U.S. patent application number 13/034895 was filed with the patent office on 2011-09-01 for s1p receptor modulating compounds and use thereof.
This patent application is currently assigned to EPIX PHARMACEUTICALS, INC.. Invention is credited to Oren Becker, Roland Burli, Victor J. Cee, Srinivasa R. Cheruku, Kimberley Gannon, Jennifer Golden, Brian Alan Lanman, Jian Lin, Mercedes Lobera, Yael Marantz, Dilara McCauley, Susana Neira, Silvia Noiman, Pini Orbach, Robert C. Penland, Ashis Saha, Nili Schutz, Sharon Shacham, Anurag Sharadendu, Xiang Yu, Zhaoda Zhang.
Application Number | 20110212940 13/034895 |
Document ID | / |
Family ID | 46328609 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212940 |
Kind Code |
A1 |
Burli; Roland ; et
al. |
September 1, 2011 |
S1P Receptor Modulating Compounds and Use Thereof
Abstract
The present invention relates to compounds of the general
formula (I) that have activity as S1P receptor modulating agents
and the use of such compounds to treat diseases associated with
inappropriate S1P receptor activity. The compounds may be used as
immunomodulators, e.g., for treating or preventing diseases such as
autoimmune and related immune disorders including systemic lupus
erythematosus, inflammatory bowel diseases such as Crohn's disease
and ulcerative colitis, type I diabetes, uveitis, psoriasis,
myasthenia gravis, rheumatoid arthritis, non-glomerular nephrosis,
hepatitis, Behcet's disease, glomerulonephritis, chronic
thrombocytopenic purpura, hemolytic anemia, hepatitis and Wegner's
granuloma; and for treating other conditions.
Inventors: |
Burli; Roland; (Pasadena,
CA) ; Cee; Victor J.; (Thousand Oaks, CA) ;
Golden; Jennifer; (Simi Valley, CA) ; Lanman; Brian
Alan; (Oak Park, CA) ; Neira; Susana;
(Thousand Oaks, CA) ; Saha; Ashis; (Stow, MA)
; Schutz; Nili; (Tel-Aviv, IL) ; Yu; Xiang;
(Acton, MA) ; McCauley; Dilara; (Cambridge,
MA) ; Lobera; Mercedes; (Bolton, MA) ;
Marantz; Yael; (Kadima, IL) ; Lin; Jian;
(Walpole, MA) ; Cheruku; Srinivasa R.; (Lexington,
MA) ; Orbach; Pini; (Needham, MA) ;
Sharadendu; Anurag; (Washington, DC) ; Penland;
Robert C.; (Watertown, MA) ; Gannon; Kimberley;
(Watertown, MA) ; Shacham; Sharon; (Newton,
MA) ; Noiman; Silvia; (Herzliyya, IL) ;
Becker; Oren; (Mavaseret Zion, IL) ; Zhang;
Zhaoda; (Andover, MA) |
Assignee: |
EPIX PHARMACEUTICALS, INC.
Lexington
MA
|
Family ID: |
46328609 |
Appl. No.: |
13/034895 |
Filed: |
February 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11726351 |
Mar 21, 2007 |
7919519 |
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13034895 |
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11491766 |
Jul 24, 2006 |
7855193 |
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11726351 |
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60739466 |
Nov 23, 2005 |
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60753806 |
Dec 22, 2005 |
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60784549 |
Mar 21, 2006 |
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Current U.S.
Class: |
514/210.18 ;
514/210.2; 514/233.5; 514/307; 514/422; 514/469; 544/111; 544/153;
546/147; 546/196; 546/284.1; 548/205; 548/252; 548/525; 548/953;
549/469 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 9/10 20180101; C07D 413/14 20130101; A61P 37/00 20180101; C07D
409/14 20130101; C07D 405/10 20130101; A61P 19/02 20180101; C07D
491/048 20130101; A61P 11/06 20180101; A61P 17/06 20180101; A61P
35/02 20180101; A61P 43/00 20180101; C07D 307/81 20130101; A61P
1/16 20180101; A61P 7/06 20180101; C07D 307/79 20130101; A61P 9/00
20180101; A61P 37/06 20180101; A61P 25/00 20180101; A61P 29/00
20180101; C07D 403/10 20130101; A61P 17/02 20180101; C07D 405/04
20130101; A61P 3/10 20180101; A61P 1/00 20180101; C07D 405/14
20130101; A61P 13/12 20180101; C07D 413/10 20130101; C07D 471/04
20130101; C07D 417/10 20130101; A61P 35/00 20180101; C07D 417/14
20130101; C07D 307/80 20130101 |
Class at
Publication: |
514/210.18 ;
548/953; 546/196; 546/284.1; 548/205; 546/147; 514/307; 548/525;
514/422; 549/469; 514/469; 544/111; 544/153; 514/233.5; 548/252;
514/210.2 |
International
Class: |
A61K 31/397 20060101
A61K031/397; C07D 405/10 20060101 C07D405/10; A61K 31/4525 20060101
A61K031/4525; C07D 405/14 20060101 C07D405/14; A61K 31/443 20060101
A61K031/443; C07D 413/14 20060101 C07D413/14; A61K 31/427 20060101
A61K031/427; C07D 409/14 20060101 C07D409/14; A61K 31/4725 20060101
A61K031/4725; A61K 31/4025 20060101 A61K031/4025; C07D 307/80
20060101 C07D307/80; A61K 31/343 20060101 A61K031/343; A61K 31/5377
20060101 A61K031/5377; C07D 413/10 20060101 C07D413/10; A61K 31/41
20060101 A61K031/41; A61P 35/00 20060101 A61P035/00; A61P 37/06
20060101 A61P037/06; A61P 29/00 20060101 A61P029/00; A61P 3/10
20060101 A61P003/10; A61P 25/00 20060101 A61P025/00; A61P 17/06
20060101 A61P017/06; A61P 35/02 20060101 A61P035/02; A61P 11/00
20060101 A61P011/00; A61P 11/06 20060101 A61P011/06; A61P 17/02
20060101 A61P017/02; A61P 9/00 20060101 A61P009/00; A61P 9/10
20060101 A61P009/10; A61P 13/12 20060101 A61P013/12; A61P 1/16
20060101 A61P001/16; A61P 7/06 20060101 A61P007/06 |
Claims
1.-38. (canceled)
39. A method of treating a S1P-1 receptor-mediated condition
selected from the group consisting of transplant rejection, ovarian
cancer, peritoneal cancer, endometrial cancer, cervical cancer,
breast cancer, colorectal cancer, uterine cancer, stomach cancer,
small intestine cancer, thyroid cancer, lung cancer, kidney cancer,
pancreas cancer and prostrate cancer; acute lung diseases,
autoimmune disease, inflammatory diseases; rheumatoid arthritis;
lupus; insulin dependent diabetes; non-insulin dependent diabetes;
multiple sclerosis; psoriasis; ulcerative colitis; inflammatory
bowel disease; Crohn's disease; acute and chronic lymphocytic
leukemias; lymphomas; adult respiratory distress syndrome, acute
inflammatory exacerbation of chronic lung diseases, asthma, surface
epithelial cell injury, transcorneal freezing, cutaneous burns,
cardiovascular diseases, ischemia, vasoconstriction in cerebral
arteries, autoimmune and related immune disorders, systemic lupus
erythematosus, uveitis, myasthenia gravis, non-glomerular
nephrosis, hepatitis, Behcet's disease, glomerulonephritis, chronic
thrombocytopenic purpura, hemolytic anemia, hepatitis and Wegner's
granuloma, comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of the formula,
##STR00462## wherein A is phenyl optionally substituted with one,
two or three substituents selected from the group consisting of
halogen, hydroxyl, SR.sup.2, S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2,
NHS(O).sub.2R.sup.2, COR.sup.2, CO.sub.2R.sup.2, cyano, amino,
C.sub.1-5 alkylamino, arylamino, heteroarylamino, C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen-substituted
C.sub.1-6 alkyl, and halogen-substituted C.sub.1-5 alkoxy; R.sup.2
is selected from the group consisting of hydrogen, hydroxyl, amino,
alkylamino, arylamino, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5
alkylthio, halogen-substituted C.sub.1-6 alkyl, halogen-substituted
C.sub.1-5 alkoxy, aryl, and heteroaryl; B and C are selected from
the group consisting of ##STR00463## optionally substituted with 1
to 5 substituents selected from the group consisting of C.sub.1-6
alkyl, C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen, hydroxyl,
cyano, halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; R.sup.13 is selected from the group consisting of
H, C.sub.1-6alkyl, C.sub.1-5alkylthio, C.sub.1-5alkoxy, halogen,
hydroxyl, cyano, halogen-substituted C.sub.1-6alkyl, and
halogen-substituted C.sub.1-5alkoxy; X is selected from the group
consisting of WC(O)OR.sup.6a, WP(O)R.sup.6bR.sup.6c, WS(O).sub.2OH,
WCONHSO.sub.3H and 1H-tetrazol-5-yl; W is a direct bond, oxygen or
C.sub.1-4 alkyl having one or more substituents independently
selected from the group consisting of halogen, hydroxyl, cyano,
amino, alkylamino, arylamino, heteroarylamino groups, C.sub.1-4
alkoxy and CO.sub.2H; R.sup.6a is hydrogen or C.sub.1-4 alkyl;
R.sup.6b and R.sup.6c are independently hydrogen, hydroxyl,
C.sub.1-4 alkyl or halogen substituted C.sub.1-4 alkyl; Y is
formula (a) where the left and right asterisks indicate the point
of attachment ##STR00464## wherein Q is selected from the group
consisting of a direct bond, C.dbd.O, C.dbd.S, SO.sub.2, C.dbd.ONR
and (CR.sup.10R.sup.11).sub.m; m is 0, 1, 2 or 3; R.sup.7 and
R.sup.8 are independently selected from the group consisting of
hydrogen, halogen, amino, C.sub.1-5 alkylamino, hydroxyl, cyano,
C.sub.1-6 alkyl, C.sub.1-6 hydroxyalkyl (e.g., hydroxy-terminated
alkyl), C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; R.sup.9
is selected from the group consisting of hydrogen, halogen,
hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; R.sup.10 and R.sup.11 are independently selected
from the group consisting of hydrogen, halogen, hydroxyl, cyano,
C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio,
halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; Z.sup.1 is independently selected from the group
consisting of O, NR.sup.3, S, S(O), S(O).sub.2NR.sup.3,
(CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S, C.dbd.N--R.sup.3, and a
direct bond; Z.sup.2 is independently selected from the group
consisting of O, NR.sup.3, S, S(O), S(O).sub.2, S(O).sub.2NR.sup.3,
(CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S, C.dbd.N--R.sup.3, and a
direct bond; R.sup.3 is selected from the group consisting of
hydrogen, hydroxyl, SO.sub.2, C.dbd.O, C.dbd.S, C.dbd.NH, C.sub.1-6
alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy, aryl and
heteroaryl, or when Z.sup.2 is a direct bond, R.sup.3 is a
C.sub.3-C.sub.6 ring optionally containing a heteroatom; R.sup.4
and R.sup.5 are independently selected from the group consisting of
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy, aryl and heteroaryl, or
together form C.dbd.O; n is 0, 1, 2 or 3; and R.sup.1 is selected
from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy,
C.sub.1-5 alkylamino, aryl, heteroaryl, and heterocycle, wherein
the C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylamino, aryl,
and heteroaryl groups are optionally substituted with a substituent
selected from the group consisting of hydroxyl, halogen, cyano,
amino, alkylamino, aryl amino, and heteroarylamino, wherein the
aryl and heteroaryl groups are optionally substituted with 1 to 5
substituents selected from the group consisting of hydroxyl,
halogen, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, and C.sub.3-6 cycloalkyl, and wherein the heterocyclic
groups are optionally substituted with one or more substituents
selected from the group consisting of halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato,
phosphinato, cyano, amino, acylamino, amidino, imino, sulfhydryl,
alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, azido,
heterocyclyl, aryl, and heteroaryl; or a pharmaceutically
acceptable salt thereof.
40. The compound of claim 39, wherein A is substituted with one,
two or three substituents selected from the group consisting of
halogen, hydroxyl, SR.sup.2, S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2,
NHS(O).sub.2R.sup.2, COR.sup.2, CO.sub.2R.sup.2, cyano, amino,
C.sub.1-5 alkylamino/arylamino/heteroarylamino, C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen-substituted
C.sub.1-6 alkyl, and halogen-substituted C.sub.1-5 alkoxy.
41. The compound of claim 39, wherein the pharmaceutically
acceptable salt is selected from the group consisting of
hydrochloride, maleate, citrate, fumarate, succinate, tartarate,
mesylate, sodium, potassium, magnesium, and calcium salts.
42. The method according to claim 39, wherein the condition is
ovarian cancer, peritoneal cancer, endometrial cancer, cervical
cancer, breast cancer, colorectal cancer, uterine cancer, stomach
cancer, small intestine cancer, thyroid cancer, lung cancer, kidney
cancer, pancreas cancer or prostate cancer.
43. A method of treating a S1P-1 receptor-mediated condition
selected from the group consisting of transplant rejection, ovarian
cancer, peritoneal cancer, endometrial cancer, cervical cancer,
breast cancer, colorectal cancer, uterine cancer, stomach cancer,
small intestine cancer, thyroid cancer, lung cancer, kidney cancer,
pancreas cancer and prostrate cancer; acute lung diseases,
autoimmune disease, inflammatory diseases; rheumatoid arthritis;
lupus; insulin dependent diabetes; non-insulin dependent diabetes;
multiple sclerosis; psoriasis; ulcerative colitis; inflammatory
bowel disease; Crohn's disease; acute and chronic lymphocytic
leukemias; lymphomas; adult respiratory distress syndrome, acute
inflammatory exacerbation of chronic lung diseases, asthma, surface
epithelial cell injury, transcorneal freezing, cutaneous burns,
cardiovascular diseases, ischemia, vasoconstriction in cerebral
arteries, autoimmune and related immune disorders, systemic lupus
erythematosus, uveitis, myasthenia gravis, non-glomerular
nephrosis, hepatitis, Behcet's disease, glomerulonephritis, chronic
thrombocytopenic purpura, hemolytic anemia, hepatitis and Wegner's
granuloma, comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of the formula.
##STR00465## wherein the benzofuranyl ring is optionally
substituted with 1 to 5 substituents selected from the group
consisting of C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, halogen, hydroxyl, cyano, halogen-substituted C.sub.1-6
alkyl and halogen-substituted C.sub.1-5 alkoxy; A is phenyl
optionally substituted with one, two or three substituents selected
from the group consisting of halogen, hydroxyl, SR.sup.2,
S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2, NHS(O).sub.2R.sup.2,
COR.sup.2, CO.sub.2R.sup.2, cyano, amino, C.sub.1-5 alkylamino,
arylamino, heteroarylamino, C.sub.1-6 alkyl, C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl, and
halogen-substituted C.sub.1-5 alkoxy; R.sup.2 is selected from the
group consisting of hydrogen, hydroxyl, amino, alkylamino,
arylamino, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio,
halogen-substituted C.sub.1-6 alkyl, halogen-substituted C.sub.1-5
alkoxy, aryl and heteroaryl; X is --C(O)OR.sup.6a, where R.sup.6a
is hydrogen or C.sub.1-4 alkyl; Y is a residue of formula (a)
##STR00466## wherein Q is (CR.sup.10R.sup.11).sub.m; m is 0, 1, 2,
3 or 4; R.sup.7 and R.sup.8 are independently hydrogen, hydroxyl,
or lower alkyl; R.sup.9 is selected from the group consisting of
hydrogen, halogen, hydroxyl, and cyano; Z.sup.1 and Z.sup.2 are
independently O or (CR.sup.4R.sup.5).sub.n; R.sup.4 and R.sup.5 are
independently hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl,
or C.sub.1-5 alkoxy; n is 0, 1, 2 or 3; R.sup.1 is selected from
the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy,
C.sub.1-5 alkylamino, aryl heteroaryl, and heterocycle, wherein the
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6
cycloalkyl, C.sub.1-5 alkoxy, and C.sub.1-5 alkylamino groups are
optionally substituted with a substituent selected from the group
consisting of hydroxyl, halogen, cyano, amino, alkylamino,
arylamino, and heteroarylamino, wherein the aryl and heteroaryl
groups are optionally substituted with one to five substituents
selected from the group consisting of hydroxyl, halogen, cyano,
C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, and
C.sub.3-6 cycloalkyl, and wherein the heterocyclic groups are
optionally substituted with one or more substituents selected from
the group consisting of halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato,
cyano, amino, acylamino, amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, azido, heterocyclyl, aryl, and
heteroaryl; or a pharmaceutically acceptable salt thereof.
44. The compound of claim 43, wherein A is substituted with one,
two or three substituents selected from the group consisting of
halogen, hydroxyl, SR.sup.2, S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2,
NHS(O).sub.2R.sup.2, COR.sup.2, CO.sub.2R.sup.2, cyano, amino,
C.sub.1-5 alkylamino/arylamino/heteroarylamino, C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen-substituted
C.sub.1-6 alkyl, and halogen-substituted C.sub.1-5 alkoxy.
45. The compound of claim 43, wherein the pharmaceutically
acceptable salt is selected from the group consisting of
hydrochloride, maleate, citrate, fumarate, succinate, tartarate,
mesylate, sodium, potassium, magnesium, and calcium salts.
46. The method according to claim 43 wherein the condition is
ovarian cancer, peritoneal cancer, endometrial cancer, cervical
cancer, breast cancer, colorectal cancer, uterine cancer, stomach
cancer, small intestine cancer, thyroid cancer, lung cancer, kidney
cancer, pancreas cancer or prostate cancer.
47. A method of treating a S1P-1 receptor-mediated condition
selected from the group consisting of transplant rejection, ovarian
cancer, peritoneal cancer, endometrial cancer, cervical cancer,
breast cancer, colorectal cancer, uterine cancer, stomach cancer,
small intestine cancer, thyroid cancer, lung cancer, kidney cancer,
pancreas cancer and prostrate cancer; acute lung diseases,
autoimmune disease, inflammatory diseases; rheumatoid arthritis;
lupus; insulin dependent diabetes; non-insulin dependent diabetes;
multiple sclerosis; psoriasis; ulcerative colitis; inflammatory
bowel disease; Crohn's disease; acute and chronic lymphocytic
leukemias; lymphomas; adult respiratory distress syndrome, acute
inflammatory exacerbation of chronic lung diseases, asthma, surface
epithelial cell injury, transcorneal freezing, cutaneous burns,
cardiovascular diseases, ischemia, vasoconstriction in cerebral
arteries, autoimmune and related immune disorders, systemic lupus
erythematosus, uveitis, myasthenia gravis, non-glomerular
nephrosis, hepatitis, Behcet's disease, glomerulonephritis, chronic
thrombocytopenic purpura, hemolytic anemia, hepatitis and Wegner's
granuloma, comprising administering to a patient in need thereof a
therapeutically effective amount of a compound selected from the
group consisting of:
3-(4-(5-Benzylbenzofuran-2-yl)-3-fluorobenzylamino)-2-methylpropanoic
acid;
4-Amino-2-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobu-
tanoic acid;
4-Amino-3-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid; and pharmaceutically acceptable salts or hydrates thereof
48. The method according to claim 47 wherein the condition is
ovarian cancer, peritoneal cancer, endometrial cancer, cervical
cancer, breast cancer, colorectal cancer, uterine cancer, stomach
cancer, small intestine cancer, thyroid cancer, lung cancer, kidney
cancer, pancreas cancer or prostate cancer.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of copending
U.S. Ser. No. 11/726,351, filed Mar. 21, 2007, which is a
continuation-in-part of U.S. Ser. No. 11/491,766, filed Jul. 24,
2006, now U.S. Pat. No. 7,855,193, which claims priority to U.S.
Ser. No. 60/739,466, filed Nov. 23, 2005, U.S. Ser. No. 60/753,806,
filed Dec. 22, 2005, and U.S. Ser. No. 60/784,549, filed Mar. 21,
2006, and this application also claims the benefit of priority
under 35 U.S.C. 119(e) to copending U.S. Provisional Application
No. 60/784,549, filed on Mar. 21, 2006, the entire contents of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compounds that have
activity as S1P receptor modulating agents and the use of such
compounds to treat diseases associated with inappropriate S1P
receptor activity.
BACKGROUND OF THE INVENTION
[0003] Sphingosine-1-phosphate (S1P) has been demonstrated to
induce many cellular effects, including those that result in
platelet aggregation, cell proliferation, cell morphology, tumor
cell invasion, endothelial cell chemotaxis and endothelial cell in
vitro angiogenesis. S1P receptors are therefore good targets for
therapeutic applications such as wound healing and tumor growth
inhibition. S1P signals cells in part via a set of G
protein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5
(formerly called EDG-1, EDG-5, EDG-3, EDG-6, and EDG-8,
respectively). These receptors share 50-55% amino acid and cluster
identity with three other receptors (LPA1, LPA2, and LPA3 (formerly
EDG-2, EDG-4 and EDG-7) for the structurally-related
lysophosphatidic acid (LPA).
[0004] A conformational shift is induced in the G-Protein Coupled
Receptor (GPCR) when the ligand binds to that receptor, causing GDP
to be replaced by GTP on the .alpha.-subunit of the associated
G-proteins and subsequent release of the G-proteins into the
cytoplasm. The .alpha.-subunit then dissociates from the
.beta..gamma.-subunit, and each subunit can then associate with
effector proteins, which activate second messengers leading to a
cellular response. Eventually the GTP on the G-proteins is
hydrolyzed to GDP, and the subunits of the G-proteins re-associate
with each other and then with the receptor. Amplification plays a
major role in the general GPCR pathway. The binding of one ligand
to one receptor leads to the activation of many G-proteins, each
capable of associating with many effector proteins, leading to an
amplified cellular response.
[0005] S1P receptors make good drug targets, because individual
receptors are both tissue- and response-specific. Tissue
specificity of the S1P receptors is important, because development
of an agonist or antagonist selective for one receptor localizes
the cellular response to tissues containing that receptor, limiting
unwanted side effects. Response specificity of the S1P receptors is
also important because it allows for development of agonists or
antagonists that initiate or suppress certain cellular responses
without affecting other things. For example, the response
specificity of the S1P receptors could allow for an S1P mimetic
that initiates platelet aggregation without affecting cell
morphology.
[0006] S1P is formed as a metabolite of sphingosine in its reaction
with sphingosine kinase, and is abundantly stored in platelet
aggregates where high levels of sphingosine kinase exist and
sphingosine lyase is lacking. S1P is released during platelet
aggregation, accumulates in serum and is also found in malignant
ascites. S1P biodegradation most likely proceeds via hydrolysis by
ectophosphohydrolases, specifically the sphingosine 1-phosphate
phosphohydrolases.
SUMMARY OF THE INVENTION
[0007] The present invention relates to the use of new compositions
which include S1P modulators, e.g., agonists, partial agonists,
inverse agonists and antagonists, and their use in treating,
preventing or curing various S1P receptor-related conditions. The
invention features compounds which are S1P receptor modulators; in
an embodiment, such compounds include those having the formula
##STR00001##
[0008] and pharmaceutically acceptable salts thereof, wherein
R.sup.1, Z.sup.2, C, B, A, Z.sup.1, Y and X are defined herein.
DETAILED DESCRIPTION OF THE INVENTION
[0009] In one embodiment, the present invention relates to
compounds of formula I.
##STR00002##
[0010] In formula I, A may be an aryl or heteroaryl group,
optionally substituted with one, two or three substituents which
may include halogen, hydroxyl, SR.sup.2, S(O).sub.2R.sup.2,
S(O).sub.2NR.sup.2, NHS(O).sub.2R.sup.2, COR.sup.2,
CO.sub.2R.sup.2, cyano, amino, C.sub.1-5
alkylamino/arylamino/heteroarylamino, C.sub.1-6alkyl, C.sub.1-5
alkylthio, C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl,
and halogen-substituted C.sub.1-5 alkoxy. Optionally two adjacent
substituents of A may, taken with Z.sup.1 and the ring A to which
they are attached, form a fused ring that may optionally contain
one or more hetero atoms. R.sup.2 may be selected independently
from hydrogen, hydroxyl, amino, alkylamino/arylamino, C.sub.1-6
alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; or
aryl/heteroaryl. A may desirably be a C.sub.5-6 cyclic ring
(alicyclic or aromatic) optionally having one or more
heteroatoms.
[0011] B and C are an at least partially aromatic bicyclic ring
system, e.g., bicycloaryl, bicycloheteroaryl, dihydrobicyclic or
tetrahydrobicyclic aryl and heteroaryl. The bicyclic ring system
may be substituted with 1 to 5 substituents, e.g., C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen, hydroxyl, cyano,
halogen-substituted C.sub.1-6alkyl and halogen-substituted
C.sub.1-5 alkoxy.
[0012] Z.sup.1 and Z.sup.2 may be independently selected from O,
NR.sup.3, S, S(O), S(O).sub.2, S(O).sub.2NR.sup.3,
(CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S, C.dbd.N--R.sup.3, or a
direct bond. R.sup.3 may be hydrogen, hydroxyl, C.sub.1-6 alkyl,
C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; aryl or
heteroaryl. R.sup.4 and R.sup.5 may independently be hydrogen,
halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkoxy,
C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl and
halogen substituted C.sub.1-5 alkoxy; aryl or heteroaryl or
together form "C.dbd.O"; n may be 0, 1, 2 or 3. In an embodiment
where Z.sup.2 is a direct bond, R.sub.3 may be a C.sub.3-C.sub.6
ring optionally containing a heteroatom.
[0013] R.sup.1 may be C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy, C.sub.1-5
alkylamino, aryl or heteroaryl. R.sup.1 may optionally be
substituted with, e.g., hydroxyl, halogen, cyano, amino,
alkylamino, aryl amino, heteroarylamino groups, and the aryl and
heteroaryl groups may optionally be substituted with 1-5
substituents, e.g., hydroxyl, halogen, cyano, C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, C.sub.3-6 cycloalkyl.
[0014] X may be WC(O)OR.sup.6a, WP(O)R.sup.6bR.sup.6c,
WS(O).sub.2OH, WCONHSO.sub.3H or 1H-tetrazol-5-yl. W may be a
direct bond, oxygen or C.sub.1-4 alkyl with substituents
independently selected from the group consisting of: halogen,
hydroxyl, cyano, amino, alkylamino, arylamino, heteroarylamino
groups, C.sub.1-4 alkoxy and; R.sup.6a may be hydrogen or
C.sub.1-4alkyl; R.sup.6b and R.sup.6c may be hydrogen, hydroxyl,
C.sub.1-4alkyl or halogen substituted C.sub.1-4alkyl.
[0015] Y may be a residue of formula (a) where the left and right
asterisks indicate the point of attachment:
##STR00003##
[0016] wherein Q may be a direct bond, C.dbd.O, C.dbd.S, SO.sub.2,
C.dbd.ONR or (CR.sup.10R.sup.11).sub.m; m may be 0, 1, 2 or 3;
R.sup.7 and R.sup.8 may be independently hydrogen, halogen, amino,
C.sub.1-5 alkylamino, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6
hydroxyalkyl (e.g., hydroxy-terminated alkyl), C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl and
halogen-substituted C.sub.1-5 alkoxy; or R.sup.7 and R.sup.8 may be
joined together with the atoms to which they are attached to form a
4 to 7-membered ring, optionally having a hetero atom. R.sup.9 may
be hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkylthio, C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy; R.sup.10 and R.sup.11 may
individually be hydrogen, halogen, hydroxyl, cyano, C.sub.1-6
alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl or halogen-substituted C.sub.1-5 alkoxy.
[0017] In another embodiment, the invention includes compounds
having the formula
##STR00004##
[0018] and pharmaceutically acceptable salts thereof. In formula I,
A may be a C.sub.1-6 cyclic ring (alicyclic or aromatic) that may
have one or more heteroatoms. Where A is an aryl or heteroaryl
group, A may be optionally substituted with one, two or three
substituents which may include halogen, hydroxyl, S,
S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2, NHS(O).sub.2R.sup.2,
COR.sup.2, CO.sub.2R.sup.2, cyano, amino, C.sub.1-5
alkylamino/arylamino/heteroarylamino, C.sub.1-6 alkyl, C.sub.1-5
alkylthio, C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl,
and halogen-substituted C.sub.1-5 alkoxy. Optionally two adjacent
substituents may, taken with Z.sup.1 and the ring to which they are
attached, form an alicyclic or heterocyclic ring, e.g. piperidinyl.
R.sup.2 may be hydrogen, hydroxyl, amino, alkylamino/arylamino,
C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio,
halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; or aryl/heteroaryl.
[0019] B and C are an at least partially aromatic bicyclic ring
system, e.g., bicycloaryl, bicycloheteroaryl, dihydrobicyclic or
tetrahydrobicyclic aryl and heteroaryl. The bicyclic ring system
may be substituted with 1 to 5 substituents, e.g., C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, halogen, hydroxyl, cyano,
halogen-substituted C.sub.1-6alkyl and halogen-substituted
C.sub.1-5 alkoxy; Z.sup.1 and Z.sup.2 may be independently selected
from O, NR.sup.3, S, S(O), S(O).sub.2, S(O).sub.2NR.sup.3,
(CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S, C.dbd.N--R.sup.3, or a
direct bond. R.sup.3 may be hydrogen, hydroxyl, S(O).sub.2,
C.dbd.O, C.dbd.S, C.dbd.NH, C.sub.1-6 alkyl, C.sub.1-5 alkoxy,
C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl and
halogen-substituted C.sub.1-5 alkoxy; aryl or heteroaryl. R.sup.4
and R.sup.5 may independently be hydrogen, halogen, hydroxyl,
cyano, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio,
halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; aryl or heteroaryl or together form "C.dbd.O"; n
may be 0, 1, 2 or 3. R.sup.1 may be C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy,
C.sub.1-5 alkylamino, aryl or heteroaryl. R.sub.1 may optionally be
substituted with, e.g., hydroxyl, halogen, cyano, amino,
alkylamino, aryl amino, heteroarylamino groups, and the aryl and
heteroaryl groups may optionally be substituted with 1-5
substituents, e.g., hydroxyl, halogen, cyano, C.sub.1-6 alkyl,
C.sub.1-5 alkylthio, C.sub.1-5 alkoxy, C.sub.3-6 cycloalkyl.
[0020] X may be WC(O)OR.sup.6a, WP(O)R.sup.6bR.sup.6c,
WS(O).sub.2OH, WCONHSO.sub.3H or 1H-tetrazol-5-yl. W may be a
direct bond, oxygen or C.sub.1-4 alkyl with substituents
independently selected from the group consisting of: halogen,
hydroxyl, cyano, amino, alkylamino, arylamino, heteroarylamino
groups, C.sub.1-4 alkoxy and COO.sub.2H; R.sup.4a may be hydrogen
or C.sub.1-4alkyl; R.sup.6b and R.sup.6c may be hydrogen, hydroxyl,
C.sub.1-4alkyl or halogen substituted C.sub.1-4alkyl. Y has the
formula:
##STR00005##
[0021] wherein Q may be a direct bond, C.dbd.O, C.dbd.S, SO.sub.2,
C.dbd.ONR or (CR.sup.10R.sup.11).sub.m; m may be 0, 1, 2 or 3, and
R.sub.7-R.sub.8 may be hydrogen, halogen, amino, C.sub.1-5
alkylamino, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl and
halogen-substituted C.sub.1-5 alkoxy; or R.sup.7 and R.sup.8 may be
joined together with the atoms to which they are attached to form a
4 to 7 member ring. R.sup.9 may be hydrogen, halogen, hydroxyl,
cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5 alkoxy,
halogen-substituted C.sub.1-6 alkyl or halogen-substituted
C.sub.1-5 alkoxy; R.sup.10 and R.sup.11 may individually be
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy.
[0022] In another embodiment, the present invention relates to a
compound having the formula
##STR00006##
[0023] wherein
[0024] A is an optionally substituted aryl or heteroaryl group;
[0025] B and C are an at least partially aromatic bicyclic ring
system;
[0026] X is selected from the group consisting of WC(O)OR.sup.6a,
WP(O)R.sup.6bR.sup.6c, WS(O).sub.2OH, WCONHSO.sub.3H or
1H-tetrazol-5-yl; where W is a direct bond, oxygen or C.sub.1-4
alkyl having one or more substituents independently selected from
the group consisting of halogen, hydroxyl, cyano, amino,
alkylamino, arylamino, heteroarylamino groups, C.sub.1-4 alkoxy and
COO.sub.2H; R.sup.6a is hydrogen or C.sub.1-4alkyl; and R.sup.6b
and R.sup.6c are independently hydrogen, hydroxyl, C.sub.1-4alkyl
or halogen substituted C.sub.1-4alkyl;
[0027] Y is residue of formula (a) where the left and right
asterisks indicate the point of attachment
##STR00007##
[0028] wherein
[0029] Q is selected from C.dbd.O, C.dbd.S, SO.sub.2, C.dbd.ONR and
(CR.sup.10R.sup.11).sub.m;
[0030] m is 0, 1, 2 or 3;
[0031] R.sup.7 and R.sup.8 are independently selected from the
group consisting of hydrogen, halogen, amino, C.sub.1-5 alkylamino,
hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 hydroxyalkyl (e.g.,
hydroxy-terminated alkyl), C.sub.1-5 alkylthio, C.sub.1-5 alkoxy,
halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; or R.sup.7 and R.sup.8 may be joined together
with the atoms to which they are attached to form a 4 or 5-membered
ring; and
[0032] R.sup.9 is selected from the group consisting of hydrogen,
halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy;
[0033] R.sup.10 and R.sup.11 are independently selected from the
group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6
alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl or halogen-substituted C.sub.1-5 alkoxy and
[0034] Z.sup.1 is selected from O, NR.sup.3, S, S(O), S(O).sub.2,
S(O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S and
C.dbd.N--R.sup.3; and Z.sup.2 is selected from O, NR.sup.3, S,
S(O), S(O).sub.2, S(O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n,
C.dbd.O, C.dbd.S and C.dbd.N--R.sup.3; wherein
[0035] R.sup.3 is selected from the group consisting of hydrogen,
hydroxyl, SO.sub.2, C.dbd.O, C.dbd.S, C.dbd.NH, C.sub.1-6 alkyl,
C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; aryl or
heteroaryl, or when Z.sup.2 is a direct bond, R.sub.3 is a
C.sub.3-C.sub.6 ring optionally containing a heteroatom;
[0036] R.sup.4 and R.sup.5 are independently selected from the
group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6
alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; aryl or
heteroaryl or together form C.dbd.O; and
[0037] n is 0, 1, 2 or 3; and
[0038] R.sup.1 is selected from the group consisting of C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
C.sub.1-5 alkoxy, C.sub.1-5 alkylamino, aryl or heteroaryl.
[0039] In another embodiment, the invention includes compounds of
formula (II):
##STR00008##
[0040] wherein A may be an aryl or heteroaryl group; X is
--C(O)OR.sup.6a, where R.sup.6a is hydrogen or C.sub.1-4alkyl; Y is
a residue of formula (a)
##STR00009##
[0041] wherein Q is (CR.sup.10R.sup.11).sub.m is 0, 1, 2, 3 or 4;
R.sup.7 and R.sup.8 may independently be hydrogen, hydroxyl, lower
alkyl; or R.sup.7 and R.sup.8, taken with the atoms to which they
are attached, form a ring; R.sup.9 is selected from, e.g.,
hydrogen, halogen, hydroxyl, or cyano; and Z.sup.1 and Z.sup.2 are
independently O or (CR.sup.4R.sup.5).sub.n, where R.sup.4 and
R.sup.5 are independently hydrogen, halogen, hydroxyl, cyano,
C.sub.1-6 alkyl, C.sub.1-5 alkoxy; n is 0, 1, 2 or 3; and R.sup.1
is selected from, e.g., C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy,
C.sub.1-5 alkylamino, aryl or heteroaryl; or a pharmaceutically
acceptable salts thereof.
[0042] The aryl or heteroaryl group may be substituted with one,
two or three substituents such as halogen, hydroxyl, S,
S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2, NHS(O).sub.2R.sup.2,
COR.sup.2, CO.sub.2R.sup.2, cyano, amino, C.sub.1-5
alkylamino/arylamino/heteroarylamino, C.sub.1-6 alkyl, C.sub.1-5
alkylthio, C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl,
or halogen-substituted C.sub.1-5 alkoxy (where R.sup.2 is, e.g., of
hydrogen, hydroxyl, amino, alkylamino/arylamino, C.sub.1-6 alkyl,
C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; or
aryl/heteroaryl; or optionally, two adjacent substituents on A may,
taken with Z.sup.1 and the ring to which they are attached, form an
alicyclic or heterocyclic ring. R.sup.2 may be selected from
hydrogen, hydroxyl, amino, alkylamino/arylamino, C.sub.1-6 alkyl,
C.sub.1-5 alkoxy, C.sub.1-5 alkylthio, halogen-substituted
C.sub.1-6 alkyl and halogen-substituted C.sub.1-5 alkoxy; or
aryl/heteroaryl.
[0043] The benzofuranyl ring may be substituted with 1 to 5
substituents, e.g., of C.sub.1-6 alkyl, C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen, hydroxyl, cyano, halogen-substituted
C.sub.1-6 alkyl or halogen-substituted C.sub.1-5 alkoxy. R.sup.1
may be C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.3-6 cycloalkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylamino, aryl
or heteroaryl; R.sub.1 may optionally substituted with, e.g.,
hydroxyl, halogen, cyano, amino, alkylamino, arylamino, or
heteroarylamino groups. (The aryl and heteroaryl groups may be
substituted with one to five substituents such as hydroxyl,
halogen, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, and C.sub.3-6 cycloalkyl.
[0044] The present invention relates, in one embodiment, to
compounds according to Formula I. Preferably A is a substituted or
unsubstituted aryl or heteroaryl group, which may be one
illustrated below, where R.sup.12 is hydrogen or C.sub.1-6alkyl;
and the left and right asterisks indicate the point of attachment
in formula (I);
##STR00010##
[0045] R.sup.12 may be hydrogen, hydroxyl, amino, alkylamino or
arylamino, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5 alkylthio,
halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy; aryl or heteroaryl; more preferably hydrogen.
[0046] B and C preferably are substituted or unsubstituted aryl or
heteroaryl, e.g.,
##STR00011##
[0047] In the groups shown in the two tables directly above, the
asterisks indicate that the group depicted may be attached to the
molecule as shown, or "inverted". The groups depicted immediately
above this text may desirably be present in the molecule in the
orientation illustrated.
[0048] wherein R.sup.12 is hydrogen or C.sub.1-6 alkyl; and the
left and right asterisks indicate the point of attachment in
formula (I); W.sub.1, W.sub.2, W.sub.3 or W.sub.4 may be C, N,
C--OH, C--OR.sup.13 or C--R.sup.13; R.sup.13 is hydrogen or
C.sub.1-6alkyl, C.sub.1-5alkylthio, C.sub.1-5alkoxy, halogen,
hydroxyl, cyano, halogen-substituted C.sub.1-6alkyl and
halogen-substituted C.sub.1-5alkoxy.
[0049] Z.sup.1 and Z.sup.2 are preferably CH.sub.2, O, S or a
direct bond. R.sup.3 is preferably methyl. R.sup.4 and R.sup.5 are
preferably hydrogen or methyl. n is preferably for 2. X may be
combined with Y, e.g.,
##STR00012## ##STR00013##
[0050] Optionally, two adjacent substituents on the ring A with
Z.sup.1 to form a fused ring, that may contain one or more hetero
atoms, and wherein X may be combined with Y, e.g.,
##STR00014##
[0051] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is an aryl or heteroaryl group,
optionally substituted with one, two or three substituents selected
from halogen, hydroxyl, SR.sup.2, S(O).sub.2R.sup.2,
S(O).sub.2NR.sup.2, NHS(O).sub.2R.sup.2, COR.sup.2,
CO.sub.2R.sup.2, cyano, amino, C.sub.1-5 alkylamino, arylamino,
heteroarylamino, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, halogen-substituted C.sub.1-6 alkyl, and
halogen-substituted C.sub.1-5 alkoxy; wherein R.sup.2 is selected
independently, in each instance, from hydrogen, hydroxyl, amino,
alkylamino, arylamino, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5
alkylthio, halogen-substituted C.sub.1-6 alkyl, halogen-substituted
C.sub.1-5 alkoxy, aryl and heteroaryl.
[0052] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is 1,4-disubstituted phenyl,
additionally optionally substituted with one, two or three
substituents selected from halogen, hydroxyl, SR.sup.2,
S(O).sub.2R.sup.2, S(O).sub.2NR.sup.2, NHS(O).sub.2R.sup.2,
COR.sup.2, CO.sub.2R.sup.2, cyano, amino, C.sub.1-5 alkylamino,
arylamino, heteroarylamino, C.sub.1-6 alkyl, C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen-substituted C.sub.1-6 alkyl, and
halogen-substituted C.sub.1-5 alkoxy; wherein R.sup.2 is selected
independently, in each instance, from hydrogen, hydroxyl, amino,
alkylamino, arylamino, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5
alkylthio, halogen-substituted C.sub.1-6 alkyl, halogen-substituted
C.sub.1-5 alkoxy, aryl and heteroaryl.
[0053] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is an aryl or heteroaryl group,
optionally substituted with one, two or three halogen atoms.
[0054] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is an phenyl, optionally
substituted with one, two or three halogen atoms.
[0055] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is an phenyl, optionally
substituted with one, two or three fluorine atoms.
[0056] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is fluorophenyl.
[0057] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is
##STR00015##
[0058] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is a heteroaryl group,
optionally substituted with one, two or three halogen atoms.
[0059] In another embodiment of the invention, in conjunction with
the above and below embodiments, A is a heteroaryl group.
[0060] In another embodiment of the invention, in conjunction with
the above and below embodiments, B and C together are
bicycloheteroaryl optionally substituted with 1 to 5 substituents
selected from C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, halogen, hydroxyl, cyano, halogen-substituted
C.sub.1-6alkyl and halogen-substituted C.sub.1-5 alkoxy.
[0061] In another embodiment of the invention, in conjunction with
the above and below embodiments, B and C together are a
bicycloheteroaryl selected from:
##STR00016## ##STR00017##
[0062] any of which are optionally substituted with 1 to 5
substituents selected from C.sub.1-6 alkyl, C.sub.1-5 alkylthio,
C.sub.1-5 alkoxy, halogen, hydroxyl, cyano, halogen-substituted
C.sub.1-6alkyl and halogen-substituted C.sub.1-5 alkoxy.
[0063] In another embodiment of the invention, in conjunction with
the above and below embodiments, B and C together are a
bicycloheteroaryl selected from:
##STR00018## ##STR00019##
[0064] any of which are substituted with 1 to 5 substituents
selected from C.sub.1-6 alkyl, halogen, and halogen-substituted
C.sub.1-6alkyl.
[0065] In another embodiment of the invention, in conjunction with
the above and below embodiments, B and C together are a
bicycloheteroaryl selected from:
##STR00020## ##STR00021##
[0066] In another embodiment of the invention, in conjunction with
the above and below embodiments, B and C together are a
bicycloheteroaryl selected from:
##STR00022## ##STR00023##
[0067] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is selected from C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl,
C.sub.1-5 alkoxy, C.sub.1-5 alkylamino, aryl and heteroaryl; any of
which are optionally substituted with hydroxyl, halogen, cyano,
amino, alkylamino, arylamino or heteroarylamino, wherein the aryl
and heteroaryl groups may optionally be substituted with 1-5
substituents independently selected from hydroxyl, halogen, cyano,
C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5 alkoxy and
C.sub.3-6 cycloalkyl.
[0068] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is selected from phenyl
and heteroaryl; both of which are optionally substituted with
halogen.
[0069] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is phenyl optionally
substituted with halogen.
[0070] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is heteroaryl optionally
substituted with halogen.
[0071] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is phenyl.
[0072] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is heteroaryl.
[0073] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is 5- or 6-membered
unsaturated ring including one atom selected from N, O and S, and
0, 1, 2 or 3 additional N atoms.
[0074] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.1 is selected from
pyridinyl, pyrimidine, thiazolyl, oxazolyl, furanyl and
thiophenyl.
[0075] In another embodiment of the invention, in conjunction with
the above and below embodiments, X is WC(O)OR.sup.6a,
WP(O)R.sup.6bR.sup.6c, WS(O).sub.2OH, WCONHSO.sub.3H or
1H-tetrazol-5-yl. W is a direct bond, oxygen or C.sub.1-4 alkyl
with substituents independently selected from halogen, hydroxyl,
cyano, amino, alkylamino, arylamino, heteroarylamino and C.sub.1-4
alkoxy; and R.sup.6a is hydrogen or C.sub.1-4alkyl; R.sup.6b and
R.sup.6c are independently selected from hydrogen, hydroxyl,
C.sub.1-4alkyl and halogen substituted C.sub.1-4alkyl.
[0076] In another embodiment of the invention, in conjunction with
the above and below embodiments, X is CO.sub.2H.
[0077] In another embodiment of the invention, in conjunction with
the above and below embodiments, Y is
##STR00024##
[0078] wherein R.sup.9 is selected from hydrogen, halogen,
hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkylthio, C.sub.1-5
alkoxy, halogen-substituted C.sub.1-6 alkyl and halogen-substituted
C.sub.1-5 alkoxy.
[0079] In another embodiment of the invention, in conjunction with
the above and below embodiments, Y is
##STR00025##
[0080] wherein R.sup.9 is selected from hydrogen, halogen and
hydroxyl.
[0081] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.9 is hydrogen.
[0082] In another embodiment of the invention, in conjunction with
the above and below embodiments, R.sup.9 is hydroxyl.
[0083] In another embodiment of the invention, in conjunction with
the above and below embodiments, Z.sup.1 is CR.sup.4R.sup.5;
wherein R.sup.4 and R.sup.5 are independently hydrogen, halogen,
hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5 alkoxy, C.sub.1-5
alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy.
[0084] In another embodiment of the invention, in conjunction with
the above and below embodiments, Z.sup.1 is CR.sup.4R.sup.5;
wherein R.sup.4 and R.sup.5 are independently hydrogen, halogen,
C.sub.1-6 alkyl or halogen-substituted C.sub.1-6 alkyl.
[0085] In another embodiment of the invention, in conjunction with
the above and below embodiments, Z.sup.1 is CH.sub.2.
[0086] In another embodiment of the invention, in conjunction with
the above and below embodiments,
[0087] Z.sup.2 is selected from O, NR.sup.3, S, S(O), S(O).sub.2,
S(O).sub.2NR.sup.3, (CR.sup.4R.sup.5).sub.n, C.dbd.O, C.dbd.S, and
C.dbd.N--R.sup.3;
[0088] R.sup.3 is hydrogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy;
[0089] R.sup.4 and R.sup.5 are independently selected from
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy, or together form
"C.dbd.O"; and
[0090] n is 1, 2 or 3.
[0091] In another embodiment of the invention, in conjunction with
the above and below embodiments,
[0092] Z.sup.2 is selected from O, NR.sup.3, S, S(O), S(O).sub.2,
S(O).sub.2NR.sup.3, CR.sup.4R.sup.5, C.dbd.O, C.dbd.S, and
C.dbd.N--R.sup.3;
[0093] R.sup.3 is hydrogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy; and
[0094] R.sup.4 and R.sup.5 are independently selected from
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy.
[0095] In another embodiment of the invention, in conjunction with
the above and below embodiments Z.sup.2 is selected from O,
NR.sup.3, S, CR.sup.4R.sup.5, C.dbd.O, C.dbd.S, and
C.dbd.N--R.sup.3;
[0096] R.sup.3 is hydrogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl or
halogen-substituted C.sub.1-5 alkoxy; and
[0097] R.sup.4 and R.sup.5 are independently selected from
hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-5
alkoxy, C.sub.1-5 alkylthio, halogen-substituted C.sub.1-6 alkyl
and halogen-substituted C.sub.1-5 alkoxy.
[0098] In another embodiment of the invention, in conjunction with
the above and below embodiments Z.sup.2 is selected from O, S,
CH.sub.2, C.dbd.O, C.dbd.S and C.dbd.N--OH.
[0099] The specification and claims contain listing of species
using the language "selected from . . . and . . . " and "is . . .
or . . . " (sometimes referred to as Markush groups). When this
language is used in this application, unless otherwise stated it is
meant to include the group as a whole, or any single members
thereof, or any subgroups thereof. The use of this language is
merely for shorthand purposes and is not meant in any way to limit
the removal of individual elements or subgroups as needed.
[0100] In one aspect, the present invention provides methods for
modulating S1P-1 receptor mediated biological activity. The present
invention also provides methods for using S1P-1 modulators (i.e.,
agonists or antagonists) in treating or preventing diseases such as
ovarian cancer, peritoneal cancer, endometrial cancer, cervical
cancer, breast cancer, colorectal cancer, uterine cancer, stomach
cancer, small intestine cancer, thyroid cancer, lung cancer, kidney
cancer, pancreas cancer and prostate cancer; acute lung diseases,
adult respiratory distress syndrome ("ARDS"), acute inflammatory
exacerbation of chronic lung diseases such as asthma, surface
epithelial cell injury such as transcorneal freezing or cutaneous
burns, and cardiovascular diseases such as ischemia in a subject in
need of such treatment or prevention.
[0101] In another aspect, the invention provides methods for using
S1P-1 modulators in treating or preventing disorders such as, but
not limited to, vasoconstriction in cerebral arteries, autoimmune
and related immune disorders including systemic lupus
erythematosus, inflammatory bowel diseases such as Crohn's disease
and ulcerative colitis, type I diabetes, uveitis, psoriasis,
myasthenia gravis, rheumatoid arthritis, non-glomerular nephrosis,
hepatitis, Behcet's disease, glomerulonephritis, chronic
thrombocytopenic purpura, hemolytic anemia, hepatitis and Wegner's
granuloma.
[0102] In still another aspect, the invention provides methods for
using S1P-1 modulators to treat or prevent a disease or disorder in
a subject, comprising administering to a subject in need of such
treatment or prevention a therapeutically effective amount of an
S1P-1 modulator, e.g., an agonist, that stimulates the immune
system. In certain embodiments, the subject is afflicted by an
infectious agent. In other embodiments, the subject is
immunocompromised.
[0103] In still another aspect, the present invention provides a
method of modulating an S1P-1 receptor-mediated biological activity
in a cell. A cell expressing the S1P-1 receptor is contacted with
an amount of an S1P-1 receptor modulator sufficient to modulate the
S1P-1 receptor mediated biological activity.
[0104] In yet another aspect, the present invention provides a
method for modulating an S1P-1 receptor mediated biological
activity in a subject. In such a method, an amount of a modulator
of the S1P-1 receptor effective to modulate an S1P-1
receptor-mediated biological activity is administered to the
subject.
[0105] In yet another aspect, the present invention provides a
method for treating, preventing or ameliorating an S1P-1 receptor
mediated condition in a subject. In such a method, an amount of a
modulator of the S1P-1 receptor effective to modulate an S1P-1
receptor-mediated biological activity is administered to the
subject. The S1P-1 receptor mediated condition may be, e.g.,
transplant rejection (solid organ transplant and islet cells);
transplant rejection (tissue); cancer; autoimmune/inflammatory
diseases; rheumatoid arthritis; lupus; insulin dependent diabetes
(Type I); non-insulin dependent diabetes (Type II); multiple
sclerosis; psoriasis; ulcerative colitis; inflammatory bowel
disease; Crohn's disease; acute and chronic lymphocytic leukemias
and lymphomas.
[0106] The features and other details of the invention will now be
more particularly described. It will be understood that particular
embodiments described herein are shown by way of illustration and
not as limitations of the invention. The principal features of this
invention can be employed in various embodiments without departing
from the scope of the invention. All parts and percentages are by
weight unless otherwise specified.
Definitions
[0107] For convenience, certain terms used in the specification and
examples are collected here.
[0108] "Treating", includes any effect, e.g., lessening, reducing,
modulating, or eliminating, that results in the improvement of the
condition, disease, disorder, etc.
[0109] "Alkyl" includes saturated aliphatic groups, e.g.,
straight-chain alkyl groups such as methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, nonyl, and decyl; branched-chain
alkyl groups (e.g., isopropyl, tert-butyl, and isobutyl);
cycloalkyl (alicyclic) groups like cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl, and cyclooctyl); alkyl-substituted
cycloalkyl groups; and cycloalkyl-substituted alkyl groups.
[0110] "Alkyl" groups may also optionally include heteroatoms,
i.e., where oxygen, nitrogen, sulfur or phosphorous atoms replaces
one or more hydrocarbon backbone carbon atoms, particularly where
the substitution does not adversely impact the efficacy of the
resulting compound.
[0111] Straight or branched alkyl groups may have six or fewer
carbon atoms in their backbone (e.g., C.sub.1-C.sub.6 for straight
chain, C.sub.3-C.sub.6 for branched chain), and more preferably
four or fewer. Preferred cycloalkyl groups have from three to eight
carbon atoms in their ring structure, and more preferably five or
six carbons in the ring structure. "C.sub.1-C.sub.6" includes alkyl
groups containing one to six carbon atoms.
[0112] "Substituted alkyls" refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,
sulfhydryl, alkylthio, thiocarboxylate, sulfates, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, or heterocyclyl.
[0113] "Aryl" includes groups with aromaticity, including 5- and
6-membered unconjugated (i.e., single-ring) aromatic groups that
may include from zero to four heteroatoms, as well as conjugated
(i.e., multicyclic) systems having at least one ring that is
aromatic. Examples of aryl groups include benzene, phenyl, tolyl
and the like. Multicyclic aryl groups include tricyclic and
bicyclic systems, e.g., naphthalene, benzoxazole, benzodioxazole,
benzothiazole, benzoimidazole, benzothiophene,
methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,
benzofuran, purine, benzofuran, deazapurine, indolizine, tetralin,
and methylenedioxyphenyl.
[0114] Aryl groups having heteroatoms in the ring structure may
also be referred to as "aryl heterocycles", "heterocycles,"
"heteroaryls" or "heteroaromatics"; e.g., pyrrole, furan,
thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine. The aromatic ring can be substituted at one or more
ring positions with, for example, halogen, hydroxyl, alkoxy,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,
aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,
arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, cyano, amino, acylamino, amidino, imino, sulfhydryl,
alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0115] An "alkylaryl" or an "aralkyl" moiety is an alkyl
substituted with an aryl group (e.g., phenylmethyl(benzyl)).
[0116] "Alkenyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
that contain at least one double bond. For example, the term
"alkenyl" includes straight-chain alkenyl groups (e.g., ethenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
and decenyl), branched-chain alkenyl groups, cycloalkenyl groups
such as cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,
and cyclooctenyl; alkyl or alkenyl-substituted cycloalkenyl groups,
and cycloalkyl or cycloalkenyl-substituted alkenyl groups.
[0117] "Alkenyl" groups may also optionally include heteroatoms,
i.e., where oxygen, nitrogen, sulfur or phosphorous atoms replaces
one or more hydrocarbon backbone carbon atoms, particularly where
the substitution does not adversely impact the efficacy of the
resulting compound.
[0118] Straight or branched alkenyl groups may have six or fewer
carbon atoms in their backbone (e.g., C.sub.2-C.sub.6 for straight
chain, C.sub.3-C.sub.6 for branched chain.) Preferred cycloalkenyl
groups have from three to eight carbon atoms in their ring
structure, and more preferably have five or six carbons in the ring
structure. The term "C.sub.2-C.sub.6" includes alkenyl groups
containing two to six carbon atoms.
[0119] "Substituted alkenyls" refers to alkenyl moieties having
substituents replacing a hydrogen on one or more hydrocarbon
backbone carbon atoms. Such substituents can include alkyl groups,
alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino, acylamino, amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, or
heterocyclyl.
[0120] "Alkynyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
which contain at least one triple bond. For example, "alkynyl"
includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,
butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl),
branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl
substituted alkynyl groups.
[0121] "Alkynyl" groups may also optionally include heteroatoms,
i.e., where oxygen, nitrogen, sulfur or phosphorous atoms replaces
one or more hydrocarbon backbone carbon atoms, particularly where
the substitution does not adversely impact the efficacy of the
resulting compound
[0122] Straight or branched chain alkynyls group may have six or
fewer carbon atoms in their backbone (e.g., C.sub.2-C.sub.6 for
straight chain, C.sub.3-C.sub.6 for branched chain). The term
"C.sub.2-C.sub.6" includes alkynyl groups containing two to six
carbon atoms.
[0123] "Substituted alkynyls" refers to alkynyl moieties having
substituents replacing a hydrogen on one or more hydrocarbon
backbone carbon atoms. Such substituents can include alkyl groups,
alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, or heterocyclyl.
[0124] Unless the number of carbons is otherwise specified, "lower
alkyl" includes an alkyl group, as defined above, but having from
one to ten, more preferably from one to six, carbon atoms in its
backbone structure. "Lower alkenyl" and "lower alkynyl" have chain
lengths of, for example, 2-5 carbon atoms.
[0125] "Acyl" includes compounds and moieties which contain the
acyl radical (CH.sub.3CO--) or a carbonyl group. "Substituted acyl"
includes acyl groups where one or more of the hydrogen atoms are
replaced by for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0126] "Acylamino" includes moieties wherein an acyl moiety is
bonded to an amino group. For example, the term includes
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
"Alkylamino" includes moieties wherein an alkyl moiety is bonded to
an amino group; "dialkylamino", "arylamino", "diarylamino", and
"alkylarylamino" are analogously named. In some embodiments,
"amino" may include acylamino and/or alkylamino groups.
[0127] "Alkoxyalkyl", "alkylaminoalkyl" and "thioalkoxyalkyl"
include alkyl groups, as described above, which further include
oxygen, nitrogen or sulfur atoms replacing one or more hydrocarbon
backbone carbon atoms, e.g., oxygen, nitrogen or sulfur atoms.
[0128] "Alkoxy" includes alkyl, alkenyl, and alkynyl groups
covalently linked to an oxygen atom. Examples of alkoxy groups
include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy
groups. Examples of "substituted alkoxy" groups include halogenated
alkoxy groups. Substituted alkoxy groups can include alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, or heterocyclyl substituents.
Examples of halogen-substituted alkoxy groups include
fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,
dichloromethoxy, and trichloromethoxy.
[0129] The terms "heterocyclyl" or "heterocyclic group" include
closed ring structures, e.g., 3- to 10-, or 4- to 7-membered rings
which include one or more heteroatoms. Heterocyclyl groups can be
saturated or unsaturated and include pyrrolidine, oxolane,
thiolane, piperidine, piperizine, morpholine, lactones, lactams
such as azetidinones and pyrrolidinones, sultams, sultones, and the
like.
[0130] Heterocyclic rings may be substituted at one or more
positions with such substituents as described above, as for
example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate, phosphonato, phosphinato, cyano, amino, acylamino,
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic or
heteroaromatic moiety.
[0131] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0132] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl" which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0133] The term "ester" includes compounds and moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl,
alkenyl, or alkynyl groups are as defined above.
[0134] The term "thioether" includes compounds and moieties which
contain a sulfur atom bonded to two different carbon or
heteroatoms. Examples of thioethers include, but are not limited to
alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term
"alkthioalkyls" include compounds with an alkyl, alkenyl, or
alkynyl group bonded to a sulfur atom which is bonded to an alkyl
group. Similarly, the term "alkthioalkenyls" and alkthioalkynyls"
refer to compounds or moieties wherein an alkyl, alkenyl, or
alkynyl group is bonded to a sulfur atom which is covalently bonded
to an alkynyl group.
[0135] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-.
[0136] The term "halogen" includes fluorine, bromine, chlorine,
iodine, etc. The term "perhalogenated" generally refers to a moiety
wherein all hydrogens are replaced by halogen atoms.
[0137] "Heteroatom" includes atoms of any element other than carbon
or hydrogen. Examples of heteroatoms include nitrogen, oxygen,
sulfur and phosphorus.
[0138] "At least partially aromatic bicyclic ring system", means a
bicyclic ring system where either or both of the rings forming the
bicycle are aromatic.
[0139] It will be noted that the structure of some of the compounds
of the invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(e.g., all enantiomers and diastereomers) are included within the
scope of the invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by classical separation
techniques and by stereochemically controlled synthesis.
Furthermore, the structures and other compounds and moieties
discussed in this application also include all tautomers thereof.
Alkenes can include either the E- or Z-geometry, where
appropriate.
[0140] "Combination therapy" (or "co-therapy") includes the
administration of a S1P receptor modulator of the invention and at
least a second agent as part of a specific treatment regimen
intended to provide the beneficial effect from the co-action of
these therapeutic agents. The beneficial effect of the combination
includes, but is not limited to, pharmacokinetic or pharmacodynamic
co-action resulting from the combination of therapeutic agents.
Administration of these therapeutic agents in combination typically
is carried out over a defined time period (usually minutes, hours,
days or weeks depending upon the combination selected).
"Combination therapy" may, but generally is not, intended to
encompass the administration of two or more of these therapeutic
agents as part of separate monotherapy regimens that incidentally
and arbitrarily result in the combinations of the present
invention. "Combination therapy" is intended to embrace
administration of these therapeutic agents in a sequential manner,
that is, wherein each therapeutic agent is administered at a
different time, as well as administration of these therapeutic
agents, or at least two of the therapeutic agents, in a
substantially simultaneous manner. Substantially simultaneous
administration can be accomplished, for example, by administering
to the subject a single capsule having a fixed ratio of each
therapeutic agent or in multiple, single capsules for each of the
therapeutic agents. Sequential or substantially simultaneous
administration of each therapeutic agent can be effected by any
appropriate route including, but not limited to, oral routes,
intravenous routes, intramuscular routes, and direct absorption
through mucous membrane tissues. The therapeutic agents can be
administered by the same route or by different routes. For example,
a first therapeutic agent of the combination selected may be
administered by intravenous injection while the other therapeutic
agents of the combination may be administered orally.
Alternatively, for example, all therapeutic agents may be
administered orally or all therapeutic agents may be administered
by intravenous injection. The sequence in which the therapeutic
agents are administered is not narrowly critical. "Combination
therapy" also can embrace the administration of the therapeutic
agents as described above in further combination with other
biologically active ingredients and non-drug therapies (e.g.,
surgery or radiation treatment.) Where the combination therapy
further comprises a non-drug treatment, the non-drug treatment may
be conducted at any suitable time so long as a beneficial effect
from the co-action of the combination of the therapeutic agents and
non-drug treatment is achieved. For example, in appropriate cases,
the beneficial effect is still achieved when the non-drug treatment
is temporally removed from the administration of the therapeutic
agents, perhaps by days or even weeks.
[0141] An "anionic group," as used herein, refers to a group that
is negatively charged at physiological pH. Preferred anionic groups
include carboxylate, sulfate, sulfonate, sulfinate, sulfamate,
tetrazolyl, phosphate, phosphonate, phosphinate, or
phosphorothioate or functional equivalents thereof. "Functional
equivalents" of anionic groups are intended to include
bioisosteres, e.g., bioisosteres of a carboxylate group.
Bioisosteres encompass both classical bioisosteric equivalents and
non-classical bioisosteric equivalents. Classical and non-classical
bioisosteres are known in the art (see, e.g., Silverman, R. B. The
Organic Chemistry of Drug Design and Drug Action, Academic Press,
Inc.: San Diego, Calif., 1992, pp. 19-23). A particularly preferred
anionic group is a carboxylate.
[0142] The term "heterocyclic group" is intended to include closed
ring structures in which one or more of the atoms in the ring is an
element other than carbon, for example, nitrogen, or oxygen or
sulfur. Heterocyclic groups can be saturated or unsaturated and
heterocyclic groups such as pyrrole and furan can have aromatic
character. They include fused ring structures such as quinoline and
isoquinoline. Other examples of heterocyclic groups include
pyridine and purine. Heterocyclic groups can also be substituted at
one or more constituent atoms with, for example, a halogen, a lower
alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower
alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, --CF.sub.3,
--CN, or the like.
[0143] An "S1P-modulating agent" includes compound or compositions
capable of inducing a detectable change in S1P receptor activity in
vivo or in vitro, e.g., at least 10% increase or decrease in S1P
activity as measured by a given assay such as the bioassay
described hereinbelow.
[0144] "EC.sub.50 of an agent" included that concentration of an
agent at which a given activity, including binding of sphingosine
or other ligand of an S1P receptor and/or a functional activity of
a S1P receptor (e.g., a signaling activity), is 50% maximal for
that S1P receptor. Stated differently, the EC.sub.50 is the
concentration of agent that gives 50% activation, when 100%
activation is set at the amount of activity of the S1P receptor
which does not increase with the addition of more ligand/agonist
and 0% activation is set at the amount of activity in the assay in
the absence of added ligand/agonist.
[0145] "Purified" and like terms relate to the isolation of a
molecule or compound in a form that is substantially free of
contaminants normally associated with the molecule or compound in a
native or natural environment.
[0146] An "effective amount" includes an amount sufficient to
produce a selected effect. For example, an effective amount of an
S1P receptor antagonist is an amount that decreases the cell
signaling activity of the S1P receptor.
[0147] "Immunomodulation" includes effects on the functioning of
the immune system, and includes both the enhancement of an immune
response as well as suppression of the immune response.
[0148] The compounds of the invention and the other
pharmacologically active agent may be administered to a patient
simultaneously, sequentially or in combination. It will be
appreciated that when using a combination of the invention, the
compound of the invention and the other pharmacologically active
agent may be in the same pharmaceutically acceptable carrier and
therefore administered simultaneously. They may be in separate
pharmaceutical carriers such as conventional oral dosage forms
which are taken simultaneously. The term "combination" further
refers to the case where the compounds are provided in separate
dosage forms and are administered sequentially.
[0149] The compounds of the invention may be administered to
patients (animals and humans) in need of such treatment in dosages
that will provide optimal pharmaceutical efficacy. It will be
appreciated that the dose required for use in any particular
application will vary from patient to patient, not only with the
particular compound or composition selected, but also with the
route of administration, the nature of the condition being treated,
the age and condition of the patient, concurrent medication or
special diets then being followed by the patient, and other factors
which those skilled in the art will recognize, with the appropriate
dosage ultimately being at the discretion of the attendant
physician.
[0150] An appropriate dosage level will generally be about 0.001 to
50 mg per kg patient body weight per day, which may be administered
in single or multiple doses. Preferably, the dosage level will be
about 0.01 to about 25 mg/kg per day; more preferably about 0.05 to
about 10 mg/kg per day. For example, in the treatment or prevention
of a disorder of the central nervous system, a suitable dosage
level is about 0.001 to 10 mg/kg per day, preferably about 0.005 to
5 mg/kg per day, and especially about 0.01 to 1 mg/kg per day. The
compounds may be administered on a regimen of 1 to 4 times per day,
preferably once or twice per day.
[0151] It will be appreciated that the amount of the compound of
the invention required for use in any treatment will vary not only
with the particular compounds or composition selected but also with
the route of administration, the nature of the condition being
treated, and the age and condition of the patient, and will
ultimately be at the discretion of the attendant physician.
[0152] The compositions and combination therapies of the invention
may be administered in combination with a variety of pharmaceutical
excipients, including stabilizing agents, carriers and/or
encapsulation formulations as described herein.
[0153] Aqueous compositions of the present invention comprise an
effective amount of the compounds of the invention, dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous
medium.
[0154] "Pharmaceutically or pharmacologically acceptable" include
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate. "Pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0155] For human administration, preparations should meet
sterility, pyrogenicity, general safety and purity standards as
required by FDA Office of Biologics standards.
[0156] The compositions and combination therapies of the invention
will then generally be formulated for parenteral administration,
e.g., formulated for injection via the intravenous, intramuscular,
subcutaneous, intralesional, or even intraperitoneal routes. The
preparation of an aqueous composition that contains a composition
of the invention or an active component or ingredient will be known
to those of skill in the art in light of the present disclosure.
Typically, such compositions can be prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a liquid
prior to injection can also be prepared; and the preparations can
also be emulsified.
[0157] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
[0158] Solutions of active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0159] Therapeutic or pharmacological compositions of the present
invention will generally comprise an effective amount of the
component(s) of the combination therapy, dissolved or dispersed in
a pharmaceutically acceptable medium. Pharmaceutically acceptable
media or carriers include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well known in the
art. Supplementary active ingredients can also be incorporated into
the therapeutic compositions of the present invention.
[0160] The preparation of pharmaceutical or pharmacological
compositions will be known to those of skill in the art in light of
the present disclosure. Typically, such compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection; as tablets or other solids for oral
administration; as time release capsules; or in any other form
currently used, including cremes, lotions, mouthwashes, inhalants
and the like.
[0161] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0162] The preparation of more, or highly, concentrated solutions
for intramuscular injection is also contemplated. In this regard,
the use of DMSO as solvent is preferred as this will result in
extremely rapid penetration, delivering high concentrations of the
active compound(s) or agent(s) to a small area.
[0163] The use of sterile formulations, such as saline-based
washes, by surgeons, physicians or health care workers to cleanse a
particular area in the operating field may also be particularly
useful. Therapeutic formulations in accordance with the present
invention may also be reconstituted in the form of mouthwashes, or
in conjunction with antifungal reagents. Inhalant forms are also
envisioned. The therapeutic formulations of the invention may also
be prepared in forms suitable for topical administration, such as
in cremes and lotions.
[0164] Suitable preservatives for use in such a solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium bisulfite,
sodium metabisulfite, sodium thiosulfite, thiourea and the like.
Suitable wetting and clarifying agents include polysorbate 80,
polysorbate 20, poloxamer 282 and tyloxapol. Suitable
viscosity-increasing agents include dextran 40, dextran 70,
gelatin, glycerin, hydroxyethylcellulose,
hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like.
[0165] Upon formulation, therapeutics will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed.
[0166] In this context, the quantity of active ingredient and
volume of composition to be administered depends on the host animal
to be treated. Precise amounts of active compound required for
administration depend on the judgment of the practitioner and are
peculiar to each individual.
[0167] A minimal volume of a composition required to disperse the
active compounds is typically utilized. Suitable regimes for
administration are also variable, but would be typified by
initially administering the compound and monitoring the results and
then giving further controlled doses at further intervals. For
example, for parenteral administration, a suitably buffered, and if
necessary, isotonic aqueous solution would be prepared and used for
intravenous, intramuscular, subcutaneous or even intraperitoneal
administration. One dosage could be dissolved in 1 mL of isotonic
NaCl solution and either added to 1000 mL of hypodermolysis fluid
or injected at the proposed site of infusion, (see for example,
Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038
and 1570-1580).
[0168] In certain embodiments, active compounds may be administered
orally. This is contemplated for agents which are generally
resistant, or have been rendered resistant, to proteolysis by
digestive enzymes. Such compounds are contemplated to include
chemically designed or modified agents; dextrorotatory peptides;
and peptide and liposomal formulations in time release capsules to
avoid peptidase and lipase degradation.
[0169] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0170] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0171] The preparation of more, or highly, concentrated solutions
for direct injection is also contemplated, where the use of DMSO as
solvent is envisioned to result in extremely rapid penetration,
delivering high concentrations of the active agents to a small
area.
[0172] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0173] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure.
[0174] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations;
time-release capsules; and any other form currently used, including
cremes.
[0175] Additional formulations suitable for other modes of
administration include suppositories. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%.
[0176] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders.
[0177] In certain defined embodiments, oral pharmaceutical
compositions will comprise an inert diluent or assimilable edible
carrier, or they may be enclosed in hard or soft shell gelatin
capsule, or they may be compressed into tablets, or they may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0178] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange
flavor.
[0179] The pharmaceutical compositions of this invention may be
used in the form of a pharmaceutical preparation, for example, in
solid, semisolid or liquid form, which contains one or more of the
compound of the invention, as an active ingredient, in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral or parenteral applications. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, and any
other form suitable for use. The carriers which can be used are
water, glucose, lactose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form,
and in addition auxiliary, stabilizing, thickening and coloring
agents and perfumes may be used. The active object compound is
included in the pharmaceutical composition in an amount sufficient
to produce the desired effect upon the process or condition of the
disease.
[0180] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g., conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g., water, to form a solid preformulation composition containing
a homogeneous mixture of a compound of the invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the invention. The
tablets or pills of the novel composition can be coated or
otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0181] The liquid forms in which the compositions of the invention
may be incorporated for administration orally or by injection
include aqueous solution, suitably flavored syrups, aqueous or oil
suspensions, and emulsions with acceptable oils such as cottonseed
oil, sesame oil, coconut oil or peanut oil, or with a solubilizing
or emulsifying agent suitable for intravenous use, as well as
elixirs and similar pharmaceutical vehicles. Suitable dispersing or
suspending agents for aqueous suspensions include synthetic and
natural gums such as tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or
gelatin.
[0182] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. Preferably the compositions
are administered by the oral or nasal respiratory route for local
or systemic effect. Compositions in preferably sterile
pharmaceutically acceptable solvents may be nebulized by use of
inert gases. Nebulized solutions may be breathed directly from the
nebulizing device or the nebulizing device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0183] For treating clinical conditions and diseases noted above,
the compound of this invention may be administered orally,
topically, parenterally, by inhalation spray or rectally in dosage
unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques.
[0184] The compounds of the present invention are high affinity
agonists (or antagonists) at various S1P receptors. The compounds
of the invention are also expected to evoke lymphopenia when
introduced into rodents, non human primate or humans. Thus the
compounds of the invention can be used as immune modulators, and
are useful in treating or preventing pathologies mediated by
lymphocyte actions, including acute or chronic rejection of tissue
grafts such as organ transplants, and autoimmune diseases.
Autoimmune diseases that may be treated with compounds of the
invention include: systemic lupus erythematosus, multiple
sclerosis, Behcet's disease, glomerulonephritis, rheumatoid
arthritis, inflammatory bowel diseases such as Crohn's disease and
ulcerative colitis, type I diabetes, uveitis, psoriasis, myasthenia
gravis, Hashimoto's thyroiditis, autoimmune hemolytic anemia,
autoimmune thrombocytopenic purpura, hepatitis and Wegner's
granuloma.
[0185] The compounds of the invention are useful also in treating
inflammatory disorders, including atopic asthma, inflammatory
glomerular injury and ischemia-reperfusion injury.
[0186] Lysophospholipids, S1P and lysophosphatidic acid (LPA),
stimulate cellular proliferation and affect numerous cellular
functions by signaling through G protein-coupled endothelial
differentiation gene-encoded (S1P) receptors. Accordingly, the S1P
receptor modulators of the invention are anticipated to have
utility in immunomodulation, e.g., in anti-angiogenesis therapy,
such as in neoplastic disease treatment.
[0187] In one embodiment of the invention, a pharmaceutical
composition comprising one or more of the S1P receptor agonists of
the present invention is administered to a mammalian species,
including humans, to enhance wound repair, improve neuronal
function or enhance an immune response of that species. It has also
been reported that S1P inhibits fibrosis in various organs.
Accordingly, the S1P receptor agonists of the invention can be used
to prevent/treat diseases associated with organ fibrosis, such as
pulmonary fibrosis, interstitial pneumonia, chronic hepatitis,
hepatic cirrhosis, chronic renal insufficiency or kidney glomerular
sclerosis. In one embodiment, a composition comprising an S1P
receptor agonist of the present invention is used to treat wounds,
including burns, cuts, lacerations, surgical incisions, bed sores,
and slow-healing ulcers such as those seen in diabetics.
[0188] In addition, S1P modulating compounds of the invention are
believed to mobilize lymphocytes and increase their homing to
secondary lymphoid tissues. Thus the present compounds can be used
to direct lymphocytes away from transplanted organs, e.g.,
allografts, or healthy cells, e.g., pancreatic islets as in type I
diabetes, myelin sheathing (multiple sclerosis), or other tissues
that may be subjected to an undesirable immunoresponse, and thus
decrease damage to such tissues from the immune system.
[0189] In another embodiment, the S1P receptor-modulating compounds
of the invention are administered to a subject to treat or prevent
a disorder of abnormal cell growth and differentiation. These
disorders include Alzheimer's disease, aberrant corpus luteum
formation, osteoporosis, anovulation, Parkinson's disease, and
cancer. In one embodiment, an S1P antagonist is administered to a
patient to treat a disease associated with abnormal growth.
[0190] In one embodiment, the compounds of the invention are used
as immunomodulators to alter immune system activities and prevent
damage to healthy tissue that would otherwise occur in autoimmune
diseases and in organ transplantation. In particular, the compounds
can be administered to patients as part of the treatment associated
with organ transplantation, including pancreas, pancreatic islets,
kidney, heart and lung transplantations. The S1P modulators can be
administered alone or in combination with known immunosuppressants
such as cyclosporine, tacrolimus, rapamycin, azathioprine,
cyclophosphamide, methotrexate and corticosteroids such as
cortisone, des-oxymetasone, betametasone, desametasone,
flunisolide, prednisolone, prednisone, amcinomide, desonide,
methylprednisolone, triamcinolone, and alclometasone.
[0191] S1P also acts as a survival factor in many cell types. In
particular, compounds of the invention having S1P antagonistic
activity are anticipated to be useful in protecting cells and
tissues from hypoxic conditions. In accordance with one embodiment,
compounds of the invention are administered to a patient judged to
be or actually in need of treatment, to treat cells and tissues
exposed to hypoxic conditions, including injury sustained as a
result of ischemia. In accordance with one embodiment, compounds of
the invention that show S1P receptor antagonist activity can be
used to treat ischemia reperfusion type injury. Interference with
the supply of oxygenated blood to tissues is defined as ischemia.
The effects of ischemia are known to be progressive, so that over
time cellular vitality continues to deteriorate and tissues become
necrotic. Total persistent ischemia, with limited oxygen perfusion
of tissues, results in cell death and eventually in
coagulation-induced necrosis despite reperfusion with arterial
blood. Evidence indicates that a significant proportion of the
injury associated with ischemia is a consequence of the events
associated with reperfusion of ischemic tissues, hence the term
reperfusion injury.
[0192] Pharmaceutical compositions comprising the compounds of the
invention may be administered to an individual in need by any
number of routes, including topical, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means. The oral
route is typically employed for most conditions requiring the
compounds of the invention. Preference is given to intravenous
injection or infusion for the acute treatments. For maintenance
regimens the oral or parenteral, e.g., intramuscular or
subcutaneous, route is preferred. In accordance with one embodiment
a composition is provided that comprises a compound of invention
and albumin, e.g., a compound of the present invention, a
pharmaceutically acceptable carrier and 0.1-1.0% albumin. Albumin
functions as a buffer and improves the solubility of the
compounds.
[0193] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention. In
accordance with one embodiment, a kit is provided for treating a
patient in need of immunomodulation, including instructions for use
of the kit. In this embodiment the kit comprises one or more of the
S1P modulators of the invention, and may also include one or more
known immunosuppressants. These pharmaceuticals can be packaged in
a variety of containers, e.g., vials, tubes, microtiter well
plates, bottles, and the like. Other reagents can be included in
separate containers and provided with the kit; e.g., positive
control samples, negative control samples, buffers, cell culture
media, etc. Preferably, the kits will also include instructions for
use.
[0194] The activity of compounds of the invention may be determined
by using an assay for detecting S1P receptor activity (such as the
[.gamma.-35 S]GTP binding assay) and assaying for activity in the
presence of S1P and the test compound. More particularly, in the
method described by Traynor et al., 1995, Mol. Pharmacol. 47:
848-854, incorporated herein by reference, G-protein coupling to
membranes can be evaluated by measuring the binding of labeled
GTP.
[0195] For example, samples comprising membranes isolated from
cells expressing an S1P polypeptide can be incubated in a buffer
promoting binding of the polypeptide to ligand (i.e. S1P), in the
presence of radiolabeled GTP and unlabeled GDP (e.g., in 20 mM
HEPES, pH 7.4, 100 mM NaCl, and 10 mM MgCl.sub.2, 80 pM
.sup.35S-GTP.sub..gamma.S and 3 .mu.M GDP), with and without a
candidate modulator. The assay mixture is incubated for a suitable
period of time to permit binding to and activation of the receptor
(e.g., 60 minutes at 30.degree. C.), after which time unbound
labeled GTP is removed (e.g., by filtration onto GF/B filters).
Bound, labeled GTP can be measured by liquid scintillation
counting. A decrease of 10% or more in labeled GTP binding as
measured by scintillation counting in a sample containing a
candidate modulator, relative to a sample without the modulator,
indicates that the candidate modulator is an inhibitor of S1P
receptor activity.
[0196] A similar GTP-binding assay can be performed without the
presence of the ligand (S1P) to identify agents that act as
agonists. In this case, ligand-stimulated GTP binding is used as a
standard. An agent is considered an agonist if it induces at least
50% of the level of GTP binding induced by S1P when the agent is
present at 10 .mu.m or less, and preferably will induce a level
which is the same as or higher than that induced by the ligand.
[0197] GTPase activity can be measured by incubating cell membrane
extracts containing an S1P receptor with .gamma..sup.32P-GTP.
Active GTPase will release the label as inorganic phosphate, which
can be detected by separation of free inorganic phosphate in a 5%
suspension of activated charcoal in 20 mM H.sub.3PO.sub.4, followed
by scintillation counting. Controls would include assays using
membrane extracts isolated from cells not expressing an S1P
receptor (e.g., mock-transfected cells), in order to exclude
possible non-specific effects of the candidate modulator. In order
to assay for the effect of a candidate modulator on S1P-regulated
GTPase activity, cell membrane samples can be incubated with the
ligand (S1P), with and without the modulator, and a GTPase assay
can be performed as described above. A change (increase or
decrease) of 10% or more in the level of GTP binding or GTPase
activity relative to samples without modulator is indicative of S1P
modulation by a candidate modulator.
[0198] S1P receptor activity may also be measured by an S1P
receptor (e.g., hS1P1, hS1P3, rS1P1, rS1P3 and parental cell)
Ca.sup.2+ Flux protocol as described below:
[0199] 1. Material [0200] a. FLIPR buffer: 1.times.HBSS; 10 mM
HEPES [0201] b. Cell growth media: [0202] i. human and rat S1P1 and
S1P3: F12-Ham's media; 10% FBS (qualified); 1.times.Pen/Strep/Glu;
300 ug/mL Hygromycin; 400 ug/mL Geneticin [0203] ii. Parental cell:
human and rat S1P1 and S1P3: F12-Ham's media; 10% FBS (qualified);
1.times.Pen/Strep/Glu; 300 ug/mL Hygromycin; [0204] c. Cell seeding
media; F12-Ham's media; 10% FBS (Charcoal/dextran stripped);
1.times.Pen/Strep/Glu; [0205] d. Cell dissociation buffer: Versene
from Invitrogen [0206] e. Agonist (S1P) dissolving buffer: 0.4%
(w/v) fatty-acid free BSA (Sigma #A8806) in FLIPR buffer [0207] f.
FLIPR dye: BD PBX Calcium assay kit; Cat#641077 is composed of
Calcium indicator (Cat#850000) and 100.times.PBX signal enhancer
(cat#850001). The 100.times.PBX signal enhancer is diluted into
FLIPR buffer 1:100 and the calcium indicator is then added at
1:1000 ratio. [0208] g. Cell plates (96-well): Greiner Cat#655090
[0209] h. Compound plates (96-well): Costar #3365 [0210] i. S1P
stock solution preparation: S1P is purchased from CalBioChem
(Catalog #970471; 1 mg vial; custom prep using methanol, &
nitrogen gas drying inside glass vial; Storage @-20.degree. C.).
Dissolve 1 vial of S1P into 26.4 ml of agonist dissolving buffer in
a 50 ml centrifuge tube; Remove label from bottle of S1P, open and
drop entire bottle into tube. Sonicate at 37.degree. for 1/2 hour.
Clear solution of 100 .mu.M will result. This stock solution is
aliquoted and stored at -80 C
[0211] 2. Cell Line Maintenance [0212] a. V5 tags were added at the
N-terminus of hS1P1, hS1P3, rS1P1 and rS1P3. All four genes were
transfected into CHO K1 cells which stably express Gqi5. [0213] b.
hS1P1 and hS1P3 were established as stable clones and rS1P1 and
rS1P3 were sorted by anti V5 tag and established as stable pools
after sorting. [0214] c. Parental cell line CHO/K1 Gqi5 is used as
the control. [0215] d. All the cells are maintained in cell growth
media and splitted twice a week using Versene. [0216] e. All the
cell lines are used under passage 30.
[0217] 3. Assay Protocol [0218] a. Cell seeding: Cells are lifted
from the flask by Versene and seeded in cell plates at 50K/well in
cell seeding media. Cells are grown overnight at 37 degree. [0219]
b. Cell loading: Cell seeding media is discarded. Cells are loaded
with 50 ul of FLIPR dye at RT for 90 min. Signal is stable for up
to 5 h after dye loading. [0220] c. Agonist (S1P) preparation:
Frozen stock of S1P is thawed out and sonicated at 37 c for 30
minutes every time before use. The stock is then diluted into FLIPR
buffer at proper concentration. [0221] d. Compound preparation:
Compounds are dissolved in DMSO. A 3.times., 10 point dilution of
the compounds are carried out in DMSO. Then the compounds are
diluted into assay buffer 133.times. so that the DMSO concentration
is 0.75%. [0222] e. Activity measurement: The fluorescence signal
change of the cells upon compound addition is monitored in
FLIPRtetra. 25 ul of compound is transferred into the cell plates
(50 ul of FLIPR dye; DMSO concentration: 0.25%). Signal is recorded
for 90 sec after compound addition. Then 50 ul of 500 nM S1P is
added in the cell plate, and signal is recorded for 90 seconds upon
addition.
[0223] 4. Data Analysis [0224] a. Peak value is calculated for each
compound/S1P addition [0225] b. The peak value of S1P at 200 nM is
used as high control (100%), and the peak value of buffer only is
used as low control (0%). [0226] c. Data is normalized against high
and low controls using the following equation:
[0226] POC.sub.--S=100*(RAW-LO)/(HI-LO) [0227] d. Peak value is
plotted against the concentration of compound. [0228] e. Curve is
fitted using the 4 parameter fit:
[0228] Y=(A+(B/(1+((x/C) D)))) [0229] where: [0230] Y is POC_S (or
POC) [0231] X is compound concentration [0232] A is the minimum
(EC50min or IC50min) [0233] B is the maximum (EC50max or IC50max)
[0234] C is the inflection point (EC50IP or IC.sub.50IP) [0235] D
is the hill slope (EC50 slope or IC50 slope).
[0236] Identified S1P receptor agonists and antagonists can be used
to treat a variety of human diseases and disorders, including, but
not limited to the treatment of infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
ulcers; asthma; allergy; benign prostatic hypertrophy; migraine;
vomiting; psychotic and neurological disorders, including anxiety,
schizophrenia, manic depression, depression, delirium, dementia,
and severe mental retardation.
[0237] Pain is a complex subjective sensation reflecting real or
potential tissue damage and the affective response to it. Acute
pain is a physiological signal indicating a potential or actual
injury. Chronic pain can either be somatogenetic (organic) or
psychogenic. Chronic pain is frequently accompanied or followed by
vegetative signs, which often result in depression.
[0238] Somatogenetic pain may be of nociceptive origin,
inflammatory or neuropathic. Nociceptive pain is judged to be
commensurate with ongoing activation of somatic or visceral
pain-sensitive nerve fibers. Neuropathic pain results from
dysfunction in the nervous system; it is believed to be sustained
by aberrant somatosensory processes in the peripheral nervous
system, the CNS, or both. Chronic pain results in individual
suffering and social economic costs of tremendous extent. Existing
pharmacological pain therapies are widely unsatisfying both in
terms of efficacy and of safety.
[0239] In one embodiment, S1P modulators of the present invention
are used as immunomodulators to suppress the immune system and
prevent damage to healthy tissue that would otherwise occur in
autoimmune diseases and in organ transplantation. The compounds can
be administered to patients as part of the treatment associated
with organ transplantation, including pancreas, pancreatic islets,
kidney, heart and lung transplantations. The S1P modulators can be
administered alone or in combination with known immunosuppressants
such as cyclosporine, tacrolimus, azatioprine, desoxymetasone,
cyclophosphamide, cortisone, betametasone, FK 506 (a fungal
macrolide immunosuppressant), desametasone, flunisolide,
prednisolone, prednisone, amcinomide desonide, methylprednisolone,
triamcinolone, alclometasone and methotrexate.
[0240] The dosage to be used is, of course, dependent on the
specific disorder to be treated, as well as additional factors
including the age, weight, general state of health, severity of the
symptoms, frequency of the treatment and whether additional
pharmaceuticals accompany the treatment. The dosages are in general
administered several times per day and preferably one to three
times per day. The amounts of the individual active compounds are
easily determined by routine procedures known to those of ordinary
skill in the art
[0241] S1P also acts as a survival factor in many cell types. S1P
receptor modulators are anticipated to have activity in protecting
cells and tissues from hypoxic conditions. In accordance with one
embodiment compounds of the invention are administered to treat
cells and tissues exposed to hypoxic conditions, including injury
sustained as a result of ischemia. In accordance with one
embodiment, the S1P modulators having antagonistic activity can be
used to treat ischemia reperfusion type injury. Interference with
the supply of oxygenated blood to tissues is defined as ischemia.
The effects of ischemia are known to be progressive, such that over
time cellular vitality continues to deteriorate and tissues become
necrotic. Total persistent ischemia, with limited oxygen perfusion
of tissues, results in cell death and eventually in
coagulation-induced necrosis despite reperfusion with arterial
blood.
[0242] The compounds of the invention and the other
pharmacologically active agent may be administered to a patient
simultaneously, sequentially or in combination. It will be
appreciated that when using a combination of the invention, the
compound of the invention and the other pharmacologically active
agent may be in the same pharmaceutically acceptable carrier and
therefore administered simultaneously. They may be in separate
pharmaceutical carriers such as conventional oral dosage forms
which are taken simultaneously. The term "combination" further
refers to the case where the compounds are provided in separate
dosage forms and are administered sequentially.
[0243] The compounds of the invention may be administered to
patients (animals and humans) in need of such treatment in dosages
that will provide optimal pharmaceutical efficacy. It will be
appreciated that the dose required for use in any particular
application will vary from patient to patient, not only with the
particular compound or composition selected, but also with the
route of administration, the nature of the condition being treated,
the age and condition of the patient, concurrent medication or
special diets then being followed by the patient, and other factors
which those skilled in the art will recognize, with the appropriate
dosage ultimately being at the discretion of the attendant
physician.
[0244] An appropriate dosage level will generally be about 0.001 to
50 mg per kg patient body weight per day, which may be administered
in single or multiple doses. Preferably, the dosage level will be
about 0.01 to about 25 mg/kg per day; more preferably about 0.05 to
about 10 mg/kg per day. For example, in the treatment or prevention
of a disorder of the central nervous system, a suitable dosage
level is about 0.001 to 10 mg/kg per day, preferably about 0.005 to
5 mg/kg per day, and especially about 0.01 to 1 mg/kg per day. The
compounds may be administered on a regimen of 1 to 4 times per day,
preferably once or twice per day.
[0245] It will be appreciated that the amount of the compound of
the invention required for use in any treatment will vary not only
with the particular compounds or composition selected but also with
the route of administration, the nature of the condition being
treated, and the age and condition of the patient, and will
ultimately be at the discretion of the attendant physician.
[0246] The compositions and combination therapies of the invention
may be administered in combination with a variety of pharmaceutical
excipients, including stabilizing agents, carriers and/or
encapsulation formulations as described herein.
[0247] Aqueous compositions of the present invention comprise an
effective amount of the compounds of the invention, dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous
medium.
[0248] "Pharmaceutically or pharmacologically acceptable" include
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate. "Pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0249] For human administration, preparations should meet
sterility, pyrogenicity, general safety and purity standards as
required by FDA Office of Biologics standards.
[0250] The compositions and combination therapies of the invention
will then generally be formulated for parenteral administration,
e.g., formulated for injection via the intravenous, intramuscular,
subcutaneous, intralesional, or even intraperitoneal routes. The
preparation of an aqueous composition that contains a composition
of the invention or an active component or ingredient will be known
to those of skill in the art in light of the present disclosure.
Typically, such compositions can be prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a liquid
prior to injection can also be prepared; and the preparations can
also be emulsified.
[0251] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
[0252] Solutions of active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0253] Therapeutic or pharmacological compositions of the present
invention will generally comprise an effective amount of the
component(s) of the combination therapy, dissolved or dispersed in
a pharmaceutically acceptable medium. Pharmaceutically acceptable
media or carriers include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well known in the
art. Supplementary active ingredients can also be incorporated into
the therapeutic compositions of the present invention.
[0254] The preparation of pharmaceutical or pharmacological
compositions will be known to those of skill in the art in light of
the present disclosure. Typically, such compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection; as tablets or other solids for oral
administration; as time release capsules; or in any other form
currently used, including cremes, lotions, mouthwashes, inhalants
and the like.
[0255] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0256] The preparation of more, or highly, concentrated solutions
for intramuscular injection is also contemplated. In this regard,
the use of DMSO as solvent is preferred as this will result in
extremely rapid penetration, delivering high concentrations of the
active compound(s) or agent(s) to a small area.
[0257] The use of sterile formulations, such as saline-based
washes, by surgeons, physicians or health care workers to cleanse a
particular area in the operating field may also be particularly
useful. Therapeutic formulations in accordance with the present
invention may also be reconstituted in the form of mouthwashes, or
in conjunction with antifungal reagents. Inhalant forms are also
envisioned. The therapeutic formulations of the invention may also
be prepared in forms suitable for topical administration, such as
in cremes and lotions.
[0258] Suitable preservatives for use in such a solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium bisulfite,
sodium metabisulfite, sodium thiosulfite, thiourea and the like.
Suitable wetting and clarifying agents include polysorbate 80,
polysorbate 20, poloxamer 282 and tyloxapol. Suitable
viscosity-increasing agents include dextran 40, dextran 70,
gelatin, glycerin, hydroxyethylcellulose,
hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like.
[0259] Upon formulation, therapeutics will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed.
[0260] In this context, the quantity of active ingredient and
volume of composition to be administered depends on the host animal
to be treated. Precise amounts of active compound required for
administration depend on the judgment of the practitioner and are
peculiar to each individual.
[0261] A minimal volume of a composition required to disperse the
active compounds is typically utilized. Suitable regimes for
administration are also variable, but would be typified by
initially administering the compound and monitoring the results and
then giving further controlled doses at further intervals. For
example, for parenteral administration, a suitably buffered, and if
necessary, isotonic aqueous solution would be prepared and used for
intravenous, intramuscular, subcutaneous or even intraperitoneal
administration. One dosage could be dissolved in 1 mL of isotonic
NaCl solution and either added to 1000 mL of hypodermolysis fluid
or injected at the proposed site of infusion, (see for example,
Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038
and 1570-1580).
[0262] In certain embodiments, active compounds may be administered
orally. This is contemplated for agents which are generally
resistant, or have been rendered resistant, to proteolysis by
digestive enzymes. Such compounds are contemplated to include
chemically designed or modified agents; dextrorotatory peptides;
and peptide and liposomal formulations in time release capsules to
avoid peptidase and lipase degradation.
[0263] Pharmaceutically acceptable salts include acid addition
salts and which are formed with inorganic acids such as, for
example, hydrochloric, hydrobromic, boric, phosphoric, sulfuric
acids or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, maleic, fumaric, citric, succinic, mesylic, mandelic,
succinic, benzoic, ascorbic, methanesulphonic, a-keto glutaric,
a-glycerophosphoric, glucose-1-phosphoric acids and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, magnesium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Other examples of pharmaceutically acceptable salts include
quaternary derivatives, and internal salts such as N-oxides.
[0264] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0265] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0266] The preparation of more, or highly, concentrated solutions
for direct injection is also contemplated, where the use of DMSO as
solvent is envisioned to result in extremely rapid penetration,
delivering high concentrations of the active agents to a small
area.
[0267] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0268] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure.
[0269] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations;
time-release capsules; and any other form currently used, including
cremes.
[0270] Additional formulations suitable for other modes of
administration include suppositories. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%.
[0271] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders.
[0272] In certain defined embodiments, oral pharmaceutical
compositions will comprise an inert diluent or assimilable edible
carrier, or they may be enclosed in hard or soft shell gelatin
capsule, or they may be compressed into tablets, or they may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0273] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange
flavor.
[0274] The pharmaceutical compositions of this invention may be
used in the form of a pharmaceutical preparation, for example, in
solid, semisolid or liquid form, which contains one or more of the
compound of the invention, as an active ingredient, in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral or parenteral applications. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, and any
other form suitable for use. The carriers which can be used are
water, glucose, lactose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form,
and in addition auxiliary, stabilizing, thickening and coloring
agents and perfumes may be used. The active object compound is
included in the pharmaceutical composition in an amount sufficient
to produce the desired effect upon the process or condition of the
disease.
[0275] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g., conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g., water, to form a solid preformulation composition containing
a homogeneous mixture of a compound of the invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the invention. The
tablets or pills of the novel composition can be coated or
otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0276] The liquid forms in which the compositions of the invention
may be incorporated for administration orally or by injection
include aqueous solution, suitably flavored syrups, aqueous or oil
suspensions, and emulsions with acceptable oils such as cottonseed
oil, sesame oil, coconut oil or peanut oil, or with a solubilizing
or emulsifying agent suitable for intravenous use, as well as
elixirs and similar pharmaceutical vehicles. Suitable dispersing or
suspending agents for aqueous suspensions include synthetic and
natural gums such as tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or
gelatin.
[0277] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. Preferably the compositions
are administered by the oral or nasal respiratory route for local
or systemic effect. Compositions in preferably sterile
pharmaceutically acceptable solvents may be nebulized by use of
inert gases. Nebulized solutions may be breathed directly from the
nebulizing device or the nebulizing device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0278] For treating clinical conditions and diseases noted above,
the compound of this invention may be administered orally,
topically, parenterally, by inhalation spray or rectally in dosage
unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques.
[0279] The following examples are given for the purpose of
illustrating the invention, but not for limiting the scope or
spirit of the invention.
[0280] Compounds of the invention may be prepared as described in
the following schemes.
##STR00026##
##STR00027##
##STR00028##
##STR00029##
##STR00030##
##STR00031##
##STR00032##
##STR00033##
##STR00034##
EXAMPLES
[0281] Compounds were prepared using the general procedures as
described below:
##STR00035##
A: General Procedure for C--C Bond Coupling with Rieke Reagents
[0282] 5-bromobenzofuran (1.0 mmol) was dissolved in a THF solution
of Rieke reagent (0.5M, 2.9 mmol) in a microwave reaction tube.
Pd(PtBu.sub.3).sub.2 (0.05 mmol) was added to this solution. The
mixture was purged with N.sub.2 gas for 3-5 min and heated at
100.degree. C. for 30 min under microwave irradiation (Personal
Chemistry Emrys.TM. Optimizer microwave reactor). Upon completion
of the reaction, the reaction mixture was diluted with ethyl
acetate, washed with 1N HCl aqueous solution, brine, filtered
through Celite. The filtrate was dried over Na.sub.2SO.sub.4 and
concentrated. The residue was purified by silica gel column
chromatography (ISCO system) to give a pure product.
B: General Procedure for N--C Bond Coupling Reaction
[0283] 5-Bromobenzofuran (1.0 mmol), piperidine (1.2 mmol),
Pd(dppf)Cl.sub.2 (0.03 mmol), dppf (0.045 mmol) and sodium
tert-butoxide (1.5 mmol) was mixed in toluene (2 mL). The mixture
was purged with N.sub.2 gas for 3-5 min and heated at 120.degree.
C. for 30 min under microwave irradiation (Personal Chemistry
Emrys.TM. Optimizer microwave reactor). Upon completion of the
reaction, the reaction mixture was directly loaded on silica gel
column and purified on ISCO system (5% EtOAc in hexanes) to give a
pure product.
C: General Preparative Procedure for Formation of Benzofuran
Boronic Acids
[0284] A solution of n-BuLi (1.2 mmol, 2.5M solution in hexanes)
was added dropwise to a solution of benzofuran compounds (1.0 mmol)
in anhydrous THF (20 mL) at -78.degree. C. The resulting mixture
was stirred at -78.degree. C. for 20 min, and treated with
B(.sup.iPrO).sub.3 (1.5 mmol). The reaction mixture was allowed to
warm up slowly to room temperature and stirred for 1 h. The
reaction was cooled in ice-bath and quenched with 2N HCl or
saturate NH.sub.4Cl and extracted with Et.sub.2O. The combined
organic extracts were washed with brine, dried and concentrated
under reduced pressure to yield a desired benzofuran boronic acid
without further purification for next step.
D: General Procedure of Coupling Boronic Acids with Aryl
Halides
[0285] A mixture of benzofuran boronic acid (1.1 mmol), aryl halide
(1.0 mmol), triethylamine (20 mmol) and
bis(triphenylphosphine)palladium(II) chloride (0.05 mmol) in
ethanol (30 mL) was irradiated in a microwave instrument at
100.degree. C. for 20 min. The reaction mixture was cooled, and the
solvent was removed. The residue was treated with water and
extracted with ethyl acetate. The organic layer was dried and
concentrated in vacuo (the aqueous work-up is optional).
Purification by silica gel chromatography gave the desired
product.
E: General Procedure of Reductive Amination
[0286] A mixture of aldehyde (1.0 mmol), acetic acid (1.5 mmol) and
azetidine-3-carboxylic acid or piperidine-4-carboxylic acid
(1.2-1.5 mmol) in DCM/MeOH (1:1, 10 mL) was stirred at room
temperature for 1 h. Sodium cyanoborohydride (0.5 mmol) was added
and the reaction mixture was stirred for 2-3 h at room temperature.
After concentration of solvent under reduced pressure, the
resulting residue was dissolved in DMSO, filtered and purified by
reverse phase preparative HPLC (Phenomenex reverse phase Luna 5.mu.
C18(2) column, 60.times.21.2 mm ID, mobile phase: A=0.05% TFA in
water; B=0.05% TFA in acetonitrile. The flow rate was 10-12 mL/min)
to yield the desired final product with purity greater than 95%.
All final products were obtained as the TFA salts except for
Compound 59. Alternatively, the crude mixture of reductive
amination can be purified by trituration with MeOH and water.
Compound 1
1-(4-(5-Phenylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
1-(2,2-Diethoxyethoxy)-4-phenylbenzene (step 1 in Scheme 1)
##STR00036##
[0288] A mixture of 4-phenylphenol (5 g, 29.4 mmol),
bromoacetaldehyde diethyl acetal (4.56 mL, 29.4 mmol) and KOH (1.94
g, 29.4 mmol) in DMSO (15 mL) was stirred at reflux for 6 h. The
reaction mixture was allowed to cool down to room temperature and
poured over ice containing 0.60 g of KOH and diluted to 100 mL with
water. The solution was extracted with Et.sub.2O (20 mL.times.3);
the combined extracts were washed with 1N NaOH solution, water and
brine, dried, and concentrated under reduced pressure to yield 7.97
g (94%) of a yellow oil that was used without further purification:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.56-7.50 (m, 4H), 7.41
(t, 2H), 7.30 (t, 1H), 7.00 (dt, 2H), 4.86 (t, 1H), 4.05 (d, 2H),
3.82-3.74 (m, 2H), 3.69-3.62 (m, 2H).
5-Phenylbenzofuran (step 2 in Scheme 1)
##STR00037##
[0290] A mixture of 1-(2,2-diethoxyethoxy)-4-phenylbenzene (3.52 g
, 12.3 mmol) and polyphosphoric acid (2.95 g, 29.4 mmol) in benzene
(60 mL) was stirred at reflux for 2 h. The reaction mixture was
cooled to room temperature, decanted from the PPA and filtered
through a plug of silica gel, which was washed with hexanes. The
filtrate and the wash were combined anc concentrated under reduced
pressure to yield 2.00 g of the crude benzofuran: .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 7.79 (dd, 1H), 7.66 (d, 1H), 7.63-7.60 (m,
2H), 7.58-7.51 (m, 2H), 7.45 (t, 2H), 7.36-7.33 (m, 1H), 6.82 (dd,
1H).
5-Phenylbenzofuran-2-yl-2-boronic acid (step 3 in Scheme 1)
##STR00038##
[0292] A solution of n-BuLi (2.0 mL, 2.5M solution in hexanes) was
added dropwise to a solution of 5-phenylbenzofuran (816 mg, 4.21
mmol) in anhydrous THF (20 mL) at -78.degree. C. The resulting
mixture was stirred at -78.degree. C. for 20 min, and treated with
B(.sup.iPrO).sub.3 (1.46 mL, 6.31 mmol). The reaction mixture was
allowed to warm up slowly to room temperature and stirred for 1 h.
The reaction was quenched with 2N HCl and extracted with Et.sub.2O.
The combined extracts were washed with brine, dried and
concentrated under reduced pressure to yield 1.2 g of crude boronic
acid, that was used without further purification: .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 7.83 (dd, 1H), 7.64-7.55 (m, 4H),
7.48-7.42 (m, 3H), 7.38-7.32 (m 1H).
4-(5-Phenylbenzofuran-2-yl)benzaldehyde (step 4 in Scheme 1)
##STR00039##
[0294] A solution of 5-phenylbenzofuran-2-yl-2-boronic acid (527
mg, 2.22 mmol), 4-bromobenzaldehyde (315 mg, 1.70 mmol),
palladiumdichlorobis(triphenylphosphine) (60 mg, 0.085 mmol) and
triethylamine (4.74 mL, 34 mmol) in EtOH was irradiated in the
microwave at 100.degree. C. for 1200 s. The precipitated that
formed was filtered and rinsed with ethanol to yield 217 mg of
desired benzaldehyde: NMR (400 MHz, CD.sub.3OD) .delta. 10.06 (s,
1H), 8.05 (d, 2H), 7.98 (d, 2H), 7.82 (br s, 1H), 7.65-7.52 (m,
4H), 7.48 (dd, 2H), 7.37 (t, 1H). MS (ESI) m/z: Calculated: 298.10.
Observed: 299.1 (M.sup.++1).
1-(4-(5-Phenylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic acid
(step 5 in Scheme 1)
##STR00040##
[0296] A mixture of 4-(5-phenylbenzofuran-2-yl)benzaldehyde (49 mg,
0.14 mmol) and azetidine-3-carboxylic acid (30 mg, 0.28 mmol) in
MeOH (1 mL) was stirred at room temperature for 1 h. Sodium
cyanoborohydride (60 mg, 0.28 mmol) was added in two portions and
the reaction mixture was stirred for 16 h. Concentration of the
solvent under reduced pressure yielded a yellow solid that was
dissolved in DMSO (3 mL) and filtered to give a yellow solution
that was purified by HPLC to yield 3 mg of desired product [hS1P1
EC.sub.50=1200 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.03
(d, 2H), 7.84 (br s, 1H), 7.66-7.58 (m, 6H), 7.45 (t, 2H),
7.36-7.32 (m, 2H), 4.47 (s, 2H), 4.40-4.32 (m, 4H), 3.72 (m, 1H).
MS (ESI) m/z: Calculated: 383.15. Observed: 383.9 (M.sup.++1).
Compound 2
1-((4-(5-Butylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
1-(2,2-Diethoxyethoxy)-4-butylbenzene
##STR00041##
[0298] The title compound was prepared as Example Compound 1 (step
1 in Scheme 1) in the general method described above (90% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.07 (d, J=8.8, 2H), 6.83
(d, J=8.8, 2H), 4.83 (t, J=5.1, 1H), 3.98 (d, J=5.1, 2H), 3.80-3.72
(m, 2H), 3.67-3.59 (m, 2H), 2.54 (t, J=7.7, 2H), 1.59-1.51 (m, 2H),
1.36-1.30 (m, 2H), 1.24 (t, J=7.0, 6H), 0.91 (t, J=7.3, 3H).
5-Butylbenzofuran
##STR00042##
[0300] The title compound was prepared as Example Compound 1 (step
2 in Scheme 1) in the general method described above (91% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.58 (d, J=2.2, 1H),
7.41-7.36 (m, 2H), 7.11 (dd, J=8.5, 1.8, 1H), 6.70 (dd, J=2.2, 1.1,
1H), 2.70 (t, J=7.7, 2H), 1.67-1.60 (m, 2H), 1.42-1.32 (m, 2H),
0.93 (t, J=7.3, 3H).
5-Butylbenzofuran-2-yl-2-boronic acid
##STR00043##
[0302] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) in the general method described above (67% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.31 (m, 2H),
7.22-7.14 (m, 2H), 2.70 (t, J=7.7, 2H), 1.67-1.59 (m, 2H),
1.41-1.32 (m, 2H), 0.93 (t, J=7.3, 3H).
4-(5-Butylbenzofuran-2-yl)benzaldehyde
##STR00044##
[0304] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (72% yield):
NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.00 (d, J=8.4,
2H), 7.94 (d, J=8.4, 2H), 7.45-7.41 (m, 2H), 7.17-7.15 (m, 2H),
2.71 (t, J=7.7, 2H), 1.68-1.61 (m, 2H), 1.41-1.33 (m, 2H), 0.94 (t,
J=7.3, 3H).
1-((4-(5-Butylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00045##
[0306] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (42% yield)
[hS1P1 EC.sub.50=200 nM, 510 nM, 867 nM]: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.98 (d, J=8.4, 2H), 7.55 (d, J=8.4, 2H),
7.43-7.41 (m, 2H), 7.23 (s, 1H), 7.15 (d, J=8.8, 1H), 4.40 (s, 2H),
4.25-4.23 (m, 4H), 3.52-3.46 (m, 1H), 2.71 (t, J=7.7, 2H),
1.67-1.61 (m, 2H), 1.41-1.33 (m, 2H), 0.95 (t, J=7.3, 3H). MS (ESI)
m/z: Calculated: 363.18. Observed: 364.0 (M.sup.++1).
Compound 3
1-(4-(5-Butoxybenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
1-(2,2-Diethoxyethoxy)-4-butoxybenzene
##STR00046##
[0308] The title compound was prepared as Example Compound 1 (step
1 in Scheme 1) in the general method described above (84% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 6.86-6.80 (m, 4H), 4.81
(t, J=5.1, 1H), 3.96 (d, J=5.1, 2H), 3.90 (t, J=6.6, 2H), 3.79-3.72
(m, 2H), 3.67-3.59 (m, 2H), 1.77-1.70 (m, 2H), 1.52-1.43 (m, 2H),
1.24 (t, J=7.0, 6H), 0.96 (t, J=7.4, 3H).
5-Butoxybenzofuran
##STR00047##
[0310] The title compound was prepared as Example Compound 1 (step
2 in Scheme 1) in the general method described above (81% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.58 (d, J=2.2, 1H), 7.38
(d, J=9.2, 1H), 7.05 (d, J=2.5, 1H), 6.90 (dd, J=2.5, 8.8, 1H),
6.69 (br d, J=2.2, 1H), 3.99 (t, J=6.6, 2H), 1.82-1.75 (m, 2H),
1.56-1.47 (m, 2H), 0.99 (t, J=7.3, 3H).
5-Phenylbenzofuran-2-yl-2-boronic acid (step 3 in Scheme 1)
##STR00048##
[0312] A solution of n-BuLi (2.5 mL, 2.5M solution in hexanes) was
added dropwise to a solution of 5-butoxybenzofuran (1.0 g, 5.21
mmol) in anhydrous THF (20 mL) at -78.degree. C. The resulting
mixture was stirred at -78.degree. C. for 20 min, and treated with
B(.sup.iPrO).sub.3 (1.80 mL, 7.8 mmol). The reaction mixture was
allowed to warm up slowly to room temperature and stirred for 1 h.
The reaction was quenched with 2N HCl and extracted with Et.sub.2O.
The combined extracts were washed with brine, dried and
concentrated under reduced pressure to yield 1.2 g of crude boronic
acid, that was used without further purification: (98% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.37 (d, 1H), 7.30 (d,
1H), 7.06 (s, 1H), 6.98 (d, 1H), 4.44 (s, 2H), 1.81-1.71 (m, 2H),
1.58-1.50 (m, 2H), 1.00 (t, 3H).
4-(5-Butoxybenzofuran-2-yl)benzaldehyde (step 4 in Scheme 1)
##STR00049##
[0314] A solution of 5-phenylbenzofuran-2-yl-2-boronic acid (702
mg, 3.0 mmol), 4-bromobenzaldehyde (427 mg, 2.30 mmol),
palladiumdichlorobis(triphenylphosphine) (80 mg, 0.11 mmol) and
triethylamine (6.5 mL, 45 mmol) in EtOH (2 mL) was irradiated in
the microwave at 100.degree. C. for 1200 s. The precipitate that
formed was filtered and rinsed with ethanol to yield 620 mg of
crude product, which upon column chromatography afforded 375 mg of
the desired compound (43%): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 10.03 (s, 1H), 8.05 (d, 2H), 7.98 (d, 2H), 7.82 (d, 1H),
7.18 (d, 1H), 7.16 (d, 1H), 6.94 (s, 1H), 4.44 (s, 2H), 1.81-1.71
(m, 2H), 1.58-1.50 (m, 2H), 1.00 (t, 3H). MS (ESI) m/z: Calculated:
294.34. Observed: 295.2 (M.sup.++1).
1-(4-(5-Butoxybenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid (step 5 in Scheme 1)
##STR00050##
[0316] A mixture of 4-(5-butoxybenzofuran-2-yl)benzaldehyde (70 mg,
0.30 mmol), azetidine-3-carboxylic acid (46 mg, 0.45 mmol) and
acetic acid (0.50 mmol) in MeOH-DCM (3:1; 2 mL) was stirred at room
temperature for 1 h. Sodium triacetoxyborohydride (211 mg, 1.00
mmol) was added and the reaction mixture was stirred for 16 h.
Concentration of the solvent under reduced pressure yielded a
yellow solid that was dissolved in DMSO (3 mL) and filtered to give
a yellow solution that was purified by HPLC to afford 6 mg of
desired product (5% yield) [hS1P1 EC.sub.50=520 nM]: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 7.97 (d, 2H), 7.55 (d, 2H), 7.40 (d,
1H), 7.21 (s, 1H), 7.10 (d, 1H), 6.92-6.89 (dd, 1H), 4.44 (s, 2H),
4.37 (q, 4H), 4.00 (t, 2H), 3.72-3.64 (m, 1H), 1.81-1.71 (m, 2H),
1.58-1.50 (m, 2H), 1.00 (t, 3H). MS (ESI) m/z: Calculated: 379.45.
Observed: 380.3 (M.sup.++1).
Compound 4
1-((4-(5-Benzylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
1-(4-(2,2-Diethoxyethoxy)benzyl)benzene
##STR00051##
[0318] The title compound was prepared as Example Compound 1 (step
1 in Scheme 1) in the general method described above (84% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.30-7.25 (m, 2H),
7.20-7.15 (m, 3H), 7.09 (d, J=8.8, 2H), 6.84 (d, J=8.8, 2H), 4.82
(t, J=5.5, 1H), 3.98 (d, J=5.5, 2H), 3.92 (s, 2H), 3.79-3.72 (m,
2H), 3.66-3.59 (m, 2H), 1.24 (t, 7.1, 3H).
5-Benzylbenzofuran
##STR00052##
[0320] The title compound was prepared as Example Compound 1 (step
2 in Scheme 1) in the general method described above (89% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.58 (d, J=2.2, 1H),
7.42-7.40 (m, 2H), 7.31-7.7.26 (m, 3H), 7.25-7.12 (m, 3H), 6.70 (m,
1H), 4.08 (s, 2H).
5-Benzylbenzofuran-2-yl-2-boronic acid
##STR00053##
[0322] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) in the general method described above (66% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.44 (m, 1H), 7.42 (d,
J=8.4, 1H), 7.32-7.26 (m, 4H), 7.25-7.19 (m, 3H), 4.81 (s, 2H),
4.08 (s, 2H).
4-(5-Benzylbenzofuran-2-yl)benzaldehyde
##STR00054##
[0324] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (76% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 7.99 (d,
J=8.4, 2H), 7.94 (d, J=8.4, 2H), 7.46-7.41 (m, 2H), 7.32-7.17 (m,
6H), 7.13 (br s, 1H), 4.08 (s, 2H).
1-((4-(5-Benzylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00055##
[0326] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (62% yield)
[hS1P1 EC.sub.50=400 nM, 460 nM, 420 nM, 107 nM]: .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 7.98 (d, J=8.4, 2H), 7.55 (d, J=8.4, 2H),
7.45-7.42 (m, 2H), 7.28-7.15 (m, 7H), 4.44 (s, 2H), 4.37-4.22 (m,
4H), 4.06 (s, 2H), 3.72-3.64 (m, 1H). MS (ESI) m/z: Calculated:
397.17. Observed: 398.0 (M.sup.++1).
Compound 5
1-((4-(7-Benzylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
1-(2-(2,2-Diethoxyethoxy)benzyl)benzene
##STR00056##
[0328] The title compound was prepared as Example Compound 1 (step
1 in Scheme 1) in the general method described above (99% yield):
NMR (400 MHz, CDCl.sub.3) .delta. 7.27-7.21 (m, 4H), 7.19-7.15 (m,
2H), 7.08 (br d, J=5.9, 1H), 6.90-6.83 (m, 2H), 4.78 (t, J=5.1,
1H), 4.00-3.98 (m, 4H), 3.76-3.69 (m, 2H), 3.63-3.56 (m, 2H), 1.22
(t, J=7.0, 6H).
7-Benzylbenzofuran
##STR00057##
[0330] The title compound was prepared as Example Compound 1 (step
2 in Scheme 1) in the general method described above (84% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.62 (d, J=2.2, 1H), 7.45
(d, J=7.7, 1H), 7.36 (s, 1H), 7.29-7.26 (m, 3), 7.25-7.13 (m, 2),
7.05 (d, J=7.4, 1H), 6.76 (d, J=2.2, 1H), 4.27 (s, 2H).
7-Benzylbenzofuran-2-yl-2-boronic acid
##STR00058##
[0332] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) in the general method described above (67% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.50 (dd, J=7.7, 1H),
7.36 (s, 1H), 7.29-7.25 (m, 4H), 7.18-7.09 (m, 3H), 4.29 (s,
2H).
4-(7-Benzylbenzofuran-2-yl)benzaldehyde
##STR00059##
[0334] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (72% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.04 (s, 1H), 8.00-7.94
(m, 4H), 7.50 (d, J=9.9, 1H), 7.47-7.27 (m, 4H), 7.24-7.17 (m, 3H),
7.11 (d, J=7.3, 1H), 4.33 (s, 2H).
1-((4-(7-Benzylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00060##
[0336] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (81% yield)
[hS1P1 EC.sub.50>1 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.97 (d, J=8.0, 2H), 7.56 (d, J=8.0, 2H), 7.48 (d, J=7.7,
1H), 7.34-7.24 (m, 5H), 7.19-7.10 (m, 3H), 4.44 (s, 2H), 4.32-4.25
(m, 6H), 3.66-3.56 (m, 1H). MS (ESI) m/z: Calculated: 397.17.
Observed: 397.9 (M.sup.++1).
Compound 6
1-(4-(5-cyclohexylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
5-cyclohexylbenzofuran (step 1 in Scheme 2)
##STR00061##
[0338] 5-bromobenzofuran (500 mg, 2.55 mmol) was dissolved in a
TI-IF solution of cyclohexyl zinc(II) bromide (0.5M, 15 mL, 7.40
mmol) in a microwave reaction tube. Pd(P.sup.tBu.sub.3).sub.2 (65
mg, 0.128 mmol, 0.05 eqv.) was added to this solution. The mixture
was purged with N.sub.2 gas for 3-5 min and heated at 100.degree.
C. for 30 min under microwave irradiation. Upon completion of the
reaction, the reaction mixture was diluted with ethyl acetate,
washed with 1N HCl aqueous solution, brine, filtered through
Celite. The filtrate was dried over Na.sub.2SO.sub.4 and
concentrated. The residue was purified by silica gel column
chromatography (ISCO system, 5% EtOAc in hexanes) to give 0.217 g
desired product (43% yield): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.57 (d, 1H), 7.41 (d, 2H), 7.15 (d, 1H), 6.72 (d, 1H),
2.58 (m, 1H), 1.92-1.74 (m, 4H), 1.51-1.35 (m, 4H), 1.31-1.25 (m,
2H).
5-cyclohexylbenzofuran-2-ylboronic acid (step 2 in Scheme 2)
##STR00062##
[0340] A solution of n-BuLi (360 .mu.L, 0.9 mmol, 2.5M solution in
hexanes) was added dropwise to a solution of 5-cyclohexylbenzofuran
(150 mg, 0.75 mmol) in anhydrous THF (5 mL) at -78.degree. C. The
resulting mixture was stirred at -78.degree. C. for 40 min, and
treated with B(.sup.iPrO).sub.3 (260 .mu.L, 1.13 mmol). The
reaction mixture was allowed to warm up slowly to room temperature
and stirred for 1 h. TLC indicated the completion of reaction. The
reaction was cooled in ice-bath and quenched with 2N HCl (3 mL) and
extracted with Et.sub.2O. The combined organic extracts were washed
with brine, dried and concentrated under reduced pressure to yield
a desired boronic acid (0.156 g, 85% yield) without further
purification for next step. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.46 (s, 1H), 7.43 (d, 1H), 7.32 (s, 1H), 7.25 (d, 1H),
2.62 (m, 1H), 1.93-1.85 (m, 4H), 1.78-1.75 (m, 4H), 1.34-1.22 (m,
2H).
4-(5-cyclohexylbenzofuran-2-yl)benzaldehyde (step 3 in Scheme
2)
##STR00063##
[0342] A mixture of 5-cyclohexylbenzofuran-2-ylboronic acid (75 mg,
0.37 mmol), 4-bromobenzaldehyde (62 mg, 0.34 mmol), triethylamine
(1.1 mL, 7.5 mmol) and bis(triphenylphosphine)palladium(II)
chloride (13 mg, 0.05 mmol) in ethanol (11 mL) was irradiated in a
microwave instrument at 100.degree. C. for 20 min. The reaction
mixture was cooled, and the solvent was removed. The residue was
purification by silica gel chromatography on ISCO system gave the
title compound (52 mg, 46% yield): >95% purity by LCMS, ESI-MS:
305.2 (M+H.sup.+). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03
(s, 1H), 8.00 (d, 2H), 7.95 (d, 2H), 7.46 (d, 2H), 7.19 (d, 1H),
7.16 (s, 1H), 2.63-2.58 (m, 1H), 1.94-1.76 (m, 4H), 1.53-1.42 (m,
4H), 1.38-1.25 (m, 2H). MS (ESI) m/z: Calculated: 304.38. Observed:
305.2 (M.sup.++1).
1-(4-(5-cyclohexylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (step 4 of Scheme 2)
##STR00064##
[0344] A mixture of 4-(5-cyclohexylbenzofuran-2-yl) benzaldehyde
(30 mg, 0.1 mmol), acetic acid (9 .mu.L, 0.15 mmol) and
azetidine-3-carboxylic acid (15 mg, 0.15 mmol) in DCM/MeOH (1:1, 2
mL) was stirred at room temperature for 1 h. Sodium
cyanoborohydride (3.1 mg, 0.05 mmol) was added and the reaction
mixture was stirred for 3 h at room temperature. After
concentration of solvent under reduced pressure, the resulting
residue was dissolved in hot MeOH and filtered. The filtrate and
the white solid, which was redissolved in hot DMSO, were both
purified by reverse phase preparative HPLC (Phenomenex reverse
phase Luna 5.mu. C18(2) column, 60.times.21.2 mm ID) to yield the
desired final product (16 mg, 42% yield) as a white powder [hS1P1
EC.sub.50=970 nM, 400 nM, 440 nM, 421 nM]: >95% purity by LCMS,
ESI-MS: 459.1 (M+H).sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.95 (d, 2H), 7.56 (d, 2H), 7.45 (d, 1H), 7.42 (d, 1H),
7.24 (s, 1H), 7.19 (dd, 1H), 4.45 (s, 2H), 4.34 (dd, 4H), 3.69 (m,
1H), 2.64-2.57 (d, 1H), 1.89 (t, 4H), 1.58-1.40 (m, 4H), 1.38-1.26
(m, 2H).
Compound 7
1-(4-(5-cyclohexylbenzofuran-2-yl)benzyl)piperidine-4-carboxylic
acid
##STR00065##
[0346] A mixture of 4-(5-cyclohexylbenzofuran-2-yl)benzaldehyde (22
mg, 0.07 mmol), acetic acid (7 .mu.L, 0.11 mmol) and
piperidine-4-carboxylic acid (14 mg, 0.11 mmol) in DCM/MeOH (1:1,
1.6 mL) was stirred at room temperature for 1 h. Sodium
cyanoborohydride (2.3 mg, 0.05 mmol) was added and the reaction
mixture was stirred for 4 h at room temperature. After
concentration of solvent under reduced pressure, the resulting
residue was dissolved in DMSO, filtered and purified by reverse
phase preparative HPLC (Phenomenex reverse phase Luna 5.mu. C18(2)
column, 60.times.21.2 mm ID) to yield the desired final product
(15.4 mg, 51%) [hS1P1 EC.sub.50=1600 nM, >25000 nM]: >95%
purity by LCMS, ESI-MS: 418.1 (M+H).sup.+, .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.00 (d, J=8.0 Hz, 2H), 7.59 (d, J=8.0 Hz, 2H),
7.45 (d, J=1.6 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.26 (s, 1H), 7.21
(dd, J=8.4 Hz, J=1.6 Hz), 4.35 (s, 2H), 3.57 (d, J=11.6 Hz, 2H),
3.07 (t, J=12 Hz, 2H), 2.64-2.53 (m, 2H), 2.24 (d, 2H), 1.19-1.86
(m, 4H), 1.79 (t, 2H), 1.58-1.42 (m, 4H), 1.38-1.26 (m, 2H).
Compound 8
1-((4-(5-Butylbenzofuran-2-yl)phenyl)methyl)piperidine-4-carboxylic
acid
##STR00066##
[0348] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above except using
piperidine-4-carboxylic acid (57% yield) [hS1P1 EC.sub.50=3100 nM,
>25000 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.00 (d,
J=8.1, 2H), 7.59 (d, J=8.1, 2H), 7.43-7.41 (m, 2H), 7.25 (s, 1H),
7.15 (d, J=8.8, 1H), 4.35 (s, 2H), 3.57 (br d, J=11.7, 2H), 3.07
(br t, J=12.5, 2H), 2.71 (t, J=7.7, 2H), 2.70-2.59 (m, 1H), 2.25
(br d, J=14.6, 2H), 1.93-1.79 (m, 2H), 1.67-1.61 (m, 2H), 1.43-1.33
(m, 2H), 0.95 (t, J=7.3, 3H). MS (ESI) m/z: Calculated: 391.21.
Observed: 392.0 (M.sup.++1).
Compound 9
1-((4-(5-Benzylbenzofuran-2-yl)phenyl)methyl)piperidine-4-carboxylic
acid
##STR00067##
[0350] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above except using
piperidine-4-carboxylic acid [hS1P1 EC.sub.50=4800 nM, 25000 nM]:
NMR (400 MHz, CD.sub.3OD) .delta. 7.99 (br d, J=8.0, 2H), 7.58 (br
d, J=8.0, 2H), 7.44-7.42 (m, 2H), 7.28-7.16 (m, 7H), 4.34 (br s,
2H), 4.05 (br s, 2H), 3.57 (br d, J=11.7, 2H), 3.05 (br t, J=12.4,
2H), 2.65-2.62 (m, 1H), 2.23 (br d, J=13.5, 2H), 1.89-1.80 (m, 2H).
MS (ESI) m/z: Calculated: 425.20. Observed: 426.0 (M.sup.++1).
Compound 10
1-((4-(5-isobutylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid (Scheme 2)
5-isobutylbenzofuran (step 1 in Scheme 2)
##STR00068##
[0352] 5-bromobenzofuran (500 mg, 2.56 mmol) was dissolved in THF
solution of isobutylzinc(II) bromide (0.5M, 15 mL, 7.40 mmol) in a
microwave reaction tube. Pd(P.sup.tBu.sub.3).sub.2 (65 mg, 0.128
mmol, 0.05 eqv.) was added to this solution. The mixture was purged
with N.sub.2 gas for 3-5 min and heated at 100.degree. C. for 30
min under microwave irradiation. Upon completion of the reaction,
the reaction mixture was diluted with ethyl acetate, washed with 1N
HCl aqueous solution, brine, filtered through Celite. The filtrate
was dried over Na.sub.2SO.sub.4 and concentrated. The residue was
purified by silica gel column chromatography (ISCO system, 5% EtOAc
in hexanes) to give 0.331 g desired product (74% yield): .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.59 (s, 1H), 7.35 (d, 1H), 7.07
(d, 1H), 6.70 (s, 1H), 2.59 (d, 2H), 1.9 (m, 1H), 0.9 (d, 6H).
5-isobutylbenzofuran-2-ylboronic acid (step 2 in Scheme 2)
##STR00069##
[0354] A solution of n-BuLi (912 .mu.L, 2.28 mmol, 2.5M solution in
hexanes) was added dropwise to a solution of 5-isobutylbenzofuran
(331 mg, 1.9 mmol) in anhydrous THF (12 mL) at -78.degree. C. The
resulting mixture was stirred at -78.degree. C. for 40 min, and
treated with B(.sup.iPrO).sub.3 (658 .mu.L, 2.85 mmol). The
reaction mixture was allowed to warm up slowly to room temperature
and stirred for 1 h. TLC indicated the completion of reaction. The
reaction was cooled in ice-bath and quenched with 2N HCl (6 mL) and
extracted with Et.sub.2O. The combined organic extracts were washed
with brine, dried and concentrated under reduced pressure to yield
a crude benzofuran boronic acid (0.76 g) without further
purification for next step.
4-(5-isobutylbenzofuran-2-yl)benzaldehyde (step 3 in Scheme 2)
##STR00070##
[0356] A mixture of 5-isobutylbenzofuran-2-ylboronic acid (70 mg,
0.33 mmol), 4-bromobenzaldehyde (61 mg, 0.33 mmol), triethylamine
(1.7 mL, 12.6 mmol) and bis(triphenylphosphine)palladium(II)
chloride (12 mg, 0.017 mmol) in ethanol (10 mL) was irradiated in a
microwave instrument at 100.degree. C. for 20 min. The reaction
mixture was cooled, and the solvent was removed. The residue was
treated with water and extracted with ethyl acetate. The organic
layer was dried and concentrated in vacuo (the aqueous work-up is
optional). Purification by silica gel chromatography on ISCO system
gave the title compound (59 mg, 65% yield): >99% purity by LCMS,
ESI-MS: 279.2 (M+H).sup.+.
1-((4-(5-isobutylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid (step 4 in Scheme 2)
##STR00071##
[0358] A mixture of 4-(5-isobutylbenzofuran-2-yl)benzaldehyde (30
mg, 0.11 mmol), acetic acid (10 .mu.L, 0.15 mmol) and
azetidine-3-carboxylic acid (16 mg, 0.16 mmol) in DCM/MeOH (1:1, 2
mL) was stirred at room temperature for 1 h. Sodium
cyanoborohydride (3.4 mg, 0.054 mmol) was added and the reaction
mixture was stirred for 3 h at room temperature. After
concentration of solvent under reduced pressure, the resulting
residue was dissolved in an aliquot of DMSO and purified by reverse
phase preparative HPLC (Phenomenex reverse phase Luna 5.mu. C18(2)
column, 60.times.21.2 mm ID) to yield the desired final product
(25.6 mg, 65% yield) as a colorless film [hS1P1 EC.sub.50=270 nM,
490 nM, 383 nM]: >95% purity by LCMS, ESI-MS: 364.0 (M+H).sup.+,
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.99 (d, 2H), 7.55 (d,
2H), 7.42 (d, 1H), 7.39 (s, 1H), 7.24 (s, 1H), 7.12 (dd, 1H), 4.44
(s, 2H), 4.33 (d, 4H), 3.68 (m, 1H), 2.57 (d, 2H), 1.90 (m, 1H),
0.92 (d, 6H).
Compound 11
1-((4-(5-phenethylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00072##
[0360] The title compound was prepared in the same manner as
Example Compound 6 [hS1P1 EC.sub.50=580 nM, 25000 nM]: >95%
purity by LCMS, ESI-MS: 411.9 (M+H).sup.+, .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.99 (d, 2H), 7.55 (d, 2H), 7.41 (d, 1H), 7.38
(s, 1H), 7.24-7.21 (m, 3H), 7.17-7.14 (m, 4H), 4.44 (s, 2H), 4.34
(d, 4H), 3.70 (m, 1H), 3.01-2.90 (m, 4H).
Compound 12
1-(4-(5-(pyridin-3-yl)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
3-(benzofuran-5-yl)pyridine (step 1 in Scheme 2 except using Suzuki
coupling)
##STR00073##
[0362] A solution of 5 pyridin-3-ylboronic acid (390 mg, 3.18
mmol), 5-bromobenzofuran (500 mg, 2.54 mmol),
palladiumdichlorobis(triphenylphosphine) (111 mg, 0.16 mmol) and
triethylamine (8.8 mL, 63.5 mmol) in EtOH was irradiated in the
microwave at 100.degree. C. for 1200 s. Removal of the solvents
followed by dissolving in CH.sub.2Cl.sub.2 and filtering gave the
residue after concentration of the solvent under reduced pressure.
The compound was purifided on ISCO to afford 316 mg of the title
compound as a light yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.89 (s, 1H), 8.60 (d, 1H), 7.90 (d, 1H), 7.80 (s, 1H),
7.69 (s, 1H), 7.55 (d, 1H), 7.50 (d, 1H) 7.38 (dd, 1H), 6.85 (dd,
1H). MS (ESI) m/z: Calculated: 195.07. Observed: 196.30
(M.sup.++1).
5-(pyridin-3-yl)benzofuran-2-ylboronic acid (step 2 in Scheme
2)
##STR00074##
[0364] A solution of n-BuLi (0.76 mL, 2.5M solution in hexanes) was
added dropwise to a solution of 3-(benzofuran-5-yl)pyridine (310
mg, 1.59 mmol) in anhydrous THF (10 mL) at -78.degree. C. The
resulting mixture was stirred at -78.degree. C. for 30 min, and
treated with B(.sup.iPrO).sub.3 (0.55 mL, 2.39 mmol). The reaction
mixture was allowed to warm up slowly to room temperature and
stirred for 1 h. The reaction was quenched with 2N HCl and
extracted with Et.sub.2O. The aqueous layer was neutralized with 5N
NaOH(PH=6) followed by extraction with THF:ether (1:1) three times.
The combined extracts were washed with brine, dried and
concentrated under reduced pressure to yield 241 mg of the crude
boronic acid, which was used without further purification.
4-(5-(pyridin-3-yl)benzofuran-2-yl)benzaldehyde (step 3 in Scheme
2)
##STR00075##
[0366] The title compound was prepared as Example Compound 6 in the
general method described above (44% yield): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.06 (s, 1H), 8.91 (br s, 1H), 8.61 (br s,
1H), 8.07 (d, 2H), 7.98 (d, 2H), 7.93 (d, 1H), 7.65 (d, 1H), 7.55
(d, 1H), 7.82 (m, 1H), 7.39 (m, 1H), 7.27 (m, 1H). MS (ESI) m/z:
Calculated: 299.09. Observed: 300.30 (M.sup.++1).
1-(4-(5-(pyridin-3-yl)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (step 4 in Scheme 2)
##STR00076##
[0368] The title compound was prepared as Example Compound 6 in the
general method described above (22% yield) [hS1P1 EC.sub.50=3600,
>1 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 9.11 (br s,
1H), 8.70 (m, 2H), 8.06 (m, 3H), 7.98 (m, 1H), 7.74 (m, 2H), 7.60
(d, 2H), 7.44 (s, 1H), 4.47 (s, 2H), 4.40-4.38 (m, 4H), 3.72 (m,
1H). MS (ESI) m/z: Calculated: 384.20. Observed: 385.00
(M.sup.++1).
Compound 13
1-(4-(5-isobutylbenzofuran-2-yl)benzyl)piperidine-4-carboxylic
acid
##STR00077##
[0370] A mixture of 4-(5-isobutylbenzofuran-2-yl)benzaldehyde (22
mg, 0.08 mmol), acetic acid (7 .mu.L, 0.12 mmol) and
piperidine-4-carboxylic acid (15 mg, 0.12 mmol) in DCM/MeOH (1:1,
1.4 mL) was stirred at room temperature for 1 h. Sodium
cyanoborohydride (2.5 mg, 0.04 mmol) was added and the reaction
mixture was stirred for 4 h at room temperature. After
concentration of solvent under reduced pressure, the resulting
residue was dissolved in an aliquot of DMSO and purified by reverse
phase preparative HPLC (Phenomenex reverse phase Luna 5.mu. C18(2)
column, 60.times.21.2 mm ID) to yield the desired final product
(16.9 mg, 55%) [hS1P1 EC.sub.50=1700 nM, >25000]: >95% purity
by LCMS, ESI-MS: 392.0 (M+H).sup.+, .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.01 (d, 2H), 7.59 (d, 2H), 7.43 (d, 1H), 7.39
(s, 1H), 7.26 (s, 1H), 7.13 (dd, 1H), 4.36 (s, 2H), 3.58 (m, 2H),
3.10 (m, 2H), 2.65 (m, 1H), 2.57 (d, 2H), 1.90 (m, 1H), 0.92 (d,
6H).
Compound 14
1-((4-(5-Benzylbenzofuran-2-yl)2-fluorophenyl)methyl)azetidine-3-carboxyli-
c acid
4-(5-Benzylbenzofuran-2-yl)-2-fluorobenzaldehyde
##STR00078##
[0372] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (67% yield):
NMR (400 MHz, CDCl.sub.3) .delta. 10.35 (s, 1H), 7.92 (dd, J=8.1,
7.0, 2H), 7.69 (d, J=8.5, 1H), 7.63 (d, J=11.4, 1H), 7.46-7.42 (m,
2H), 7.33-7.19 (m, 6H), 7.13 (s, 1H), 4.09 (s, 2H).
1-(4-(5-Benzylbenzofuran-2-yl)-2-fluorophenyl)methyl)azetidine-3-carboxyli-
c acid
##STR00079##
[0374] The title compound was prepared as Example Compound 1 (step
5 Scheme 1) in the general method described above (54% yield)
[hS1P1 EC.sub.50=620 nM, 334 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.78 (d, J=8.1, 1H), 7.73 (d, J=9.9, 1H), 7.58 (t, J=7.7,
1H), 7.46-7.44 (m, 2H), 7.29-7.16 (m, 7H), 4.39 (s, 2H), 4.17-4.15
(m, 4H), 4.06 (s, 2H), 3.72-3.64 (m, 1H). MS (ESI) m/z: Calculated:
415.16. Observed: 416.0 (M.sup.++1).
Compound 15
1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxyl-
ic acid
4-(5-Benzylbenzofuran-2-yl)-3-fluorobenzaldehyde
##STR00080##
[0376] The title compound was prepared as Example Compound 1 (step
4 Scheme 1) in the general method described above (65% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.01 (s, 1H), 8.20 (t,
J=7.7, 1H), 7.77 (d, J=8.0, 1H), 7.68 (d, J=11.3, 1H), 7.47-7.45
(m, 2H), 7.37-7.20 (m, 7H), 4.10 (s, 2H).
1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxyl-
ic acid
##STR00081##
[0378] The title compound was prepared as Example Compound 1 (step
5 Scheme 1) in the general method described above (56% yield)
[hS1P1 EC.sub.50=160 nM, 38 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 8.09 (t, J=7.9, 1H), 7.47-7.45 (m, 2H), 7.40-7.37 (m, 2H),
7.28-7.16 (m, 7H), 4.34 (s, 2H), 4.17-4.15 (m, 4H), 4.07 (s, 2H),
3.53-3.45 (m, 1H). MS (ESI) m/z: Calculated: 415.16. Observed:
415.9 (M.sup.++1).
Compound 16
1-(4-(5-Butoxybenzofuran-2-yl)phenyl)methyl)piperidine-4-carboxylic
acid
##STR00082##
[0380] A mixture of 4-(5-butoxybenzofuran-2-yl)benzaldehyde (50 mg,
0.20 mmol), piperidine-4-carboxylic acid (41 mg, 0.31 mmol) and
acetic acid (0.50 mmol) in MeOH-DCM (3:1 ; 2 mL) was stirred at
room temperature for 1 h. Sodium triacetoxyborohydride (135 mg,
0.64 mmol) was added and the reaction mixture was stirred for 16 h.
Concentration of the solvent under reduced pressure yielded a
yellow solid that was dissolved in DMSO (3 mL) and filtered to give
a yellow solution that was purified by HPLC to afford the desired
product [hS1P1 EC.sub.50=17000 nM]: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.98 (d, 1H), 7.97 (d, 1H), 7.58 (d, 2H), 7.40
(d, 1H), 7.24 (s, 1H), 7.11 (d, 1H), 6.90 (dd, 1H), 4.35 (s, 2H),
4.00 (dd, 2H), 3.55 (m, 2H), 3.3 (m, 1H), 3.10 (m, 2H), 2.2 (m,
2H), 1.8 (m, 2H), 1.52 (m, 2H), 1.28 (m, 2H), 1.00 (dd, 3H). MS
(ESI) m/z: Calculated: 407.21. Observed: 407.90 (M.sup.++1).
Compound 17
1-((6-(5-cyclohexylbenzofuran-2-yl)pyridin-3-yl)methyl)azetidine-3-carboxy-
lic acid
##STR00083##
[0382] The title compound was prepared in the same manner as
Example Compound 6 except using 6-bromo-3-pyridinecarboxaldehyde in
step-3 (Scheme 2) [hS1P1 EC.sub.50=720 nM, 302 nM]: >95% purity
by LCMS, ESI-MS: 391.1 (M+H).sup.+, .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.81 (d, 1H), 7.94 (d, 1H), 7.65 (d, 1H), 7.59
(s, 1H), 7.50 (m, 2H), 7.35 (m, 1H), 4.44 (s, 2H), 4.45 (s, 2H),
4.34 (dd, 4H), 3.69 (m, 1H), 2.64-2.57 (d, 1H), 1.89 (t, 4H),
1.58-1.41 (m, 4H), 1.38-1.26 (m, 2H).
Compound 18
1-(4-(5-(6-methylpyridin-2-yl)benzofuran-2-yl)benzyl)azetidine-3-carboxyli-
c acid (Scheme 2)
##STR00084##
[0384] The title compound was prepared in the same manner as
Example Compound 6 except using (6-methylpyridin-2-yl) zinc (II)
bromide in step-1 (Scheme 2) [hS1P1 EC.sub.50=2900 nM]: >95%
purity by LCMS, ESI-MS: 391.1 (M+H).sup.+, .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.42 (t, 1H), 8.22 (d, 1H), 8.07-8.10 (m, 3H),
7.77-7.88 (m, 3H), 7.62 (d, 2H), 7.50 (dd, 1H), 4.48 (s, 2H), 4.36
(d, 4H), 3.71 (m, 1H), 2.85 (s, 3H).
Compound 19
1-(4-(5-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
1-(2,2-Diethoxy-ethoxy)-4-phenoxy-benzene
##STR00085##
[0386] The title compound was prepared as Example Compound 1 (step
1 Scheme 1) in the general method described above.
5-Phenoxy-benzofuran
##STR00086##
[0388] The title compound was prepared as Example Compound 1 (step
2 Scheme 1) in the general method described above (65% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.63 (d, 1H), 7.45 (d,
1H), 7.29 (m, 2H), 7.22 (d, 1H), 7.00-7.08 (m, 4H), 6.71 (m
1H).
5-phenoxybenzofuran-2-ylboronic acid
##STR00087##
[0390] The title compound was prepared as Example Compound 1 (step
3 Scheme 1) in the general method described above (74% yield).
4-(5-phenoxybenzofuran-2-yl)benzaldehyde
##STR00088##
[0392] The title compound was prepared as Example Compound 1 (step
4 Scheme 1) in the general method described above (65% yield):
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.05 (s, 1H), 8.13 (d,
2H), 8.03 (d, 2H), 7.70 (d, 1H), 7.66 (br s, 1H), 7.39 (m, 4H),
7.10 (m, 2H), 7.00 (dd, 1H). MS (ESI) m/z: Calculated: 314.10.
Observed: 315.10 (M.sup.++1).
1-(4-(5-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
##STR00089##
[0394] The title compound was prepared as Example Compound 1 (step
5 Scheme 1) in the general method described above (7% yield) [hS1P1
EC.sub.50=510 nM, 92 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.90 (d, 2H), 7.55 (m, 3H), 7.32 (m, 2H), 7.27 (s, 1H), 7.22 (d,
1H), 7.03 (m, 4H), 4.47 (s, 2H), 4.34 (m, 4H), 3.62 (m, 1H). MS
(ESI) m/z: Calculated: 399.20. Observed: 399.90 (M.sup.++1).
Compound 20
1-((4-(5-Isopentylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
5-Isopentylbenzofuran
##STR00090##
[0396] The title compound was prepared as Example Compound 6 (step
1 in Scheme 2) in the general method described above (75% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.58 (d, J=2.0, 1H),
7.41-7.39 (m, 2H), 7.11 (dd, J=8.2, 2.0, 1H), 6.70 (br s, 1H),
2.72-2.68 (m, 2H), 1.62-1.51 (m, 3), 0.94 (d, J=6.6, 6H).
5-Isopentylbenzofuran-2-yl-2-boronic acid
##STR00091##
[0398] The title compound was prepared as Example Compound 6 (step
2 in Scheme 2) in the general method described above (53% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.38 (m, 2H), 7.30
(s, 1H), 7.18 (d, J=8.5, 1H), 2.72-2.68 (m, 2H), 1.60-1.50 (m, 3),
0.94 (d, J=6.6, 6H).
4-(5-Isopentylbenzofuran-2-yl)benzaldehyde
##STR00092##
[0400] The title compound was prepared as Example Compound 6 (step
3 in Scheme 2) in the general method described above (79% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.00 (d,
J=8.5, 2H), 7.95 (d, J=8.5, 2H), 7.46-7.42 (m, 2H), 7.18-7.15 (m,
2H), 2.73-2.69 (m, 2H), 1.62-1.54 (m, 3), 0.95 (d, J=6.2, 6H).
1-((4-(5-Isopentylbenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00093##
[0402] The title compound was prepared as Example Compound 6 (step
4 in Scheme 2) in the general method described above (63% yield)
[hS1P1 EC.sub.50=630 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.98 (d, J=8.3, 2H), 7.55 (d, J=8.3, 2H), 7.43-7.41 (m, 2H), 7.23
(s, 1H), 7.15 (d, J=8.8, 1H), 4.44 (s, 2H), 4.38-4.30 (m, 4H),
3.73-3.65 (m, 1H), 2.73-2.69 (m, 2H), 1.62-1.52 (m, 3), 0.96 (d,
J=7.6, 6H). MS (ESI) m/z: Calculated: 377.2. Observed: 377.9
(M.sup.++1).
Compound 21
1-((4-(6-Butoxybenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
1-(2,2-Diethoxyethoxy)-3-butoxybenzene
##STR00094##
[0404] The title compound was prepared as Example Compound 1 (step
1 in Scheme 1) in the general method described above (86% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.15 (t, J=7.4),
6.52-6.49 (m, 3H), 4.83 (t, J=5.1, 1H), 3.99 (d, J=5.1, 2H), 3.93
(t, J=6.6, 2H), 3.80-3.72 (m, 2H), 3.67-3.60 (m, 2H), 1.79-1.72 (m,
2H), 1.53-1.43 (m, 2H), 1.25 (t, J=7.3, 6H), 0.97 (t, J=7.3,
3H).
6-Butoxybenzofuran
##STR00095##
[0406] The title compound was prepared as Example Compound 1 (step
2 in Scheme 1) in the general method described above (83% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.52 (d, J=2.2, 1H), 7.44
(d, J=8.5, 1H), 7.03 (d, J=2.2, 1H), 6.87 (dd, J=8.8, 2.5, 1H),
6.69-6.68 (m, 1H), 4.00 (t, J=6.6, 2H), 1.83-1.76 (m, 2H),
1.56-1.47 (m, 2H), 0.99 (t, J=7.4, 3H).
6-Butoxybenzofuran-2-yl-2-boronic acid
##STR00096##
[0408] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) in the general method described above (76% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.52-7.42 (m, 2H), 7.00
(br s, 1H), 6.90-6.85 (m, 1H), 4.00 (t, J=6.6, 2H), 1.82-1.78 (m,
2H), 1.56-1.48 (m, 2H), 0.98 (t, J=7.3, 3H).
4-(6-Butoxybenzofuran-2-yl)benzaldehyde
##STR00097##
[0410] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (62% yield):
NMR (400 MHz, CDCl.sub.3) .delta. 9.94 (s, 1H), 7.94-7.89 (m, 4H),
7.45 (d, J=8.5, 2H), 7.10 (s, 1H), 7.05 (br d, J=2.2, 1H), 6.89
(dd, J=8.5, 2.2, 1H), 4.02 (t, J=6.2), 1.85-1.78 (m, 2H), 1.57-1.52
(m, 2H), 1.00 (t, J=7.3, 3H).
1-((4-(6-Butoxybenzofuran-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00098##
[0412] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (46% yield)
[hS1P1 EC.sub.50=2000 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.94 (d, J=8.4, 2H), 7.53 (d, J=8.4, 2H), 7.47 (d, J=8.5,
1H), 7.21 (s, 1H), 7.11 (br d, J=2.2, 1H), 6.88 (dd, J=8.5, 2.2),
4.43 (s, 2H), 4.34-4.32 (m, 4H), 4.04 (t, J=6.2), 3.71-3.63 (m,
1H), 1.81-1.76 (m, 2H), 1.57-1.52 (m, 2H), 1.01 (t, J=7.3, 3H). MS
(ESI) m/z: Calculated: 379.18. Observed: 379.8 (M.sup.++1).
Compound 22
1-((2-(5-butoxybenzofuran-2-yl)thiazol-5-yl)methyl)azetidine-3-carboxylic
acid
2-(5-butoxybenzofuran-2-yl)thiazole-5-carbaldehyde
##STR00099##
[0414] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above except using
2-bromothiazole-5-carbaldehyde (29% yield): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.07 (s, 1H), 8.46 (dd, 1H), 7.45 (dd, 2H),
7.03 (dd, 2H), 4.01 (dd, 2H), 1.74 (m, 2H), 1.54 (m, 2H), 1.01 (t,
3H). MS (ESI) m/z: Calculated: 301.10. Observed: 302.10
(M.sup.++1).
1-((2-(5-butoxybenzofuran-2-yl)thiazol-5-yl)methyl)azetidine-3-carboxylic
acid
##STR00100##
[0416] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (36% yield)
[hS1P1 EC.sub.50=3200 nM, 1100 nM]: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.06 (br s, 1H), 7.344 (m, 2H), 7.18 (m, 1H),
7.01 (ddd, 1H), 4.79 (s, 2H), 4.36 (m, 4H), 3.98 (m, 2H), 3.69 (m,
1H), 1.75 (m, 2H), 1.50 (m, 2H), 1.00 (t, 3H). MS (ESI) m/z:
Calculated: 386.13. Observed: 386.90 (M.sup.++1).
Compound 23
1-((4-(5-Butoxybenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxyl-
ic acid
4-(5-Butoxybenzofuran-2-yl)-3-fluorobenzaldehyde
##STR00101##
[0418] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (36% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.02 (s, 1H), 8.18 (t,
J=7.7, 1H), 7.73 (d, J=8.0, 1H), 7.66 (d, J=11.2, 1H), 7.44-7.39
(m, 2H), 7.09 (d, J=2.4, 1H), 6.92 (dd, J=2.4, 8.8, 1H), 4.01 (t,
J=6.2), 1.81-1.76 (m, 2H), 1.57-1.51 (m, 2H), 1.01 (t, J=7.2,
3H).
1-((4-(5-Butoxybenzofuran-2-yl)3-fluorophenyl)methyl)azetidine-3-carboxyli-
c acid
##STR00102##
[0420] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (51% yield)
[hS1P1 EC.sub.50=520 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
8.08 (t, J=7.7, 1H), 7.44-7.37 (m, 3H), 7.25 (d, J=3.7, 1H), 7.14
(d, J=2.2, 1H), 6.94 (dd, J=8.8, 2.2), 4.35 (s, 2H), 4.18-4.15 (m,
4H), 4.01 (t, J=6.2), 3.45-3.37 (m, 1H), 1.82-1.75 (m, 2H),
1.57-1.49 (m, 2H), 1.00 (t, J=7.2, 3H). MS (ESI) m/z: Calculated:
397.17. Observed: 397.9 (M.sup.++1).
Compound 24
1-((4-(5-Butoxybenzofuran-2-yl)-3-methoxyphenyl)methyl)azetidine-3-carboxy-
lic acid
4-(5-butoxybenzofuran-2-yl)-3-methoxybenzaldehyde
##STR00103##
[0422] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (65% yield):
.sup.1H NMR (400 MHz, CD.sub.3Cl) .delta. 10.03 (s, 1H), 8.22 (d,
1H), 7.59 (s, 1H), 7.50 (s, 1H), 7.45 (d, 1H), 7.41 (s, 1H), 7.08
(d, 1H), 6.93 (d, 1H), 4.16 (s, 3H), 4.05 (t, 2H), 1.84 (m, 2H),
1.61 (m, 2H), 1.04 (t, 3H). MS (ESI) m/z: Calculated: 324.14.
Observed: 324.9 (M.sup.++1).
1-((4-(5-Butoxybenzofuran-2-yl)-3-methoxyphenyl)methyl)azetidine-3-carboxy-
lic acid
##STR00104##
[0424] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (36% yield)
[hS1P1 EC.sub.50=420 nM, 700 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 8.04 (d, 1H), 7.39 (s, 1H), 7.38 (s, 1H), 7.21 (s, 1H),
7.15 (d, 1H), 7.08 (s, 1H), 6.83 (d, 1H), 4.44 (s, 2H), 4.38 (m,
7H), 4.02 (m, 2H), 3.62 (m, 1H), 1.82 (m, 2H), 1.63 (m, 2H), 1.01
(t, 3H). MS (ESI) m/z: Calculated: 409.19. Observed: 409.9
(M.sup.++1).
Compound 25
1-((5-(5-butoxybenzofuran-2-yl)thiophen-2-yl)methyl)azetidine-3-carboxylic
acid
5-(5-butoxybenzofuran-2-yl)thiophene-2-carbaldehyde
##STR00105##
[0426] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above except using
5-bromothiophene-2-carbaldehyde (32% yield): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 9.92 (s, 1H), 7.73 (d, 1H), 7.51 (dd, 1H), 7.39
(d, 1H), 7.96 (m, 2H), 6.94 (dd, 1H), 3.98 (dd, 2H), 1.80 (m, 2H),
1.70 (m, 2H), 1.01 (t, 3H).
1-((5-(5-butoxybenzofuran-2-yl)thiophen-2-yl)methyl)azetidine-3-carboxylic
acid
##STR00106##
[0428] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (27% yield)
[hS1P1 EC.sub.50=1600 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.49 (br s, 1H), 7.35 (m, 2H), 7.03 (d, 2H), 6.89 (dd, 1H),
4.67 (s, 2H), 4.35 (m, 4H), 3.98 (m, 2H), 3.67 (m, 1H), 1.73 (m,
2H), 1.51 (m, 2H), 0.99 (t, 3H). MS (ESI) m/z: Calculated: 385.13.
Observed: 385.70 (M.sup.++1).
Compound 26
1-((6-(5-Butoxylbenzofuran-2-yl)pyridin-3-yl)methyl)azetidine-3-carboxylic
acid
4-(5-Butoxybenzofuran-2-yl)pyridine-3-carboxaldehyde
##STR00107##
[0430] The title compound was prepared in the same manner as
described in step 4 (Scheme 1) by using
6-bromo-3-pyridinecarboxaldehyde (48%): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.10 (s, 1H), 9.08 (s, 1H), 8.24 (d, 1H), 8.01
(d, 1H), 7.56 (s, 1H), 7.47 (d, 1H), 7.02 (s, 1H), 6.99 (d, 1H),
4.03 (q, 4H), 1.84-1.77 (m, 2H), 1.50-1.48 (m, 2H), 1.00 (t, 3H).
MS (ESI) m/z: Calculated: 295.33. Observed: 296.2 (M.sup.++1).
1-((6-(5-Butoxylbenzofuran-2-yl)pyridin-3-yl)methyl)azetidine-3-carboxylic
acid
##STR00108##
[0432] The title compound was prepared as in step 5 (Scheme 1) of
the general method described earlier (68% yield) [hS1P1
EC.sub.50=2600 nM, 201 nM]: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.86 (s, 1H), 8.19 (d, 1H), 7.90 (d, 1H), 7.49 (s, 1H),
7.47 (d, 1H), 7.23 (d, 1H), 6.86 (s, 1H), 3.80 (q, 2H), 4.52-4.40
(m, 4H), 3.80 (t, 2H), 3.52-3.47 (m, 1H), 1.68-1.66 (m, 2H),
1.44-1.37 (m, 2H), 0.94 (t, 3H). MS (ESI) m/z: Calculated: 380.44.
Observed: 381.0 (M.sup.++1).
Compound 27
1-(4-(5-cyclohexylbenzofuran-2-yl)3-fluorophenyl)methyl)azetidine-3-carbox-
ylic acid
5-cyclohexylbenzofuran (step 1 in Scheme 2)
##STR00109##
[0434] 5-bromobenzofuran (500 mg, 2.55 mmol) was dissolved in a THF
solution of cyclohexyl zinc(II) bromide (0.5M, 15 mL, 7.40 mmol) in
a microwave reaction tube. Pd(P.sup.tBu.sub.3).sub.2 (65 mg, 0.128
mmol, 0.05 eq.) was added to this solution. The mixture was purged
with N.sub.2 gas for 3-5 min and heated at 100.degree. C. for 30
min under microwave irradiation. Upon completion of the reaction,
the reaction mixture was diluted with ethyl acetate, washed with 1N
HCl aqueous solution, brine, filtered through Celite. The filtrate
was dried over Na.sub.2SO.sub.4 and concentrated. The residue was
purified by silica gel column chromatography (ISCO system, 5% EtOAc
in hexanes) to give 0.217 g desired product (43% yield): .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.57 (d, 1H), 7.41 (d, 2H), 7.15
(d, 1H), 6.72 (d, 1H), 2.58 (m, 1H), 1.92-1.74 (m, 4H), 1.51-1.35
(m, 4H), 1.31-1.25 (m, 2H).
5-cyclohexylbenzofuran-2-ylboronic acid (step 2 in Scheme 2)
##STR00110##
[0436] A solution of n-BuLi (360 .mu.L, 0.9 mmol, 2.5M solution in
hexanes) was added dropwise to a solution of 5-cyclohexylbenzofuran
(150 mg, 0.75 mmol) in anhydrous THF (5 mL) at -78.degree. C. The
resulting mixture was stirred at -78.degree. C. for 40 min, and
treated with B(.sup.iPrO).sub.3 (260 .mu.L, 1.13 mmol). The
reaction mixture was allowed to warm up slowly to room temperature
and stirred for 1 h. TLC indicated the completion of reaction. The
reaction was cooled in ice-bath and quenched with 2N HCl (3 mL) and
extracted with Et.sub.2O. The combined organic extracts were washed
with brine, dried and concentrated under reduced pressure to yield
a desired boronic acid (0.156 g, 85% yield) without further
purification for next step. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.46 (s, 1H), 7.43 (d, 1H), 7.32 (s, 1H), 7.25 (d, 1H),
2.62 (m, 1H), 1.93-1.85 (m, 4H), 1.78-1.75 (m, 4H), 1.34-1.22 (m,
2H).
4-(5-cyclohexylbenzofuran-2-yl)2-fluorobenzaldehyde (step 3 in
Scheme 2)
##STR00111##
[0438] A mixture of 5-cyclohexylbenzofuran-2-ylboronic acid (75 mg,
0.30 mmol), 4-bromo-2-fluorobenzaldehyde (48 mg, 0.24 mmol),
triethylamine (1.1 mL, 7.5 mmol) and
bis(triphenylphosphine)palladium(II) chloride (12 mg, 0.05 mmol) in
ethanol (11 mL) was irradiated in a microwave instrument at
100.degree. C. for 20 min. The reaction mixture was cooled, and the
solvent was removed. The residue was treated with water and
extracted with ethyl acetate. The organic layer was dried and
concentrated in vacuo (the aqueous work-up is optional).
Purification by silica gel chromatography on ISCO system gave the
title compound (51 mg, 49% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.04 (s, 1H), 8.00-7.97 (m, 2H), 7.46 (s, 1H),
7.43 (d, 2H), 7.32 (s, 1H), 7.25 (d, 1H), 2.62 (m, 1H), 1.95-1.77
(m, 4H), 1.58-1.56 (m, 4H), 1.46-1.44 (m, 2H). MS (ESI) m/z:
Calculated: 322.27. Observed: 323.2 (M.sup.++1).
1-(4-(5-cyclohexylbenzofuran-2-yl)3-fluorophenyl)methyl)azetidine-3-carbox-
ylic acid (step 4 in Scheme 2)
##STR00112##
[0440] A mixture of
4-(5-cyclohexylbenzofuran-2-yl)3-fluorobenzaldehyde (40 mg, 0.12
mmol), acetic acid (10 .mu.L, 0.15 mmol) and azetidine-3-carboxylic
acid (15 mg, 0.15 mmol) in DCM/MeOH (1:1, 2 mL) was stirred at room
temperature for 1 h. Sodium cyanoborohydride (3.0 mg, 0.05 mmol)
was added and the reaction mixture was stirred for 3 h at room
temperature. After concentration of solvent under reduced pressure,
the resulting residue was dissolved in hot MeOH and filtered. The
filtrate and the white solid, which was redissolved in hot DMSO,
were both purified by reverse phase preparative HPLC (Phenomenex
reverse phase Luna 5.mu. C18(2) column, 60.times.21.2 mm ID) to
yield the desired final product (12 mg, 42% yield) as a white
powder [hS1P1 EC.sub.50=160 nM, 361 nM]: >95% purity by LCMS,
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.12 (d, 1H), 7.47-7.38
(m, 4H), 7.28-7.20 (m, 2H), 4.66 (s, 2H), 4.34 (m, 4H), 3.72 (m,
1H), 2.61 (m, 1H), 1.95-1.82 (m, 4H), 1.60-1.56 (m, 4H), 1.42-1.40
(m, 2H). MS (ESI) m/z: Calculated: 407.48. Observed: 408.2
(M.sup.++1).
Compound 28
1-((4-(5-(thiophen-2-yl)benzofuran-2-yl)phenyl)methyl)azetidine-3-carboxyl-
ic acid
5-(Thiophen-2-yl)benzofuran
##STR00113##
[0442] The title compound was prepared as Example Compound 6 (step
1 in Scheme 2) in the general method described above except using
thiophen-2-ylboronic acid (55% yield). .sup.1H NMR (400 MHz,
CD.sub.3Cl) .delta. 7.82 (s, 1H), 7.62 (s, 1H), 7.55-7.03 (m, 5H),
6.79 (d, 1H).
5-(Thiophen-2-yl)benzofuran-2-yl-boronic acid
##STR00114##
[0444] The title compound was prepared as Example Compound 6 (step
2 in Scheme 2) in the general method described above (77% yield).
.sup.1H NMR (400 MHz, CD.sub.3Cl) .delta. 7.92 (s, 1H), 7.88 (s,
1H), 7.66-7.34 (m, 4H), 7.08 (d, 1H).
4-(5-(thiophen-2-yl)benzofuran-2-yl)benzaldehyde
##STR00115##
[0446] The title compound was prepared as Example Compound 6 (step
3 in Scheme 2) in the general method described above (61% yield):
.sup.1H NMR (400 MHz, CD.sub.3Cl) .delta. 10.01 (s, 1H), 8.19 (d,
1H), 8.01 (d, 1H), 7.82 (s, 1H), 7.62-7.24 (m, 7H), 7.16 (dd, 1H).
MS (ESI) m/z: Calculated: 304.06. Observed: 304.9 (M.sup.++1).
1-((4-(5-(thiophen-2-yl)benzofuran-2-yl)phenyl)methyl)azetidine-3-carboxyl-
ic acid
##STR00116##
[0448] The title compound was prepared as Example Compound 6 (step
4 in Scheme 2) in the general method described above (31% yield)
[hS1P1 EC.sub.50=1800 nM, 25000 nM]: .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.01 (d, 2H), 7.87 (s, 1H), 7.64-7.44 (m,
7H), 7.19 (dd, 1H), 4.25 (m, 2H), 3.55 (m, 5H). MS (ESI) m/z:
Calculated: 389.11. Observed: 389.9 (M.sup.++1).
Compound 29
3-(6-(5-benzylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoic
acid
2-(3-bromophenyl)ethanamine (step 1 in Scheme 7)
##STR00117##
[0450] A suspension of LiAlH.sub.4 (3.04 g, 80 mmole) in dry THF
(100 mL) was cooled to -5.degree. C. Concentrated H.sub.2SO.sub.4
(3.9 g, 40 mmole) was added dropwise, and the resulting mixture was
stirred at -5.degree. C. for 1 hour. A solution of
3-bromo-benzenacetonitrile (9.80 g, 50 mmole) in THF (5 mL) was
added dropwise, and the reaction was allowed to warm to room
temperature when the addition was complete. The reaction was
stirred at room temperature for 1 hour, and then cooled back to
0.degree. C. and quenched by the addition of a 1:1 THF: H.sub.2O
mixture (12.4 mL). Et.sub.2O was added (50 mL), followed by a 3.6 M
solution of NaOH (24.4 mL). The mixture was filtered through
Celite, and the solids were washed well with additional Et.sub.2O.
The organic phase was dried over Na.sub.2SO.sub.4, filtered, and
concentrated in vacuo to provide the title compound (9.7 g, 97%).
The crude compound was used in subsequent steps. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.38-7.30 (m, 2H), 7.20-7.10 (m, 2H), 2.96
(t, 2H), 2.72 (t, 2H), 1.35 (br s, 2H). MS (ESI) m/z: Calculated:
199. Observed: 200/202 (M.sup.++1).
N-(3-bromophenethyl)-2,2,2-trifluoroacetamide (step 2 in Scheme
7)
##STR00118##
[0452] A mixture of 3-bromobenzeneethanamine (9.70 g, 48.5 mmole)
and 2,6-lutidine (5.8 mL, 50.0 mmole) in dry CH.sub.2Cl.sub.2 (150
mL) was cooled to 0.degree. C. Trifluoroacetic anhydride (5.6 mL,
40 mmole) was added dropwise; the reaction was then warmed to room
temperature and allowed to stir for 24 hours. Water (120 mL) was
added to the reaction, the phases were separated, and the aqueous
layer was extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The
combined organic phases were washed successively with 1N HCl (100
mL) and saturated NaHCO.sub.3 (100 mL), and then dried over
Na.sub.2SO.sub.4, filtered, and concentrated in vacuo to provide
the tile compound (12.3 g, 86%). The crude compound was used in
subsequent steps. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.40
(d, J=8.0 Hz, 1H), 7.36 (s, 1H), 7.21 (t, J=7.6 Hz, 1H), 7.12 (t,
J=7.6 Hz, 1H), 6.31 (br s, 1H), 3.59 (q, J=6.8 Hz, 2H), 2.87 (t,
J=7.2 Hz, 2H).
1-(6-bromo-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethanone
and
1-(8-bromo-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethanone
(step 3 in Scheme 7)
##STR00119##
[0454] A mixture of glacial acetic acid (68 mL) and concentrated
sulfuric acid (45 mL) was added to a mixture of
N-(3-bromophenethyl)-2,2,2-trifluoroacetamide (12.3 g, 41.54 mmol)
and paraformaldehyde (2.0 g). The reaction was stirred at room
temperature for 24 hours, and then poured into 300 mL of cold
water. The aqueous solution was extracted with EtOAc (3.times.150
mL). The combined organic phases were washed with saturated
NaHCO.sub.3 (200 mL) and water (2.times.200 mL). The organic phase
was then dried over Na.sub.2SO.sub.4, filtered and concentrated in
vacuo. The residue was purified on ISCO column (20% EtOAc/Hexane)
to provide a mixture of the title compounds (9.6 g, 75%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.46 (dd, J=2.0 Hz, J=8.0 Hz,
0.33H), 7.38-7.31 (m, 1.33H), 7.15-7.09 (m, 0.67H), 7.05-6.98 (m,
0.67H), 4.75, 4.73, 4.69 (3.times.s, 2H), 3.90-3.80 (m, 2H),
3.00-2.90 (m, 2H). MS (ESI) m/z: Calculated: 306.98. Observed:
308/310 (M.sup.++1).
6-(5-benzylbenzofuran-2-yl)-1,2,3,4-tetrahydroisoquinoline (step 4
in Scheme 7)
##STR00120##
[0456] A solution of 5-benzylbenzofuran-2-ylboronic acid (252 mg,
1.0 mmole) in ethanol (3 mL) was added to a mixture of
1-(6-bromo-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethanone
and
1-(8-bromo-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethanone
(308 mg, 1.0 mmole), Pd(PPh.sub.3).sub.4, toluene, and 2 M
Na.sub.2CO.sub.3(3.5 mL). The resulting mixture was heated at
reflux overnight. The reaction was concentrated in vacuo, and the
residue was diluted with water. The aqueous phase was extracted
with EtOAc (3.times.50 mL). The combined organic phases were washed
with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated
in vacuo. The residue was purified on ISCO column (5% to 10%
MeOH/CH.sub.2Cl.sub.2) to provide the title compounds (189 mg,
56%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.60 (m, 2H), 7.39
(dd, 1H), 7.37 (s, 1H), 7.25 (m, 5H), 7.10 (dd, 2H), 6.90 (s, 1H),
4.10 (s, 2H), 3.40 (s, 2H), 3.18 (m, 2H), 2.94 (m, 2H). MS (ESI)
m/z: Calculated: 339.16. Observed: 340.10 (M.sup.++1).
Tert-butyl
3-(6-(5-benzylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)-
propanoate (step 5 in Scheme 6)
##STR00121##
[0458] 6-(5-benzylbenzofuran-2-yl)-1,2,3,4-tetrahydroisoquinoline
(67 mg, 0.2 mmol) was dissolved in methanol (2 mL). DIEA (0.35 mL)
and acrylic acid tert-butyl ester (51 mg, 0.4 mmol) were added. The
mixture was headed to 90.degree. C. for 30 minutes using microwave
irradiation. All the solvents was evaporated and the crude product
of tert-butyl
3-(6-(5-benzylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoate
was used in the next step without further purification. MS (ESI)
m/z: Calculated: 467.25. Observed: 468.30 (M.sup.++1).
3-(6-(5-benzylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoic
acid (step 6 in Scheme 7)
##STR00122##
[0460] To a solution of tert-butyl
3-(6-(5-benzylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoate
(40 mg, 0.086 mmole) in CH.sub.2Cl.sub.2 (1 mL) was added TFA (1
mL). The mixture was stirred at room temperature for 3 hours. All
the solvents were evaporated. The mixture was purified by reverse
phase preparative HPLC to give the title compound (14 mg, 40%)
[hS1P1 EC.sub.50=160 nM, 261 nM]. NMR (400 MHz, CD.sub.3OD) .delta.
7.77 (m, 2H), 7.42 (dd, 1H), 7.40 (s, 1H), 7.20-7.30 (m, 5H), 7.10
(m, 3H), 4.50 (s, 2H), 4.04 (s, 2H), 3.64 (dd, 2H), 3.55 (dd, 2H),
3.26 (dd, 2H), 2.90 (dd, 2H). MS (ESI) m/z: Calculated: 411.18.
Observed: 412.10 (M.sup.++1).
Compound 30
1-(4-(5-cyclopentylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
5-Cyclopentylbenzofuran
##STR00123##
[0462] The title compound was prepared as Example Compound 6 (step
1 in Scheme 2) in the general method described above (67% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.58 (d, J=2.2, 1H), 7.45
(br d, J=1.8, 1H), 7.41 (d, J=8.8, 1H), 7.18 (dd, J=8.8, 1.8, 1H),
6.71 (dd, J=1.1, 2.2, 1H), 3.13-3.05 (m, 1H), 2.14-2.07 (m, 2H),
1.88-1.58 (m, 6H).
5-Cyclopentylbenzofuran-2-yl-2-boronic acid
##STR00124##
[0464] The title compound was prepared as Example Compound 6 (step
2 in Scheme 2) in the general method described above (yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.50-7.45 (m, 2H),
7.43-7.39 (m, 1H), 7.31 (s, 1H), 3.12-3.05 (m, 1H), 2.14-2.06 (m,
2H), 1.80-1.60 (m, 6H).
4-(5-Cyclopentylbenzofuran-2-yl)benzaldehyde
##STR00125##
[0466] The title compound was prepared as Example Compound 6 (step
3 in Scheme 2) in the general method described above (95% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.00 (d,
J=8.0, 2H), 7.94 (d, J=8.0, 2H), 7.51-7.44 (m, 3H), 7.15 (s, 1H),
3.14-3.06 (m, 1H), 2.20-2.10 (m, 2H), 1.88-1.62 (m, 6H).
1-(4-(5-cyclopentylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (step 4 in Scheme 2)
##STR00126##
[0468] The title compound was prepared as Example Compound 6 (step
4 in Scheme 2) in the general method described earlier for
reductive amination (71% yield) [hS1P1 EC.sub.50=210 nM]. .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 8.02 (d, 2H), 7.57 (d, 2H), 7.49
(s, 1H), 7.44 (d, 1H), 7.25 (d, 2H), 4.56 (s, 2H), 4.30 (m, 4H),
3.62 (m, 1H), 3.11 (m, 1H), 2.25-2.12 (m, 2H), 1.90-1.66 (m, 6H).
MS (ESI) m/z: Calculated: 375.46. Observed: 375.9 (M.sup.++1).
Compound 31
1-(3-fluoro-4-(5-(piperidin-1-yl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
1-(benzofuran-5-yl)piperidine (step 1 of Scheme 3)
##STR00127##
[0470] 5-bromobenzofuran (2 g, 10 mmol), piperidine (1.2 mL, 12
mmol), Pd(dppf)Cl.sub.2 (245 mg, 0.3 mmol), dppf (250 mg, 0.45
mmol) and sodium tert-butoxide (1.44 g, 15 mmol) was mixed in
toluene (10 mL). The mixture was purged with N.sub.2 gas for 3-5
min and heated at 120.degree. C. for 30 min under microwave
irradiation (Personal Chemistry Emrys.TM. Optimizer microwave
reactor). Upon completion of the reaction, the reaction mixture was
directly loaded on silica gel column and purified on ISCO system
(<2% EtOAc in hexanes) to give 0.539 g desired product (27%
yield): ESI-MS: 202.3 (M+H).sup.+, .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.58 (s, 1H), 7.40 (d, 1H), 7.15 (s, 1H), 7.00
(d, 1H), 6.65 (s, 1H), 3.10 (m, 4H), 1.70 (m, 4H), 1.48 (m, 2H).
Note: the title compound appeared to be very volatile. The
evaporation of solvent should be carried out very carefully.
5-(piperidin-1-yl)benzofuran-2-ylboronic acid (step 2 of Scheme
3)
##STR00128##
[0472] A solution of n-BuLi (334 .mu.L, 0.83 mmol, 2.5 M solution
in hexanes) was added dropwise to a solution of
1-(benzofuran-5-yl)piperidine (140 mg, 0.70 mmol) in anhydrous THF
(5 mL) at -78.degree. C. The resulting mixture was stirred at
-78.degree. C. for 40 min, and treated with B(.sup.iPrO).sub.3 (241
.mu.L, 1.04 mmol). The reaction mixture was allowed to warm up
slowly to room temperature and stirred for 1 h. TLC indicated the
completion of reaction. The reaction was cooled in ice-bath and
quenched with saturated NH.sub.4Cl (1.5 mL) and extracted with
Et.sub.2O. The separated aqueous layer was neutralized to
pH.about.5. The solution turned cloudy, which was extracted with
ethyl acetate (.times.3). The combined organic extracts were
concentrated in vacuo yielding the desired boronic acid as brown
solids (0.16 g, 94% yield) without further purification for next
step. ESI-MS: 246.3 (M+H).sup.+.
3-fluoro-4-(5-(piperidin-1-yl)benzofuran-2-yl)benzaldehyde (step 3
of Scheme 3)
##STR00129##
[0474] A mixture of 5-(piperidin-1-yl)benzofuran-2-ylboronic acid
(50 mg, 0.204 mmol), 4-bromo-3-fluorobenzaldehyde (37 mg, 0.184
mmol), triethylamine (0.56 mL, 4.1 mmol) and
bis(triphenylphosphine)palladium(II) chloride (14 mg, 0.02 mmol) in
ethanol (5 mL) was irradiated in a microwave instrument at
100.degree. C. for 20 min. The reaction mixture was cooled, and the
solvent was removed. The residue was purification by silica gel
chromatography on ISCO system yielding the title compound (15 mg,
15% yield). ESI-MS: 324.2 (M+H).sup.+, .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.00 (s, 1H), 8.19 (t, 1H), 7.75 (d, 1H), 7.67
(d, 1H), 7.43 (d, 1H), 7.35 (d, 1H), 7.14-7.11 (m, 2H), 3.13 (m,
4H), 1.77 (m, 4H), 1.59 (m, 2H).
1-(3-fluoro-4-(5-(piperidin-1-yl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid trifluoroacetic acid salt(step 4 of Scheme 3)
##STR00130##
[0476] A mixture of
3-fluoro-4-(5-(piperidin-1-yl)benzofuran-2-yl)benzaldehyde (9 mg,
0.028 mmol), acetic acid (2.5 .mu.L, 0.042 mmol) and
azetidine-3-carboxylic acid (4.2 mg, 0.042 mmol) in DCM/MeOH (2:1,
0.9 mL) was stirred at room temperature for 1 h. Sodium
cyanoborohydride (1.0 mg, 0.014 mmol) was added and the reaction
mixture was stirred for 3 h at room temperature. After
concentration of solvent under reduced pressure, the resulting
residue was dissolved in DMSO, and purified by reverse phase
preparative HPLC (Phenomenex reverse phase Luna 5.mu. C18 (2)
column, 60.times.21.2 mm ID, mobile phase: A=0.05% TFA in water;
B=0.05% TFA in acetonitrile. The flow rate was 12 mL/min. The
gradient time was 2% B to 52% B over 25 min.) to yield the desired
final product (10.3 mg, 70% yield) as a white powder
(ditrifluoroacetic acid salt) [hS1P1 EC.sub.50=860 nM, 307 nM]:
>95% purity by LCMS, ESI-MS: 409.1 (M+H).sup.+, .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 8.17 (t, 1H), 8.02 (d, 1H), 7.81 (d, 1H),
7.66 (dd, 1H), 7.49-7.47 (m, 3H), 4.50 (s, 2H), 4.39 (dd, 4H),
3.72-3.70 (m, 5H), 2.08 (m, 4H), 1.84 (m, 2H).
Compound 32
1-((6-(5-benzylbenzofuran-2-yl)pyridin-3-yl)methyl)azetidine-3-carboxylic
acid
6-(5-benzylbenzofuran-2-yl)nicotinaldehyde
##STR00131##
[0478] The title compound was prepared as the Example Compound 1
(step 4 in Scheme 1) in the general method described above except
using 6-bromo-3-pyridinecarboxaldehyde (53% yield): .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 10.13 (s, 1H), 9.15 (s, 1H), 8.36
(d, 1H), 8.14 (m, 1H), 7.76 (d, 1H), 7.62 (m, 2H), 7.29 (m, 6H),
4.07 (s, 2H). MS (ESI) m/z: Calculated: 313.11. Observed: 314.20
(M.sup.++1).
1-((6-(5-benzylbenzofuran-2-yl)pyridin-3-yl)methyl)azetidine-3-carboxylic
acid
##STR00132##
[0480] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (33% yield)
[hS1P1 EC.sub.50=2600 nM, 516 nM]: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.68 (s, 1H), 8.01 (br s, 2H), 7.45 (m, 3H),
7.16 (m, 6H), 4.50 (s, 2H), 4.35 (m, 4H), 4.04 (s, 2H), 3.70 (m,
1H). MS (ESI) m/z: Calculated: 398.16. Observed: 399.00
(M.sup.++1).
Compound 33
1-((4-(5-benzylbenzofuran-2-yl)-3-methoxyphenyl)methyl)azetidine-3-carboxy-
lic acid
4-(5-benzylbenzofuran-2-yl)-3-methoxybenzaldehyde
##STR00133##
[0482] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (60% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.22 (d,
1H), 7.64-7.44 (m, 11H), 4.16 (m, 5H). MS (ESI) m/z: Calculated:
342.13. Observed: 342.9 (M.sup.++1).
1-((4-(5-benzylbenzofuran-2-yl)-3-methoxyphenyl)methyl)azetidine-3-carboxy-
lic acid
##STR00134##
[0484] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (50% yield)
[hS1P1 EC.sub.50=1000 nM, 1900 nM, 196 nM]: .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.98 (d, 1H), 7.42-7.07 (m, 11H), 4.18 (m,
2H), 3.82 (m, 5H), 3.57 (m, 1H), 3.14 (m, 4H). MS (ESI) m/z:
Calculated: 427.18. Observed: 427.9 (M.sup.++1).
Compound 34
1-(4-(5-(piperidin-1-yl)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
4-(5-(piperidin-1-yl)benzofuran-2-yl)benzaldehyde (step 3 of Scheme
3)
##STR00135##
[0486] A mixture of 5-(piperidin-1-yl)benzofuran-2-ylboronic acid
(90 mg, 0.367 mmol), 4-bromobenzaldehyde (62 mg, 0.330 mmol),
triethylamine (1.0 mL, 7.3 mmol) and
bis(triphenylphosphine)palladium(II) chloride (12.8 mg, 0.02 mmol)
in ethanol (9 mL) was irradiated in a microwave instrument at
100.degree. C. for 20 min. The reaction mixture was cooled, and the
solvent was removed. The residue was purification by silica gel
chromatography on ISCO system yielding the title compound (31 mg,
28% yield). ESI-MS: 306.4 (M+H).sup.+, .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.02 (s, 1H), 7.95 (dd, 4H), 7.42 (d, 1H),
7.11 (m, 2H), 7.07 (dd, 1H), 3.13 (t, 4H), 1.78-1.74 (m, 4H),
1.62-1.56 (m, 2H).
1-(4-(5-(piperidin-1-yl)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (step 4 of Scheme 3)
##STR00136##
[0488] A mixture of
4-(5-(piperidin-1-yl)benzofuran-2-yl)benzaldehyde (31 mg, 0.102
mmol), acetic acid (9 .mu.L, 0.15 mmol) and azetidine-3-carboxylic
acid (12.3 mg, 0.122 mmol) in DCM/MeOH (2:1, 1.5 mL) was stirred at
room temperature for 1 h. Sodium cyanoborohydride (3.2 mg, 0.051
mmol) was added and the reaction mixture was stirred for 3 h at
room temperature. After concentration of solvent under reduced
pressure, the resulting residue was dissolved in DMSO, and purified
by reverse phase preparative HPLC (Phenomenex reverse phase Luna
5.mu. C18 (2) column, 60.times.21.2 mm ID, mobile phase: A=0.05%
TFA in water; B=0.05% TFA in acetonitrile) to yield the desired
final product (29.1 mg, 57% yield) as a white powder
(ditrifluoroacetic acid salt) [hS1P1 EC.sub.50=1500 nM]: >95%
purity by LCMS, ESI-MS: 391.1 (M+H).sup.+, .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.05 (t, 3H), 7.79 (d, 1H), 7.65-7.62 (m, 3H),
7.47 (s, 1H), 4.48 (m, 2H), 4.38-4.32 (m, 4H), 3.73-3.70 (m, 5H),
2.15 (m, 4H), 1.16 (m, 2H).
Compound 35
6-(5-benzylbenzofuran-2-yl)-2-(2-carboxyethyl)-3,4-dihydroisoquinolinium
2,2,2-trifluoroacetate (step 6 in Scheme 7)
##STR00137##
[0490] The title compound was isolated by reverse phase preparative
HPLC during the purification of Compound 29. NMR (400 MHz,
CD.sub.3OD) .delta. 9.17 (s, 1H), 8.04 (m, 2H), 7.90 (d, 1H), 7.50
(m, 3H), 7.23 (m, 6H), 4.28 (dd, 2H), 4.16 (dd, 2H), 4.08 (s, 2H),
3.34 (m, 2H), 3.03 (m, 2H). MS (ESI) m/z: Calculated: 410.18.
Observed: 410.30 (M.sup.+).
Compound 36
1-((4-(5-benzylbenzofuran-2-yl)-3-chlorophenyl)methyl)azetidine-3-carboxyl-
ic acid
4-(Ethoxycarbonyl)-2-chlorophenyl trifluoromethanesulfonate
##STR00138##
[0492] Trifluoroacetic anhydride (4.6 mL, 27.2 mmol) was added
dropwise to a solution of ethyl 3-chloro-4-hydroxybenzoate (5.02 g,
25.0 mmol) and pyridine (2.2 mL, 27.5 mmol) in DCM (31 mL) at
-10.degree. C. The reaction mixture was stirred for 1 h at
-10.degree. C., allowed to warm up to rt and stirred for an
additional 2 h. The reaction mixture was quenched with H.sub.2O,
and the resulting biphasic mixture was stirred for 15 min. The
layers were separated and the organic layer was washed with 0.2 N
HCl, water and brine. The final organic layer was dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure to yield
6.8 g of a white solid, containing a mixture of triflate and
remaining phenol. The mixture was redissolved in DCM and passed
through a plug of silica gel to afford 3.8 g (45%) of pure triflate
and 3 g of product impure with starting material. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.21 (d, J=1.8, 1H), 8.03 (dd, J=8.5, 1.8,
1H), 7.43 (d, J=8.5, 1H), 4.42 (q, J=7.3, 2H), 1.41 (t, J=7.3,
3H).
Ethyl 4-(5-benzylbenzofuran-2-yl)-3-chlorobenzoate (step 2 in
Scheme 5)
##STR00139##
[0494] The title compound was prepared as Example Compound 40 (step
2 of Scheme 5) in the general method described above (94% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.15-8.12 (m, 2H), 8.00
(br d, J=8.4, 1H), 7.62 (s, 1H), 7.45-7.44 (m, 2H), 7.32-7.19 (m,
6H), 4.42 (q, J=7.3, 2H), 4.09 (s, 2H), 1.42 (t, J=7.3, 3H).
(4-(5-benzylbenzofuran-2-yl)-3-chlorophenyl)methanol (step 3 in
Scheme 5)
[0495] The title compound was prepared as Example Compound 40 (step
3 of Scheme 5) in the general method described above (66 mg of a
1:1 mixture of primary alcohol and aldehyde that was used without
further purification).
4-(5-Benzylbenzofuran-2-yl)-3-chlorobenzaldehyde (step 4 in Scheme
5)
##STR00140##
[0497] The title compound was prepared as Example Compound 40 (step
4 of scheme 5) in the general method described above (63% for the
two steps): .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.00 (s,
1H), 8.24 (d, J=8.4, 1H), 7.99 (d, J=1.4, 1H), 7.86 (dd, J=8.0,
1.5), 7.69 (s, 1H), 7.47-7.45 (m, 2H), 7.32-7.19 (m, 6H), 4.10 (s,
2H).
1-(4-(5-Benzylbenzofuran-2-yl)-3-chlorobenzyl)azetidine-3-carboxylic
acid (step 5 in Scheme 5)
##STR00141##
[0499] The title compound was prepared as Example Compound 40 (step
5 in Scheme 5) in the general method described above (42% yield)
[hS1P1 EC.sub.50=199 nM]: .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 8.05 (d, J=7.8, 1H), 7.76-7.72 (m, 1H), 7.59-7.54 (m, 4H),
7.27-7.16 (m, 6H), 4.46-4.36 (m, 2H), 4.32-4.16 (m, 4H), 4.03 (s,
2H), 3.64-3.58 (m, 1H). MS (ESI) m/z: Calculated: 431.13. Observed:
431.9 (M.sup.++1).
Compound 37
3-(6-(5-cyclopentylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propan-
oic acid
Tert-butyl 3-(6-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)propanoate
(step 1 in Scheme 8)
##STR00142##
[0501] A solution of 1,2,3,4-tetrahydroisoquinolin-6-ol
hydrobromide (345 mg, 1.5 mmol), tert-butyl acrylate (0.44 mL, 3.0
mmol) and N-ethyl-N-isopropylpropan-2-amine (2.6 mL, 15.0 mmol) in
MeOH was irradiated in the microwave at 90.degree. C. for 1800 s.
Removal of the solvents gave the residue which was purified on ISCO
column (2% to 5% MeOH/CH.sub.2Cl.sub.2) to provide the title
compounds (332 mg, 80%). NMR (400 MHz, CD.sub.3OD) .delta. 6.85 (d,
1H), 6.55 (dd, 1H), 6.54 (s, 1H), 3.55 (s, 2H), 2.83 (m, 4H), 2.76
(m, 2H), 2.54 (dd, 2H), 1.45 (s, 9H). MS (ESI) m/z: Calculated:
277.17. Observed: 277.90 (M.sup.++1).
Tert-butyl
3-(6-(trifluoromethylsulfonyloxy)-3,4-dihydroisoquinolin-2(1H)--
yl)propanoate (step 2 in Scheme 8)
##STR00143##
[0503] Trifluorosulfonic anhydride (87 .mu.L, 0.52 mmol) was added
to the solution of tert-butyl
3-(6-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)propanoate (111 mg,
0.4 mmol) in pyridine (5 mL) at 0.degree. C. The reaction mixture
was stirred for 1 hour at room temperature , concentrated, purified
on ISCO column (2% to 5% MeOH/CH.sub.2Cl.sub.2) to provide the
title compounds (93 mg, 57%). .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.20 (d, 1H), 7.12 (s, 1H), 7.10 (s, 1H), 3.68 (s, 2H),
2.94 (dd, 2H), 2.83 (dd, 2H), 2.78 (dd, 2H), 2.54 (dd, 2H), 1.44
(s, 9H). MS (ESI) m/z: Calculated: 409.12. Observed: 409.80
(M.sup.++1).
Tert-butyl
3-(6-(5-cyclopentylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H-
)-yl)propanoate (step 3 in Scheme 8)
##STR00144##
[0505] A mixture of 5-cyclopentylbenzofuran-2-ylboronic acid (78
mg, 0.34 mmol), tert-butyl
3-(6-(trifluoromethylsulfonyloxy)-3,4-dihydroisoquinolin-2(1H)-yl)propano-
ate (93 mg, 0.23 mmol), triethylamine (0.95 mL, 6.8 mmol) and
bis(triphenylphosphine) palladium(II) chloride (16 mg, 0.02 mmol)
in ethanol (5 mL) was irradiated in a microwave instrument at
100.degree. C. for 20 min. The reaction mixture was cooled, and the
solvent was removed. The residue was purification by silica gel
chromatography on ISCO system yielding the title compound (34 mg,
34% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.59 (m, 2H),
7.40 (d, 2H), 7.15 (dd, 1H), 7.07 (d, 1H), 6.91 (s, 1H), 3.70 (s,
2H), 3.08 (m, 1H), 2.96 (dd, 2H), 2.85 (dd, 2H), 2.78 (dd, 2H),
2.54 (dd, 2H), 2.11 (m, 2H), 1.84 (m, 2H), 1.68 (m, 4H), 1.46 (s,
9H). MS (ESI) m/z: Calculated: 445.26. Observed: 446.00
(M.sup.++1).
3-(6-(5-cyclopentylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propan-
oic acid (step 4 in Scheme 8)
##STR00145##
[0507] To a stirring solution of tert-butyl
3-(6-(5-cyclopentylbenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propa-
noate (25 mg, 0.056 mmole) in CH.sub.2Cl.sub.2 (0.5 mL) was added
TFA (0.5 mL). The mixture was stirred at room temperature for 3
hours. Under reduced pressure, solvents and excess of TFA were
removed affording a yellow oil which was rinsed with a mixture of
CH.sub.2Cl.sub.2/Hexane (1:4) followed by ether. The solvents were
removed under vacuum to give the title compound (19 mg, 90%) [hS1P1
EC.sub.50=4170 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.82
(m, 2H), 7.47 (s, 1H), 7.40 (d, 1H), 7.30 (d, 1H), 7.20 (d, 1H),
7.19 (s, 1H), 4.54 (br, 2H), 3.69 (br, 2H), 3.60 (dd, 2H), 3.28 (m,
2H), 3.10 (m, 1H), 2.96 (dd, 2H), 2.10 (m, 2H), 1.85 (m, 2H), 1.74
(m, 2H), 1.65 (m, 2H). MS (ESI) m/z: Calculated: 389.2. Observed:
390.20 (M.sup.++1).
Compound 38
1-(4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorobenzyl)azetidine-3-ca-
rboxylic acid
5-hydroxy benzofuran (step 1 of Scheme 4)
##STR00146##
[0509] To an ice-cooled solution of 5-methylbenzofuran (0.5 g, 3.37
mmol) in DCM (7 mL) was added boron tribromide (3.4 mL, 3.37 mmol,
1M in DCM). The light brown solution was stirred at 0.degree. C.
for 1 h, another equivalent of boron tribromide (3.4 mL) was then
added. The mixture was stirred at room temperature for 2 h. TLC
analysis indicated the completion of the reaction. The mixture was
poured into ice and the pH was adjusted to 7 with Na.sub.2CO.sub.3.
The aqueous was extracted with DCM (.times.2). The combined organic
layers were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated. The resulting light brown sold gave the satisfactory
purity without further purification for next step: 0.36 g (79.6%
yield), .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.59 (d, J=2.0
Hz, 1H), 7.35 (d, J=9.2 Hz, 1H), 7.01 (d, J=2.4 Hz, 1H), 6.82 (dd,
J=8.8 Hz, J=2.8 Hz, 1H), 6.67 (m, 1H), 4.73 (s, 1H).
5-(cyclopentylmethoxy)benzofuran (step 2 of Scheme 4)
##STR00147##
[0511] DEAD (362 mg, 2.09 mmol) was slowly added to a solution of
5-hydroxybenzofuran (200 mg, 1.49 mmol), triphenylphosphine (547
mg, 2.09 mmol) and cyclopentyl-methanol (203 mg, 2.0 2 mmol) in 3
mL of THF. The mixture was stirred at room temperature for 16
hours. The solvent was removed and the residue was purified by ISCO
column chromatography using 0-5% AcOEt in Hexanes. The title
compound was obtained as a white solid (0.208 g, 65% yield): 84%
purity by HPLC; .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.58 (d,
1H), 7.38 (d, J=8.4 Hz, 1H), 7.06 (s, 1H), 6.91 (d, J=9.2 Hz, 1H),
6.69 (m, 1H), 3.82 (d, 2H), 2.39 (m, 1H), 1.85 (m, 2H), 1.63 (m,
4H), 1.39 (m, 2H).
5-(cyclopentylmethoxy)benzofuran-2-ylboronic acid (step 3 of Scheme
1)
##STR00148##
[0513] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) by the general method C described above (94.7%
yield): .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.39 (d, J=9.2
Hz, 1H), 7.30 (s, 1H), 7.07 (d, 1H), 6.99 (dd, J=9.2 Hz, J=2.4 Hz,
1H), 3.82 (d, J=7.0 Hz, 2H), 2.39 (m, 1H), 1.86 (m, 2H), 1.63 (m,
4H), 1.39 (m, 2H).
4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorobenzaldehyde
(step 4 of Scheme 1)
##STR00149##
[0515] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) by the general method D described above (53% yield):
ESI-MS: 339.3 (M+H).sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 10.0 (s, 1H), 8.20 (t, 1H), 7.30 (s, 1H), 7.77 (d, 1H),
7.68 (d, 1H), 7.43 (d, 1H), 7.36 (d, 1H), 7.09 (s, 1H), 6.99 (dd,
1H), 3.88 (d, J=7.0 Hz, 2H), 2.39 (m, 1H), 1.86 (m, 2H), 1.63 (m,
4H), 1.39 (m, 2H).
1-(4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorobenzyl)azetidine-3-ca-
rboxylic acid (step 5 of Scheme 1)
##STR00150##
[0517] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) by the general method E described above (79% yield)
[hS1P1 EC.sub.50=803 nM]: ESI-MS: 423.9 (M+H).sup.+, .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 8.11 (t, 1H), 7.45-7.40 (m, 3H), 7.28
(d, 1H), 7.15 (d, 1H), 6.95 (dd, 1H), 4.46 (s, 2H), 4.36-4.34 (m,
4H), 3.88 (d, J=7.4 Hz, 2H), 3.68 (m, 1H), 2.38 (m, 1H), 1.85 (m,
2H), 1.65 (m, 4H), 1.43 (m, 2H).
Compound 39
1-(4-(5-(cyclopentylmethoxy)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
4-(5-(cyclopentylmethoxy)benzofuran-2-yl)benzaldehyde (step 4 of
Scheme 1)
##STR00151##
[0519] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) by the general method D described above (33% yield):
ESI-MS: 321.2 (M+H).sup.+, NMR (400 MHz, CD.sub.3OD) .delta. 10.0
(s, 1H), 7.98 (dd, 4H), 7.43 (d, 1H), 7.14 (s, 1H), 7.07 (d, 1H),
6.96 (dd, 1H), 3.88 (d, J=7.0 Hz, 2H), 2.41 (m, 1H), 1.86 (m, 2H),
1.63 (m, 4H), 1.39 (m, 2H).
1-(4-(5-(cyclopentylmethoxy)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (step 5 of Scheme 1)
##STR00152##
[0521] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) by the general method E described above (76% yield):
ESI-MS: 405.9 (M+H).sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.97 (d, 2H), 7.55 (d, 2H), 7.40 (d, 1H), 7.23 (s, 1H),
7.11 (d, 1H), 6.95 (dd, 1H), 4.44 (s, 2H), 4.35-4.33 (m, 4H), 3.88
(d, J=7.0 Hz, 2H), 3.69 (m, 1H), 2.38 (m, 1H), 1.87 (m, 2H), 1.65
(m, 4H), 1.43 (m, 2H).
Compound 40
1-((4-(5-Benzylbenzofuran-2-yl)-3-cyanophenyl)methyl)azetidine-3-carboxyli-
c acid
4-(ethoxycarbonyl)-2-cyanophenyl trifluoromethanesulfonoate
##STR00153##
[0523] The title compound was prepared as Example Compound 36 in
the general method described above (92% yield): .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.61 (s, 1H), 8.40 (d, 1H), 7.96 (d, 1H),
4.23 (q, 2H), 1.21 (t, 3H).
Ethyl-4-(5-benzylbenzofuran-2-yl)-3-cyanobenzoate (step 4, Scheme
1)
##STR00154##
[0525] The title compound was prepared as Example Compound 1 (step
4, Scheme 1) in the general method described above (26% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.42 (s, 1H), 8.31 (d,
1H), 8.19 (d, 1H), 7.81 (s, 1H), 7.42 (d, 2H), 7.32-7.17 (m, 6H),
4.38 (q, 2H), 4.06 (s, 2H), 1.41 (t, 3H).
2-(5-Benzylbenzofuran-2-yl)-5-(hydroxymethyl)benzonitrile (step 3,
scheme 5)
##STR00155##
[0527] A solution of
Ethyl-4-(5-benzylbenzofuran-2-yl)-3-cyanobenzoate (0.05 g, 0.13
mmol), sodium borohydride (0.01 g, 0.26 mmol) and calcium chloride
(0.015 g, 0.13 mmol) in ethanol (2.5 mL) were stirred at room
temperature for 1 hour. Water was added and the aqueous layer was
extracted with ethyl acetate (.times.2, 10 mL). Organic layer was
washed with water and brine and dried over sodium sulphate in 75%
yield: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.06 (d, 1H), 7.79
(s, 1H), 7.64 (d, 1H), 7.63 (s, 1H), 7.43-7.21 (m, 8H), 4.78 (s,
2H), 4.06 (s, 3H).
2-(5-Benzylbenzofuran-2-yl)-5-formylbenzonitrile (step 4, Scheme
5)
##STR00156##
[0529] A suspension of
2-(5-Benzylbenzofuran-2-yl)-5-(hydroxymethyl)benzonitrile (0.03 g,
0.09 mmol), Molecular sieves 4 A (0.2 g), TPAP (0.0016 mg, 0.004
mmol) and N-morpholino oxide (0.02 g, 0.18 mmol) in acetonitrile
was stirred for 1 hour and then filtered through celite to obtain
title compound in 93% yield (step 4, Scheme 5): .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 10.05 (s, 1H), 8.25 (s, 1H), 8.24 (d, 1H),
8.14 (d, 1H), 7.84 (s, 1H), 7.45 (m, 2H), 7.38-7.18 (m, 6H), 4.06
(s, 2H).
1-((4-(5-Benzylbenzofuran-2-yl)-3-cyanophenyl)methyl)azetidine-3-carboxyli-
c acid (step 5, Scheme 1)
##STR00157##
[0531] The title compound was prepared as Example Compound 1 (step
5, Scheme 1) in the general method described above (28% yield):
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.18 (d, 1H), 8.09 (s,
1H), 7.82 (d, 1H), 7.64 (s, 1H), 7.61 (s, 1H), 7.59 (d, 1H),
7.38-7.18 (m, 6H), 4.85 (bs, 2H), 4.42 (s, 2H), 4.38-4.25 (m, 4H),
4.06 (s, 2H), 3.74-3.66 (m, 1H). MS (ESI) m/z: Calculated: 422.16.
Observed: 423.0 (M.sup.++1).
Compound 41
1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)pyrrolidine-3-carboxy-
lic acid
##STR00158##
[0533] The title compound was prepared as racemic mixture according
the reductive amination procedure as described in step 5 of
Scheme-1 (60% yield) [hS1P1 EC.sub.50=315 nM]. .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 8.11 (t, 1H), 7.47-7.44 (m, 4H), 7.27-7.19
(m, 7H), 4.45 (s, 2H), 4.05 (s, 2H), 3.73-3.52 (m, 2H), 3.48-3.34
(m, 3H), 251-2.38 (m, 2H). MS (ESI) m/z: Calculated: 429.48.
Observed: 430.0 (M.sup.++1).
Compound 42
1-(4-(5-cyclopentylbenzofuran-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
4-(5-cyclopentylbenzofuran-2-yl)-3-fluorobenzaldehyde (step 3 in
Scheme 2)
##STR00159##
[0535] A solution of 5-cyclopentylbenzofuran-2-ylboronic acid (276
mg, 1.2 mmol), 4-bromobenzaldehyde (162 mg, 0.80 mmol),
palladiumdichlorobis(triphenylphosphine) (56 mg, 0.08 mmol) and
triethylamine (2.2 mL, 16 mmol) in EtOH (5 mL) was irradiated in
the microwave at 100.degree. C. for 20 min. The reaction mixture
was cooled, and the solvent was removed. The residue was
purification by silica gel chromatography on ISCO system yielding
the title compound (34 mg, 34% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.0 (s, 1H), 8.21 (dd, 1H), 7.77 (d, 1H), 7.66
(d, 1H), 7.51 (s, 1H), 7.46 (d, 1H), 7.38 (d, 1H), 7.28 (d, 1H),
3.11 (m, 1H), 2.12 (m, 2H), 1.84 (m, 2H), 1.72 (m, 2H), 1.64 (m,
2H).
1-(4-(5-cyclopentylbenzofuran-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid (step 5 in Scheme 1)
##STR00160##
[0537] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (20 mg, 18%
yield): .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.09 (dd, 1H),
7.51 (s, 1H), 7.44 (d, 1H), 7.40 (s, 1H), 7.37 (d, 1H), 7.29 (m,
2H), 4.34 (s, 2H), 4.14 (m, 4H), 3.39 (m, 1H), 3.11 (m, 1H), 2.12
(m, 2H), 1.85 (m, 2H), 1.74 (m, 2H), 1.65 (m, 2H). MS (ESI) m/z:
Calculated: 393.17. Observed: 393.90 (M.sup.++1).
Compound 43
1-((4-(5-Benzylbenzofuran-2-yl)-3-methylphenyl)methyl)azetidine-3-carboxyl-
ic acid
Methyl 4-(5-benzylbenzofuran-2-yl)-3-methylbenzoate (step 2 in
Scheme 5)
##STR00161##
[0539] The title compound was prepared as Example Compound 40 (step
2, Scheme 5) in the general method described above (52% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.89 (m, 2H), 7.61 (d,
1H), 7.42-7.17 (m, 8H), 6.95 (s, 1H), 4.06 (s, 2H), 3.82 (s, 3H),
2.61 (s, 3H).
(4-(5-Benzylbenzofuran-2-yl)-3-methylphenyl)methanol (step 3 in
Scheme 5)
##STR00162##
[0541] The title compound was prepared as Example Compound 40 (step
3, Scheme 5) in the general method described above (86% yield): NMR
(400 MHz, CDCl.sub.3) .delta. 7.94 (s, 1H), 7.82 (d, 1H), 7.48-7.07
(m, 9H), 6.85 (s, 1H), 4.67 (brs, 1H), 4.06 (s, 4H), 2.58 (s,
3H).
4-(5-Benzylbenzofuran-2-yl)-3-methylbenzaldehyde (step 4 in Scheme
5)
##STR00163##
[0543] The title compound was prepared as Example Compound 40 (step
4, Scheme 5) in the general method described above (90% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.07 (d,
1H), 7.81 (m, 2H), 7.46-7.17 (m, 8H), 7.01 (s, 1H), 4.08 (s, 2H),
2.63 (s, 3H).
1-((4-(5-Benzylbenzofuran-2-yl)-3-methylphenyl)methyl)azetidine-3-carboxyl-
ic acid (step 5 in Scheme 1)
##STR00164##
[0545] The title compound was prepared as Example Compound 1 (step
5, Scheme 1) in the general method described above (62% yield)
[hS1P1 EC.sub.50=241 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.92 (d, 1H), 7.51-7.17 (m, 10H), 7.03 (s, 1H), 4.84 (bs, 2H), 4.41
(s, 2H), 4.37-4.22 (m, 4H), 4.08 (s, 2H), 3.68-3.61 (m, 1H), 2.63
(s, 3H). MS (ESI) m/z: Calculated: 411.18. Observed: 411.9
(M.sup.++1).
Compound 44
3-(6-(5-butoxybenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoic
acid
Tert-butyl
3-(6-(5-butoxybenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)-
propanoate (step 3 in Scheme 8)
##STR00165##
[0547] The title compound was prepared as Example Compound 37 (step
3 in Scheme 8) in the general method described above (57 mg, 50%
yield): NMR (400 MHz, CDCl.sub.3) .delta. 7.57 (m, 2H), 7.36 (d,
1H), 7.06 (d, 1H), 7.00 (d, 1H), 6.88 (s, 1H), 6.85 (d, 1H), 3.99
(dd, 2H), 3.68 (s, 2H), 2.96 (dd, 2H), 2.85 (dd, 2H), 2.78 (dd,
2H), 2.53 (dd, 2H), 1.80 (m, 2H), 1.56 (m, 2H), 1.45 (s, 9H), 1.00
(t, 3H). MS (ESI) m/z: Calculated: 449.26. Observed: 449.90
(M.sup.++1).
3-(6-(5-butoxybenzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoic
acid (step 4 in Scheme 8)
##STR00166##
[0549] The title compound was prepared as Example Compound 37 (step
4 in Scheme 8) in the general method described above (25 mg, 75%
yield) [hS1P1 EC.sub.50=4440 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.79 (m, 2H), 7.38 (d, 1H), 7.30 (d, 1H), 7.17 (s, 1H),
7.09 (d, 1H), 6.88 (dd, 1H), 4.54 (br s, 2H), 4.00 (dd, 2H), 3.68
(m, 2H), 3.60 (dd, 2H), 3.21 (m, 1H), 2.95 (dd, 2H), 1.78 (m, 2H),
1.53 (m, 2H), 1.00 (t, 2H). MS (ESI) m/z: Calculated: 393.19.
Observed: 394.20 (M.sup.++1).
Compound 45
3-(5-(5-benzylbenzofuran-2-yl)-2,3-dihydro-1H-inden-2-ylamino)-propanoic
acid
##STR00167##
[0551] The title compound was prepared according the reductive
amination procedure as described in step 5 of Scheme-1 (60% yield)
[hS1P1 EC.sub.50=807 nM]. .sup.1H NMR (400 MHz, CD.sub.3Cl.sub.3)
.delta. 7.80-7.74 (m, 2H), 7.54 (d, 1H), 7.40-7.22 (m, 7H), 7.18
(d, 1H), 6.92 (s, 1H), 4.79 (s, 1H), 4.02 (s, 2H), 3.28-2.92 (m,
4H), 2.73 (t, 2H), 2.48-2.30 (m, 2H). MS (ESI) m/z: Calculated:
411.49. Observed: 412.7 (M.sup.++1).
Compound 46
3-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methylamino)-3-methylbutano-
ic acid
##STR00168##
[0553] The title compound was prepared as Example Compound 1 (step
5, in Scheme 1) in the general method described above but using
3-amino-3-methylbutanoic acid instead of azetidine-3-carboxylic
acid (46% yield) [hS1P1 EC.sub.50>25000 nM]: .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 8.11 (t, J=7.8, 1H), 7.50-7.17 (m, 12H),
4.28 (s, 2H), 4.07 (s, 2H), 1.51 (s, 6H). MS (ESI) m/z: Calculated:
431.19. Observed: 432.0 (M.sup.++1).
Compound 47
1-((4-(5-cyclopentylbenzofuran-2-yl)-3-methoxyphenyl)methyl)azetidine-3-ca-
rboxylic acid
4-(5-cyclopentylbenzofuran-2-yl)-3-methoxybenzaldehyde
##STR00169##
[0555] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (56% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.04 (s, 1H), 8.21 (d,
1H), 7.77 (d, 1H), 7.59-7.19 (m, 5H), 4.04 (s, 3H), 3.11 (m, 1H),
2.15-1.77 (m, 4H), 1.58-1.56 (m, 4H).
##STR00170##
[0556] The title compound was prepared as Example Compound 1 (step
5, in Scheme 1) in the general method described earlier for
reductive amination (71% yield) [hS1P1 EC.sub.50=1070 nM]. .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 8.16 (d, 1H), 7.45 (s, 1H),
7.41-7.36 (m, 2H), 7.26-7.17 (m, 3H), 4.85 (bs, 2H), 4.41 (s, 2H),
4.32 (m, 4H), 4.04 (s, 3H), 3.62 (m, 1H), 3.11 (m, 1H), 2.25-2.12
(m, 2H), 1.90-1.66 (m, 6H). MS (ESI) m/z: Calculated: 405.19.
Observed: 405.9 (M.sup.++1).
Compound 48
1-((4-(5-Benzylbenzofuran-2-yl)-3,5-difluorophenyl)methyl)azetidine-3-carb-
oxylic acid
4-(5-Benzylbenzofuran-2-yl)-3,5-difluorobenzaldehyde
##STR00171##
[0558] The title compound was prepared as Example Compound 1 (step
4, Scheme 1) in the general method described above (66% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.04 (s, 1H), 7.66 (s,
1H), 7.45 (d, 1H), 7.41-7.17 (m, 8H), 4.08 (s, 2H).
1-((4-(5-Benzylbenzofuran-2-yl)-3,5-difluorophenyl)methyl)azetidine-3-carb-
oxylic acid
##STR00172##
[0560] The title compound was prepared as Example Compound 1 (step
5, Scheme 1) in the general method described above (62% yield)
[hS1P1 EC.sub.50=89 nM]: .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.55 (s, 1H), 7.47 (d, 1H), 7.41-7.12 (m, 8H), 4.42 (s,
2H), 4.37-4.22 (m, 7H), 4.06 (s, 2H), 3.72-3.64 (m, 1H). MS (ESI)
m/z: Calculated: 433.15. Observed: 433.9 (M.sup.++1).
Compound 49
1-(4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorobenzyl)azetidine-3-ca-
rboxylic acid
5-(cyclopropylmethoxy)benzofuran (step 2 of Scheme 4)
##STR00173##
[0562] The title compound was prepared as Example Compound 38 (step
2 in Scheme 4) by the general method described above (49% yield):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.60 (d, 1H), 7.38 (d,
1H), 7.05 (s, 1H), 6.94 (d, 1H), 6.69 (m, 1H), 3.84 (d, 2H), 1.31
(m, 1H), 0.66 (m, 2H), 0.37 (m, 2H).
5-(cyclopropylmethoxy)benzofuran-2-ylboronic acid (step 3 of Scheme
1)
##STR00174##
[0564] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) by the general method C described above (98% yield):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.39 (d, 1H), 7.29 (s,
1H), 7.06 (d, 1H), 7.00 (dd, 1H), 3.83 (d, J=6.9 Hz, 2H), 1.30 (m,
1H), 0.66 (m, 2H), 0.38 (m, 2H).
4-(5-(cyclopropylmethoxy)benzofuran-2-yl)-3-fluorobenzaldehyde
(step 4 of Scheme 1)
##STR00175##
[0566] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) by the general method D described above (50% yield):
ESI-MS: 311.2 (M+H).sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 10.01 (s, 1H), 8.20 (t, 1H), 7.78 (d, 1H), 7.69 (d, 1H),
7.44 (d, 1H), 7.36 (d, 1H), 7.08 (s, 1H), 7.01 (d, 1H), 3.85 (d,
J=7.1 Hz, 2H), 1.32 (m, 1H), 0.68 (m, 2H), 0.38 (m, 2H).
1-(4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorobenzyl)azetidine-3-ca-
rboxylic acid
##STR00176##
[0568] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) by the general method E described above (68% yield)
[hS1P1 EC.sub.50=109 nM]: ESI-MS: 395.9 (M+H).sup.+, .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 8.01 (t, 1H), 7.35-7.30 (m, 3H), 7.17
(d, 1H), 7.04 (d, 1H), 6.87 (dd, 1H), 4.37 (s, 2H), 4.28-4.25 (m,
4H), 3.76 (d, J=6.7 Hz, 2H), 3.60 (m, 1H), 1.18 (m, 2H), 0.54-0.51
(m, 2H), 0.28-0.26 (m, 2H).
Compound 50
1-((4-(5-Butoxybenzofuran-2-yl)-3-chlorophenyl)methyl)azetidine-3-carboxyl-
ic acid
2-Chloro-4-formylphenyl trifluoromethanesulfonate
##STR00177##
[0570] The title compound was prepared as Example Compound 36 in
the general method described above but using
3-chloro-4-hydroxybenzaldehyde instead of ethyl
3-chloro-4-hydroxybenzoate (92% yield): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.00 (s, 1H), 8.06 (d, J=1.8, 1H), 7.88 (dd,
J=8.4, 1.8, 1H), 7.55 (d, J=8.4, 1H).
4-(5-Butoxybenzofuran-2-yl)-3-chlorobenzaldehyde
##STR00178##
[0572] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (72% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.00 (s, 1H), 8.25 (d,
J=8.0, 1H), 7.99 (d, J=1.4, 1H), 7.86 (dd, J=8.4, 1.5), 7.70 (s,
1H), 7.42 (d, J=8.8), 7.10 (d, J=2.6, 1H), 6.99 (dd, J=8.8, 2.5),
4.01 (t, J=6.5, 2H), 1.84-1.77 (m, 2H), 1.54-1.49 (m, 2H), 1.00 (t,
J=7.3, 3H).
1-((4-(5-Butoxybenzofuran-2-yl)-3-chlorophenyl)methyl)azetidine-3-carboxyl-
ic acid
##STR00179##
[0574] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (66% yield)
[hS1P1 EC.sub.50=266 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
8.13 (d, J=8.4, 1H), 7.70 (d, J=1.8, 1H), 7.57 (s, 1H), 7.53 (dd,
J=8.4, 1.8, 1H), 7.42 (d, J=9.1, 1H), 7.15 (d, J=2.5 1H), 6.95 (dd,
J=9.1, 2.5), 4.45 (s, 2H), 4.40-4.32 (m, 4H), 4.00 (t, J=6.5, 2H),
3.74-3.66 (m, 1H), 1.81-1.74 (m, 2H), 1.58-1.49 (m, 2H), 1.00 (t,
J=7.3, 3H). Calculated: 413.14. Observed: 413.9 (M.sup.++1).
Compound 51
1-((3-chloro-4-(5-cyclopentylbenzofuran-2-yl)phenyl)methyl)azetidine-3-car-
boxylic acid
Ethyl 3-chloro-4-(5-cyclopentylbenzofuran-2-yl)benzoate
##STR00180##
[0576] The title compound was prepared as Example Compound 40 (step
2 in Scheme 5) in the general method described above (73% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.14 (d, J=7.8, 2H), 8.00
(d, J=8.1, 1H), 7.64 (s, 1H), 7.51-7.44 (m, 3H), 4.42 (q, J=7.0,
2H), 3.12-3.08 (m, 1H), 2.16-2.08 (m, 2H), 1.84-1.58 (m, 6H), 1.42
(t, J=7.3, 3H).
(3-Chloro-4-(5-cyclopentylbenzofuran-2-yl)phenyl)methanol
##STR00181##
[0578] The title compound was prepared as Example Compound 40 (step
3 in Scheme 5) in the general method described above (142 mg of a
1:1 mixture of primary alcohol and aldehyde that was used without
further purification).
3-Chloro-4-(5-cyclopentylbenzofuran-2-yl)benzaldehyde
##STR00182##
[0580] The title compound was prepared as Example Compound 40 (step
4 of Scheme 5) in the general method described above (61% for the
two steps): .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.00 (s,
1H), 8.25 (d, J=8.1, 1H), 7.99 (s, 1H), 7.86 (d, J=8.1), 7.71 (s,
1H), 7.52 (s, 1H), 7.47 (d, J=8.8, 1H), 7.27 (d, J=8.8, 1H),
3.16-3.06 (m, 1H), 2.18-2.06 (m, 2H), 1.88-1.60 (m, 6H).
1-((3-chloro-4-(5-cyclopentylbenzofuran-2-yl)phenyl)methyl)azetidine-3-car-
boxylic acid
##STR00183##
[0582] The title compound was prepared as Example Compound 40 (step
5 of Scheme 5) in the general method described above (60% yield)
[hS1P1 EC.sub.50=558 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
8.16 (d, J=8.1, 1H), 7.70 (d, J=1.5, 1H), 7.61 (s, 1H), 7.55-7.53
(m, 2H), 7.45 (d, J=8.8, 1H), 7.28 (dd, J=8.4, 1.5), 4.45 (s, 2H),
4.40-4.34 (m, 4H), 3.72-3.64 (m, 1H), 3.16-3.10 (m, 1H), 2.15-2.06
(m, 2H), 1.87-1.66 (m, 6H). Calculated: 409.14. Observed: 409.9
(M.sup.++1).
Compound 52
3-(N-((4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorophenyl)methyl)-N--
(2-hydroxyethyl)amino)propanoic acid
##STR00184##
[0584] The title compound was prepared as Example Compound 1 (step
5, Scheme 1) in the general method described above except using
3-(2-hydroxyethylamino)propanoic acid (13% yield): .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 7.57 (d, 1H), 7.55-7.47 (m, 2H), 7.26
(m, 2H), 7.22 (s, 1H), 6.96 (d, 1H), 4.82 (bs, 3H), 4.42 (s, 2H),
4.06 (s, 2H), 3.92-3.65 (m, 4H), 2.75-2.33 (m, 4H), 1.95-1.31 (m,
9H). MS (ESI) m/z: Calculated: 455.21. Observed: 455.9
(M.sup.++1).
Compound 53
1-((3-fluoro-4-(5-morpholinobenzofuran-2-yl)phenyl)methyl)azetidine-3-carb-
oxylic acid
4-(benzofuran-5-yl)morpholine
##STR00185##
[0586] The title compound was prepared as Example Compound 31 (step
1 Scheme 3) in the general method described above (52% yield)
[hS1P1 EC.sub.50=2090 nM]: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.46 (s, 1H), 7.36 (d, 1H), 6.95 (s, 1H), 6.81 (d, 1H),
6.58 (s, 1H), 3.78 (m, 4H), 2.95 (m, 4H).
5-morpholinobenzofuran-2-yl-2-boronic acid
##STR00186##
[0588] The title compound was prepared as Example Compound 31 (step
2 scheme 3) in the general method described above (72% yield): MS
(ESI) m/z: Calculated: 247.1. Observed: 248.1 (M.sup.++1).
3-fluoro-4-(5-morpholinobenzofuran-2-yl)benzaldehyde
##STR00187##
[0590] The title compound was prepared as Example Compound 31 (step
3 Scheme 3) in the general method described above (52% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.01 (s, 1H), 7.76 (d,
1H), 7.61 (d, 1H), 7.55-7.06 (m, 5H), 3.86 (m, 4H), 3.15 (m,
4H).
1-((3-fluoro-4-(5-morpholinobenzofuran-2-yl)phenyl)methyl)azetidine-3-carb-
oxylic acid
##STR00188##
[0592] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (28% yield):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.03 (t, 1H), 7.56 (d,
1H), 7.53 (d, 1H), 7.43 (d, 1H), 7.22 (d, 1H), 7.19 (d, 1H) 7.05
(dd, 1H), 4.50 (s, 2H), 4.39 (dd, 4H), 3.72-3.70 (m, 6H), 2.08 (m,
4H), 1.84 (m, 2H). MS (ESI) m/z: Calculated: 410.1. Observed: 411.1
(M.sup.++1).
Compound 54
4-((4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)morpholine-2-carboxy-
lic acid
##STR00189##
[0594] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above but using
morpholine-2-carboxylic acid instead of azetidine-3-carboxylic acid
(57% yield) [hS1P1 EC.sub.50=1860 nM]: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.09 (t, J=7.8, 1H), 7.46-7.42 (m, 4H),
7.28-7.14 (m, 7H), 4.38 (br d, J=9.5, 1H), 4.30-4.21 (m, 2H),
4.13-4.04 (m, 1H), 4.06 (s, 2H), 3.83 (br t, J=10.6, 1H), 3.53 (br
d, J=12.4, 1H), 3.30-3.22 (m, 1H), 3.13-3.00 (m, 2H). MS (ESI) m/z:
Calculated: 445.17. Observed: 445.90 (M.sup.++1).
Compound 55
4-((4-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3-fluorophenyl)methyl)morpho-
line-2-carboxylic acid
##STR00190##
[0596] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above but using
morpholine-2-carboxylic acid instead of azetidine-3-carboxylic acid
(57% yield) [hS1P1 EC.sub.50>25000 nM]: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.06 (t, J=7.8, 1H), 7.44-7.42 (m, 3H), 7.24
(d, J=3.3, 1H), 7.13 (d, J=2.5, 1H), 6.95-6.92 (m, 1H), 4.38 (dd,
J=9.5, 2.6, 1H), 4.21-4.09 (m, 3H), 3.88 (d, J=7.0, 2H), 3.81 (t,
J=10.2, 1H), 3.45 (br d, J=11.4, 1H), 3.15 (br d, J=12.4, 1H),
3.03-2.91 (m, 2H), 2.41-2.34 (m, 1H), 1.90-1.83 (m, 2H), 1.71-1.57
(m, 4H), 1.46-1.37 (m, 2H). MS (ESI) m/z: Calculated: 453.20.
Observed: 453.90 (M.sup.++1).
Compound 56
1-(5-(5-benzylbenzofuran-2-yl)-2,3-dihydro-1H-inden-2-yl
azetidine-3-carboxylic acid
##STR00191##
[0598] The title compound was prepared according the reductive
amination procedure as described in step 5 of Scheme-1 (69% yield).
.sup.1H NMR (400 MHz, CD.sub.3Cl.sub.3) .delta. 7.78-7.68 (m, 2H),
7.45-7.22 (m, 8H), 7.22 (d, 1H), 6.94 (s, 1H), 4.73 (s, 1H), 4.05
(s, 2H), 3.52-3.20 (m, 2H), 3.29-2.62 (m, 7H), 2.48-2.31 (m, 2H).
MS (ESI) m/z: Calculated: 423.5. Observed: 423.7 (M.sup.++1).
Compound 57
3-(6-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)--
yl)propanoic acid
Tert-butyl
3-(6-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3,4-dihydroisoquin-
olin-2(1H)-yl)propanoate (step 3 in Scheme 8)
##STR00192##
[0600] The title compound was prepared as Example Compound 37 (step
3 in Scheme 8) in the general method described above (73 mg, 40%
yield) [hS1P1 EC.sub.50=877 nM]: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.57 (m, 2H), 7.36 (d, 1H), 7.06 (d, 1H), 7.01 (d, 1H),
6.85 (m, 2H), 3.85 (d, 2H), 3.69 (s, 2H), 2.96 (dd, 2H), 2.86 (dd,
2H), 2.79 (dd, 2H), 2.54 (dd, 2H), 2.38 (m, 1H), 1.66 (m, 2H), 1.59
(m, 4H), 1.45 (s, 9H), 1.39 (t, 2H). MS (ESI) m/z: Calculated:
475.27. Observed: 475.90 (M.sup.++1).
3-(6-(5-(cyclopentylmethoxy)benzofuran-2-yl)-3,4-dihydroisoquinolin-2(1H)--
yl)propanoic acid (step 4 in Scheme 8)
##STR00193##
[0602] The title compound was prepared as Example Compound 37 (step
4 in Scheme 8) in the general method described above (19 mg, 72%
yield): .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.78 (m, 2H),
7.38 (d, 1H), 7.30 (d, 1H), 7.17 (s, 1H), 7.09 (d, 1H), 6.88 (dd,
1H), 4.53 (s, 2H), 3.87 (d, 2H), 3.68 (m, 2H), 3.58 (dd, 2H), 3.27
(m, 1H), 2.93 (dd, 2H), 2.37 (m, 1H), 1.86 (m, 2H), 1.63 (m, 4H),
1.41 (m, 2H). MS (ESI) m/z: Calculated: 419.21. Observed: 420.2
(M.sup.++1).
Compound 58
3-(4-(5-cyclopentylbenzofuran-2-yl)-3-fluorobenzylamino)propanoic
acid (Step 5 in Scheme 1)
##STR00194##
[0604] The title compound was prepared as Example Compound 1 (Step
5 in Scheme 1) in the general method described above (4.1 mg, 4.6%
yield) [hS1P1 EC.sub.50=1070 nM]: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 8.10 (dd, 1H), 7.50 (s, 1H), 7.42 (m, 3H), 7.25 (m, 2H),
4.30 (s, 2H), 3.25 (m, 2H), 3.10 (m, 1H), 2.25 (dd, 2H), 2.18 (m,
2H), 1.84 (m, 2H), 1.65 (m, 4H). MS (ESI) m/z: Calculated: 381.17.
Observed: 381.80 (M.sup.++1).
Compound 59
3-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenoxy)propane-1,2-diol
4-(5-benzylbenzofuran-2-yl)-3-fluorophenol (step 4 in Scheme 1)
##STR00195##
[0606] The title compound was prepared as the Example Compound 1
(step 4 in Scheme 1). in the general method described above except
using 4-bromo-3-fluorophenol. The compound was used without further
purification for the next step reaction.
3-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenoxy)propane-1,2-diol
(step 2 in Scheme 9)
##STR00196##
[0608] A mixture of 4-(5-benzylbenzofuran-2-yl)-3-fluorophenol (22
mg, 0.069 mmol), and 3-bromopropane-1,2-diol (48 mg, 0.31 mmol) and
2 N NaOH (200 .mu.L) in i-PrOH (1 mL) was heated at 90.degree. C.
for overnight. After concentration of solvents under reduced
pressure, the resulting residue was dissolved in DMSO and purified
by reverse phase preparative HPLC (Phenomenex reverse phase Luna
5.mu. C18 (2) column, 60.times.21.2 mm ID, mobile phase: A=0.05%
TFA in water; B=0.05% TFA in acetonitrile) to yield the desired
final product (4.4 mg, 16% yield) as a white powder: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 7.89 (m, 1H), 7.40 (m, 2H), 7.23 (m,
4H), 7.10 (m, 2H), 6.99 (m, 1H), 6.89 (m, 2H), 4.10 (m, 1H), 4.04
(s, 2H), 3.96 (m, 2H), 3.65 (m, 2H). MS (ESI) m/z: Calculated:
392.14. Observed: 393.20 (M.sup.++1).
Compound 60
1-((3-Fluoro-4-(5-(1-(methylsulfonyl)piperidine-4-yl)benzofuran-2-yl)pheny-
l)methyl)azetidine-3-carboxylic acid
4-(4-(2,2-Diethoxyethoxy)-1-(methylsulfonyl)piperidine
##STR00197##
[0610] The title compound was prepared as Example Compound 1 (step
1 in Scheme 1) in the general method described above (70% yield):
NMR (400 MHz, CDCl.sub.3) .delta. 7.07 (d, 2H), 6.82 (d, 2H), 4.81
(t, 1H), 3.96 (d, 4H), 3.90 (t, 2H), 3.79-3.72 (m, 2H), 3.67-3.59
(m, 2H), 2.81 (s, 3H), 2.77 (t, 2H), 2.61 (m, 1H), 1.77-1.70 (m,
2H), 1.21 (t, 6H).
4-(Benzofuran-5-yl)-1-(methylsulfonyl)piperidine
##STR00198##
[0612] The title compound was prepared as Example Compound 1 (step
2 in Scheme 1) in the general method described above (20% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45 (d, 1H), 7.42 (s,
1H), 7.13 (d, 1H), 6.62 (s, 1H), 4.03 (m, 4H), 2.83 (s, 3H), 2.78
(t, 1H), 1.82-1.75 (m, 4H).
5-(1-(Methylsulfonyl)piperidin-4-yl)benzofuran-2-yl-2-boronic
acid
##STR00199##
[0614] The title compound was prepared as Example Compound 1 (step
3 in Scheme 1) in the general method described above (84% yield):
MS (ESI) m/z: Calculated: 323.1. Observed: 324.1 (M.sup.++1).
3-Fluoro-4-(5-(1-(methylsulfonyl)piperidin-4-yl)benzofuran-2-yl)benzaldehy-
de
##STR00200##
[0616] The title compound was prepared as Example Compound 1 (step
4 in Scheme 1) in the general method described above (62% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.21 (t,
1H), 7.79 (d, 1H), 7.72 (d, 1H), 7.51-7.21 (m, 4H), 3.98 (m, 4H),
2.84 (s, 3H), 2.76 (m, 1H), 2.05-1.81 (m, 4H).
1-((3-Fluoro-4-(5-(1-(methylsulfonyl)piperidine-4-yl)benzofuran-2-yl)pheny-
l)methyl)azetidine-3-carboxylic acid
##STR00201##
[0618] The title compound was prepared as Example Compound 1 (step
5 in Scheme 1) in the general method described above (70% yield):
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.21 (t, 1H), 7.84 (d,
1H), 7.77 (d, 1H), 7.51-7.21 (m, 4H), 4.85 (bs, 2H), 4.46 (s, 2H),
3.98 (m, 4H), 3.68 (m, 1H), 3.62 (m, 4H), 2.84 (s, 3H), 2.76 (m,
1H), 1.91-1.71 (m, 4H). MS (ESI) m/z: Calculated: 486.1. Observed:
486.9 (M.sup.++1).
Compound 61
1-(3-fluoro-4-(5-(tetrahydro-2H-pyran-4-yl)benzofuran-2-yl)phenyl)
methyl)azetidine-3-carboxylic acid
4-(benzofuran-5-yl)-tetrahydro-2H-pyran-4-ol (step 1 in Scheme
6)
##STR00202##
[0620] To a suspension of Mg (550 mg, 23.0 mmol) in dry THF (15
mL), under nitrogen atmosphere was added 5-bromobenzofuran (3.9 g,
20.0 mmol) in one portion. A crystal of iodine was added and then
the contents were refluxed for 3 h. The reaction was then allowed
to attain ambient temperature, and then cooled to -40.degree. C.
Pyran-4-one (3.0 g, 30.0 mmol) was added drop-wise and the
resulting solution was allowed to reach room temperature. The
reaction mixture was quenched by addition of 1N HCl (5 mL) and then
was diluted with ether (30 mL). It was washed with water
(2.times.15 mL) and the combined organic extract was washed with
brine (15 mL), dried and concentrated under reduced pressure to
give the crude carbinol as colorless oil. Purification by column
chromatography using 5% EtOAc-hexanes afforded the desired product
as white solid (41%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.74 (d, 1H), 7.64 (d, 1H), 7.51-7.44 (m, 2H), 6.78 (d, 1H),
4.00-3.88 (m, 4H), 2.25 (t, 2H), 1.78-1.74 (m, 2H).
5-(Tetrahydro-2H-pyran-4-yl)benzofuran (step 2 in Scheme 6)
##STR00203##
[0622] To a solution of
4-(benzofuran-5-yl)-tetrahydro-2H-pyran-4-ol (109 mg, 0.5 mmol) in
DCM (5 mL) at 0.degree. C. under nitrogen atmosphere was added
triethylsilane (175 mg, 1.5 mmol) followed by TFA (570 mg, 5.0
mmol). After stirring for 15 min at the same temperature, the
cooling bath was removed, and allowed the reaction mixture to reach
room temperature. It was further stirred at room temperature for 6
h and then poured into crushed ice-water mixture (10 mL). It was
extracted with DCM (3.times.10 mL), and the combined organic layer
was washed with brine (10 mL), dried and evaporated. The crude
compound was purified by column chromatography using 5%
EtOAc-hexanes to afford the desired product (88%). .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.61 (d, 1H), 7.44 (d, 2H), 7.15 (d, 1H),
6.73 (d, 1H), 4.11 (dd, 2H), 3.56 (t, 2H), 2.89-2.81 (m, 1H),
1.93-1.79 (m, 4H).
5-(tetrahydro-2H-pyran-4-yl-benzofuran-2-yl-2-boronic acid (step 3
in Scheme 1)
##STR00204##
[0624] The title compound was prepared in the same manner as
described in step 3 of Scheme 1 (86%): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.48-7.41 (m, 2H), 7.28 (d, 1H), 7.20 (d, 1H),
4.10 (t, 2H), 3.60 (t, 2H), 2.98-2.94 (m, 1H), 1.97-1.80 (m, 4H).
MS (ESI) m/z: Calculated: 324.35. Observed: 325.1 (M.sup.++1).
3-Fluoro-4-(5-(tetrahydro-2H-pyran-4-yl-benzofuran-2-yl)benzaldehyde
(step 4 in Scheme 1)
##STR00205##
[0626] The title compound was prepared in the same manner as
described in step 4 of Scheme 1 (68%): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.01 (s, 1H), 8.04 (t, 1H), 7.80 (t, 1H), 7.74
(d, 1H), 7.52 (t, 2H), 7.40 (d, 1H), 7.22 (s, 1H), 4.10 (t, 2H),
3.60 (t, 2H), 2.98-2.94 (m, 1H), 1.97-1.80 (m, 4H). MS (ESI) m/z:
Calculated: 324.35. Observed: 325.1 (M.sup.++1).
1-(3-fluoro-4-(5-(tetrahydro-2H-pyran-4-yl)benzofuran-2-yl)phenyl)methyl)a-
zetidine-3-carboxylic acid (step 5 in Scheme 1)
##STR00206##
[0628] The title compound was prepared according the reductive
amination procedure as described in step 5 of Scheme-1 (73% yield)
[hS1P1 EC.sub.50=207 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
8.12 (t, 1H), 7.54 (s, 1H), 7.49 (d, 1H), 7.43 (s, 1H), 7.41 (d,
1H), 7.31-7.27 (m, 2H), 4.46 (s, 2H), 4.39-4.31 (m, 4H), 4.05 (d,
2H), 3.71-3.64 (m, 1H), 3.59 (t, 2H), 1.95-1.89 (m, 4H). MS (ESI)
m/z: Calculated: 409.45. Observed: 410.0 (M.sup.++1).
[0629] The following examples are given for the purpose of
illustrating the invention, but not for limiting the scope or
spirit of the invention.
[0630] Compounds of the invention may be prepared as described in
the following schemes.
##STR00207##
##STR00208##
##STR00209##
##STR00210##
##STR00211##
##STR00212##
##STR00213##
##STR00214##
##STR00215##
##STR00216## ##STR00217##
##STR00218##
##STR00219##
##STR00220##
[0631] Compounds were prepared using the general procedures as
described below:
##STR00221##
Compound 62
(3R)-1-(4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)piperidine-3-car-
boxylic acid
##STR00222##
[0633] Synthesized according to Scheme 1 and general procedure E to
give
(3R)-1-((4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)piperidine-3-c-
arboxylic acid [hS1P1 EC.sub.50=5451 nM]. MS (ESI) m/z: Calculated:
443.2. Observed: 444.1 (M.sup.++1).
Compound 63
(3S)-1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)piperidine-3-ca-
rboxylic acid
##STR00223##
[0635] Synthesized according to Scheme 1 and general procedure E to
give
(3S)-1-((4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)piperidine-3-c-
arboxylic acid [hS1P1 EC.sub.50=3340 nM]. MS (ESI) m/z: Calculated:
443.2. Observed: 444.1 (M.sup.++1).
Compound 64
1-(3-Fluoro-4-(5-phenylthio)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
(4-(2,2-Diethoxyethoxy)phenyl)(phenyl)sulfane (step 2 in Scheme
A-4)
##STR00224##
[0637] A mixture of 4-(phenylthio)phenol (4.7 g, 23 mmol),
bromoacetaldehyde diethyl acetal (4.3 mL, 28 mmol) and
K.sub.2CO.sub.3 (3.82 g, 28 mmol) in DMF (40 mL) was stirred at
reflux for 6 h. The reaction mixture was allowed to cool down to
room temperature and poured over ice and diluted to 100 mL with
water. The solution was extracted with Et.sub.2O (20 mL.times.3);
the combined extracts were washed with 1N NaOH solution, water and
brine, dried, and concentrated under reduced pressure to yield a
yellow oil that was used without further purification: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.42 (d, 2H), 7.27-7.06 (m, 5H), 6.92
(d, 2H), 4.84 (t, 1H), 4.02 (d, 2H), 3.84-3.68 (m, 4H), 1.13 (m,
6H).
5-(Phenylthio)benzofuran (step 3 in Scheme A-4)
##STR00225##
[0639] A mixture of (4-(2,2-Diethoxyethoxy)phenyl)(phenyl)sulfane
(2.7 g , 8.5 mmol) and polyphosphoric acid (2.7 g, 27 mmol) in
benzene (70 mL) was stirred at reflux for 2 h. The reaction mixture
was cooled to room temperature, decanted from the PPA and filtered
through a plug of silica gel, which was washed with hexanes. The
filtrate and the wash were combined anc concentrated under reduced
pressure to yield 1.7 g (89%) of benzofuran: .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.74 (d, 1H), 7.64 (dd, 1H), 7.49-7.39 (m, 2H),
7.29-7.16 (m, 5H), 6.74 (dd, 1H).
5-(phenylthio)benzofuran-2-ylboronic acid (step 4 in Scheme
A-4)
##STR00226##
[0641] A solution of n-BuLi (1.5 mL, 2.5M solution in hexanes) was
added dropwise to a solution of 5-(phenylthio)benzofuran (0.7 g,
3.1 mmol) in anhydrous THF (20 mL) at -78.degree. C. The resulting
mixture was stirred at -78.degree. C. for 20 minute, and treated
with B(.sup.iPrO).sub.3 (1.1 mL, 4.64 mmol). The reaction mixture
was allowed to warm up slowly to room temperature and stirred for 1
hour. The reaction was quenched with 2N HCl and extracted with
Et.sub.2O. The combined extracts were washed with brine, dried and
concentrated under reduced pressure to yield 0.81 g of crude
boronic acid, that was used without further purification: .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.82 (dd, 1H), 7.58-7.42 (m, 2H),
7.38-7.17 (m, 6H).
3-Fluoro-4-(5-(phenylthio)benzofuran-2-yl)benzaldehyde (step 5 in
Scheme A-4)
##STR00227##
[0643] A solution of 5-(phenylthio)benzofuran-2-ylboronic acid (610
mg, 2.26 mmol), 4-bromobenzaldehyde (38 mg, 1.87 mmol),
palladiumdichlorobis(triphenylphosphine) (66 mg, 0.094 mmol) and
triethylamine (5 mL, 68 mmol) in EtOH (5 mL) was irradiated in the
microwave at 110.degree. C. for 1200 seconds. The crude reaction
mixture was purified via chromatography (9/1 Hexane//Ethyl acetate)
to yield the desired benzaldehyde. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.05 (s, 1H), 8.21 (t, 1H), 7.75 (d, 1H), 7.72
(s, 1H), 7.68 (d, 1H), 7.52-7.16 (m, 8H). MS (ESI) m/z: Calculated:
348.06. Observed: 349.1 (M.sup.++1).
1-(3-Fluoro-4-(5-phenylthio)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (step 6 in Scheme A-4)
##STR00228##
[0645] A mixture of
3-Fluoro-4-(5-(phenylthio)benzofuran-2-yl)benzaldehyde (350 mg,
1.04 mmol), azetidine-3-carboxylic acid (120 mg, 0.28 mmol) and
acetic acid (0.12 mL, 2.0 mmol) in CH.sub.2Cl.sub.2 (10 mL) was
stirred at room temperature for 1 hour. Sodium
triacetoxyborohydride (340 mg, 1.6 mmol) was added in two portions
and the reaction mixture was stirred for 16 h. Concentration of the
solvent under reduced pressure yielded a yellow solid that was
dissolved in DMSO (3 mL) and filtered to give a yellow solution
that was purified by HPLC to yield the desired product [hS1P1
EC.sub.50=142 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.17
(t, 1H), 7.78 (s, 1H), 7.59 (d, 1H), 7.47-7.39 (m, 3H), 7.36-7.17
(m, 6H), 4.46 (s, 2H), 4.39 (m, 4H), 3.74 (m, 1H). MS (ESI) m/z:
Calculated: 433.11. Observed: 433.9 (M.sup.++1).
Compound 65
1-(3-Fluoro-4-(5-phenylsulfinyl)benzofuran-2-yl)benzyl)azetidine-3-carboxy-
lic acid (step 7 in Scheme A-4)
##STR00229##
[0647] A mixture of
1-(3-Fluoro-4-(5-phenylthio)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (50 mg, 0.12 mmol) and m-chloroperbenzoic acid (35 mg, 0.12
mmol) in Chloroform (9 mL) and MeOH (1 mL) was stirred at
-20.degree. C. for 1 hour. Concentration of the solvent under
reduced pressure yielded a white solid that was dissolved in DMSO
(3 mL) and filtered to give a yellow solution that was purified by
HPLC to yield the desired product [hS1P1 EC.sub.50=67 nM]. .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 8.17 (t, 1H), 7.96 (s, 1H), 7.94
(d, 1H), 7.75-7.43 (m, 9H), 4.45 (s, 2H), 4.39 (m, 4H), 3.74 (m,
1H). MS (ESI) m/z: Calculated: 449.11. Observed: 449.9
(M.sup.++1).
Compound 66
1-(4-(5-(Cyclopropylmethoxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidi-
ne-3-carboxylic acid
5-((Cyclopropylmethoxy)methyl)benzofuran (step 1 in Scheme
A-3-a)
##STR00230##
[0649] A mixture of benzofuran-5-ylmethanol (1.0 g, 6.8 mmol), and
sodium hydride (0.25 g, 10.0 mmol) in DMF (20 mL) was stirred at
0.degree. C. for 30 minutes. Cyclopropylmethyl bromide (0.97 g, 7.0
mmol) was the added and resultant reaction mixture was slowly
warmed to room temperature and left stirring over night. Reaction
mixture was diluted to 100 mL with water. The solution was
extracted with Et.sub.2O (20 mL.times.3); the combined extracts
were washed with 1N NaOH solution, water and brine, dried, and
concentrated under reduced pressure to yield the desired product
after purification on silica (4/1 hexane/ethyl acetate). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.61 (d, 2H), 7.44 (d, 1H), 7.27
(d, 1H), 6.76 (s, 1H), 4.62 (s, 2H) 3.37 (d, 2H), 1.41 (m, 1H),
0.58 (m, 2H), 0.21 (m, 2H).
5-((Cyclopropylmethoxy)methyl)benzofuran-2-ylboronic acid (step 2
in Scheme A-3-a)
##STR00231##
[0651] The title compound was prepared as Example Compound A (step
3) in the general method described above and used without further
purification for the next step: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.72 (m, 1H), 7.52 (m, 1H), 7.32 (m, 1H), 7.21 (m, 1H),
4.62 (s, 2H) 3.37 (d, 2H), 1.41 (m, 1H), 0.58 (m, 2H), 0.21 (m,
2H).
4-(5-(Cyclopropylmethoxy)methyl)benzofuran-2-yl)-3-fluorobenzaldehyde
(step 3 in Scheme A-3-a)
##STR00232##
[0653] The title compound was prepared as Example Compound A (step
4) in the general method described above. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 10.02 (s, 1H), 8.24 (t, 1H), 7.78 (d, 1H),
7.69 (d, 1H), 7.63 (s, 1H), 7.54 (d, 1H), 7.36 (m, 2H), 4.62 (s,
2H) 3.38 (d, 2H), 1.41 (m, 1H), 0.58 (m, 2H), 0.21 (m, 2H).
1-(4-(5-(Cyclopropylmethoxy)methyl)benzofuran-2-yl)-3-fluorobenzyl)azetidi-
ne-3-carboxylic acid (step 4 in Scheme A-3-a)
##STR00233##
[0655] The title compound was prepared as Example Compound A (step
5) in the general method described above. Compound was isolated as
free base via triturating [hS1P1 EC.sub.50=75 nM]. .sup.1H NMR (400
MHz, DMSO-d.sub.6): 7.79 (t, 1H), 7.43 (m, 2H), 7.18 (m, 4H), 4.41
(s, 2H), 3.35 (m, 4H), 3.14 (d, 2H), 3.13 (m, 1H), 0.93 (m, 1H),
0.38 (m, 2H), 0.05 (m, 2H). MS (ESI) m/z: Calculated: 409.17.
Observed: 409.9 (M.sup.++1).
Compound 67
1-(4-(5-(Cyclobutoxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-ca-
rboxylic acid
5-(Cyclobutoxymethyl)benzofuran (step 1 in Scheme A-3-b)
##STR00234##
[0657] A mixture of cyclobutanol (1.0 mL, 14.0 mmol) and sodium
hydride (0.5 g, 14.0 mmol) in DMF (20 mL) was stirred at 0.degree.
C. for 30 minutes. 5-(Bromomethyl)benzofuran (2.9 g, 14.0 mmol) was
the added and resultant reaction mixture was slowly warmed to room
temperature and left stirring over night. Reaction mixture was
diluted to 100 mL with water. The solution was extracted with
Et.sub.2O (20 mL.times.3); the combined extracts were washed with
1N NaOH solution, water and brine, dried, and concentrated under
reduced pressure to yield the desired product after purification on
silica (4/1 hexane/ethyl acetate). NMR (400 MHz, CDCl.sub.3)
.delta. 7.58 (d, 2H), 7.42 (d, 1H), 7.27 (d, 1H), 6.78 (s, 1H),
4.54 (s, 2H) 4.03 (m, 1H), 2.21 (m, 2H), 1.94 (m, 2H), 1.76 (m,
1H), 1.52 (m, 1H).
5-(Cyclobutoxymethyl)benzofuran-2-ylboronic acid (step 2 in Scheme
A-3-b)
##STR00235##
[0659] The title compound was prepared as Example Compound A (step
3) in the general method C described above and used without
purification for the next step: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.62 (m, 1H), 7.42 (m, 1H), 7.32 (m, 1H), 7.21 (m, 1H),
4.48 (s, 2H), 4.03 (m, 1H), 2.24 (m, 2H), 2.02 (m, 2H), 1.76 (m,
1H), 1.56 (m, 1H).
4-(5-(Cyclobutoxymethyl)benzofuran-2-yl)-3-fluorobenzaldehyde (step
3 in Scheme A-3-b)
##STR00236##
[0661] The title compound was prepared as Example Compound A (step
4) in the general method D described above (67% yield in two
steps): .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 10.01 (s, 1H),
8.21 (t, 1H), 7.76 (d, 1H), 7.63 (d, 1H), 7.61 (s, 1H), 7.46 (d,
1H), 7.39 (m, 2H), 4.51 (s, 2H) 4.04 (m, 1H), 2.22 (m, 2H), 2.01
(m, 2H), 1.74 (m, 1H), 1.53 (m, 1H).
1-(4-(5-(Cyclobutoxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-ca-
rboxylic acid (step 4 in Scheme A-3-b)
##STR00237##
[0663] The title compound was prepared as Example Compound A (step
5) in the general method E described above [hS1P1 EC.sub.50=25 nM].
The product was isolated as free base via triturating: .sup.1H NMR
(400 MHz, DMSO-d.sub.6): 7.91 (t, 1H), 7.61 (m, 2H), 7.23 (m, 4H),
4.42 (s, 2H), 3.94 (m, 1H), 3.61 (s, 2H), 3.37 (m, 1H), 3.35 (m,
4H), 2.18 (m, 2H), 1.82 (m, 2H), 1.61 (m, 1H), 1.45 (m, 1H). MS
(ESI) m/z: Calculated: 409.17. Observed: 410.0 (M.sup.++1).
Compound 68
1-(4-(benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-carboxylic
acid
4-(benzofuran-2-yl)-3-fluorobenzaldehyde (step 4 in Scheme A-1)
##STR00238##
[0665] The title compound was prepared as Example Compound A (step
4) in the general method D described above (42% yield): .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 10.01 (s, 1H), 8.22 (t, 1H),
7.79-7.17 (m, 7H).
1-(4-(benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-carboxylic acid
(step 5 in Scheme A-1)
##STR00239##
[0667] The title compound was prepared as Example Compound A (step
5) in the general method E described above. The product [hS1P1
EC.sub.50=706 nM] was isolated as free base via triturating:
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 7.96 (t, 1H), 7.74 (d,
1H), 7.62 (d, 1H), 7.39-7.28 (m, 5H), 4.34 (s, 2H), 4.16 (m, 4H),
3.49 (m, 1H). MS (ESI) m/z: Calculated: 325.11. Observed: 326.0
(M.sup.++1).
Compound 69
1-(3-Fluoro-4-(5-(phenoxymethyl)benzofuran-2-yl)benzyl)azetidine-3-carboxy-
lic acid
5-(Phenoxymethyl)benzofuran (step 1 in Scheme A-3-b)
##STR00240##
[0669] The title compound was prepared as Example Compound 5 (step
1): .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.63 (d, 2H), 7.47
(d, 1H), 7.29 (d, 1H), 7.27 (m, 2H), 6.99 (m, 3H), 6.79 (s, 1H),
5.18 (s, 2H).
5-(Phenoxymethyl)benzofuran-2-ylboronic acid (step 2 in Scheme
A-3-b)
##STR00241##
[0671] The title compound was prepared as Example Compound A (step
3) in the general method C described above and used without further
purification for the next step: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.73 (m, 2H), 7.64 (m, 1H), 7.49 (m, 3H), 6.99 (m, 3H),
5.16 (s, 2H).
3-Fluoro-4-(5-(phenoxymethyl)benzofuran-2-yl)benzaldehyde (step 3
in Scheme A-3-b)
##STR00242##
[0673] The title compound was prepared as Example Compound A (step
4) in the general method D described above. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.01 (s, 1H), 8.22 (t, 1H), 7.78 (d, 1H), 7.75
(s, 1H), 7.69 (d, 1H), 7.58 (d, 1H), 7.42-7.27 (m, 2H), 7.02 (m,
5H), 5.18 (s, 2H).
1-(3-Fluoro-4-(5-(phenoxymethyl)benzofuran-2-yl)benzyl)azetidine-3-carboxy-
lic acid (step 4 in Scheme A-3-b)
##STR00243##
[0675] The title compound was prepared as Example Compound A (step
5) in the general method described above. Compound [hS1P1
EC.sub.50=87 nM] was isolated as free base via triturating: .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.87 (t, 1H), 7.78 (s, 1H),
7.63 (d, 1H), 7.43 (d, 1H), 7.39-7.26 (m, 5H), 7.06 (m, 2H), 6.97
(t, 1H), 5.18 (s, 2H), 4.46 (s, 2H), 4.39 (m, 4H), 3.74 (m, 1H). MS
(ESI) m/z: Calculated: 431.15. Observed: 431.9 (M.sup.++1).
Compound 70
1-(4-(5-(Cyclohexyloxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3--
carboxylic acid
5-(Cyclohexyloxymethyl)benzofuran (step 1 in Scheme A-3-b)
##STR00244##
[0677] The title compound was prepared as Example Compound 5 (step
1): .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.61 (d, 2H), 7.48
(d, 1H), 7.35 (d, 1H), 6.78 (s, 1H), 4.62 (s, 2H) 3.41 (m, 1H),
1.99 (m, 2H), 1.78 (m, 2H), 1.58 (m, 1H), 1.41 (m, 2H), 1.23 (m,
3H).
5-(Cyclohexyloxymethyl)benzofuran-2-ylboronic acid (step 2 in
Scheme A-3-b)
##STR00245##
[0679] The title compound was prepared as Example Compound A (step
3) in the general method C described above and used as crude for
the next step: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.71 (m,
2H), 7.58 (m, 1H), 7.47 (m, 1H), 4.63 (s, 2H) 3.42 (m, 1H), 1.99
(m, 2H), 1.78 (m, 2H), 1.58 (m, 1H), 1.41 (m, 3H), 0.95 (m,
3H).
4-(5-(Cyclohexyloxymethyl)benzofuran-2-yl)-3-fluorobenzaldehyde
(step 3 in Scheme A-3-b)
##STR00246##
[0681] The title compound was prepared as Example Compound A (step
4) in the general method D described above. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 10.01 (s, 1H), 8.21 (t, 1H), 7.78 (d, 1H),
7.64 (d, 1H), 7.61 (s, 1H), 7.54 (d, 1H), 7.39 (m, 2H), 4.63 (s,
2H) 3.39 (m, 1H), 1.98 (m, 2H), 1.78 (m, 2H), 1.34 (m, 2H), 1.21
(m, 4H).
1-(4-(5-(Cyclohexyloxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3--
carboxylic acid (step 4 in Scheme A-3-b)
##STR00247##
[0683] The title compound was prepared as Example Compound A (step
5) in the general method E described above. The product [hS1P1
EC.sub.50=91 nM] was isolated as free base via triturating: NMR
(400 MHz, DMSO-d.sub.6): 7.84 (t, 1H), 7.62 (s, 1H), 7.58 (d, 1H),
7.24 (m, 4H), 4.58 (s, 2H), 3.63 (m, 1H), 3.61 (s, 2H), 3.37 (m,
1H), 3.35 (m, 4H), 1.78 (m, 2H), 1.61 (m, 2H) 1.41 (m, 1H), 1.18
(m, 5H). MS (ESI) m/z: Calculated: 437.20. Observed: 437.9
(M.sup.++1).
Compound 71
1-(4-(5-(Cyclopentyloxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-
-carboxylic acid
5-(Cyclopentyloxymethyl)benzofuran (step 1 in Scheme A-3-b)
##STR00248##
[0685] The title compound was prepared as Example Compound 5 (step
1). NMR (400 MHz, CDCl.sub.3) .delta. 7.57 (d, 2H), 7.45 (d, 1H),
7.27 (d, 1H), 6.77 (s, 1H), 4.53 (s, 2H), 4.01 (m, 1H), 1.76 (m,
4H), 1.57 (m, 2H), 1.24 (m, 1H), 0.94 (m, 1H).
5-(Cyclopentyloxymethyl)benzofuran-2-ylboronic acid (step 2 in
Scheme B-3-b)
##STR00249##
[0687] The title compound was prepared as Example Compound A (step
3) in the general method C described above and used without further
purification for the next step: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.72 (m, 1H), 7.48 (m, 1H), 7.38 (m, 1H), 7.21 (m, 1H),
4.58 (s, 2H), 4.03 (m, 1H), 1.78 (m, 4H), 1.56 (m, 2H), 1.24 (m,
1H), 0.96 (m, 1H).
4-(5-(Cyclopentyloxymethyl)benzofuran-2-yl)-3-fluorobenzaldehyde
(step 3 in Scheme A-3-b)
##STR00250##
[0689] The title compound was prepared as Example Compound A (step
4) in the general method D described above. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 10.01 (s, 1H), 8.23 (t, 1H), 7.86 (d, 1H),
7.66 (d, 1H), 7.61 (s, 1H), 7.52 (d, 1H), 7.39 (m, 2H), 4.73 (s,
2H) 4.03 (m, 1H), 1.78 (m, 4H), 1.61 (m, 2H), 1.21 (m, 1H), 0.95
(m, 1H).
1-(4-(5-(Cyclopentyloxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-
-carboxylic acid (step 4 in Scheme A-3)
##STR00251##
[0691] The title compound was prepared as Example Compound A (step
5) in the general method E described above. .sup.1H NMR (400 MHz,
CD.sub.3OD): 8.14 (t, 1H), 7.48 (s, 1H), 7.52 (d, 1H), 7.41 (d,
2H), 7.37 (m, 2H), 4.58 (s, 2H), 4.43 (s, 2H), 4.39 (m, 4H), 4.04
(m, 1H), 3.63 (m, 1H), 1.78 (m, 4H), 1.58 (m, 2H) 1.21 (m, 2H). MS
(ESI) m/z: Calculated: 423.18. Observed: 424.0 (M.sup.++1).
Compound 72
1-(3-Fluoro-4-(5-phenylsulfonyl)benzofuran-2-yl)benzyl)azetidine-3-carboxy-
lic acid
(3-Fluoro-4-(5-phenylsulfonyl)benzofuran-2-yl)phenyl)methanol (step
1 in Scheme A-5)
##STR00252##
[0693] A mixture of
1-(3-Fluoro-4-(5-phenylthio)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid (50 mg, 0.12 mmol) and m-chloroperbenzoic acid (100 mg, 0.36
mmol) in chloroform (10 mL) and MeOH (1 mL) was stirred at
-20.degree. C. for 1 h and slowly warmed to room temperature
overnight. Concentration of the solvent under reduced pressure
yielded a white solid that was purified by chromatography (1/9
ethyl acetate/hexanes) to yield the desired product: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.22 (s, 1H), 8.03 (dd, 2H), 7.71 (d,
1H), 7.61 (d, 1H), 7.58-7.43 (m, 3H), 7.22 (m, 4H), 4.77 (s, 2H).
MS (ESI) m/z: Calculated: 382.07. Observed: 383.0 (M.sup.++1).
3-Fluoro-4-(5-phenylsulfonyl)benzofuran-2-yl)benzaldehyde (step 2
in Scheme A-5)
##STR00253##
[0695] A suspension of
(3-Fluoro-4-(5-phenylsulfonyl)benzofuran-2-yl)phenyl)methanol (46
mg, 0.12 mmol), Molecular sieves 4 A (0.25 g), TPAP (0.0021 mg,
0.06 mmol) and N-morpholino oxide (0.029 g, 0.24 mmol) in
acetonitrile was stirred for 1 hour and then filtered through
celite to yield the title compound: NMR (400 MHz, CDCl.sub.3)
.delta. 10.02 (s, 1H), 8.36 (s, 1H), 8.20 (t, 1H), 8.01 (dd, 2H),
7.79 (d, 1H), 7.71 (d, 1H), 7.62 (d, 1H), 7.56 (m, 5H), 4.77 (s,
2H).
1-(3-Fluoro-4-(5-phenylsulfonyl)benzofuran-2-yl)benzyl)azetidine-3-carboxy-
lic acid (step 3 in Scheme A-5)
##STR00254##
[0697] A mixture of
3-Fluoro-4-(5-(phenylthio)benzofuran-2-yl)benzaldehyde (49 mg, 0.14
mmol) and azetidine-3-carboxylic acid (30 mg, 0.28 mmol) in MeOH (1
mL) was stirred at room temperature for 1 hour. Sodium
cyanoborohydride (60 mg, 0.28 mmol) was added in two portions and
the reaction mixture was stirred for 16 h. Concentration of the
solvent under reduced pressure yielded a yellow solid that was
dissolved in DMSO (3 mL) and filtered to give a yellow solution
that was purified by HPLC to yield 11.5 mg (22%) of the desired
product: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.39 (s, 1H),
8.16 (t, 1H), 8.01 (d, 2H), 7.96 (d, 1H), 7.76 (d, 1H), 7.62-7.41
(m, 6H), 4.46 (s, 2H), 4.38 (m, 4H), 3.64 (m, 1H). MS (ESI) m/z:
Calculated: 465.10. Observed: 465.9 (M.sup.++1).
Compound 73
1-(3-fluoro-4-(5-(4-fluorobenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
5-(4-fluorobenzyl)benzofuran
##STR00255##
[0699] The title compound was prepared as Compound B (step 1 in
Scheme A-2) in the general method A described above (85% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.59 (d, J=2.3, 1H), 7.42
(d, J=8.5, 1H), 7.37 (br s, 1H), 7.17-7.05 (m, 3H), 7.00-6.93 (m,
2H), 6.70 (dd, J=2.2, 0.7, 1H), 4.04 (s, 2H).
5-(4-fluorobenzyl)benzofuran-2-ylboronic acid
##STR00256##
[0701] The title compound was prepared as Compound B (step 2 in
Scheme A-2) in the general method C described above (64% yield):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.41 (m, 2H), 7.30
(br s, 1H), 7.18-7.13 (m, 3H), 6.99-6.95 (m, 2H), 4.04 (s, 2H).
3-fluoro-4-(5-(4-fluorobenzyl)benzofuran-2-yl)benzaldehyde
##STR00257##
[0703] The title compound was prepared as Compound B (step 3 in
Scheme A-2) in the general method D described above. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 10.00 (d, J=1.8, 1H), 8.20 (t, J=7.7,
1H), 7.77 (dd, J=8.0, 1.5, 1H), 7.67 (dd, J=11.4, 1.5, 1H),
7.48-7.43 (m, 2H), 7.36 (dd, J=3.6, 0.8, 1H), 7.19-7.15 (m, 3H),
7.00-6.96 (m, 2H), 4.06 (s, 2H).
1-(4-(5-Benzylbenzofuran-2-yl)-3-chlorobenzyl)azetidine-3-carboxylic
acid
##STR00258##
[0705] The title compound was prepared as Compound B (step 4 in
Scheme A-2) in the general method E described above [hS1P1
EC.sub.50=53 nM]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.12 (d,
J=8.0, 1H), 7.49-7.40 (m, 4H), 7.29-7.20 (m, 4H), 7.02-6.97 (m,
2H), 4.47 (br s, 2H), 4.41-4.33 (m, 4H), 4.06 (s, 2H), 3.75-3.66
(m, 1H). MS (ESI) m/z: Calculated: 433.15. Observed: 434.0
(M.sup.++1).
Compound 74
1-(4-(5-(Cyclohexylmethyl)benzofuran-2-yl)-3-fluorobenzyl)azetidine-3-carb-
oxylic acid
5-(Cyclohexylmethyl)benzofuran
##STR00259##
[0707] The title compound was prepared as Compound B (step 1 in
Scheme A-2) in the general method A described above. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.58 (d, J=2.2, 1H), 7.39 (d, J=8.4,
1H), 7.34 (br s, 1H), 7.07 (dd, J=8.2, 1.5, 1H), 6.70 (dd, J=2.2,
0.8, 1H), 2.57 (d, J=7.3, 2H), 1.70-1.60 (m, 5H), 1.58-1.48 (m,
1H), 1.22-1.15 (m, 3H), 1.00-0.94 (m, 2H).
5-(4-Fluorobenzyl)benzofuran-2-ylboronic acid
##STR00260##
[0709] The title compound was prepared as Compound B (step 2 in
Scheme A-2) in the general method C described above. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.41-7.39 (m, 2H), 7.31 (s, 1H), 7.14
(dd, J=8.1, 1.5, 1H), 2.57 (d, J=7.9, 2H), 1.70-1.52 (m, 6H),
1.25-0.94 (m, 5H)
3-Fluoro-4-(5-(4-fluorobenzyl)benzofuran-2-yl)benzaldehyde
##STR00261##
[0711] The title compound was prepared as Compound B (step 3 in
Scheme A-2) in the general method D described above. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 10.00 (d, J=1.8, 1H), 8.20 (t, J=7.3,
1H), 7.77 (dd, J=8.0, 1.4, 1H), 7.67 (dd, J=11.2, 1.6, 1H),
7.45-7.37 (m, 3H), 7.15 (dd, J=8.4, 1.5, 1H), 2.58 (d, J=6.9, 2H),
1.71-1.50 (m, 6H), 1.26-0.94 (m, 5H).
1-(4-(5-Benzylbenzofuran-2-yl)-3-chlorobenzyl)azetidine-3-carboxylic
acid
##STR00262##
[0713] The title compound was prepared as Compound B (step 4 in
Scheme A-2) in the general method E described above [hS1P1
EC.sub.50=400 nM]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.12
(d, J=8.0, 1H), 7.46-7.40 (m, 4H), 7.27 (d, J=3.5, 1H), 7.16 (br d,
J=10.2, 1H), 4.46 (br s, 2H), 4.36-4.34 (m, 4H), 3.71-3.63 (m, 1H),
2.58 (d, J=7.1, 2H), 1.76-1.55 (m, 6), 1.30-1.06 (m, 5H). MS (ESI)
m/z: Calculated: 421.21. Observed: 422.0 (M.sup.++1).
Compound 75
1-(3-Fluoro-4-(5-(2-fluorobenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
5-(2-Fluorobenzyl)benzofuran
##STR00263##
[0715] The title compound was prepared as Example Compound B (step
1 in Scheme A-2) in the general method A described above. .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 7.68 (s, 1H), 7.40 (d, J=10.4 Hz,
1H), 7.38 (d, J=8.0 Hz, 1H), 6.97-7.22 (m, 5H), 6.74 (S, 1H), 4.06
(s, 2H).
5-(2-Fluorobenzyl)benzofuran-2-ylboronic acid
##STR00264##
[0717] The title compound was prepared as Example Compound B (step
2 in Scheme A-2) in the general method C described above. The
compound was used for the next step without further
purification.
3-Fluoro-4-(5-(2-fluorobenzyl)benzofuran-2-yl)benzaldehyde
##STR00265##
[0719] The title compound was prepared as Example Compound B (step
3 in Scheme A-2) in the general method D described above. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 9.99 (s, 1H), 8.19 (t, J=7.2 Hz,
1H), 7.76 (d, J=8.0 Hz, 1H), 7.67 (d, J=11.2 Hz, 1H), 7.47 (d,
J=9.2 Hz, 2H), 7.35 (d, J=3.6 Hz, 1H), 7.25 (m, 3H), 7.26 (m, 2H),
4.10 (s, 2H).
1-(3-Fluoro-4-(5-(2-fluorobenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
##STR00266##
[0721] The title compound was prepared as Example Compound B (step
4 in Scheme A-2) in the general method E described above [hS1P1
EC.sub.50=129 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.12
(dd, J=7.6, 8.4 Hz, 1H), 7.49 (m, 2H), 7.43 (m, 2H), 7.24 (m, 4H),
7.07 (m, 2H), 4.47 (s, 2H), 4.37 (m, 4H), 4.10 (s, 2H), 3.71 (m,
1H). .sup.19F NMR (376 MHz, CD.sub.3OD) .delta. -77.6 (TFA),
-113.1, -120.6. MS (ESI) m/z: Calculated: 433.15. Observed: 433.9
(M.sup.++1).
Compound 76
1-(3-Fluoro-4-(5-(3-fluorobenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
5-(3-Fluorobenzyl)benzofuran
##STR00267##
[0723] The title compound was prepared as Example Compound B (step
1 in Scheme A-2) in the general method A described above. .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 7.68 (s, 1H), 7.41 (d, J=6.8 Hz,
1H), 7.40 (d, J=8.4 Hz, 1H), 7.26 (m, 1H), 7.13 (d, J=8.0 Hz, 1H),
7.02 (d, J=8.0 Hz, 1H), 6.89 (m, 2H), 6.76 (m, 1H), 4.04 (s,
2H).
5-(3-Fluorobenzyl)benzofuran-2-ylboronic acid
##STR00268##
[0725] The title compound was prepared as Example Compound B (step
2 in Scheme A-2) in the general method C described above. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.53 (s, 1H), 7.48 (d, J=8.4
Hz, 1H), 7.38 (s, 1H), 7.31 (m, 1H), 7.21 (d, J=5.2 Hz, 1H), 7.20
(m, 2H), 6.98 (m, 1H), 4.04 (s, 2H).
3-Fluoro-4-(5-(3-fluorobenzyl)benzofuran-2-yl)benzaldehyde
##STR00269##
[0727] The title compound was prepared as Example Compound B (step
3 in Scheme A-2) in the general method D described above. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 10.03 (s, 1H), 8.20 (t, J=7.6 Hz,
1H), 7.79 (dd, J=8.4, 1.2 Hz, 1H), 7.69 (dd, J=11.6, 1.2 Hz, 1H),
7.49 (d, J=8.4 Hz, 1H), 7.47 (d, J=6.0 Hz, 1H), 7.37 (d, J=3.6 Hz,
1H), 7.26 (m, 1H), 7.21 (dd, J=8.8, 1.2 Hz, 1H), 7.01 (d, J=8.4 Hz,
1H), 6.92 (m, 2H), 4.08 (s, 2H).
1-(3-Fluoro-4-(5-(3-fluorobenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
##STR00270##
[0729] The title compound was prepared as Example Compound B (step
4 in Scheme A-2) in the general method E described above [hS1P1
EC.sub.50=169 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.10
(t, J=8.0 Hz, 1H), 7.49 (dd, J=9.6, 9.6 Hz, 2H), 7.43 (dd, J=8.0,
11.2 Hz, 1H), 7.40 (dd, J=3.2, 10.8 Hz, 1H), 7.28 (m, 3H), 7.06 (d,
J=7.6 Hz, 1H), 6.92 (m, 2H), 4.45 (s, 2H), 4.34 (m, 4H), 4.08 (s,
2H), 3.68 (m, 1H). .sup.19F NMR (376 MHz, CD.sub.3OD) .delta. -77.4
(TFA), -113.1, -116.2. MS (ESI) m/z: Calculated: 433.15. Observed:
433.9 (M.sup.++1).
Compound 77
1-(3-Fluoro-445-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
methyl
1-(3-fluoro-4-(5-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxyl-
ate
##STR00271##
[0731] The title compound was prepared as Example Compound A (step
5 in Scheme A-1) in the general method D described above except
using methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (54%
yield). The product was isolated as a free base via triturating: MS
(ESI) m/z: Calculated: 431.15. Observed: 432.00 (M.sup.++1).
1-(3-Fluoro-4-(5-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
##STR00272##
[0733] To a solution of ester (110 mg, 0.25 mmol) in 2 mL THF was
added lithium hydroxide hydrate (30 mg, 1.25 mmol) in 1 mL water.
The mixture was stirred until completion. The solvents were
removed, and the solid was suspended in 2 mL water. 3 equivalents
2N HCl was then added to neutralize the base, and the mixture was
sonicated. 4 mL 1 M pH 6 phosphate buffer was added and the
reaction was sonicated. The slurry was filtered and the solid
rinsed with water and EtOH and dried in vacuo to give the desired
product as the white solid [hS1P1 EC.sub.50=13 nM] .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 7.89 (s, 1H), 7.65 (s, 1H), 7.30 (m,
6H), 6.97 (m, 4H), 3.58 (m, 3H), 3.40 (m, 4H). .sup.19F NMR (376
MHz, DMSO-d.sub.6) .delta. -113.7. MS (ESI) m/z: Calculated:
417.14. Observed: 417.90 (M.sup.++1).
Compound 78
5-(1-(4-(5-Benzylbenzofuran-2-yl)-3-fluorobenzyl)azetidin-3-yl)-2H-tetrazo-
le
5-(1-Benzhydrylazetidin-3-yl)-2H-tetrazole (step 1 in Scheme
A-8)
##STR00273##
[0735] 1-Benzhydrylazetidine-3-carbonitrile (2.48 g, 10 mmol),
trimethylsilyl azide (2.30 g, 20 mmol) and dibutyltin oxide (498
mg, 2.0 mmol) were dissolved in toluene (20 mL) and heated to
reflux for 32 h. The reaction mixture was then cooled to room
temperature and applied directly to ISCO system with 10% methanol
in dichloromethane to give the product. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.54 (m, 4H), 7.31 (m, 6H), 5.11 (s, 1H), 4.47
(m, 1H), 4.05 (t, J=7.2 Hz, 2H), 3.91 (t, J=7.2 Hz, 2H). MS (ESI)
m/z: Calculated: 291.15. Observed: 291.90 (M.sup.++1).
5-(Azetidin-3-yl)-2H-tetrazole hydrochloride (step 2 in Scheme
A-8)
##STR00274##
[0737] To a solution of (500 mg, 1.72 mmol) in MeOH (5 mL) and
EtOAc (5 mL) was added 1N HCl in Et.sub.2O (0.5 mL) and 10%
palladium on carbon (500 mg). The mixture was stirred under
hydrogen atmosphere for 72 h. The catalyst was removed by
filtration and the solvent was removed under reduced pressure to
give the crude compound which was used for the next step without
further purification. MS (ESI) m/z: Calculated: 125.13. Observed:
126.2 (M.sup.++1).
5-(1-(4-(5-Benzylbenzofuran-2-yl)-3-fluorobenzyl)azetidin-3-yl)-2H-tetrazo-
le
##STR00275##
[0739] The title compound was prepared as Example Compound A (step
5 in Scheme A-1) in the general method E described above except
using 5-(azetidin-3-yl)-2H-tetrazole hydrochloride [hS1P1
EC.sub.50=2090 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.13
(t, J=8.8 Hz, 1H), 7.48 (m, 4H), 7.25 (m, 7H), 4.63 (m, 4H), 4.51
(m, 3H), 4.07 (s, 2H). .sup.19F NMR (376 MHz, CD.sub.3OD) .delta.
-77.4 (TFA), -113.1. MS (ESI) m/z: Calculated: 439.18. Observed:
440.00 (M.sup.++1).
Compound 80
1-(4-(2-Benzylbenzofuran-5-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
(5-Bromobenzofuran-2-yl)(phenyl)methanone (step 1 in Scheme
A-6)
##STR00276##
[0741] 5-Bromosalicyaldehyde (2.01 g, 10 mmole), cesium carbonate
(3.26 g, 10 mmole) and acetonitrile (100 mL) were combined and
heated to reflex for 30 minutes. The mixture was cooled to
0.degree. C. and a solution of 2-bromo-1-phenylethanone (1.99 g, 10
mmole) in acetonitrile (20 mL) was added. The cooling bath was
removed, the mixture stirred at room temperature for 5 h and the
precipitate collected the desired product as a white solid: NMR
(400 MHz, DMSO-d.sub.6) .delta. 8.07 (d, J=2.0 Hz, 1H), 7.98 (d,
J=7.6 Hz, 2H), 7.68-7.78 (m, 4H), 7.60 (dd, J=7.6, 8.0 Hz, 2H).
2-Benzyl-5-bromobenzofuran (step 2 in Scheme A-6)
##STR00277##
[0743] (5-bromobenzofuran-2-yl)(phenyl)methanone (2.0 g, 6.6 mmol),
sodium cyanoborohydride (3.2 g, 51 mmol), zinc iodide (2.6 g, 13.2
mmol) and 1,2-dichloroethane (50 mL) were combined and heated to
reflux for overnight. The mixture was cooled, quenched with
saturated ammonium chloride, acidified with concentrated HCl and
stirred for 30 minutes. The layers were separated, the aqueous
layer was extracted with dichloromethane (2.times.100 mL) and the
combined organic layers were washed with water (50 mL) and brine
(50 mL), dried over sodium sulfate and concentrated. The residue
was purified by silica gel column chromatography (ISCO system, 20%
dichloromethane in hexanes) to give the product as a white solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.59 (s, 1H), 7.27-7.34
(m, 7H), 6.32 (s, 1H), 4.10 (s, 2H).
4-(2-Benzylbenzofuran-5-yl)-3-fluorobenzaldehyde (step 3 in Scheme
A-6)
##STR00278##
[0745] A solution of 2-fluoro-4-formylphenylboronic acid (58 mg,
0.348 mmol), 2-benzyl-5-bromobenzofuran (50 mg, 0.174 mmol),
tetrakis(triphenylphosphine) palladium(0) (20 mg, 0.0174 mmol) and
sodium carbonate (106 mg, 1.0 mmol) in 1,4-dioxane (4 mL) and water
(1 mL) was heated at 100.degree. C. for 12 h. The mixture was
partitioned between brine (20 mL) and EtOAc (20 mL). The organic
layer was dried. Removal of solvents gave the residue which was
purified by silica gel column chromatography (ISCO system, 30%
dichloromethane in hexanes) to give the product as a off white
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.0 (s, 1H),
7.61-7.75 (m, 5H), 7.27-7.51 (m, 7H), 6.45 (s, 1H), 4.14 (s,
2H).
1-(4-(2-Benzylbenzofuran-5-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
##STR00279##
[0747] The title compound was prepared as Example Compound A (step
5 in Scheme A-2) in the general method E described above as a white
TFA salt [hS1P1 EC.sub.50=59 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.66 (s, 1H), 7.61 (t, J=8.8 Hz, 1H), 7.48 (d, J=8.4 Hz,
1H), 7.29-7.39 (m, 7H), 7.25 (m, 1H), 6.50 (s, 1H), 4.45 (s, 2H),
4.35 (m, 4H), 4.13 (s, 2H), 3.71 (m, 1H). .sup.19F NMR (376 MHz,
CD.sub.3OD) .delta. -77.4 (TFA), -118.3. MS (ESI) m/z: Calculated:
415.16. Observed: 416.00 (M.sup.++1).
Compound 81
1-(4-(5-Benzylbenzo[b]thiophen-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
5-Benzylbenzothiophene (step 1 in Scheme A-7)
##STR00280##
[0749] 5-Bromobenzothiophene (2.13 g, 10 mmol) was dissolved in a
THF solution of benzyl zinc(II) bromide (0.5M, 10 mL, 20 mmol) in a
microwave reaction tube. Pd(P.sup.tBu.sub.3).sub.2 (255 mg, 0.5
mmol) was added to this solution. The mixture was purged with
N.sub.2 gas for 3-5 minutes and heated at 100.degree. C. for 30
minutes under microwave irradiation. Upon completion of the
reaction, the reaction mixture was diluted with ethyl acetate (150
mL), washed with 1N HCl aqueous solution, brine, filtered through
Celite. The filtrate was dried over Na.sub.2SO.sub.4 and
concentrated. The residue was purified by silica gel column
chromatography (ISCO system, 5% EtOAc in hexanes) to give the
desired product (65% yield): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.79 (d, J=8.4 Hz, 1H), 7.63 (s, 1H), 7.40 (d, J=5.6 Hz,
1H), 7.18-7.30 (m, 7H), 4.10 (s, 2H).
4-(5-Benzylbenzo[b]thiophen-2-yl)-3-fluorobenzaldehyde (step 2 in
Scheme A-7)
##STR00281##
[0751] A mixture of 4-bromo-3-fluorobenzaldehyde (40 mg, 0.198
mmol), 5-benzylbenzothiophene (44 mg, 0.198 mmol), potassium
acetate (5 mg, 0.05 mmol), tetrakis(triphenylphosphine)
palladium(0) (11 mg, 0.010 mmol) and N,N-dimethylacetamide (5 mL)
was heated at 150.degree. C. for 12 h. The solvent was evaporated
in vacuo and the residue was triturated with water (50 mL) and
extracted with dichloromethane (2.times.100 mL). The organic phase
was dried over sodium sulfate, filtered and concentrated. The
residue was purified by silica gel column chromatography (ISCO
system, 5% EtOAc in hexanes) to give the desired product. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 9.90 (s, 1H), 7.64-7.85 (m, 6H),
7.22-7.33 (m, 6H), 4.11 (s, 2H).
1-(4-(5-Benzylbenzo[b]thiophen-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
##STR00282##
[0753] The title compound was prepared as Example Compound A (step
5 in Scheme A-2) in the general method E described above [hS1P1
EC.sub.50=11 nM]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.79
(t, J=8.4 Hz, 1H), 7.69 (m, 2H), 7.59 (s, 1H), 7.30 (m, 2H),
7.06-7.20 (m, 6H), 4.35 (s, 2H), 4.27 (m, 4H), 3.99 (s, 2H), 3.61
(m, 1H). .sup.19F NMR (376 MHz, CD.sub.3OD) .delta. -77.4 (TFA),
-113.5. MS (ESI) m/z: Calculated: 431.14. Observed: 431.90
(M.sup.++1).
Compound 82
3-(4-(5-Benzylbenzofuran-2-yl)-3-fluorobenzylamino)-2-methylpropanoic
acid
3-(4-(5-Benzylbenzofuran-2-yl)-3-fluorobenzylamino)-2-methylpropanoic
acid (Step 1 of Scheme A-13)
##STR00283##
[0755] In a similar manner as described in general procedure E,
3-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-2-methylpropanoic
acid (white solid [hS1P1 EC.sub.50=1300 nM]) was obtained as a TFA
salt by using 4-(5-phenylbenzofuran-2-yl)-3-fluorobenzaldehyde (80
mg, 0.24 mmol), DL-3-aminoisoburic acid (49.9 mg, 0.48 mmol),
sodium cyanoborohydride (30.4 mg, 0.48 mmol), acetic acid (30
methanol (3.5 mL) and dichloromethane (2.5 mL). .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 8.11 (t, J=8.0 Hz, 1H), 7.49-7.43 (m, 4H),
7.29-7.14 (m, 7H), 4.31 (s, 2H), 4.07 (s, 2H), 3.32-3.28 (m, 1H),
3.10 (dd, J=4.6, 12.2 Hz, 1H), 2.88 (m, 1H), 1.29 (d, J=7.2 Hz,
3H); .sup.19F NMR (376 MHz, CD.sub.3OD) .delta. -77.5 (TFA),
-113.5. MS (ESI) m/z: Calculated (without TFA): 417.47. Observed:
417.9 (M.sup.++1).
Compound 83
4-Amino-2-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid
4-Amino-2-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid (Step 1 of Scheme A-13)
##STR00284##
[0757] In a similar manner as described in general procedure E,
4-amino-2-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid (white solid [hS1P1 EC.sub.50=2300 nM]) was obtained as a TFA
salt by using 4-(5-phenylbenzofuran-2-yl)-3-fluorobenzaldehyde
(83.9 mg, 0.25 mmol), DL-asparagine monohydrate (76.3 mg, 0.51
mmol), acetic acid (30 .mu.L), sodium cyanoborohydride (32 mg, 0.51
mmol), methanol (3.5 mL) and dichloromethane (2.5 mL). .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 8.10 (t, J=8.0 Hz, 1H), 7.48-7.44 (m,
4H), 7.29-7.15 (m, 7H), 4.39 (q, J=12.8 Hz, 2H), 4.26 (b, 1H), 4.07
(s, 1H), 3.60 (q, J=6.8 Hz, 1H), 3.13-2.88 (m, 2H); .sup.19F NMR
(376 MHz, CD.sub.3OD) .delta. -77.5 (TFA), -113.5. MS (ESI) m/z:
Calculated (without TFA): 446.47. Observed: 446.9 (M.sup.++1).
Compound 84
1-((6-(5-Benzylbenzofuran-2-yl)-5-fluoropyridin-3-yl)methyl)azetidine-3-ca-
rboxylic acid
5-(Bromomethyl)-2-chloro-3-fluoropyridine (Step 1 of Scheme
A-11)
##STR00285##
[0759] Benzoyl peroxide (100 mg, 41 mmol) was added to a refluxing
mixture of 2-chloro-3-fluoro-5-methylpyridine (5.0 g, 34.35 mmol)
and NBS (6.73 g, 37.79 mmol) in CCl.sub.4 (180 mL). After stirring
the mixture for 15 minutes, an additional amount of benzoyl
peroxide (400 mg, 1.65 mmol) was added in four portions over a
period of 1 hour and the stirring was continued for 1 hour. The
reaction mixture was cooled to room temperature, filtered and the
solid was washed with dichloromethane. The combined filtrates was
washed with water, dried over anhydrous sodium sulfate and
concentrated on a rotary evaporator. The crude product was purified
by silica gel column chromatography (hexane/ethyl acetate) to
afford 5-(bromomethyl)-2-chloro-3-fluoropyridine: .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.23 (d, J=2.0 Hz, 1H), 7.55 (dd, J=2.0,
8.8 Hz, 1H), 4.45 (s, 2H).
6-Chloro-5-fluoronicotinaldehyde (Step 2 of Scheme A-11)
##STR00286##
[0761] A mixture of 5-(bromomethyl)-2-chloro-3-fluoropyridine (2.0
g, 8.91 mmol) and hexamethylenetetramine (2.75 g, 19.6 mmol) in 50%
aqueous acetic acid (53 ml) was heated to reflux. After 1 hour, the
mixture was cooled to room temperature, neutralized carefully with
solid NaHCO.sub.3 (73.7 g), diluted with water (400 mL) and
extracted with dichloromethane (2.times.100 mL). The combined
organic extracts were dried over anhydrous sodium sulfate,
concentrated on a rotary evaporator and purified by silica gel
column chromatography (hexane/ethyl acetate) to afford
6-chloro-5-fluoronicotinaldehyde as pale-yellow solid: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 10.12 (d, J=2.4 Hz, 1H), 8.72 (d,
J=1.6 Hz, 1H), 7.94 (dd, J=2.0, 8.0 Hz, 1H); .sup.19F NMR (376 MHz,
CDCl.sub.3) .delta. -116.3.
1-((6-Chloro-5-fluoropyridin-3-yl)methyl)azetidine-3-carboxylic
acid (Step 3 of Scheme A-11)
##STR00287##
[0763] In a similar manner as described in general procedure E,
1-((6-chloro-5-fluoropyridin-3-yl)methyl)azetidine-3-carboxylic
acid (203 mg, 0.83 mmol, white solid) was prepared by using
6-chloro-5-fluoronicotinaldehyde (284 mg, 1.78 mmol),
azetidine-3-carboxylic acid (184 mg, 1.82 mmol), sodium
cyanoborohydride (112 mg, 1.78 mmol), acetic acid (0.155 mL),
methanol (6 mL) and dichloromethane (6 mL). MS (ESI) m/z:
Calculated (without TFA): 244.65. Observed: 245.1 (M.sup.++1).
1-((6-(5-Benzylbenzofuran-2-yl)-5-fluoropyridin-3-yl)methyl)azetidine-3-ca-
rboxylic acid (Step 4 of Scheme A-11)
##STR00288##
[0765] In a similar manner as described in general procedure D,
1-((6-(5-benzylbenzofuran-2-yl)-5-fluoropyridin-3-yl)methyl)azetidine-3-c-
arboxylic acid was obtained as TFA salt (pale-yellow solid [hS1P1
EC.sub.50=470 nM]) by using 5-benzylbenzofuran-2-ylboronic acid
(272 mg, 1.08 mmol),
1-((6-chloro-5-fluoropyridin-3-yl)methyl)azetidine-3-carboxylic
acid (203 mg, 0.83 mmol), palladium acetate (9.3 mg, 41.5 .mu.mol),
2-(di-t-butylphosphino)biphenyl (24.8 mg, 83 .mu.mol) and THF (15
mL). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.59 (s, 1H), 7.91
(d, J=11.7 Hz, 1H), 7.56-7.51 (m, 3H), 7.30-7.12 (m, 6H), 4.55 (s,
2H), 4.40 (m, 4H), 4.08 (s, 2H), 3.72 (m, 1H); .sup.19F NMR (376
MHz, CD.sub.3OD) .delta. -77.5 (TFA), -120.8. MS (ESI) m/z:
Calculated (without TFA): 416.44. Observed: 416.9 (M.sup.++1).
Compound 85
4-Amino-3-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid
4-Amino-3-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid (Step 1 of Scheme A-13)
##STR00289##
[0767] In a similar manner as described in general procedure E,
4-amino-3-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzylamino)-4-oxobutanoic
acid (white solid [hS1P1 EC.sub.50=1880 nM]) was obtained as a TFA
salt by using 4-(5-phenylbenzofuran-2-yl)-3-fluorobenzaldehyde (80
mg, 0.24 mmol), DL-3,4-diamino-4-oxobutanoic acid (32 mg, 0.48
mmol), acetic acid (30 .mu.L), sodium cyanoborohydride (30.4 mg,
0.48 mmol), methanol (3.5 mL) and dichloromethane (2.5 mL). NMR
(400 MHz, DMSO-d.sub.6) .delta. 7.91 (t, J=8.0 Hz, 1H), 7.62 (s,
1H), 7.54-7.52 (m, 2H), 7.43 (d, J=12.8 Hz, 1H), 7.34 (d, J=7.6 Hz,
1H), 7.29-7.14 (m, 6H), 4.02 (s, 2H), 3.95-3.75 (m, 2H), 3.54 (b,
1H), 2.65-2.40 (m, 2H); .sup.19F NMR (376 MHz, DMSO-d6) .delta.
-77.5 (TFA), -117.4. MS (ESI) m/z: Calculated (without TFA):
446.47. Observed: 446.9 (M.sup.++1).
Compound 86
1-(3-Fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid
5-(4-Methylbenzyl)benzofuran (Step 1 of Scheme A-2)
##STR00290##
[0769] In a similar manner as described in general procedure A,
5-(4-methylbenzyl)benzofuran was obtained as colorless oil by using
(4-methylbenzyl)zinc(II) chloride (14.7 mL of 0.5 M solution in
THF, 7.35 mmol), 5-bromobenzofuran (500 mg, 2.53 mmol) and
Pd(tBu.sub.3).sub.2 (64.8 mg, 0.127 mmol). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.55 (d, J=2.0 Hz, 1H), 7.40-7.37 (m, 2H),
7.12-7.06 (m, 5H), 6.66 (m, 1H), 4.02 (s, 2H), 2.30 (s, 3H).
5-(4-Methylbenzyl)benzofuran-2-ylboronic acid (Step 2 of Scheme
A-2)
##STR00291##
[0771] In a similar manner as described in general procedure C,
5-(4-methylbenzyl)benzofuran (253 mg, 1.138 mmol) was converted to
5-(4-methylbenzyl)benzofuran-2-ylboronic acid as white solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.44-7.38 (m, 2H), 7.30
(s, 1H), 7.19 (d, J=8.4 Hz, 1H), 7.10 (s, 4H), 5.04 (b, 2H), 4.04
(s, 2H), 2.32 (s, 3H).
Methyl
1-(3-fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-
-carboxylate (Step 1 of Scheme B-12)
##STR00292##
[0773] In a similar manner as described in general procedure D,
5-(4-methylbenzyl)-benzofuran-2-ylboronic acid (150 mg, 0.56 mmol)
was reacted with methyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (155 mg, 0.51
mmol) to give methyl
1-(3-fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbo-
xylate as white solid: MS (ESI) m/z: Calculated: 443.51. Observed:
444.0 (M.sup.++1).
1-(3-Fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid (Step 2 of Scheme B-12)
##STR00293##
[0775] In a similar manner as described in general procedure H,
methyl
1-(3-fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbo-
xylate (100 mg, 0.225 mmol) was hydrolyzed to give
1-(3-Fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbo-
xylic acid [hS1P1 EC.sub.50=12 nM] as white foam: .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 7.87 (t, J=8.0 Hz, 1H), 7.52 (d, J=8 Hz,
1H), 7.48 (d, J=1.2 Hz, 1H), 7.25 (d, J=6.8 Hz, 1H), 7.23 (s, 1H),
7.21 (d, J=3.2 Hz, 1H), 7.17 (dd, J=1.2, 8.8 Hz, 1H), 7.11 (d,
J=7.6 Hz, 2H), 7.07 (d, J=8.0 Hz, 2H). 3.96 (s, 2H), 3.58 (s, 2H),
3.39 (m, 2H), 3.18 (m, 3H), 2.22 (s, 3H); .sup.19F NMR (376 MHz,
DMSO-D6) .delta. -113.7. MS (ESI) m/z: Calculated: 429.48.
Observed: 429.9 (M.sup.++1).
Compound 87
1-(4-(5-Benzyl-2H-indazol-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid
5-Bromo-2-nitrobenzaldehyde (Step 1 of Scheme A-10)
##STR00294##
[0777] To concentrate nitric acid (10 mL) in concentrated sulfuric
acid (120 mL) at 5.degree. C. was added 5-bromo-2-nitrobenzaldehyde
(11.71 mL, 100 mmol) dropwise. The reaction mixture was allowed to
warm to room temperature and stirred overnight. The reaction
mixture was poured onto ice and the resulting precipitates removed
by filtration, dissolved in dichloromethane, dried over anhydrous
sodium sulfate and concentrated at reduced pressure. The residue
was purified by silica gel column chromatography (hexane/ethyl
acetate) to give 5-bromo-2-nitrobenzaldehyde: .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.42 (s, 1H), 8.07 (d, J=2.0 Hz, 1H), 8.03 (d,
J=8.6 Hz, 1H), 7.88 (dd, J=2.0, 8.6 Hz, 1H).
(E)-Ethyl 4-(5-bromo-2-nitrobenzylideneamino)-3-fluorobenzoate
(Step 2 of Scheme A-10)
##STR00295##
[0779] A mixture of 5-bromo-2-nitrobenzaldehyde (2.79 g, 12.13
mmol) and ethyl 4-amino-3-fluorobenzoate (2.22 g, 12.12 mmol) in
ethanol (60 mL) was stirred at reflux for 2 h. After the solvent
was removed under reduced pressure, the reaction mixture was
purified by silica gel column chromatography to give (E)-ethyl
4-(5-bromo-2-nitrobenzylideneamino)-3-fluorobenzoate as pale yellow
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.01 (s, 1H), 8.47
(d, J=2.0 Hz, 1H), 8.02 (d, J=8.6 Hz, 1H), 7.91 (dd, J=1.6, 2.3,
9.0 Hz, 1H), 7.85 (dd, J=1.6, 11.0 Hz, 1H), 7.80 (dd, J=2.3, 8.6
Hz, 1H), 7.24 (t, J=8.0 Hz, 1H), 4.40 (q, J=7.0 Hz, 2H), 1.42 (t,
J=7.0 Hz, 3H); .sup.19F NMR (376 MHz, CDCl.sub.3) .delta.
-125.6.
Ethyl 4-(5-bromo-2H-indazol-2-yl)-3-fluorobenzoate (Step 3 of
Scheme A-10)
##STR00296##
[0781] A mixture of (E)-ethyl
4-(5-bromo-2-nitrobenzylideneamino)-3-fluorobenzoate (432 mg, 1.09
mmol) and triethyl phosphate (1.5 mL, 9.0 mmol) was irradiated in a
microwave instrument at 150.degree. C. for 1.5 h. The cooled
reaction mixture was purified by silica gel column chromatography
(hexane/ethyl acetate) to give ethyl
4-(5-bromo-2H-indazol-2-yl)-3-fluorobenzoate as pale yellow solid.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.56 (d, J=2.0 Hz, 1H),
8.25 (t, J=7.8 Hz, 1H), 8.02-7.90 (m, 2H), 7.89 (d, J=0.8 Hz, 1H),
7.66 (d, J=9.2 Hz, 1H), 7.40 (dd, J=1.6, 9.2 Hz, 1H), 4.43 (q,
J=7.2 Hz, 2H), 1.44 (t, J=7.2 Hz, 3H); .sup.19F NMR (376 MHz,
CDCl.sub.3) .delta. -123.4. MS (ESI) m/z: Calculated: 363.18.
Observed: 363.2 (M.sup.++1).
Ethyl 4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate and benzyl
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate (Step 4 of Scheme
A-10)
##STR00297##
[0783] In a similar manner as described in general procedure A, a
mixture (171 mg) of ethyl
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate and benzyl
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate was obtained by using
ethyl 4-(5-bromo-2H-indazol-2-yl)-3-fluorobenzoate (135 mg, 0372
mmol), 0.5 M solution of benzylzinc(II) bromide in THF (2.16 mL,
1.08 mmol) and Pd(tBu.sub.3P).sub.2 (9.5 mg, 19 .mu.mol). Ethyl
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate: MS (ESI) m/z:
Calculated: 374.41. Observed: 375.3 (M.sup.++1).
[0784] Benzyl 4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate: MS
(ESI) m/z: Calculated: 436.48. Observed: 437.3 (M.sup.++1).
(4-(5-Benzyl-2H-indazol-2-yl)-3-fluorophenyl)methanol (step 5 of
Scheme A-10)
##STR00298##
[0786] To a mixture (171 mg) of ethyl
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate and benzyl
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzoate in dichloromethane
(10 mL) was added 1.0 M solution of DIBAL-H (1.37 mL, 1.37 mmol) in
dichloromethane slowly at -78.degree. C. The mixture stirred for 1
hour at -78.degree. C. followed by quenching at -78.degree. C. with
0.5 mL of a saturated solution of ammonium chloride. Hydrochloric
acid (2N, 0.6 mL) was added, the cooling bath removed, and the
mixture stirred for 1 hour. The mixture was subsequently extracted
with dichloromethane and the extracts were dried over anhydrous
sodium sulfate. The solvent was removed under reduced pressure to
give (4-(5-benzyl-2H-indazol-2-yl)-3-fluorophenyl)methanol. MS
(ESI) m/z: Calculated: 332.37. Observed: 333.3 (M.sup.++1).
4-(5-Benzyl-2H-indazol-2-yl)-3-fluorobenzaldehyde (Step 6 of Scheme
A-10)
##STR00299##
[0788] To a mixture of
(4-(5-benzyl-2H-indazol-2-yl)-3-fluorophenyl)methanol (100 mg, 0.30
mmol), 4-methylmorpholine N-oxide (43 mg, 0.36 mmol) and activated
molecular sieves (200 mg) in dichloromethane (10 mL) was added TPAP
(10.6 mg, 0.03 mmol) at room temperature. After stirred overnight,
the reaction mixture was filtered, concentrated and purified by
silica gel column chromatography (hexane/ethyl acetate) to give
4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzaldehyde as white solid:
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 10.0 (d, J=1.6 Hz, 1H),
8.55 (m, 1H), 8.41 (t, J=7.4 Hz, 1H), 7.84 (m, 2H), 7.68 (d, J=9.0
Hz, 1H), 7.46 (s, 1H), 7.34-7.30 (m, 2H), 7.27-7.19 (m, 4H), 4.06
(s, 2H); .sup.19F NMR (376 MHz, CD.sub.3OD) .delta. -122.3. MS
(ESI) m/z: Calculated: 330.36. Observed: 331.2 (M.sup.++1).
1-(4-(5-Benzyl-2H-indazol-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid (Step 7 of Scheme A-10)
##STR00300##
[0790] In a similar manner as described in general procedure E,
1-(4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid (white foam [hS1P1 EC.sub.50=94 nM]) was obtained as TFA salt
by using 4-(5-benzyl-2H-indazol-2-yl)-3-fluorobenzaldehyde (108 mg,
0.328 mmol), azetidine-3-carboxylic acid (66 mg, 0.657 mmol),
acetic acid (45 .mu.L), sodium cyanoborohydride (41 mg, 0.66 mmol),
methanol (7.5 mL) and dichloromethane (4.5 mL). .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 8.61 (d, J=2.0 Hz, 1H), 8.09 (t, J=8.2 Hz,
1H), 7.61-7.49 (m, 4H), 7.30-7.18 (m, 6H), 4.52 (s, 2H), 4.43-4.35
(m, 4H), 4.05 (s, 2H), 3.76-3.67 (m, 1H); .sup.19F NMR (376 MHz,
CD.sub.3OD) .delta. -77.5 (TFA), -124.3. MS (ESI) m/z: Calculated
(without TFA): 415.46. Observed: 416.2 (M.sup.++1).
##STR00301##
##STR00302##
##STR00303##
##STR00304##
##STR00305##
##STR00306##
F: General Procedure of Coupling Boronic Acids with Aryl
Halides
[0791] Bis(di-tert-Butyl(phenyl)phosphine)palladium dichloride
(0.0285 mmol), methyl or ethyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (0.474 mmol),
boronic acid (0.617 mmol), potassium acetate (0.949 mmol) were
combined in a sealable tube and diluted with EtOH. The mixture was
flushed with nitrogen and heated to 80.degree. C. for several
hours. The reaction was cooled and partitioned between EtOAc and 1N
NaOH. The organic layer was washed with brine, dried over sodium
sulfate, filtered, and concentrated. The residue was purified by
silica gel chromatography to give the desired product.
G: General Procedure for Alkyne Cyclization
[0792] PdCl.sub.2(PPh.sub.3).sub.2 (0.129 mmol), copper(I) iodide
(0.129 mmol), 2-halophenol or 2-haloaniline (1.29 mmol), and methyl
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (1.54 mmol)
were combined in a sealable tube and 3 mL DMF and 3 mL TEA was
added. Argon was bubbled through the solvent for 3 min, and the
homogeneous brown reaction was sealed and heated to 100.degree. C.
After completion of the reaction, the reaction was concentrated in
vacuo and adsorbed onto 5 g silica gel and purified by silica gel
chromatography to give the desired product.
H: General Procedure for Ester Hydrolysis
[0793] To a solution of ester (0.428 mmol) in 2 mL THF was added
lithium hydroxide hydrate (1.29 mmol) in 1 mL water. The lt. yellow
reaction was stirred until completion. The THF was removed, and the
solid was suspended in 2 mL water. HCl (3 equiv, 2N) was added to
neutralize the base, and the mixture was sonicated. Phosphate
buffer (4 mL, 1M, pH 6) was added and the reaction was sonicated.
The slurry was filtered and the solid rinsed with water and EtOH
and dried in vacuo to give the desired product.
I: General Procedure of Reductive Amination
[0794] A mixture of aldehyde (1.0 mmol), acetic acid (1.5-2 mmol)
and azetidine-3-carboxylic acid or piperidine-4-carboxylic acid
(1-3 mmol) in DCM/MeOH (1:1, 10 mL) was stirred at room temperature
for 1 h. Sodium cyanoborohydride (0.5-1.0 mmol) was added and the
reaction mixture was stirred for 2-3 h at room temperature. The
reaction mixture was filtered, and the resulting residue was rinsed
with DCM. The solid was suspended in 0.5-1.0M pH 6 phosphate buffer
with sonication, filtered, and rinsed with water followed by EtOH
to give the desired product.
Common Intermediates
Intermediate 1
Methyl 3-hydroxyazetidine-3-carboxylate
1-Benzhydrylazetidin-3-ol
##STR00307##
[0796] NaOH (5N aqueous, 26.1 mL, 131 mmol) was added to a mixture
of 1-benzhydrylazetidin-3-ol hydrochloride (30.00 g, 109 mmol) in
water (150 mL). The mixture was allowed to stir for 15 min,
extracted with AcOEt, dried over MgSO.sub.4 to give
1-benzhydrylazetidin-3-ol. 1H NMR (300 MHz, CDCl.sub.3) .delta. ppm
7.35-7.44 (m, 4H), 7.22-7.32 (m, 4H), 7.13-7.22 (m, 2H), 4.40-4.52
(m, 1H), 4.35 (s, 1H), 3.47-3.60 (m, 2H), 2.80-2.97 (m, 2H). MS
(ESI) m/z: Calculated; 239.1. Observed: 340.1 (M.sup.++1).
1-Benzhydrylazetidin-3-one
##STR00308##
[0798] A three-neck flask was charged with oxalyl dichloride (10.6
mL, 119 mmol) and DCM (100.00 ml, 306 mmol), and the solution was
cooled to -78.degree. C. To the stirred solution was added, via
dropping funnel, dimethylsulfoxide (16.9 mL, 238 mmol) in DCM (50
mL) over 30 min. The reaction was stirred at -78.degree. C. for an
additional 5 min, and then 1-benzhydrylazetidin-3-ol (25.90 g, 108
mmol) in DCM (50 mL) and DMSO (10 mL) was added dropwise over 5 min
(T was maintained <-60.degree. C.). The solution was stirred at
-78.degree. C. for an additional 20 min, and Et.sub.3N (75.3 mL,
541 mmol) was added slowly. The reaction was allowed to reach room
temperature over 30 min and water (200 mL) was added. The mixture
was extracted with EtOAc, washed with brine, dried over MgSO.sub.4
and evaporated. Purification by flash chromatography using 2%
Et.sub.3N/hexanes gave 1-benzhydrylazetidin-3-one. 1H NMR (300 MHz,
CDCl.sub.3) .delta. ppm 7.41-7.52 (m, 4H), 7.26-7.35 (m, 4H),
7.16-7.26 (m, 2H), 4.59 (s, 1 H), 3.90-4.07 (m, 4H).
1-Benzhydryl-3-(trimethylsilyloxy)azetidine-3-carbonitrile
##STR00309##
[0800] Trimethylsilyl cyanide (4.5 mL, 34 mmol) was added to a
solution of 1-benzhydrylazetidin-3-one (4.00 g, 17 mmol) in DCM (85
mL) followed by addition of a solution of tetrabutylammonium
cyanide (0.45 g, 1.7 mmol) in DCM (85 mL). The solution was allowed
to stir at room temperature for 1 h, treated with water, extracted
with DCM, dried over MgSO.sub.4, and evaporated to give
1-benzhydryl-3-(trimethylsilyloxy)azetidine-3-carbonitrile. 1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.15-7.28 (m, 4H), 7.03-7.14 (m,
4H), 6.92-7.03 (m, 2H), 4.15 (s, 1H), 3.34-3.62 (m, 2H), 2.72-2.94
(m, 2H), 0.00 (s, 9H). MS (ESI) m/z: Calculated; 336.2. Observed:
337.1 (M.sup.++1).
1-Benzhydryl-3-hydroxyazetidine-3-carboxylic acid
##STR00310##
[0802] 1-Benzhydryl-3-(trimethylsilyloxy)azetidine-3-carbonitrile
(2.85 g, 8.47 mmol) was taken up in a 1:1 mixture of 1,4-dioxane
(30 mL) and 60% aqueous sulfuric acid (30 mL). The mixture was
heated to 95.degree. C. for 1 h. The solvent was removed and the
residue was taken to pH 7 using 5N NaOH. The solid was isolated by
filtration, rinsed with Et.sub.2O to give
1-benzhydryl-3-hydroxyazetidine-3-carboxylic acid. 1H NMR (300 MHz,
DMSO-d.sub.6) .delta. ppm 7.36-7.47 (m, 4H), 7.22-7.33 (m, 4H),
7.13-7.23 (m, 2H), 4.52 (s, 1H), 3.46 (d, J=8.3 Hz, 2 H), 3.02 (d,
J=8.2 Hz, 2H). MS (ESI) m/z: Calculated; 283.1. Observed: 284.1
(M.sup.++1).
Methyl 1-benzhydryl-3-hydroxyazetidine-3-carboxylate
##STR00311##
[0804] Sulfuric acid (4.00 mL, 47.3 mmol) was added to a mixture of
1-benzhydryl-3-hydroxyazetidine-3-carboxylic acid (2.13 g, 7.50
mmol) in MeOH (20 mL). The mixture was heated to 80.degree. C. for
18 h, diluted with EtOAc, extracted with water, 1N NaOH, dried over
MgSO.sub.4, and evaporated. The solid was rinsed with Et.sub.2O to
give methyl 1-benzhydryl-3-hydroxyazetidine-3-carboxylate. 1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.43-7.49 (m, 4H), 7.24-7.31 (m,
4H), 7.15-7.23 (m, 2H), 4.54 (s, 1H), 3.90 (s, 3H), 3.59-3.72 (m,
2H), 3.24-3.39 (m, 2H). MS (ESI) m/z: Calculated; 297.14. Observed:
298.1 (M.sup.++1).
Methyl 3-hydroxyazetidine-3-carboxylate
##STR00312##
[0806] A reactor was charged with methyl
1-benzhydryl-3-hydroxyazetidine-3-carboxylate (1.6 g, 5.4 mmol),
10% Pd/C (0.300 g, 2.8 mmol), and glacial acetic acid (0.300 mL,
5.2 mmol) in MeOH (30 mL). The mixture was allowed to stir for 3 h
under 50 psig of H.sub.2. The crude was filtered through Celite and
rinsed with MeOH. After evaporation, the solid was rinsed with
Et.sub.2O to give methyl 3-hydroxyazetidine-3-carboxylate as the
acetic acid salt. 1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 3.74
(d, J=9.1 Hz, 2H), 3.69 (s, 3H), 3.44 (d, J=8.9 Hz, 2H), 1.88 (s,
3H).
Intermediate 2
Methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
##STR00313##
[0808] Azetidine-3-carboxylic acid (43 g, 421 mmol),
4-bromo-3-fluorobenzaldehyde (81.4 g, 401 mmol), methyl
orthoformate (219 mL, 2005 mmol), and AcOH (34 mL, 601 mmol), was
added to DCM (700 mL) at rt under N.sub.2 atm. The mixture was
stirred for 15 min, at which point sodium triacetoxyborohydride
(127 g, 601 mmol) was added portionwise (exothermic). After 2 h,
solvent swap with MeOH (257 g, 8019 mmol), and sulfuric acid (79 g,
802 mmol) was added slowly (exothermic). The mixture was heated at
reflux for 18 h. Solvent was removed and the mixture was extracted
using DCM and water. The organic layer was purified using Biotage
column (isopropanol/heptane) affording methyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate as a clear oil.
MS (ESI) m/z: Calculated: 301.0. Observed: 302.0 (M.sup.++1).
Intermediate 3
Ethyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
##STR00314##
[0810] Synthesized in an analogous fashion as methyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate, but MeOH from
step 1 was removed and EtOH was used for step 2. MS (ESI) m/z:
Calculated: 315.0. Observed: 316.0 (M.sup.++1).
Intermediate 4
Methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate
Methyl
1-(3-fluoro-4-(2-(trimethylsilyl)ethynyl)benzyl)azetidine-3-carboxy-
late
##STR00315##
[0812] Methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
(25.00 g, 82.7 mmol), copper (I) iodide (3.14 g, 16.5 mmol),
(trimethylsilyl)acetylene (81.9 mL, 579 mmol),
bis(triphenylphosphine)palladium(ii) chloride (5.81 g, 8.27 mmol),
and Hunig's base (115 mL, 662 mmol) was added in a sealable tube
along with 100 mL THF. The reaction is sealed and heated to
80.degree. C. under vigorous stirring for 24 h. The mixture was
cooled to room temperature, filtered and evaporated. The resulting
oil was purified using Biotage (75 L, 0-50% EtOAc/hexanes)
affording methyl
1-(3-fluoro-4-(2-(trimethylsilyl)ethynyl)benzyl)azetidine-3-carboxylate
as a transparent brown oil. MS (ESI) m/z: Calculated: 319.1.
Observed: 320.1 (M.sup.++1).
Methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate
##STR00316##
[0814] Methyl
1-(3-fluoro-4-(2-(trimethylsilyl)ethynyl)benzyl)azetidine-3-carboxylate
(20.9 g, 65 mmol), and cesium fluoride (11 g, 72 mmol), was added
to DMF (50 mL). MeOH (100 mL) was added. After 2 h, MeOH was
removed and the mixture was extracted with DCM and water. The
organic layer is washed with brine and dried over magnesium
sulfate. The solvent was removed and the material purified using
Biotage (75 L, 7-100% EtOAc/hexanes) affording methyl
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate as light yellow
oil. MS (ESI) m/z: Calculated: 247.1. Observed: 248.0
(M.sup.++1).
Intermediate 5
2-Fluoro-4-formylbenzoyl chloride
1-Bromo-4-(diethoxymethyl)-2-fluorobenzene
##STR00317##
[0816] To a solution of 3-fluoro-4-bromobenzaldehyde (20.0 g, 98.5
mmol) in dry EtOH (120 mL) was added acetyl chloride (2.04 mL, 29.6
mmol) followed by the addition of triethyl orthoformate (6.55 mL,
39.4 mmol) and the contents were heated to 70.degree. C. for 3 h.
The contents were cooled to room temperature and shifted to a
rotary evaporator and subjected to reduced pressure (280 mm Hg)
with bath temperature 65.degree. C. for 45 min. The pressure was
further lowered to remove all the solvent. To this mixture, fresh
Ethanol (60 mL), acetyl chloride (1.5 mL), triethyl orthoformate
(5.0 mL) and heated to 70.degree. C. for 2 h. The solvent was
removed under the reduced pressure and diluted with EtOAc (200 mL),
washed with saturated sodium bicarbonate (3.times.100 mL), brine
and dried over anhydrous sodium sulfate. The solvent was evaporated
and the residue purified by silica gel column (basified with 5%
Et.sub.3N, eluent: EtOAc/hexanes, 1/20) to afford
1-bromo-4-(diethoxymethyl)-2-fluorobenzene as a colorless oil. 1H
NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.03 (t, J=8.1 Hz, 1H),
7.36-7.33 (m, 2H), 5.54 (s, 1H), 3.63-3.52 (m, 4H), 1.25 (m,
6H).
2-Fluoro-4-formylbenzoic acid
##STR00318##
[0818] To a solution of 1-bromo-4-(diethoxymethyl)-2-fluorobenzene
(10.12 g, 36.53 mmol) in dry THF (90 mL) cooled to -78.degree. C.
was added n-butyllithium (2.5 M in hexanes, 16.5 mL, 43.83 mmol)
was added dropwise over a period of 10 min. The contents were
further stirred for 30 min and CO.sub.2 was bubbled through the
mixture for 0.5 h. (exothermic). The cooling bath was removed and
the contents warmed to room temperature. The mixture was treated
with aqueous NaOH (1N, 100 mL) and washed with EtOAc. The aqueous
layer was acidified to pH 2 with HCl (5N) and the free acid was
extracted with EtOAc (3.times.75 mL). The combined organic layers
were washed with water and brine, dried over MgSO.sub.4 and
evaporated. The residue was dissolved in ether (30 mL), TFA (1.5
mL) and water (2.0 mL) and stirred overnight. The volatiles were
removed under reduced pressure and co-evaporated with toluene. The
residue was then treated with diethyl ether (75 mL) and filtered.
The filter cake was dried under vacuum without further purification
to give 2-fluoro-4-formylbenzoic acid as a white solid. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. ppm 13.48 (s, 1H), 10.06 (s, 1H), 8.06
(t, J=7.4 Hz, 1H), 7.84-7.79 (m, 2H). MS (ESI) m/z: Calculated:
168.0. Observed: 167.0 (M.sup.--1).
2-Fluoro-4-formylbenzoyl chloride
##STR00319##
[0820] To a slurry of 2-fluoro-4-formylbenzoic acid (0.531 g, 3.16
mmol) 10 mL DCM was added oxalyl chloride (0.168 mL, 1.89 mmol) and
catalytic DMF (2 drops). The reaction was allowed to stir under a
positive pressure of argon with a needle outlet to air. After 2 h,
a quenched aliquot of the reaction (MeOH) was determined to contain
no acid. The reaction was concentrated in vacuo and dried on a hood
pump for 10 min to give a yellow solid, which was used without
further purification.
Intermediate 6
4-(2-Bromoacetyl)-3-fluorobenzaldehyde
1-Bromo-4-(diethoxymethyl)-2-fluorobenzene
##STR00320##
[0822] To a solution of 4-bromo-3-fluorobenzaldehyde (1.00 g, 4.9
mmol) in dioxane (10.0 mL) was added
tributyl(1-ethoxyvinyl)stannane (1.7 mL, 5.2 mmol) and
Pd(PPh.sub.3).sub.2Cl.sub.2 (0.069 g, 0.099 mmol). The resulting
solution was purged with argon for 2 min and then heated
(microwave) in a sealed tube at 130.degree. C. for 30 min. The
cooled reaction solution was filtered through a plug of silica gel
(eluting with 80 mL EtOAc), and the filtrate was concentrated in
vacuo. Chromatographic purification of the residue (ISCO, 40 g,
0-30% EtOAc/Hex) afforded
1-bromo-4-(diethoxymethyl)-2-fluorobenzene as a light yellow oil.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 9.98 (s, 1H), 7.81
(t, J=7.5 Hz, 1H), 7.65 (d, J=7.5 Hz, 1H), 7.57 (d, J=11.0 Hz, 1H),
4.90 (d, J=2.0 Hz, 1H), 4.58 (s, 1H), 3.94 (q, J=7.0 Hz, 2H), 1.42
(t, J=6.8 Hz, 3H).
4-(2-Bromoacetyl)-3-fluorobenzaldehyde
##STR00321##
[0824] N-Bromosuccinimide (505 mg, 2837 .mu.mol) was added in one
portion to a solution of 1-bromo-4-(diethoxymethyl)-2-fluorobenzene
(551 mg, 2837 .mu.mol) in 3:1 THF--H.sub.2O (6.0 mL) at 25.degree.
C., and the resulting solution was stirred at 25.degree. C. for 10
min. The solution was partitioned between EtOAc (50 mL) and brine
(8 mL). The organic layer was separated, dried over sodium sulfate,
and concentrated in vacuo. Chromatographic purification of the
residue (ISCO, 4 g, 0-100% EtOAc/hexanes) furnished
4-(2-bromoacetyl)-3-fluorobenzaldehyde as a white solid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. ppm 10.08 (d, J=1.8 Hz, 1H),
8.06-8.12 (m, 1H), 7.79 (dd, J=8.0, 1.4 Hz, 1H), 7.69 (dd, J=10.6,
1.4 Hz, 1H), 4.52 (d, J=2.3 Hz, 2H).
Compound 88
1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazetidine--
3-carboxylic acid
Methyl
1-((4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)-3-hydroxyaze-
tidine-3-carboxylate
##STR00322##
[0826] Synthesized according to Scheme B1 and general procedure I
from 4-(5-benzylbenzofuran-2-yl)-3-fluorobenzaldehyde (0.100 g,
0.30 mmol) and methyl 3-hydroxyazetidine-3-carboxylate (0.058 g,
0.30 mmol) to give methyl
1-((4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)-3-hydroxyaz-
etidine-3-carboxylate. 1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm
7.91 (t, J=7.9 Hz, 1H), 7.40-7.61 (m, 2H), 7.15-7.36 (m, 8H), 6.26
(s, 1H), 4.04-4.10 (m, 1H), 4.04 (s, 2H), 3.70 (s, 3H), 3.68-3.69
(m, 1H), 3.63 (d, J=7.9 Hz, 1H), 3.12-3.18 (m, 4H). MS (ESI) m/z:
Calculated: 445.2. Observed: 446.1 (M.sup.++1).
1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazetidine--
3-carboxylic acid
##STR00323##
[0828] Synthesized according to general procedure H from methyl
1-(4-(5-benzylbenzofuran-2-yl)-3-fluorobenzyl)-3-hydroxyazetidine-3-carbo-
xylate (0.078 g, 0.18 mmol) to give
1-((4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazetidine-
-3-carboxylic acid [hS1P1 EC.sub.50=105 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.05 (t, J=7.5 Hz, 1H), 7.54-7.65 (m,
3H), 7.44-7.54 (m, 1H), 7.36 (s, 1H), 7.23-7.33 (m, 5H), 7.13-7.23
(m, 1H), 4.43-4.58 (m, 4H), 3.64-4.23 (m, 4H). MS (ESI) m/z:
Calculated: 431.2. Observed: 432.1 (M.sup.++1).
Compound 89
1-((4-(5-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazeti-
dine-3-carboxylic acid
Methyl
1-((4-(5-benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)-3-hydro-
xyazetidine-3-carboxylate
##STR00324##
[0830] Synthesized according to Scheme B1 and general procedure I
from 4-(5-benzylbenzo[d]thiazol-2-yl)-3-fluorobenzaldehyde (0.057
g, 0.16 mmol) and methyl 3-hydroxyazetidine-3-carboxylate (0.031 g,
0.16 mmol) to give methyl
1-((4-(5-benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazet-
idine-3-carboxylate. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm
8.37 (t, J=7.7 Hz, 1H), 7.93 (s, 1H), 7.84 (d, J=8.2 Hz, 1H),
7.21-7.35 (m, 8H), 4.15 (s, 2H), 3.93-4.07 (m, 7H), 3.52-3.67 (m,
2H). MS (ESI) m/z: Calculated: 462.1. Observed: 463.1
(M.sup.++1).
1-((4-(5-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazeti-
dine-3-carboxylic acid
##STR00325##
[0832] Synthesized according to general procedure H from methyl
1-(4-(5-benzylbenzo[d]thiazol-2-yl)-3-fluorobenzyl)-3-hydroxyazetidine-3--
carboxylate (0.035 g, 0.076 mmol) to give
1-((4-(5-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)-3-hydroxyazet-
idine-3-carboxylic acid [hS1P1 EC.sub.50=27 nM]. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 7.23 (br. t, J=8.0 Hz, 1H),
6.61-6.66 (br. s, 2H), 6.21-6.34 (m, 2H), 5.85-6.06 (m, 6),
3.29-3.20 (m, 4H), 2.92-2.97 (br. m, 2H), 2.86 (br. s, 2H). MS
(ESI) m/z: Calculated: 448.1. Observed: 449.2 (M.sup.++1).
Compound 90
1-((3-Fluoro-4-(5-(1-phenylethyl)benzofuran-2-yl)phenyl)methyl)azetidine-3-
-carboxylic acid
Benzofuran-5-yl(phenyl)methanol
##STR00326##
[0834] To a solution of 1-benzofuran-5-carbaldehyde (2.70 g, 18.5
mmol) in 50 mL THF under N.sub.2 was added phenylmagnesium bromide
3.0M solution in diethyl ether (7.39 mL, 22.2 mmol). The reaction
was allowed to stir for 1 h, then was quenched with NH.sub.4Cl
sat'd aq., extracted with diethyl ether, washed with brine, dried
over anhyd. sodium sulfate, filtered, and concentrated in vacuo to
a solid to give benzofuran-5-yl(phenyl)methanol which was used
without further purification. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 7.94 (d, J=2.0 Hz, 1H), 7.64 (s, 1H), 7.49 (d, J=8.5
Hz, 1H), 7.36-7.42 (m, 2H), 7.26-7.33 (m, 3H), 7.19 (t, J=7.3 Hz,
1H), 6.92 (d, J=1.5 Hz, 1H), 5.88 (d, J=4.0 Hz, 1H), 5.77-5.83 (m,
1H).
Benzofuran-5-yl(phenyl)methanone
##STR00327##
[0836] To a solution of benzofuran-5-yl(phenyl)methanol (4.0 g, 18
mmol) and triethylamine (7.3 mL, 54 mmol) in 30 mL 1:1 DCM/DMSO
under nitrogen at 0.degree. C. was added a solution of SO.sub.3*py
(8.5 g, 54 mmol) in 20 mL DMSO dropwise via addition funnel. The
reaction was allowed to stir 5 h at 0.degree. C. and was quenched
by addition of water and 250 mL Et.sub.2O. The organic layer was
washed with water, 1N HCl, 1N NaOH, brine, and was dried over
sodium sulfate, filtered, and concentrated. The resulting solid
benzofuran-5-yl(phenyl)methanone was used without further
purification. MS (ESI) m/z: Calculated: 222.1. Observed: 223.0
(M.sup.++1).
1-(Benzofuran-5-yl)-1-phenylethanol
##STR00328##
[0838] To a solution of benzofuran-5-yl(phenyl)methanone (1.0 g,
4.5 mmol) in 20 mL THF under nitrogen at 0.degree. C. was added
methylmagnesium bromide 3.0M in diethyl ether (2.2 mL, 6.7 mmol)
dropwise via syringe. The reaction was allowed to warm to ambient
temperature slowly. The reaction was quenched with sat'd aq.
NH.sub.4Cl and DCM. The aqueous layer was extracted 2.times.DCM,
and the combined organics were dried over anhyd sodium sulfate,
filtered, and concentrated in vacuo to give
1-(benzofuran-5-yl)-1-phenylethanol as an oil, which was used
without further purification. MS (ESI) m/z: Calculated: 238.1.
Observed: 238.8 (M.sup.++1).
5-(1-Phenylethyl)benzofuran
##STR00329##
[0840] To a solution of 1-(benzofuran-5-yl)-1-phenylethanol (1.1 g,
4.6 mmol) in 20 mL DCM at 0.degree. C. under nitrogen was added
triethylsilane (0.88 mL, 5.5 mmol) followed by trifluoroacetic acid
(0.39 mL, 5.1 mmol) dropwise from a syringe. Each drop of TFA
resulted in a yellow color which persisted slightly upon complete
addition. After 1.5 h at 0.degree. C., and was quenched by sat. aq.
NaHCO.sub.3. The aqueous layer was extracted 2.times.DCM, and the
combined organics were dried over anhyd. sodium sulfate, filtered,
and concentrated in vacuo to give an oil. The material was further
purified by silica gel chromatography, ISCO, 40 g, 0-10%
EtOAc/hexanes to give 0.94 g of a mixture of
5-(1-phenylethyl)benzofuran and 5-(1-phenylvinyl)benzofuran. This
material was taken up in 1 mL DCM at 0.degree. C. and
triethylsilane (0.629 mL, 3.94 mmol) was added followed by
trifluoroacetic acid (1.52 mL, 19.7 mmol) dropwise via syringe. The
reaction became yellow upon complete addition. After 15 min, the
reaction was quenched at 0.degree. C. by addition of 1 N NaOH and
diluted with DCM. The aq. layer was extracted 1.times.DCM, and the
combined organics were dried over anhyd sodium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel chromatography, ISCO, 80 g, 0-15% EtOAc/hexanes. The
product-containing fractions were concentrated in vacuo to give
5-(1-phenylethyl)benzofuran as a clear and colorless oil. 1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 7.94 (d, J=2.0 Hz, 1H), 7.53
(s, 1H), 7.48 (d, J=8.5 Hz, 1H), 7.24-7.34 (m, 4H), 7.12-7.22 (m,
2H), 6.89 (d, J=2.0 Hz, 1H), 4.26 (q, J=7.5 Hz, 1H), 1.62 (d, J=7.0
Hz, 3H).
5-(1-Phenylethyl)benzofuran-2-ylboronic acid
##STR00330##
[0842] To a solution of 5-(1-phenylethyl)benzofuran (0.713 g, 3.21
mmol) in 32 mL THF at -78.degree. C. was added 1-butyllithium (1.54
mL, 3.85 mmol) slowly dropwise. The reaction was allowed to stir
for 25 min, at which point triisopropyl borate (1.08 mL, 4.72 mmol)
was added slowly dropwise. After 0.5 h, the bath was removed and
the reaction was allowed to warm to ambient temperature. After 0.5
h, 50 mL 2N HCl was added. The mixture was extracted 2.times.MTBE,
and the combined organics were washed with brine, dried over anhyd.
sodium sulfate, filtered, and concentrated in vacuo to give an oil.
Treatment with 30 mL hexanes and sonication for 5 min resulted in a
white solid which was collected by filtration and dried in vacuo to
give 5-(1-phenylethyl)benzofuran-2-ylboronic acid. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.49-8.57 (m, 2H), 7.58 (s, 1H), 7.46 (d,
J=8.5 Hz, 1H), 7.40 (s, 1H), 7.12-7.32 (m, 6H), 4.26 (q, J=7.5 Hz,
1H), 1.62 (d, J=7.0 Hz, 3H).
1-((3-Fluoro-4-(5-(1-phenylethyl)benzofuran-2-yl)phenyl)methyl)azetidine-3-
-carboxylic acid
##STR00331##
[0844] According to Scheme B2 and general procedure F, methyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (0.241 g, 0.797
mmol) and 5-(1-phenylethyl)benzofuran-2-ylboronic acid (0.212 g,
0.797 mmol) were employed to give methyl
1-((3-fluoro-4-(5-(1-phenylethyl)benzofuran-2-yl)phenyl)methyl)azetidine--
3-carboxylate. According to Scheme B2 and general procedure H,
methyl
1-((3-fluoro-4-(5-(1-phenylethyl)benzofuran-2-yl)phenyl)-methyl)azetidine-
-3-carboxylate (0.275 g, 0.620 mmol) provided
1-((3-fluoro-4-(5-(1-phenylethyl)benzofuran-2-yl)phenyl)methyl)azetidine--
3-carboxylic acid as a white solid [hS1P1 EC.sub.50=99 nM]. 1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 7.89 (t, J=8.0 Hz, 1H), 7.59
(s, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.20-7.35 (m, 8H), 7.12-7.20 (m,
1H), 4.28 (q, J=7.0 Hz, 1H), 3.60 (s, 2H), 3.18-3.46 (m, 5H), 1.64
(d, J=7.5 Hz, 3H). MS (ESI) m/z: Calculated: 429.2. Observed: 430.2
(M.sup.++1).
Compound 91
1-((4-(5-(Difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)methyl)az-
etidine-3-carboxylic acid
5-(2-Phenyl-1,3-dithiolan-2-yl)benzofuran
##STR00332##
[0846] To a solution of benzofuran-5-yl(phenyl)methanone (2.45 g,
11.0 mmol), glacial acetic acid (1.27 mL, 22.0 mmol) and
1,2-ethanedithiol (1.39 mL, 16.5 mmol) in 30 mL DCM at ambient
temperature was added boron trifluoride diethyletherate (1.38 mL,
11.0 mmol). The reaction was allowed to stir overnight, and was
quenched by the addition of 1N NaOH. The aq. layer was extracted
1.times.DCM, and the combined organics were dried over sodium
sulfate, filtered, and concentrated. Purification by silica gel
chromatography, ISCO, 0-20% EtOAc/hexanes provided
5-(2-phenyl-1,3-dithiolan-2-yl)benzofuran as a clear/colorless oil.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 7.99 (d, J=2.0 Hz,
1H), 7.84 (d, J=2.0 Hz, 1H), 7.48-7.57 (m, 3H), 7.38-7.45 (m, 1H),
7.29-7.36 (m, 2H), 7.24 (t, J=7.3 Hz, 1H), 6.96 (d, J=2.0 Hz, 1H)
3.43 (s, 4H).
5-(Difluoro(phenyl)methyl)benzofuran
##STR00333##
[0848] To a solution of Selectfluor.TM. fluorinating reagent (3.70
g, 10.5 mmol) in 30 mL HF*py at 0.degree. C. in a nalgene bottle
was added a solution of 5-(2-phenyl-1,3-dithiolan-2-yl)benzofuran
(1.56 g, 5.23 mmol) in 15 mL DCM (5 mL rinse) slowly via pipette.
The dark red reaction was allowed to stir for 15 min, at which
point it was quenched with ice and basified with 1N and 10N NaOH.
Et.sub.2O (250 mL) was added and the organic layer was washed
2.times.1N HCl, 1.times. brine, dried over sodium sulfate,
filtered, and concentrated in vacuo. The resulting material was
purified by silica gel chromatography, ISCO, 0-20% EtOAc/hexanes to
give 5-(difluoro(phenyl)methyl)benzofuran. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.10 (d, J=2.0 Hz, 1H), 7.83 (s, 1H),
7.71 (d, J=9.0 Hz, 1H), 7.48-7.58 (m, 5H), 7.45 (d, J=8.5 Hz, 1H),
7.04 (s, 1H).
Ethyl
1-((4-(5-(difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)met-
hyl)azetidine-3-carboxylate
##STR00334##
[0850] To a solution of 5-(difluoro(phenyl)methyl)benzofuran (0.684
g, 2.8 mmol) in 28 mL THF at -78.degree. C. under nitrogen was
added butyllithium, 2.5M solution in hexanes (1.3 mL, 3.4 mmol)
dropwise. The clear solution was allowed to stir for 30 min, at
which point triisopropyl borate (0.97 mL, 4.2 mmol) was added.
After 30 min, the bath was removed and the reaction allowed
reaching ambient temperature. After 30 min, 28 mL 2N HCl was added,
and the reaction was diluted with MTBE. The organic layer was
washed with brine and dried with anhyd sodium sulfate, filtered,
and concentrated to a ]clear oil. Hexanes was added to give a white
solid, which was collected by filtration. A portion of the
unpurified material was carried forward as follows: ethyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (0.150 g, 0.474
mmol) and 5-(difluoro(phenyl)methyl)benzofuran-2-ylboronic acid
(0.273 g, 0.949 mmol) were reacted according to Scheme B2 and
general procedure F to give ethyl
1-((4-(5-(difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)me-
thyl)azetidine-3-carboxylate. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 7.94 (t, J=8.0 Hz, 1H), 7.89 (s, 1H), 7.76 (d, J=8.5
Hz, 1H), 7.45-7.60 (m, 6H), 7.38 (d, J=3.0 Hz, 1H), 7.26-7.33 (m,
2H), 4.10 (q, J=7.0 Hz, 2H), 3.63 (s, 2H), 3.43-3.51 (m, 2H),
3.20-3.38 (m, 3H), 1.19 (t, J=7.0 Hz, 3H).
1-((4-(5-(Difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)methyl)az-
etidine-3-carboxylic acid
##STR00335##
[0852] Synthesized according to Scheme B2 and general procedure H
using ethyl
1-((4-(5-(difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)me-
thyl)azetidine-3-carboxylate (0.181 g, 0.377 mmol) to give
1-((4-(5-(difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)methyl)a-
zetidine-3-carboxylic acid as a white solid [hS1P1 EC.sub.50=13
nM]. 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 7.93 (t, J=8.0 Hz,
1H), 7.89 (s, 1H), 7.76 (d, J=8.5 Hz, 1H), 7.45-7.61 (m, 6H), 7.38
(d, J=3.0 Hz, 1H), 7.26-7.33 (m, 2H), 3.62 (s, 2H), 3.39-3.48 (m,
2H), 3.17-3.32 (m, 3H). MS (ESI) m/z: Calculated: 451.1. Observed:
452.2 (M.sup.++1).
Compound 92
1-((4-(6-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxyl-
ic acid
6-Benzylbenzofuran-2-ylboronic acid
##STR00336##
[0854] To a purple mixture of benzofuran-6-ol (1.85 g, 13.8 mmol)
and N,N-diisopropylethylamine 99% (7.21 mL, 41.4 mmol) in 50 mL DCM
was added
1,1,1-trifluoro-n-phenyl-n-((trifluoromethyl)sulfonyl)methanesulfonamide
(4.93 g, 13.8 mmol). The reaction became light blue. After 2 h, the
reaction was light yellow. The reaction was quenched with saturated
aq. sodium bicarbonate, and the aqueous layer was extracted with
DCM. The combined organics were dried over anhyd sodium sulfate,
filtered, and concentrated in vacuo. The resulting material was
purified by silica gel chromatography, 0-15% EtOAc/hexanes, to give
benzofuran-6-yl trifluoromethanesulfonate as a semi-solid. A
portion of the unpurified material was carried on as follows. A
mixture of 9-benzyl-9-bora-bicyclo[3.3.1]nonane 0.5M in THF (23 mL,
11 mmol), benzofuran-6-yl trifluoromethanesulfonate (1.50 g, 5.6
mmol), potassium phosphate (3.6 g, 17 mmol), benzofuran-6-yl
trifluoromethanesulfonate (1.50 g, 5.6 mmol), S-Phos (0.19 g, 0.45
mmol), Pd(OAc).sub.2 (0.051 g, 0.23 mmol) was flushed with argon,
sealed, and heated to 60.degree. C. overnight. The green/gray
reaction was cooled and filtered through celite rinsing with
Et.sub.2O. The filtrate was concentrated and adsorbed onto 15 g
silica gel and dried. The material was purified by silica gel
chromatography, ISCO, 80 g, 0-10% EtOAc/hexanes to give
6-benzylbenzofuran. A portion of this material was processed as
follows. To a solution of 6-benzylbenzofuran (0.663 g, 3.18 mmol)
in 30 mL THF at -78.degree. C. was added 1-butyllithium 2.5 M in
hexanes (1.53 mL, 3.82 mmol) slowly dropwise. The reaction was
allowed to stir for 25 min, at which point triisopropyl borate
(1.08 mL, 4.68 mmol) was added slowly dropwise. After 0.5 h, the
bath was removed and the reaction was allowed to warm to ambient
temperature. After 10 min, 50 mL 2N HCl was added. The mixture was
diluted with MTBE, and the organics were washed with brine, dried
over anhyd. MgSO.sub.4, filtered, and concentrated in vacuo to give
an oil. The oil was treated with hexanes to give a solid, and the
material was filtered and rinsed with hexanes and dried in vacuo to
give 6-benzylbenzofuran-2-ylboronic acid as a white solid. 1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 8.47 (s, 2H), 7.57 (d, J=8.0
Hz, 1H), 7.42 (s, 1H), 7.39 (s, 1H), 7.24-7.33 (m, 4H), 7.15-7.22
(m, 1H), 7.10 (d, J=8.0 Hz, 1H), 4.06 (s, 2H).
Ethyl
1-((4-(6-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-ca-
rboxylate
##STR00337##
[0856] Synthesized according to Scheme B2 and general procedure F
using ethyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
(0.150 g, 0.474 mmol) and 6-benzylbenzofuran-2-ylboronic acid
(0.155 g, 0.617 mmol) to give ethyl
1-((4-(6-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxy-
late. MS (ESI) m/z: Calculated: 443.2. Observed: 444.1
(M.sup.++1).
1-((4-(6-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxyl-
ic acid
##STR00338##
[0858] Synthesized according to Scheme B2 and general procedure H
using ethyl
1-((4-(6-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-c-
arboxylate (0.190 g, 0.428 mmol) to give
1-((4-(6-benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxy-
lic acid as a white solid [hS1P1 EC.sub.50=7 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.89 (t, J=8.0 Hz, 1H), 7.60 (d, J=8.0
Hz, 1H), 7.50 (s, 1H), 7.21-7.34 (m, 7H), 7.15-7.21 (m, 2H), 4.07
(s, 2H), 3.60 (s, 2H), 3.37-3.49 (m, 2H), 3.15-3.30 (m, 3H). MS
(ESI) m/z: Calculated: 415.2. Observed: 416.1 (M.sup.++1).
Compound 93
1-((3-Fluoro-4-(5-(pyridin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azetid-
ine-3-carboxylic acid
Benzofuran-5-yl(pyridin-2-yl)methanol
##STR00339##
[0860] Under argon at -78.degree. C., a mixture of 2-bromopyridine
(1.11 g, 7.05 mmol) in THF (30 mL) was dropwise treated with n-BuLi
(1.05 equiv. 2.7 mL of 2.5M BuLi in hexanes) over a period of 10
min, and stirred for 10 min. The resulting brown solution was
treated dropwise with a solution of benzofuran-5-carbaldehyde
(0.937 g, 6.41 mmol) in THF (10 mL) and continued to stir for 15
min at -78.degree. C. The mixture was treated with MeOH (3 mL),
followed by H.sub.2O (20 mL) and warmed to 4.degree. C. The mixture
was treated with EtOAc, the layers separated, and the aqueous layer
extracted 1.times. with EtOAc. The combined organic layers were
dried over MgSO.sub.4 and evaporated, which resulted in a dark
yellow oil which was used without further purification.
2-(Benzofuran-5-ylmethyl)pyridine
##STR00340##
[0862] A solution of benzofuran-5-yl(pyridin-2-yl)methanol (500 mg,
2220 .mu.mol) in DCM (10 mL) at 0.degree. C. was treated with
tribromophosphine (314 .mu.L, 3330 .mu.mol) and stirred for 1.5 h.
The mixture was diluted with CHCl.sub.3, washed with sat. aqueous
KHCO.sub.3, dried over MgSO.sub.4, and evaporated. A suspension of
the crude material and 10% Pd--C (50 mg) in EtOAc (5 mL) and MeOH
(3 mL) was stirred under 1 atm H.sub.2 at 24.degree. C. for 1 h.
The solids were filtered off (Celite) and washed with EtOAc. The
filtrate was evaporated and purified by flash chromatography
(hexanes to EtOAc to 5% MeOH in DCM) to give
2-(benzofuran-5-ylmethyl)pyridine as a yellow foam. .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. ppm 8.71 (br. s, 1H), 8.17 (t, J=7.6
Hz, 1H), 7.70-7.64 (m, 3H), 7.50-7.48 (m, 2H), 7.29 (d, J=8.1 Hz,
1H), 6.76 (s, 1H), 4.71 (s, 2H). MS (ESI) m/z: Calculated: 209.1.
Observed: 210.0 (M.sup.++1).
5-(Pyridin-2-ylmethyl)benzofuran-2-ylboronic acid
##STR00341##
[0864] A solution of 2-(benzofuran-5-ylmethyl)pyridine (66 mg, 315
.mu.mol) in THF (2 mL) was cooled to -78.degree. C. (under argon),
treated with 2.5 M n-BuLi (in hexanes, 2.5 equiv, 0.315 mL) and
stirred for 2 min, warmed to 0.degree. C. for 30 min, cooled to
-78.degree. C., and treated with triisopropyl borate (178 .mu.L,
946 .mu.mol). After 5 min, cooling was removed, the mixture slowly
warmed to room temperature and stirred for 2 h. The mixture was
diluted with EtOAc, washed with 1.times. with brine, dried over
MgSO.sub.4 and evaporated yielding yellow solids used without
further purification. MS (ESI) m/z: Calculated: 253.1. Observed:
254.1 (M.sup.++1).
Ethyl
1-((3-fluoro-4-(5-(pyridin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)-
azetidine-3-carboxylate
##STR00342##
[0866] In a sealed flask, a mixture of
5-(pyridin-2-ylmethyl)benzofuran-2-ylboronic acid (214 mg, 846
.mu.mol) and potassium acetate (166 mg, 1691 .mu.mol) was as set
under argon, treated with
bis{di(.sup.tbutyl)phenyl}palladium(II)dichloride (32 mg, 51
.mu.mol), followed by a solution of ethyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (267 mg, 846
.mu.mol) in EtOH (5 mL). The resulting suspension was degassed
again and heated to 80.degree. C. for 2.5 h. The mixture was cooled
to .about.10.degree. C. and dropwise treated with H.sub.2O (15 mL).
The mixture was extracted 2.times. with EtOAc. The combined organic
layers were dried over MgSO.sub.4 and evaporated. Purification by
flash chromatography (DCM to DCM/MeOH=4:1) gave ethyl
1-((3-fluoro-4-(5-(pyridin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azeti-
dine-3-carboxylate as a yellow foam.
1-((3-Fluoro-4-(5-(pyridin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azetid-
ine-3-carboxylic acid
##STR00343##
[0868] A mixture of ethyl
1-(3-fluoro-4-(5-(pyridin-2-ylmethyl)benzofuran-2-yl)benzyl)azetidine-3-c-
arboxylate (31 mg, 70 .mu.mol) in THF (2 mL) was treated with 1M
LiOH in H.sub.2O (0.4 mL) and stirred at 24.degree. C. for 3 h,
neutralized with 0.1M aqueous HCl, and evaporated. Purification by
SFC (supercritical flash chromatography) resulted in title compound
as its diethylammonium salt [hS1P1 EC.sub.50=31 nM]. 1H NMR (400
MHz, CD.sub.3OD) .delta. ppm 8.48 (d, J=0.8 Hz, 1H), 8.02 (t, J=7.9
Hz, 1H), 7.78 (t, J=5.9 Hz, 1H), 7.54 (s, 1H), 7.48 (d, J=10.0 Hz,
1H), 7.33-7.22 (m, 6H), 4.25 (s, 2H), 3.95 (s, 2H), 3.82 (t, J=8.6
Hz, 2H), 3.68 (t, J=8.5 Hz, 2H), 3.36-3.34 (m, 1H, partially
overlapping with CD.sub.3OH signal), 3.05 (q, J=7.4 Hz, 2H), 1.31
(t, J=7.2 Hz, 3H). MS (ESI) m/z: Calculated: 416.2. Observed: 417.2
(M.sup.++1).
Compound 94
1-((3-Fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azetid-
ine-3-carboxylic acid
Benzofuran-5-yl(thiazol-2-yl)methanol
##STR00344##
[0870] Under argon at -78.degree. C., a mixture of thiazole (1.13
g, 13.2 mmol) in THF (50 mL) was dropwise treated with 2.5M n-BuLi
(1.05 equiv., 5.04 mL, in hexanes) over a period of 10 min and
stirred for 45 min. The resulting yellow mixture was treated
dropwise with a solution of benzofuran-5-carbaldehyde (1.76 g, 12.0
mmol) in THF (10 mL). Cooling was removed after addition. When the
mixture reached room temperature, it was treated with EtOAc, washed
with 1M aqueous HCl and brine, dried over MgSO.sub.4, and
evaporated, to give benzofuran-5-yl(thiazol-2-yl)methanol as a
yellow oil. 1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.71-7.75 (m,
2H), 7.64 (d, J=2.2 Hz, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.39 (dd,
J=8.6, 1.7 Hz, 1H), 7.29 (d, J=3.1 Hz, 1H), 6.76-6.75 (m, 1H), 6.17
(s, 1H).
2-(Benzofuran-5-ylmethyl)thiazole
##STR00345##
[0872] Under argon at 24.degree. C., crude
benzofuran-5-yl(thiazol-2-yl)methanol (2.50 g, 10.8 mmol) was
dissolved in 1,2-dichloroethane (40 mL), treated with
triethylsilane (3.14 g, 27.0 mmol) and trifluoroacetic acid (1.25
mL, 16.2 mmol), and heated to reflux for 20 h. The mixture was
diluted with EtOAc and ice, washed 1.times. with saturated aqueous
NaHCO.sub.3, brine, dried over MgSO.sub.4, and evaporated.
Purification by flash chromatography (hexanes to hexanes/EtOAc=3:2)
gave 2-(benzofuran-5-ylmethyl)thiazole as a yellow oil. 1H NMR (400
MHz, CDCl.sub.3) .delta. ppm 7.71 (d, J=3.3 Hz, 1H), 7.61 (d, J=2.3
Hz, 1H), 7.55 (br. s, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.27-7.24 (m,
1H), 7.19 (d, J=3.3 Hz, 1H), 6.74-6.73 (m, 1H), 4.44 (s, 2 H). MS
(ESI) m/z: Calculated: 215.0. Observed: 216.1 (M.sup.++1).
5-(Thiazol-2-ylmethyl)benzofuran-2-ylboronic acid
##STR00346##
[0874] A solution of 2-(benzofuran-5-ylmethyl)thiazole (0.68 g, 3
mmol) in THF (20 mL) was cooled to -78.degree. C. (under argon),
treated with 2.5 M n-BuLi (3.0 equiv, 3.6 mL, in hexanes), and
stirred for 2 min, warmed to 0.degree. C. for 30 min, cooled to
-78.degree. C., and treated with triisopropyl borate (1 mL, 6
mmol). After 5 min, cooling was removed and the mixture slowly
warmed to room temperature and stirred for 2 h. The mixture was
diluted with EtOAc, washed 1.times. with brine, dried over
MgSO.sub.4 and evaporated to give
5-(thiazol-2-ylmethyl)benzofuran-2-ylboronic acid as a yellow
solid. MS (ESI) m/z: Calculated: 259.1. Observed: 260.4
(M.sup.++1).
Ethyl
1-((3-fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methyla-
zetidine-3-carboxylate
##STR00347##
[0876] In a sealed flask, a mixture of
5-(thiazol-2-ylmethyl)benzofuran-2-ylboronic acid (373 mg, 1440
.mu.mol) and potassium acetate (283 mg, 2879 .mu.mol) was set under
argon, treated with bis{di(.sup.tbutyl)phenyl}palladium(II)
dichloride (53.7 mg, 86.4 .mu.mol), followed by a solution of ethyl
1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate (455 mg, 1440
.mu.mol) in EtOH (10 mL). The resulting suspension was degassed
again and heated to 80.degree. C. for 2.5 h. The mixture was cooled
to 24.degree. C., treated with H.sub.2O (15 mL) and extracted
2.times. with EtOAc. The combined organic layers were washed with
brine, dried over MgSO.sub.4 and evaporated. Purification by flash
chromatography (DCM to DCM/MeOH=19:1) gave ethyl
1-((3-fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methylazetid-
ine-3-carboxylate as a yellow foam. MS (ESI) m/z: Calculated:
450.5. Observed: 451.2 (M.sup.++1).
1-((3-Fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azetid-
ine-3-carboxylic acid
##STR00348##
[0878] A mixture of ethyl
1-((3-fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azeti-
dine-3-carboxylate (290 mg, 644 .mu.mol) in THF (5 mL) was treated
with a solution of LiOH (27 mg) in H.sub.2O (2 mL) and stirred at
24.degree. C., neutralized with 0.1M aqueous HCl, and concentrated.
The resulting solids were collected by filtration and purified by
RP-HPLC to give
1-((3-fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azeti-
dine-3-carboxylic acid as a white solid [hS1P1 EC.sub.50=76 nM]. 1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 7.93-7.91 (m, 1H), 7.73 (s,
1H), 7.66-7.58 (m, 3H), 7.32-7.28 (m, 4H), 4.44 (s, 2H), 3.65 (br.
s, 2H), 3.46 (br. s, 2H), 3.30-3.26 (m, 3H). MS (ESI) m/z:
Calculated: 422.1. Observed: 423.2 (M.sup.++1).
Compound 95
1-((3-Fluoro-4-(5-(1-phenylpropyl)benzofuran-2-yl)phenyl)methyl)azetidine--
3-carboxylic acid
1-(Benzofuran-5-yl)-1-phenylpropan-1-ol
##STR00349##
[0880] To a solution of ethylmagnesium chloride (2M in
tetrahydrofuran, 10.63 mL, 21.26 mmol) under nitrogen at room
temperature was added zinc chloride (0.5M in tetrahydrofuran, 5.67
mL, 2.84 mmol) via syringe. The mixture was stirred at room
temperature for 1 h. The reaction mixture was brought to 0.degree.
C. and benzofuran-5-yl(phenyl)methanone (3.15 g, 14.17 mmol) in
tetrahydrofuran (10 mL) was added via syringe. The reaction mixture
was stirred at 0.degree. C. for 2.5 h. The reaction mixture was
cooled to room temperature and quenched with saturated aqueous
NH.sub.4Cl. EtOAc was added, and the aqueous layer was separated
and extracted with EtOAc (2.times.). The combined organic layers
were washed with brine, dried (MgSO.sub.4), and concentrated to
give a crude oil. The crude product was purified by silica flash
chromatography (0-100% DCM/hexanes) to give
1-(benzofuran-5-yl)-1-phenylpropan-1-ol as an off-white oil. 1H NMR
(400 MHz, Chloroform-d) .delta. ppm 7.69 (1H, d, J=1.6 Hz), 7.60
(1H, d, J=2.2 Hz), 7.39-7.45 (3H, m), 7.28-7.34 (3H, m), 7.22 (1H,
tt, J=7.2, 1.4 Hz), 6.73 (1H, dd, J=2.2, 1.0 Hz), 2.38 (2H, q,
J=7.3 Hz), 2.09 (1H, s), 0.90 (3H, t, J=7.3 Hz).
5-(1-Phenylpropyl)benzofuran
##STR00350##
[0882] To a stirred solution of
1-(benzofuran-5-yl)-1-phenylpropan-1-ol (3.14 g, 12.4 mmol) and
triethylsilane (2.39 mL, 14.9 mmol) in DCM (10 mL) under nitrogen
at 0.degree. C. was added trifluoroacetic acid (4.62 mL, 62.2 mmol)
dropwise via syringe. The reaction mixture was stirred at 0.degree.
C. for 3 h. Additional triethylsilane (2.39 mL, 14.9 mmol) followed
by trifluoroacetic acid (4.62 mL, 62.2 mmol) were added via
syringe, and the reaction was brought to room temperature and
stirred for 2 h. The reaction mixture was quenched with 1M aqueous
NaOH. DCM was added, and the aqueous layer was separated and
extracted again with DCM. The combined organic layers were dried
(MgSO.sub.4) and concentrated to give a crude oil. The crude
product was purified by silica flash chromatography to give
5-(1-phenylpropyl)-benzofuran as a transparent oil. MS (ESI) m/z:
Calculated: 236.1. Observed: 237.2 (M.sup.++1).
5-(1-Phenylpropyl)benzofuran-2-ylboronic acid
##STR00351##
[0884] Synthesized according to Scheme 1 and general procedure C
using 5-(1-phenylpropyl)benzofuran (2.30 g, 9.73 mmol) to give
5-(1-phenylpropyl)benzofuran-2-ylboronic acid. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.50 (2H, s), 7.60 (1H, s), 7.45 (1H, d,
J=8.6 Hz), 7.39 (1H, s), 7.23-7.34 (5H, m), 7.14 (1H, t, J=7.1 Hz),
3.91 (1H, t, J=7.7 Hz), 2.08 (2H, dq, J=7.7, 7.2 Hz), 0.83 (3H, t,
J=7.2 Hz).
Methyl
1-((3-fluoro-4-(5-(1-phenylpropyl)benzofuran-2-yl)phenyl)methyl)aze-
tidine-3-carboxylate
##STR00352##
[0886] Synthesized according to Scheme B2 and general procedure F
using methyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate
(1.76 g, 5.81 mmol) and 5-(1-phenylpropyl)-benzofuran-2-ylboronic
acid (1.63 g, 5.81 mmol) to give methyl
1-((3-fluoro-4-(5-(1-phenylpropyl)benzofuran-2-yl)phenyl)methyl)azetidine-
-3-carboxylate. MS (ESI) m/z: Calculated: 457.2. Observed: 458.3
(M.sup.++1).
1-((3-Fluoro-4-(5-(1-phenylpropyl)benzofuran-2-yl)phenyl)methyl)azetidine--
3-carboxylic acid
##STR00353##
[0888] Synthesized according to Scheme B2 and general procedure H
using methyl
1-((3-fluoro-4-(5-(1-phenylpropyl)benzofuran-2-yl)phenyl)methyl)az-
etidine-3-carboxylate (151 mg, 330 .mu.mol) to give
1-((3-fluoro-4-(5-(1-phenylpropyl)benzofuran-2-yl)phenyl)methyl)azetidine-
-3-carboxylic acid as a white solid. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 7.89 (1H, dd, J=8.0, 8.0 Hz), 7.62 (1H, s), 7.53 (1H,
d, J=8.6 Hz), 7.22-7.35 (8H, m), 7.15 (1H, t, J=7.1 Hz), 3.93 (1H,
t, J=7.7 Hz), 3.60 (2H, s), 3.37-3.45 (2H, m), 3.16-3.24 (3H, m),
2.09 (2H, dq, J=7.7, 7.1 Hz), 0.84 (3H, t, J=7.1 Hz). MS (ESI) m/z:
Calculated: 443.2. Observed: 444.2 (M.sup.++1).
Compound 96
1-(3-(5-Benzylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
Methyl 1-(3-bromobenzyl)azetidine-3-carboxylate
##STR00354##
[0890] Synthesized according to Scheme B2, Step 1 from
3-bromobenzaldehyde and azetidine-3-carboxylic acid to give methyl
1-(3-bromobenzyl)azetidine-3-carboxylate as a clear oil. MS (ESI)
m/z: Calculated: 283.0. Observed: 284 (M.sup.++1).
Methyl
1-(3-(5-benzylbenzofuran-2-yl)benzyl)azetidine-3-carboxylate
##STR00355##
[0892] Synthesized according to Scheme B2, Step 2 and general
procedure F from 5-benzylbenzofuran-2-ylboronic acid and methyl
1-(3-bromobenzyl)azetidine-3-carboxylate to give methyl
1-(3-(5-benzylbenzofuran-2-yl)benzyl)azetidine-3-carboxylate as a
light yellow oil. MS (ESI) m/z: Calculated: 411.2. Observed: 412
(M.sup.++1).
1-(3-(5-Benzylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid
##STR00356##
[0894] Synthesized according to Scheme B2, Step 3 and general
procedure H from methyl
1-(3-(5-benzylbenzofuran-2-yl)benzyl)azetidine-3-carboxylate to
give 1-(3-(5-benzylbenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid as a white solid [hS1P1 EC.sub.50=4834 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.76-7.85 (m, 2H), 7.54 (d, J=8.4 Hz,
1H), 7.49 (s, 1H), 7.45 (t, J=7.6 Hz, 1H), 7.38 (s, 1H), 7.30-7.35
(m, 1H), 7.25-7.30 (m, 4H), 7.16-7.21 (m, 2H), 4.04 (s, 2H), 3.74
(s, 2H), 3.54 (s, 2H), 3.31 (s, 4H). MS (ESI) m/z: Calculated:
397.2. Observed: 398 (M.sup.++1).
Compound 97
[0895]
1-((3-Fluoro-4-(5-(2-phenylpropan-2-yl)benzofuran-2-yl)phenyl)methy-
l)azetidine-3-carboxylic acid
1-(2,2-Diethoxyethoxy)-4-(2-phenylpropan-2-yl)benzene
##STR00357##
[0897] Synthesized according to Scheme 1, Step 1 from
(4-(2-phenylisopropyl)phenol) and 2-bromo-1,1-diethoxyethane to
give 1-(2,2-diethoxyethoxy)-4-(2-phenylpropan-2-yl)benzene as a
yellow-orange oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm
7.19-7.29 (m, 4H), 7.14-7.19 (m, 1H), 7.13 (d, J=9.0 Hz, 2H), 6.82
(d, J=8.5 Hz, 2H), 4.82 (t, J=5.3 Hz, 1H), 3.98 (d, J=5.0 Hz, 2H),
3.70-3.81 (m, 2H), 3.57-3.68 (m, 2H), 1.65 (s, 6H), 1.24 (t, J=7.0
Hz, 6H).
5-(2-Phenylpropan-2-yl)benzofuran
##STR00358##
[0899] Synthesized according to Scheme 1, Step 2 from
1-(2,2-diethoxyethoxy)-4-(2-phenylpropan-2-yl)benzene to give
5-(2-phenylpropan-2-yl)benzofuran as a light yellow oil. 1H NMR
(400 MHz, CHLOROFORM-d) .delta. ppm 7.58 (d, J=2.0 Hz, 1H), 7.52
(s, 1H), 7.36 (d, J=8.5 Hz, 1H), 7.22-7.30 (m, 4H), 7.14-7.20 (m,
1H), 7.11 (d, J=8.5 Hz, 1H), 6.71 (s, 1H), 1.74 (s, 6H).
5-(2-Phenylpropan-2-yl)benzofuran-2-ylboronic acid
##STR00359##
[0901] Synthesized according to Scheme 1, Step 3 from
5-(2-phenylpropan-2-yl)benzofuran to give
5-(2-phenylpropan-2-yl)benzofuran-2-ylboronic acid as a white
solid. 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.50 (s, 2H),
7.59 (d, J=1.8 Hz, 1H), 7.39-7.44 (m, 2 H), 7.20-7.30 (m, 4H), 7.15
(m, J=6.7 Hz, 1H), 7.09 (dd, J=8.6, 2.0 Hz, 1H), 3.31 (s, 6H).
Ethyl
1-(3-fluoro-4-(5-(2-phenylpropan-2-yl)benzofuran-2-yl)benzyl)azetidi-
ne-3-carboxylate
##STR00360##
[0903] Synthesized according to Scheme B2, Step 2 and general
procedure F from 5-(2-phenylpropan-2-yl)benzofuran-2-ylboronic acid
and ethyl 1-(4-bromo-3-fluorobenzyl)azetidine-3-carboxylate to give
ethyl
1-(3-fluoro-4-(5-(2-phenylpropan-2-yl)benzofuran-2-yl)benzyl)azetidine-3--
carboxylate as a yellow foam. MS (ESI) m/z: Calculated: 471.2.
Observed: 472 (M.sup.++1).
1-((3-Fluoro-4-(5-(2-phenylpropan-2-yl)benzofuran-2-yl)phenyl)methyl)azeti-
dine-3-carboxylic acid
##STR00361##
[0905] Synthesized according to Scheme B2, Step 3 and general
procedure H from ethyl
1-(3-fluoro-4-(5-(2-phenylpropan-2-yl)benzofuran-2-yl)benzyl)azetidine-3--
carboxylate to give
1-((3-fluoro-4-(5-(2-phenylpropan-2-yl)benzofuran-2-yl)phenyl)methyl)azet-
idine-3-carboxylic acid as a white solid [hS1P1 EC.sub.50=365 nM].
1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 7.89 (t, J=8.2 Hz, 1H),
7.61 (d, J=1.8 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 7.22-7.31 (m, 7H),
7.15-7.19 (m, 1H), 7.12 (dd, J=8.8, 2.0 Hz, 1H), 3.60 (s, 2H), 3.42
(s, 1H), 3.32 (s, 4H), 3.21 (s, 1H), 1.70 (s, 6H). MS (ESI) m/z:
Calculated: 443.2. Observed: 444 (M.sup.++1).
##STR00362##
Compound 98
1-((3-Fluoro-4-(5-(phenylmethyl)furo[2,3-b]pyridin-2-yl)phenyl)methyl)-3-a-
zetidinecarboxylic acid
5-Benzyl-2-methoxypyridine
##STR00363##
[0907] A sealable tube was charged with S-phos (0.22 g, 0.53 mmol),
palladium acetate (0.060 g, 0.27 mmol), potassium phosphate (8.5 g,
40 mmol) and 5-bromo-2-methoxypyridine (1.8 mL, 13 mmol) under
argon. 9-Benzyl-9-bora-bicyclo[3.3.1]nonane 0.5M in THF (53 mL, 27
mmol) was added, the vessel was sealed and heated to 60.degree. C.
overnight. The mixture was diluted with diethyl ether and was
filtered through a pad of celite. Evaporation of the filtrate and
purification by flash chromatography (0-40% EtOAc/hexanes) gave
5-benzyl-2-methoxypyridine. MS (ESI) m/z: Calculated: 199.1.
Observed: 200.1 (M.sup.++1).
5-Benzyl-pyridin-2-ol
##STR00364##
[0909] 5-Benzyl-2-methoxypyridine (2.6 g, 13 mmol) in 15 mL AcOH
was added 15 mL 48% HBr. The mixture was heated to 140.degree. C.
for 3 h, cooled and poured onto ice. The mixture was basified to pH
6-7 and the resulting solid was collected by filtration, rinsed
with water, and dried in vacuo to give 5-benzylpyridin-2-ol as an
off-white solid. MS (ESI) m/z: Calculated: 185.1. Observed: 186.0
(M.sup.++1).
5-Benzyl-3-iodopyridin-2-ol
##STR00365##
[0911] To a yellow solution of 5-benzylpyridin-2-ol (1.10 g, 5.94
mmol) in 22 mL AcOH under Ar was added 1.5 mL TFA, followed by
N-iodosuccinimide (1.34 g, 5.94 mmol). The red homogeneous solution
was allowed to stir overnight, poured onto ice and neutralized with
conc. NH.sub.4OH. The solids were collected by filtration, rinsed
with water, treated with MeOH/DCM, dried over sodium sulfate, and
evaporated. The resulting brown solid was purified by silica gel
chromatography (ISCO, 80 g, 0-70% 90/10 DCM/MeOH in DCM) to give
5-benzyl-3-iodopyridin-2-ol as a yellow/brown solid. MS (ESI) m/z:
Calculated: 311.0. Observed: 311.9 (M.sup.++1).
Methyl
1-((3-fluoro-4-(5-(phenylmethyl)furo[2,3-b]pyridin-2-yl)phenyl)meth-
yl)-3-azetidinecarboxylate
##STR00366##
[0913] Synthesized according to Scheme B3 and general procedure G
from 5-benzyl-3-iodopyridin-2-ol (0.400 g, 1.29 mmol) and methyl
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (0.381 g, 1.54
mmol): light yellow solid. MS (ESI) m/z: Calculated: 430.2.
Observed: 431.1 (M.sup.++1).
1-((3-Fluoro-4-(5-(phenylmethyl)furo[2,3-b]pyridin-2-yl)phenyl)methyl)-3-a-
zetidinecarboxylic acid
##STR00367##
[0915] Synthesized according to Scheme B3 and general procedure H
from methyl
1-(4-(5-benzylfuro[2,3-b]pyridin-2-yl)-3-fluorobenzyl)azetidine-3--
carboxylate (0.146 g, 0.339 mmol): white solid [hS1P1 EC.sub.50=27
nM]. 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.26 (d, J=1.5 Hz,
1H), 7.97 (d, J=2.0 Hz, 1H), 7.92 (t, J=8.0 Hz, 1H), 7.25-7.35 (m,
7H), 7.16-7.25 (m, 1H), 4.09 (s, 2H), 3.62 (s, 2H), 3.37-3.48 (m,
2H), 3.26-3.36 (m, 1H), 3.16-3.25 (m, 2H). MS (ESI) m/z:
Calculated: 416.2. Observed: 417.2 (M.sup.++1).
Compound 99
1-((3-Fluoro-4-(5-(phenylmethyl)-1H-indol-2-yl)phenyl)methyl)-3-azetidinec-
arboxylic acid
Methyl
1-(4-(5-benzyl-1H-indol-2-yl)-3-fluorobenzyl)azetidine-3-carboxylat-
e
##STR00368##
[0917] To a sealed flask was added 4-benzyl-2-iodobenzenamine (0.20
g, 0.65 mmol) and methyl
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (0.32 g, 1.3
mmol) in THF (15 mL). To this solution was added
PdCl.sub.2(PPh.sub.3).sub.2 (0.045 g, 0.065 mmol), CuI (0.025 g,
0.13 mmol), and ethyldiisopropylamine (0.90 mL, 5.2 mmol). The
flask was flushed with Ar, sealed and placed in a preheated oil
bath at 100.degree. C. for 3 h. The mixture was concentrated under
reduced pressure to afford a dark oil which was adsorbed onto
silica and purified (30% EtOAc/hexanes to 100% EtOAc) to afford
title compound as a yellow oil. MS (ESI) m/z: Calculated: 428.1.
Observed: 429.1 (M.sup.++1).
1-((3-Fluoro-4-(5-(phenylmethyl)-1H-indol-2-yl)phenyl)methyl)-3-azetidinec-
arboxylic acid
##STR00369##
[0919] To a solution of methyl
1-(4-(5-benzyl-1H-indol-2-yl)-3-fluorobenzyl)azetidine-3-carboxylate
(0.090 g, 0.21 mmol) in THF (1.0 mL) and water (1.0 mL) was added
lithium hydroxide (0.025 g, 1.1 mmol). The solution was stirred at
rt for 1 h, or until starting material was no longer observed by
LCMS. The solvent was removed under reduced pressure, and 0.5M
phosphate buffer (3.0 mL, pH 6.0) was added (pH was ca. 9-10). The
mixture was acidified with 1N HCl to pH 6.0 and sonicated for 5
min. The aqueous solution was extracted with EtOAc (3.times.15 mL),
and the separated organic extracts were dried over MgSO.sub.4,
filtered and concentrated to afford a brown oil. The oil was
dissolved in AcOH (3.0 mL), and the excess AcOH was removed under
reduced pressure. Ether (15 mL) was added, and the resulting
precipitate was collected by filtration. The brown solids were
washed with water (10 mL) and chloroform (15 mL) to afford title
compound as a yellow solid [hS1P1 EC.sub.50=1336 nM]. MS (ESI) m/z:
Calculated: 414.1. Observed: 415.1 (M.sup.++1).
Compound 100
1-((3-Fluoro-4-(5-(pyrimidin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azet-
idine-3-carboxylic acid
(4-(Bromomethyl)phenoxy)(tert-butyl)dimethylsilane
##STR00370##
[0921] 4-Hydroxybenzaldehyde (8.86 mL, 81.9 mmol) was added as a
solution in THF (50 mL) to a suspension of sodium hydride (2.36 g,
98.3 mmol) in THF (100 mL) via syringe. The mixture was stirred
until gas evolution stopped and chloro-tert-butyldimethylsilane
(18.5 g, 123 mmol) was added as a solution in THF (50 mL). The
reaction was stirred for 1.5 h and quenched with 1N NaOH. The
mixture was extracted with EtOAc twice and the extracts were washed
with water once, brine once, dried over MgSO.sub.4 and concentrated
in vacuo to give an oil.
[0922] The oil was dissolved in EtOH (200 mL) and cooled to
0.degree. C. Sodium borohydride (3.41 g, 90.1 mmol) was added and
the mixture was stirred at 0.degree. C. for 30 min. The reaction
was quenched with satd. NH.sub.4Cl and extracted with Et.sub.2O
twice. The organic extracts were dried over MgSO.sub.4 and
concentrated to give an oil, which was purified by column
chromatography to an oil.
[0923] Methanesulfonyl chloride (0.56 mL, 7.06 mmol) was added
dropwise to a solution of triethylamine (1.79 mL, 12.8 mmol) and
the above oil (1.53 g, 6.42 mmol) in DCM (25 mL) at 0.degree. C.
The mixture was stirred for 1 h at 0.degree. C., Et.sub.2O was
added and the mixture was washed with water once, 1N HCl once,
satd. NaHCO.sub.3 once, dried over MgSO.sub.4 and concentrated in
vacuo to give an oil. This oil was dissolved in acetone (50 mL) and
lithium bromide (2.79 g, 32.1 mmol) was added. The mixture was
stirred at reflux for 15 min, cooled to room temperature, diluted
with Et.sub.2O, and washed with water twice, sodium thiosulfate
once, dried over MgSO.sub.4, and concentrated to give
(4-(bromomethyl)phenoxy)(tert-butyl)dimethylsilane as a
light-yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 7.26
(2H, d, J=8.5 Hz), 6.79 (2H, d, J=8.0 Hz), 4.48 (2H, s), 0.98 (9H,
s), 0.19 (6H, m).
2-(4-(tert-Butyldimethylsilyloxy)benzyl)pyrimidine
##STR00371##
[0925] Zinc (0.33 g, 4.97 mmol) and iodine (0.008 g, 0.033 mmol)
were added to an oven-dried flask (25 mL). The mixture was heated
under vacuum with a heat gun for 10 min, cooled to RT, placed under
argon atmosphere, suspended in 3 mL DMF (dry, degassed by bubbling
nitrogen through it for 10 min), and cooled to 0.degree. C.
(4-(Bromomethyl)phenoxy)(tert-butyl)dimethylsilane (1.00 g, 3.31
mmol) was added as a solution in 3 mL DMF, the mixture was stirred
at 0.degree. C. for 30 min, then room temperature for 30 min. Next,
2-bromopyrimidine (526 mg, 3.31 mmol), Pd.sub.2 dba.sub.3 (0.091
mg, 0.099 mmol), and S-Phos (0.163 g, 0.40 mmol) were added. The
flask was purged with argon and heated for 1 h at 60.degree. C.,
cooled to RT, filtered through Celite, and concentrated in vacuo.
The resulting solid was purified by flash chromatography to give
title compound as a light yellow oil. MS (ESI) m/z: Calculated:
300.2. Observed: 301.5 (M.sup.++1).
4-(Pyrimidin-2-ylmethyl)phenol
##STR00372##
[0927] Tetrabutylammonium fluoride (1.68 mL, 1.68 mmol, 1M in THF)
was added to a solution of
2-(4-(tert-butyldimethylsilyloxy)benzyl)pyrimidine (0.50 g, 1.68
mmol) in THF (5 mL) at 0.degree. C. The mixture was stirred for 30
min, diluted with water, and extracted with DCM three times. The
combined organic extracts were dried over MgSO.sub.4, concentrated
in vacuo, and purified by column chromatography to give
4-(pyrimidin-2-ylmethyl)phenol as an oil. MS (ESI) m/z: Calculated:
186.1. Observed: 187.3 (M.sup.++1).
2-Iodo-4-(pyrimidin-2-ylmethyl)phenol
##STR00373##
[0929] Bis(pyridine)iodonium tetrafluoroborate (379 mg, 1.02 mmol)
was added to a solution of 4-(pyrimidin-2-ylmethyl)phenol (200 mg,
1.07 mmol) in 9:1 DCM:TFA (6 mL) at 0.degree. C. The mixture was
stirred for 1 h, partitioned between EtOAc and water, the layers
were separated, and the organic layer was washed with satd. sodium
thiosulfate. The organic layer was dried over MgSO.sub.4,
concentrated in vacuo, and purified by column chromatography to
give 2-iodo-4-(pyrimidin-2-ylmethyl)phenol. MS (ESI) m/z:
Calculated: 312.0. Observed: 313.0 (M.sup.++1).
Methyl
1-((3-fluoro-4-(5-(pyrimidin-2-ylmethyl)benzofuran-2-yl)phenyl)meth-
yl)azetidine-3-carboxylate
##STR00374##
[0931] Synthesized according to Scheme B3 and general procedure G
from 2-iodo-4-(pyrimidin-2-ylmethyl)phenol (0.230 g, 0.74 mmol) and
methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (0.220
g, 0.89 mmol). MS (ESI) m/z: Calculated: 431.2. Observed: 432.5
(M.sup.++1).
1-((3-Fluoro-4-(5-(pyrimidin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azet-
idine-3-carboxylic acid
##STR00375##
[0933] Synthesized according to Scheme B3 and general procedure H
from methyl
1-((3-fluoro-4-(5-(pyrimidin-2-ylmethyl)benzofuran-2-yl)phenyl)met-
hyl)azetidine-3-carboxylate (0.180 g, 0.42 mmol): white solid
[hS1P1 EC.sub.50=87 nM]. 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
8.74 (d, J=4.9 Hz, 2H), 7.90 (t, J=8.0 Hz, 1H), 7.61 (s, 1H), 7.55
(d, J=8.4 Hz, 1H), 7.35 (t, J=4.9 Hz, 1H), 7.21-7.32 (m, 4H), 4.30
(s, 2H), 3.61 (s, 2H), 3.38-3.46 (m, 2H), 3.19-3.26 (m, 3H). MS
(ESI) m/z: Calculated: 417.2. Observed: 418.5 (M.sup.++1).
Compound 101
1-((3-fluoro-4-(5-(pyridin-3-ylmethyl)benzofuran-2-yl)phenyl)methyl)azetid-
ine-3-carboxylic acid
3-(4-Methoxybenzyl)pyridine
##STR00376##
[0935] Zinc (0.976 g, 14.9 mmol) and iodine (0.025 g, 0.100 mmol)
were added to a flame-dried flask (25 mL) and heated with a heat
gun under vacuum for 10 min. The flask was cooled to 0.degree. C.
and DMF (5 mL) was added. 1-(Bromomethyl)-4-methoxybenzene (1.43
mL, 9.95 mmol) was added as a solution in DMF (5 mL) and the
mixture was stirred for 20 min at 0.degree. C., then RT for 20 min.
S-Phos (0.490 g, 1.19 mmol), Pd.sub.2 dba.sub.3 (0.273 g, 0.299
mmol), and 3-bromopyridine (0.976 mL, 9.95 mmol) were added and the
mixture was heated 60.degree. C. for 1 h. The mixture was cooled to
RT, filtered through celite, concentrated in vacuo, and purified by
column chromatography to give 3-(4-methoxybenzyl)pyridine as a
light-yellow oil. MS (ESI) m/z: Calculated: 199.1. Observed: 200.3
(M.sup.++1).
3-(3-Iodo-4-methoxybenzyl)pyridine
##STR00377##
[0937] Bis(pyridine)iodonium tetrafluoroborate (1.35 g, 3.64 mmol)
was added to a solution of 3-(4-methoxybenzyl)pyridine (720 mg,
3.64 mmol) in 9:1 TFA:DCM (20 mL) and the mixture was stirred for 1
h at 0.degree. C. The ice bath was removed and the reaction was
stirred for 2 h. EtOAc was added and the mixture was washed with
water twice, satd. sodium thiosulfate twice, dried over MgSO.sub.4,
concentrated in vacuo, and purified by column chromatography to
give 3-(3-iodo-4-methoxybenzyl)pyridine as a yellow oil. MS (ESI)
m/z: Calculated: 325.0. Observed: 326.3 (M.sup.++1).
2-Iodo-4-(pyridin-3-ylmethyl)phenol
##STR00378##
[0939] Boron tribromide (11.1 mL, 11.1 mmol, 1M in DCM) was added
to a solution of 3-(3-iodo-4-methoxybenzyl)pyridine (720 mg, 2.21
mmol) in DCM (3 mL) at 0.degree. C., and the mixture was stirred
for 1 h. The mixture was quenched by the careful addition of water,
the layers were separated, and the organic layer was washed with
water three times. The combined organic extracts were dried over
MgSO.sub.4, concentrated in vacuo, and purified by column
chromatography to give 2-iodo-4-(pyridin-3-ylmethyl)phenol as a
yellow oil. MS (ESI) m/z: Calculated: 311.0. Observed: 312.3
(M.sup.++1).
Methyl-1-((3-fluoro-4-(5-(pyridin-3-ylmethyl)benzofuran-2-yl)phenyl)methyl-
)azetidine-3-carboxylate
##STR00379##
[0941] Synthesized according to Scheme B3 and general procedure G
from 2-iodo-4-(pyridin-3-ylmethyl)phenol (0.305 g, 0.98 mmol) and
methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (0.220
g, 0.89 mmol): yellow solid. MS (ESI) m/z: Calculated: 430.2.
Observed: 431.5 (M.sup.++1).
1-((3-Fluoro-4-(5-(pyridin-3-ylmethyl)benzofuran-2-yl)phenyl)methyl)azetid-
ine-3-carboxylic acid
##STR00380##
[0943] Synthesized according to Scheme B3 and general procedure H
from methyl
1-((3-fluoro-4-(5-(pyridin-3-ylmethyl)benzofuran-2-yl)phenyl)methy-
l)azetidine-3-carboxylate (0.068 g, 0.16 mmol): white solid. 1H NMR
(400 MHz, MeOH) .delta. ppm 8.47 (s, 1H), 8.39 (d, J=4.5 Hz, 1H),
8.11 (dd, J=8.2, 7.6 Hz, 1H), 7.73 (d, 6.4 Hz, 1H), 7.50-7.55 (m,
2H), 7.43 (s, 1H), 7.35-7.42 (m, 2H), 7.29 (d, J=2.9 Hz, 1H), 7.25
(d, J=8.6 Hz, 1H), 4.40 (s, 2H), 4.20-4.31 (m, 4H), 4.15 (s, 2H),
3.53 (dt, J=8.8, 7.8 Hz, 1H). MS (ESI) m/z: Calculated: 416.2.
Observed: 417.5 (M.sup.++1).
Compound 102
1-((4-(5-Benzoylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxy-
lic acid
(4-Hydroxy-3-iodophenyl)(phenyl)methanone
##STR00381##
[0945] A solution of (4-hydroxyphenyl)(phenyl)methanone (2.0 g, 10
mmol) in saturated aqueous ammonium hydroxide (130 mL) was stirred
at 25.degree. C. for 15 min, and then treated with a solution of
potassium iodide (8.2 g, 49 mmol) and iodine (2.6 g, 10 mmol) in
water (260 mL). After 30 min, the mixture was adjusted to pH 2 with
concentrated aqueous HCl and extracted with EtOAc (500 mL). The
organic extract was sequentially washed with sat. aqueous sodium
thiosulfate (100 mL) and brine (100 mL), dried over sodium sulfate,
filtered, and concentrated in vacuo. Chromatographic purification
of the residue (ISCO, 40 g, 0->80% EtOAc/hexanes) gave
(4-hydroxy-3-iodophenyl)(phenyl)methanone as a white solid. MS
(ESI) m/z: Calculated: 324.0. Observed: 325 (M.sup.++1).
Methyl
1-(4-(5-benzoylbenzofuran-2-yl)-3-fluorobenzyl)azetidine-3-carboxyl-
ate
##STR00382##
[0947] Synthesized according to Scheme B3, Step 3 and general
procedure G from (4-hydroxy-3-iodophenyl)(phenyl)methanone and
methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate:
yellow-orange solid. MS (ESI) m/z: Calculated: 443.2. Observed: 444
(M.sup.++1).
1-((4-(5-Benzoylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxy-
lic acid
##STR00383##
[0949] Synthesized according to Scheme B3, Step 4 and general
procedure H from methyl
1-(4-(5-benzoylbenzofuran-2-yl)-3-fluorobenzyl)azetidine-3-carboxylate:
off-white solid [hS1P1 EC.sub.50=80 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.13 (s, 1H), 7.97 (t, J=8.0 Hz, 1H),
7.80-7.85 (m, 2H), 7.75-7.80 (m, 2H), 7.70 (t, J=7.4 Hz, 1H), 7.59
(t, J=7.5 Hz, 2H), 7.45 (d, J=3.1 Hz, 1H), 7.32 (d, J=4.1 Hz, 1H),
7.30 (s, 1H), 3.63 (s, 2H), 3.40-3.48 (m, 2H), 3.32 (s, 2H),
3.19-3.27 (m, 2H). MS (ESI) m/z: Calculated: 429.1. Observed: 430
(M.sup.++1).
Compound 103
(E/Z)-1-((3-Fluoro-4-(5-((hydroxyimino)(phenyl)methyl)benzofuran-2-yl)phen-
yl)methyl)azetidine-3-carboxylic acid
##STR00384##
[0951] Hydroxylamine, 50 wt. %, solution in water (146 .mu.L, 2375
.mu.mol) was added to a solution of
1-((4-(5-benzoylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carbox-
ylic acid (34.0 mg, 79 .mu.mol) in acetic acid (0.560 mL), and the
resulting yellow solution was stirred at 60.degree. C. for 15 h. 1
M pH 6 phosphate buffer (6.0 mL) was added to the resulting
solution, and the mixture was sonicated for 1 min. The resulting
white slurry was filtered, and the collected solid was washed with
water (12 mL) and EtOH (10 mL) to afford title compound as a light
yellow solid [hS1P1 EC.sub.50=16 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 11.34 (s, 0.5H), 11.25 (s, 0.5H),
7.87-8.00 (m, 1H), 7.72 (d, J=8.5 Hz, 1H), 7.61-7.68 (m, 1H), 7.59
(s, 1H), 7.44-7.52 (m, 2H), 7.42 (dd, J=7.3, 3.3 Hz, 1H), 7.30-7.39
(m, 3H), 7.24-7.30 (m, 2H), 3.62 (d, J=5.5 Hz, 2H), 3.39-3.48 (m,
3H), 3.23 (d, J=4.5 Hz, 2H). MS (ESI) m/z: Calculated: 444.2.
Observed: 445 (M.sup.++1).
Compound 104
(E/Z)-1-((3-Fluoro-4-(5-((methoxyimino)(phenyl)methyl)benzofuran-2-yl)phen-
yl)methyl)azetidine-3-carboxylic acid, trifluoroacetic acid
salt
##STR00385##
[0953] O-Methyl hydroxylamine hydrochloride, 25% in water (257
.mu.L, 848 .mu.mol) was added to a solution of
1-((4-(5-benzoylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carbox-
ylic acid (36.4 mg, 85 .mu.mol) and sodium acetate (70 mg, 848
.mu.mol) in acetic acid (700 .mu.L; 0.13M), and the resulting
yellow solution was stirred at 70.degree. C. for 17 h. The solution
was filtered through a cotton plug and concentrated in vacuo. The
residue was taken up in MeOH, filtered through a cotton plug, and
purified by HPLC (Phenomenex C18, 5-100% CH.sub.3CN/H.sub.2O+0.1%
TFA) to provide
(E/Z)-1-(3-fluoro-4-(5-((methoxyimino)(phenyl)methyl)benzofuran-2-yl)phen-
yl)methyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt as
a white solid. 1H NMR (400 MHz, MeOH-d.sub.4) .delta. ppm 8.18-8.23
(m, 0.5H), 8.16 (t, J=7.0 Hz, 0.5H), 7.68 (d, J=1.6 Hz, 0.5H),
7.64-7.67 (m, 0.5H), 7.59-7.62 (m, 0.5H), 7.55-7.59 (m, 0.5H),
7.44-7.51 (m, 4H), 7.40-7.44 (m, 2H), 7.37-7.40 (m, 1H), 7.30-7.37
(m, 2H), 4.51 (s, 1H), 4.50 (s, 1H), 4.34-4.44 (m, 4H), 3.96 (s,
1.5H), 3.96 (s, 1.5H), 3.67-3.79 (m, 1H). MS (ESI) m/z: Calculated:
458.2. Observed: 459 (M.sup.++1).
Compound 105
(E/Z)-1-((3-Fluoro-4-(5-((ethoxyimino)(phenyl)methyl)benzofuran-2-yl)pheny-
l)methyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt
##STR00386##
[0955] O-Ethyl hydroxylamine hydrochloride (86 mg, 885 .mu.mol) was
added to a solution of
1-((4-(5-benzoylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carbox-
ylic acid (38.0 mg, 88 .mu.mol) and sodium acetate (73 mg, 885
.mu.mol) in acetic acid (700 .mu.L; 0.13M) and water (200 .mu.L)
and the resulting yellow solution was stirred at 70.degree. C. for
17 h. The solution was concentrated in vacuo, and the residue was
taken up in MeOH, filtered through a cotton plug, and purified by
HPLC (Phenomenex C18, 5-100% CH.sub.3CN/H.sub.2O+0.1% TFA) to
provide
(E/Z)-1-((4-(5-((ethoxyimino)(phenyl)methyl)benzofuran-2-yl)-3-fluorophen-
yl)methyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt as
a white solid. 1H NMR (400 MHz, MeOD-d.sub.4) .delta. ppm 8.18-8.23
(m, 0.5H), 8.14-8.18 (m, 0.5H), 7.66-7.69 (m, 0.5H), 7.66 (d, J=3.7
Hz, 0.5H), 7.58-7.62 (m, 0.5H), 7.55-7.58 (m, 0.5H), 7.44-7.51 (m,
4H), 7.40-7.44 (m, 1H), 7.31-7.40 (m, 4H), 4.51 (s, 1H), 4.50 (s,
1H), 4.34-4.44 (m, 4H), 4.24-4.27 (m, 1H), 4.20-4.24 (m, 1H),
3.68-3.77 (m, 1H), 1.30 (t, J=7.0 Hz, 3H). MS (ESI) m/z:
Calculated: 472.2. Observed: 473 (M.sup.++1).
Compound 106
(.+-.)-1-((3-Fluoro-4-(5-(hydroxy(phenyl)methyl)benzofuran-2-yl)phenyl)met-
hyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt
(.+-.)-Methyl
1-((3-fluoro-4-(5-(hydroxy(phenyl)methyl)benzofuran-2-yl)phenyl)methyl)-a-
zetidine-3-carboxylate
##STR00387##
[0957] To solution of methyl
1-((4-(5-benzoylbenzofuran-2-yl)-3-fluorophenyl)methyl)-azetidine-3-carbo-
xylate (190 mg, 428 .mu.mol) in MeOH (5.0 mL; 0.14M) and THF (1.0
mL) was added sodium borohydride (130 mg, 3438 .mu.mol) at
0.degree. C., and the resulting mixture was stirred at 0.degree. C.
for 10 min, then at 25.degree. C. for 30 min. Saturated aqueous
ammonium chloride (10 mL) was added, and the resulting mixture was
extracted twice with EtOAc (30 mL). The combined extracts were
washed with brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. Chromatographic purification of the residue
(ISCO, 4 g, 0-100% EtOAc/hexanes) furnished (.+-.)-methyl
1-((3-fluoro-4-(5-(hydroxy(phenyl)methyl)benzofuran-2-yl)phenyl)methyl)az-
etidine-3-carboxylate as a clear oil. MS (ESI) m/z: Calculated:
445.2. Observed: 446 (M.sup.++1).
(.+-.)-1-((3-Fluoro-4-(5-(hydroxy(phenyl)methyl)benzofuran-2-yl)phenyl)met-
hyl)azetidine-3-carboxylic acid, trifluoroacetic acid salt
##STR00388##
[0959] Lithium hydroxide monohydrate (17.6 mg, 420 .mu.mol) in
water (2.0 mL) was added to a solution of (.+-.)-methyl
1-((3-fluoro-4-(5-(hydroxy(phenyl)methyl)benzofuran-2-yl)phenyl)methyl)az-
etidine-3-carboxylate (62.3 mg, 140 .mu.mol) in THF (3.0 mL) at
25.degree. C., and the resulting solution was stirred for 1.5 h. 2N
HCl (0.210 mL) was added, and the resulting solution was
concentrated in vacuo. The residue was taken up in DMSO (3.0 mL),
filtered through cotton plug, and purified by HPLC (Phenomenex C18,
5-50-100% CH.sub.3CN/H.sub.2O+0.1% TFA) to furnish
1-((3-fluoro-4-(5-(hydroxy(phenyl)methyl)benzofuran-2-yl)phenyl)-methyl)a-
zetidine-3-carboxylic acid, trifluoroacetic acid salt as a white
solid. 1H NMR (400 MHz, MeOH-d.sub.4) .delta. ppm 8.13 (t, J=8.0
Hz, 1H), 7.70 (d, J=1.8 Hz, 1H), 7.51 (d, J=8.6 Hz, 1H), 7.40-7.46
(m, 4H), 7.38 (dd, J=8.6, 1.8 Hz, 1H), 7.30-7.36 (m, 3H), 7.21-7.27
(m, 1H), 5.91 (s, 1H), 4.47 (s, 2H), 4.38 (d, J=3.1 Hz, 2H), 4.36
(d, J=1.2 Hz, 2H), 3.65-3.75 (m, 1H). MS (ESI) m/z: Calculated:
431.2. Observed: 432 (M.sup.++1).
Compound 107
1-((4-(5-Benzyl-7-chlorofuro[2,3-c]pyridin-2-yl)-3-fluorophenyl)methyl)aze-
tidine-3-carboxylic acid
2-Chloro-6-iodopyridin-3-ol
##STR00389##
[0961] Iodine (2.38 mL, 46.3 mmol) was added to a solution of
2-chloro-3-pyridinol (5.00 g, 38.6 mmol) in water (60 mL)
containing potassium carbonate (18.7 g, 135 mmol). The resulting
solution was allowed to stir for 2 h at room temperature. The
mixture was treated with sodium thiosulfate, acidified to pH 2
using 12M aqueous HCl, and extracted with EtOAc. The organic phase
was dried over MgSO.sub.4 and evaporated. Purification by flash
chromatography using EtOAc/hexanes gave
2-chloro-6-iodopyridin-3-ol. 1H NMR (300 MHz, CDCl.sub.3) .delta.
ppm 7.56 (d, J=8.2 Hz, 1H), 7.02 (d, J=8.3 Hz, 1H). MS (ESI) m/z:
Calculated; 254.9. Observed: 255.8 (M.sup.++1).
6-Benzyl-2-chloropyridin-3-ol
##STR00390##
[0963] A mixture of K.sub.3PO.sub.4 (12 g, 55 mmol), S-Phos (0.45
g, 1.1 mmol), and palladium acetate (0.21 g, 0.91 mmol) in THF (55
mL) was prepared in a sealed tube. The mixture was treated with
2-chloro-6-iodopyridin-3-ol (4.67 g, 18 mmol) and
.beta.-benzyl-9-BBN (0.5M solution in THF, 73 mL, 37 mmol) and
heated to 80.degree. C. for 2 h. The crude mixture was diluted with
EtOAc, washed with 2M aqueous NaOH, brine, dried over MgSO.sub.4,
and evaporated. Purification by flash chromatography
(EtOAc/hexanes) gave 6-benzyl-2-chloropyridin-3-ol. 1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 7.11-7.41 (m, 6H), 6.94 (d, J=8.2 Hz,
1H), 5.49 (br. s, 1H), 4.06 (s, 2H). MS (ESI) m/z: Calculated;
219.1. Observed: 220.0 (M.sup.++1).
6-Benzyl-2-chloro-4-iodopyridin-3-ol
##STR00391##
[0965] Iodine (5.87 g, 23.1 mmol) was added to a solution of
6-benzyl-2-chloropyridin-3-ol (2.54 g, 11.6 mmol) in water (45 mL)
containing potassium carbonate (3.20 g, 23.1 mmol). The mixture was
allowed to stir for 3 h at room temperature, treated with sodium
thiosulfate, and acidified to pH 2 using 12M aqueous HCl. The
mixture was extracted with EtOAc. The organic phase was dried over
MgSO.sub.4 and evaporated. Purification by flash chromatography
using EtOAc/hexanes gave 6-benzyl-2-chloro-4-iodopyridin-3-ol. 1H
NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 10.36 (br. s, 1H), 7.68 (s,
1H), 7.15-7.35 (m, 5H), 3.93 (s, 2H). MS (ESI) m/z: Calculated;
345.0. Observed: 345.9 (M.sup.++1).
Methyl
1-((4-(5-benzyl-7-chlorofurofluorophenyl)methyl)azetidine-3-carboxy-
late
##STR00392##
[0967] Synthesized according to Scheme B3 and general procedure G
from 6-benzyl-2-chloro-4-iodopyridin-3-ol (1.00 g, 2.89 mmol) and
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (0.859 g, 3.47
mmol). 1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 8.03 (t, J=7.8 Hz,
1H), 6.98-7.40 (m, 9H), 4.22 (s, 2H), 3.72 (s, 3H), 3.66 (s, 2H),
3.52-3.61 (m, 2H), 3.26-3.40 (m, 3H). MS (ESI) m/z: calculated;
464.1. Observed: 465.0 (M.sup.++1).
1-((4-(5-Benzyl-7-chlorofuro[2,3-c]pyridin-2-yl)-3-fluorophenyl)methyl)aze-
tidine-3-carboxylic acid
##STR00393##
[0969] Synthesized according to Scheme B3 and general procedure H
from methyl
1-((4-(5-benzyl-7-chlorofurofluorophenyl)methyl)azetidine-3-carbox-
ylate [hS1P1 EC.sub.50=2583 nM]. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 7.95 (br. t, J=8.0 Hz, 1H), 7.59 (br. s, 1H), 7.16-7.45
(m, 8H), 4.15 (br. s, 2H), 3.63 (br. s, 2H), 3.42 (br. s, 2H),
3.17-3.26 (m, 3H). MS (ESI) m/z: Calculated; 450.1. Observed: 451.0
(M.sup.++1).
Compound 108
1-((4-(5-Benzylfuro[2,3-c]pyridin-2-yl)-3-fluorophenyl)methyl)azetidine-3--
carboxylic acid
Methyl
1-((4-(5-benzylfuro[2,3-c]pyridin-2-yl)-3-fluorophenyl)methyl)azeti-
dine-3-carboxylate
##STR00394##
[0971] A mixture of methyl
1-(4-(5-benzyl-7-chlorofuro[2,3-c]pyridin-2-yl)-3-fluorobenzyl)azetidine--
3-carboxylate (0.320 g, 0.69 mmol), cyclohexene (1.00 mL, 9.9
mmol), palladium (10 wt. % on activated carbon, 0.320 g, 3.0 mmol)
in EtOH (6.0 mL) was prepared in a sealed tube and heated under
N.sub.2 to 85.degree. C. for 4 h. The mixture was filtered through
Celite, and the filtrate evaporated purified by flash
chromatography using EtOAc/hexanes to give title compound. 1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 8.82 (s, 1H), 7.96 (t, J=7.7 Hz,
1H), 6.99-7.38 (m, 9H), 4.25 (s, 2H), 3.72 (s, 3H), 3.65 (s, 2H),
3.48-3.59 (m, 2H), 3.33-3.39 (m, 3H). MS (ESI) m/z: Calculated;
430.2. Observed: 431.2 (M.sup.++1).
1-((4-(5-Benzylfuro[2,3-c]pyridin-2-yl)-3-fluorophenyl)methyl)azetidine-3--
carboxylic acid
##STR00395##
[0973] Synthesized according to Scheme B3 and general procedure H
from methyl
1-((4-(5-benzylfuro[2,3-c]pyridin-2-yl)-3-fluorophenyl)methyl)azet-
idine-3-carboxylate [hS1P1 EC.sub.50=409 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.88 (br. s, 1H), 7.96 (br. t, J=8.0 Hz,
1H), 7.57 (s, 1H), 7.16-7.34 (m, 8H), 4.17 (br. s, 2H), 3.62 (br.
s, 2H), 3.40-3.45 (m, 2H), 3.20-3.25 (m, 3H). MS (ESI) m/z:
Calculated; 416.2. Observed: 417.1 (M.sup.++1).
Compound 109
1-((4-(5-Benzylfuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)methyl)azetidine-3--
carboxylic acid
6-Benzylpyridin-3-ol
##STR00396##
[0975] In a sealed tube, a mixture of K.sub.3PO.sub.4 (16 g, 78
mmol), S-Phos (0.64 g, 1.6 mmol), and palladium acetate (0.29 g,
1.3 mmol) in THF (70 mL) was treated with 6-bromopyridin-3-ol (4.50
g, 26 mmol) and .beta.-benzyl-9-BBN (0.5M solution in THF, 103 mL,
52 mmol). The mixture was heated to 80.degree. C. for 18 h and
diluted with EtOAc. The organic phase was washed with 2M aqueous
NaOH, brine, dried over MgSO.sub.4, and concentrated. Purification
by flash chromatography using EtOAc/hexanes gave
6-benzylpyridin-3-ol. 1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm
9.66 (br. s, 1H), 8.03 (t, J=1.8 Hz, 1H), 7.13-7.30 (m, 4H), 7.07
(d, J=1.9 Hz, 2H), 3.95 (s, 2H). MS (ESI) m/z: Calculated; 185.1.
Observed: 186.1 (M.sup.++1).
6-Benzyl-2-iodopyridin-3-ol
##STR00397##
[0977] Iodine (3.21 g, 12.6 mmol) was added to a solution of
6-benzylpyridin-3-ol (2.34 g, 12.6 mmol) and sodium carbonate
anhydrous (1.06 mL, 25.3 mmol) in water (60 mL) and THF (60 mL).
The mixture was allowed to stir for 1 h at room temperature,
treated with sodium thiosulfate, and acidified pH 3 using 5M
aqueous HCl. Extractive workup and flash chromatography using
EtOAc/hexanes gave 6-benzyl-2-iodopyridin-3-ol. 1H NMR (300 MHz,
DMSO-d.sub.6) .delta. ppm 10.62 (s, 1H), 7.13-7.34 (m, 5H),
6.99-7.11 (m, 2H), 3.95 (s, 2H). MS (ESI) m/z: Calculated; 311.0.
Observed: 311.9 (M.sup.++1).
Methyl
1-((4-(5-benzylfuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)methyl)azeti-
dine-3-carboxylate
##STR00398##
[0979] Synthesized according to Scheme B3 and general procedure G
from 6-benzyl-2-iodopyridin-3-ol (0.500 g, 1.61 mmol) and methyl
1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate (0.477 g, 1.93
mmol). 1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.93 (t, J=7.9 Hz,
1H), 7.65 (d, J=8.5 Hz, 1H), 7.37 (d, J=3.1 Hz, 1H), 7.10-7.33 (m,
7H), 7.04 (d, J=8.5 Hz, 1H), 4.27 (s, 2H), 3.72 (s, 3H), 3.65 (s,
2H), 3.48-3.60 (m, 2H), 3.30-3.40 (m, 3H). MS (ESI) m/z:
Calculated; 430.2. Observed: 431.1 (M.sup.++1).
1-((4-(5-Benzylfuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)methyl)azetidine-3--
carboxylic acid
##STR00399##
[0981] Synthesized according to Scheme B3 and general procedure H
from methyl
1-(4-(5-benzylfuro[3,2-b]pyridin-2-yl)-3-fluorobenzyl)azetidine-3--
carboxylate [hS1P1 EC.sub.50=409 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.82-8.07 (m, 2H), 7.20-7.42 (m, 8H),
7.14-7.23 (m, 1H), 4.19 (s, 2H), 3.62 (s, 2H), 3.37-3.49 (m, 2H),
3.14-3.29 (m, 3H). MS (ESI) m/z: Calculated: 416.2. Observed: 417.1
(M.sup.++1).
Compound 110
1-((4-(6-Benzyl-5-chlorofuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)methyl)aze-
tidine-3-carboxylic acid
5-Benzyl-6-chloropyridin-3-ol
##STR00400##
[0983] Starting material (5-bromo-6-chloropyridin-3-ol) is
available from a published 3-step procedure: Synthesis, 1990,
499-501. To an oven-dried flask was added zinc (2.45 g, 37.4 mmol)
and a crystal of iodine. The mixture was heated with a heat gun for
10 min under vacuum and then allowed to cool under Ar atmosphere.
After charging the flask with degassed DMF (10 mL), the mixture was
cooled to 0.degree. C., and benzyl bromide (2.96 mL, 24.9 mmol) was
added. After stirring the resulting mixture at 0.degree. C. for 30
min, a mixture of Pd.sub.2(dba).sub.3 (0.571 g, 0.624 mmol), S-Phos
(1.02 g, 2.49 mmol) and 5-bromo-6-chloropyridin-3-ol (2.60 g, 12.5
mmol) was added to the flask quickly, and then the reaction was
flushed with Ar and placed in an oil bath at 60.degree. C. for 4 h
or until starting material was gone by LCMS. The reaction was
cooled to rt and filtered through a fine fritted funnel. The
filtrate was concentrated to afford a yellow oil that was adsorbed
onto silica and purified by flash chromatography (25% EtOAc/Hex,
ramped to 100% EtOAc). The product was further purified after the
column by recrystallization from hexane and minimal cold chloroform
to afford a white solid. MS (ESI) m/z: Calculated: 219.1. Observed:
220.0 (M.sup.++1).
5-Benzyl-6-chloro-2-iodopyridin-3-ol
##STR00401##
[0985] To a solution of K.sub.2CO.sub.3 (1.6 g, 12 mmol) in water
(25.0 mL) was added
[0986] 5-benzyl-6-chloropyridin-3-ol (0.73 g, 3.3 mmol) and I.sub.2
(0.21 mL, 4.0 mmol). The mixture became homogeneous and yellow
after stirring for 16 h. The mixture was washed with sodium
thiosulfate (100 mL), and then treated cautiously with conc. HCl
until the pH was 2 by pH paper. The solution was extracted with
EtOAc (3.times.50 mL), and the combined organic extracts were dried
over MgSO.sub.4, filtered and concentrated to afford a pale yellow
solid. The solid was purified by triturating with minimal DCM and
ether to give a beige solid. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. ppm 10.90-11.09 (1H, br s), 7.30-7.37 (2H, m), 7.17-7.28
(3H, m), 6.99 (1H, s), 3.95 (2H, s); MS (ESI) m/z: Calculated:
344.9. Observed: 345.9 (M.sup.++1).
Methyl
1-((4-(6-benzyl-5-chlorofuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)met-
hyl)azetidine-3-carboxylate
##STR00402##
[0988] In a sealed flask was combined
5-benzyl-6-chloro-2-iodopyridin-3-ol (1.50 g, 4.3 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (0.30 g, 0.43 mmol), copper(I) iodide
(0.17 g, 0.87 mmol), N-ethyl-N-isopropylpropan-2-amine (6.1 mL, 35
mmol), methyl 1-(4-ethynyl-3-fluorobenzyl)azetidine-3-carboxylate
(1.2 g, 4.8 mmol) and DMF (10 mL, 129 mmol). The flask was flushed
with argon, sealed and placed in an oil bath for 16 h at 80.degree.
C. The reaction was concentrated to remove solvent, and the
resulting black mixture was adsorbed onto silica. Flash
chromatography (100% EtOAc) afforded a yellow oil that was further
purified on a Varian HF Mega Bond Elut SCX column (eluted with 2M
ammonia in MeOH). Trituration with of the resulting solids with
ether afforded an off-white solid. .sup.1H NMR (300 MHz,
CHLOROFORM-d) .delta. ppm 7.83-7.90 (1H, m), 7.49 (1H, s),
7.27-7.40 (5H, m), 7.22-7.24 (1H, m), 7.12-7.19 (2H, m), 4.20 (2H,
s), 3.72 (3H, s), 3.65 (2H, s), 3.52-3.59 (2H, m), 3.32-3.38 (3H,
m); MS (ESI) m/z: Calculated: 464.1. Observed: 465.1
(M.sup.++1).
1-((4-(6-benzyl-5-chlorofuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)methyl)aze-
tidine-3-carboxylic acid
##STR00403##
[0990] To a solution of LiOH (0.022 g, 0.90 mmol) in water (3.0 mL)
was added methyl
1-((4-(6-benzyl-5-chlorofuro[3,2-b]pyridin-2-yl)-3-fluorophenyl)methyl)az-
etidine-3-carboxylate (0.084 g, 0.18 mmol) in THF (2.0 mL). The
solution was stirred for 30 min and then the solvent was removed
under reduced pressure to afford a white suspension. To the mixture
was added 0.5M phosphate buffer, pH 6.0 (3 mL), and then 1 N HCl
was added until the pH was 6.0 as indicated by pH paper. The
resulting suspension was filtered, and the off white solid was
collected and dried under vacuum at 50.degree. C. .sup.1H NMR (400
MHz, DMSO-d.sub.6 with 1 drop of TFA-d) .delta. ppm 8.04-8.21 (2H,
m), 7.44-7.70 (3H, m), 7.18-7.40 (5H, m), 4.45-4.60 (2H, m),
4.13-4.40 (6H, m), 3.60-3.76 (1H, m); MS (ESI) m/z: Calculated:
450.1. Observed: 451.1 (M.sup.++1).
Compound 111
1-((4-(6-Benzylbenzo[d]oxazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-carb-
oxylic acid
4-Benzyl-2-iodobenzenamine
##STR00404##
[0992] To a solution of 4-benzylbenzenamine (3.00 g, 16.4 mmol) in
16 mL MeOH was added iodine (2.49 g, 9.82 mmol) followed by
hydrogen peroxide, 30% in water (1.67 mL, 16.4 mmol). The dark
solution was allowed to stir overnight. The reaction was
partitioned between water/brine and EtOAc. The organic layer was
washed 1.times. brine, dried over MgSO.sub.4, filtered, and
concentrated. The resulting oil was purified by ISCO (120 g, 0-10%
EtOAc/hexanes) to give 4-benzyl-2-iodobenzenamine as a red oil
which slowly solidified to a red solid. MS (ESI) m/z: Calculated:
309.0. Observed: 310.0 (M.sup.++1).
N-(4-Benzyl-2-iodophenyl)-2-fluoro-4-formylbenzamide
##STR00405##
[0994] To a slurry of 2-fluoro-4-formylbenzoyl chloride (1.5 mmol)
in 5 mL THF was added N-ethyl-N-isopropylpropan-2-amine (0.39 mL,
2.2 mmol) and 4-benzyl-2-iodobenzenamine (0.41 g, 1.3 mmol). After
30 min, the reaction was treated with 1N HCl and Et.sub.2O. The
organic layer was washed 1.times.1N HCl, 2.times.sat'd aq.
NaHCO.sub.3, and 1.times. brine. The org. layer was dried over
MgSO.sub.4, filtered, and concentrated in vacuo. The resulting
material was purified by ISCO, 40 g, 0-20% EtOAc/hexanes to give
N-(4-benzyl-2-iodophenyl)-2-fluoro-4-formylbenzamide as a white
solid. MS (ESI) m/z: Calculated: 459.0. Observed: 457.9
(M.sup.--1).
4-(6-Benzylbenzo[d]oxazol-2-yl)-3-fluorobenzaldehyde
##STR00406##
[0996] 1,10-Phenanthroline (0.016 g, 0.087 mmol), cuprous iodide
(0.008 g, 0.044 mmol), cesium carbonate (0.21 g, 0.65 mmol), and
N-(4-benzyl-2-iodophenyl)-2-fluoro-4-formylbenzamide (0.200 g, 0.44
mmol) were combined under argon. Dioxane (2 mL) was added and the
reaction was sealed and heated to 90.degree. C. for 24 h. The
reaction was cooled and partitioned between EtOAc and water. The
organics were washed with brine, dried over sodium sulfate,
filtered, and concentrated. The residue was purified by ISCO, 40 g,
0-30% EtOAc/hexanes to give
4-(6-benzylbenzo[d]oxazol-2-yl)-3-fluorobenzaldehyde as a white
solid. MS (ESI) m/z: Calculated: 331.1. Observed: 332.1
(M.sup.++1).
1-((4-(6-Benzylbenzo[d]oxazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-carb-
oxylic acid
##STR00407##
[0998] Synthesized according to Scheme B4 and general procedure I
using 4-(6-benzylbenzo[d]oxazol-2-yl)-3-fluorobenzaldehyde as a
white solid [hS1P1 EC.sub.50=8 nM]. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.12 (t, J=7.8 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.69
(s, 1 H), 7.26-7.38 (m, 7H), 7.16-7.25 (m, 1H), 4.11 (s, 2H), 3.65
(s, 2H), 3.19-3.48 (m, 5H). MS (ESI) m/z: Calculated: 416.2.
Observed: 417.2 (M.sup.++1).
Compound 112
1-((4-(5-Benzylbenzo[d]oxazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-carb-
oxylic acid
4-Benzyl-2-nitrophenol
##STR00408##
[1000] To a solution of 4-benzylphenol (10.0 g, 54.3 mmol) in 300
mL AcOH at ambient temperature was added slowly dropwise a solution
of nitric acid, red fuming (2.28 mL, 54.3 mmol) in 100 mL AcOH over
1-2 h. The reaction was allowed to stir for 3 h, poured onto ice,
and the resulting solid was collected by filtration, rinsing with
water to give 4-benzyl-2-nitrophenol as a yellow solid. MS (ESI)
m/z: Calculated: 229.1. Observed: 227.8 (M.sup.++1).
2-Amino-4-benzylphenol
##STR00409##
[1002] 10% Palladium on carbon, Pearlman, (50% wet) (2.3 g, 2.2
mmol) and 4-benzyl-2-nitrophenol (5.00 g, 22 mmol) were combined
under nitrogen and diluted with 80 mL MeOH delivered via syringe.
The vessel was pressurized to 40 psi and shaken in a Parr shaker
for .about.24 h. The resulting material was flushed with nitrogen
and filtered through Celite, rinsing with MeOH. The filtrate was
concentrated in vacuo to give 2-amino-4-benzylphenol as a brown
solid. MS (ESI) m/z: Calculated: 199.1. Observed: 200.0
(M.sup.++1).
N-(5-Benzyl-2-hydroxyphenyl)-2-fluoro-4-formylbenzamide
##STR00410##
[1004] To a mixture of 2-fluoro-4-formylbenzoic acid (0.600 g, 3.57
mmol) in 15 mL DCM was added oxalyl dichloride (0.374 mL, 4.28
mmol) and N,N-dimethylformamide (0.00261 g, 0.0357 mmol) (a few
drops). The mixture was allowed to stir for several h, treated with
additional (COCl).sub.2 (0.100 mL), stirred further for 1 h,
concentrated in vacuo, and the semi-solid was suspended in 15 mL
THF. N,N-diisopropylethylamine (0.808 mL, 4.64 mmol) was added
followed by 2-amino-4-benzylphenol (0.711 g, 3.57 mmol). After 4 h,
the reaction was treated with EtOAc, water, and 1N HCl. The organic
layer was washed 1.times. brine, dried over sodium sulfate,
filtered, and concentrated. The material was purified by silica gel
chromatography to give title compound as a solid. MS (ESI) m/z:
Calculated: 349.1. Observed: 347.7 (M.sup.--1).
5-Benzyl-2-(4-(dimethoxymethyl)-2-fluorophenyl)benzo[d]oxazole
##STR00411##
[1006] A mixture of
N-(5-benzyl-2-hydroxyphenyl)-2-fluoro-4-formylbenzamide (0.500 g,
1.43 mmol), p-toluenesulfonic acid monohydrate (0.817 g, 4.29 mmol)
in 14 mL toluene in a flask fitted with a water-cooled reflux
condensor under nitrogen was heated to 115.degree. C. After 3 h the
reaction was cooled, and diluted with DCM and MeOH to give a
solution and 15 g Si-carbonate (derivitized silica gel, silicycle)
was added and the mixture dried. The material was purified by
silica gel chromatography, 0-50% EtOAc/hexanes to give title
compound as an off-white solid. MS (ESI) m/z: Calculated: 377.1.
Observed: 378.1 (M.sup.++1).
5-Benzyl-2-(4-(dimethoxymethyl)-2-fluorophenyl)benzo[d]oxazole
##STR00412##
[1008]
5-Benzyl-2-(4-(dimethoxymethyl)-2-fluorophenyl)benzo[d]oxazole
(0.160 g, 0.424 mmol) was dissolved in 2 mL THF and 1 mL 5N HCl was
added. The mixture was stirred for 1 h, diluted with DCM, and
quenched with 10N NaOH until basic. The aqueous was extracted
2.times.DCM. Combined organics were dried over sodium sulfate,
filtered, and concentrated to give
4-(5-benzylbenzo[d]oxazol-2-yl)-3-fluorobenzaldehyde as an orange
solid. 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 10.09 (s, 1H),
8.43 (t, J=7.3 Hz, 1H), 7.90-8.04 (m, 2H), 7.72-7.83 (m, 2H), 7.38
(d, J=8.5 Hz, 1H), 7.26-7.33 (m, 4H), 7.17-7.24 (m, 1H), 4.11 (s,
2H).
1-((4-(5-Benzylbenzo[d]oxazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-carb-
oxylic acid
##STR00413##
[1010] Synthesized according to Scheme B4 and general procedure I
using 4-(5-benzylbenzo[d]oxazol-2-yl)-3-fluorobenzaldehyde [hS1P1
EC.sub.50=19 nM] as a white solid. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.13 (t, J=8.0 Hz, 1H), 7.67-7.74 (m, 2H), 7.26-7.39
(m, 7 H), 7.16-7.24 (m, 1H), 4.09 (s, 2H), 3.66 (s, 2H), 3.39-3.51
(m, 2H), 3.20-3.36 (m, 3H). MS (ESI) m/z: Calculated: 416.2.
Observed: 417.0 (M.sup.++1).
Compound 113
1-((4-(5-Benzylbenzo[d]oxazol-2-yl)phenyl)methyl)azetidine-3-carboxylic
acid
##STR00414##
[1012] Synthesized in a manner analogous to
1-((4-(5-benzylbenzo[d]oxazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-car-
boxylic acid, except employing 4-formylbenzoyl chloride. white
solid [hS1P1 EC.sub.50=364 nM]. 1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.12 (d, J=8.0 Hz, 2H), 7.61-7.72 (m, 2H), 7.49 (d,
J=8.0 Hz, 2H), 7.25-7.35 (m, 5H), 7.15-7.24 (m, 1H), 4.08 (s, 2H),
3.64 (s, 2H), 3.19-3.48 (m, 5H). MS (ESI) m/z: Calculated: 398.2.
Observed: 399.2 (M.sup.++1).
Compound 114
1-((4-(6-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-car-
boxylic acid
S-4-Benzyl-2-nitrophenyl dimethylcarbamothioate
##STR00415##
[1014] To a yellow solution of 4-benzyl-2-nitrophenol (2.0 g, 8.7
mmol) in 20 mL DMF at 0.degree. C. was added sodium hydride (0.38
g, 9.6 mmol). The reaction became deep orange, and after 30 min
dimethylthiocarbamoyl chloride (1.2 g, 9.6 mmol) was added. The
reaction was stirred over the weekend at room temperature. Water
was added, and the mixture was extracted with DCM 3 times. The
combined extracts were washed with 0.5M NaHCO.sub.3, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo to
give a brown oil. The oil was heated to 160.degree. C. for a total
of 2 h. The reaction was cooled, and the resulting oil was loaded
directly onto a 120 g ISCO column and eluted with 0-30-50%
EtOAc/hexanes, to give S-4-benzyl-2-nitrophenyl
dimethylcarbamothioate. MS (ESI) m/z: Calculated: 316.1. Observed:
317.1 (M.sup.++1).
S-2-Amino-4-benzylphenyl dimethylcarbamothioate
##STR00416##
[1016] To a solution of S-4-benzyl-2-nitrophenyl
dimethylcarbamothioate (1.58 g, 4.99 mmol) in 25 mL 5:1
acetone/water under nitrogen was added zinc, nanosize activated
powder (1.63 g, 25.0 mmol) and ammonium chloride (2.67 g, 49.9
mmol). The reaction became hot and was cooled with an ice bath. The
heterogeneous reaction was allowed to stir for .about.4 h. The
reaction was allowed to stand overnight and was partitioned between
200 mL EtOAc and water. The organic layer was washed with brine,
and dried over sodium sulfate, filtered, and concentrated to give
S-2-amino-4-benzylphenyl dimethylcarbamothioate which was used
without further purification. MS (ESI) m/z: Calculated: 286.1.
Observed: 287.0 (M.sup.++1).
2-(2-(2-Amino-4-benzylphenyl)disulfanyl)-5-benzylbenzenamine
##STR00417##
[1018] To a slurry of S-2-amino-4-benzylphenyl
dimethylcarbamothioate (1.43 g, 4.99 mmol) in 15 mL ethylene glycol
was added potassium hydroxide (0.840 g, 15.0 mmol) (solid, finely
crushed). The reaction was heated to 60.degree. C. under nitrogen.
10 mL 2-BuOH was added as cosolvent and the resulting solution was
heated for 2 h. The reaction was fitted with a water-cooled reflux
condensor and heated to 90.degree. C. for 3 h. The reaction was
cooled, and concentrated in vacuo. The material was partitioned
between Et.sub.2O and water. The organic layer was dried over
sodium sulfate, filtered, and concentrated in vacuo to give an oil,
which was purified by silica gel chromatography, ISCO, 0-30%
EtOAc/hexanes to give
2-(2-(2-amino-4-benzylphenyl)disulfanyl)-5-benzylbenzenamine as a
brown oil. MS (ESI) m/z: Calculated: 428.1. Observed: 429.0
(M.sup.++1).
4-(5-Benzylbenzo[d]thiazol-2-yl)-3-fluorobenzaldehyde
##STR00418##
[1020] 2-Fluoro-4-formylbenzoyl chloride (3.16 mmol) was dissolved
in 5 mL THF, and a solution of N,N-diisopropylethylamine (0.879 mL,
5.05 mmol) and
2-(2-(2-amino-4-benzylphenyl)disulfanyl)-5-benzylbenzenamine (0.541
g, 1.26 mmol) in 5 mL THF was added dropwise via syringe, 1 mL THF
rinse. The mixture became brown and then a precipitate formed. The
mixture was allowed to stir overnight. The reaction was diluted
with EtOAc and 1N NaOH. The orgn. layer was washed 1.times. brine,
dried over anhyd. sodium sulfate, filtered, and concentrated in
vacuo to give a brown oil. The resulting brown oil was treated with
16 mL EtOH, 2 mL water, and 8 mL conc. HCl followed by tin II
chloride dihydrate. (1.71 g, 7.57 mmol). The heterogeneous reaction
was fitted with a water cooled reflux condensor and was heated to
reflux overnight. The reaction was cooled to 0.degree. C. and
basified with 10N NaOH. The reaction was partitioned between water
and DCM. The aqueous layer was extracted with DCM 3 times, and the
combined organics were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The material was treated with
10% MeOH in DCM and adsorbed onto 5 g silica gel and passed through
a Redi-Sep.RTM. pre-packed silica gel column (80 g) using 0-15%
EtOAc/hexane. The product-containing fractions were concentrated to
afford title compound as a pale yellow solid. MS (ESI) m/z:
Calculated: 347.1. Observed: 348.0 (M.sup.++1).
1-((4-(5-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-car-
boxylic acid
##STR00419##
[1022] Synthesized according to Scheme B4 and general procedure I
using 4-(5-benzylbenzo[d]thiazol-2-yl)-3-fluorobenzaldehyde: light
yellow solid [hS1P1 EC.sub.50=29 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.27 (t, J=8.0 Hz, 1H), 8.07 (d, J=8.5
Hz, 1H), 7.95 (s, 1H), 7.27-7.44 (m, 7H), 7.16-7.25 (m, 1H), 4.13
(s, 2H), 3.65 (s, 2H), 3.40-3.50 (m, 2H), 3.20-3.30 (m, 3H). MS
(ESI) m/z: Calculated: 432.1. Observed: 433.0 (M.sup.++1).
Compound 115
1-((4-(5-Benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorophenyl)methyl)azetidine--
3-carboxylic acid
4-Benzyl-2-nitrobenzenamine
##STR00420##
[1024] 4-Aminodiphenylmethane (5.00 g, 27 mmol) was added in
portions to acetic anhydride (26 mL, 273 mmol) with rapid stirring.
A solid mass formed which was liberated by the addition of
.about.15 mL additional acetic anhydride. The reaction was allowed
to cool to ambient temp, and nitric acid (2.0 mL, 41 mmol) was
added slowly dropwise via addition funnel over 30 min. The
homogeneous red mixture was allowed to stir overnight and poured
into a rapidly stirring solution of 30 mL water, 7 mL conc. HCl,
and 24 mL EtOH (exothermic!). The reaction was allowed to cool and
was then heated to reflux for .about.4 h, cooled and poured onto
ice and neutralized with 10N NaOH to pH 8-9. The aqueous layer was
extracted 3.times.DCM and dried over sodium sulfate, filtered, and
concentrated. The resulting dark red oil was purified by silica gel
chromatography, ISCO 120 g, 0-20% EtOAc/hexanes, to give
4-benzyl-2-nitrobenzenamine as a red oil. MS (ESI) m/z: Calculated:
228.1. Observed: 229.0 (M.sup.++1).
N-(4-Benzyl-2-nitrophenyl)-4-(dimethoxymethyl)-2-fluorobenzamide
##STR00421##
[1026] 2-Fluoro-4-formylbenzoic acid (0.300 g, 1.78 mmol) was
slurried in 10 mL DCM and catalytic DMF was added, followed by
oxalyl chloride (0.317 mL, 3.57 mmol). The reaction was allowed to
stir several h. Upon consumption of the acid, the reaction was
concentrated in vacuo and diluted with 7 mL THF.
4-Benzyl-2-nitrobenzenamine (0.611 g, 2.68 mmol) and
diisopropylethylamine (0.622 mL, 3.57 mmol) was added as a solution
in 5 mL THF. The reaction was allowed to stir overnight and worked
up with EtOAc/saturated sodium bicarbonate, dried over sodium
sulfate, filtered, and concentrated. The residue was treated with
p-toluenesulfonic acid monohydrate (0.170 g, 0.892 mmol) and MeOH.
After 1 h, the mixture was concentrated and adsorbed onto silica
gel and purified by chromatography to give
N-(4-benzyl-2-nitrophenyl)-4-(dimethoxymethyl)-2-fluorobenzamide.
MS (ESI) m/z: Calculated: 424.1. Observed: 425.2 (M.sup.++1).
(4-(5-Benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorophenyl)methanol
##STR00422##
[1028] To a yellow-orange solution of
N-(4-benzyl-2-nitrophenyl)-4-(dimethoxymethyl)-2-fluorobenzamide
(0.466 g, 1.1 mmol) in 5 mL 3:2 AcOH/EtOH was added iron powder
-325 mesh (0.078 mL, 11 mmol). The reaction was fitted with a
reflux condensor and was stirred rapidly at 120.degree. C. bath.
The reaction became a nearly solid mass after 10 min, and 3 mL 2:1
AcOH/EtOH was added to promote stirring. After 3 h, the mixture was
bright yellow. The reaction was diluted with water, EtOAc, and
brine, and the layers were separated. The aqueous layer was
extracted 2.times.EtOAc, and the combined organics were washed
2.times.1N NaOH and 1.times. brine, dried over sodium sulfate,
filtered, and concentrated to an orange oil. The material was
purified by silica gel chromatography, ISCO, 40 g, 0-40% 90/10
DCM/MeOH in DCM to give title compound. MS (ESI) m/z: Calculated:
332.1. Observed: 333.1 (M.sup.++1).
4-(5-Benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorobenzaldehyde
##STR00423##
[1030] To a solution of
(4-(5-benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorophenyl)methanol
(0.055 g, 0.17 mmol) and triethylamine (0.12 mL, 0.83 mmol) in 1.5
mL 1:1 DCM/DMSO under nitrogen at 0.degree. C. was added a solution
of SO.sub.3*py (0.13 g, 0.83 mmol) in 0.75 mL DMSO. The reaction
was allowed to stir for 2 h and was then diluted with EtOAc and
water. The organic layer was washed 1.times. water, 1.times. brine,
dried over anhydrous sodium sulfate, filtered, and concentrated.
The residue was purified by ISCO, 12 g, 0-40% EtOAc/hexanes, to
give 4-(5-benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorobenzaldehyde as
a light yellow oil. MS (ESI) m/z: Calculated: 330.1. Observed:
331.0 (M.sup.++1).
1-((4-(5-Benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorophenyl)methyl)azetidine--
3-carboxylic acid
##STR00424##
[1032] Synthesized according to Scheme B4 and general procedure I
using 4-(5-benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorobenzaldehyde to
give
1-((4-(5-benzyl-1H-benzo[d]imidazol-2-yl)-3-fluorophenyl)methyl)azetidine-
-3-carboxylic acid as a white solid [hS1P1 EC.sub.50=4954 nM]. MS
(ESI) m/z: Calculated: 415.2. Observed: 416.2 (M.sup.++1).
Compound 116
1-((3-Fluoro-4-(5-phenoxybenzo[d]oxazol-2-yl)phenyl)methyl)azetidine-3-car-
boxylic acid
2-Nitro-4-phenoxyphenol
##STR00425##
[1034] To a solution of 4-phenoxyphenol (10.0 g, 54 mmol) in 100 mL
AcOH was added slowly dropwise nitric acid (69-70%, 3.2 mL, 54
mmol) over about 3 min from an addition funnel. The reaction became
warm and was cooled with a water bath. After 1 h, the reaction was
poured onto ice and allowed to warm to rt. A gummy solid resulted,
which was partitioned between MTBE and water. The organic layer was
washed 1.times. water, 1.times. brine, dried over sodium sulfate,
filtered, and concentrated to a dark oil. This was adsorbed onto 36
g silica gel and dried, and purified in two parts by silica gel
chromatography, ISCO, 0-10% EtOAC/hexanes. Product-containing
fractions were combined and concentrated to give
2-nitro-4-phenoxyphenol as an orange oil. MS (ESI) m/z: Calculated:
231.1. Observed: 230.0 (M.sup.--1).
2-Amino-4-phenoxyphenol
##STR00426##
[1036] 10% Palladium on carbon, 50% water wet (1.43 g, 1.34 mmol)
and 2-nitro-4-phenoxyphenol (3.10 g, 13.4 mmol) were combined under
nitrogen and 30 mL MeOH was added. The mixture was exposed to
H.sub.2 from a balloon and was stirred rapidly overnight. In the
morning the reaction was flushed with nitrogen and filtered and
concentrated in vacuo to give 2-amino-4-phenoxyphenol as a light
brown solid. MS (ESI) m/z: Calculated: 201.1. Observed: 202.1
(M.sup.++1).
3-Fluoro-4-(5-phenoxybenzo[d]oxazol-2-yl)benzaldehyde
##STR00427##
[1038] To a slurry of 2-fluoro-4-formylbenzoic acid (0.500 g, 3.0
mmol) in 8 mL DCM was added 2 drops N,N-dimethylformamide (0.011 g,
0.15 mmol), followed by oxalyl dichloride (0.39 mL, 4.5 mmol).
After 30 min, the reaction became clear, light yellow, and
homogeneous. The reaction was evaporated and dried in vacuo. The
resulting solid was suspended in 10 mL THF, and
2-amino-4-phenoxyphenol (0.60 g, 3.0 mmol) was added as a solid
followed by Hunig's base (0.67 mL, 3.9 mmol). The resulting brown
solution became warm, and was allowed to stir for overnight. The
reaction was diluted with DCM and 1N HCl. The aq. layer was
extracted 1.times.DCM, and the organics were dried over sodium
sulfate, filtered, and concentrated to give a brown solid. This was
treated with 4-methylbenzenesulfonic acid hydrate (0.85 g, 4.5
mmol) and 10 mL toluene. The reaction was fitted with a
water-cooled reflux condensor and drying tube, and placed in a
120.degree. C. bath for .about.4 h, cooled and partitioned between
DCM and 1N NaOH with stirring. The reaction was filtered, and the
aq. layer was extracted 1.times.DCM. The combined organics were
dried over sodium sulfate, filtered, and concentrated to give an
orange oil which was purified by ISCO, 0-30% EtOAc/hexanes to give
title compound as an off-white solid. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 10.10 (s, 1H), 8.45 (t, J=7.5 Hz, 1H),
7.94-8.02 (m, 2H), 7.90 (d, J=9.0 Hz, 1H), 7.54 (d, J=2.0 Hz, 1H),
7.37-7.46 (m, 2H), 7.22 (dd, J=8.8, 2.3 Hz, 1H), 7.15 (t, J=7.3 Hz,
1H), 7.04 (d, J=8.5 Hz, 2H).
1-((3-Fluoro-4-(5-phenoxybenzo[d]oxazol-2-yl)phenyl)methyl)azetidine-3-car-
boxylic acid
##STR00428##
[1040] Synthesized according to Scheme B4 and general procedure I
using 3-fluoro-4-(5-phenoxybenzo[d]oxazol-2-yl)benzaldehyde (0.035
g, 0.11 mmol) to give
1-(3-fluoro-4-(5-phenoxybenzo[d]oxazol-2-yl)benzyl)azetidine-3-carboxylic
acid as a white solid [hS1P1 EC.sub.50=12 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.15 (t, J=7.8 Hz, 1H), 7.84 (d, J=8.5
Hz, 1H), 7.48 (d, J=2.0 Hz, 1H), 7.31-7.44 (m, 4H), 7.10-7.20 (m,
2H), 7.03 (d, J=8.5 Hz, 2H), 3.67 (s, 2H), 3.40-3.51 (m, 2H),
3.21-3.35 (m, 3H). MS (ESI) m/z: Calculated: 418.1. Observed: 419.2
(M.sup.++1).
Compound 117
1-((3-Fluoro-4-(5-(phenylthio)benzo[d]oxazol-2-yl)phenyl)
methyl)azetidine-3-carboxylic acid
5-(Phenylthio)benzo[d]oxazole
##STR00429##
[1042] A 150 mL pressure bottle was charged with
5-bromobenzo[d]oxazole (2.43 g, 12.3 mmol),
N,N-diisopropylethylamine (4.28 mL, 24.5 mmol), and 24 mL dioxane.
N.sub.2 was bubbled through the solution for 3 min, at which point
tris(dibenzylidineacetone)palladium(0) (0.281 g, 0.307 mmol),
4,5-bis(diphenylphosphino)-9,9-dimethyl-9H-xanthene (0.355 g, 0.614
mmol), and benzenethiol (1.26 mL, 12.3 mmol) were added. The dark
brown solution was sealed and heated to 100.degree. C. overnight.
The mixture was diluted with EtOAc and NaOH, and the organic layer
was extracted 2.times.1N NaOH. The organic layer was dried over
anhydrous sodium sulfate, filtered, and concentrated to give an
orange oil, which was purified by ISCO, 0-100% 10% EtOAc/hexanes in
hexanes to give 5-(phenylthio)benzo[d]oxazole as an orange oil. MS
(ESI) m/z: Calculated: 227.0. Observed: 228.0 (M.sup.++1).
2-Amino-4-(phenylthio)phenol hydrochloride
##STR00430##
[1044] A solution of 5-(phenylthio)benzo[d]oxazole (1.04 g, 4.6
mmol) and conc. HCl (0.97 mL, 11 mmol) under nitrogen in 4.6 mL
EtOH in a 50 mL rbf fitted with a water-cooled reflux condensor was
placed in a 100.degree. C. bath. After 2 h, the mixture was cooled,
concentrated in vacuo and dried to give a yellow solid. The
material was sonicated in 10 mL DCM, filtered, rinsing with DCM,
and dried to give 2-amino-4-(phenylthio)phenol hydrochloride as an
off-white solid. 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 10.72
(s, 1H), 8.75 (br. s., 1H), 7.28-7.35 (m, 2H), 7.12-7.25 (m, 5H),
6.99 (d, J=8.0 Hz, 1H), 4.32 (br. s., 2H).
2-Fluoro-4-formyl-N-(2-hydroxy-5-(phenylthio)phenyl)benzamide
##STR00431##
[1046] To a slurry of 2-fluoro-4-formylbenzoic acid (0.600 g, 3.57
mmol) in 10 mL anhydrous DCM under nitrogen was added 3 drops DMF,
followed by oxalyl chloride (0.380 mL, 4.28 mmol). The reaction was
allowed to stir under N.sub.2. The reaction was allowed to stir for
4 h and concentrated in vacuo to a yellow oil. This material was
dissolved in 10 mL THF and was added via pipette to a slurry of
2-amino-4-(phenylthio)phenol hydrochloride (0.906 g, 3.57 mmol) and
diisopropylethylamine (1.55 mL, 8.92 mmol) in THF at 0.degree. C.
The bright yellow slurry was allowed to stir overnight. The mixture
was treated with 1N HCl and DCM, and the organic layer was
extracted 1.times.DCM. The organics were dried, filtered, and
concentrated in vacuo. The orange oil was dissolved in 5 mL DCM,
and a thick yellow precipitate resulted. This was collected by
filtration, rinsing with DCM to give
2-fluoro-4-formyl-N-(2-hydroxy-5-(phenylthio)phenyl)benzamide as a
bright yellow solid. MS (ESI) m/z: Calculated: 367.1. Observed:
366.0 (M.sup.--1).
3-Fluoro-4-(5-(phenylthio)benzo[d]oxazol-2-yl)benzaldehyde
##STR00432##
[1048] A mixture of
2-fluoro-4-formyl-N-(2-hydroxy-5-(phenylthio)phenyl)benzamide
(0.180 g, 0.490 mmol) and pyridinium p-toluenesulfonate (0.123 g,
0.490 mmol) in 5.0 mL toluene was heated to 130.degree. C. in a
sealed tube for a total of 6 h. The reaction was cooled and was
partitioned between 1N NaOH and EtOAc. The organic layer was washed
1.times. brine, dried over sodium sulfate, filtered, and
concentrated in vacuo to give a solid that was purified by silica
gel chromatography, 0-30-60% EtOAc/hexanes to give
3-fluoro-4-(5-(phenylthio)benzo[d]oxazol-2-yl)benzaldehyde as a
yellow solid. MS (ESI) m/z: Calculated: 349.1. Observed: 350.0
(M.sup.++1).
1-((3-Fluoro-4-(5-(phenylthio)benzo[d]oxazol-2-yl)phenyl)methyl)azetidine--
3-carboxylic acid
##STR00433##
[1050] Synthesized according to Scheme B4 and general procedure I
using 3-fluoro-4-(5-(phenylthio)benzo[d]oxazol-2-yl)benzaldehyde
(0.116 g, 0.33 mmol): white solid [hS1P1 EC.sub.50=3 nM]. 1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 8.15 (t, J=7.8 Hz, 1H),
7.82-7.91 (m, 2 H), 7.48 (d, J=8.5 Hz, 1H), 7.25-7.42 (m, 7H), 3.67
(s, 2H), 3.40-3.50 (m, 2H), 3.18-3.38 (m, 3H). MS (ESI) m/z:
Calculated: 434.1. Observed: 435.2 (M.sup.++1).
Compound 118
1-((4-(6-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-car-
boxylic acid
6-Benzylbenzo[d]thiazol-2-amine
##STR00434##
[1052] A mixture of 4-benzylbenzenamine (10.37 g, 56.6 mmol) and
ammonium thiocyanate (3.30 mL, 56.6 mmol) in AcOH (100 mL) at
12-18.degree. C. was treated dropwise with Br.sub.2 (2.93 mL).
During the addition, the temperature was kept below 18.degree. C.
After completion of the addition, the mixture was stirred at
24.degree. C. for 2 h and the solvent was partially evaporated. The
precipitates were collected by filtration, partially dissolved in
400 mL EtOAc at reflux temperature, and cooled to -50.degree. C.
The solids were collected by filtration, washed with cold EtOAc,
and dried in vacuo to give light yellow solids (16.17 g). This
material was recrystallized from acetone to give
6-benzylbenzo[d]thiazol-2-amine as a beige solid. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.89 (br. s, 2H), 7.67 (s, 1H), 7.35 (d,
J=9.2 Hz, 1H), 7.29-7.20 (m, 6H), 3.98 (s, 2H). MS (ESI) m/z:
Calculated: 240.3. Observed: 241.1 (M.sup.++1).
4-(6-Benzylbenzo[d]thiazol-2-yl)-3-fluorobenzaldehyde
##STR00435##
[1054] A solution of 12.3 g KOH in H.sub.2O (15 mL) was mixed with
ethylene glycol (8 mL). 6-benzylbenzo[d]thiazol-2-amine (3.00 g,
12.5 mmol) was added to the mixture under N.sub.2 atmosphere. The
mixture was stirred at 135.degree. C. for 6 h, added to a 100 mL
volume of ice and acidified to pH .about.6 using 5M aqueous HCl.
The resulting suspension was extracted 2.times. with DCM. The
organic layers were washed 1.times. with brine, dried over
MgSO.sub.4, and evaporated to give beige solids (1.81 g, used
without further purification). A solution of the crude material
(0.695 g, 3.23 mmol) in THF (5 mL) was added to a mixture of
2-fluoro-4-formylbenzoyl chloride (0.55 g) in THF (5 mL) and
Hunig's base (0.844 mL, 4.84 mmol) and stirred for 17 h at
24.degree. C. The mixture was diluted with EtOAc, washed 1.times.
with saturated aqueous NaHCO.sub.3, 1.times. with brine, dried over
MgSO.sub.4 and evaporated to give a red foam (0.916 g, used for the
next step). The crude material was treated with EtOH (16 mL), water
(2 mL), and concentrated aqueous HCl (8 mL) followed by SnCl.sub.2
(1.67 g, 8798 .mu.mol) and heated to reflux for 6 h
(heterogeneous). The mixture was cooled to 0.degree. C., basified
with 10M aqueous NaOH, and partitioned between water and DCM. The
aqueous layer was extracted with DCM (3.times.) and the combined
organic layers were dried over MgSO.sub.4 and evaporated.
Purification by flash chromatography (hexanes/EtOAc=5:1) gave
4-(6-Benzylbenzo[d]thiazol-2-yl)-3-fluorobenzaldehyde as a yellow
solid. 1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 10.06 (s, 1H), 8.67
(t, J=7.0 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.83 (d, J=8.0 Hz, 1H),
7.76-7.73 (m, 2H), 7.43-7.40 (m, 1H), 7.33-7.31 (m, 2H), 7.26-7.23
(m, 3H), 4.16 (s, 2H). MS (ESI) m/z: Calculated: 347.4. Observed:
348.1 (M.sup.++1).
1-((4-(6-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)
methyl)azetidine-3-carboxylic acid
##STR00436##
[1056] At 24.degree. C., a mixture of
4-(6-benzylbenzo[d]thiazol-2-yl)-3-fluorobenzaldehyde (190 mg, 547
.mu.mol), azetidine-3-carboxylic acid (166 mg, 1641 .mu.mol),
acetic acid (0.095 mL, 1641 .mu.mol) in MeOH (2 mL) and DCM (2 mL)
was stirred for 1 h. The light yellow solution was treated with
sodium borocyanohydride (34 mg, 547 mmol). The mixture was stirred
for 12 h. The solids were filtered off and washed 3.times. with
DCM, suspended in 4 mL of an aqueous buffered solution (phosphate
buffer pH 6) and sonicated for 10 min. The mixture was filtered and
the solids washed with water and dried in vacuo to yield
1-((4-(6-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)azeti-
dine-3-carboxylic acid as a white solid [hS1P1 EC.sub.50=149 nM].
1H NMR (400 MHz, DMSO-d6) .delta. ppm 8.27 (t, J=8.4 Hz, 1H), 8.04
(s, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.44 (d, J=6.6 Hz, 1H), 7.34-7.29
(m, 6H), 7.21-7.20 (m, 1H), 4.11 (s, 2H), 3.83-3.82 (br. m, 1H),
3.63 (s, 2H), 3.41 (t, J=6.8 Hz, 2H), 3.21 (t, J=7.0 Hz, 2H). MS
(ESI) m/z: Calculated: 432.5. Observed: 433.2 (M.sup.++1).
Compound 119
1-(4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3-c-
arboxylic acid, trifluoroacetic acid salt
4-Benzylpyridin-2-amine
##STR00437##
[1058] A mixture of 4-benzylpyridine (14.1 mL, 88.6 mmol), sodium
amide (5.71 g, 146 mmol), and p-cymene (105 mL) was heated to
165.degree. C. After 1 d, the mixture was allowed to cool; water
(30 mL) and concentrated hydrochloric acid (30 mL) were
sequentially added, the aqueous layer was separated, and the
organic layer was extracted with 60 mL of 2N HCl (aq). The aqueous
extracts were then combined, washed with ether (50 mL), and made
strongly basic with solid potassium hydroxide, during which time a
brown oil separated. The oil was extracted into DCM (2.times.150
mL). The combined extracts were then dried over sodium sulfate,
filtered, and concentrated to give a brown oil. Column
chromatography (ISCO, 80 g, 50->100% EtOAc/Hex) afforded
4-benzylpyridin-2-amine as a tan solid. MS (ESI) m/z: Calculated:
184.1. Observed: 185 (M.sup.++1).
4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzaldehyde
##STR00438##
[1060] A solution of 4-benzylpyridin-2-amine (175 mg, 949 .mu.mol)
and 4-(2-bromoacetyl)-3-fluorobenzaldehyde (232.5 mg, 949 mmol) in
EtOH (3.0 mL) was heated at reflux for 2 h, then cooled to
25.degree. C. and concentrated in vacuo. The residue was taken up
in THF (8.0 mL), concentrated aqueous hydrochloric acid (788 .mu.L,
1576 .mu.mol) was added, and the resulting mixture was stirred for
20 min. NaOH (1M, aq; 5.2 eq) was subsequently added to the
reaction solution, which was then partitioned between EtOAc (20 mL)
and brine (3 mL). The organic layer was dried over sodium sulfate
and concentrated in vacuo. Chromatographic purification of the
residue (ISCO, 4 g, 0-100% EtOAc/Hex+2% Et.sub.3N) furnished
4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzaldehyde as
a white solid. MS (ESI) m/z: Calculated: 330.1. Observed: 331
(M.sup.++1).
1-(4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3-c-
arboxylic acid, trifluoroacetic acid salt
##STR00439##
[1062]
4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzaldehyde
(47.3 mg, 143 .mu.mol) was dissolved in 1.0 mL DCM, and 1.0 mL MeOH
was added to the resulting solution, followed by
azetidine-3-carboxylic acid (43 mg, 430 mop, and acetic acid (25
.mu.L, 430 .mu.mol). The mixture was stirred rapidly for 1 h, and
sodium cyanoborohydride (9.0 mg, 143 .mu.mol) was then added in one
portion. After 15 h, the reaction solution was concentrated in
vacuo and the residue was taken up in methanol (3.0 mL), filtered
through a cotton plug, and purified by HPLC (Phenomenex C18, 1-100%
CH3CN/H2O+0.1% TFA) to give
1-(4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3--
carboxylic acid, trifluoroacetic acid salt as a clear oil [hS1P1
EC.sub.50=54 nM]. 1H NMR (400 MHz, MeOH-d.sub.4) .delta. ppm 8.71
(d, J=6.8 Hz, 1H), 8.59 (d, J=2.0 Hz, 1H), 8.05 (t, J=7.9 Hz, 1H),
7.69 (s, 1H), 7.52-7.64 (m, 1H), 7.38-7.43 (m, 2H), 7.36 (d, J=6.1
Hz, 3H), 7.33 (dd, J=6.1, 1.6 Hz, 1H), 7.28-7.32 (m, 1H), 4.55 (s,
2H), 4.36-4.47 (m, 4H), 4.28 (s, 2H), 3.76 (t, J=8.2 Hz, 1H). MS
(ESI) m/z: Calculated: 415.2. Observed: 416 (M.sup.++1).
Compound 120
1-(4-(6-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3-c-
arboxylic acid, trifluoroacetic acid salt
5-Benzylpyridin-2-amine
##STR00440##
[1064] 9-Benzyl-9-bora-bicyclo[3.3.1]nonane (0.5M solution in THF,
12727 .mu.L, 6363 .mu.mol) was added to a mixture of
5-iodopyridin-2-amine (700 mg, 3182 .mu.mol), potassium phosphate
(2026 mg, 9545 .mu.mol), Pd.sub.2dba.sub.3 (58 mg, 64 .mu.mol), and
X-Phos (61 mg, 127 .mu.mol) suspended in H.sub.2O (1 mL) in a
sealable reaction vial. The vial was flushed with argon gas and
then sealed and heated (microwave) at 120.degree. C. for 30 min.
The crude mixture was diluted with EtOAc, and the resulting
solution was sequentially washed with 1M NaOH and brine. The
organic layer was dried over sodium sulfate and concentrated in
vacuo. The residue was taken up in MeOH (5 mL), concentrated
aqueous HCl (0.30 mL) was added, and the resulting solution was
stirred 10 min. Solid NaOH (120 mg) was then added, and the
resulting solution was concentrated onto silica gel.
Chromatographic purification of the product (ISCO, 12 g, 0-10%
MeOH/CH.sub.2Cl.sub.2) furnished 5-benzylpyridin-2-amine as a
yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 7.95 (s,
1H), 7.28 (t, J=7.5 Hz, 2H), 7.23 (d, J=11.0 Hz, 2H), 7.17 (t,
J=8.5 Hz, 2H), 6.44 (d, J=8.5 Hz, 1H), 4.34 (s, 2H), 3.80-3.86 (m,
2H).
6-Benzyl-2-(4-(diethoxymethyl)-2-fluorophenyl)H-imidazo[1,2-a]pyridine
##STR00441##
[1066] A solution of 5-benzylpyridin-2-amine (340.9 mg, 1850
.mu.mol) and 4-(2-bromoacetyl)-3-fluorobenzaldehyde (453.4 mg, 1850
.mu.mol) in ethanol (6.0 mL) was heated at reflux for 5 h. The
solution was cooled to 25.degree. C., triethylamine (260 .mu.L) was
added, and the resulting solution was concentrated onto silica gel
and purified by column chromatography (ISCO, 4 g, 0->100%
EtOAc/Hex, both +2% triethylamine) to furnish crude
6-benzyl-2-(4-(diethoxymethyl)-2-fluorophenyl)H-imidazo[1,2-a]pyridine
as an orange oil. This oil was taken up in THF (10.0 mL),
concentrated aqueous HCl (1.24 mL, 2487 .mu.mol) was added, and the
resulting mixture was stirred for 20 min at 25.degree. C. NaOH (1M,
aq; 5.2 eq) was then added to the reaction solution, and the
resulting mixture was partitioned between EtOAc (40 mL) and brine
(5 mL). The organic layer was dried over sodium sulfate and
concentrated in vacuo. Chromatographic purification of the residue
(ISCO, 4 g, 0-100% EtOAc/hexanes+2% Et.sub.3N) provided
4-(6-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzaldehyde as
a yellow oil. MS (ESI) m/z: Calculated: 330.1. Observed: 331
(M.sup.++1).
1-(4-(6-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3-c-
arboxylic acid, trifluoroacetic acid salt
##STR00442##
[1068]
4-(6-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzaldehyde
(100.5 mg, 304 .mu.mol) was dissolved in 2.0 mL DCM, and 2.0 mL
MeOH was added, followed by azetidine-3-carboxylic acid (92.3 mg,
913 .mu.mol), and acetic acid (52.7 .mu.L, 913 .mu.mol). The
mixture was stirred rapidly for 1 h, at which point sodium
cyanoborohydride (19.1 mg, 304 .mu.mol) was added in one portion.
After 2.5 d, the mixture was diluted with 2 mL DCM and the slurry
was filtered, rinsing with DCM. The filtrate was concentrated in
vacuo to afford a white foam, which was taken up in MeOH (3.0 mL),
filtered through a cotton plug, and purified by HPLC (Phenomenex
C18, 5-50-100% CH.sub.3CN/H.sub.2O+0.1% TFA) to afford
1-(4-(6-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3--
carboxylic acid, trifluoroacetic acid salt as a clear oil. 1H NMR
(400 MHz, MeOH) .delta. ppm 8.66 (s, 1H), 8.59 (s, 1H), 8.07 (t,
J=7.8 Hz, 1H), 7.87 (s, 2H), 7.53-7.62 (m, 2H), 7.32-7.40 (m, 4H),
7.28 (t, J=6.8 Hz, 1H), 4.55 (s, 2H), 4.35-4.48 (m, 4H), 4.18 (s,
2H), 3.70-3.81 (m, 1H). MS (ESI) m/z: Calculated: 415.2. Observed:
416 (M.sup.++1).
Compound 121
1-((2-(5-Benzylbenzofuran-2-yl)pyrimidin-5-yl)methyl)azetidine-3-carboxyli-
c acid
2-(5-Benzylbenzofuran-2-yl)pyrimidine-5-carbaldehyde
##STR00443##
[1070] A 25 mm tube was charged with trifuran-2-ylphosphine (0.0737
g, 0.317 mmol), Pd.sub.2 dba.sub.3 (0.0411 g, 0.0397 mmol),
5-benzylbenzofuran-2-ylboronic acid (0.500 g, 1.98 mmol), CuTC
(0.492 g, 2.58 mmol), 2-(methylthio)pyrimidine-5-carbaldehyde
(0.306 g, 1.98 mmol) under argon. The reactants were diluted in 8
mL THF, sealed, and heated to 50.degree. C. overnight. The mixture
was filtered through Celite rinsing with 200 mL EtOAc. The green
solution was concentrated and adsorbed onto 5 g silica gel, and
purified by ISCO, 0-20% EtOAc/hexanes to give
2-(5-benzylbenzofuran-2-yl)pyrimidine-5-carbaldehyde as a light
yellow solid. MS (ESI) m/z: Calculated: 314.1. Observed: 315.1
(M.sup.++1).
1-((2-(5-Benzylbenzofuran-2-yl)pyrimidin-5-yl)methyl)azetidine-3-carboxyli-
c acid
##STR00444##
[1072] Synthesized according general procedure I from
2-(5-benzylbenzofuran-2-yl)pyrimidine-5-carbaldehyde (0.110 g, 0.35
mmol): white solid [hS1P1 EC.sub.50=1323 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.79 (s, 2H), 7.70 (s, 1H), 7.57-7.65 (m,
2H), 7.25-7.37 (m, 5H), 7.14-7.23 (m, 1H), 4.06 (s, 2H), 3.62 (s,
2H), 3.40-3.51 (m, 2H), 3.17-3.30 (m, 3H). MS (ESI) m/z:
Calculated: 399.2. Observed: 400.2 (M.sup.++1).
Compound 122
1-((6-(5-Benzylbenzofuran-2-yl)-2-methylpyridin-3-yl)methyl)azetidine-3-ca-
rboxylic acid
6-(5-Benzylbenzofuran-2-yl)-2-methylnicotinaldehyde
##STR00445##
[1074] In a sealed flask, a mixture of
5-benzylbenzofuran-2-ylboronic acid (525 mg, 2.1 mmol) and
potassium acetate (0.41 g, 4.2 mmol) was set under argon, treated
with bis{di(.sup.tbutyl)phenyl}palladium(II) dichloride (0.078 g,
0.12 mmol), followed by a solution of
6-chloro-2-methylnicotinaldehyde (0.29 g, 1.9 mmol) in EtOH (10
mL). The resulting suspension was degassed again and heated to
80.degree. C. for 2 h. The mixture was cooled to 24.degree. C.,
treated with EtOAc and washed with saturated aqueous NaHCO.sub.3
and brine (each 1.times.). The combined organic layers were dried
over MgSO.sub.4 and evaporated. Purification by flash
chromatography (hexanes to hexanes/EtOAc=9:1) gave
6-(5-Benzylbenzofuran-2-yl)-2-methylnicotinaldehyde as a pale
yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 10.33
(s, 1H), 8.27 (d, J=8.0 Hz, 1H), 7.97-7.95 (m, 2H), 7.51-7.49 (m,
2H), 7.31-7.29 (m, 2H), 7.24-7.21 (m, 3H), 4.10 (s, 2H), 3.07 (s,
3H). MS (ESI) m/z: Calculated: 327.4. Observed: 328.1
(M.sup.++1).
1-((6-(5-Benzylbenzofuran-2-yl)-2-methylpyridin-3-yl)methyl)azetidine-3-ca-
rboxylic acid
##STR00446##
[1076] A suspension of
6-(5-benzylbenzofuran-2-yl)-2-methylnicotinaldehyde (160 mg, 489
.mu.mol) and 3-azetidinecarboxylic acid (49.4 mg, 489 .mu.mol) in
MeOH (8 mL) and glacial acetic acid (44.0 mg, 733 .mu.mol) was
stirred at 24.degree. C. for 1 h, treated with sodium
borocyanohydride (15.4 mg, 244 .mu.mol), and stirred for 2 h. The
mixture was diluted with 30 mL of 1M HCl in Et.sub.2O and
evaporated. Purification of the residue by RP-HPLC to give title
compound as an off-white solid [hS1P1 EC.sub.50=420 nM]. 1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 7.72 (s, 2H), 7.55-7.53 (m,
2H), 7.43 (s, 1H), 7.28-7.18 (m, 6H), 4.04 (s, 2H), 3.59 (s, 2H),
3.45-3.23 (m, 8H). MS (ESI) m/z: Calculated: 412.5. Observed: 413.3
(M.sup.++1).
Compound 123
1-(1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethyl)azetidine-3-carboxy-
lic acid
1-(4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)ethanol
##STR00447##
[1078] To a mixture of
4-(5-benzylbenzofuran-2-yl)-3-fluorobenzaldehyde (0.559 g, 2 mmol)
in THF at 0.degree. C. was added methyl-magnesium bromide (1.4M
solution in toluene/THF=3:1, 2.40 mL, 3 mmol) dropwise over 5 min.
The mixture was allowed to stir for 20 min at 0.degree. C., treated
with NH.sub.4Cl, extracted with EtOAc, dried over MgSO.sub.4, and
evaporated. The crude product was purified by flash chromatography
(EtOAc/hexanes) to give
1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethanol. 1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 7.98 (t, J=8.0 Hz, 1H), 7.38-7.46 (m,
2H), 7.10-7.34 (m, 9H), 4.77-5.04 (m, 1H), 4.08 (s, 2H), 1.85 (d,
J=3.8 Hz, 1H), 1.46-1.60 (m, 3H). MS (ESI) m/z: Calculated; 346.1.
Observed: 347.1 (M.sup.++1).
1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethanone
##STR00448##
[1080] Dess-Martin reagent (0.411 g, 0.970 mmol) was added to a
solution of 1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethanol
(0.280 g, 0.808 mmol) in DCM (25 mL) and stirred at room
temperature for 1 h. The mixture was treated with saturated aqueous
NaHCO.sub.3, stirred for 5 min, and extracted with DCM. The
combined organic layers were dried over MgSO.sub.4 and evaporated.
Purification by flash chromatography using EtOAc/hexanes gave
1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethanone. 1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 8.11 (t, J=7.7 Hz, 1H), 7.68-7.88 (m,
2H), 7.41-7.48 (m, 2H), 7.12-7.35 (m, 7H), 4.09 (s, 2H), 2.63 (s,
3H). MS (ESI) m/z: Calculated; 344.4. Observed: 345.0
(M.sup.++1).
1-(1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethyl)azetidine-3-carboxy-
lic acid
##STR00449##
[1082] A mixture of azetidine-3-carboxylic acid (0.015 g, 0.15
mmol), 1-(4-(5-benzylbenzofuran-2-yl)-3-fluorophenyl)ethanone
(0.050 g, 0.15 mmol), acetic acid (0.013 mL, 0.22 mmol) in methanol
(3.0 mL) was stirred at room temperature for 1 h. Sodium
triacetoxyborohydride (0.037 g, 0.17 mmol) was added and the
mixture was stirred for 1 h at room temperature. Evaporation and
purification by flash chromatography (5% AcOH in CHCl.sub.3 and
MeOH). The resulting solid was suspended in an aqueous buffered
solution (pH 6, phosphate buffer) and sonicated. The solids were
collected by filtration, washed with water and Et.sub.2O to give
title compound [hS1P1 EC.sub.50=313 nM]. 1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.89 (t, J=7.9 Hz, 1H), 7.51-7.59 (m,
2H), 7.14-7.34 (m, 9H), 4.04 (s, 2H), 2.92-3.50 (m, 6H partially
overlapping with HDO signal), 1.11 (d, J=6.3 Hz, 3H). MS (ESI) m/z:
Calculated; 429.2. Observed: 430.2 (M.sup.++1).
Activity of Compounds of the Invention
[1083] The compounds of the invention made according to the
synthesis noted above were assayed for their ability to modulate
the S1P-1 receptor. Compounds were evaluated for the ability to
induce S1P1-specific receptor internalization using standard
in-vitro receptor internalization assays and their utility as
immunoregulatory agents was demonstrated by their activity as
agonists of the S1P1 receptor measured in the receptor
internalization assay (>50% of S1P control at 10 nM or 300 nM).
The compounds accordingly are expected to be useful as S1P-1
receptor modulators, e.g., in the treatment of a variety of S1P-1
receptor-mediated clinical conditions. Such conditions include
transplant rejection (solid organ transplant and islet cells);
transplant rejection (tissue); cancer; autoimmune/inflammatory
diseases; rheumatoid arthritis; lupus; insulin dependent diabetes
(Type I); non-insulin dependent diabetes (Type II); multiple
sclerosis; psoriasis; ulcerative colitis; inflammatory bowel
disease; Crohn's disease; acute and chronic lymphocytic leukemias
and lymphomas.
[1084] To further demonstrate the suitability of compounds of the
invention as S1P-1 receptor modulators for treating conditions such
as transplant rejection; cancer; autoimmune/inflammatory diseases;
rheumatoid arthritis; lupus; diabetes; multiple sclerosis;
psoriasis; ulcerative colitis; inflammatory bowel disease; Crohn's
disease; acute and chronic lymphocytic leukemias and lymphomas
where immunosuppression is central (of which reduction of
lymphopenia is therefore a well-established indicator), compounds
of the invention were evaluated in laboratory animals as described
below.
Protocol
Mice
[1085] C57BL/6J mice (B6, Jackson Laboratories, Bar Harbor, Me.)
were maintained in a specific pathogen-free environment under a
microisolator containment system. Both adult male and female
age-matched mice were used for all experiments, which were reviewed
and approved by the Animal Care and Use Committee at the University
of Virginia. Whenever the protocol stated Mice were anesthetized
via intraperitoneal injections of ketamine hydrochloride (125
mg/kg; Sanofi Winthrop Pharmaceuticals, New York, N.Y.), xylazine
(12.5 mg/kg TranquiVed; Phoenix Scientific, St. Joseph, Mo.), and
atropine sulfate (0.025 mg/kg; Fujisawa USA, Deerfield, Ill.).
Flow Cytometry Preparation and Analysis
[1086] Blood was harvested from at least six mice for each time
point of 0, 4, 8, 24, 48, 72 h following one day, 3 days or 7 days
daily dosing with the test compound. Following terminal bleeds
brain and certain other tissues were harvested from all animals
undergoing treatment. Cell counts were determined from whole blood,
yielding cell counts in thousands of cells per microliter
(K/IL).
[1087] To identify and quantify lymphocyte subsets, cell
suspensions were analyzed by flow cytometry. Following red blood
cell lysis, cells were stained with anti-mouse monoclonal
antibodies against CD3, CD4, CD8, CD19, and NK1.1 (BD Biosciences,
San Jose, Calif.). Cells were analyzed via four-color flow
cytometry on a FACSCalibur (BD Biosciences) in the University of
Virginia Cancer Center Core Facility. Lymphocyte subsets, including
B cells, total T cells, CD4 T cells, CD8 T cells, double-positive
thymocytes, double-negative thymocytes, NK cells, and NK/T cells,
were analyzed. The size of each cell population was calculated as
the product of the total lymphocyte count recorded by the Hemavet
or hemocytometer and the percentage of positive lymphocytes
recorded by the flow cytometer. All data were analyzed with BD
Biosciences Cell Quest analysis software.
Statistical Analysis
[1088] Statistical significance was determined using Student's
t-test to compare all time points to -24 hour group.
[1089] The compounds tested:
TABLE-US-00001 ##STR00450## 1-(4-(5-Butoxybenzofuran-2-
yl)phenyl)methyl)azetidine-3-carboxylic acid ##STR00451##
1-((4-(5-Benzylbenzofuran-2-
yl)phenyl)methyl)azetidine-3-carboxylic acid ##STR00452##
1-(4-(5-cyclohexylbenzofuran-2- yl)benzyl)azetidine-3-carboxylic
acid ##STR00453## 1-((4-(5-isobutylbenzofuran-2-
yl)phenyl)methyl)azetidine-3-carboxylic acid ##STR00454##
1-((4-(5-Benzylbenzofuran-2-yl)-3- fluorophenyl)methyl)azetidine-3-
carboxylic acid ##STR00455## l-((4-(5-cyclohexylbenzofuran-2-yl)3-
fluorophenyl)methyl)azetidine-3- carboxylic acid ##STR00456##
1-(3-fluoro-4-(5-(piperidin-1-
yl)benzofuran-2-yl)benzyl)azetidine-3- carboxylic acid
trifluoroacetic acid salt ##STR00457##
1-(4-(5-(cyclopentylmethoxy)benzofuran-
2-yl)-3-fluorobenzyl)azetidine-3- carboxylic acid ##STR00458##
1-(4-(5-(cyclopropylmethoxy)benzofuran-
2-yl)-3-fluorobenzyl)azetidine-3- carboxylic acid ##STR00459##
1-(4-(5-butoxybenzofuran-2-yl)-3-
chlorobenzyl)azetidine-3-carboxylic acid ##STR00460##
3-(6-(5-cyclopentylbenzofuran-2-yl)-3,4-
dihydroisoquinolin-2(1H)-yl)propanoic acid ##STR00461##
1-(4-(5-benzylbenzofuran-2-yl)-3-
fluorobenzyl)pyrrolidine-3-carboxylic acid
[1090] showed a reduction in lymphopenia ranging from 35% to 90%
compared to baseline at dosages of 0.3 to 10 mg/kg. The final two
compounds in the above table did not show lymphopenia reduction
under the conditions tested. As such, the compounds of the
invention are expected to be useful drugs for treating conditions
such as transplant rejection; cancer; autoimmune/inflammatory
diseases; rheumatoid arthritis; lupus; diabetes; multiple
sclerosis; psoriasis; ulcerative colitis; inflammatory bowel
disease; Crohn's disease; acute and chronic lymphocytic leukemias
and lymphomas where immunosuppression is central.
[1091] Rat Lymphopenia Study Protocol
[1092] Animals:
[1093] Female Lewis rats (150-175 gms, 6-8 wks) are received from
Charles River Laboratories and allowed to acclimatize for at least
one week before being placed on study.
[1094] Procedure:
[1095] 1) Rats (n=4/group) are administered compound or vehicle
(12.5% captisol in water) orally (PO, 10 mL/kg) at time 0.
[1096] 2) At various time points following dosing (1, 4, 8, or 24
hrs), animals are sacrificed by CO2 inhalation.
[1097] 3) Using a 20G needle and 1 cc syringe, blood is collected
by cardiac puncture.
[1098] 4) Approximately 500 uL of blood is placed in a microtainer
tube containing EDTA (BD #365973), and the sample is mixed
thoroughly.
[1099] 5) Differential cell counts are performed using an Advia 120
hematology system by Bayer.
[1100] The following compounds exhibited hS1P1:hS1P3 EC.sub.50
selectivity ratios of better than 1:100:
[1101]
1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-c-
arboxylic acid; [1102]
1-(4-(5-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxylic acid;
[1103]
1-((6-(5-Butoxylbenzofuran-2-yl)pyridin-3-yl)methyl)azetidine-3-carboxyli-
c acid; [1104]
1-((4-(5-benzylbenzofuran-2-yl)-3-methoxyphenyl)methyl)azetidine-3-carbox-
ylic acid; [1105]
1-(3-fluoro-4-(5-(tetrahydro-2H-pyran-4-yl)benzofuran-2-yl)phenyl)methyl)-
azetidine-3-carboxylic acid; [1106]
(E/Z)-1-((3-Fluoro-4-(5-((hydroxyimino)(phenyl)methyl)benzofuran-2-yl)phe-
nyl)methyl)azetidine-3-carboxylic acid; [1107]
1-((3-Fluoro-4-(5-(phenylmethyl)furo[2,3-b]pyridin-2-yl)phenyl)methyl)-3--
azetidinecarboxylic acid; [1108]
1-((3-Fluoro-4-(5-(phenylthio)benzo[d]oxazol-2-yl)phenyl)methyl)azetidine-
-3-carboxylic acid; [1109]
1-((3-Fluoro-4-(5-(pyridin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azeti-
dine-3-carboxylic acid; [1110]
1-((3-Fluoro-4-(5-(pyrimidin-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)aze-
tidine-3-carboxylic acid; [1111]
1-((3-Fluoro-4-(5-(thiazol-2-ylmethyl)benzofuran-2-yl)phenyl)methyl)azeti-
dine-3-carboxylic acid; [1112]
1-((4-(5-(Difluoro(phenyl)methyl)benzofuran-2-yl)-3-fluorophenyl)methyl)a-
zetidine-3-carboxylic acid; [1113]
1-((4-(5-Benzylbenzo[d]thiazol-2-yl)-3-fluorophenyl)methyl)azetidine-3-ca-
rboxylic acid; [1114]
1-((4-(6-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl)azetidine-3-carboxy-
lic acid; [1115]
1-(3-Fluoro-4-(5-(4-methylbenzyl)benzofuran-2-yl)benzyl)azetidine-3-carbo-
xylic acid; [1116]
1-(3-Fluoro-4-(5-(phenoxymethyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid; [1117]
1-(3-Fluoro-4-(5-phenoxybenzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid; [1118]
1-(3-Fluoro-4-(5-phenylsulfinyl)benzofuran-2-yl)benzyl)azetidine-3-carbox-
ylic acid; [1119]
1-(3-Fluoro-4-(5-phenylthio)benzofuran-2-yl)benzyl)azetidine-3-carboxylic
acid; [1120]
1-(4-(5-(Cyclobutoxymethyl)benzofuran-2-yl)-3-fluoro-benzyl)azetidine-3-c-
arboxylic acid; [1121]
1-(4-(5-Benzylbenzo[b]thiophen-2-yl)-3-fluorobenzyl)azetidine-3-carboxyli-
c acid; and [1122]
1-(4-(7-Benzyl-1H-imidazo[1,2-a]pyridin-2-yl)-3-fluorobenzyl)azetidine-3--
carboxylic acid, trifluoroacetic acid salt.
EQUIVALENTS
[1123] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of the invention.
Various substitutions, alterations, and modifications may be made
to the invention without departing from the spirit and scope of the
invention. Other aspects, advantages, and modifications are within
the scope of the invention. The contents of all references, issued
patents, and published patent applications cited throughout this
application are hereby incorporated by reference. The appropriate
components, processes, and methods of those patents, applications
and other documents may be selected for the invention and
embodiments thereof.
* * * * *