary, buccal, subdermal, intradermal, transdermal or
parenteral, e.g., rectal, depot, subcutaneous, intravenous,
intraurethral, intramuscular, intranasal, ophthalmic solution or an
ointment, the oral route being preferred.
[0133] For parenteral administration, the carrier will typically
comprise sterile water, although other ingredients that aid
solubility or serve as preservatives can also be included.
Furthermore, injectable suspensions can also be prepared, in which
case appropriate liquid carriers, suspending agents and the like
can be employed.
[0134] For topical administration, the compounds of the present
invention can be formulated using bland, moisturizing bases such as
ointments or creams.
[0135] If a solid carrier is used for oral administration, the
preparation can be tabletted, placed in a hard gelatin capsule in
powder or pellet form or it can be in the form of a troche or
lozenge. If a liquid carrier is used, the preparation can be in the
form of a syrup, emulsion, soft gelatin capsule or sterile
injectable liquid such as an aqueous or non-aqueous liquid
suspension or solution.
[0136] Injectable dosage forms generally include aqueous
suspensions or oil suspensions which can be prepared using a
suitable dispersant or wetting agent and a suspending agent
Injectable forms can be in solution phase or in the form of a
suspension, which is prepared with a solvent or diluent. Acceptable
solvents or vehicles include sterilized water, Ringer's solution,
or an isotonic aqueous saline solution. Alternatively, sterile oils
can be employed as solvents or suspending agents. Preferably, the
oil or fatty acid is non-volatile, including natural or synthetic
oils, fatty acids, mono-, di- or tri-glycerides.
[0137] For injection, the formulation can also be a powder suitable
for reconstitution with an appropriate solution as described above.
Examples of these include, but are not limited to, freeze dried,
rotary dried or spray dried powders, amorphous powders, granules,
precipitates, or particulates. For injection, the formulations can
optionally contain stabilizers, pH modifiers, surfactants,
bioavailability modifiers and combinations of these. The compounds
can be formulated for parenteral administration by injection such
as by bolus injection or continuous infusion. A unit dosage form
for injection can be in ampoules or in multi-dose containers.
[0138] The formulations of the invention can be designed to provide
quick, sustained, or delayed release of the active ingredient after
administration to the patient by employing procedures well known in
the art. Thus, the formulations can also be formulated for
controlled release or for slow release.
[0139] Compositions contemplated by the present invention can
include, for example, micelles or liposomes, or some other
encapsulated form, or can be administered in an extended release
form to provide a prolonged storage and/or delivery effect.
Therefore, the formulations can be compressed into pellets or
cylinders and implanted intramuscularly or subcutaneously as depot
injections. Such implants can employ known inert materials such as
silicones and biodegradable polymers, e.g.,
polylactide-polyglycolide. Examples of other biodegradable polymers
include poly(orthoesters) and poly(anhydrides).
[0140] For nasal administration, the preparation can contain a
compound of the invention which inhibits the enzymatic activity of
the focal adhesion kinase, dissolved or suspended in a liquid
carrier, preferably an aqueous carrier, for aerosol application.
The carrier can contain additives such as solubilizing agents,
e.g., propylene glycol, surfactants, absorption enhancers such as
lecithin (phosphatidylcholine) or cyclodextrin, or preservatives
such as parabens.
[0141] For parenteral application, particularly suitable are
injectable solutions or suspensions, preferably aqueous solutions
with the active compound dissolved in polyhydroxylated castor
oil.
[0142] Dosage forms can be administered daily, or more than once a
day, such as twice or thrice daily. Alternatively dosage forms can
be administered less frequently than daily, such as every other
day, or weekly, if found to be advisable by a prescribing
physician.
[0143] An embodiment of the invention also encompasses prodrugs of
a compound of the invention which on administration undergo
chemical conversion by metabolic or other physiological processes
before becoming active pharmacological substances. Conversion by
metabolic or other physiological processes includes without
limitation enzymatic (e.g, specific enzymatically catalyzed) and
non-enzymatic (e.g., general or specific acid or base induced)
chemical transformation of the prodrug into the active
pharmacological substance. In general, such prodrugs will be
functional derivatives of a compound of the invention which are
readily convertible in vivo into a compound of the invention.
Conventional procedures for the selection and preparation of
suitable prodrug derivatives are described, for example, in Design
of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.
[0144] In another embodiment, there are provided methods of making
a composition of a compound described herein including formulating
a compound of the invention with a pharmaceutically acceptable
carrier or diluent. In some embodiments, the pharmaceutically
acceptable carrier or diluent is suitable for oral administration.
In some such embodiments, the methods can further include the step
of formulating the composition into a tablet or capsule. In other
embodiments, the pharmaceutically acceptable carrier or diluent is
suitable for parenteral administration. In some such embodiments,
the methods further include the step of lyophilizing the
composition to form a lyophilized preparation.
[0145] The compounds of the invention can be used therapeutically
in combination with i) one or more other S1P.sub.1 inhibitors
and/or ii) one or more other types of protein kinase inhibitors
and/or one or more other types of therapeutic agents which can be
administered orally in the same dosage form, in a separate oral
dosage form (e.g., sequentially or non-sequentially) or by
injection together or separately (e.g., sequentially or
non-sequentially).
[0146] Accordingly, in another embodiment the invention provides
combinations, comprising: [0147] a) a compound of the invention as
described herein; and [0148] b) one or more compounds comprising:
[0149] i) other compounds of the present invention, [0150] ii)
other medicaments adapted for treatment of a malcondition for which
activation of S1P.sub.1 is medically indicated, for example
multiple sclerosis, transplant rejection, or adult respiratory
distress syndrome.
[0151] Combinations of the invention include mixtures of compounds
from (a) and (b) in a single formulation and compounds from (a) and
(b) as separate formulations. Some combinations of the invention
can be packaged as separate formulations in a kit. In some
embodiments, two or more compounds from (b) are formulated together
while a compound of the invention is formulated separately.
[0152] The dosages and formulations for the other agents to be
employed, where applicable, will be as set out in the latest
edition of the Physicians' Desk Reference, incorporated herein by
reference.
Methods of Treatment
[0153] In certain embodiments, the present invention encompasses
orally bioavailable compounds that specifically agonize S1P.sub.1
without binding (S1P.sub.2, S1P.sub.3 and S1P.sub.4), or having
significant specificity over (S1P.sub.5), other EDG receptors. A
selective S1P.sub.1 agonist can be used to treat diseases with an
autoimmune, hyperactive immune-response, angiogenesis or
inflammatory components, but would not be limited to such
conditions. Selective S1P.sub.1 agonists have advantages over
current therapies by increasing the therapeutic window because of
reduced toxicity due to engagement of other EDG receptors.
[0154] In certain embodiments, the present invention encompasses
compounds that bind with high affinity and specificity to the
S1P.sub.1 receptor in an agonist manner. Upon ligation of the
S1P.sub.1 receptor with agonist, signaling proceeds through
G.sub..alpha.i, inhibiting the generation of cAMP by adenylate
cyclase.
[0155] In certain embodiments, the present invention provides a
method for activating or agonizing (i.e., to have an agonic effect,
to act as an agonist) a sphingosine-1-phosphate receptor subtype,
such as S1P.sub.1, with a compound of the invention. The method
involves contacting the receptor with a suitable concentration of
an inventive compound to bring about activation of the receptor.
The contacting can take place in vitro, for example in carrying out
an assay to determine the S1P receptor activation activity of an
inventive compound undergoing experimentation related to a
submission for regulatory approval.
[0156] In certain embodiments, the method for activating an S1P
receptor, such as S1P.sub.1, can also be carried out in vivo, that
is, within the living body of a mammal, such as a human patient or
a test animal. The inventive compound can be supplied to the living
organism via one of the routes as described above, e.g., orally, or
can be provided locally within the body tissues, for example by
injection of a tumor within the organism. In the presence of the
inventive compound, activation of the receptor takes place, and the
effect thereof can be studied.
[0157] An embodiment of the present invention provides a method of
treatment of a malcondition in a patient for which activation of an
S1P receptor, such as S1P.sub.1, is medically indicated, wherein
the patient is administered the inventive compound in a dosage, at
a frequency, and for a duration to produce a beneficial effect on
the patient. The inventive compound can be administered by any
suitable means, examples of which are described above.
Preparation of Certain Embodiments
##STR00029##
[0159] The (S)-enantiomer was prepared in the same manner outlined
in Scheme 1 using RuCl(p-cymene)[(S,S)-Ts-DPEN] in step (ii).
Racemic material can be prepared in the same manner outlined in
Scheme 1 using NaBH.sub.4 in (ii).
##STR00030##
[0160] The (R)-enantiomer was prepared in the same manner outlined
in Scheme 2 starting from
(S)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile.
##STR00031##
##STR00032##
##STR00033##
##STR00034##
[0161] The (S)-enantiomer can be prepared using
(S)-2-methylpropane-2-sulfinamide in step (i).
##STR00035##
##STR00036##
##STR00037##
[0162] The (S)-enantiomer can be prepared using protected
(R)-1-amino-N-hydroxy-2,3-dihydro-1H-indene-4-carboximidamide in
step (i).
##STR00038##
##STR00039##
##STR00040##
##STR00041##
##STR00042##
EXAMPLES
General Methods
[0163] .sup.1H NMR (400 MHz) and .sup.13C NMR (100 MHz) were
obtained in solution of deuteriochloroform (CDCl.sub.3),
deuteriomethanol (CD.sub.3OD) or dimethyl sulfoxide--D.sub.6
(DMSO). NMR spectra were processed using Mestrec 5.3.0 and 6.0.1.
.sup.13C NMR peaks that are bracketed are two rotomers of the same
carbon. Mass spectra (LCMS) were obtained using an Agilent
1100/6110 HPLC system equipped with a Thompson ODS-A, 100A, 5.mu.
(50.times.4.6 mm) column using water with 0.1% formic acid as the
mobile phase A, and acetonitrile with 0.1% formic acid as the
mobile phase B. The gradient was 20-100% with mobile phase B over
2.5 min then held at 100% for 2.5 mins. The flow rate was 1 mL/min.
Unless otherwise indicated, the LCMS data provided uses this
method. For more hydrophobic compounds, the following gradient was
used, denoted as Method 1: 40-95% over 0.5 min, hold at 95% for 8.5
min, with a flow rate of 1 mL/min. Final compounds were checked for
purity using Method 2: 5% for 1 min, 5-95% over 9 min, then hold at
95% for 5 min, with a flow rate of 1 mL/min. Enantiomeric excess
was determined by integration of peaks that were separated on a
Chiralpak AD-H, 250.times.4.6 mm column at a flow rate of 1 mL/min
and an isocratic mobile phase. Unless otherwise indicated, the
chiral data provided uses this method. Alternatively, chiral
separations were performed under the following conditions, denoted
as Chiral Method 1: Chiralpak AY-H, 250.times.4.6 mm column at a
flow rate of 1 mL/min and an isocratic mobile phase. Chiral Method
2: Chiralcel OZ-3, 150.times.4.6 mm column at a flow rate of 0.75
ml/min and an isocratic mobile phase. The pyridine, dichloromethane
(DCM), tetrahydrofuran (THF), and toluene used in the procedures
were from Aldrich Sure-Seal bottles kept under nitrogen (N.sub.2).
All reactions were stirred magnetically and temperatures are
external reaction temperatures. Chromatographies were carried out
using a Combiflash Rf flash purification system (Teledyne Isco)
equipped with Redisep (Teledyne Isco) silica gel (SiO.sub.2)
columns. Preparative HPLC purifications were done on Varian
ProStar/PrepStar system using water containing 0.05%
trifluoroacetic acid as mobile phase A, and acetonitrile with 0.05%
trifluoroacetic acid as mobile phase B. The gradient was 10-80%
with mobile phase B over 12 min, hold at 80% for 2 min, and then
return to 10% over 2 min with flow rate of 22 mL/min. Other methods
similar to this may have been employed. Fractions were collected
using a Varian Prostar fraction collector and were evaporated using
a Savant SpeedVac Plus vacuum pump. Compounds with salt-able
centers were presumed to be the trifluoroacetic acid (TFA) salt.
Microwave heating was performed using a Biotage Initiator microwave
reactor equipped with Biotage microwave vessels. The following
abbreviations are used: ethyl acetate (EA), triethylamine (TEA),
diethyl amine (DEA), diispropyl ethyl amine (DIEA),
hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC), isopropanol (IPA),
dimethylformamide (DMF), dimethyl acetamide (DMA). Norit is
activated charcoal.
Experimental Procedures
5-oxo-5,6,7,8-tetrahydronaphthalene-1-carbonitrile (INT-1)
##STR00043##
[0165] To a stirred solution of
5-bromo-3,4-dihydronaphthalen-1(2H)-one (9.95 g, 44.2 mmol) in NMP
(50 mL) was added Zn(CN).sub.2 (10.38 g, 88.4 mmol). The mixture
was degassed twice by bubbling N.sub.2 through the solution for 30
min then evacuated. Pd(Ph.sub.3).sub.4 (0.5 g, 0.44 mmol) was added
and the mixture was heated to 110.degree. C. under N.sub.2. After 5
h, the mixture was cooled to room temperature and poured onto ice
(600 mL), using water (300 mL) to complete the transfer. After the
ice had melted, the solution was filtered and the resulting solid
was collected, suspended in DCM, and filtered again. The solid was
collected, washed with water, and purified by column chromatography
(EA/hex) to provide 6.9 g (91%) of
5-oxo-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-1 as a white
solid. LCMS-ESI (m/z) calculated for C.sub.11H.sub.9NO: 171.2;
found 172.1 [M+H].sup.+, t.sub.R=2.95 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.26 (dd, J=7.9, 1.4 Hz, 1H), 7.82 (dd, J=7.6,
1.4 Hz, 1H), 7.44 (t, J=7.8 Hz, 1H), 3.20 (t, J=6.1 Hz, 2H), 2.72
(dd, J=7.2, 6.1 Hz, 2H), 2.30-2.17 (m, 2H). .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 196.22, 147.39, 137.18, 133.39, 131.59, 127.19,
116.93, 112.94, 38.48, 28.05, 22.28.
(R)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
(INT-2)
##STR00044##
[0167] To a stirred solution of
5-oxo-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-1 (3.0 g,
17.5 mmol) in 5:1 HCO.sub.2:NEt.sub.3 (24 mL) was added
RuCl(p-cymene)[(R,R)-Ts-DPEN] (0.13 g, 0.26 mmol). The mixture was
stirred at 30.degree. C. for 15 h then partitioned between EA and
H.sub.2O. The combined organic layers were dried over
Na.sub.2SO.sub.4 and chromatographed (EA/hex) to provide 2.99 g
(99%) of (R)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
INT-2 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.11H.sub.11NO: 173.2; found 174.1 [M+H].sup.+, 156.1
[M-NH.sub.4].sup.+, t.sub.R=2.60 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.71 (d, J=7.8 Hz, 1H), 7.54 (dt, J=8.7, 4.4
Hz, 1H), 7.34-7.26 (m, 1H), 4.85-4.71 (m, 2H), 3.48 (s, 1H),
3.13-2.96 (m, 1H), 2.90 (ddd, J=17.7, 7.8, 5.6 Hz, 1H), 2.15-1.95
(m, 2H), 1.97-1.76 (m, 2H). Chiral HPLC:
(R)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile was
eluted with 5% IPA/hexane: 99.1% ee, t.sub.R=15.3 min.
[0168] (S)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
INT-3 was prepared in an analogous fashion using INT-1 and
RuCl(p-cymene)[(S,S)-Ts-DPEN]. Chiral HPLC: 99.4% ee, t.sub.R for
the (S)-enantiomer=17.99 min.
[0169] General Procedure 1. Preparation of Amide Oximes
[0170] To (R)- or (S)-cyanides (1 eq) in EtOH (0.56 M) was added
hydroxylamine hydrochloride (3 eq) and either NaHCO.sub.3 or TEA (3
eq) and the reaction mixture heated at 85.degree. C. for 1-2 h. The
organic soluble amide oximes were isolated by removal of the
solvent and partitioning between water and DCM. The water soluble
amide oximes were chromatographed or used directly in the
cyclization. Pure amide oximes can be obtained by recrystallization
from alcoholic solvents.
(R)--N,5-dihydroxy-5,6,7,8-tetrahydronaphthalene-1-carboximidamide
(INT-4)
##STR00045##
[0172] Prepared using General Procedure 1. To a stirring solution
of (R)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-2
(79.1 mg, 0.46 mmol) in EtOH (2 mL) was added hydroxylamine
hydrochloride (34.9 mg, 0.50 mmol) and sodium bicarbonate (42.2 mg,
0.50 mmol). The mixture was heated at 70.degree. C. for 18 h. The
product was purified by chromatography (MeOH/DCM) to provide 27.3
mg (29%)
(R)--N,5-dihydroxy-5,6,7,8-tetrahydronaphthalene-1-carboximidamide
INT-4 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.11H.sub.11NO: 173.2; found 174.1 [M+H].sup.+, 156.1
[M-NH.sub.4].sup.+, t.sub.R=2.60
min.(S)--N,5-dihydroxy-5,6,7,8-tetrahydronaphthalene-1-carboximidamide
INT-5 was prepared in an analogous fashion from
(S)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
INT-3.
[0173] General Procedure 2. Cyclization to Oxadiazole Amines
[0174] A solution of the appropriate acid (1 eq), HOBt (1.3 eq),
and EDC (1.3 eq) in DMF (0.08
[0175] M in acid) was stirred at room temperature under an
atmosphere of N.sub.2. After the complete formation of the
HOBt-acid complex (1-3 h), the (R)- or (S)-amide oxime (1.1 eq) was
added to the mixture. After complete formation of the coupled
intermediate (ca. 0.5-2 h), the mixture was heated to 75-95.degree.
C. until the cyclization was complete (8-12 h). The reaction
mixture was diluted with saturated NaHCO.sub.3 and extracted with
EA. The combined organic extracts were dried, concentrated, and
could be purified by chromatography (EA/hexanes), preparative HPLC
or recrystallization.
(R)-5-(3-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-y-
l)-2-isopropoxybenzonitrile (Compound I)
##STR00046##
[0177] Prepared using General Procedure 2. To a stirring solution
of 3-cyano-4-isopropoxybenzoic acid (16.7 mg, 0.08 mmol) in DMF (1
mL) were added HOBt (14.3 mg, 0.11 mmol) and EDCI (20.3 mg, 0.11
mmol). After stirring for 30 min,
(R)--N,5-dihydroxy-5,6,7,8-tetrahydronaphthalene-1-carboximidamide
INT-4 (27.3 mg, 0.09 mmol) was added as a solution in DMF (1.5 mL).
After stirring at room temperature for an additional 60 min, the
mixture was heated to 90.degree. C. for 15 h. The mixture was
diluted with EA and washed with NaHCO.sub.3. The combined organic
layers were dried, concentrated, chromatographed (EA/hexanes) to
provide 12.72 mg (42.4%)
(R)-5-(3-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5--
yl)-2-isopropoxybenzonitrile 1 as a white solid. LCMS-ESI (m/z)
calculated for C.sub.22H.sub.21N.sub.3O.sub.3: 375.4; found 376.1
[M+H].sup.+, t.sub.R=3.73 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.42 (d, J=2.2 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H), 7.97
(dd, J=7.7, 1.3 Hz, 1H), 7.66 (d, J=7.2 Hz, 1H), 7.38 (t, J=7.7 Hz,
1H), 7.12 (d, J=9.0 Hz, 1H), 4.91-4.83 (m, 1H), 4.79 (dq, J=12.0,
6.0 Hz, 1H), 3.20 (dt, J=17.8, 5.4 Hz, 1H), 3.01 (dt, J=13.3, 6.4
Hz, 1H), 2.13-1.81 (m, 4H), 1.79 (d, J=7.2 Hz, 1H), 1.47 (d, J=5.6
Hz, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 172.70, 169.48,
162.75, 140.10, 137.4, 134.13, 133.88, 131.68, 129.96, 126.18,
125.97, 116.82, 115.26, 113.54, 103.95, 72.73, 68.47, 31.62, 28.50,
21.73, 18.57. Chiral HPLC:
(R)-5-(3-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5--
yl)-2-isopropoxybenzonitrile was eluted with 10% IPA/hexane: 99.4%
ee, t.sub.R=40.85 min.
[0178]
(S)-5-(3-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadia-
zol-5-yl)-2-isopropoxybenzonitrile 2 was prepared in an analogous
fashion from
(S)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-5.
Chiral HPLC: 99.1% ee, t.sub.R for the (S)-enantiomer=38.19
min.
(S)-5-azido-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
(INT-6)
##STR00047##
[0180] A stirring solution of
(R)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-2
(3.00 g, 17.32 mmol) in toluene (16 mL) under N.sub.2, was cooled
to 0.degree. C. DPPA (9.53 g, 34.64 mmol) was added, followed by
dropwise addition of DBU (3.16 mL, 20.78 mmol) over 20 min. The
mixture was stirred at 0.degree. C. for 4 h then slowly warmed to
room temperature over 2 h and then concentrated. The resulting
crude mixture was diluted with EA and washed with NaHCO.sub.3. The
combined organic layers were washed with brine, dried over
Na.sub.2SO.sub.4, and chromatographed (EA/hexane) to provide 2.49 g
(72.6%) of (S)-5-azido-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
INT-6 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.11H.sub.10N.sub.4: 198.2; found 156.1 [M-N.sub.3].sup.+,
t.sub.R=3.65 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.60
(dd, J=7.6, 1.2 Hz, 1H), 7.56 (dd, J=7.8, 0.6 Hz, 1H), 7.33 (t,
J=7.7 Hz, 1H), 4.59 (t, J=4.4 Hz, 1H), 3.08 (dt, J=18.0, 5.1 Hz,
1H), 2.99-2.83 (m, 1H), 2.15-1.96 (m, 3H), 2.00-1.81 (m, 1H).
.sup.13C NMR (101 MHz, DMSO) .delta. 141.05, 135.58, 133.47,
132.66, 126.58, 117.43, 113.21, 58.74, 28.28, 27.54, 18.27.
[0181] (R)-5-azido-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
INT-7 was prepared in an analogous fashion from
(S)-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carbonitrile
INT-3.
(S)-tert-butyl (5-cyano-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
(INT-8)
##STR00048##
[0183] To a stirring solution of
(S)-5-azido-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-6 (3.1
g, 15.63 mmol) in MeOH (50 mL) were added 10% Pd/C (500 mg),
(Boc).sub.2O (6.83 g, 31.27 mmol) and Et.sub.3N (3.16 g, 31.27
mmol). The reaction mixture was purged and flushed with H.sub.2
(3.times.) and stirred under H.sub.2. After 3 h the mixture was
filtered through celite, rinsing with MeOH. The MeOH filtrate was
concentrated, dissolved in EA and washed with NaHCO.sub.3 and
brine. The organic layer was dried (MgSO.sub.4), concentrated and
chromatographed (EA/hexanes). The resulting material was
crystallized from hexanes to provide 3.45 g (81%) of (S)-tert-butyl
(5-cyano-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate INT-8 as a
white solid. LCMS-ESI (m/z) calculated for
C.sub.16H.sub.20N.sub.2O.sub.2: 272.34; found 156.1
[M-NHBoc].sup.+, t.sub.R=3.77 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.60 (d, J=7.8 Hz, 1H), 7.53 (d, J=7.5 Hz, 1H),
7.31-7.20 (m, 1H), 4.86 (s, 1H), 4.75 (d, J=8.9 Hz, 1H), 3.06-2.85
(m, 2H), 2.07 (dt, J=11.3, 5.1 Hz, 1H), 1.91 (s, 2H), 1.86-1.71 (m,
1H), 1.48 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) .delta.
155.38, 140.70, 139.12, 133.03, 131.63, 126.41, 117.62, 112.42,
79.62, 48.25, 29.75, 28.28, 27.77, 19.36. Chiral HPLC:
(S)-tert-butyl (5-cyano-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
was eluted with 2.5% EtOH/hexanes: 92.4% ee, t.sub.R=14.22 min.
[0184] (R)-tert-butyl
(5-cyano-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate INT-9 was
prepared in an analogous fashion from
(R)-5-azido-5,6,7,8-tetrahydronaphthalene-1-carbonitrile INT-7.
Chiral HPLC: 99.6% ee, t.sub.R for the (R)-enantiomer=11.60
min.
(S)-tert-butyl
(5-(N-hydroxycarbamimidoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
(INT-10)
##STR00049##
[0186] Prepared using General Procedure 1. To a stirring solution
of (S)-tert-butyl
(5-cyano-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate INT-8 (3.10 g,
11.38 mmol) in EtOH (25 mL) was added hydroxylamine hydrochloride
(2.77 g, 39.84 mmol) and NEt.sub.3 (3.17 mL, 22.77 mmol). After
heating at 85.degree. C. for 15 h, the mixture was concentrated,
redissolved in DCM, and washed with NaHCO.sub.3. The combined
organic layers were dried, concentrated and chromatographed
(MeOH/DCM) to provide 3.56 g crude (S)-tert-butyl
(5-(N-hydroxycarbamimidoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
INT-10 (58% product by UV area), which was used in the next
reaction without further purification. LCMS-ESI (m/z) calculated
for C.sub.16H.sub.23N.sub.3O.sub.3: 305.37; found 306.2
[M+H].sup.+, t.sub.R=2.00 min.
[0187] (R)-tert-butyl
(5-(N-hydroxycarbamimidoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
INT-11 was prepared in an analogous fashion from (R)-tert-butyl
(5-cyano-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate INT-9.
(S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)carbamate (INT-12)
##STR00050##
[0189] Prepared using General Procedure 2. To a stirring solution
of 3-cyano-4-isopropoxybenzoic acid (556 mg, 2.72 mmol) in DMF (10
mL) was added HOBt (476.6 mg, 3.53 mmol) and EDCI (677.9 mg, 3.53
mmol). After stirring for 30 min, (S)-tert-butyl
(5-(N-hydroxycarbamimidoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
INT-10 (3.56 g crude, approximately 2.99 mmol) was added. After
stirring at room temperature for an additional 90 min, the mixture
was heated to 90.degree. C. for 15 h. The mixture was diluted with
EA and washed with NaHCO.sub.3. The combined organic layers were
dried, concentrated, and chromatographed (EA/hexanes) to provide
1.89 g (46%) (S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)carbamate INT-12 as a white solid. LCMS-ESI (m/z)
calculated for C.sub.27H.sub.30N.sub.4O.sub.4: 474.6; no M/Z
observed, t.sub.R=4.23 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.42 (d, J=2.1 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H), 7.92
(dd, J=7.7, 1.1 Hz, 1H), 7.56 (d, J=7.6 Hz, 1H), 7.32 (dd, J=20.3,
12.6 Hz, 1H), 7.12 (d, J=9.0 Hz, 1H), 4.94 (d, J=6.0 Hz, 1H),
4.88-4.72 (m, 1H), 3.23-3.08 (m, 1H), 3.07-2.94 (m, 1H), 2.06 (d,
J=12.6 Hz, 1H), 1.97-1.78 (m, 3H), 1.53-1.43 (m, 15H). .sup.13C NMR
(101 MHz, CDCl.sub.3) .delta. 172.64, 169.40, 162.69, 155.43,
138.65, 137.55, 134.04, 133.83, 131.68, 129.55, 126.08, 125.97,
116.74, 115.20, 113.53, 103.87, 79.44, 72.69, 48.97, 29.73, 28.40,
21.68, 19.71, 14.14.
[0190] (R)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl-1,2,3,4-tetrahydr-
onaphthalen-1-yl)carbamate INT-13 was prepared in an analogous
fashion from (R)-tert-butyl
(5-(N-hydroxycarbamimidoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
INT-11.
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)-
-2-isopropoxybenzonitrile (Compound 4)
##STR00051##
[0192] To a stirring solution of (S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)carbamate INT-12 (0.90 g, 1.9 mmol) in dioxanes
(10 mL) was added 4N HCl/dioxanes (2.5 mL). After stirring at
60.degree. C. for 5.5 h, the mixture was concentrated to provide
0.8 g (100%) of the HCl salt of
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadia-
zol-5-yl)-2-isopropoxybenzonitrile 4 as a white solid. An
analytically pure sample was purified by preparative HPLC and the
free amine was prepared by partitioning between NaHCO.sub.3 and EA.
LCMS-ESI (m/z) calculated for C.sub.22H.sub.22N.sub.4O.sub.2:
374.4; found 358.1 [M-NH.sub.2].sup.+, t.sub.R=2.45 min. .sup.1H
NMR (400 MHz, DMSO) .delta. 8.63 (s, 2H), 8.50 (d, J=2.2 Hz, 1H),
8.39 (dd, J=9.0, 2.3 Hz, 1H), 7.95 (dd, J=7.7, 1.0 Hz, 1H), 7.85
(d, J=7.5 Hz, 1H), 7.56 (d, J=9.2 Hz, 1H), 7.49 (t, J=7.7 Hz, 1H),
4.98 (hept, J=6.0 Hz, 1H), 4.55 (t, J=5.3 Hz, 1H), 3.11 (dt,
J=17.9, 5.5 Hz, 1H), 2.94 (dt, J=13.9, 6.1 Hz, 1H), 2.18-1.88 (m,
3H), 1.88-1.71 (m, 1H), 1.38 (d, J=6.0 Hz, 6H). .sup.13C NMR (101
MHz, DMSO) .delta. 172.91, 168.60, 162.52, 137.73, 134.60, 134.09,
133.80, 132.08, 130.30, 126.29, 126.06, 115.92, 115.24, 114.93,
102.49, 72.54, 66.34, 48.02, 27.57, 26.54, 21.47, 17.68. Chiral
HPLC:
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile was eluted with 8% EtOH/hexanes, with
0.3% DEA (Chiral Method 1): 94.2% ee, t.sub.R=42.7 min.
[0193]
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazo-
l-5-yl)-2-isopropoxybenzonitrile 3 was prepared in an analogous
fashion from (R)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl) carbamate INT-13. Chiral HPLC (Chiral Method 1):
99.9% ee, t.sub.R for the (R)-enantiomer=39.72 min.
[0194] General Procedure 3. Preparation of Tetrahydronaphthalene
Ureas
[0195] To a stirring solution of CDI (1.2 eq) in DCM (0.16M) were
added either the solution of (R)- or (S)-tetrahydronapthalene amine
(1 eq) and Et.sub.3N (3 eq) in DCM (0.01M). After stirring for 15
h, this solution was added to a second solution containing the
appropriate amine (3 eq) and Et.sub.3N (3 eq) in DCM (0.4M), at
room temperature. The resulting mixture was stirred at room
temperature for 4 h until all of starting material was consumed.
The solvent was evaporated and the pure product isolated after
preparative HPLC.
[0196] Compounds 5-20 were prepared using General Procedure 3.
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)-3-hydroxyazetidine-1-carboxamide (Compound
8)
##STR00052##
[0198] Prepared using General Procedure 3: LCMS-ESI (m/z)
calculated for C.sub.26H.sub.27N.sub.5O.sub.4: 473.5; found 474.2
[M+H].sup.+, t.sub.R=3.21 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.41 (d, J=2.2 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H), 7.91
(dd, J=7.7, 1.2 Hz, 1H), 7.53 (t, J=6.1 Hz, 1H), 7.32 (dd, J=20.1,
12.4 Hz, 1H), 7.12 (d, J=9.0 Hz, 1H), 5.13 (d, J=8.0 Hz, 1H), 4.79
(dt, J=12.2, 6.1 Hz, 1H), 4.68 (tt, J=6.7, 4.4 Hz, 1H), 4.35 (d,
J=8.7 Hz, 1H), 4.25-4.14 (m, 2H), 3.85 (dd, J=8.8, 4.1 Hz, 2H),
3.14 (t, J=12.1 Hz, 1H), 3.10-2.92 (m, 1H), 2.16-1.96 (m, 1H),
1.99-1.64 (m, 4H), 1.48 (d, J=6.1 Hz, 6H). Chiral HPLC:
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)-3-hydroxyaz etidine-1-carboxamide
was eluted with 15% water/MeOH, (Chiral Method 2): 91.4% ee,
t.sub.R=15.52 min.
[0199]
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)-3-hydroxyazetidine-1-carboxamide 7
was prepared in an analogous fashion. Chiral HPLC: 99.94% ee,
t.sub.R=17.17 min (Chiral Method 2).
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)pyrrolidine-1-carboxamide (Compound 12)
##STR00053##
[0201] Prepared using General Procedure 3: LCMS-ESI (m/z)
calculated for C.sub.27H.sub.29N.sub.5O.sub.3: 471.55; found 472.2
[M+H].sup.+, t.sub.R=3.78 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.41 (d, J=2.1 Hz, 1H), 8.32 (dd, J=8.9, 2.2 Hz, 1H), 7.90
(dd, J=7.6, 1.1 Hz, 1H), 7.59 (d, J=7.6 Hz, 1H), 7.33 (t, J=7.7 Hz,
1H), 7.12 (d, J=9.0 Hz, 1H), 5.25-5.12 (m, 1H), 4.79 (hept, J=6.0
Hz, 1H), 4.45 (t, J=21.8 Hz, 1H), 3.36 (t, J=6.4 Hz, 4H), 3.23-3.09
(m, 1H), 3.02 (dt, J=18.0, 6.0 Hz, 1H), 2.16-1.99 (m, 1H),
2.01-1.79 (m, 7H), 1.47 (d, J=6.1 Hz, 6H).
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)morpholine-4-carboxamide (Compound 15)
##STR00054##
[0203] Prepared using General Procedure 3: LCMS-ESI (m/z)
calculated for C.sub.27H.sub.29N.sub.5O.sub.4: 487.5; found 488.2
[M+H].sup.+, t.sub.R=3.58 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.40 (d, J=2.2 Hz, 1H), 8.32 (dd, J=8.9, 2.2 Hz, 1H), 7.91
(dd, J=7.7, 1.1 Hz, 1H), 7.55 (t, J=10.0 Hz, 1H), 7.33 (t, J=7.7
Hz, 1H), 7.12 (d, J=9.0 Hz, 1H), 5.19 (dd, J=12.9, 5.3 Hz, 1H),
4.86-4.75 (m, 1H), 4.72 (d, J=8.1 Hz, 1H), 3.76-3.63 (m, 4H), 3.36
(dd, J=11.6, 7.1 Hz, 4H), 3.16 (dt, J=16.7, 5.4 Hz, 1H), 3.02 (dt,
J=12.6, 5.9 Hz, 1H), 2.14-1.98 (m, 2H), 1.94-1.81 (m, 2H), 1.47 (d,
J=6.1 Hz, 6H).
(R)--N--((R)-5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3-
,4-tetrahydronaphthalen-1-yl)-3-(dimethylamino)pyrrolidine-1-carboxamide
(Compound 20)
##STR00055##
[0205] Prepared using General Procedure 3: To a stirring solution
of CDI ((9.5 mg, 0.06 mmol) in DCM (1 mL) were added dropwise a
solution of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile HCl salt 3 (20 mg, 0.05 mmol) and
Et.sub.3N (20.3 .mu.L, 0.15 mmol) in DCM (1 mL). After stirring for
15 h at room temperature, this solution was added dropwise to
another solution containing (R)-3-dimethylaminopyrrolidine (18.6
mg, 0.15 mmol)) in DCM (1 mL) at room temperature. The reaction was
stirred at room temperature for 6.5 h. The solvent was evaporated
and the pure product was isolated by preparative HPLC to afford
17.5 mg (70%) of
(R)--N--((R)-5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,-
3,4-tetrahydronaphthalen-1-yl)-3-(dimethylamino)
pyrrolidine-1-carboxamide 20. LCMS-ESI (m/z) calculated for
C.sub.29H.sub.34N.sub.6O.sub.3: 514.6; found 515.3 [M+H].sup.+,
t.sub.R=2.56 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.39
(d, J=2.2 Hz, 1H), 8.31 (dt, J=8.7, 4.3 Hz, 1H), 7.91 (d, J=7.6 Hz,
1H), 7.52 (d, J=7.6 Hz, 1H), 7.34 (t, J=7.7 Hz, 1H), 7.12 (d, J=9.2
Hz, 1H), 5.13 (d, J=6.9 Hz, 1H), 4.86-4.73 (m, 1H), 4.64 (d, J=8.2
Hz, 1H), 3.91 (dd, J=10.4, 7.3 Hz, 1H), 3.80-3.56 (m, 3H), 3.41
(dd, J=17.5, 8.3 Hz, 1H), 3.15 (d, J=18.0 Hz, 1H), 3.10-2.93 (m,
1H), 2.85 (s, 6H), 2.46 (m, 2H), 2.05 (dd, J=9.2, 4.8 Hz, 1H), 1.88
(m, 3H), 1.45 (dd, J=13.9, 6.1 Hz, 6H).
[0206] General Procedure 4. Preparation of Tetrahydronapthalene
Amides via Acid Chlorides
[0207] To a stirring solution of (R)- or (S)-tetrahydronapthalene
amine HCl (1 eq) in DCM were added an acid chloride (2 eq) and
NEt.sub.3 (2 eq). The reaction was stirred at room temperature for
1 h. The solvent was evaporated and mixture was purified by
preparative HPLC.
[0208] Compounds 21-25 were prepared using General Procedure 4.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)acetamide (Compound 21)
##STR00056##
[0210] Prepared using General Procedure 4: To a stirring solution
of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile HCl 3 (20 mg, 0.05 mmol) in DCM (0.5 mL)
were added acetyl chloride (7 .mu.L, 0.10 mmol) and NEt.sub.3 (14
.mu.L, 0.10 mmol). After stirring for 1 h, the solvent was
evaporated and the residue was purified by preparative HPLC to
provide 11.3 mg (56%) of
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)acetamide 21. LCMS-ESI (m/z) calculated
for C.sub.24H.sub.24N.sub.4O.sub.3: 416.5; found 417.2 [M+H].sup.+,
t.sub.R=3.56 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.40
(d, J=2.2 Hz, 1H), 8.32 (dd, J=8.9, 2.2 Hz, 1H), 7.92 (dd, J=7.7,
1.2 Hz, 1H), 7.48 (d, J=7.6 Hz, 1H), 7.33 (t, J=7.7 Hz, 1H), 7.12
(d, J=9.0 Hz, 1H), 5.83 (d, J=8.6 Hz, 1H), 5.36-5.21 (m, 1H), 4.79
(hept, J=6.0 Hz, 1H), 3.16 (dt, J=17.9, 6.0 Hz, 1H), 3.03 (dt,
J=18.2, 6.3 Hz, 1H), 2.10-1.99 (m, 4H), 1.97-1.79 (m, 3H), 1.47 (d,
J=6.1 Hz, 6H).
[0211]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)acetamide 22 was prepared in an
analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile HCl-4.
[0212] General Procedure 5. Preparation of Tetrahydronapthalene
Sulfamides
[0213] To a solution of (R)- or (S)-tetrahydronapthalene amine HCl
(1 eq) in dioxane were added sulfamide (5 eq) and DIEA (3 eq). The
reaction was stirred at 110.degree. C. for 18 h. The solvent was
evaporated and mixture was purified by preparative HPLC.
[0214] Compounds 26 and 27 were prepared using General Procedure
5.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)sulfamide (Compound 26)
##STR00057##
[0216] Prepared using General Procedure 5: To a stirring solution
of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile HCl 3 (50 mg, 0.12 mmol) in dioxane (3
mL) were added sulfamide (58 mg, 0.61 mmol) and DIEA (47.2 .mu.L,
0.37 mmol) and the mixture was heated to 110.degree. C. for 14 h.
The solvent was evaporated and the residue was purified by
preparative HPLC to provide 22.8 mg (42%) of
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)sulfamide 26. LCMS-ESI (m/z) calculated
for C.sub.22H.sub.23N.sub.5O.sub.4S: 453.5; found 454.1
[M+H].sup.+, t.sub.R=3.47 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.41 (d, J=2.2 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H), 7.97
(dd, J=7.7, 1.2 Hz, 1H), 7.72 (d, J=7.7 Hz, 1H), 7.38 (t, J=7.8 Hz,
1H), 7.12 (d, J=9.0 Hz, 1H), 4.78 (ddd, J=13.4, 11.7, 5.7 Hz, 2H),
4.59 (d, J=19.8 Hz, 2H), 4.55 (d, J=8.2 Hz, 1H), 3.19 (dt, J=18.0,
5.6 Hz, 1H), 3.02 (dt, J=18.2, 7.2 Hz, 1H), 2.23-2.03 (m, 2H), 1.92
(dt, J=12.4, 6.3 Hz, 2H), 1.48 (d, J=6.1 Hz, 6H).
[0217]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)sulfamide 27 was prepared in an
analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile HC14.
[0218] General Procedure 6. Preparation of Tetrahydronaphthalene
Sulfonamides via Sulfonyl Chlorides
[0219] To a solution of (R)- or (S)-tetrahydronapthalene amine HCl
(1 eq) in DCM (0.05M) was added TEA (2 eq) and the appropriate
sulfonyl chloride (1-2 eq.) at room temperature. The reaction
mixture was stirred at room temperature for 18 h. The solvent was
evaporated and the product isolated after preparative HPLC
purification.
[0220] Compounds 28-33 were prepared using General Procedure 6.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)methanesulfonamide (Compound 28)
##STR00058##
[0222] Prepared using General Procedure 6: To a stirring solution
of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 3 (20 mg, 0.05 mmol) in
DCM (1 mL) at 0.degree. C. was added TEA (20 .mu.L, 0.15 mmol) and
methanesulfonyl chloride (4.5 .mu.L, 0.06 mmol). The mixture was
allowed to warm to room temperature over 2 h. The solvent was
evaporated and crude mixture was purified by preparative HPLC to
afford 12.8 mg (58%) of
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)methanesulfonamide 28. LCMS-ESI (m/z)
calculated for C.sub.23H.sub.24N.sub.4O.sub.4S: 452.5; found 453.1
[M+H].sup.+, t.sub.R=3.68 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.40 (t, J=3.5 Hz, 1H), 8.32 (dd, J=8.9, 2.2 Hz, 1H), 7.96
(dd, J=7.7, 1.1 Hz, 1H), 7.67 (d, J=7.7 Hz, 1H), 7.38 (t, J=7.7 Hz,
1H), 7.16-7.07 (m, 1H), 4.88-4.70 (m, 1H), 4.54 (d, J=8.4 Hz, 1H),
3.19 (dt, J=18.0, 5.9 Hz, 1H), 3.10 (d, J=5.0 Hz, 3H), 3.09-2.95
(m, 1H), 2.14 (qt, J=14.5, 7.3 Hz, 1H), 2.06-1.83 (m, 3H), 1.47 (t,
J=5.5 Hz, 6H).
[0223]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)methanesulfonamide 29 was prepared
in an analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 4.
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)-2-methoxyethanesulfonamide (Compound
31)
##STR00059##
[0225] Prepared via General Procedure 6 using cyclopropanesulfonyl
chloride. LCMS-ESI (m/z) calculated for
C.sub.25H.sub.26N.sub.4O.sub.4S: 478.6; found 479.1 [M+H].sup.+,
t.sub.R=3.84 min.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)-2-methoxyethanesulfonamide (Compound
32)
##STR00060##
[0227] Prepared via General Procedure 6 using
2-methoxyethanesulfonyl chloride. LCMS-ESI (m/z) calculated for
C.sub.25H.sub.28N.sub.4O.sub.5S: 496.58; found 519.1 [M+Na].sup.+,
t.sub.R=3.83 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.41
(d, J=2.2 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H), 7.99-7.92 (m, 1H),
7.74 (d, J=7.6 Hz, 1H), 7.37 (t, J=7.7 Hz, 1H), 7.12 (d, J=9.0 Hz,
1H), 4.85-4.71 (m, 2H), 4.68-4.60 (m, 1H), 3.93-3.76 (m, 2H),
3.47-3.31 (m, 5H), 3.17 (dt, J=18.0, 6.0 Hz, 1H), 3.02 (dt, J=18.1,
6.7 Hz, 1H), 2.20-1.82 (m, 4H), 1.48 (d, J=6.1 Hz, 6H). Chiral
HPLC:
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)-2-methoxyethanesulfonamide 32 was eluted
with 10% water/MeOH, (Chiral Method 2): 99.98% ee, t.sub.R=21.07
min.
[0228]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydro naphthalen-1-yl)-2-methoxyethanesulfonamide 33 was
prepared in an analogous fashion from 4. Chiral HPLC: 99.04% ee,
t.sub.R=18.57 min (Chiral Method 2).
(R)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1-
,2,3,4-tetrahydronaphthalen-1-yl)sulfamoyl)acetate (INT-14)
##STR00061##
[0230] Prepared using General Procedure 6: To a stirring solution
of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 3 (0.15 g, 0.37 mmol) in
DCM (5 mL) were added TEA (76 .mu.L, 0.55 mmol) and
methyl-2-(chlorosulfonyl)acetate (76 mg, 0.44 mmol). Additional TEA
and methyl-2-(chlorosulfonyl)acetate were added to drive the
reaction to completion over 24 h. The crude reaction mixture was
partitioned between DCM and saturated NaHCO.sub.3. The organic
layer was dried over Na.sub.2SO.sub.4, concentrated, and purified
by column chromatography (EA/hexanes) to give 0.11 g (57%) of
(R)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tet-
rahydronaphthalen-1-yl)sulfamoyl)acetate INT-14. LCMS-ESI (m/z)
calculated for C.sub.25H.sub.26N.sub.4O.sub.6S: 510.6; found 511.1
[M+H].sup.+, t.sub.R=3.73 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.42 (d, J=2.2 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H),
8.01-7.93 (m, 1H), 7.74 (d, J=7.8 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H),
7.12 (d, J=9.0 Hz, 1H), 5.00 (d, J=8.4 Hz, 1H), 4.81 (dq, J=18.3,
5.9 Hz, 2H), 4.25-4.00 (m, 2H), 3.83 (s, 3H), 3.20 (dt, J=18.1, 5.9
Hz, 1H), 3.12-2.97 (m, 1H), 2.22-2.01 (m, 2H), 2.02-1.83 (m, 2H),
1.48 (d, J=6.1 Hz, 6H).
[0231] (S)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tet-
rahydronaphthalen-1-yl)sulfamoyl)acetate INT-15 was prepared in an
analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 4.
[0232] General Procedure 7. Preparation of Tetrahydronapthalene
Sulfonamide Acids
[0233] To a stirring solution of (R)- or (S)-tetrahydronapthalene
sulfonamide ester (1 eq) in MeOH (0.2 M) was added 6N NaOH (2 eq)
at room temperature. The reaction was stirred at room temperature
for 6 h. The crude reaction was diluted with water, acidified with
1N HCl and extracted with DCM and EA. The organic layer was dried
over Na.sub.2SO.sub.4, concentrated, and isolated after preparative
HPLC purification.
[0234] Compounds 34 and 35 were prepared using General Procedure
7.
(R)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4--
tetrahydronaphthalen-1-yl)sulfamoyl)acetic acid (Compound 34)
##STR00062##
[0236] Prepared using General Procedure 7: To a stirring solution
of (R)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tet-
rahydronaphthalen-1-yl)sulfamoyl) acetate INT-14 (0.082 g, 0.16
mmol) in MeOH (1.5 mL) was added 6N NaOH (0.08 mL). The reaction
was stirred at room temperature for 6 h. The crude reaction was
diluted with water, acidified with 1N HCl and extracted with DCM
and EA. The organic layer was dried over Na.sub.2SO.sub.4,
concentrated, and isolated after preparative HPLC purification to
give 0.057 g (72%) of
(R)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-
-tetrahydronaphthalen-1-yl)sulfamoyl)acetic acid 34. An
analytically pure sample was prepared by preparative HPLC
purification. LCMS-ESI (m/z) calculated for
C.sub.24H.sub.26N.sub.4O.sub.6S: 496.5; found 520.1 [M+Na].sup.+,
t.sub.R=3.47 min.
[0237]
(S)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1-
,2,3,4-tetrahydronaphthalen-1-yl) sulfamoyl)acetic acid 35 was
prepared in an analogous fashion from (S)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tet-
rahydronaphthalen-1-yl)sulfamoyl)acetate INT-15.
[0238] General Procedure 8. Preparation of Tetrahydronapthalene
Sulfonamide Alcohols
[0239] To a stirring solution of either (R)- or
(S)-tetrahydronapthalene sulfonamide ester (1 eq) in THF (0.06 M)
was added sodium borohydride (2.5 eq) at room temperature. The
reaction mixture was heated to 75.degree. C. and methanol (1 eq)
was added dropwise. After 1 h, the reaction was cooled and
concentrated and purified by preparative HPLC.
[0240] Compounds 36 and 37 were prepared using General Procedure
8.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)-2-hydroxyethanesulfonamide (Compound
37)
##STR00063##
[0242] Prepared using General Procedure 8: To a stirring solution
of (R)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tet-
rahydronaphthalen-1-yl)sulfamoyl)acetate INT-14 (0.025 g, 0.05
mmol) in THF (25 mL) was added sodium borohydride (0.05 g, 0.12
mmol) at room temperature. The reaction was heated to 75.degree. C.
and methanol (0.02 mL, 0.05 mmol) was added. After 1 h, the
reaction was cooled and concentrated and purified by preparative
HPLC to give 16.0 mg (66%) of
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)-2-hydroxyethanesulfonamide 37. LCMS-ESI
(m/z) calculated for C.sub.24H.sub.26N.sub.4O.sub.5S: 482.6; found
505.1 [M+Na].sup.+, t.sub.R=3.46 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.39 (dd, J=5.4, 2.4 Hz, 1H), 8.35-8.25 (m,
1H), 7.95 (dt, J=7.7, 3.9 Hz, 1H), 7.67 (d, J=7.5 Hz, 1H),
7.43-7.32 (m, 1H), 7.12 (t, J=7.5 Hz, 1H), 4.80 (m, 3H), 4.12 (t,
J=5.2 Hz, 2H), 3.46-3.28 (m, 2H), 3.17 (dt, J=18.0, 5.9 Hz, 1H),
3.02 (dt, J=18.1, 6.8 Hz, 1H), 2.68 (s, 1H), 2.12 (m, 1H),
2.07-1.82 (m, 3H), 1.47 (d, J=6.1 Hz, 6H). .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 172.82, 169.22, 162.81, 137.73, 136.90, 134.12,
133.89, 132.15, 130.28, 126.46, 126.33, 116.65, 115.22, 113.57,
103.93, 72.78, 57.32, 56.17, 52.54, 30.60, 28.06, 21.72, 19.04.
Chiral HPLC:
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)-2-hydroxyethane sulfonamide 37 was eluted
with 15% water/MeOH, (Chiral Method 2): 99.82% ee, t.sub.R=22.23
min.
[0243]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)-2-hydroxyethanesulfonamide 36 was
prepared in an analogous fashion from (S)-methyl
2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tet-
rahydronaphthalen-1-yl)sulfamoyl)acetate INT-15. Chiral HPLC: 91.7%
ee, t.sub.R=19.83 min (Chiral Method 2).
[0244] General Procedure 9. Preparation of Tetrahydronapthalene
Sulfonamide Amides
[0245] To a stirring solution of either (R)- or
(S)-tetrahydronapthalene sulfonamide acid (1 eq) in DMF (0.25 M)
were added EDC and N-hydroxybenzotriazole. After 5 min, the amine
was added and the reaction mixture was stirred 18 h at room
temperature. The crude reaction was diluted with NaHCO.sub.3 added
extracted with EA. The combined organic layers were dried over
Na.sub.2SO.sub.4, and purified by preparative HPLC.
[0246] Compounds 38-43 were prepared using General Procedure 9.
(R)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4--
tetrahydronaphthalen-1-yl)sulfamoyl)-N,N-dimethylacetamide
(Compound 40)
##STR00064##
[0248] Prepared using General Procedure 9: To a stirring solution
of
(R)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-
-tetrahydronaphthalen-1-yl)sulfamoyl) acetic acid 34 (15 mg, 0.05
mmol) in DMF (0.5 mL) was added N-hydroxybenzotriazole (6.1 mg,
0.05 mmol) and EDC (8.7 mg, 0.05 mmol). After 5 min, dimethylamine
(40 wt % solution in THF, 50 .mu.L, 0.09 mmol) was added and the
reaction mixture was stirred 18 h at room temperature. The crude
reaction was diluted with sat NaHCO.sub.3 and extracted with EA.
The combined organic layers were dried over Na.sub.2SO.sub.4 and
purified by preparative HPLC to give 4.41 mg (28%) of
(R)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,-
3,4-tetrahydronaphthalen-1-yl)sulfamoyl)-N,N-dimethylacetamide 40.
LCMS-ESI (m/z) calculated for C.sub.26H.sub.29N.sub.5O.sub.5S:
523.6; found 546.2 [M+Na].sup.+, t.sub.R=3.58 min. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.41 (d, J=2.1 Hz, 1H), 8.33 (dd, J=8.9,
2.2 Hz, 1H), 7.96 (dd, J=7.7, 1.2 Hz, 1H), 7.82 (d, J=7.5 Hz, 1H),
7.38 (t, J=7.8 Hz, 1H), 7.12 (d, J=9.0 Hz, 1H), 5.36 (t, J=17.3 Hz,
1H), 4.88-4.73 (m, 2H), 4.27 (d, J=14.6 Hz, 1H), 4.07 (d, J=14.6
Hz, 1H), 3.24-3.09 (m, 4H), 3.09-2.97 (m, 4H), 2.23-2.08 (m, 2H),
2.10-1.84 (m, 2H), 1.47 (d, J=6.1 Hz, 6H). .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 172.75, 169.37, 163.15, 162.77, 137.79, 136.92,
134.13, 133.90, 132.50, 130.18, 126.36, 126.17, 116.79, 115.25,
113.55, 103.96, 72.74, 55.46, 53.05, 38.22, 35.98, 29.84, 28.10,
21.73, 19.10.
[0249]
((S)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)--
1,2,3,4-tetrahydronaphthalen-1-yl)sulfamoyl)-N,N-dimethylacetamide
41 was prepared in an analogous fashion from
(S)-2-(N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-
-tetrahydronaphthalen-1-yl) sulfamoyl) acetic acid 35.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)ethenesulfonamide INT-16
##STR00065##
[0251] To a stirred solution of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 3 (100 mg, 0.24 mmol) in
DCM (5 mL) at 0.degree. C. were added TEA (170 .mu.L, 1.2 mmol) and
2-chloroethanesulfonyl chloride (76 .mu.L, 0.73 mmol). The reaction
mixture was warmed to room temperature and stirred for 30 min. The
solvent was removed and the product was purified by chromatography
(EA/hexane) to give 83.0 mg (75%) of
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)ethenesulfonamide INT-16 as a white solid.
LCMS-ESI (m/z) calculated for C.sub.24H.sub.24N.sub.4O.sub.4S:
464.5; found 465.1 [M+H].sup.+, t.sub.R=3.83 min. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.41 (d, J=2.2 Hz, 1H), 8.32 (dd, J=8.9,
2.2 Hz, 1H), 7.96 (dd, J=7.7, 1.2 Hz, 1H), 7.62 (t, J=11.0 Hz, 1H),
7.38 (t, J=7.7 Hz, 1H), 7.12 (d, J=9.0 Hz, 1H), 6.66 (dt, J=26.0,
13.0 Hz, 1H), 6.38 (d, J=16.5 Hz, 1H), 6.03 (dd, J=10.1, 5.2 Hz,
1H), 4.86-4.73 (m, 1H), 4.68-4.57 (m, 1H), 4.50 (d, J=8.3 Hz, 1H),
3.18 (dt, J=18.1, 5.8 Hz, 1H), 3.02 (dt, J=18.1, 6.8 Hz, 1H),
2.15-1.96 (m, 2H), 1.97-1.79 (m, 2H), 1.48 (d, J=6.1 Hz, 6H).
[0252]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)ethenesulfonamide INT-17 was
prepared in an analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 4.
[0253] General Procedure 10. Preparation of Tetrahydronaphthalene
Sulfonamides via Michael Addition
[0254] To a stirring solution of either the (R)- or
(S)-tetrahydronapthalene vinyl sulfonamide (1 eq) in DMF (0.1M)
were added TEA (5 eq) and the appropriate amine (5 eq). The
reaction mixture was stirred at room temperature for 18 h. The
products were purified by preparative HPLC.
[0255] Compounds 44-47 were prepared using General Procedure
10.
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)-2-(dimethylamino)ethanesulfonamide
(Compound 44)
##STR00066##
[0257] Prepared using General Procedure 10. To a solution of
((R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4--
tetrahydronaphthalen-1-yl)ethenesulfonamide INT-16 (40 mg, 0.09
mmol) in DMF (1.0 mL) was added 2N methylamine in THF (0.22 mL,
0.43 mmol) and the reaction mixture was stirred at room temperature
for 18 h. The crude product was purified by preparative HPLC to
give 24.6 mg (54%) of the TFA salt of
(R)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1-
,2,3,4-tetrahydronaphthalen-1-yl)-2-(dimethylamino)ethanesulfonamide
44 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.26H.sub.31N.sub.5O.sub.4S: 509.6; found 510.2 [M+H].sup.+,
t.sub.R=2.61 min. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.41-8.32 (m,
1H), 8.33-8.26 (m, 1H), 7.92 (t, J=6.9 Hz, 1H), 7.61 (t, J=7.2 Hz,
1H), 7.35 (dd, J=14.5, 7.1 Hz, 1H), 7.11 (d, J=9.1 Hz, 1H),
4.86-4.64 (m, 2H), 3.61 (ddt, J=27.2, 13.7, 7.8 Hz, 4H), 3.23-3.06
(m, 1H), 3.10-2.91 (m, 1H), 2.93 (d, J=30.3 Hz, 6H), 2.09 (ddd,
J=28.4, 16.5, 12.3 Hz, 1H), 1.95 (ddd, J=15.1, 8.3, 3.5 Hz, 3H),
1.46 (t, J=6.0 Hz, 6H).
[0258]
(S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)-2-(dimethylamino) ethanesulfonamide
45 was prepared in analogous fashion from
((S)--N-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4--
tetrahydronaphthalen-1-yl)ethenesulfonamide INT-17.
(R)-2-isopropoxy-5-(3-(5-((2-(methylsulfonyl)ethyl)amino)-5,6,7,8-tetrahyd-
ronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (Compound
48)
##STR00067##
[0260] To a solution of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 3 (20 mg, 0.05 mmol) in
DMA (0.5 mL) was added TEA (136 .mu.L, 0.97 mmol) and
methylvinylsulfone (52 mg, 0.5 mmol). The reaction was heated to
80.degree. C. for 24 h. The crude reaction mixture was purified by
preparative HPLC to give
(R)-2-isopropoxy-5-(3-(5-(2-(methylsulfonyl)ethyl)amino)-5,6,7,8-tetrahyd-
ronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)benzonitrile 48. LCMS-ESI
(m/z) calculated for C.sub.25H.sub.28N.sub.4O.sub.4S: 480.6; found
481.2 [M+H].sup.+, t.sub.R=2.58 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.37 (t, J=9.1 Hz, 1H), 8.31 (dd, J=8.9, 2.2
Hz, 1H), 8.08 (d, J=7.1 Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 7.42 (t,
J=7.7 Hz, 1H), 7.12 (d, J=9.1 Hz, 1H), 4.85-4.73 (m, 1H), 4.53 (t,
J=5.0 Hz, 1H), 3.63 (dd, J=15.3, 4.0 Hz, 2H), 3.61-3.50 (m, 2H),
3.33-3.17 (m, 1H), 3.18-3.04 (m, 1H), 3.04 (d, J=8.6 Hz, 3H), 2.16
(ddd, J=29.6, 18.8, 12.2 Hz, 2H), 2.00 (dd, J=36.4, 18.4 Hz, 2H),
1.47 (d, J=6.1 Hz, 6H).
[0261]
(S)-2-isopropoxy-5-(3-(5-(2-(methylsulfonyl)ethyl)amino)-5,6,7,8-te-
trahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)benzonitrile,
compound 49 was prepared in an analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile hydrochloride 4.
(R)-methyl
2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydronaphthalen-1-yl)amino)acetate (INT-18)
##STR00068##
[0263] To a stirring solution of
(R)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile 3 (119 mg, 0.32 mmol) in CH.sub.3CN (5.0
mL) was added methyl bromoacetate (53.5 .mu.L, 0.35 mmol) and
K.sub.2CO.sub.3 (138 mg, 1.27 mmol). After stirring for 18 h, the
mixture was diluted with brine and washed with NaHCO.sub.3. The
organic layer was dried with Na.sub.2SO.sub.4 and concentrated. The
resulting crude solid was purified by chromatography (MeOH/DCM) to
provide 113.1 mg (79%) of (R)-methyl
2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetra-
hydronaphthalen-1-yl)amino) acetate INT-18. LCMS-ESI (m/z)
calculated for C.sub.25H.sub.26N.sub.4O.sub.4: 446.5; found 447.2
[M+H].sup.+, t.sub.R=2.52 min.
[0264] (S)-methyl
2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetra-
hydronaphthalen-1-yl)amino)acetate INT-19 was prepared in an
analogous fashion from
(S)-5-(3-(5-amino-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl-
)-2-isopropoxybenzonitrile 4.
(S)-methyl
2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,-
4-oxadiazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetate
(INT-20)
##STR00069##
[0266] To a stirred solution of (S)-methyl
2-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrah-
ydronaphthalen-1-yl)amino)acetate INT-19 (128.0 mg, 0.29 mmol) in
DCM (6.0 mL) was added Boc anhydride (125.1 mg, 0.57 mmol) and TEA
(120 .mu.L, 0.86 mmol). After stirring for 18 h, the mixture was
concentrated. The resulting crude solid was purified by
chromatography (EA/hexanes) to provide 119 mg (76%) of (S)-methyl
2-((tert-butoxycarbonyl)
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)amino)acetate INT-20. LCMS-ESI (m/z) calculated
for C.sub.30H.sub.34N.sub.4O.sub.6: 546.61; no M/Z observed,
t.sub.R=4.32 min.
[0267] (R)-methyl 2-((tert-butoxycarbonyl)
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)amino)acetate INT-21 was prepared in an analogous
fashion from (R)-methyl
2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetra-
hydronaphthalen-1-yl)amino)acetate INT-18.
[0268] (S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl) (2-hydroxyethyl)carbamate (INT-22)
##STR00070##
[0269] To a stirring solution of (S)-methyl
2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetra-
hydronaphthalen-1-yl)amino)acetate INT-20 (35 mg, 0.06 mmol) in THF
(6.0 mL) at 75.degree. C. was added sodium borohydride (6 mg, 0.16
mmol). After stirring for 0.5 h, MeOH (7.7 .mu.L, 0.19 mmol) was
added and the mixture was heated for an additional 1.5 h. The
mixture was concentrated and the resulting solid was purified by
chromatography (EA/hexanes) to provide 16 mg (48%) of
(S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-hydroxyethyl)carbamate INT-22. LCMS-ESI (m/z)
calculated for C.sub.29H.sub.34N.sub.4O.sub.5: 518.6; found 419.2
[M-Boc+H].sup.+, t.sub.R=4.10 min.
[0270] (R)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-hydroxyethyl)carbamate INT-23 was prepared in
prepared in an analogous fashion from (R)-methyl
2-((tert-butoxycarbonyl)
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)amino)acetate INT-21.
[0271] General Procedure 11: Boc Deprotection of
Tetrahydronaphthalene Amines
[0272] To a stirring solution of Boc protected (R)- or
(S)-tetrahydronaphthalene amine in dioxane was added 4N
HCl/dioxanes (4-10 eq). The reaction mixture was heated at
50.degree. C. for 18 h. The reaction mixture was concentrated and
the resulting solid was purified by preparative HPLC.
[0273] Compounds 50-53 were prepared using General Procedure
11.
(R)-5-(3-(5-((2-hydroxyethyl)amino)-5,6,7,8-tetrahydronaphthalen-1-yl)-1,2-
,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile (Compound 50)
##STR00071##
[0275] Prepared using General Procedure 11. To a stirred solution
of (R)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl) (2-hydroxyethyl)carbamate INT-23 (15 mg, 0.03
mmol) in dioxane (1 mL) was added 4N HCl/dioxanes (116 .mu.L, 0.116
mmol). After heating at 50.degree. C. for 18 h, the mixture was
concentrated and the resulting solid was purified by preparative
HPLC to provide 9.53 mg (79%) of
(R)-5-(3-(5-((2-hydroxyethyl)amino)-5,6,7,8-tetrahydronaphthalen-1-yl)-
-1,2,4-oxadiazol-5-yl)-2-isopropoxy benzonitrile 50. LCMS-ESI (m/z)
calculated for C.sub.24H.sub.26N.sub.4O.sub.3: 418.5; found 419.2
[M+H].sup.+, t.sub.R=2.52 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.39 (d, J=2.2 Hz, 1H), 8.32 (dd, J=8.9, 2.2 Hz, 1H), 8.04
(d, J=7.7 Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 7.39 (t, J=7.7 Hz, 1H),
7.12 (d, J=9.1 Hz, 1H), 4.88-4.70 (m, 1H), 4.54 (s, 1H), 3.78 (d,
J=12.1 Hz, 2H), 3.62 (s, 2H), 3.24 (dt, J=18.0, 5.6 Hz, 1H),
3.18-2.89 (m, 3H), 2.16 (d, J=5.2 Hz, 2H), 2.10-1.75 (m, 2H), 1.47
(d, J=6.1 Hz, 6H).
[0276]
(S)-5-(3-(5-((2-hydroxyethyl)amino)-5,6,7,8-tetrahydronaphthalen-1--
yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile 51 was prepared
in an analogous fashion from (S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-hydroxyethyl)carbamate INT-22.
(S)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadi-
azol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic acid
(INT-24)
##STR00072##
[0278] To a stirring solution of (S)-methyl
2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetra-
hydronaphthalen-1-yl)amino)acetate INT-20 (93.2 mg, 0.17 mmol) in
MeOH (2 ml) was added 10 drops of 1N NaOH. The mixture was stirred
at 50.degree. C. for 2 h, then diluted with H.sub.2O and
neutralized with 1N HCl. The aqueous solution was extracted with
DCM and EA. The combined organic layers were dried over
Na.sub.2SO.sub.4 and concentrated to provide 61 mg (67%) of
(S)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1-
,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic
acid INT-24 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.29H.sub.32N.sub.4O.sub.6: 532.6; found 358.1
[M-2-((tert-butoxycarbonyl)amino)acetic acid].sup.+, t.sub.R=3.97
min.
[0279]
(R)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,-
4-oxadiazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic
acid INT-25 was prepared in an analogous fashion from (S)-methyl
2-(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrah-
ydronaphthalen-1-yl)amino)acetate INT-21.
(S)-2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-te-
trahydronaphthalen-1-yl)amino)acetic acid (Compound 53)
##STR00073##
[0281] Prepared using General Procedure 11. A solution of
(S)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxad-
iazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic acid
INT-24 (30 mg, 0.06 mmol) in 4N HCl/dioxanes (200 .mu.l, 50 mmol)
was stirred at room temperature for 18 h. The mixture was
concentrated and the residue was purified by preparative HPLC to
provide 21 mg (67%) of
(S)-2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-t-
etrahydronaphthalen-1-yl)amino)acetic acid 53 as the TFA salt.
[0282] LCMS-ESI (m/z) calculated for
C.sub.24H.sub.24N.sub.4O.sub.4: 432.5; found 358.1 [M-2-aminoacetic
acid].sup.+, t.sub.R=2.65 min.
[0283]
(R)-2-((5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2-
,3,4-tetrahydro naphthalen-1-yl)amino) acetic acid 52 was prepared
in an analogous fashion from
(R)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxad-
iazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic acid
INT-25.
[0284] General Procedure 12: Preparation of Tetrahydronaphthalene
Amino Amides
[0285] To the Boc-protected (R)- or (S)-tetrahydronaphthalene
aminoacid DMF were added N-hydroxybenzotriazole (2 eq) and EDC (2
eq). After 10 min, the appropriate amine (10 eq) was added and the
reaction mixture was stirred for 18 h at room temperature. The
crude reaction mixture was diluted with NaHCO.sub.3 and extracted
with EA. The combined organic layers were dried over
Na.sub.2SO.sub.4 and purified by preparative HPLC.
[0286] Compounds 54 and 55 were prepared using General Procedure
12.
(S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate
(INT-26)
##STR00074##
[0288] Prepared using General Procedure 12. To a stirring solution
of
(S)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxad-
iazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic acid
INT-24 (30 mg, 0.06 mmol) in DMF (0.5 mL) were added
N-hydroxybenzotriazole (15.21 mg, 0.11 mmol) and EDC (21.63 mg,
0.11 mmol). After 10 min, pyrrolidine (46 .mu.L, 0.56 mmol) was
added and the reaction mixture was stirred 18 h at room
temperature. The crude reaction was diluted with sat NaHCO.sub.3
added extracted with EA. The combined organic layers were dried
over Na.sub.2SO.sub.4 and purified by preparative HPLC to give 26.9
mg (82%) of (S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate.
LCMS-ESI (m/z) calculated for C.sub.33H.sub.39N.sub.5O.sub.5:
585.7; found 358.1 [M-tert-butyl
(2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate].sup.+, t.sub.R=4.18
min.
[0289] (R)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate INT-27
was prepared in an analogous fashion from
(R)-2-((tert-butoxycarbonyl)(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxad-
iazol-3-yl)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)acetic acid
INT-25.
(R)-2-isopropoxy-5-(3-(5-((2-oxo-2-(pyrrolidin-1-yl)ethyl)amino)-5,6,7,8-t-
etrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)benzonitrile
(Compound 54)
##STR00075##
[0291] Prepared using General Procedure 11. A solution of
(R)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl) (2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate INT-27
(18 mg, 0.03 mmol) in 4N HCl/dioxanes (1 mL) was stirred at room
temperature for 18 h. The reaction mixture was concentrated and
purified by preparative HPLC to provide 12 mg (68%) of
(R)-2-isopropoxy-5-(3-(5-((2-oxo-2-(pyrrolidin-1-yl)ethyl)amino)-5,6,7,8--
tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)benzonitrile 54 as
the TFA salt. LCMS-ESI (m/z) calculated for
C.sub.28H.sub.31N.sub.5O.sub.3: 485.6; found 486.2 [M+H].sup.+,
t.sub.R=2.60 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.40
(d, J=2.2 Hz, 1H), 8.33 (dd, J=8.9, 2.2 Hz, 1H), 8.07 (d, J=7.6 Hz,
1H), 7.77 (d, J=7.6 Hz, 1H), 7.44 (t, J=7.7 Hz, 1H), 7.13 (d, J=9.1
Hz, 1H), 4.87-4.68 (m, 2H), 3.98-3.80 (m, 2H), 3.53-3.03 (m, 7H),
2.17 (ddd, J=17.7, 10.4, 5.5 Hz, 3H), 2.04-1.79 (m, 5H), 1.48 (d,
J=6.1 Hz, 6H).
[0292]
(S)-2-isopropoxy-5-(3-(5-((2-oxo-2-(pyrrolidin-1-yl)ethyl)amino)-5,-
6,7,8-tetrahydronaphthalen-1-yl)-1,2,4-oxadiazol-5-yl)benzonitrile
55 was prepared in an analogous fashion from (S)-tert-butyl
(5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1,2,3,4-tetrahyd-
ronaphthalen-1-yl)(2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate
INT-26.
4-bromo-2,3-dihydro-1H-inden-1-ol (INT-28)
##STR00076##
[0294] To a stirring solution of 4-bromoindanone (3 g, 14.2 mmol)
in anhydrous EtOH (30 mL) were added sodium borohydride (0.36 g,
9.5 mmol) and silica gel (2 g) at 0.degree. C. The reaction was
stirred at 0.degree. C. for 20 min and was allowed to stir at room
temperature for 2 h. The reaction mixture was quenched with
saturated NaHCO.sub.3 and concentrated to remove EtOH. The aqueous
layer was extracted with EA and the organic phase was dried over
MgSO.sub.4. After concentration, the crude product was purified by
chromatography (EA/hexane) to yield
4-bromo-2,3-dihydro-1H-inden-1-ol INT-28 (2.56 g, 85%) as white
solid. LCMS-ESI (m/z) calculated for C.sub.9H.sub.9BrO: 213.1;
found 195.0 [M-H.sub.2O].sup.+, t.sub.R=3.07 min. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.35 (d, J=7.9, 1H), 7.27 (d, J=7.4, 1H),
7.05 (t, J=7.7, 1H), 5.23 (t, J=6.2, 1H), 3.00 (ddd, J=16.6, 8.8,
4.6, 1H), 2.84-2.66 (m, 1H), 2.45 (dddd, J=13.2, 8.4, 7.0, 4.6,
1H), 1.96-1.70 (m, 2H).
(4-bromo-2,3-dihydro-1H-inden-1-yloxy)(tert-butyl)dimethylsilane
(INT-29)
##STR00077##
[0296] To a solution of 4-bromo-2,3-dihydro-1H-inden-1-ol INT-28
(2.56 g, 12.0 mm) in DMF (5 mL) were added TBDMSC1 (2.17 g, 14.4
mmol) and imidazole (2 g, 30.0 mmol) and the reaction mixture
stirred at room temperature overnight. The reaction mixture was
diluted with water and extracted with EA. The organic layers were
washed with water and brine, and dried over MgSO.sub.4. The crude
product was purified by chromatography (EA/hexane) to afford
(4-bromo-2,3-dihydro-1H-inden-1-yloxy)(tert-butyl)dimethylsilane
INT-29 (3.3 g, 84%) as a clear oil. LCMS-ESI (m/z) calculated for
C.sub.15H.sub.23BrOSi: 327.3; found 195.0 [M-OTBS].sup.+,
t.sub.R=3.07 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.20
(d, J=7.8 Hz, 1H), 7.06 (d, J=7.4 Hz, 1H), 6.92 (t, J=7.7 Hz, 1H),
5.13 (t, J=7.0 Hz, 1H), 2.85 (ddd, J=16.4, 9.1, 2.9 Hz, 1H), 2.57
(dt, J=16.5, 8.3 Hz, 1H), 2.36-2.17 (m, 1H), 1.76 (dtd, J=12.8,
8.8, 7.1 Hz, 1H), 0.83-0.72 (m, 9H), 0.05--0.06 (m, 6H).
tert-butyldimethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dih-
ydro-1H-inden-1-yloxy)silane (INT-30)
##STR00078##
[0298] A solution of
(4-bromo-2,3-dihydro-1H-inden-1-yloxy)(tert-butyl)dimethylsilane
INT-29 (50 mg, 0.15 mmol),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (42 mg,
0.16 mmol), and potassium acetate (45 mg, 0.45 mmol) in anhydrous
1,4-dioxane (2 mL) was degassed by passing N.sub.2 through the
solution for 5 min. PdCl.sub.2(dppf).CH.sub.2Cl.sub.2 was then
added and the reaction mixture was heated at 85.degree. C.
overnight. The solvent was removed under vacuum, the residue was
diluted with EA (10 mL), and filtered through celite to remove
solids. The filtrate was washed with water and brine and dried over
MgSO.sub.4. The crude product was purified by chromatography
(EA/hexanes) to afford
tert-butyldimethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-di-
hydro-1H-inden-1-yl-oxy)silane INT-30 (26 mg, 45%) as a white
semi-solid. LCMS-ESI (m/z) calculated for
C.sub.21H.sub.35BO.sub.3Si: 374.4; found 245.0 [M-OTBS].sup.+,
t.sub.R=3.07 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.57-7.43 (m, 1H), 7.21 (dd, J=11.0, 4.2 Hz, 1H), 7.08-7.01 (m,
1H), 5.06 (t, J=7.0 Hz, 1H), 3.11 (ddd, J=16.8, 8.9, 3.0 Hz, 1H),
2.72 (dt, J=16.8, 8.3 Hz, 1H), 2.22 (dddd, J=12.6, 7.9, 7.1, 3.1
Hz, 1H), 1.71 (dtd, J=12.6, 8.8, 7.0 Hz, 1H), 1.21-1.10 (m, 12H),
0.81-0.71 (m, 9H), 0.03--0.07 (m, 6H).
5-(4,5-dihydrooxazol-2-yl)-2-isopropoxybenzonitrile (INT-31)
##STR00079##
[0300] To a stirring suspension of 3-cyano-4-isopropoxybenzoic acid
(1.0 g, 4.8 mmol) in DCM (20 mL) was added oxalyl chloride (3.7 g,
29.2 mmol) followed by two drops DMF. The reaction mixture was
stirred at 50.degree. C. for 2 h. The mixture was concentrated and
the residue re-dissolved in DCM (10 mL). Ethanolamine (0.6 g, 9.7
mmol) and TEA (1.45 g, 14.4 mmol) were added and the reaction
mixture was stirred overnight at room temperature. The resulting
solid was filtered, washed with water, and dried to afford 1.0 g
(83%) of 3-cyano-N-(2-hydroxyethyl)-4-isopropoxybenzamide which was
used in the next step without purification. LCMS-ESI (m/z)
calculated for C.sub.13H.sub.16N.sub.2O.sub.3: 248.3; found 249.0
[M+H].sup.+, t.sub.R=2.41 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.02-7.79 (m, 2H), 6.97-6.87 (m, 1H), 6.71 (s, 1H), 4.65
(dt, J=12.1, 6.1 Hz, 1H), 3.82-3.70 (m, 2H), 3.56 (dd, J=10.2, 5.5
Hz, 2H), 1.96 (d, J=10.0 Hz, 1H), 1.40-1.29 (m, 6H).
[0301] 3-cyano-N-(2-hydroxyethyl)-4-isopropoxybenzamide was
dissolved in DCM (30 mL) and thionyl chloride (1.43 g, 12 mmol) was
added at 0.degree. C. The reaction mixture was stirred at room
temperature for 1 h and then quenched at 0.degree. C. with water
(200 .mu.L) and 6N NaOH solution (1 mL). The mixture was stirred
for 30 min. The aqueous layers were extracted with DCM and the
combined organic extracts were washed with brine and dried over
MgSO.sub.4 to afford 570 mg (61% for two steps) of
5-(4,5-dihydrooxazol-2-yl)-2-isopropoxybenzonitrile INT-31.
LCMS-ESI (m/z) calculated for C.sub.13H.sub.14N.sub.2O.sub.2:
230.3; found 231.0 [M+H].sup.+, t.sub.R=2.50 min. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.17-7.86 (m, 2H), 6.91 (d, J=8.9 Hz, 1H),
4.65 (dt, J=12.2, 6.1 Hz, 1H), 4.37 (dd, J=14.3, 4.9 Hz, 2H), 3.98
(t, J=9.5 Hz, 2H), 1.36 (t, J=5.5 Hz, 6H).
5-(5-bromooxazol-2-yl)-2-isopropoxybenzonitrile (INT-32)
##STR00080##
[0303] A stirring solution of
5-(4,5-dihydrooxazol-2-yl)-2-isopropoxybenzonitrile INT-31 (420 mg,
1.82 mmol), N-bromosuccinamide (990 mg, 5.56 mmol) and
azoisobutyronitrile (14.9 mg, 0.09 mmol) in carbon tetrachloride
(20 mL) was heated at 80.degree. C. under N.sub.2 for 18 h. The
reaction mixture was cooled to room temperature and the solids were
removed by filtration. The filtrate was washed with sodium
thiosulfate (20 mL) and brine (20 mL), and dried over MgSO.sub.4.
The product was purified by chromatography (EA/hexanes) to afford
300 mg (55%) of 5-(5-bromooxazol-2-yl)-2-isopropoxybenzonitrile
INT-32 as a yellow solid. LCMS-ESI (m/z) calculated for
C.sub.13H.sub.11BrN.sub.2O.sub.2: 307.1; found 309.0 [M+2].sup.+,
t.sub.R=3.79 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.19-7.93 (m, 2H), 7.08-6.85 (m, 2H), 4.81-4.47 (m, 1H), 1.38 (dd,
J=6.6, 3.0 Hz, 6H).
[0304] General Procedure 13: Coupling of Heterocyclic Bromide to
Indanol Boronate
[0305] A 20 mL microwave vial was charged sequentially with
heterocyclic bromide (1 eq), (R)--(S)-- or racemic indanol
dioxaborolane (1 eq), DME/H.sub.2O (3:1, 0.05 M) and potassium
carbonate (3 eq). The mixture was degassed by bubbling N.sub.2 gas
through the stirring solution for 10 min. Pd(PPh.sub.3).sub.4 (0.07
eq) was added and the mixture degassed for additional 2 min. The
vial was capped and subjected to microwave irradiation at
100.degree. C. until reaction completed (40-60 min). Additional
bromide was added if needed. The vial was cooled to room
temperature, diluted with EA (10.times. volume), washed with water
and brine, dried over MgSO.sub.4, and concentrated. The crude
product was purified by silica gel column chromatography
(EA/hexanes).
5-(5-(1-(tert-butyldimethylsilyloxy)-2,3-dihydro-M-inden-4-yl)oxazol-2-yl)-
-2-isopropoxybenzonitrile (INT-33)
##STR00081##
[0307] Prepared using General Procedure 13. A 20 mL microwave vial
was charged with 5-(5-bromooxazol-2-yl)-2-isopropoxybenzonitrile
INT-32 (200 mg, 0.65 mmol),
tert-butyldimethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-di-
hydro-1H-inden-1-yloxy)silane INT-30 (243 mg, 0.65 mmol), potassium
carbonate (269 mg, 1.95 mmol) and a 3:1 mixture of dimethylethylene
glycol/H.sub.2O (10 mL). The reaction mixture was degassed by
bubbling N.sub.2 gas through the stirring solution for 10 min.
Pd(PPh.sub.3).sub.4 was added and the solution degassed for
additional 2 min. The vial was subjected to microwave irradiation
at 100.degree. C. for 40 min. The vial was cooled to 0.degree. C.
and the resulting solid obtained was collected by filtration,
washed with ice water, and dried to afford 290 mg (94%) of
5-(5-(1-(tert-butyldimethylsilyloxy)-2,3-dihydro-1H-inden-4-yl)
oxazol-2-yl)-2-isopropoxybenzonitrile INT-33 as a light yellow
solid. LCMS-ESI (m/z) calculated for
C.sub.28H.sub.34N.sub.2O.sub.3Si: 474.7; found 475.2 [M+H].sup.+,
t.sub.R=5.90 min (Method 1). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.16-7.96 (m, 2H), 7.57-7.42 (m, 1H), 7.24-7.12 (m, 3H),
6.90 (t, J=10.4 Hz, 1H), 5.14 (t, J=7.0 Hz, 1H), 4.57 (dt, J=12.3,
6.1 Hz, 1H), 3.04 (ddd, J=16.1, 9.1, 3.1 Hz, 1H), 2.78 (dt, J=16.1,
8.1 Hz, 1H), 2.43-2.24 (m, 1H), 1.84 (ddd, J=15.8, 12.8, 8.9 Hz,
1H), 1.27 (t, J=5.8 Hz, 6H), 0.86-0.61 (m, 9H), 0.06--0.14 (m,
6H).
5-(5-(1-hydroxy-2,3-dihydro-M-inden-4-yl)oxazol-2-yl)-2-isopropoxybenzonit-
rile (Compound 56)
##STR00082##
[0309] To a solution of
5-(5-(1-(tert-butyldimethylsilyloxy)-2,3-dihydro-1H-inden-4-yl)oxazol-2-y-
l)-2-isopropoxybenzonitrile INT-33 (350 mg, 0.737 mmol) in
anhydrous THF (2 mL) was added a 1M solution of tetrabutylammonium
fluoride in THF (3.6 mL, 3.6 mmol) at 0.degree. C. The reaction
mixture was allowed to stir at room temperature for 16 h before
quenching with brine (5 mL). The THF was removed under vacuum, the
residue was diluted with water (5 mL), and the aqueous layer was
extracted with EA. The combined extracts were washed with brine,
dried over MgSO.sub.4, and purified by chromatography to afford 220
mg (63%) of
5-(5-(1-hydroxy-2,3-dihydro-1H-inden-4-yl)oxazol-2-yl)-2-isopropoxybenzon-
itrile 56 as a light yellow solid. LCMS-ESI (m/z) calculated for
C.sub.22H.sub.20N.sub.2O.sub.3: 360.4; found 343.0 [M-OH].sup.+,
t.sub.R=2.30 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.30
(d, J=2.2 Hz, 1H), 8.26 (dd, J=8.9, 2.2 Hz, 1H), 7.75 (d, J=7.6 Hz,
1H), 7.48 (d, J=7.3 Hz, 1H), 7.42 (t, J=7.6 Hz, 1H), 7.10 (d, J=8.9
Hz, 1H), 5.35 (d, J=4.8 Hz, 1H), 4.78 (dt, J=12.2, 6.1 Hz, 1H),
3.30 (ddd, J=16.4, 8.7, 4.8 Hz, 1H), 3.13-2.94 (m, 1H), 2.64 (dddd,
J=13.3, 8.4, 7.1, 4.8 Hz, 1H), 2.17-2.08 (m, 1H), 1.86 (s, 1H),
1.60 (s, 1H), 1.46 (dd, J=13.9, 6.0 Hz, 6H).
[0310] General Procedure 14. Preparation of Indane Amines via
Chloride Displacement
[0311] To a stirring solution of indane alcohol (1 eq) in DCM (1
mL) was added thionyl chloride (2 eq.) at 0.degree. C. The reaction
mixture was stirred at room temperature for 3 h. The solvent was
evaporated and the crude chloride re-dissolved in dimethyl
acetamide (1 mL). Diisopropyl ethylamine (3 eq.) and the
appropriate amine (3 eq.) were added and the reaction mixtures were
stirred at 70.degree. C. overnight. The reaction mixtures were
quenched with water (200 .mu.L) and purified by preparative
HPLC.
[0312] Compounds 57, 58, and 61-64 were prepared using General
Procedure 14.
5-(5-(1-(2-hydroxyethylamino)-2,3-dihydro-M-inden-4-yl)oxazol-2-yl)-2-isop-
ropoxybenzonitrile (Compound 57)
##STR00083##
[0314] Prepared using General Procedure 14. To a stirring solution
of
5-(5-(1-hydroxy-2,3-dihydro-1H-inden-4-yl)oxazol-2-yl)-2-isopropoxybenzon-
itrile 56 (50 mg, 0.1 mmol) in DCM (3 mL) was added thionyl
chloride (25 mg, 0.21 mmol) at 0.degree. C. The reaction mixture
was stirred at room temperature for 3 h. The solvent was evaporated
and the crude chloride re-dissolved in dimethyl acetamide (3 mL).
Isopropyl ethylamine (40.8 mg, 0.316 mmol) and ethanolamine (19.3
mg, 0.31 mmol) were added and the reaction mixture heated at
70.degree. C. overnight. The reaction mixture was quenched with
NaHCO.sub.3 and extracted with EA. The combined organic extracts
were washed with brine and then dried over MgSO.sub.4. The product
was purified by chromatography (10% MeOH/DCM) to afford 25 mg (60%)
of
5-(5-(1-(2-hydroxyethylamino)-2,3-dihydro-1H-inden-4-yl)oxazol-2-
-yl)-2-isopropoxybenzonitrile 57. LCMS-ESI (m/z) calculated for
C.sub.24H.sub.25N.sub.3O.sub.3: 403.5; found 404.1 [M+H].sup.+,
t.sub.R=2.41 min. .sup.1H NMR (400 MHz, DMSO) .delta. 8.18 (t,
J=2.3 Hz, 1H), 8.08 (dd, J=9.0, 2.3 Hz, 1H), 7.70 (d, J=7.7 Hz,
1H), 7.44 (d, J=17.4 Hz, 1H), 7.42-7.32 (m, 1H), 7.30-7.11 (m, 2H),
4.70 (dt, J=12.2, 6.1 Hz, 2H), 4.39 (s, 1H), 3.40 (t, J=5.0 Hz,
2H), 3.18-2.95 (m, 2H), 2.93-2.75 (m, 1H), 2.73-2.54 (m, 2H),
2.38-2.16 (m, 1H), 1.98-1.78 (m, 1H), 1.15 (d, J=6.0 Hz, 6H).
.sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 161.35, 159.17, 151.04,
146.60, 139.78, 132.16, 127.43, 125.59, 125.07, 123.99, 120.49,
116.10, 113.90, 103.77, 72.60, 62.95, 61.51, 48.70, 33.27, 31.29,
29.91, 22.02.
5-(5-(1-((R)-1-hydroxypropan-2-ylamino)-2,3-dihydro-M-inden-4-yl)oxazol-2--
yl)-2-isopropoxybenzonitrile (Compound 58)
##STR00084##
[0316] Prepared using General Procedure 14. LCMS-ESI (m/z)
calculated for: C.sub.25H.sub.27N.sub.3O.sub.3: 417.5; found 418.4
[M+H].sup.+, t.sub.R=2.49 min.
(R)--N--((R)-4-bromo-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfinam-
ide (INT-34)
##STR00085##
[0318] To a stirring solution of 4-bromo-2,3-dihydro-1H-inden-1-one
(5.0 g, 23.6 mmol) and (R)-2-methylpropane-2-sulfinamide (3.15 g,
26.0 mmol) in toluene (40 mL) was added titanium tetraethoxide (8.1
g, 35.5 mmol) and the reaction mixture was heated at 60.degree. C.
for 18 h under N.sub.2. To this mixture was added THF (40 mL) and
the resulting solution was cooled to -78.degree. C. Sodium
borohydride (3.5 g, 94.7 mmol) was added in one portion. The
reaction mixture was stirred at -78.degree. C. for 15 min, and then
warmed to room temperature and stirred at this temperature for 2 h.
The reaction mixture was cooled to 0.degree. C. before quenching
with brine and sodium potassium tartrate. EA was added and the
mixture was stirred at room temperature overnight during which time
Ti salts precipitated. The organic layers were decanted, and washed
successively with saturated NH.sub.4Cl, water, and brine. The
organic layers were dried over MgSO.sub.4, filtered through a pad
of MgSO.sub.4, and concentrated to produce
(R)--N--((R)-4-bromo-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfina-
mide INT-34 as a solid (3.14 g, 42%) which was used in the next
step without purification. LCMS-ESI (m/z) calculated
C.sub.13H.sub.18BrNOS: 317.3; found 318.0 [M+H].sup.+, t.sub.R=3.59
min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.46 (d, J=7.5, 1H),
7.34 (d, J=7.9, 1H), 7.05 (t, J=7.7, 1H), 4.96-4.77 (m, 1H), 3.39
(d, J=6.8, 1H), 3.06-2.86 (m, 1H), 2.82-2.60 (m, 1H), 2.50-2.29 (m,
1H), 2.05-1.81 (m, 1H), 1.16 (s, 9H).
[0319]
(S)--N--((S)-4-bromo-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-s-
ulfinamide INT-35 can be made in an analogous fashion using
(S)-2-methylpropane-2-sulfinamide.
(R)-4-bromo-2,3-dihydro-1H-inden-1-amine (INT-36)
##STR00086##
[0321] To crude
(R)--N--((R)-4-bromo-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfina-
mide INT-34 (3.14 g, 9.9 mol) in MeOH (10 mL) was added 4N HCl in
dioxane (7.5 mL, 30 mmol) and the resulting yellow suspension was
stirred at room temperature for 2 h. The crude reaction mixture
diluted with MeOH (5 mL), cooled to 0.degree. C., and filtered to
remove Ti by-products. The filtrate was concentrated and the
resulting solid refluxed in acetonitrile (60 mL) for 30 min and
then cooled to 0.degree. C. The resulting white solid was collected
to produce the HCl salt of (R)-4-bromo-2,3-dihydro-1H-inden-1-amine
INT-36 (1.55 g, 63%) which was used in the next step without
purification. LCMS-ESI (m/z) calculated for C.sub.9H.sub.10BrN:
212.1; found 197.0 [M-NH].sup.+, t.sub.R=0.75 min. .sup.1H NMR (400
MHz, DMSO) .delta. 8.60 (s, 1H), 7.67 (d, J=7.5 Hz, 1H), 7.57 (d,
J=7.9 Hz, 1H), 7.39-7.07 (m, 1H), 4.81 (dd, J=7.9, 5.6 Hz, 1H),
3.25-2.64 (m, 3H), 2.59-2.32 (m, 1H), 2.21-1.69 (m, 1H).
[0322] (S)-4-bromo-2,3-dihydro-1H-inden-1-amine INT-37 can be made
in an analogous fashion from
(S)--N--((S)-4-bromo-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfina-
mide INT-35.
(R)-tert-butyl 4-bromo-2,3-dihydro-1H-inden-1-ylcarbamate
(INT-38)
##STR00087##
[0324] To crude (R)-4-bromo-2,3-dihydro-1H-inden-1-amine HCl INT-36
(1.55 g, 6.2 mmol) in DCM (10 mL) at 0.degree. C. was added TEA
(1.38 g, 13.7 mmol) followed by Boc anhydride (1.49 g, 6.8 mmol)
and the reaction mixture stirred at room temperature overnight. The
reaction mixture was washed with brine, and the organic layers were
dried over MgSO.sub.4 and filtered. The product was purified by
chromatography (EA/hexanes) to afford (R)-tert-butyl
4-bromo-2,3-dihydro-1H-inden-1-ylcarbamate INT-38 (1.63 g, 84%) as
an off-white solid. LCMS-ESI (m/z) calculated for
C.sub.14H.sub.18BrNO.sub.2: 312.20; found 197.0 [M-NHBoc].sup.+,
t.sub.R=3.97 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.31
(d, J=7.9 Hz, 1H), 7.23-7.13 (m, 1H), 7.02 (t, J=7.7 Hz, 1H),
5.30-5.07 (m, 1H), 4.69 (d, J=7.5 Hz, 1H), 2.93 (ddd, J=16.5, 9.0,
3.4 Hz, 1H), 2.75 (dt, J=16.5, 8.2 Hz, 1H), 2.60-2.43 (m, 1H), 1.73
(dq, J=13.1, 8.4 Hz, 1H), 1.41 (s, 9H).
[0325] (S)-tert-butyl 4-bromo-2,3-dihydro-1H-inden-1-ylcarbamate
INT-39 can be made in an analogous fashion from
(S)-4-bromo-2,3-dihydro-1H-inden-1-amine INT-37.
(R)-tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate (INT-40)
##STR00088##
[0327] A solution of (R)-tert-butyl
4-bromo-2,3-dihydro-1H-inden-1-ylcarbamate INT-38 (300 mg, 0.96
mmol) and
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (268
mg, 1.0 mmol), potassium acetate (283 mg, 2.88 mmol) in anhydrous
1,4-dioxane (5 mL) was degassed by passing N.sub.2 through the
solution for 5 min. PdCl.sub.2(dppf).DCM (157 mg, 0.19 mmol) was
added and the reaction mixture was heated at 85.degree. C.
overnight. The solvent was removed under vacuum and the residue
dissolved in EA (10 mL) and filtered through celite to remove the
solids. The filtrate was washed with water and brine, dried over
MgSO.sub.4, and purified by chromatography (EA/hexanes) to afford
(R)-tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-40 (265 mg, 77%) as white semi-solid. LCMS-ESI (m/z)
calculated for C.sub.20H.sub.30BNO.sub.4: 359.3; found 383.0
[M+Na].sup.+, t.sub.R=4.26 min. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.71 (d, J=7.3 Hz, 1H), 7.42 (t, J=7.9 Hz, 1H), 7.24 (dd,
J=9.7, 5.2 Hz, 1H), 5.19 (dd, J=15.9, 7.9 Hz, 1H), 4.72 (d, J=8.5
Hz, 1H), 3.28 (ddd, J=17.0, 8.8, 3.6 Hz, 1H), 2.99 (dt, J=16.8, 8.4
Hz, 1H), 2.69-2.44 (m, 1H), 1.77 (ddd, J=16.4, 12.8, 8.6 Hz, 1H),
1.51 (s, 9H), 1.39-1.31 (m, 12H).
[0328] (S)-tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-41 can be made in an analogous fashion from
(S)-tert-butyl 4-bromo-2,3-dihydro-1H-inden-1-ylcarbamate
INT-39.
(R)-tert-butyl
4-(2-(3-cyano-4-isopropoxyphenyl)oxazol-5-yl)-2,3-dihydro-1H-inden-1-ylca-
rbamate (INT-42)
##STR00089##
[0330] Prepared using General Procedure 13. A 20 mL microwave vial
was charged with (R)-tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-32 (58.4 mg, 0.16 mmol),
5-(5-bromooxazol-2-yl)-2-isopropoxybenzonitrile INT-40 (50 mg, 0.16
mmol), potassium carbonate (68 mg, 0.5 mmol) and a 3:1 mixture of
dimethylethylene glycol/H.sub.2O (2 mL). The reaction mixture was
degassed by bubbling N.sub.2 gas through the stirring solution for
10 min. Pd(PPh.sub.3).sub.4 ((3.9 mg, 0.004 mmol) was added and the
solution degassed for additional 2 min. The vial was subjected to
microwave irradiation at 100.degree. C. for 30 min. The solvent was
removed and the residue dissolved in EA (10 mL), washed with brine,
and then dried over MgSO.sub.4. The product was purified by
chromatography (EA/hexanes) to afford 50 mg (67%) of (R)-tert-butyl
4-(2-(3-cyano-4-isopropoxyphenyl)oxazol-5-yl)-2,3-dihydro-1H-inden-1-ylca-
rbamate INT-42 as an off-white solid. LCMS-ESI (m/z) calculated for
C.sub.27H.sub.29N.sub.3O.sub.4: 459.5; found 460.2 [M+H].sup.+,
t.sub.R=4.1 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.32-8.03 (m, 2H), 7.60 (dd, J=8.6, 4.1 Hz, 1H), 7.32-7.22 (m, 3H),
7.00 (d, J=8.9 Hz, 1H), 5.19 (dd, J=15.5, 7.5 Hz, 1H), 4.82-4.56
(m, 2H), 3.12 (ddd, J=16.3, 9.0, 3.5 Hz, 1H), 2.95 (dt, J=16.3, 8.1
Hz, 1H), 2.70-2.51 (m, 1H), 1.83 (dq, J=13.1, 8.2 Hz, 1H), 1.43 (s,
9H), 1.41-1.35 (m, 6H).
[0331] (S)-tert-butyl
4-(2-(3-cyano-4-isopropoxyphenyl)oxazol-5-yl)-2,3-dihydro-1H-inden-1-ylca-
rbamate INT-43 can be made in an analogous fashion from
(S)-tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-41.
(R)-5-(5-(1-amino-2,3-dihydro-1H-inden-4-yl)oxazol-2-yl)-2-isopropoxy-benz-
onitrile hydrochloride (Compound 59)
##STR00090##
[0333] To a stirring solution of (R)-tert-butyl
4-(2-(3-cyano-4-isopropoxyphenyl)oxazol-5-yl)-2,3-dihydro-1H-inden-1-ylca-
rbamate INT-42 (48 mg, 0.1 mmol) in 1,4-dioxane (1 mL) was added a
4N HCl solution in 1,4-dioxane (1 mL). The reaction mixture was
heated at 55-65.degree. C. for 48 h. The cooled reaction mixture
was diluted with Et.sub.2O (10 mL). The resulting solid was
collected and dried under high vacuum to yield 32 mg (78%) of
(R)-5-(5-(1-amino-2,3-dihydro-1H-inden-4-yl)oxazol-2-yl)-2-isopropoxybenz-
onitrile hydrochloride 59 as a white solid. LCMS-ESI (m/z)
calculated for C.sub.22H.sub.21N.sub.3O.sub.2: 359.4; found 343.1
[M-NH.sub.2].sup.+, t.sub.R=2.40 min. .sup.1H NMR (400 MHz, DMSO)
.delta. 8.55 (br s, 2H), 8.43 (dd, J=6.5, 2.4 Hz, 1H), 8.32 (ddd,
J=6.7, 6.1, 2.9 Hz, 1H), 8.00 (t, J=13.5 Hz, 1H), 7.72 (s, 1H),
7.66 (d, J=7.5 Hz, 1H), 7.49 (dd, J=8.5, 6.3 Hz, 2H), 4.93 (dt,
J=12.1, 6.0 Hz, 1H), 4.81 (s, 1H), 3.43-3.25 (m, 1H), 3.23-3.04 (m,
1H), 2.67-2.55 (m, 1H), 2.11 (ddd, J=14.2, 9.0, 5.9 Hz, 1H), 1.36
(dd, J=13.8, 7.0 Hz, 6H).
[0334]
(S)-5-(5-(1-amino-2,3-dihydro-1H-inden-4-yl)oxazol-2-yl)-2-isopropo-
xybenzonitrile hydrochloride INT-44 can be made in an analogous
fashion from (S)-tert-butyl
4-(2-(3-cyano-4-isopropoxyphenyl)oxazol-5-yl)-2,3-dihydro-1H-inden-1-ylca-
rbamate INT-43.
1-oxo-2,3-dihydro-1H-indene-4-carbonitrile (INT-45)
##STR00091##
[0336] To a stirring solution of 4-bromo-2,3-dihydro-1H-inden-1-one
(100.0 g, 0.48 mol) in 150 mL of 1-methyl-2-pyrrolidine (NMP) was
added zinc cyanide (111.8 g, 0.95 mol) and
tetrakis(triphenylphosphine)palladium [Pd(PPh.sub.3).sub.4] (2.75
g, 0.024 mol). The solution was degassed with N.sub.2 and the
reaction mixture heated at 95.degree. C. for 7 h. Upon cooling, the
reaction mixture was poured onto ice water (3.5 L). The compound
and inorganic Zn salts precipitated. The solid was collected and
partitioned between DCM and water. The organic layers were filtered
to remove the Zn salts, and the filtrate was concentrated and
crystallized from a 4:1 mixture of EtOH and MeOH (400 mL) to give
45.5 g (60%) of 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile INT-45
as a light yellow solid. LCMS-ESI (m/z) calculated for
C.sub.10H.sub.7NO: 157.2; found 158.1 [M+H].sup.+, t.sub.R=2.67
min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.00-7.90 (m, 1H),
7.86 (dd, J=7.5, 1.1, 1H), 7.50 (t, J=7.6, 1H), 3.40-3.19 (m, 2H),
2.90-2.61 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) .delta.
204.70, 157.90, 138.38, 137.88, 128.44, 128.28, 116.31, 111.70,
36.01, 25.49.
(.+-.)-1-hydroxy-2,3-dihydro-1H-indene-4-carbonitrile (INT-46)
##STR00092##
[0338] To a stirring suspension of
1-oxo-2,3-dihydro-1H-indene-4-carbonitrile INT-45 (1.2 g, 7.64
mmol) and silica gel (catalytic) in EtOH at 0.degree. C. was added
NaBH.sub.4 (237.2 mg, 7.64 mmol). The reaction was allowed to warm
to room temperature and stirred for 2 h. The solvent was removed
under reduced pressure, and the product was purified by
chromatography (EA/hexane) to afford 1.02 g (82%) of
1-hydroxy-2,3-dihydro-1H-indene-4-carbonitrile INT-46 as a white
solid. LCMS-ESI (m/z) calculated for C.sub.10H.sub.9NO; 159.2;
found 160.1 [M+H].sup.+, t.sub.R=2.39 min.
N,1-dihydroxy-2,3-dihydro-1H-indene-4-carboximidamide (INT-47)
##STR00093##
[0340] Prepared using General Procedure 1. To hydroxylamine
hydrochloride (0.87 g, 12.5 mmol) and sodium carbonate (1.32 g,
12.5 mmol) in EtOH (20 mL) was added
1-hydroxy-2,3-dihydro-1H-indene-4-carbonitrile INT-46 (1.80 g, 11.3
mmol) in one portion and the solution was heated to reflux. After
16 h, the reaction was cooled and filtered to remove the solids.
The EtOH was removed and the compound was purified by
chromatography (MeOH/DCM) to give 1.74 g (90%) of
N,1-dihydroxy-2,3-dihydro-1H-indene-4-carboximidamide INT-47 as a
white foam. LCMS-ESI (m/z) calculated for
C.sub.10H.sub.12N.sub.2O.sub.2: 192.1; found: 193.1 [M+H].sup.+,
t.sub.R=0.56 min. .sup.1H NMR (400 MHz, MeOD) .delta. 10.30 (s,
1H), 9.97 (s, 1H), 7.72-7.58 (m, 1H), 7.46-7.37 (m, 2H), 5.22 (t,
J=6.5, 1H), 3.17-3.03 (m, 1H), 2.99-2.83 (m, 1H), 2.49 (dddd,
J=11.4, 8.0, 7.0, 4.4, 1H), 2.02-1.88 (m, 1H).
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-ol
(Compound 60)
##STR00094##
[0342] Prepared using General Procedure 2. A solution of
2-(3,4-diethoxyphenyl)acetic acid (180.0 mg, 0.80 mmol) in DMF (3
mL) was treated with HOBt (197.8 mg, 1.46 mmol) and EDC (207.3 mg,
1.08 mmol) at room temperature. The reaction was stirred for 2 h
until the complete formation of the HOBt-acid complex.
N-1-dihydroxy-2,3-dihydro-1H-indene-4-carboximidamide INT-47 (185.1
mg, 0.96 mmol) was added and the mixture was stirred at room
temperature for 2 h and then heated to 80.degree. C. for 16 h. The
reaction mixture was diluted with NaHCO.sub.3 and extracted with
EA. The organic phase was dried over MgSO.sub.4 and crude product
was purified by chromatography (EA/hexanes) to produce
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-ol
60 (190 mg, 62%) as an off-white solid. LCMS-ESI (m/z) calculated
for C.sub.22H.sub.24N.sub.2O.sub.4: 380.1; found 381.1 [M+H].sup.+,
t.sub.R=3.45 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.02-7.86 (m, 1H), 7.46 (d, J=7.5 Hz, 1H), 7.29 (t, J=7.6 Hz, 1H),
6.81 (ddd, J=21.0, 13.6, 5.1 Hz, 3H), 5.21 (t, J=5.6 Hz, 1H), 4.13
(s, 2H), 4.01 (dq, J=14.1, 7.0 Hz, 4H), 3.34 (ddd, J=17.5, 8.7, 4.6
Hz, 1H), 3.16-2.92 (m, 1H), 2.53-2.38 (m, 1H), 1.91 (qdd, J=8.7,
6.6, 5.5 Hz, 2H), 1.36 (td, J=7.0, 4.6 Hz, 6H).
2-((4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-
-yl)amino)ethanol (Compound 61)
##STR00095##
[0344] Prepared using General Procedure 14 using
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-ol
60 and 2-aminoethanol.
(2R)-2-((4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-in-
den-1-yl)amino)propan-1-ol (Compound 62)
##STR00096##
[0346] Prepared using General Procedure 14 from
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-ol
60 and (R)-2-aminopropan-1-ol.
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-N-(2-(methylsulfonyl)ethyl-
)-2,3-dihydro-1H-inden-1-amine (Compound 63)
##STR00097##
[0348] Prepared using General Procedure 14 from
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-ol
60 and 2-(methylsulfonyl)ethanamine.
2-((4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-
-yl)amino)-N-methylethanesulfonamide (Compound 64)
##STR00098##
[0350] Prepared using General Procedure 14 from
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-ol
60 and 2-amino-N,N-dimethylethanesulfonamide.
(R)--N-(4-cyano-2,3-dihydro-1H-indene-1-ylidene)-2-methylpropane-2-sulfina-
mide (INT-48)
##STR00099##
[0352] To 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile INT-45 (42.5
g, 0.27 mol) and (R)-2-methylpropane-2-sulfinamide (36.0 g, 0.30
mol) in toluene (530 mL) was added titanium tetraethoxide (84.1 mL,
92.5 g, 0.40 mol) and the reaction mixture was heated at 60.degree.
C. for 12 h under N.sub.2. The crude
(R)--N-(4-cyano-2,3-dihydro-1H-indene-1-ylidene)-2-methylpropan-
e-2-sulfinamide INT-48 was used directly in the next experiment.
LCMS-ESI (m/z) calculated for C.sub.14H.sub.16N.sub.2OS: 260.3;
found 261.1 [M+H].sup.+, t.sub.R=3.19 min.
(R)--N--((R)-4-cyano-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfinam-
ide (INT-49)
##STR00100##
[0354] To a flask containing the crude suspension of
(R)--N-(4-cyano-2,3-dihydro-1H-indene-1-ylidene)-2-methylpropane-2-sulfin-
amide INT-48 under N.sub.2 was added THF (1.0 L) and the reaction
mixture cooled to -78.degree. C. Sodium borohydride (40.9 g, 1.08
mol) was added portion-wise over 30 mins. (The internal temperature
did not rise during the addition.) The reaction mixture was stirred
at -78.degree. C. for 30 mins, half out of the bath for 30 mins,
then warmed to 0.degree. C. over 1 h. The 0.degree. C. reaction
mixture was placed in an ice bath and quenched with brine (100 mL)
followed by saturated sodium potassium tartrate (420 mL) and the Ti
salts precipitated. The reaction mixture was diluted with EA (1.5
L) and stirred at room temperature overnight. The organic layers
were decanted and washed successively with saturated NH.sub.4Cl,
water, and brine. The organic layers were dried over MgSO.sub.4 and
filtered through a pad of MgSO.sub.4. The filtrate was concentrated
to produce 52.9 g of crude
(R)--N--((R)-4-cyano-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfina-
mide INT-49 as a brown oil, which was used directly in the next
step. LCMS-ESI (m/z) calculated for C.sub.14H.sub.18N.sub.2OS:
262.3; found 263.1 [M+H].sup.+, t.sub.R=2.99 min. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.89 (d, J=7.7, 1H), 7.56 (t, J=6.8, 1H),
7.36 (t, J=7.7, 1H), 4.97 (q, J=7.5, 1H), 3.50 (d, J=7.6, 1H), 3.22
(ddd, J=16.9, 8.8, 3.9, 1H), 3.01 (dt, J=22.4, 6.9, 1H), 2.70-2.53
(m, 1H), 2.15-1.95 (m, 1H), 1.33-1.20 (m, 9H).
(R)-1-amino-2,3-dihydro-1H-indene-1-yl)-4-carbonitrile (INT-50)
##STR00101##
[0356] To crude
(R)--N--((R)-4-cyano-2,3-dihydro-1H-inden-1-yl)-2-methylpropane-2-sulfina-
mide INT-49 (52.9 g, 0.20 mol) in MeOH (200 mL) was added 4N HCl in
dioxane (152.0 mL, 0.60 mol) and the resulting yellow suspension
was stirred at room temperature for 1.5 h. The crude reaction
mixture was diluted with MeOH (500 mL) and filtered to remove some
Ti by-products. The filtrate was concentrated and the resulting
solid was refluxed in acetonitrile (500 mL). The resulting white
solid was collected to produce 13.0 g (31% over 3 steps) of the HCl
salt of (R)-1-amino-2,3-dihydro-1H-indene-1-yl)-4-carbonitrile
INT-50. LCMS-ESI (m/z) calculated for C.sub.10H.sub.10N.sub.2:
158.2; found 142.0 [M-NH.sub.2].sup.+, t.sub.R=0.84 min. .sup.1H
NMR (400 MHz, DMSO) .delta. 8.61 (s, 3H), 7.96 (d, J=7.7, 1H), 7.83
(d, J=7.5, 1H), 7.52 (t, J=7.7, 1H), 4.80 (s, 1H), 3.23 (ddd,
J=16.6, 8.7, 5.2, 1H), 3.05 (ddd, J=16.6, 8.6, 6.3, 1H), 2.62-2.51
(m, 1H), 2.15-2.01 (m, 1H). .sup.13C NMR (101 MHz, DMSO) .delta.
148.09, 141.15, 132.48, 130.32, 127.89, 117.27, 108.05, 54.36,
39.08, 29.64. The free base can be prepared by extraction with 1N
NaHCO.sub.3 and DCM. LCMS-ESI (m/z) calculated for
C.sub.10H.sub.10N.sub.2: 158.2; found 142.0 [M-NH.sub.2].sup.+,
t.sub.R=0.83 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.52-7.38 (m, 2H), 7.23 (dd, J=17.4, 9.8, 1H), 4.35 (t, J=7.6, 1H),
3.11 (ddd, J=16.8, 8.7, 3.2, 1H), 2.89 (dt, J=16.9, 8.5, 1H), 2.53
(dddd, J=12.8, 8.1, 7.3, 3.2, 1H), 1.70 (dtd, J=12.8, 8.8, 8.0,
1H). .sup.13C NMR (101 MHz, DMSO) .delta. 150.16, 146.67, 130.19,
128.74, 127.38, 117.77, 107.42, 56.86, 38.86, 29.14. Chiral HPLC:
(R)-1-amino-2,3-dihydro-1H-indene-1-yl)-4-carbonitrile was eluted
using 5% EtOH in hexanes, plus 0.05% TEA: 95% ee, t.sub.R=23.02
min.
[0357] The (S)-enantiomer INT-51 was prepared in an analogous
sequence (INT-48, INT-49, and INT-50) using
(S)-2-methylpropane-2-sulfinamide in the first step. t.sub.R for
(S)-enantiomer=20.17 min.
(R)-tert-butyl 4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate
(INT-52)
##STR00102##
[0359] To (R)-1-amino-2,3-dihydro-1H-indene-1-yl)-4-carbonitrile
HCl INT-50 (11.6 g, 59.6 mmol) in DCM (100 mL) at 0.degree. C. was
added TEA (12.0 mL, 131.0 mmol). To the resulting solution was
added a solution of Boc anhydride (14.3 g, 65.6 mmol) in DCM (30
mL) and the reaction mixture stirred at room temperature for 1.5 h.
The reaction mixture was washed with brine, and the organic layers
were dried over MgSO.sub.4 and filtered. Additional DCM was added
to a total volume of 250 mL and Norit (4.5 g) was added. The
product was refluxed for 15 mins and the hot mixture filtered
through a pad of celite/silica. The filtrate was concentrated and
recrystallized from EA (50 mL) and hexane (150 mL) to produce 12.93
g (84%) of (R)-tert-butyl
4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate INT-52 as an off-white
solid. LCMS-ESI (m/z) calculated for
C.sub.15H.sub.18N.sub.2O.sub.2: 258.3; found 281.1 [M+Na].sup.+,
t.sub.R=3.45 min. Elemental Analysis determined for
C.sub.15H.sub.18N.sub.2O.sub.2; C calculated=69.74%; found=69.98%.
H calculated=7.02%; found=7.14%. N calculated=10.84%; found=10.89%.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.64-7.49 (m, 2H), 7.34
(dt, J=7.7, 3.8, 1H), 5.36-5.20 (m, 1H), 4.78 (d, J=6.8, 1H), 3.20
(ddd, J=16.9, 8.9, 3.3, 1H), 3.02 (dt, J=25.4, 8.4, 1H), 2.82-2.53
(m, 1H), 1.88 (dq, J=13.2, 8.6, 1H), 1.55-1.44 (m, 9H). .sup.13C
NMR (101 MHz, DMSO) .delta. 155.52, 146.68, 146.32, 130.89, 128.70,
127.63, 117.51, 107.76, 77.98, 55.09, 31.88, 29.11, 28.19. Chiral
HPLC: (R)-tert-butyl 4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate was
eluted using 2.5% EtOH in hexanes: >99.9% ee, t.sub.R=19.36
min.
[0360] The (S)-enantiomer INT-53 was prepared in an analogous
fashion using
(S)-1-amino-2,3-dihydro-1H-indene-1-yl)-4-carbonitrile HCl INT-51.
t.sub.R for (S)-enantiomer=28.98 min.
(R)-tert-butyl
4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H-inden-1-ylcarbamate
(INT-54)
##STR00103##
[0362] Prepared using General Procedure 1. To (R)-tert-butyl
4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate INT-52 (15.0 g, 58.2
mmol) in EtOH (100 mL) was added hydroxylamine hydrochloride (12.1
g, 174.2 mmol) and TEA (17.6 mL, 174.2 mmol) and the reaction
mixture was heated at 85.degree. C. for 2 h. The solvents were
removed and the resulting white solid was partitioned between water
and DCM. The organic layers were dried over Na.sub.2SO.sub.4,
concentrated, and recrystallized from isopropanol (50 mL) to afford
14.4 g (85%) of (R)-tert-butyl
4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H-inden-1-ylcarbamate
INT-54 as white crystalline solid. LCMS-ESI (m/z) calculated for
C.sub.15H.sub.21N.sub.3O.sub.3: 291.4; found 292.1 [M+H].sup.+,
t.sub.R=2.04 min. .sup.1H NMR (400 MHz, DMSO) .delta. 9.53 (s, 1H),
7.38-7.32 (m, 1H), 7.32-7.12 (m, 3H), 5.68 (s, 2H), 4.97 (q, J=8.5,
1H), 3.07 (ddd, J=16.6, 8.7, 2.6, 1H), 2.86 (dt, J=16.8, 8.4, 1H),
2.30 (ddd, J=12.6, 7.6, 3.6, 1H), 1.75 (dq, J=12.3, 9.0, 1H), 1.44
(s, 9H).
[0363] (S)-tert-butyl
4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H-inden-1-ylcarbamate
INT-55 was prepared in an analogous fashion from (R)-tert-butyl
4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate INT-53.
(R)-tert-butyl
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate (INT-56)
##STR00104##
[0365] Prepared using General Procedure 2. A solution of
2-(3,4-diethoxyphenyl)acetic acid (150.0 mg, 0.67 mmol) in DMF (3
mL) was treated with HOBt (164.8 mg, 1.22 mmol) and EDC (172.7 mg,
0.9 mmol) at room temperature. The reaction was stirred for 2 h
until the complete formation of the HOBt-acid complex.
(R)-tert-butyl
4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H-inden-1-ylcarbamate
INT-54 (233.8 mg, 0.8 mmol) was added and stirred at room
temperature for 2 h and then mixture was heated to 80.degree. C.
for 16 h. The reaction was diluted with NaHCO.sub.3 (10 mL) and
extracted with EA (3.times.10 ml). The organic phase was dried over
MgSO.sub.4 and the crude product was purified by a chromatography
(EA/hexanes) to produce (R)-tert-butyl
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-56 (187 mg, 58%) as off-white solid and used directly
in the next step. LCMS-ESI (m/z) calculated for
C.sub.27H.sub.33N.sub.3O.sub.5: 479.2; found 502.2 [M+Na].sup.+,
t.sub.R=4.11 min. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.91
(d, J=7.6 Hz, 1H), 7.39 (d, J=7.5 Hz, 1H), 7.27 (t, J=7.7 Hz, 1H),
6.81 (ddd, J=20.2, 12.8, 5.1 Hz, 3H), 5.18 (d, J=8.4 Hz, 1H), 4.69
(d, J=8.3 Hz, 1H), 4.15 (d, J=6.1 Hz, 2H), 4.06-3.93 (m, 4H), 3.32
(ddd, J=17.4, 8.8, 3.4 Hz, 1H), 3.14-2.91 (m, 1H), 2.65-2.40 (m,
1H), 1.75 (dq, J=12.9, 8.4 Hz, 1H), 1.36 (ddd, J=40.2, 26.9, 22.6
Hz, 15H).
[0366] (S)-tert-butyl
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-57 was prepared in an analogous fashion from
(R)-tert-butyl
4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H-inden-1-ylcarbamate
INT-55.
(R)-4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-
-amine hydrochloride (Compound 65)
##STR00105##
[0368] To (R)-tert-butyl
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-56 (150 mg, 0.312 mmol) in dioxane (1 mL) was added
4N HCl in dioxane (1 mL). The mixture was stirred at room
temperature for 6 h, and product precipitated. The reaction mixture
was diluted with Et.sub.2O and the solid collected by filtration to
produce of
(R)-4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden--
1-amine hydrochloride 65 (125 mg, 96%) as an off-white solid.
LCMS-ESI (m/z): calcd for C.sub.22H.sub.25N.sub.3O.sub.3: 379.2;
found 402.1 [M+Na].sup.+, t.sub.R=2.38 min. .sup.1H NMR (400 MHz,
DMSO) .delta. 8.40 (s, 1H), 8.01 (d, J=7.6 Hz, 1H), 7.78 (d, J=7.2
Hz, 1H), 7.53 (t, J=7.7 Hz, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.90 (dt,
J=8.2, 5.1 Hz, 2H), 4.81 (s, 1H), 4.35 (s, 2H), 4.01 (p, J=6.9 Hz,
4H), 3.36 (s, 2H), 3.22-3.04 (m, 1H), 2.55-2.43 (m, 2H), 2.05 (dd,
J=14.0, 8.4 Hz, 1H), 1.32 (td, J=7.0, 4.0 Hz, 6H).
[0369]
(S)-4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H--
inden-1-amine hydrochloride INT-58 can be prepared in an analogous
fashion from (S)-tert-butyl
4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl-
carbamate INT-57.
(R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-cyano-2,3-dihydro-1H-inden-1-yl)car-
bamate (INT-59)
##STR00106##
[0371] To (R)-tert-butyl 4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate
INT-52 (0.700 g, 2.7 mmol) was added anhydrous DMF (10 mL) and the
reaction mixture was stirred in a 0.degree. C. ice bath under
N.sub.2. Sodium hydride (0.541 g, 13.5 mmol) was added and the
mixture was stirred at 0.degree. C. for 2 h. After 2 h,
(2-bromoethoxy)-tert-butyldimethylsilane (1.43 g, 5.9 mmol) was
added and the reaction mixture was allowed to warm to room
temperature for 1 h. The reaction was cooled to 0.degree. C. and
quenched with MeOH followed by saturated NaHCO.sub.3. The mixture
was extracted with EA and brine. The combined organic layers were
dried over MgSO.sub.4, filtered, and concentrated to produce a
brown oil. The crude product was purified by silica gel flash
chromatography (20% EA/Hexanes) to afford 0.868 g (77%) of
(R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-cyano-2,3-dihydro-1H-inden-1-yl)car-
bamate INT-59 as a yellow oil. LCMS-ESI (m/z) calculated for
C.sub.23H.sub.36N.sub.2O.sub.3Si: 416.6; found 317.1
[M+H-Boc].sup.+, t.sub.R=4.05 min. .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO) .delta. 7.50 (m, 1H), 7.37 (m, 1H), 7.26 (m,
1H), 5.78 (m, 1H), 4.02 (m, 2H), 3.51 (m, 2H), 3.29 (m, 1H), 2.97
(m, 1H), 2.26 (m, 2H), 1.40 (s, 9H), 0.83 (s, 9H), 0.09 (s,
6H).
(S)-tert-butyl
(4-cyano-2,3-dihydro-1H-inden-1-yl)(2-(dimethylamino)-2-oxoethyl)carbamat-
e (INT-60)
##STR00107##
[0373] (S)-tert-butyl
(4-cyano-2,3-dihydro-1H-inden-1-yl)(2-(dimethylamino)-2-oxoethyl)carbamat-
e INT-60 was prepared analogously to INT-59 from (5)-tert-butyl
4-cyano-2,3-dihydro-1H-inden-1-ylcarbamate INT-53 and
2-chloro-N,N-dimethylacetamide.
(R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-(N-hydroxy-carbamimidoyl)-2,3-dihyd-
ro-1H-inden-1-yl)carbamate (INT-61)
##STR00108##
[0375] Prepared using General Procedure 1. To (R)-tert-butyl
2-(tert-butyldimethyl
silyloxy)ethyl(4-cyano-2,3-dihydro-1H-inden-1-yl)carbamate INT-59
(0.800 g, 1.9 mmol) in EtOH (8 mL) was added hydroxylamine
hydrochloride (0.400 g, 5.8 mmol) and Na.sub.2CO.sub.3 (0.610 g,
5.8) and the reaction mixture was heated at 85.degree. C. for 12 h.
Once cooled to room temperature, the reaction mixture was filtered
using EtOH to rinse the filter cake. The filtrate was concentrated
under reduced pressure and washed with EA and brine. The combined
organic layers were dried over MgSO.sub.4, filtered, and
concentrated to produce 0.860 g (100%) of (R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-(N-hydroxycarbamimidoyl)-2,3-dihydr-
o-1H-inden-1-yl)carbamate INT-61 as a light yellow oil. LCMS-ESI
(m/z) calculated for C.sub.23H.sub.39N.sub.3O.sub.4Si: 449.7; found
350.2 [M+H-Boc].sup.+, t.sub.R=1.97 min.
(S)-tert-butyl
(2-(dimethylamino)-2-oxoethyl)(4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H--
inden-1-yl)carbamate (INT-62)
##STR00109##
[0377] (S)-tert-butyl
(2-(dimethylamino)-2-oxoethyl)(4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H--
inden-1-yl)carbamate INT-62 was prepared from (S)-tert-butyl
(4-cyano-2,3-dihydro-1H-inden-1-yl)(2-(dimethylamino)-2-oxoethyl)carbamat-
e INT-60 using General Procedure 1 and in an analogous fashion to
INT-61.
(R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-(5-(4-phenyl-5-(trifluoromethyl)thi-
ophen-2-yl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl)carbamate
(INT-63)
##STR00110##
[0379] Prepared using General Procedure 2. To a solution of
4-phenyl-5-(trifluoromethyl)thiophene-2-carboxylic acid (0.109 g,
0.4 mmol) in DMF (3.0 mL) was added HOBt (0.088 g, 0.57 mmol) and
EDC (0.109 g, 0.57 mmol) at room temperature. The reaction mixture
was stirred for 0.5 h until the complete formation of the HOBt-acid
complex. (R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-(N-hydroxycarbamimidoyl)-2,3-dihydr-
o-1H-inden-1-yl)carbamate INT-61 (0.200 g, 0.44 mmol) was added and
the mixture was stirred at room temperature for 0.5 h until the
formation of the intermediate (R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-(N-(4-phenyl-5-(trifluoromethyl)thi-
ophene-2-carbonyloxy)carbamimidoyl)-2,3-dihydro-1H-inden-1-yl)
carbamate was observed. The reaction mixture was heated at
85.degree. C. for 4 h. Upon cooling, the mixture was extracted with
DCM and brine. The combined organic layers were dried over
MgSO.sub.4, filtered, and concentrated under reduced pressure to
produce a brown oil. The crude product was purified by silica gel
flash chromatography (MeOH/DCM) to yield 0.108 g (40%) of
(R)-tert-butyl 2-(tert-butyldimethylsilyloxy)
ethyl(4-(5-(4-phenyl-5-(trifluoromethyl)thiophen-2-yl)-1,2,4-oxa-diazol-3-
-yl)-2,3-dihydro-1H-inden-1-yl)carbamate INT-63 as a light yellow
oil. LCMS-ESI (m/z) calculated for
C.sub.35H.sub.42F.sub.3N.sub.3O.sub.4SSi: 685.9; found 411.0
[M+H-tert-butyl
2-(tert-butyldimethylsilyloxy)ethylcarbamate].sup.+, t.sub.R=4.01
min.
(S)-tert-butyl
(4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-y-
l)(2-(dimethylamino)-2-oxoethyl)carbamate (INT-64)
##STR00111##
[0381] (S)-tert-butyl
(4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-y-
l)(2-(dimethylamino)-2-oxoethyl)carbamate INT-64 was prepared using
General Procedure 2, analogously to INT-63, from (S)-tert-butyl
(2-(dimethylamino)-2-oxoethyl)(4-(N-hydroxycarbamimidoyl)-2,3-dihydro-1H--
inden-1-yl)carbamate INT-62 and 2-(3,4-diethoxyphenyl)acetic
acid.
(R)-2-(4-(5-(4-phenyl-5-(trifluoromethyl)thiophen-2-yl)-1,2,4-oxadiazol-3--
yl)-2,3-dihydro-1H-inden-1-ylamino)ethanol (Compound 67)
##STR00112##
[0383] To (R)-tert-butyl
2-(tert-butyldimethylsilyloxy)ethyl(4-(5-(4-phenyl-5-(trifluoromethyl)thi-
ophen-2-yl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl)carbamate
INT-63 (0.108 g, 0.16 mmol) dissolved in DCM (1.5 mL) was added 2N
HCl in ether (1.45 mL, 2.9 mmol). The solution was stirred at room
temperature for 12 h. The solvent was removed under a stream of
nitrogen and the product dried under vacuum to afford 0.052 g (65%)
of
(R)-2-(4-(5-(4-phenyl-5-(trifluoromethyl)thiophen-2-yl)-1,2,4-oxadiazol-3-
-yl)-2,3-dihydro-1H-inden-1-ylamino) ethanol 67 as the HCl salt.
LCMS-ESI (m/z): calcd for C.sub.24H.sub.20F.sub.3N.sub.3O.sub.2S:
471.5; found 472.1 [M+H].sup.+, t.sub.R=7.43 min (Method 2).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.62 (s, 1H), 8.19 (d,
J=7.6, 1H), 8.03 (d, J=7.5, 1H), 7.87 (t, J=1.5, 1H), 7.53-7.40 (m,
6H), 4.86 (d, J=4.8, 1H), 3.88 (s, 2H), 3.74-3.50 (m, 1H), 3.41
(ddd, J=13.3, 9.4, 4.4, 1H), 3.06 (m, 1H), 2.98 (m, 1H), 2.67-2.42
(m, 2H). .sup.13C NMR (100 MHz, DMSO) .delta. 169.22, 168.07,
145.68, 144.75, 139.39, 135.43, 132.42, 129.42, 129.37, 129.25,
128.69, 128.27, 127.62, 126.41, 123.16, 122.37, 120.47, 61.10,
56.63, 46.54, 31.66, 27.80.
(S)-2-((4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-ind-
en-1-yl)amino)-N,N-dimethylacetamide (Compound 66)
##STR00113##
[0385]
(S)-2-((4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-
-1H-inden-1-yl)amino)-N,N-dimethylacetamide 66 was prepared
analogously to compound 67 from (S)-tert-butyl
(4-(5-(3,4-diethoxybenzyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-y-
l)(2-(dimethylamino)-2-oxoethyl)carbamate INT-64.
tert-butyl 5-cyano-1H-indole-1-carboxylate (INT-65)
##STR00114##
[0387] To a flask containing 5-cyanoindole (500 mg, 3.52 mmol) in
CH.sub.3CN (5 mL) was added Boc.sub.2O (920 mg, 4.22 mmol) and DMAP
(42 mg, 0.35 mmol) and the mixture was stirred at room temperature
for 0.5 h. The mixture was concentrated, redissolved in DCM and
chromatographed (EtOAc/hexanes) to provide 766 mg (90%) of
tert-butyl 5-cyano-1H-indole-1-carboxylate INT-65 as a white solid.
LCMS-ESI (m/z) calculated for C.sub.14H.sub.14N.sub.2O.sub.2:
242.27; found 243.1 [M+H].sup.+, t.sub.R=3.93 min.
tert-butyl 5-(N-hydroxycarbamimidoyl)-1H-indole-1-carboxylate
(INT-66)
##STR00115##
[0389] Prepared using General Procedure 1. To a flask containing
tert-butyl 5-cyano-1H-indole-1-carboxylate INT-65 (200 mg, 0.73
mmol) was added EtOH (6 mL), hydroxylamine hydrochloride (177 mg,
2.54 mmol) and Na.sub.2CO.sub.3 (154 mg, 1.45 mmol). The mixture
was stirred at 75.degree. C. overnight then concentrated,
re-dissolved in DCM and washed with NaHCO.sub.3. The combined
organic layers were dried over Na.sub.2SO.sub.4 and concentrated to
provide 222 mg of crude tert-butyl
5-(N-hydroxycarbamimidoyl)-1H-indole-1-carboxylate INT-66 as a
white solid which was used directly in the next experiment.
LCMS-ESI (m/z) calculated for C.sub.14H.sub.17N.sub.3O.sub.3:
275.3; found 276.1 [M+H].sup.+, t.sub.R=2.25 min.
tert-butyl
5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1H-indo-
le-1-carboxylate (INT-67)
##STR00116##
[0391] Prepared using General Procedure 2. A flask containing
3-cyano-4-isopropoxybenzoic acid (135 mg, 0.66 mmol), HOBt (130 mg,
0.85 mmol) and EDC (164 mg, 0.85 mmol) in DMF (2.5 mL) was stirred
for 1.5 h at room temperature under an atmosphere of N.sub.2. A
solution of crude tert-butyl
5-(N-hydroxycarbamimidoyl)-1H-indole-1-carboxylate INT-66 (199 mg,
0.72 mmol) in DMF (2.5 mL) was added to the mixture. After 1 h at
room temperature, the mixture was heated to 75.degree. C. and
stirred overnight. The reaction mixture was diluted with
NaHCO.sub.3 and extracted with EtOAc. The combined organic extracts
were dried over Na.sub.2SO.sub.4 and concentrated. The resulting
crude material was chromatographed (EtOAc/hexanes) to provide 174
mg (59%) of tert-butyl
5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1H-indole-1-carbo-
xylate INT-67 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.25H.sub.24N.sub.4O.sub.4: 444.5; found 445.1 [M+H].sup.+,
t.sub.R=3.67 min (Method 1).
5-(3-(1H-indol-5-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
(Compound 68)
##STR00117##
[0393] To a flask containing tert-butyl
5-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-1H-indole-1-carbo-
xylate INT-67 (75 mg, 0.17 mmol) was added dioxane (2 mL) followed
by 4N HCl in dioxane (0.5 mL, 2 mmol). The reaction mixture was
stirred overnight at room temperature then heated at 50.degree. C.
overnight. Additional 4N HCl/dioxane (0.5 mL, 2 mmol) was added and
the mixture was heated at 50.degree. C. for an additional 2 h to
complete the deprotection. The reaction mixture was diluted with
EtOAc and washed with NaHCO.sub.3. The combined organic layers were
washed with brine, dried over Na.sub.2SO.sub.4, and concentrated.
The material was purified by chromatography (EtOAc/hexanes) to
provide 17 mg (30%) of
5-(3-(1H-indol-5-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
68 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.20H.sub.16N.sub.4O.sub.2: 344.5; found 345.1 [M+H].sup.+,
t.sub.R=2.34 min (Method 1). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.51-8.47 (m, 1H), 8.45 (d, J=2.2 Hz, 1H), 8.38 (d, J=7.2
Hz, 1H), 8.35 (dd, J=8.9, 2.2 Hz, 1H), 7.99 (dd, J=8.5, 1.6 Hz,
1H), 7.51 (d, J=8.5 Hz, 1H), 7.33-7.28 (m, 1H), 7.11 (d, J=9.0 Hz,
1H), 6.72-6.64 (m, 1H), 4.79 (dt, J=12.2, 6.1 Hz, 1H), 1.47 (t,
J=5.8 Hz, 6H).
N-hydroxybenzofuran-5-carboximidamide (INT-68)
##STR00118##
[0395] Prepared using General Procedure 1. To a flask containing
benzofuran-5-carbonitrile (200 mg, 0.73 mmol) was added EtOH (6
mL), hydroxylamine hydrochloride (176.7 mg, 2.54 mmol) and
Na.sub.2CO.sub.3 (154 mg, 1.42 mmol). The mixture was stirred at
75.degree. C. overnight then concentrated, re-dissolved in DCM and
washed with NaHCO.sub.3. The combined organic layers were dried
over Na.sub.2SO.sub.4, and concentrated to provide 222 mg of crude
N-hydroxybenzofuran-5-carboximidamide INT-68 as a white solid which
was used directly in the next step without purification. LCMS-ESI
(m/z) calculated for C.sub.9H.sub.8N.sub.2O.sub.2: 176.2; found
177.1 [M+H].sup.+, t.sub.R=0.83 min.
5-(3-(benzofuran-5-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
(Compound 69)
##STR00119##
[0397] Prepared using General Procedure 2. A flask containing
3-cyano-4-isopropoxybenzoic acid (147.7 mg, 0.72 mmol), HOBt (143
mg, 0.94 mmol) and EDC (180 mg, 0.94 mmol) in DMF (2.0 mL) was
stirred for 0.5 h at room temperature under an atmosphere of
N.sub.2. A solution of N-hydroxybenzofuran-5-carboximidamide INT-68
(218 mg, 0.79 mmol) in DMF (2.0 mL) was added to the mixture. After
1 h at room temperature, the mixture was stirred at 85.degree. C.
overnight. The reaction mixture was diluted with NaHCO.sub.3 and
extracted with EA. The combined organic extracts were dried over
Na.sub.2SO.sub.4, and concentrated. The resulting crude material
was chromatographed (EA/hexanes) to provide 110 mg (44%) of
5-(3-(benzofuran-5-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
69 as a white solid. LCMS-ESI (m/z) calculated for
C.sub.20H.sub.15N.sub.3O.sub.3: 345.4; found 346.1 [M+H].sup.+,
t.sub.R=2.77 min (Method 1). .sup.1HNMR (400 MHz, CDCl.sub.3)
.delta. 8.45 (dd, J=4.5, 1.9 Hz, 2H), 8.35 (dd, J=8.9, 2.2 Hz, 1H),
8.12 (dd, J=8.6, 1.7 Hz, 1H), 7.71 (d, J=2.2 Hz, 1H), 7.63 (d,
J=8.6 Hz, 1H), 7.13 (d, J=9.0 Hz, 1H), 6.88 (dd, J=2.2, 0.8 Hz,
1H), 4.80 (s, 1H), 1.48 (d, J=6.1 Hz, 6H).
N-hydroxy-3-methylisonicotinimidamide (INT-69)
##STR00120##
[0399] Prepared using General Procedure 1. To
3-methylisonicotinonitrile (0.500 g, 4.2 mmol) in EtOH (7 mL) was
added hydroxylamine hydrochloride (0.588 g, 8.5 mmol) and
Na.sub.2CO.sub.3 (1.34 g, 12.7 mmol) and the reaction mixture was
heated at 85.degree. C. for 4 h. Once cooled to room temperature,
the reaction mixture was filtered using EtOH to rinse the filter
cake. The filtrate was concentrated under reduced pressure. The
resulting pale yellow solid was triturated with ice water (50 mL),
filtered, and the solid was washed with ice water (5 mL). The solid
was dried under reduced pressure to yield 0.47 g (74%) of
N-hydroxy-3-methylisonicotinimidamide INT-69 as a white powder.
LCMS-ESI (m/z) calculated for C.sub.7H.sub.9N.sub.3O: 151.2; found
152.1 [M+H].sup.1, t.sub.R=0.56 min. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.43-8.32 (m, 2H), 7.34 (d, J=5.0, 1H), 2.39
(s, 3H).
2-isopropoxy-5-(3-(3-methylpyridin-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile
(Compound 70)
##STR00121##
[0401] Prepared using General Procedure 2. To a solution of
3-cyano-4-isopropoxybenzoic acid (0.122 g, 0.60 mmol) in DMF (1.5
mL) was added HOBt (0.132 g, 0.86 mmol) and EDC (0.165 g, 0.86
mmol) at room temperature. The reaction was stirred for 0.5 h until
the complete formation of the HOBt-acid complex.
N-hydroxy-3-methylisonicotinimidamide INT-69 (0.100 g, 0.66 mmol)
was added and the mixture was stirred at room temperature for 0.5 h
until formation of the intermediate
N-(3-cyano-4-isopropoxybenzoyloxy)-3-methylisonicotinimidamide was
observed. The reaction mixture was then heated at 80.degree. C. for
4 h. Upon cooling, the mixture was extracted with DCM and brine.
The combined organic layers were dried over MgSO.sub.4, filtered,
and concentrated under reduced pressure to produce a brown oil. The
crude product recrystallized from MeOH (3 mL) and the resulting
crystals were filtered and washed with cold MeOH to yield product
as a white crystalline solid. To the product was added Et.sub.2O
(0.5 mL) followed by 2N HCl in Et.sub.2O (0.6 mL). The mixture was
stirred at room temperature for 10 minutes then dried under
nitrogen and subsequently under vacuum to afford 0.087 g (45%) of
2-isopropoxy-5-(3-(3-methylpyridin-4-yl)-1,2,4-oxadiazol-5-yl)benzonitril-
e 70 as the HCl salt. LCMS-ESI (m/z) calculated for
C.sub.18H.sub.16N.sub.4O.sub.2: 320.3; found 321.1 [M+H].sup.+,
t.sub.R=8.82 min (Method 2). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.80 (d, J=17.5, 2H), 8.70 (d, J=5.7, 1H), 8.44 (d, J=2.2,
1H), 8.36 (dd, J=8.9, 2.2, 1H), 7.18 (d, J=9.1, 1H), 4.83 (dt,
J=12.2, 6.1, 1H), 2.95 (s, 3H), 1.49 (d, J=6.1, 6H). .sup.13C NMR
(101 MHz, DMSO) .delta. 173.90, 166.58, 162.81, 147.11, 142.99,
137.55, 135.01, 134.79, 134.06, 125.00, 115.42, 115.17, 115.00,
102.57, 72.66, 21.48, 18.69.
4-bromo-2-((tert-butyldimethylsilyloxy)methyl)pyridine (INT-70)
##STR00122##
[0403] To a stirring solution of (4-bromopyridin-2-yl)methanol
(1.50 g, 8.0 mmol) in DCM (4 mL) was added
tert-butylchlorodimethylsilane (1.20 g, 8.0 mmol) following by TEA
(1.60 g, 12.0 mmol). The reaction mixture was stirred at room
temperature for 12 h then washed with brine and EA. The combined
organic layers were dried over MgSO.sub.4, filtered, and
concentrated to yield an amber liquid. The crude product was
purified by chromatography (EA/Hexanes) to produce 1.67 g (70%) of
4-bromo-2-((tert-butyldimethylsilyloxy)methyl)pyridine INT-70 as a
light yellow liquid. LCMS-ESI (m/z) calculated for
C.sub.12H.sub.20BrNOSi: 302.3; found 303.0 [M+H].sup.+,
t.sub.R=4.87 min (Method 1). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.30 (d, J=5.3, 1H), 7.68 (dd, J=1.9, 0.7, 1H), 7.35-7.24
(m, 1H), 4.80 (s, 2H), 0.99-0.86 (m, 9H), 0.16-0.06 (m, 6H).
2-((tert-butyldimethylsilyloxy)methyl)isonicotinonitrile
(INT-71)
##STR00123##
[0405] To a stirring solution of
4-bromo-2-((tert-butyldimethylsilyloxy)methyl)pyridine INT-70
(0.800 g, 2.6 mmol) in 3 mL of 1-methyl-2-pyrrolidine (NMP) was
added zinc cyanide (0.610 g, 5.2 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.060 g, 0.052 mmol). The
solution was degassed with N.sub.2 and the reaction mixture heated
at 95.degree. C. for 12 h. Upon cooling, the reaction mixture was
diluted with saturated NaHCO.sub.3 and extracted with DCM. The
combined organic layers were dried over MgSO.sub.4, filtered, and
concentrated. The crude product was purified by chromatography
(MeOH/DCM) to produce 0.170 g (26%) of
2-((tert-butyldimethylsilyloxy)methyl)isonicotinonitrile INT-71 as
a light yellow solid. LCMS-ESI (m/z) calculated for
C.sub.13H.sub.20N.sub.2OSi: 248.4; found 249.1 [M+H].sup.+,
t.sub.R=4.21 min (Method 1).
2-((tert-butyldimethylsilyloxy)methyl)-N-hydroxyisonicotinimidamide
(INT-72)
##STR00124##
[0407] Prepared using General Procedure 1. To
2-((tert-butyldimethylsilyloxy) methyl)isonicotinonitrile INT-71
(0.169 g, 0.68 mmol) in EtOH (8 mL) was added hydroxylamine
hydrochloride (0.142 g, 2.0 mmol) and Na.sub.2CO.sub.3 (0.216 g,
2.0 mmol) and the reaction mixture was heated at 85.degree. C. for
12 h. Once cooled to room temperature, the reaction mixture was
filtered using EtOH to rinse the filter cake. The filtrate was
concentrated under reduced pressure and washed with EA and brine.
The combined organic layers were dried over MgSO.sub.4, filtered,
and concentrated to produce 0.191 g (100%) of
2-((tert-butyldimethylsilyloxy)methyl)-N-hydroxyisonicotinimidamide
INT-72 as a light yellow oil. LCMS-ESI (m/z) calculated for
C.sub.13H.sub.23N.sub.3O.sub.2Si: 281.4; found 282.1 [M+H].sup.+,
t.sub.R=2.76 min (Method 1). .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO) .delta. 8.50 (dd, J=5.2, 0.7, 1H), 7.74 (dd,
J=1.6, 0.7, 1H), 7.51 (dd, J=5.2, 1.7, 1H), 5.98 (s, 2H), 0.96-0.89
(m, 9H), 0.14-0.07 (m, 6H).
5-(3-(2-(hydroxymethyl)pyridin-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxy-be-
nzonitrile (Compound 71)
##STR00125##
[0409] Prepared using General Procedure 2. To a solution of
3-cyano-4-isopropoxybenzoic acid (0.033 g, 0.16 mmol) in DMF (1.0
mL) was added HOBt (0.036 g, 0.23 mmol) and EDC (0.045 g, 0.23
mmol) at room temperature. The reaction was stirred for 0.5 h until
the complete formation of the HOBt-acid complex.
2-((Tert-butyldimethylsilyloxy)methyl)-N-hydroxyisonicotinimidamide
INT-72 (0.050 g, 0.18 mmol) was added and the mixture was stirred
at room temperature for 0.5 h until the formation of the
intermediate
2-((tert-butyldimethylsilyloxy)methyl)-N-(3-cyano-4-isopropoxy
benzoyloxy) isonicotinimidamide was observed. The reaction mixture
was then heated at 85.degree. C. for 4 h. To the cooled reaction
mixture MeOH (1.0 mL) was added, and the solution was filtered. The
resulting filtrate was purified by preparative HPLC to produce 5.6
mg (8%) of
5-(3-(2-(hydroxymethyl)pyridin-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybe-
nzonitrile 71 as the TFA salt. LCMS-ESI (m/z) calculated for
C.sub.18H.sub.16N.sub.4O.sub.3: 336.3; found 337.1 [M+H].sup.+,
t.sub.R=7.45 min (Method 2). .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 8.78 (d, J=5.5, 1H), 8.48 (dd, J=10.6, 8.3, 1.4, 3H), 8.23
(dd, J=5.5, 1.6, 1H), 7.48 (d, J=9.0, 1H), 4.93 (s, 2H), 4.89 (m,
1H), 1.48 (d, J=6.1, 6H).
[0410] Selected compounds and their corresponding analytical data
is shown in Table 1, where the LCMS data was collected using Method
2 (see General Methods). The enantiomeric purity was determined for
key intermediates and selected final compounds and is presumed from
the synthesis for the remaining compounds.
TABLE-US-00001 TABLE 1 LCMS COMPOUND RETENTION STRUCTURE NUMBER
TIME (min) ##STR00126## 1 9.32 ##STR00127## 2 9.32 ##STR00128## 3
6.35 ##STR00129## 4 6.34 ##STR00130## 5 9.21 ##STR00131## 6 9.20
##STR00132## 7 8.09 ##STR00133## 8 8.08 ##STR00134## 9 8.25
##STR00135## 10 8.26 ##STR00136## 11 9.53 ##STR00137## 12 9.53
##STR00138## 13 8.16 ##STR00139## 14 8.16 ##STR00140## 15 9.01
##STR00141## 16 9.03 ##STR00142## 17 8.55 ##STR00143## 18 8.56
##STR00144## 19 8.31 ##STR00145## 20 6.45 ##STR00146## 21 8.90
##STR00147## 22 8.89 ##STR00148## 23 9.37 ##STR00149## 24 9.36
##STR00150## 25 6.56 ##STR00151## 26 8.82 ##STR00152## 27 8.8
##STR00153## 28 9.41 ##STR00154## 29 9.36 ##STR00155## 30 9.87
##STR00156## 31 9.83 ##STR00157## 32 9.68 ##STR00158## 33 9.66
##STR00159## 34 8.83 ##STR00160## 35 8.84 ##STR00161## 36 8.73
##STR00162## 37 8.76 ##STR00163## 38 8.47 ##STR00164## 39 8.49
##STR00165## 40 9.09 ##STR00166## 41 9.07 ##STR00167## 42 9.00
##STR00168## 43 9.02 ##STR00169## 44 6.74 ##STR00170## 45 6.82
##STR00171## 46 6.69 ##STR00172## 47 6.58 ##STR00173## 48 6.55
##STR00174## 49 6.54 ##STR00175## 50 6.36 ##STR00176## 51 6.40
##STR00177## 52 6.13 ##STR00178## 53 6.52 ##STR00179## 54 6.71
##STR00180## 55 6.76 ##STR00181## 56 8.63 ##STR00182## 57 6.16
##STR00183## 58 6.34 ##STR00184## 59 5.85 ##STR00185## 60 8.56
##STR00186## 61 6.07 ##STR00187## 62 6.22 ##STR00188## 63 6.33
##STR00189## 64 6.43 ##STR00190## 65 6.00 ##STR00191## 66 6.23
##STR00192## 67 7.40 ##STR00193## 68 9.66 ##STR00194## 69 10.74
##STR00195## 70 8.81 ##STR00196## 71 7.44
Biological Assays
Assay Procedures
[0411] Generation of S1P.sub.1-Mediated Inhibition of cAMP Reporter
Assay
[0412] A mammalian expression plasmid containing S1P.sub.1/EDG1
cloned into pcDNA3.1 was purchased from Missouri S&T cDNA
Resource Centre. The nucleotide and amino acid sequence of human
S1P.sub.1/EDG1 are published in Hla and Maciag (J Biol Chem, 265
(1990), 9308-9313). S1P.sub.1/pcDNA3.1 was transfected into the
CRE-bla CHO K1 (Invitrogen) cell line, and stable single cell
clones were selected using standard techniques. Expression of
functional S1P.sub.1/EDG1 receptor was confirmed by cell surface
FACS with a S1P.sub.1 antibody (R&D Systems, clone 218713) and
S1P-mediated inhibition of Forskolin induced cAMP.
S1P.sub.1, CRE-Bla CHOK1 Reporter Assay--Characterization of
S1P.sub.1 Agonists
[0413] Cells were seeded into 384-well black wall/clear bottom
plates at 10.sup.4 cells/well/19.5 .mu.l assay media (DMEM-phenol
free, 0.5% charcoal/dextran stripped serum, 2 mM glutamine, 0.1 mM
NEAA, 1 mM Na-Pyruvate, 25 mM Hepes) and incubated for 18 hrs at
37.degree. C. in 5% CO.sub.2. Dose response curves (10-point) were
generated in 10 mM Hepes, 0.1% Pluronic F127, in the presence of
Forskolin. Cells were treated with 0.5 .mu.l compound in the
presence of 2 .mu.M Forskolin for 4 hrs at 37.degree. C. The
FRET-based .beta.-lactamase fluorescent substrate
(LiveBLAzer.TM.-FRET B/G Loading Kit CCC4-AM; Invitrogen) was
prepared according to manufacturer's directions, and incubated with
cells for 2 hrs at room temperature. Plates were read at
Ex:410/Em:458 and Ex:410/Em:522, and the response ratio determined.
Data was analyzed by non-linear regression to determine the EC50
for inhibition of Forskolin induced cAMP.
Specificity Over Other S1P Receptors
[0414] To assess compound specificity on other S1P receptors the
following cell lines were used: S1P.sub.2 CRE-bla CHOK1,
S1P.sub.3-G.alpha.15 NFAT-bla HEK293T (Invitrogen), S1P.sub.4-bla
TANGO U2OS (Invitrogen), S1P.sub.5-bla TANGO U2OS (Invitrogen). The
same assay set up for S1P.sub.1 was used but without Forskolin.
S1P.sub.4 and S1P.sub.5 assays were performed in FreeStyle
Expression medium (Invitrogen). S1P.sub.5 cells were incubated for
48 hrs in prior to treatment with compound.
Reported S1P.sub.1 Activity
[0415] Activity data for selected S1P.sub.1 agonists is displayed
in Table 2. The activity range is denoted as follows: ++++ denotes
agonist activity <0.05 nM. +++ denotes agonist activity between
0.05 to 0.50 nM, and ++ denotes agonist activity between 0.50-5.00
nM, and + denotes agonist activity >5.00 nM. N/A denotes not
available.
TABLE-US-00002 TABLE 2 COMPOUND S1P.sub.1 NUMBER ACTIVITY 1 +++ 2
++++ 3 ++ 4 +++ 5 +++ 6 +++ 7 +++ 8 +++ 9 +++ 10 +++ 11 +++ 12 +++
13 +++ 14 +++ 15 +++ 16 ++ 17 ++ 18 ++ 19 ++ 20 ++ 21 +++ 22 +++ 23
++ 24 +++ 25 ++ 26 +++ 27 ++++ 28 +++ 29 +++ 30 +++ 31 ++ 32 +++ 33
+++ 34 ++++ 35 ++++ 36 ++++ 37 ++++ 38 ++++ 39 ++++ 40 ++++ 41 ++++
42 ++++ 43 ++++ 44 +++ 45 ++ 46 ++ 47 +++ 48 +++ 49 +++ 50 +++ 51
+++ 52 ++++ 53 +++ 54 ++ 55 +++ 56 ++ 57 ++ 58 + 59 + 60 + 61 + 62
+ 63 + 64 + 65 + 66 + 67 + 68 ++ 69 ++ 70 ++++ 71 +++
[0416] S1P.sub.1-S1P.sub.5 data for specific compounds is presented
in Table 3. The agonist values (EC.sub.50) are reported in nM.
TABLE-US-00003 TABLE 3 COMPOUND NUMBER S1P.sub.1 S1P.sub.2
S1P.sub.3 S1P.sub.4 S1P.sub.5 8 0.143 >10000 >10000 >10000
108.9 13 0.100 >10000 >10000 >10000 77.0 29 0.065
>10000 >10000 >10000 37.8 33 0.192 >10000 >10000
616.7 260.1 37 0.024 1437 >10000 879.4 3.5 49 0.104 >10000
>10000 >10000 94.6
In Vivo Assays
Determination of Absolute Oral Bioavailability in Rats.
[0417] Pharmacokinetic studies are conducted in non-fasted female
Sprague-Dawely rats (Simonsen Laboratories or Harlan Laboratories).
Rats are housed in an ALAAC accredited facility and the research is
approved by the facilities Institutional Animal Care and Use
Committee (IACUC). The animals are acclimated to the laboratory for
at least 48 h prior to initiation of experiments.
[0418] Compounds are formulated in 5% DMSO/5% Tween20 and 90%
purified water (intravenous infusion) or 5% DMSO/5% Tween20 and 90%
0.1N HCL (oral gavage). The concentration of the dosing solutions
is verified by HPLC-UV. For intravenous dosing, compounds are
administered by an infusion pump into the jugular vein over one
minute to manually restrained animals (n=4 rats/compound). Oral
dosing is by gavage using a standard stainless steel gavage needle
(n=2-4 rats/compound). For both routes of administration, blood is
collected at eight time-points after dosing with the final sample
drawn 24 h post dose. Aliquots of the blood samples are transferred
to polypropylene 96-well plate and frozen at -20.degree. C. until
analysis.
[0419] After thawing the blood samples at room temperature, 5 .mu.L
of DMSO is added to each well. Proteins are precipitated by adding
150 .mu.L acetonitrile containing 200 nM internal standard
(4-hydroxy-3-(alpha-iminobenzyl)-1-methyl-6-phenylpyrindin-2-(1H)-one)
and 0.1% formic acid. Plates are mixed for 1 min on a plate shaker
to facilitate protein precipitation and then centrifuged at 3,000
rpm for 10 min to pellet protein. The supernatant is transferred to
a clean plate and centrifuged at 3,000 rpm for 10 min to pellet any
remaining solid material prior to LC/MS/MS analysis. Calibration
curve standards are prepared by spiking 54 compound stock in DMSO
into freshly collected EDTA rat blood. An eight point standard
curve spanning a range of 5 nM to 10,000 nM is included with each
bio-analytical run. The standards are processed identically to the
rat pharmacokinetic samples.
[0420] Concentrations in the rat pharmacokinetic samples are
determined using a standardized HPLC-LC/MS/MS method relative to
the eight point standard curve. The system consists of a Leap CTC
Pal injector, Agilent 1200 HPLC with binary pump coupled with an
Applied Biosystems 3200 QTrap. Compounds are chromatographed on a
Phenomenex Synergy Fusion RP 20.times.2 mm 2 um Mercury Cartridge
with Security Guard. A gradient method is used with mobile phase A
consisting of 0.1% formic acid in water and mobile phase B
consisting of 0.1% formic acid in acetonitrile at flow rates
varying from 0.7 to 0.8 mL/min. Ions are generated in positive
ionization mode using an electrospray ionization (ESI) interface.
Multiple reaction monitoring (MRM) methods are developed specific
to each compound. The heated nebulizer is set at 325.degree. C.
with a nebulizer current of 4.8 .mu.A. Collision energies used to
generate daughter ions range between 29 and 39 V. Peak area ratios
obtained from MRM of the mass transitions specific for each
compound are used for quantification. The limit of quantification
of the method is typically 5 nM. Data are collected and analyzed
using Analyst software version 1.4.2.
[0421] Blood and/or plasma concentration versus time data are
analyzed using non-compartmental methods (WinNonlin version 5.2;
model 200 for oral dosing and model 202 for intravenous infusion).
Absolute oral bioavailability (%) is calculated using the following
expression: (Oral AUC.times.IV Dose)/(IV AUC.times.Oral
Dose).times.100.
Lymphopenia
[0422] In mice: Female C57BL6 mice (Simonsen Laboratories, Gilroy
Calif.) are housed in an ALAAC accredited facility and the research
is approved by the facilities Institutional Animal Care and Use
Committee (IACUC). The animals are acclimated to the laboratory for
at least 5 days prior to initiation of experiments. Mice
(n=3/compound/time-point) are dosed by oral gavage with 1 mg/kg
compound formulated in a vehicle consisting of 5% DMSO/5% Tween 20
and 90% 0.1N HCl. Control mice are dosed PO with the vehicle.
Terminal whole blood samples are collected from isoflurane
anesthetized mice by cardiac puncture into EDTA. Whole blood is
incubated with rat anti-mouse CD16/CD32 (Mouse BD Fc Block,
#553141), PE-Rat anti-mouse CD45R/B220 (BD #553089), APC-Cy7-Rat
anti-mouse CD8a (BD #557654), and Alexa Fluor647-Rat anti-mouse CD4
(BD #557681) for 30 min on ice. Red blood cells are lysed using BD
Pharm Lyse Lysing buffer (#555899) and white blood cells are
analyzed by FACS. Lymphopenia is expressed as the % of white blood
cells that are CD4 or CD8 positive T cells. The overall lymphopenia
response over 24 h is estimated by calculating the area under the
effect curve (AUEC) using the linear trapezoidal rule.
[0423] In rats: Female rats (Simonsen Laboratories, Gilroy Calif.)
are housed in an ALAAC accredited facility and the research is
approved by the facilities Institutional Animal Care and Use
Committee (IACUC). The animals are acclimated to the laboratory for
at least 5 days prior to initiation of experiments. Rats
(n=3/compound/time-point) are dosed by oral gavage with 1 mg/kg
compound formulated in a vehicle consisting of 5% DMSO/5% Tween 20
and 90% 0.1N HCL. Control rats are dosed PO with the vehicle. Whole
blood is collected from isoflurane anesthetized rats via the
retro-orbital sinus and terminal samples are collected by cardiac
puncture into EDTA. Whole blood is incubated with mouse anti-rat
CD32 (BD #550271), PE-mouse anti-rat CD45R/B220 (BD #554881),
PECy5-mouse anti-rat CD4 (BD #554839), and APC-mouse anti-rat CD8a
(eBioscience #17-0084) for 30 minutes on ice. Red blood cells are
lysed using BD Pharm Lyse Lysing buffer (#555899) and white blood
cells are analyzed with a BD FACSArray. Lymphopenia is expressed as
the % of white blood cells that are CD4 or CD8 positive T cells.
The overall lymphopenia response over 24 h is estimated by
calculating the area under the effect curve (AUEC) using the linear
trapezoidal rule. In some experiments, total lymphocyte counts are
determined using a standard impediance based veterinary hematology
analyzer (IDEXX Preclinical Research Services, Sacramento,
Calif.).
Evaluation of Therapeutic Index in Rats
[0424] Studies may be conducted in non-fasted male and female
Sprague-Dawely rats (Simonsen Laboratories). Rats may be housed in
an AAALAC accredited facility and the research can be approved by
the facilities Institutional Animal Care and Use Committee (IACUC).
The animals should be acclimated to the laboratory for at least 5
days prior to initiation of experiments.
[0425] The compounds may be formulated as suspensions in a vehicle
consisting of 0.5% carboxymethyl cellulose (Acros Organics) in
purified water (pH adjusted to .about.2.2 with hydrochloric acid).
The same formulation is used in the rat lymphopenia and toxicology
studies described below. The concentration of each compound in
suspension should be verified to be within .+-.10% of the target
concentration by HPLC-UV.
[0426] Prior to the conduct of toxicology studies, the effect of
three to five daily doses of each compound on peripheral T-cell
counts of female rats may be determined (see lymphopenia
measurements in rats above). In these lymphopenia studies, blood
samples are collected onto EDTA at intervals after the final study
dose. The collection times need not be identical for each study;
however, all studies may include a sample collected 24 hours after
the final dose. The lymphopenia data is used as a biomarker to
select equally pharmacologically active doses for the subsequent
toxicology study. The low dose for the toxicology study is the dose
of each compound that resulted in a 50% reduction of T-cell count
24 h after the final dose in the lymphopenia study relative to
vehicle treated rats.
[0427] In the toxicology studies, three male and three female rats
per group are assigned to dosing groups using body weight based
randomization. A control group in each study receives vehicle. All
animals are dosed orally by gavage on 5 or 14-consecutive days at a
dose volume of 5 mL/kg/day. The animals are observed daily for any
manifestations of adverse effect. Twenty-four hours after the final
study dose, the rats are anesthetized with isoflurane and a
terminal blood sample is taken by intra-cardiac puncture for
hematology and clinical chemistry evaluation (IDEXX Laboratories,
Sacramento, Calif.). The lungs with trachea are collected, weighed,
and then prepared for histology by perfusion with 10% neutral
buffered formalin via the trachea. The internally fixed lungs are
then preserved in 10% neutral buffered formalin and submitted for
histological examination (IDEXX).
[0428] The dose of each compound resulting in a 10% increase in the
lung to terminal body weight ratio can be estimated for each
compound by linear interpolation. The therapeutic index can then be
estimated as the ratio of the dose producing 10% lung weight
increase to the dose producing 50% T-Cell depletion.
Description of the TNBS Crohn's Colitis Model in Rats
[0429] Male Sprague-Dawley rats (180-200 g) are acclimatized for
seven days and then assigned to 8 rats per group so that each group
has approximately the same mean weight. Twenty-four hours prior to
disease initiation, rats are deprived of food. Rats are
anaesthetized and weighed, then 80 mg/kg TNBS solution (50% TNBS:
50% 200 proof ethanol) is instilled into colon via a 20 g feeding
needle inserted into the anus. The rats are maintained in head down
position until recovery from anesthesia. Daily oral dosing is
initiated 2 h post TNBS-instillation for six days. Prednisolone
serves as a positive control and is administered orally daily at 10
mg/kg. Body weights are monitored daily and 24 h after the last
dose, all groups are terminated. The colon is removed, flushed of
fecal matter and examined for gross changes including strictures,
adhesions and ulcers. The colon length, weight of the distal 2 cm,
and wall thickness is recorded.
Description of Influenza A H1N1 Model in Mice
[0430] Male C57Bl/6 (6-8 weeks of age) may be acclimatized for
seven days and then assigned to 5-8 mice per group so that each
group has approximately the same mean weight. Mice may be infected
with 10.sup.4 PFUs mouse-adapted influenza A virus (A/WSN/33) via
the intra-tracheal route. Mice may then be treated with 0.2-1.5
mg/kg compound p.o. 1 hr post-infection. Forty eight hours after
infection mice may be euthanized by cervical dislocation and
bronchoalveolar lavage fluid can be collected. Quantitative
cytokine analysis may be performed via ELISA. In some experiments
whole body perfusion can be performed and lungs can be collected
for cellular enumeration of inflammatory cells. Longevity studies
may be performed by infection with 3-10.times.10.sup.4 PFUs
mouse-adapted influenza A virus over 14 days.
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