NODWELL MATTHEW (CA)
MUI ALICE (CA)
ANDERSEN RAYMOND (CA)
NODWELL MATTHEW (CA)
MUI ALICE (CA)
| WO2004035601A1 | 2004-04-29 |
MIERAU V. ET AL.: "The Dasyscyphins A-C and Niveulone, New Biologically Active Compounds from the Ascomycete", JOURNAL OF ANTIBIOTIC, vol. 59, no. 1, 2006, pages 53 - 56, XP008102165
MUKKU V. ET AL.: "New Sesquiterpene Quinols from a Micronesian Sponge, Aka sp", JOURNAL OF NATURE PRODUCT, vol. 66, 2003, pages 686 - 689, XP008102079
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KUTNEY J.P. ET AL.: "Synthetic Studies in the Veratrum Alkaloid Series. I. Introduction and the Total Synthesis of Appropriate C-Nor-D-homo Steroid Derivatives", CANADIAN JOURNAL OF CHEMISTRY, vol. 53, 1975, pages 1775 - 1795, XP008100815
LAWRIE W. ET AL.: "A Novel Aromatisation Reaction of 11-Oxolanostanes", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 1: ORGANIC AND BIO-ORGANIC CHEMISTRY (1972-1999), vol. 5, 1978, pages 471 - 479, XP008100833
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DATABASE REGISTRY [online] XP008101577, accession no. STN Database accession no. (887609-76-9)
BIBER F.Z. ET AL.: "Synthesis of an estradiol glucuronide derivative and investigation of its radiopharmaceutical potential", APPLIED RADIATION AND ISOTOPES, vol. 64, 2006, pages 778 - 788, XP025135069
What is claimed is:
1. A compound of Formula II or a salt thereof:
II wherein;
Ri and R 2 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 ORi', -CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCY 1 Y 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Yi/Y 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =O, and Y 1 /Y 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R 6 ', OH, -0-(Ci-Ci 0 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(Rg ') 2 ; where R 6 ', Rg' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =O, SH, F, Br, Cl, I, NH 2 , -NHRi 0 ', -N(R 1 ^) 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
Ri', R 2 ', R 3 ', R 4 ', R 5 ', R 7 ', Rio', Rn', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Ri, R 2 , R 3 , R 4 , Xi, X 2 , X 3 and X 4 are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula II.
2. A compound of Formula III or a salt thereof:
III wherein;
Ri and R 2 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 ORi', -CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CYiY 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCYiY 2 -, and -CYiY 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Y x IY 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =0, and Y 1 /Y 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, R 6 ', OH, -0-(Ci-C 10 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R 9 ') 2 ; where R 6 ', Rg' and R 9 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHRi 0 ', -N(Ri i') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
Ri', R 2 ', R 3 ', R 4 ', R 5 ', R 7 ', Rio', Rn', and R 12 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi", -N(R 2 ") 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Ri, R 2 , R 3 , R 4 , are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula III.
3. A compound of Formula IV or a salt thereof:
IV wherein;
Ri and R 2 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 ORi', -CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCY 1 Y 2 -, and -CYiY 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of X x IX 1 , Y 3 /Y 4 , and Y 5 /Y 6 are =O, and Yi/Y 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H; Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R^', OH, -0-(C 1 -C 1O alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R^) 2 ; where R^', R % and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(R 1 ^) 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
R 1 ', R 2 ', R 3 ', R 4 ', R 5 ', R 7 ', R 1O ', R 1 1 ', and R 12 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of X 1 , X 2 , X 3 and X 4 are X 5 or comprise X 5 as a substituent.
4. The compound of any one of claims 1 to 3 wherein R 1 and R 2 are independently selected from the group consisting of: -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR', -CHO, -CO 2 H, and -CO 2 R';
5. The compound of any one of claims 1 to 3 or a salt thereof wherein R 1 is selected from the group consisting of: methyl, ethyl, -CH 2 OH and -CH 2 OR 1 '.
6. The compound of any one of claims 1 to 4 or a salt thereof wherein R 2 is selected from the group consisting of: methyl, ethyl, -CH 2 OH and -CH 2 OR 1 '.
7. The compound of any one of claims 1 to 6 or a salt thereof wherein R 1 ' and R 2 ' in at least one Of R 1 and R 2 is selected from the group consisting of: methyl, ethyl, propyl and butyl.
8. The compound of any one of claims 1 to 7 or a salt thereof wherein R 1 is methyl or ethyl.
9. The compound of any one of claims 1 to 8 or a salt thereof wherein R 2 is methyl or ethyl.
10. The compound of any one of claims 1 to 6 or a salt thereof wherein R 1 is methyl.
11. The compound of any one of claims 1 to 6 or a salt thereof wherein R 2 is methyl.
12. A compound of Formula V or a salt thereof:
wherein;
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CYiY 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCY 1 Y 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Y 1 ZY 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =0, and Y 1 ZY 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R 6 ', OH, -0-(Ci-C 10 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R^) 2 ; where R 6 ', Rg' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(R 11 ') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide; R 5 ', R 7 ', Rio', Rn' > and Ri 2 ' > are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where Rj" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Xi, X 2 , X 3 and X 4 are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula V.
13. A compound of Formula VI or a salt thereof:
VI wherein;
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CYiY 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCYiY 2 -, and -CYiY 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Yi/Y 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =O, and Y 1 /Y 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, R 6 ', OH, -0-(C 1 -C 10 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R^) 2 ; where R 6 ', Rg' and R 9 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(Ri i') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', an d epoxide; R 5 ', R 7 ', Rio', Rn', and R 12 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Ri, R 2 , R 3 , and R 4 , are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula VI.
14. The compound of any one of claims 1 to 13 or a salt thereof wherein Q is selected from the group consisting of -CH 2 -, -CYiY 2 -, -CH 2 CH 2 -, -CYiY 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, and -CYiY 2 CY 3 Y 4 CY 5 Y 6 -; where Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Yi/Y 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =O, and Y 1 /Y 3 is an epoxide; and, at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H; and
R 5 ' is a linear, branched, or cyclic, saturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi",
-N(R 2 "> 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated one to ten carbon alkyl group.
15. The compound of any one of claims 1 to 14 or a salt thereof wherein Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 , are H or halogen.
16. The compound of any one of claims 1 to 13 or a salt thereof wherein Q is selected from the group consisting of: -CH 2 -, -CH 2 -CH 2 -, -CH=CH, -CH 2 -CH 2 -CH 2 - and -CH=CH-CH 2 -.
17. The compound of any one of claims 1 to 15 or a salt thereof wherein Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, and -CYiY 2 CY 3 Y 4 -.
18. A compound of Formula VII or a salt thereof:
VII wherein;
X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R^ , OH, -0-(C 1 -Ci O alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(RgO 2 ; where R 6 ', Rs' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =O, SH, F, Br, Cl, I, NH 2 , -NHRi 0 ', -N(Ri i') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
R 7 ', Rio', Rn', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Xi, X 2 , X 3 and X 4 are X 5 , or comprise X 5 .
19. A compound of Formula VIII or a salt thereof:
VIII wherein;
X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R(,', OH, -0-(C 1 -C 1O alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(RcZ) 2 ; where R(,', R % and Rc/ are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi 0 ', -N(R 11 ') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
R 7 ', Rio', Rn', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHRi", -N(R 2 ") 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Xi, X 2 , X 3 and X 4 are X 5 , or comprise X 5 .
20. The compound of any one of claims 1 to 19 or a salt thereof wherein R^', R 7 ', Rg', and Rc/, in at least one of Xi, X 2 , X 3 , and X 4 is selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
21. The compound of any one of claims 1 to 20 or a salt thereof wherein at least one of Xi, X 2 , and X 3 , is selected from the group consisting of: H, X 5 , Rg', OH, 0-(Ci-Cio alkyl), halogen, -CONH 2 , -C0NHR i3 ', -CO(Ri 4 ') 2 , NHR 8 ' and N(RcZ) 2 ; where R B ' and Ri 4 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 3 ", -N(RV) 2 , NO 2 and -CO 2 H where R 3 " and R 4 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
22. The compound of claim 21 or a salt thereof wherein R f1 ', Rg', R 9 ', Rn', and R 14 ', are selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
23. The compound of any one of claims 1 to 22 or a salt thereof wherein one or more of Xj , X 2 , and X 3 are selected from the group consisting of: H, X 5 , OH, 0-(C 1 -Ci O alkyl), -CONH 2 , -CONHR 13 ', and -CO(Ri 4 f ) 2 , where Rn' and Ri 4 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 3 ", -N(R 4 ^ 2 , NO 2 and -CO 2 H where R 3 " and R 4 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
24. The compound of claim 23 or a salt thereof wherein Ri 3 ', and R 14 ', are selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
25. The compounds of any one of claims 1 to 24 or salt thereof wherein one or more of Xi, X 2 , and X 3 are selected from the group consisting of: H, X 5 , OH, and OCH 3 .
26. The compound of any one of claims 1 to 25 or salt thereof wherein X 4 is selected from the group consisting of: H, X 5 , R 6 ', OH, 0-(C 1 -Ci 0 alkyl), CO 2 H and -CO 2 R 7 '.
27. The compound of claim 26 or a salt thereof wherein R 6 ', and R 7 ', are selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
28. The compound of any one of claims 1 to 27 or a salt thereof wherein X 4 is selected from the group consisting of: H, X 5 , R 6 ', OH, OCH 3 , -CO 2 H and -CO 2 R 7 '.
29 The compound of any one of claims 1 to 28 or a salt thereof wherein X 4 is selected from the group consisting of: H, X 5 , R 6 ', OH, OCH 3 , -CO 2 H and -CO 2 CH 3 .
30. The compound of any one of claims 1 to 29 or a salt thereof wherein X 5 comprises (a) a solubilizing moiety selected from the group consisting of: a moiety having one or more ionic entities at physiological pH; a moiety having multiple hydrogen bonding functionalities, such as -OH or amide; a monophosphate; a diphosphate; a triphosphate; a monosaccharide; an oligosaccharide; a polysaccharide; an oligopeptide; a polypeptide; an amino acid; an alpha amino acid a polyether and a combination thereof; and (b) a linking moiety selected from the group consisting of: -O- ; -O-C(=O)-Z-; -NH-C(=0)-Z-; -CH 2 OC(=O)-; -C(=O)O-, -C(=0)HN-; where Z is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR', -NR ' 2 , NO 2 , -CO 2 H, -CO 2 R', and epoxide and individual carbon atoms may be replaced by S, O, N, NR', or NR' 2 atoms; and each R', is independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
31. The compound of any one of claims 1 to 30 or a salt thereof wherein X 5 comprises an amide linking moiety.
32. The compound of any one of claims 1 to 30 or a salt thereof wherein X 5 comprises an ester linking moiety.
33. The compound of any one of claims 1 to 32 wherein X 5 comprises a solubilizing moiety comprising an NH 2 moiety.
34. The compound of any one of claims 1 to 33 or a salt thereof wherein X 5 comprises an amino acid.
35. The compound of any one of claims 1 to 30 or a salt thereof wherein X 5 comprises a phosphate.
36. The compound of any one of claims 1 to 30 or a salt thereof wherein X 5 comprises a polyethylene glycol moiety.
37. A compound having the structure:
38. A compound having the structure:
39. A compound having the structure:
40. A compound having the structure:
41. A compound having the structure:
42. A compound having the structure:
43. A compound having the structure:
44. A compound having the structure:
45. A compound having the structure:
46. A compound having the structure:
47. A compound having the structure:
48. A compound having the structure:
49. A compound having the structure:
50. A compound having the structure:
51. A compound having the structure:
52. A compound having the structure:
53. A compound having the structure:
54. A compound having the structure:
55. A compound having the structure:
56. A compound having the structure:
57. A compound having the structure:
58. A compound having the structure:
59. A compound having the structure:
60. A compound having the structure:
61. A compound having the structure:
62. A compound having the structure:
63. A compound having the structure:
4. A compound having the structure:
65. A compound having the structure:
6. A compound having the structure:
67. A compound having the structure:
68. A compound having the structure:
69. A compound having the structure:
70. A compound having the structure:
71. A compound having the structure:
72. A compound having the structure:
73. A pharmaceutical composition comprising a compound of any one of claims 1 to 72 or salt thereof and a pharmaceutically acceptable excipient.
74. A compound of any one of claims 1 to 72 or salt thereof or a pharmaceutical composition of claim 73 for the treatment or prophylaxis of an inflammatory, neoplastic, hematopoetic or immune disorder or condition.
75. The compound of claim 74 wherein the neoplastic condition is a blood cancer.
76. The compound of claim 74 wherein the neoplastic condition is multiple myeloma.
77. The compound of claim 74 wherein the neoplastic condition is chronic myeloid leukemia.
78. The compound of claim 74 wherein the neoplastic condition is acute myelogenous leukemia.
79. The compound of claim 74 wherein the immune disorder is an autoimmune disorder.
80. Use of a compound of any one of claims 1 to 72 or salt thereof for the treatment or prophylaxis of an inflammatory, neoplastic, hematopoetic or immune disorder or condition.
81. Use of a compound of any one of claims 1 to 72 or salt thereof for the preparation of a medicament for the treatment or prophylaxis of an inflammatory, neoplastic, hematopoetic or immune disorder or condition.
82. The use of claim 80 or 81 wherein the neoplastic condition is a blood cancer.
83. The use of claim 80 or 81 wherein the neoplastic condition is multiple myeloma.
84. The use of claim 80 or 81 wherein the neoplastic condition is chronic myeloid leukemia.
85. The use of claim 80 or 81 wherein the neoplastic condition is acute myelogenous leukemia.
86. The use of claim 80 or 81 wherein the immune disorder is an autoimmune disorder.
87. A method of prophylaxis or treatment of an immune, hematopoietic, inflammatory or neoplastic disorder or condition comprising administering to a patient in need of said prophylaxsis or treatment, an effective amount of a pharmaceutical composition according to claim 73.
88. The method of claim 87 wherein the neoplastic condition is a blood cancer.
89. The method of claim 87 wherein the neoplastic condition is multiple myeloma.
90. The method of claim 87 wherein the neoplastic condition is chronic myeloid leukemia.
91. The method of claim 87 wherein the neoplastic condition is acute myelogenous leukemia.
92. The method of claim 87 wherein the immune disorder is an autoimmune disorder. |
SHIP 1 MODULATOR PRODRUGS
Technical Field
The present invention relates to SHIP 1, a negative regulator of cell proliferation and survival and immune cell activation.
Background of the Invention
SH 2 -containing inositol 5-phosphatase (SHIP 1), selectively hydrolyzes the 5-phosphate from inositol 1,3,4,5-tetraphosphate (IP4) and phosphatalidylinositol 3,4,5-triphosphate (PIP3). United States Patent No. 6,238,903 discloses that SHIP 1 is an enzyme regulator of signaling pathways that control gene expression, cell proliferation, differentiation, activation, and metabolism, particularly of the Ras and phospholipid signaling pathways. SHIP 1 plays an important role in cytokine and immune receptor signal tansduction. SHIP 1 disrupted (SHIP I " " ) mice exhibit a myeloproliferative phenotype characterized by overproduction of granulocytes and macrophages. (Huber, M. et al. (1999) Prog Biophys MoI Biol 71:423) SHIP I " " mast cells are more prone to IgE and Steel factor induced degranulation, while SHIP 1 " " B cells are resistant to negative regulation by Fc RIIB. SHIP 1 is also involved in the pathogenesis of chronic myelogenous leukemia. (Sattler, M. et al. (1999) MoI Cell Biol 19:7473)
SHIP 1 is expressed only in blood cells and is an important negative regulator of hemopoietic cell growth/survival and immune cell activation. The specialized function of SHIP 1 has been studied in mouse and man.
Various agonists of SHIP 1 activity are known from WO 2004/035601. An example of an agonist is the sesquiterpene compound pelorol, which was first obtained from marine sponge species. Its synthesis is described in WO 2004/035601. The precise structure of pelorol is as follows, with Me representing a methyl group and relative configuration of chiral atoms (C-5, 8, 9 and 10) shown.
Pelorol
Summary
This invention is based on the discovery that the efficacy of pelorol and related compounds as modulators of SHIP 1 activity may be improved by adding solubilizing moieties to the compounds.
In illustrative embodiments of the present invention, there is provided a compound of Formula II or a salt thereof:
II wherein;
R 1 and R 2 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 ORi', -CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCY 1 Y 2 -, and -CYiY 2 CY 3 Y 4 CY 5 Y 6 -;
where Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and YG are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Y 1 ZT 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =0, and Y]AT 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R(,', OH, -0-(C 1 -Ci O alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R 9 ') 2 ; where R 6 ', Rs' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(Ri i') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
Ri', R 2 ', R 3 ', R 4 ', R 5 ', R 7 ', Rio', Rn', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi", -N(R 2 ") 2 , NO 2 and -CO 2 H where Rf and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Ri, R 2 , R 3 , R 4 , Xi, X 2 , X 3 and X 4 are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula II.
In other illustrative embodiments of the present invention, there is provided a compound of Formula III or a salt thereof:
III wherein;
R 1 and R 2 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 ORi', -CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Q is selected from the group consisting of: -CH 2 -, -CY 1 Y 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCY 1 Y 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group Of Y 1 ZY 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =0, and Y 1 ZY 3 is an epoxide; and at least one of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, R 6 ', OH, -0-(C 1 -C 10 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHR 8 ', and -N(R 9 O 2 ; where R 6 ', Rs' and R 9 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(R 1 ^) 2 , NO 2 , -CO 2 H, -CO 2 R 12 ', and epoxide;
R 1 ', R 2 ', R 3 ', R 4 ', R 5 ', R 7 ', R 1O ', Rn', and Rj 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of R 1 , R 2 , R 3 , R 4 , are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 andZor 10 of Formula III.
In other illustrative embodiments of the present invention, there is provided a compound of Formula IV or a salt thereof:
IV wherein;
Ri and R 2 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 1 ', -CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Q is selected from the group consisting of: -CH 2 -, -CYiY 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCYiY 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group Of Y 1 ZY 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =0, and Y 1 ZY 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R 6 ', OH, -0-(Ci-Ci 0 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R 9 O 2 ; where R 6 ', Rg' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi 0 ', -N(R 11 ') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
Ri', R 2 ', R 3 ', R 4 ', R 5 ', R 7 ', Rio', Rn', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and
-CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of X 1 , X 2 , X 3 and X 4 are X 5 or comprise X 5 as a substituent.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Ri and R 2 are independently selected from the group consisting of: -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR', -CHO, -CO 2 H, and -CO 2 R';
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein R 1 is selected from the group consisting of: methyl, ethyl, -CH 2 OH and -CH 2 OR 1 '.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein R 2 is selected from the group consisting of: methyl, ethyl, -CH 2 OH and -CH 2 ORi'.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Ri' and R 2 ' in at least one of Ri and R 2 is selected from the group consisting of: methyl, ethyl, propyl and butyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Ri is methyl or ethyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein R 2 is methyl or ethyl.
hi other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Ri is methyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein R 2 is methyl.
In other illustrative embodiments of the present invention, there is provided a compound of Formula V or a salt thereof:
V wherein;
Q is selected from the group consisting of: -CH 2 -, -CY 1 Y 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCYiY 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Yi/Y 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =0, and Yi/Y 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R 6 ', OH, -0-(C,-Cio alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(RQO 2 ; where R 6 ', Rs' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(R 1 ,') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
R 5 ', R 7 ', Rio', R 11 ', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where Rj" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of X 1 , X 2 , X 3 and X 4 are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula V.
In other illustrative embodiments of the present invention, there is provided a compound of Formula VI or a salt thereof:
VI wherein;
Q is selected from the group consisting of: -CH 2 -, -CY 1 Y 2 -, -CH 2 CH 2 - , -CH=CH-, -CYiY 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCYiY 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group of Y \ IY 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =O, and Y 1 /Y 3 is an epoxide; and at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, R 6 ', OH, -0-(C 1 -C 1O alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R 9 ') 2 ; where R 6 ', Rg' and R 9 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi 0 ', -N(Ri i') 2 , NO 2 , -CO 2 H, -CO 2 Ri 2 ', and epoxide;
R 5 ', R 7 ', Rio', Ri 1 ', and Ri 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Rj, R 2 , R 3 , and R 4 , are X 5 , comprise X 5 as a substituent or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula VI.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Q is selected from the group consisting of -CH 2 -, -CYiY 2 -, -CH 2 CH 2 -, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, and -CY 1 Y 2 CY 3 Y 4 CY 5 Y 6 -; where Y h Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR 5 ', SH, any one group Of YiAf 2 , Y 3 /Y 4 , and Y 5 /Y 6 are =O, and YjAf 3 is an epoxide; and, at least one of Yj, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H; and
R 5 ' is a linear, branched, or cyclic, saturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRi", -N(R 2 ") 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated one to ten carbon alkyl group.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 , are H or halogen.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Q is selected from the group consisting of: -CH 2 -, -CH 2 -CH 2 -, -CH=CH, -CH 2 -CH 2 -CH 2 - and -CH=CH-CH 2 -.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Q is selected from the group consisting of: -CH 2 -, -CYjY 2 -, -CH 2 CH 2 - , -CH=CH-, and -CYiY 2 CY 3 Y 4 -.
In other illustrative embodiments of the present invention, there is provided a compound of Formula VII or a salt thereof:
VII wherein;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R 6 ', OH, -0-(C 1 -C 10 alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R 9 O 2 ; where R 6 ', Rs' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(R 11 ') 2 , NO 2 , -CO 2 H, -CO 2 R 12 ', and epoxide;
R 7 ', Rj 0 ', R 11 ', and R 12 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where Rj" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of X 1 , X 2 , X 3 and X 4 are X 5 , or comprise X 5 .
In other illustrative embodiments of the present invention, there is provided a compound of Formula VIII or a salt thereof:
VIII wherein;
X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, X 5 , R 6 ', OH, -0-(C 1 -Ci O alkyl), -CO 2 H, -CO 2 R 7 ', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHRg', and -N(R 9 O 2 ; where R 6 ', Rg' and R 9 ' are independently X 5 , or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: X 5 , OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 10 ', -N(Ri ^) 2 , NO 2 , -CO 2 H, -CO 2 R 12 ', and epoxide;
R 7 ', Rio', Ri 1 ', and R] 2 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where Ri" and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
X 5 is a prodrug moiety and at least one of Xi, X 2 , X 3 and X 4 are X 5 , or comprise X 5 .
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein R 6 ', R 7 ', Rg', and R 9 ', in at least one of Xi, X 2 , X 3 , and X 4 is selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein at least one of Xi, X 2 , and X 3 , is selected from the group
consisting of: H, X 5 , R 6 ', OH, 0-(C 1 -Ci 0 alkyl), halogen, -CONH 2 , -CONHR 13 ', -CO(R 14 ') 2 , NHRg' and N(R^) 2 ; where Ri 3 ' and R 14 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 3 ", -N(R 4 ") 2 , NO 2 and -CO 2 H where R 3 " and R 4 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein R 6 ', Rg', R 9 ', Ri 3 ', and R 14 ', are selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein one or more of Xj, X 2 , and X 3 are selected from the group consisting of: H, X 5 , OH, 0-(Ci-Ci 0 alkyl), -CONH 2 , -CONHR 13 ', and -CO(R U ') 2 , where Rj 3 ' and R 14 ' are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR 3 ", -N(R 4 ") 2 , NO 2 and -CO 2 H where R 3 " and R 4 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein Ri 3 ', and Ri 4 ', are selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein one or more of Xi, X 2 , and X 3 are selected from the group consisting of: H, X 5 , OH, and OCH 3 .
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 4 is selected from the group consisting of: H, X 5 , R 6 ', OH, 0-(Ci-Ci 0 alkyl), CO 2 H and -CO 2 R 7 '.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein IV, and R 7 ', are selected from the group consisting of: unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl and unsubstituted butyl.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 4 is selected from the group consisting of: H, X 5 , R^', OH, OCH 3 , -CO 2 H and -CO 2 R 7 '.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 4 is selected from the group consisting of: H, X 5 , R 6 ', OH, OCH 3 , -CO 2 H and -CO 2 CH 3 .
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises (a) a solubilizing moiety selected from the group consisting of: a moiety having one or more ionic entities at physiological pH; a moiety having multiple hydrogen bonding functionalities, such as -OH or amide; a monophosphate; a diphosphate; a triphosphate; a monosaccharide; an oligosaccharide; a polysaccharide; an oligopeptide; a polypeptide; an amino acid; an alpha amino acid a polyether and a combination thereof; and (b) a linking moiety selected from the group consisting of: -O- ; -O-C(=O)-Z-; -NH-C(=O)-Z-; -CH 2 OC(=O)-; -C(=O)O-, -C(=O)HN-; where Z is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR', -NR ' 2 , NO 2 , -CO 2 H, -CO 2 R', and epoxide and individual carbon atoms may be replaced by S, O, N, NR', or NR ' 2 atoms; and each R', is independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR 1 ", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises an amide linking moiety.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises an ester linking moiety.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises a solubilizing moiety comprising an NH 2 moiety.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises an amino acid.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises a phosphate.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein wherein X 5 comprises a polyethylene glycol moiety.
In other illustrative embodiments of the present invention, there is provided a pharmaceutical composition comprising a compound of any formula described herein and a pharmaceutically acceptable excipient.
In other illustrative embodiments of the present invention, there is provided a compound of any formula described herein or a pharmaceutical composition described herein for the treatment or prophylaxis of an inflammatory, neoplastic, hematopoetic or immune disorder or condition.
In other illustrative embodiments of the present invention, there is provided a use of a compound of any formula described herein for the treatment or prophylaxis of an inflammatory, neoplastic, hematopoetic or immune disorder or condition. The use may be for preparation of a medicament.
In other illustrative embodiments of the present invention, there is provided a method of prophylaxis or treatment of an immune, hematopoietic, inflammatory or neoplastic disorder or
condition comprising administering to a patient in need of said prophylaxsis or treatment, an effective amount of a pharmaceutical composition described herein.
In other illustrative embodiments of the present invention, there is provided a use or a method as described herein wherein the neoplastic condition is a blood cancer, multiple myeloma, chronic myeloid leukemia, or acute myelogenous leukemia.
In other illustrative embodiments of the present invention, there is provided a use or a method as described herein wherein the immune disorder is an autoimmune disorder.
In other illustrative embodiments of this invention there is provided a method of making or a method of synthesizing a compound as described herein.
In other illustrative embodiments of this invention there is provided a method of prophylaxis or treatment of an immune, hematopoietic, inflammatory or neoplastic disorder or condition comprising administering to a patient in need of said prophylaxsis or treatment, an effective amount of a pharmaceutical composition as described above. Such compositions may comprise previously known compounds of a formula described herein which have not been known as biologically active compounds suitable for pharmaceutical use or not been known as particularly efficacious.
In other illustrative embodiments of this invention there is provided the use of a compound described above or pharmaceutically acceptable salt thereof for modulation of SHIP 1 activity and for preparation of agents and medicaments for the modulation of SHIP 1 activity. Such modulation may be in vitro or in vivo. Agents for in vivo use include a pharmaceutical composition of this invention as well as agents adapted for in vitro use. The modulation may be for a treatment or prophylaxis of an immune, inflammatory, or neoplastic condition or disorders as described above.
Brief Description of the Drawings
Figure 1: is a graph depicting the results of a cell based assay to test relative inhibition of TNFα by a prodrug compound, Compound 103, compared to a non-prodrug compound, Compound 100.
Figure 2: is a graph depicting the results of a cell based assay to test the inhibition of macrophage TNFα production by varying concentrations of a prodrug, Compound 106.
Figure 3: is a graph depicting the results of a cell based assay to test the inhibition of calcium influx in mast cells by a prodrug, Compound 106.
Figure 4: is a graph depicting the results of a cell based assay to test the inhibition of TNFα production in wild type (WT) and knock-out (KO) macrophages by a prodrug, Compound 108.
Figure 5 A: is a graph depicting the results of the ability of Compound 100 at varying concentrations to reduce tumor cell survival in multiple myeloma (MM) cell lines.
Figure SB: is a graph depicting the results of the ability of Compound 100 at varying concentrations to reduce tumor cell survival in multiple myeloma (MM) cell lines.
Figure 5C: is a graph depicting the results of the ability of AQX-016A at varying concentrations to reduce tumor cell survival in multiple myeloma (MM) cell lines.
Figure 6A: is a graph depicting the results of the ability of compound 100 at varying concentrations to inhibit growth of 0PM2 MM cell lines.
Figure 6B: is a graph depicting the results of the ability of compound 100 at varying concentrations to inhibit growth of MM. IS MM cell lines.
Figure 6C: is a graph depicting the results of the ability of AQX-016A at varying concentrations to inhibit growth of RPMI 8226 MM cell lines.
Figure 6D: is a graph depicting the results of the ability of AQX-016A at varying concentrations to inhibit growth of U266 MM cell lines.
Figure 6E: is a graph depicting the results of the ability of AQX-016A at varying concentrations to inhibit growth of LCC6-Her2 MM cell lines.
Figure 7A: is a graph depicting the results of the activation of SHIP enzyme in vitro of Compound 100, AQX-16A and Compound 103.
Figure 7B: is a graph depicting the results of the activation of SHIP enzyme in vitro of Compound 100 and AQX- 16 A.
Figure 7C: is a graph depicting the results of Compound 100 inhibiting TNFα production from LPS stimulated SHIP +/+ but not ' ' ' BMmφs.
Figure 7D: is a graph depicting the results of Compound 100 inhibiting LPS-induced plasma TNFα levels in mice.
Figure 8A: is a graph depicting the results of SHIP + + ( ) and SHIP " " (J) macrophages pretreated with AQX-016A or carrier 30 min prior to stimulation with 10 ng/mL of LPS at 37 0 C for 2 h and TNFα production determination by ELISA. Absolute TNFα levels for SHIP +/+ and SHIP "λ cells were 623 +/- 30 and 812 +/- 20 pg/ml, respectively. Data are expressed as mean +/ SEM and are representative of three independent experiments.
Figure 8B: is a graph depicting the results of SHIP + + and SHIP " " mast cells pre-loaded with IgE and Fura-2 and treated for 30 min with 15 μM AQX-016A or carrier. Cells were then stimulated (as indicated by the arrow) with 0 ( ■■■■ ) or 10 (— ) ng/mL DNP-HSA and intracellular calcium levels monitored over time by spectrofluorometry.
Figure 9: is a graph depicting the results of mice administered 20 mg/kg AQX-016A or 0.4 mg/kg dexamethasone orally 30 min prior to an IP injection of 2 mg/kg LPS. Blood was collected 2 h later for TNFα determination by ELISA. Each symbol indicates one mouse and data are representative of three independent experiments.
Figure 1OA: is a graph depicting the results of Compound 100 inhibiting DNFB-induced neutrophil-specific myeloperoxidase (MPO) in sensitized mice. P-value <0.02 for the Compound 100 vs the vehicle treated groups. All data are representative of three independent experiments. Data are representative of three independent experiments.
Figure 1OB: is a graph depicting the results of AQX-016A inhibiting mast cell degranulation in CDl mice sensitized to hapten DNP by cutaneous application.
Figure HA: is a graph depicting the results of SHIP enzyme initial velocities at the indicated concentration of inositol- 1,2,4,5-tetrakisphosphate (IP 4 ) substrate.
Figure HB: is a graph depicting the results of the ability of product PI-3,4-P 2 (20 μM) or Compound 100 (3 μM) to activate wild-type (WT) and C2 domain deleted (δC2) SHIP enzyme at 30 μM IP 4 .
Figure HC: is a graph depicting the results of a protein overlay assay in which recombinant C2 domain was pre-incubated for 30 min at 23°C with 4. μM of Compound 100 or EtOH control and allowed to bind to PI-3,4-P 2 immobilized on membrane strips.
Figure HD: is a graph depicting the results of bead associated radioactivity obtained from recombinant C2 domain (10 nM) coated onto Copper chelate (His-Tag) YSi SPA Scintillation Beads in the presence of 0.25% BSA and incubated with 5 μCi of [ H]-Compound 100. Data are expressed as mean +/ SEM and are representative of at least three independent experiments.
Figure HE: is a graph depicting the results of bead associated radioactivity obtained from copper chelate (His-Tag) YSi SPA Scintillation Beads coated with either wild-type (WT) or C2 domain deleted (δC2) SHIP enzyme in the presence of 0.25% BSA aliquoted into 96 well plates and incubated with 5 μCi of [ H]-Compound 100 (42 Ci/mmol) with shaking at 23°C in the dark. The amount of [ H]-Compound 100 interacting with the protein coated beads was quantified on a plate scintillation counter.
Figure 12A: is a graph depicting the results of the activity of the enzymes in the presence of Compound 100 compared to that in the vehicle control and expressed as a % change in activity relative to that observed in the vehicle control. Changes in activity of <25% were not considered significant.
Figure 12B: is a graph depicting the results of the activity of enzymes affected by Compound 100 by more the 25% as shown in Figure 12 A.
Figure 13: is a graph depicting the results of the effect of Compound 100 and vehicle control on tumour size in mice.
Figure 14: is a graph depicting the results of the effect of Compound 100 and vehicle control on tumour volume over time in mice.
Detailed Description
In this specification, the following abbreviations will appear: THF (tetrahydrofuran); n-buLi (n-butyllithium); t-buLi (tert-butyllithium); Pl^PMe (methyl triphenyl phosphonium bromide); PCC (pyridinium chlorochromate); Ac (acetyl); Me (methyl); Et (ethyl); prop, (propyl); but. (butyl); RT, rt, or, r.t. (room temperature); hr. (hour(s)); DMSO (dimethylsulfoxide); DNFB (2,4-dinitrofluorobenzene); LPS (lipopolysaccarhide); TNF-α (Tumor Necrosis Factor Alpha); TBS (tert-butyl dimethylsilyl); EA (ethyl acetate); PG (protecting group); AA (amino acid); DCM (dichloromethane); DIPC (1,3-diisopropylcarbodiimide); DMAP (Dimethylamino pyridine); TFA (Trifluoroacetic acid); PEG (Polyethylene glycol); and BOC (t-Butyl carbamate).
As used herein the phrase "alkyl" refers to a molecule comprising hydrogen and carbon having the general formula C n H 2n+1 . A "C x to C y alkyl" or a "C x -C y alkyl" refers to an alkyl having a number of carbons, the number being from x to y carbons. For example, "C 1 to C 6 alkyl" denotes that the alkyl may have 1, 2, 3, 4, 5 or 6 carbons.
All possible stereoisomers, epimers, diastereomers and enantiomers and mixtures thereof are specifically included by formulas described herein that have one or more chiral centers with a
" <λλλAλftλ " bond (hereinafter termed a stereo-bond). Stereo-bonds denote that any one or more of the possible orientations of the bond is/are specifically included or specifically excluded from a particular embodiment and all of the embodiments, when considered together, include all such combinations of inclusion and exclusion of the possible bond orientations.
The phrase "stereo-mixture" as used herein may be a mixture of equal quantities or unequal quantities of two or more different stereoisomers. Stereo-mixtures may comprise any particular stereoisomer from 0% to 100% (and all values in between) as a component of the stereo-mixture, provided that at least 2 different stereoisomers are present in the mixture. A "racemic mixture" is a stereo-mixture that has equal quantities of each of the stereoisomers contained in the mixture.
The phrase "stereo-pure compound" as used herein refers to a compound having one or more chiral centers wherein each and every molecule of the compound has the same stereochemical structure. The phrase "substantially stereo-pure compound" refers to a compound that may be a stereo-pure compound or may be a compound wherein at least 97% of the molecules have the same stereochemical structure. Substantially stereo-pure compounds may be compounds wherein at least 98% of the molecules have the same stereochemical structure or may be compounds wherein at least 99% of the molecules have the same stereochemical structure. Substantially stereo-pure compounds may be compounds wherein at least 99.5% of the molecules have the same stereochemical structure or may be compounds wherein at least 99.9% of the molecules have the same stereochemical structure.
As used herein, the structure:
As used herein, the structure:
SHIP 1 Modulating Compounds and Prodrugs
Compounds of the invention comprise a pelorol, homopelorol, or pelorol/homopelorol analog core joined to a solubilizing moiety. The core and solubilizing moiety may be joined through a linking moiety.
In some embodiments, the core is or derived from a compound of Formula I or a salt thereof,
wherein;
R 1 and R 2 are independently selected from the group consisting of: H -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR', -CHO, -CO 2 H, and -CO 2 R';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR', -CHO, -CO 2 H, and -CO 2 R';
Q is a carbon skeleton selected from the group consisting of: -CH 2 -, -CY 1 Y 2 -, -CH 2 CH 2 - , -CH=CH-, -CY 1 Y 2 CY 3 Y 4 -, -CH 2 CH 2 CH 2 -, -CH=CHCH 2 -, -CH=CHCY 1 Y 2 -, and -CYiY 2 CY 3 Y 4 CYsY 6 -; where Yi, Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of: H, F, Br, Cl, I, OH, OR', and SH; or any one group of Y(/Y 2 , Y 3 /Y 4 , and Y 5 AT 6 may be =O; or YiAf 3 may form an epoxide; and, at least one of Yi, Y 2 , Y 3 , Y 4 , Y 5 and Y 6 when present, is not H;
Xi, X 2 , X 3 , and X 4 are independently selected from the group consisting of: H, R, OH, -0-(Ci-Ci 0 alkyl), -CO 2 H, -CO 2 R', F, Br, Cl, I, -CN, -SO 3 H, -OSO 3 H, NO 2 , NH 2 , -NHR, and -N(R) 2 ; where R is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR', -NR' 2 , NO 2 , -CO 2 H, -CO 2 R', and epoxide; and R' is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 ,
-NHR", -NR " 2 , NO 2 and -CO 2 H where R" is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
Compounds of Formula I have chiral centres at C-5, C-8, C-9 and C-IO and may be chiral at C-4 depending upon whether Rj and R 2 are different. Some embodiments have the same relative configuration of chiral centres as does pelorol or are enantiomers thereof, namely. S, R, R, S; or R, S, S, R (at C-5, 8, 9 and 10 respectively). Some embodiments have the same absolute configuration as pelorol at chiral centres. Some embodiments have the same relative configuration as pelorol at C-5 and C-IO with independently variable configurations at C-8 and C-9. Some embodiments have the same relative configuration as pelorol at C-5, C-8, and C-IO with variable configuration at C-9. In all cases, the configuration at C-4 (if chiral) may be variable or may be the same relative configuration to the remaining chiral centres as is shown in examples of structures of compounds of Formula I illustrated herein.
In various embodiments the core may have more specific limitations with respect to substituents Q, R 1 , R 2 , R 3 , R 4 , X 1 X 2 , X 3 and X 4 . Any combination of the following limitations is encompassed by this invention.
(a) Q may be as defined for Formula I except that Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 , is limited to H or halogen;
(b) Q may be limited to -CH 2 -, -CH 2 -CH 2 -, -CH=CH, -CH 2 -CH 2 -CH 2 - and -CH=CH-CH 2 -;
(c) Q may be limited to or saturated moieties in the limitation of Formula I, or according to the limitations of paragraph (a) or (b) above;
(d) Q may be limited to a one or two carbon skeleton within the limitations of Formula I, or according to the limitations of any of paragraphs (a) to (c) above;
(e) one or both of Rj and R 2 may be limited to -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR', -CHO, -CO 2 H, and -CO 2 R';
(f) one or both of R 1 and R 2 may be limited to methyl, ethyl, -CH 2 OH or -CH 2 OR';
(g) R' in one or both of R 1 and R 2 according to Formula I, or the limitation of paragraph (O above, may be limited to methyl, ethyl, propyl or butyl;
(h) one or both of Rj and R 2 may be limited to methyl or ethyl;
(i) one or both of Ri and R 2 may be limited to methyl;
(J) R and R' in any one or more of Xi - X 4 . may be limited to unsubstituted methyl, ethyl, propyl or butyl;
(k) one or more of Xi - X 3 may be limited to H, R, OH, 0-(Ci -C 10 alkyl), halogen, -CONH 2 , -CONHR', -C0R' 2 , NHR or N(R) 2 where R and R' are limited as in Formula I, or R and R' may be according to paragraph (j) above;
(1) one or more of X 1 - X 3 is limited to H, OH, 0-(Ci-Ci 0 alkyl), -CONH 2 , -CONHR', and -C0R' 2 , where R and R' are as in Formula I, or R and R' may be limited according to paragraph (j) above;
(m) one or more of Xi - X 3 may be limited to H, OH, and OCH 3 ;
(n) X 4 may be limited to H, R, OH, 0-(Ci-Ci 0 alkyl), CO 2 H or -CO 2 R', with R and R' as in Formula I, or R and R' may be limited according to paragraph (i) above;
(o) X 4 may be limited to H, R, OH, OCH 3 , -CO 2 H and -CO 2 R' with R and R' limited according to paragraph (j) above; and,
(p) X 4 may be limited to H, R, OH, OCH 3 , -CO 2 H or -CO 2 CH 3 .
In some embodiments, the core is or derived from a compound of Formula IA or a salt thereof:
OH
Formula IA wherein;
Ri and R 2 are independently selected from the group consisting of: -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 ORi', CHO, -CO 2 H, and -CO 2 R 2 ';
R 3 and R 4 are independently selected from the group consisting of: H, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OR 3 ', -CHO, -CO 2 H, and -CO 2 R 4 ';
Ri', R 2 ', R 3 ', and R 4 ', are independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHRj", -N(R 2 ") 2 , NO 2 and -CO 2 H where R 1 " and R 2 " is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group;
Gi is selected from the group consisting of: 0-(Cj-C 10 alkyl) and H;
G 2 is H or Ci-Cio alkyl; and
G 3 is selected from the group consisting of: H, -OH, C 1 -C 10 alkyl and 0-(Ci-Ci O alkyl).
Ln other embodiments of Formula IA, Gi is selected from the group consisting of -O-methyl and H; G 2 is H or methyl; and G 3 is selected from the group consisting of: H, methyl and O-methyl.
Compounds of Formula IA have chiral centres at C-5, C-8, C-9 and C-IO and may be chiral at C-4 depending upon whether Ri and R 2 are different. Some embodiments have the same relative configuration of chiral centres as does pelorol or are enantiomers thereof, namely: S, R, R, S; or R, S, S, R (at C-5, 8, 9 and 10 respectively). Some embodiments have the same absolute configuration as pelorol at chiral centres. Some embodiments have the same relative configuration as pelorol at C-5 and C-IO with independently variable configurations at C-8 and C-9. Some embodiments have the same relative configuration as pelorol at C-5, C-8, and C-IO with variable configuration at C-9. In all cases, the configuration at C-4 (if chiral) may be variable or may be the same relative configuration to the remaining chiral centres as is shown in examples of structures of compounds of Formula Ia illustrated herein.
In various embodiments the pelorol analog may have more specific limitations with respect to substituents R], R 2 , R 3 , and R 4 . Any combination of the following limitations is encompassed by this invention.
(a) one or both of Ri and R 2 may be limited to methyl, ethyl, -CH 2 OH, -CH 2 ORi', or -CH 2 OR 3 ';
(b) Ri', R 2 ', R 3 ', and/or R 4 ', in one or both of Ri and R 2 according to Formula Ia, or in the limitation of paragraph (a) above, may be limited to methyl, ethyl, propyl or butyl;
(c) one or both of Ri and R 2 may be limited to methyl or ethyl;
(d) one or both of Ri and R 2 may be limited to methyl;
In some embodiments, the core is or derived from a compound of Formula 2A or a salt thereof:
Formula 2A wherein;
Gi is selected from the group consisting of: 0-(Ci-Ci O alkyl) and H;
G 2 is H or Ci-Cio alkyl; and
G 3 is selected from the group consisting of: H, -OH, C 1 -C 10 alkyl and 0-(Ci-Cio alkyl).
Some embodiments of the core described above are encompassed by the invention, hi some embodiments of the core described above, the core comprises the solubilizing moiety and linking moiety and no further modification to the core is required. Whether or not a particular core requires further modification may be determined on the basis that if it comprises a solubilizing and a linking moiety as described below, then no further modification will be required. Nevertheless, additional solubilizing moieties may or may not be desirable and therefore further modification may be applied to all embodiments of the core described above, hi some embodiments a core without a linking moiety and/or a solubilizing moiety are within the scope of the present invention.
Solubilizing moieties may be any moiety having one or more ionic entities at physiological pH or multiple hydrogen bonding functionalities such as -OH or amide. Non-limiting examples of solubilizing moieties may be selected from: monophosphates; diphosphates; triphosphates;
monosaccharides; oligosaccharides; polysaccharides; oligopeptides, including, but not limited to, dipeptides and tripeptides; polypeptides; amino acids; alpha amino acids -CH(NH 2 MAA); polyethers and combinations thereof, wherein (AA) is an amino acid side chain.
Amino acid side chains include, but are not limited to, those portions of the naturally occurring protein amino acids and non-naturally occurring amino acids that do not comprise the alpha-carbon, the alpha-amine, the alpha-carboxy group and the hydrogen bonded directly to the alpha-carbon.
Polyethers include, but are not limited to, polyethylene glycol (PEG), methylated-polyethylene glycol (MPEG), polypropylene glycol, PEG-amine and MPEG-amine. Polyethers, including all of those specifically mentioned above, may have molecular weights of from about 62 to about 20,000 or more and all possible variations in between. Such a range of molecular weights generally corresponds to the repeating oxyalkane unit have from between about 0 to about 450 repeats. Polyethers, including all of those specifically mentioned above, may have molecular weights of from about 62 to about 6,500 or more and all possible variations in between. Such a range of molecular weights generally corresponds to the repeating oxyalkane unit have from between about 0 to about 150 repeats. From between about 1 repeat to about 50 repeats of the oxyalkane unit is commonly used and this represents a molecular weight range of from about 100 to about 2500.
In particular the following structures in Table I are non-limiting examples of solubilizing moieties. In the following table, each R is independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR', -NR' 2 , NO 2 , -CO 2 H, -CO 2 R', and epoxide; and R' is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR", -NR " 2 , NO 2 and -CO 2 H where R" is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
TABLE I - SOLUBILIZING MOIETIES
Wherein each (AA) is independently
Wherein each (AA) is independently any neutral amino acid side chain
Wherein n = O, 1, 2, 3, 4, 5 or 6 Wherein n = O, 1, 2, 3, 4, 5 or 6
Wherein n = O, 1, 2, 3, 4, 5 or 6
Where n = 1 to 450
Where X6, X7, X8 and X9 are as defined herein for X \
TABLE I - SOLUBILIZING MOIETIES
In some particular embodiments, each R as set out in Table I may be independently selected from H, methyl or acyl.
Linking moieties may connect the core to a solubilizing moiety. A linking moiety is a moiety that is cleaved in vivo such that a compound of the core is produced via cleavage of the linking moiety from the core. In some embodiments, cleavage of the linking moiety may be related to the stability of the linking moiety under physiological conditions. In some embodiments, the linking moiety may be cleaved in vivo enzymatically. In some embodiments, cleavage of the
linking moiety in vivo results in the formation of a core comprising an OH moiety where the linking moiety was bonded to the core prior to cleavage. Linking moieties comprising an ester moiety may provide formation of a core comprising an OH moiety where the ester linking moiety was bonded to the core prior to cleavage. A Linking moiety may be selected from the following moieties: -O-C(=O)-Z-, -NH-C(=0)-Z-, -CH 2 OC(=O)-, -C(=O)O-, and -C(=O)HN-; where Z is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR', -NR ' 2 , NO 2 , -CO 2 H, -CO 2 R', and epoxide and individual carbon atoms may be replaced by S, O, N, NR', or NR' 2 atoms; and wherein each R' is independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR", -NR" 2 , NO 2 , -CO 2 H, -CO 2 R", and epoxide; and R" is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group. In the case of phosphates, -O- is also a suitable linking moiety. Specific non-limiting examples of linking moieties are described below In Table II, where 1 represents the point of attachment to the core and 2 represents the point of attachment to a solubilizing moiety:
TABLE II - LINKING MOIETIES
%\ 2
Phosphates only
The linking moiety and the solubilizing moiety may also be described as a single structure, termed a prodrug moiety or X 5 . Prodrug moieties provide for improved solubility of core compounds. Additionally prodrugs may provide for better activity in vivo that the core compound that is produced upon cleavage of the prodrug moiety compared to direct administration of the core.
The prodrug moiety comprises all that is added to the core such that a compound of this invention is formed. Any combination of any linking moiety as described herein bonded to any solubilizing moiety as described herein may comprise a prodrug moiety. In some embodiments, a prodrug moiety is stable and difficult to remove from the core. In some embodiments, prodrug moieties may be moieties that may be cleaved in vivo such that a compound of the core is produced via cleavage at the linking moiety thereby separating the prodrug moiety or the solubilizing moiety from the core. In some embodiments, the linking moiety may be cleaved enzymatically. In some embodiments, in vivo cleavage of the linking moiety to separate the prodrug moiety or solubilizing moiety from the core results in the formation of a core comprising an OH moiety where the prodrug moiety was bonded to the core prior to cleavage. Prodrug moieties comprising an ester moiety may provide formation of a core comprising an OH moiety where the ester prodrug moiety was bonded to the core prior to cleavage. Specific, non-limiting examples of prodrug moieties are described below in Tables III and IV. hi the following Table III, each R is independently a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or is substituted with one or more of: OH, =0, SH, F, Br, Cl, I, NH 2 , -NHR', -NR ' 2 , NO 2 , -CO 2 H, -CO 2 R', and epoxide; and R' is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group that is unsubstituted or substituted with one or more of: OH, =O, SH, F, Br, Cl, I, NH 2 , -NHR", -NR " 2 , NO 2 and -CO 2 H where R" is a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
TABLE III - PRODRUG MOIETIES
Wherein (AA) is any Wherein (AA) is any amino acid side chain amino acid side chain
Wherein each (AA) is independently any amino acid side chain
Wherein each (AA) is independently any neutral amino acid side chain; am n is 1 to 10
Wherein each (AA) is independently
Wherein each (AA) is independently any neutral amino acid side chain any neutral amino acid side chain
TABLE III - PRODRUG MOIETIES
In some particular embodiments, each R as set out in Table III may be independently selected from H, methyl or acyl.
TABLE IV - ESTER PRODRUG MOIETIES
TABLE IV - ESTER PRODRUG MOIETIES
Prodrug moieties may be added to the core by replacing at least one of X 1 , X 2 , X 3 , X 4 , R 1 , R 2 , R 3 or R 4 with a prodrug moiety, or by adding a prodrug moiety onto an existing Xi, X 2 , X 3 , X 4 , R 1 , R 2 , R 3 , or R 4 substituent by substituting an atom or group of atoms from the existing Xi, X 2 , X 3 , X 4 , Ri, R 2 , R 3 , or R 4 substituent with the prodrug moiety. The atom or group of atoms that may be substituted from the existing X 1 , X 2 , X 3 , X 4 , R 1 , R 2 , R 3 , or R 4 substituent may be at a location close to the core or distanced from the core, in the main chain of the existing Xi, X 2 , X 3 , X 4 , Rj, R 2 , R 3 , or R 4 substituent, or in a side chain of the existing X 1 , X 2 , X 3 , X 4 , R 1 , R 2 , R 3 , or R 4 substituent. Prodrug moieties may also be added or substituted on to any one or more of the carbon atoms in Q or at positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of the core. Prodrug moieties may be added to the core as an ester or an amide.
Various embodiments of the invention are described by the following Formula II or a salt thereof:
II wherein Xi, X 2 , X 3 , X 4 , Ri, R 2 , R 3 , R 4 and Q are all as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above; and
X 5 is a prodrug moiety as described herein, or any combination of any linking moiety as described herein bonded to any solubilizing moiety as described herein and at least one of Ri, R 2 , R 3 , R 4 , Xi, X 2 , X 3 and X 4 is/are substituted on, substituted with and/or substituted by X 5 and/or X 5 is a substituent on any carbon atom in Q or in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of Formula II.
Various embodiments of the invention are described by the following Formula III or a salt thereof:
III wherein Xi, X 2 , X 3 , and X 4 , are all as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above; and
X 5 is a prodrug moiety as described herein and may be a substituent on any methylene or methyl carbon of the terpenoid fragment. In various embodiments, X 5 is joined via an ester linkage (or -O- in the case of phosphates).
Various embodiments of the invention are described by the following Formula IV or a salt thereof:
IV wherein Xj, X 2 , X 3 , X 4 , Ri, R 2 , R 3 , R 4 and Q are all as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above except that one or more of Xi, X 2 , X 3 and X 4 , is substituted with, substituted in, substituted on, or substituted by X 5 ; and
X 5 is a prodrug moiety as described herein, or any combination of any linking moiety as described herein bonded to any solubilizing moiety as described herein.
Various embodiments of the invention are described by the following Formula V or a salt thereof:
wherein Xj, X 2 , X 3 , X 4 , and Q are all as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above except that one or more of Xj, X 2 , X 3 , and/or X 4 , is substituted with, substituted in, substituted on, or substituted by X 5 or may be a substituent on any methylene or methyl carbon of the terpenoid fragment; and wherein X 5 is a prodrug moiety as described. In various embodiments, X 5 is joined via an ester linkage (or -O- in the case of phosphates).
Various embodiments of the invention are described by the following Formula VI or a salt thereof:
VI wherein X 1 , X 2 , X 3 , X 4 , and Q are all as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above; and
X 5 is a prodrug moiety as described herein and may be a substituent on any methylene or methyl carbon of the terpenoid fragment. In various embodiments, X 5 is joined via an ester linkage (or -O- in the case of phosphates).
Various embodiments of the invention are described by the following Formula VII or a salt thereof:
VII wherein Xi, X 2 , X 3 , and X 4 , are all as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above except that one or more Of X 1 , X 2 , X 3 , and/or X 4 , is replaced with a prodrug moiety joined directly to the aromatic ring via an ester linkage (or an -O- in the case of phosphates) or at least one of the prodrug moieties is attached, as a substituent, via an ester linkage (or an -O- in the case of phosphates) to at least one of X 1 , X 2 , X 3 and/or X 4 .
Various embodiments of the invention are described by the following Formula VIII or a salt thereof:
VIII wherein Xi is selected from the group consisting of: a prodrug moiety, O-methyl and H; X 2 is selected from the group consisting of: a prodrug moiety, O-methyl and H; X 3 is H or methyl; and X 4 is selected from the group consisting of: H, methyl and O-methyl.
In other embodiments of Formula VIII, X 1 is selected from the group consisting of: a prodrug moiety, O-methyl and H; X 2 is selected from the group consisting of: a prodrug moiety, O-methyl and H; X 3 is H or methyl; X 4 is selected from the group consisting of: H, methyl and O-methyl; and exactly one of Xi and X 2 is a prodrug moiety.
In other embodiments of Formula VIII, Xi is a prodrug moiety and X 2 is a prodrug moiety. Xl and X2 may have the same prodrug moiety or a different prodrug moiety.
In other embodiments of Formula VIII, Xi is H and X 2 is a prodrug moiety.
In other embodiments of Formula VIII, X 3 is methyl.
In other embodiments of Formula VIII, X 4 is H.
In other embodiments of Formula VIII, Xj is H, X 2 is a prodrug moiety, X 3 is H; and X 4 is methyl.
Shown below in Table V are non-limiting examples of the stereoisomers that are specifically encompassed by any one of Formulas I to VIII as depicted above. Stereo-mixtures and racemic mixtures of any two or more of the stereoisomers of Table V, substantially stereo-pure compounds and stereo-pure compounds are also included by Formulas I to VIII as depicted above. Xi, X 2 , X 3 , X 4 , Ri, R 2 , R 3 , and R 4 as used below in Table V are as defined for the respective Formula. Q as used below in Table V may be present or CH 2 , or as defined by any of Formulas I to VIII.
Table V
Table V
Table V
In various particular embodiments, in each of Formulas II, III, IV, V, VI, VII and VIII, the prodrug moiety or X 5 is one of the ester prodrug moieties as set out in Table IV above.
The structures in following Table VI are non-limiting examples of embodiments of this invention. Although relative stereochemistry may or may not illustrated for each structure, the configuration of chiral centres may vary according to any of the embodiments based on
chirality described above and compounds of this invention include all stereoisomers and enantiomers of compounds of Formulas II, III, IV, V, VI , VII and/or VIII. In some embodiments, stereo-pure compounds, substantially stereo-pure compounds, stereo-mixtures and racemic mixtures of the compounds of Table VI are also provided. In the following structures X 5 is as described above in any one of Formulas II, III, IV, V, VI, VII or VIII. In Table VI, X 1 , X 3 , and X 4 are as defined above for Formula I and may be limited by any or all of the further limitations for these groups as defined above.
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
Wherein n = 0, 1, 2, 3, 4, 5 or 6 Wherein n = O, 1 , 2, 3, 4, 5 or 6
Wherein n = O, 1, 2, 3, 4, 5 or 6
Wherein n = O, 1, 2, 3, 4, 5 or 6
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
Wherein n = O, 1, 2, 3, 4, 5 or 6 Wherein n = O, 1, 2, 3, 4, 5 or 6
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
TABLE VI - NON-LIMITING EXAMPLES OF SHIP MODULATING PRODRUGS
Wherein n = 1 to 450 Wherein n = 1 to 450
Wherein n = 1 to 450 Wherein n = 1 to 450
Compounds of the invention are often made by preparing or purchasing a core, preparing or purchasing a prodrug moiety and chemically joining the two moieties. Other methodologies may also be used, including chemically joining a prodrug moiety to a portion of a core and chemically joining the remainder of the core. Details regarding the synthesis of compounds of the invention are described below.
Synthesis of Compounds and Assays for Activity
Pelorol may be obtained from natural sources as taught in the prior art. Solvent fractionation and/or chromatography may be employed. It is possible to modify pelorol or other available compounds such as chrysene derivatives by known chemical methodologies to add, remove, or replace substituents in order to produce compounds of Formulas II, III, IV, V, VI, VII and/or VIII. Examples of such derivatization steps as applied to different compounds of Formulas II, III, IV, V, VI, VII and/or VIII are shown in more detail below.
The presence of SHIP 1 modulating compounds in a preparation may be determined by use of a variety of assays, including by biological assays which may be readily adapted from known procedures, including cell or animal based assays which monitor changes in: nitric oxide production from activated macrophages; IgE induced mast cell degranulation; LPS induced macrophage activation; TNF-α expression or activity. In addition, standard assays for agents which mediate inflammatory activity in living subjects may be employed. Adaptation of these assays is facilitated by the availability of SHIP 1 and SHIP 1 mice and bone marrow derived macrophages. In addition, the availability of anti-SHIP 1 antibodies facilitates use of immunoassay formats. Such assays may also be used to assess activity of compounds prepared by total synthesis, as described herein.
Total Synthesis of Compounds
A synthetic scheme for making embodiments of the invention and intermediates and precursors of embodiments of the invention is provided herein. Tables (VII, VIII and DC) provide examples of embodiments, intermediates and precursors of embodiments of such a synthesis with examples of different compounds of the invention which may be prepared. The compound shown in the Tables that is identical to pelorol except that the ring adjacent the aromatic ring has six members, is termed "homopelorol". Compounds having a six-membered
ring are termed "homopelorol analogs". Compounds having a five-membered ring other than pelorol are termed herein, "pelorol analogs".
In the synthesis methods shown in Tables VII, VIII and IX, compounds of the invention and intermediates of compounds of the invention shown therein may be conveniently based on sclareolide as a starting material. Appropriate derivatives of sclareolide providing desired Rj, R 2 , R 3 and/or R 4 substituents may be employed. Nu is a nucleophile, often a lithium salt of a carbanion or lithium, and X 2 is often an activating group such as -OMe or -NHAc in the aromatic compound shown in Tables VII and VIII. G n , G x , G y and Gz in Table VII are as defined herein for Xj, X 2 , X 3 and X 4 , respectively and in all of Tables VII to IX, Xi, X 2 , X 3 , X 4 , Ri, R 2 , R 3 , R 4 and/or Q may remain as found in the starting material or be appropriately altered to provide the desired substituents for the end product. Protecting groups may be employed on Ri, R 2 , R 3 and/or R 4 or Xi, X 3 , or X 4 .
In the synthesis methods shown in Table IX, the starting compound may conveniently be a core, the synthesis of which are described in the prior art (see for example international publication number WO 2004/035601, which is incorporated herein by reference).
A general procedure for preparation of phosphate ester compounds may be found in Steinber, G.M. J. Org. Chem. (1950), 15, 637. A specific procedure for tyrosine phosphorylation may be found in Gibson, B.W. et. al. J. Am. Chem. Soc. (1987), 109, 5343. A general description of pegylating compounds may be found in Zhu et al, J. Med. Chem. (2006), 49 1373-1378. More specific and detailed examples of syntheses of compounds of the invention may be found in the examples.
TABLE VII Synthesis of Pelorol and Pelorol Analog Core
TABLE VIII Synthesis of Homopelorol and Homopelorol Analog Core
TABLE IX General Synthesis of Some Prodrugs d as a non-limiting example of the target site for addition of the prodrug moiety.
Pharmaceutical Compositions, Dosages, Administration and Indications
Compounds for use in this invention may be formulated into pharmaceutical compositions in any number of ways, which would be known to a person of skill in the art, all of which are within the scope of the invention. The person of skill in the art may be expected to select appropriate pharmaceutically acceptable salts as well as appropriate pharmaceutically acceptable excipients, diluents, and carriers.
Compounds according to the invention can be provided in therapeutically- or prophylactically-acceptable amounts, in any pharmaceutically acceptable carrier. Methods well known in the art for making such pharmaceutical formulations are found in, for example, "Remington: The Science and Practice of Pharmacy" (21 edition), ed. A. Gennaro, 2005, Mack Publishing Company, Easton, PA, incorporated by reference herein. Pharmaceutical formulations according to the present invention may, for example, contain excipients, sterile water, or saline, ethanol, methanol, dimethyl sulfoxide, polyalkylene glycols such as polyethylene glycol, propylene glycol, or other synthetic solvents, oils of vegetable origin, or hydrogenated naphthalenes.
Compounds according to the invention may include hydrophobic compounds, for example, compounds that are substantially insoluble in water, but are freely soluble in solvents such as, for example, ethanol, methanol, dimethyl sulfoxide, or chloroform, or combinations thereof. Formulations containing such hydrophobic compounds may be provided using, for example, micelles, which are formed by amphiphilic compounds under certain conditions. In aqueous solutions, micelles are capable of incorporating hydrophobic compounds in their hydrocarbon cores, or within the micelle walls. Hydrophobic compounds may also be provided by solubilization in triglycerides (oils), for example, a digestible vegetable oil. The solubilized hydrophobic compound in the oil phase may be dispersed in an aqueous solution and stabilized using emulsifying agents, if desired. Alternatively, the hydrophobic compound may be provided in oil and delivered, for example, to the gastrointestinal system where bile salts may function as in vivo emulsifiers. Hydrophobic compounds may also be provided as microemulsions which, like emulsions, are liquid dispersions of oil and water, but have smaller particles with an oil phase in a micelle-like "core." Hydrophobic compounds according to the invention may also be provided together with a polymeric carrier, for example, a carbohydrate such as starch, cellulose, dextran, cyclodextrin, methylcellulose, or hyaluronic acid, or a
polypeptide, such as albumin, collagen, or gelatin. Other modes of formulation of hydrophobic compounds may include liposomes, natural and synthetic phospholipids, or solvents, for example, dimethyl sulfoxide or alcohols.
The pharmaceutical compositions of the invention may be formulated so as to provide controlled release of the active compound(s) over a period of time. Thus, the formulations could contain, for example, an amount of the compound that would be toxic if administered as a single dose, but whose controlled release does not exceed toxic levels. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers, for example, may be used to control the release of the compounds. Other potentially useful delivery systems for modulatory compounds according to the present invention include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
A "therapeutically effective amount" of a compound is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result using a compound according to the invention. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" of a compound refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. Amounts considered sufficient will vary according to the specific compound used, the mode of administration, the stage and severity of the disease, the age, sex, weight, and health of the individual being treated, and concurrent treatments.
A preferred range for therapeutically or prophylactically effective amounts of the compounds of the invention may be 0.1 nM-O.lM, 0.1 nM-0.05M, 0.05 nM-15μM 0.01 nM-10μM, O.lμM-lμM, 0.1μM-0.6μM or 0.3μM-0.6μM. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may
be selected by medical practitioners. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
In general, compounds of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LDlOO (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.
Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients, depending on the therapeutic or prophylactic objectives. Any appropriate route of administration may be employed, for example, systemic, parenteral, intravenous, subcutaneous, transdermal, transmucosal, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, topical, surgical, or oral administration. The formulations used may vary according to the chosen route of administration. Thus, for oral administration, the formulations may be in the form of tablets or capsules; for inhalants, the formulations may be in the form of powders, nasal drops, or aerosols; for transmucosal administration, the formulations may be nasal sprays or suppositories; for transdermal administration, the formulations may be creams, ointments, salves, or gels; etc.
Therapeutically effective or prophylactically effective amounts of SHIP 1 modulators and pharmaceutical compositions of this invention may be administered to patients in need of treatment or prophylaxis for cancer (neoplastic diseases), other cell proliferative disorders, inflammatory diseases and immune diseases. Neoplastic diseases include but are not limited to: leukemias, carcinomas, sarcoma, melanomas, neuroblastoma, capillary leak syndrome and hematological malignancies. Diseases with an inflammatory component include, but are not limited to: rheumatoid arthritis, multiple sclerosis, Guillan-Barre syndrome, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, psoriasis, graft versus host disease, host
versus graft, lupus erythematosis, Alzheimer's disease and insulin-dependent diabetes mellitus. Diseases related to inappropriate activation of macrophage-related cells of the reticuloendothelial lineage include osteoporosis.
Pelorol and other compounds having the structure of Formulas I- VIII exhibit SHIP 1 agonist activity. By activating SHIP 1, such agonists are particularly useful in the treatment of inflammatory diseases such as sepsis/septic shock, colitis, inflammatory bowel syndrome, and those involving macrophage proliferation or activation; neoplastic diseases such as myeloid and lymphoid leukemias; as an immunosuppressive agent such as in transplant rejection; hematopoietic disorders; and for affecting mast cell degeneration such as in the treatment or prophylaxis of allergies.
In the following Examples and Figures, the terms "Compound #", "MN#" "AQXMN#" and "AQX-MN#" are all equivalent. For example, "Compound 100" is the same as "MNlOO" and is the same as "AQXMNlOO" and is the same as "AQX-MNlOO".
Drimane-8α,ll-diol was prepared according to Kuchkova et al; Synthesis, 1997, 1045
Bromomethoxytoluene (2) was prepared according to Chan et al; J. Med. Chem. (2001), 44, 1866
Preparation of Aldehyde (1)
Drimane-8α,l l-diol (17.5g, 72.8mmol) was dissolved in IL CH 2 CI 2 . Diisopropylethylamine
(50.7mL, 291.2mmol) was added and the solution was cooled to -15°C. A solution of Pyr-SO 3 (46.3g, 291.2mmol) in DMSO (25OmL) was added dropwise over a period of 20min, then the reaction was stirred cold for an additional 5 min. To the cold reaction was added IM HCl (50OmL), and the organic layer was partitioned. The aqueous layer was washed with an additional 20OmL CH 2 Cl 2 . The pooled organic layers were then washed with saturated NaHCO 3 , dried over Na 2 SO 4 and concentrated. The crude product was purified by column chromatography (Hex: EtOAc) to yield 10.5g aldehyde (1) (44.1mmol, 60.1% yield) as a white semisolid.
Preparation of diol (3)
Bromomethoxytoluene (2) (3.64g, 18.29mmol) was dissolved in 35mL dry THF under an argon atomosphere. This solution was cooled to -78°C, and tBuLi (21.5mL, 36.6mmol) was added dropwise via syringe. The solution was stirred for 10 min at -78 0 C, then warmed to RT for 20 min. The solution was re-cooled to -78 0 C, and a solution of aldehyde (1) (1.45g, 6.09mmol) in 6mL dry THF was added via syringe. The solution was stirred at -78 0 C 2h, after which the reaction was quenched with the addition of IM HCl. EtOAc (10OmL) was added, and the organic phase was washed with IM HCl, followed by saturated NaHCO 3 . The organic phase was dried over MgSO 4 , filtered and concentrated. The crude reaction mixture was purified by column chromatography (Hex:EtOAc) to yield diol (3) (1.94g, 88.5% yield).
1 H NMR (CDCl 3 ) δ 0.34 (td, J= 13.3, 3.6 Hz, IH), 0.77 (s, 3H), 0.82 (s, 3H), 0.90 (m, IH), 0.97 (td, 13.5, 3.6 Hz, IH), 1.02 (s, 3H), 1.13 (m, IH). 1.16 (m, IH), 1.23 (m, IH) 1.33 (m, IH), 1.40 (m, IH), 1.54 (s, 3H), 1.56 (m, IH), 1.63 (m, IH), 1.84 (dt, 12.2, 3.3Hz, IH), 2.12 (d,
8.1Hz, IH), 2.33 (s, 3H), 3.79 (s, 3H), 4.79 (d, 8.1Hz, IH), 6.61 (s, IH), 6.78 (s, IH), 6.85 (s,
IH).
13 C NMR (CDCl 3 ) δ 15.9, 18.3, 19.8, 21.50, 21.53, 26.1, 33.2, 33.5, 38.6, 40.8, 41.3, 44.0,
55.1, 55.8, 62.84, 62.85, 76.0, 110.5, 113.6, 120.7, 139.8, 149.0, 159.7
HRESEvlS calcd for C 32 H 36 O 3 Na 383.2562, found 383.2563
Preparation of xanthate (4)
Diol (3) (1.94g, 5.39mmol) was dissolved in 2OmL dry THF under an argon atmosphere. To this solution was added NaH (237mg, 60% in oil, 5.93mmol). The reaction was then heated to 50°C until the solution was clear orange. The reaction was cooled to O 0 C, and CS 2 (ImL, 16.6mmol) was added. The solution was stirred for 20 min at 0 C, then warmed to RT for an additional 20 minutes, after which MeI (ImL, 16.6mmol) was added. The reaction was stirred at RT for lhour, then concentrated to dryness. The crude mixture was dissolved in EtOAc, and washed with 3x H 2 O. The organic solution was dried over MgSO 4 , filtered and concentrated to yield a mixture of xanthate (4) and fragmentation product, ketone (approx 4:1). This product mixture was used in the next step without further purification.
1 H NMR (CDCl 3 ) δ 0.56 (td, 12.9, 3.5Hz, IH), 0.77 (s, 3H), 0.80 (s, 3H), 0.87 (dd, 12.2, 2.4 Hz, IH), 0.99 (dt 13.6, 3.8Hz, IH), 1.02 (s, 3H), 1.28 (m, IH), 1.31 (m, IH), 1.34 (m, IH), 1.45 (m, IH), 1.50 (s, 3H), 1.55 (m, IH), 1.65 (m, IH), 1.75 (m, IH), 1.78 (m, IH), 1.81 (m, IH), 2.18 (d, 5.2 Hz IH), 2.28 (s, 3H), 2.38 (s, 3H), 3.75 (s, 3H), 5.18 (d, 5.2Hz, IH), 6.5 (s, IH), 6.7 (s, IH), 6.8 (s, IH)
13 C NMR (CDCl 3 ) δ 13.0, 15.9, 18.3, 20.2, 21.3, 21.6, 26.3, 33.26, 33.30, 40.2, 41.0, 41.3, 46.0, 46.8, 55.0, 55.9, 65.1, 74.2, 110.9, 112.3, 120.9, 139.5, 149.9, 159.4, 189.7 HRESIMS calcd for C 25 H 38 O 3 S 2 Na 473.2160, found 473.2159
Preparation of alcohol (5)
Xanthate (4) and ketone were dissolved as a crude mixture in 5OmL toluene, and placed under an argon atmosphere. Bu 3 SnH (2.9mL, 10.78mmol) was added, and the solution was heated. Once at reflux, a catalytic amount of VAZO (l,l'-Azobis(cyclohexanecarbonitrile)) (approx 50mg) was added through the top of the condensor. The solution was reflux ed for lhour, then an additional amount of VAZO was added (approx 50mg). The solution was refluxed for
another 45 min, after which TLC analysis (20% EtOAcrHex) indicated the reaction to be complete. The reaction was cooled, then concentrated to dryness. Flash chromatography of the crude product yielded alcohol (5) (1.12g, 3.23mmol, 60% yield, 2 steps) as a white foam.
1 H NMR (CDCl 3 ) δ 0.78 (s, 3H), 0.85 (s, 3H), 0.87 (s, 3H), 0.90 (m, IH), 0.93 (m, IH), 0.96 (m, IH), 1.09 (td, 13.3, 3.9Hz, IH), 1.25 (s, 3H), 1.31 (m, IH), 1.35 (m, IH), 1.39 (m, IH), 1.43 (m, IH), 1.54 (m, IH), 1.64 (m, IH), 1.70 (m, IH), 1.84 (dt, 12.4, 3.1Hz, IH), 2.27 (s, 3H), 2.60 (dd, 14.7, 4.5Hz, IH), 2.70 (dd, 14.7, 5.9Hz, IH), 3.75 (s, 3H), 6.49 (s, IH), 6.63 (s, IH), 6.68 (s, IH) 13 C NMR (CDCl 3 ) 5 15.4, 18.4, 20.2, 21.4, 21.5, 24.5, 31.2, 33.2, 33.3, 39.1, 40.3, 41.7, 44.0,
55.0, 56.0, 63.0, 74.1, 111.3, 111.9, 122.1, 139.2, 145.9, 159.5 HRESIMS calcd for C 23 H 36 O 2 Na 367.2613, found 367.2615
Preparation of tetracycle (6)
Alcohol (5) (1.12g, 3,23mmol) was dissolved in 10 mL CH 2 Cl 2 and cooled to O 0 C. To this solution was added SnC^ (ImL) neat. The orange solution was then stirred for 1 hour at 0°C, followed by quenching with MeOH. The reaction was extracted into EtOAc, and washed with 2x satd NaHCO 3 . The organic phase was dried over MgSO 4 , filtered and concentrated to yield tetracycle (6) (1.05g, 3.20mmol, 99% yield). This compound was used without further purification.
1 H NMR (CDCl 3 ) δ 0.86 (s, 6H), 0.98 (m, IH), 1.02 (s, 3H), 1.06 (s, 3H), 1.17 (td, 13.5, 4.2Hz, IH), 1.24 (m, IH), 1.40 (m, 2H), 1.54 (m, 2H), 1.71 (m, 4H), 2.27 (s, 3H), 2.34 (m, IH), 2.49 (dd, 14.5, 6.2Hz, IH), 2.60 (m, IH), 3.74 (s, 3H), 6.41 (s, IH), 6.62 (s, IH) 13 C NMR (CDCl 3 ) δ 15.7, 17.9, 18.6, 19.1, 19.9, 20.7, 28.6, 32.6, 32.9, 36.5, 37.9, 38.5, 39.7,
42.1, 54.8, 56.7, 64.2, 107.9, 113.4, 117.9, 132.5, 143.8, 157.3 HRESIMS calcd for C 23 H 35 O [M+H] + 327.2688, found 327.2685
Preparation of Compound 100 (7)
Tetracycle (6) (1.05g, 3.20mmol) was dissolved in 15 mL DCM. To this solution was added a solution of BBr 3 (1.0M in DCM) (3.2OmL, 3.20mmol). The solution was stirred at RT for 2 hours, then concentrated to dryness. The brown residue was dissolved in EtOAc, then washed
with H 2 O until the pH of the aqueous layer was neutral. The crude product was purified by flash chromatography to yield Compound 100 (7) (931mg, 2.98mmol, 93% yield) as a white solid.
1 H NMR (CDCl 3 ) δ 0.88 (s, 6H), 0.97 (m, IH), 1.00 (m, IH), 1.04 (s, 3H), 1.07 (s, 3H), 1.18
(td, 13.2, 4.2Hz, IH), 1.42 (m, IH), 1.43 (m, IH), 1.53 (m, IH), 1.58 (m, IH), 1.71 (m, IH),
1.73 (m, IH), 1.74 (m, IH), 1.75 (m, IH), 2.26 (s, 3H), 2.35 (dt, 11.7, 3.0Hz, IH), 2.48 (dd,
14.35, 6.44Hz, IH), 2.59 (m, IH), 6.36 (d, 1.9Hz, IH), 6.55 (d, 1.9Hz, IH)
13 C NMR (CDCl 3 ) δ 16.1, 18.3, 18.8, 19.6, 20.4, 21.1, 29.0, 33.1, 33.4, 37.0, 39.0, 40.1, 42.6,
47.1, 57.1, 64.5, 109.9, 115.1, 133.1, 144.2, 144.7, 153.5
HRESIMS calcd for C 22 H 33 O [M+H] + 313.2531, found 313.2526
Example 2 - Synthesis of Compound 124 and Compound 125
Experimental for preparation of Compound 124 and Compound 125
Preparation of 12
Bromide 2 (1.4 Ig, 7.09mmol) was dissolved in 30ml dry THF under an argon atmosphere and cooled to -78 0 C. tBuLi (8.3ml, 1.7M in pentane, 14.2mmol) was added over a period of lOmin and the solution was warmed to rt. After 15 min, the solution was recooled to -78°C and stirred for an additional 30 min. A solution of enal 11 (521mg, 2.36mmol) in 8mL dry THF was then added to the cold solution and the reaction was stirred at -78 0 C for 30 min. IM HCl was then added and the reaction was warmed to rt. The crude product was extracted into EtOAc and washed with satd. NaHCO3. The organic phase was dried over MgSO4, filtered
and concentrated. The crude compound was purified by flash chromatography to yield alcohol 12 (451mg, 1.32mmol, 56% yield).
1 H NMR (CDCl 3 ) δ 0.88 (s, 3H), 0.91 (s, 3H), 1.12 (s, 3H), 1.17 (m, IH), 1.20 (m, IH), 1.27
(s, 3H), 1.34 (td, 12.9, 3.5 Hz, IH), 1.41-1.75 (m, 6H), 2.01 (m, 2H), 2.31 (s, 3H), 3.77 (s, 3H),
5.33 (s, IH), 6.55 (s, IH), 6.75 (s, IH), 6.83 (s, IH)
13 C NMR (CDCl 3 ) δ 18.9, 19.1, 20.4, 21.5, 21.7, 21.8, 33.3, 33.4, 34.8, 37.1, 38.9, 41.5, 52.5,
55.1, 69.6, 108.4, 111.7, 118.5, 133.3, 138.9, 143.4, 147.6, 159.6
HRESIMS calcd for C 23 H 34 O 2 Na 365.2457, found 365.2458
Preparation of 13 and 14
Alcohol 12 (450mg, 1.32mmol) was dissolved in 1OmL CH 2 Cl 2 under an argon atmosphere and cooled to -78 C. SnCl 4 (ImL) was added and the resulting yellow solution was stirred for 15min. IM HCl was added to the cold solution and the mixture was allowed to warm to rt. The layers were separated and the organic phase was washed with 2xH 2 O, dried over MgSO 4 , filtered and concentrated. The crude product was purified by flash chromatography to yield 13 and 14 (284mg, O.88mmol, 67% yield) as a 1:1 mixture.
Preparation of 15 and 16
A 1:1 mixture of epimers 15 and 16 (84mg) was dissolved in 5mL 1:1 MeOH:DMF. 10% Pd/C (32mg) was added, and the slurry was saturated with H2. The solution was stirred for 16h under a balloon of H2, after which the solid catalyst was filtered off and washed with EtOAc. The organic phase was washed with 3xH2O, dried over MgSO4, filtered and concentrated to yield 15 and 16 (80mg, 95% yield) as a 1:1 mixture.
Preparation of Compound 124 and Compound 125
A 1:1 mixture of 15 and 16 (80mg, 0.24mmol) was dissolved in 0.5mL CH 2 Cl 2 . BBr 3 (2mL, IM in CH 2 Cl 2 , 2.0mmol) was added and the solution was stirred at rt for 15 min. The reaction was quenched with slow addition of MeOH, and the crude reaction mixture was concentrated under vacuum. The crude product was purified by flash chromatography to yield a 1 : 1 mixture of Compound 124 and Compound 125. Compound 124 was fractionally crystallized from the mixture by cooling from toluene.
1 H NMR (CDCl 3 ) 50.87 (s, 3H), 0.89 (s, 3H), 1.20 (m, 2H), 1.25 (s, 3H), 1.30-1.45 (m, 7H),
1.62 (s, 3H), 1.70 (m, IH), 1.85 (dd, 12.0, 8.4Hz, IH), 2.01 (m, IH), 2.33 (s, 3H), 2.73 (dd,
15.5, 8.3Hz, IH), 2.78 (m, IH), 4.51 (s, IH), 6.39 (s, IH), 6.50 (s, IH)
13 C NMR (CDCl 3 ) δ 17.9, 19.7, 21.5, 24.1, 25.8, 32.5, 33.1, 33.5, 36.1, 36.2, 37.9, 41.9, 46.4,
47.5, 61.9, 108.4, 115.8, 133.9, 142.4, 143.3, 153.2
HRESIMS calcd for C 22 H 33 O [M+H] + 313.2531, found 313.2533
Compound 125 was fractionally crystallized from the enriched remainder from CH 3 CN. 1 H NMR (CDCl 3 ) δ 0.47 (s, 3H), 0.80 (s, 3H), 0.89 (s, 3H), 0.90 (m, IH), 1.00 (dd, 11.3, 4.4 Hz, IH), 1.17 (m, IH), 1.18 (s, 3H), 1.29 (m, IH), 1.40 (m, 2H), 1.52 (m, IH), 1.62 (m, IH), 1.70 (m, 2H), 2.33 (s, 3H), 2.52 (dt, 14.4, 5.5Hz, IH), 2.62 (d, 16.9Hz, IH), 2.97 (dd, 16.9, 8.0Hz, IH), 4.52 (s, IH), 6.35 (s, IH), 6.47 (s, IH)
13 C NMR (CDCl 3 ) δ 15.2, 18.0, 19.1, 19.5, 21.4, 30.5, 31.7, 32.8, 32.9, 34.3, 36.9, 40.7, 41.7, 47.7, 52.0, 62.1, 108.3, 115.3, 133.2, 140.7, 145.6, 153.4 HRESIMS calcd for C 22 H 33 O [M+H] + 313.2531, found 313.2533
Example 3 - Synthesis of Compound 105:
Experimental for preparation of Compound 105:
Compound 100 (7), (60.4mg, 0.193mmol), Nα-Boc-Nδ,Nω-di-Z-L-Arg-OH (157.3mg,
0.290mmol) and DMAP (~2mg) were combined in 3mL CH 2 Cl 2 . DIPC was added, and the solution was stirred for 2h at RT. The reaction was concentrated, and purified by flash chromatography to yield 8 as a white foam.
1 H NMR (CDCl 3 ) δ 0.86 (s, 6H), 0.96 (s, 2H), 1.01 (s, 3H), 1.05 (s, 3H), 1.17 (m, IH), 1.39
(m, IH), 1.43 (s, 9H), 1.50 (m, IH), 1.58 (m, IH), 1.70 (m, 2H), 1.73 (m, 2H), 1.78 (m, 2H),
1.92 (m, IH), 2.25 (s, 3H), 2.32 (m, IH), 2.47 (dd, 14.6, 6.1Hz, IH), 2.58 (m, IH), 4.04 (m,
2H), 4.47 (s, br, IH), 5.12 (s, 2H), 5.22 (2H), 6.52 (s, IH), 6.70 (s, IH), 7.27 (m, 3H), 7.35 (m, 7H), 9.24 (s, br, IH), 9.45 (s, br, IH)
13 C NMR (CDCl 3 ) δ 14.1, 16.0, 18.2, 18.7, 19.4, 20.0, 20.9, 21.0, 24.9, 28.2 (3C), 28.8, 29.3, 32.9, 33.2, 36.9, 38.5, 40.0, 42.4, 44.1, 47.3, 53.4, 56.9, 60.2, 64.2, 66.9, 68.8, 79.7, 115.5, 120.8, 127.60, 127.62, 128.2, 128.3 (2C), 128.7 (2C), 132.9, 134.6, 136.8, 144.3, 148.1, 149.3, 155.3, 155.7, 160.4, 163.7, 171.5 HRESIMS calcd for C 49 H 65 N 4 O 8 [M+H] + 837.4802, found 837.4805
Preparation of Compound 105
Compound 8 was dissolved in 3mL 70%TFA/CH 2 Cl 2 and stirred for Ih. The solvents were then evaporated, and the resulting residue was redissolved in toluene and concentrated to dryness. The resulting solid was then dissolved in 15mL MeOH and lOOmg Pd/C (10% wt) was added. The solution was saturated with H 2 and stirred overnight under a hydrogen balloon. The Pd/C was filtered off and the solution was concentrated to dryness. The resulting solid was dissolved in 1OmL H 2 O and 5OuL IM HCl was added. After stirring 5 min, the solution was lyophilized to yield Compound 105 as a white powder.
1 H NMR (CD 3 OD) δ 0.86 (s, 3H), 0.87 (s, 3H), 1.01 (m, 2H), 1.05 (s, 3H), 1.08 (s, 3H), 1.19 (td, 13.9, 4.2Hz, IH), 1.41 (m, 2H), 1.52 (m, IH), 1.66 (m, 2H), 1.72 (m, 4H), 1.85 (m, 2H), 2.13 (m, 2H), 2.28 (s, 3H), 2.39 (m, IH), 2.51 (dd, 14.6, 6.1Hz, IH), 2.64 (m, IH), 4.33 (t, 6.3Hz, IH), 6.64 (s, IH), 6.83 (s, IH)
13 C NMR (CD 3 OD) δ 16.7, 19.1, 19.3, 20.5, 20.6, 21.5, 25.7, 28.7, 29.8, 33.9, 34.0, 38.2, 40.0, 41.3, 41.6, 43.7, 48.7, 53.7, 58.3, 66.0, 116.5, 121.9, 134.5, 145.8, 149.2, 151.3, 158.8, 169.4 HRESIMS calcd for C 28 H 45 N 4 O 2 [M+H] + 469.3543, found 469.3540
Example 4 - Synthesis of Compound 106:
Experimental for preparation of Compound 106:
Compound 100 (7) (41.7mg, 0.133mmol) was dissolved in 4mL DMF. K 2 CO 3 (37mg, 0.266mmol) was added, and the solution was stirred for 10 min. Boc-Lys(Boc)-OSu (115.3mg, 0.266mmol) was added, and the solution was stirred for 18h at RT. The reaction was extracted into EtOAc, and washed with 3 x H 2 O. The organic phase was dried, filtered and concentrated. The crude product was purified by flash chromatography to yield (23) (80.6mg, 0.126mmol, 95% yield) as a white foam.
This foam was dissolved in 2mL CH 2 Cl 2 and TFA (2mL) was added. The solution was stirred at RT for 2h, then concentrated to dryness. Toluene (3mL) was added, and the solution was concentrated to dryness again. The resulting residue was dissolved in 5mL H 2 O and lOOμL IM HCl was added. The aqueous solution was then filtered through a 0.22μm syringe filter, and lyophilized to yield Compound 106-2HC1 as a white powder.
1 H NMR (CD 3 OD) δ 0.82 (s, 3H), 0.83 (s, 3H), 0.96 (m, 2H), 1.01 (s, 3H), 1.04 (s, 3H), 1.16 (td, 13.5, 4.5Hz, IH), 1.36 (m, 2H), 1.50 (m, IH), 1.59 (m, 3H), 1.69 (m, 5H), 2.04 (m, 2H), 2.24 (s, 3H), 2.36 (m, IH), 2.48 (dd, 14.7, 6.2Hz, IH), 2.60 (m, IH), 2.92 (m, 2H), 3.23 (m, IH), 4.23 (m, IH), 6.58 (s, IH), 6.76 (s, IH)
13 C NMR (CD 3 OD) 6 16.7, 19.0, 19.4, 20.5, 20.7, 21.5, 23.3, 28.1, 29.8, 31.1, 33.8, 34.0, 38.3, 40.1, 40.3, 41.4, 43.7, 53.9, 58.4, 66.1, 116.5, 121.9, 134.5, 145.9, 149.3, 151.4, 169.4 HRESIMS calcd for C 28 H 45 N 2 O 2 [M+H] + 441.3481, found 441.3484
Example 5 - Synthesis of Compound 108:
Experimental for preparation of Compound 108:
Compound 100 (7: 12.1mg, 0.039mmol) was dissolved in ImL CH 2 Cl 2 . DMAP (~lmg) was added, followed by Bromoacetyl bromide (5.1μL, 0.059mmol), and the reaction was stirred overnight. Concentration of the reaction, followed by flash chromatography yielded bromide (9) (12.9mg, 0.030mmol, 77% yield).
1 H NMR (CDCl 3 ) δ 0.86 (s, 6H), 0.97 (m, IH), 1.02 (s, 3H), 1.07 (s, 3H), 1.18 (td, 13.5, 4.5Hz, IH), 1.25 (s, IH), 1.41 (m, 2H), 1.52 (m, IH), 1.60 (m, IH), 1.71 (m, 2H), 1.77 (m, 2H), 2.29
(s, 3H), 2.35 (m, IH), 2.52 (dd, 14.6, 6.1Hz, IH), 2.62 (m, IH), 4.00 (s, 2H), 6.59 (s, IH), 6.78 (s, IH)
13 C NMR (CDCl 3 ) δ 16.1, 18.3, 18.9, 19.5, 20.1, 21.1, 25.7, 28.9, 33.1, 33.3, 37.0, 38.6, 40.1, 42.5, 47.4 57.0, 64.3, 115.3, 120.6, 133.2, 144.6, 148.1, 149.8, 166.2
79
HRESIMS calcd for C 24 H 33 O 2 BrNa 455.1562, found 455.1550
81
HRESIMS calcd for C 24 H 33 O 2 01 BrNa 457.1541, found 457.1522
Bromide 9 (6.06 g, 11 mmol) was added portionwise over a period of 30 min. to a solution of HS-PEG (35 g, MW 6000) and iV,N-diisopropylethylamine (2.7 mL) in acetonitrile (9OmL) under nitrogen at 0 0 C. After addition, the ice bath was removed and the mixture was allowed to warm to room temperature. After 3-4 hours, 2-propanol (1200 mL) was added over 30 min. After an addition 1.5 h, the resulting solid was collected on a Buchner funnel and washed with 2 X 150 mL of 2-propanol. The wet cake was then dissolved in acetonitrile (80 mL) containing 0.5% 1 Pr 2 NEt at 0-5 0 C and precipitated by addition of 2-propanol (100OmL). The resulting solid was collected and washed with 2-propanol and dried in vacuo to give Compound 108.
Example 6 - Synthesis of Compound 109:
Experimental for the synthesis of Compound 109:
General Procedure: Steinberg, G.M. J. Org. Chem. (1950), 15, 637.
Specific tyrosine phosphorylation; Gibson, B. W et al. J. Am. Chem. Soc. (1987), 109, 5343.
Compound 100 (7) (250mg, 0.80mmol) was slurried in tetrazole/MeCN solution (18mL, 8.1mmol). THF was added until the solution was clear (~10mL). To this solution was added dibenzyl N,N-diethylphosphoramidite (1.Og, 85%, 2.7mmol), and the reaction was stirred at RT for Ih. To the reaction was then added 1OmL t-butylhydroperoxide (70% in H 2 O) and the
solution was stirred vigorously for an additional 30 min. The reaction mixture was extracted into EtOAc, washed with Ix Na 2 S 2 O 5 , Ix IM HCl, then with satd NaHC0 3 . The organic layer was dried over MgSO 4 , filtered and concentrated. The crude product was purified by flash chromatography to yield 10 (280mg, 0.49mmol, 61% yield).
1 H NMR (CDCl 3 ) δ 0.88 (s, 6H), 0.96 (m, IH), 0.99 (m, IH), 1.03 (s, 3H), 1.06 (s, 3H), 1.19
(m, IH), 1.26 (m, IH), 1.43 (m, 2H), 1.53 (m, IH), 1.60 (m, IH), 1.72 (m, 3H), 2.24 (s, 3H),
2.33 (m, IH), 2.47 (dd, 14.6, 6.3Hz, IH), 2.58 (m, IH), 5.11 (s, 2H), 5.13 (s, 2H), 6.64 (s, IH),
6.84 (s, IH), 7.33 (s, 10H)
13 C NMR (CDCl 3 ) δ 16.0, 18.2, 18.7, 19.4, 20.1, 21.0, 28.8, 32.9, 33.3, 36.9, 38.6, 40.0, 42.4,
47.2, 56.9, 64.3, 69.6, 69.7, 114.3, 114.4, 119.7, 119.8, 127.9, 128.4, 133.1, 135.5, 135.6,
144.5, 148.0, 148.1, 148.5
HRESIMS calcd for C 36 H 46 O 4 P [M+H] + 573.3134, found 573.3117
Preparation of Compound 109
Compound 10 (280mg, 0.49mmol) was dissolved in MeOH (8mL), and 10% Pd/C was added
(30mg). The solution was saturated with H 2 and stirred for 18h at RT. The reaction was then filtered through a 0.45μm membrane and concentrated to dryness to yield Compound 109
(150mg, 0.38mmol, 78% yield) as a white powder.
1 H NMR (CD 3 OD) δ 0.83 (s, 6H), 0.93 (m, 2H), 1.00 (s, 6H), 1.14 (m, IH), 1.35 (m, IH), 1.38
(m, IH), 1.48 (m, IH), 1.65 (m, 5H), 2.21 (s, 3H), 2.31 (m, IH), 2.41 (dd, 14.5, 6.0Hz, IH),
2.55 (m, IH), 6.64 (s, IH), 6.82 (s, IH)
13 C NMR (CD 3 OD) δ 16.7, 19.2, 19.4, 20.7, 21.6, 29.8, 33.9, 34.0, 38.2, 40.1, 41.3, 43.7, 48.5,
49.8, 58.4, 66.0, 115.8, 121.3, 134.1, 145.4, 149.1, 150.4
HRESIMS calcd for C 22 H 33 O 4 NaP 415.2014, found 415.2028
Example 7 - Synthesis of Compound 103 and Compound 108
\
Drimane-8α,l l-diol was prepared according to Kuchkova et al; Synthesis, 1997, 1045
Bromomethoxytoluene (2) was prepared according to Chan et al; J. Med. Chem. 44, 1866
Preparation of Aldehyde (1)
Drimane-8α,l l-diol (17.5g, 72.8mmol) was dissolved in IL CH 2 Cl 2 . Diisopropylethylamine (50.7mL, 291.2mmol) was added and the solution was cooled to -15°C. A solution of PyT-SO 3 (46.3g, 291.2mmol) in DMSO (25OmL) was added dropwise over a period of 20min, then the reaction was stirred cold for an additional 5 min. To the cold reaction was added IM HCl (50OmL), and the organic layer was partitioned. The aqueous layer was washed with an additional 20OmL CH 2 Cl 2 . The pooled organic layers were then washed with saturated NaHCO 3 , dried over Na 2 Sθ 4 and concentrated. The crude product was purified by column chromatography (Hex: EtOAc) to yield 10.5g aldehyde (1) (44.1mmol, 60.1% yield) as a white semisolid.
Preparation of diol (3)
Bromomethoxytoluene (2) (3.64g, 18.29mmol) was dissolved in 35mL dry THF under an argon atmosphere. This solution was cooled to -78 0 C, and tBuLi (21.5mL, 36.6mmol) was added dropwise via syringe. The solution was stirred for 10 min at -78 C, then warmed to RT for 20 min. The solution was re-cooled to -78 0 C, and a solution of aldehyde (1) (1.45g, 6.09mmol) in 6mL dry THF was added via syringe. The solution was stirred at -78 0 C 2h, after which the reaction was quenched with the addition of IM HCl. EtOAc (10OmL) was added, and the organic phase was washed with IM HCl, followed by saturated NaHCO 3 . The organic phase was dried over MgSO 4 , filtered and concentrated. The crude reaction mixture was purified by column chromatography (Hex:EtOAc) to yield diol (3) (1.94g, 88.5% yield) as a diastereomeric mixture.
Preparation of xanthate (4)
Diol (3) (1.94g, 5.39mmol) was dissolved in 2OmL dry THF under an argon atmosphere. To this solution was added NaH (237mg, 60% in oil, 5.93mmol). The reaction was then heated to 50 C until the solution was clear orange. The reaction was cooled to O 0 C, and CS 2 (ImL, 16.6mmol) was added. The solution was stirred for 20 min at O 0 C, then warmed to RT for an additional 20 minutes, after which MeI (ImL, 16.6mmol) was added. The reaction was stirred at RT for lhour, then concentrated to dryness. The crude mixture was dissolved in EtOAc, and washed with 3x H 2 O. The organic solution was dried over MgSO 4 , filtered and concentrated to
yield a mixture of xanthate (4) and fragmentation product, ketone (12) (approx 4:1). This product mixture was used in the next step without further purification.
Preparation of alcohol (5)
Xanthate (4) and ketone (12) were dissolved as a crude mixture in 5OmL toluene, and placed under an argon atmosphere. Bu 3 SnH (2.9mL, 10.78mmol) was added, and the solution was heated. Once at reflux, a catalytic amount of VAZO (l,l'-Azobis(cyclohexanecarbonitrile)) (approx 50mg) was added through the top of the condensor. The solution was refluxed for lhour, then an additional amount of VAZO was added (approx 50mg). The solution was refluxed for another 45 min, after which TLC analysis (20% EtOAc: Hex) indicated the reaction to be complete. The reaction was cooled, then concentrated to dryness. Flash chromatography of the crude product yielded alcohol (5) (1.12g, 3.23mmol, 60% yield, 2 steps) as a white foam.
Preparation of tetracycle (6)
Alcohol (5) (1.12g, 3,23mmol) was dissolved in 10 niL CH 2 Cl 2 and cooled to O 0 C. To this solution was added SnCU (ImL) neat. The orange solution was then stirred for 1 hour at O 0 C, followed by quenching with MeOH. The reaction was extracted into EtOAc, and washed with 2x satd NaHCO 3 . The organic phase was dried over MgSO 4 , filtered and concentrated to yield tetracycle (6) (1.05g, 3.20mmol, 99% yield). This compound was used without further purification.
Preparation of Compound 100 (7)
Tetracycle (6) (1.05g, 3.20mmol) was dissolved in 15 mL DCM. To this solution was added a solution Of BBr 3 (1.0M in DCM) (3.2OmL, 3.20mmol). The solution was stirred at RT for 2 hours, then concentrated to dryness. The brown residue was dissolved in EtOAc, then washed with H 2 O until the pH of the aqueous layer was neutral. The crude product was purified by flash chromatography to yield Compound 100 (7) (931mg, 2.98mmol, 93% yield) as a white solid.
Preparation of glycine prodrug (9)
Compound 100 (7) (36.1mg, O.l lόmmol), Boc-Gly-OH (30.5mg, 0.174mmol), DMAP (~2mg) were combined in ImL CH 2 Cl 2 . 1,3-diisopropylcarbodiimide (27uL, 0.174mmol) was added,
and the solution was stirred at RT for 2 hours. The reaction was then directly submitted to flash chromatography to yield a white foam (9). This compound was dissolved in 50% TFA/ CH 2 Cl 2 for 1 hour. The solution was concentrated, redissolved in toluene, and concentrated to dryness. Et 2 O (15mL) was added, and the compound was triturated until the precipitate appeared as a uniform solid. Centrifugation of the mixture, followed by washing of the solid with Et 2 O yielded prodrug (9) (44.5mg, 0.092mmol, 80% yield) as the TFA salt.
Preparation of Pegylated prodrug (Compound 108)
To a solution of (7) (43.Og, lOOmmol) in acetonitrile at 0°C were added α-bromoacetic acid (19.46 g, 140mmol) and DMAP (610mg, 5 mmol). The reaction mixture was then treated with a solution of DCC (29.87 g, 145 mmol) in acetonitrile (200 mL) dropwise over 30 min, then stirred at 0°C for 2.5h. The white solid formed was removed by filtration and washed with acetonitrile (2 X 100 mL). The combined acetonitrile washes were then added to H 2 O (400OmL) over 15 min. After stirring for another 15 min, the resulting solid was collected and washed with H 2 O (2 X 250ml) and IPA (2 X 20OmL) and then dried in vacuo. The collected white solid (6.06 g, 11 mmol) was added portionwise over a period of 30 min. to a solution of HS-PEG (35 g, MW 6000) and N,N-diisopropylethylamine (2.7 mL) in acetonitrile (9OmL) under nitrogen at 0 0 C. After addition, the ice bath was removed and the mixture was allowed to warm to room temperature. After 3-4 hours, 2-propanol (1200 mL) was added over 30 min. After an addition 1.5 h, the resulting solid was collected on a Buchner funnel and washed with 2 X 150 mL of 2-propanol. The wet cake was then dissolved in acetonitrile (80 mL) containing 0.5% 1 Pr 2 NEt at 0-5°C and precipitated by addition of 2-propanol (100OmL). The resulting solid was collected and washed with 2-propanol and dried in vacuo to give Compound 108.
Example 8 - Synthesis of Compound 102, Compound 103 and Compound 104
Example 9 - Compound 103 inhibits TNF alpha production better than Compound 100
An assay to determine the relative inhibition of TNFα production by Compound 103 compared to Compound 100 was conducted as follows.
ill AA macrophage cells were plated at 2X10 cells/well in 24 well plates. The next day the media was changed and Compound 100, Compound 103 or cyclodextrin carrier were added to the wells at the indicated concentrations for 30 min prior to stimulation of the cells with 2 ng/mL lipopolysaccharide (LPS). LPS activation of macrophages leads to production of TNF alpha which can be detected in the culture supernatant and quantified by ELISA. The results are depicted in a graph in Figure 1.
Example 10 - Compound 106 inhibits macrophage TNFα production
An assay to determine the inhibition of macrophage TNFα by varying concentrations of Compound 106 was carried out as follows.
J2M macrophage cells were plated at 2X10 cells/well in 24 well plates. The next day the media was changed and Compound 106 or PBS carrier were added to the wells at the indicated concentrations for 30 min prior to stimulation of the cells with 2 ng/mL lipopolysaccharide (LPS). LPS activation of macrophages leads to production of TNFa which can be detected in the culture supernatant and quantified by ELISA. The results are depicted in a graph in Figure 2.
Example 11 - Compound 106 inhibits calcium influx in mast cells
An assay to determine the inhibition of calcium influx in mast cells by Compound 106 was carried out as follows.
The activation of mast cells by IgE receptor crosslinking leads to an influx of calcium into the cells, followed by degranulation and secretion of pro-inflammatory mediator. Bone marrow derived mast cells were loaded with the fluorescent calcium indicator dye Fura-2 prior to treatment for 1 hr with Compound 106 or PBS carrier. Cells were then stimulated or not with an anti IgE antibody to cross-link the IgE receptors. Calcium influx was then monitored by fluorimetry. The results are depicted in a graph in Figure 3.
Example 12 - Compound 108 inhibits TNFα production in Wild Type (WT) but not Knock-out (KO) macrophages
Peritoneal macrophages isolated from wild-type (WT) or SHIP knock-out (KO) mice were in 24 well plates in CSF-I containing media. The next day the media was changed and Compound 108 or PBS carrier were added to the wells at the indicated concentrations for 60 min prior to stimulation of the cells with 2 ng/mL lipopolysaccharide (LPS). LPS activation of macrophages leads to production of TNF alpha which can be detected in the culture supernatant and quantified by ELISA. The results are depicted in a graph in Figure 4.
Example 13 - Assay Screening of SHIP Modulators and Prodrugs thereof
Various different SHIP modulating compounds and prodrugs thereof were tested in various different assays.
Assay 1) In vitro testing in a SHIP enzyme assay. Test compounds were dissolved in a suitable solvent (e.g. EtOH, DMSO and others) and diluted into aqueous buffer (20 mM Tris HCl, pH 7.5 and 10 mM MgCl 2 ). SHIP enzyme assays were performed in 96-well microtitre plates with 10 ng of enzyme/well in a total volume of 25 μL of 20 mM Tris HCl, pH 7.5 and 10 mM MgCl 2 . SHIP enzyme was incubated with test extracts (provided in solvent) or vehicle for 15 min at 23°C before the addition of 100 μM inositol- 1,3,4,5-tetrakisphosphate (Echelon Biosciences Inc, Salt Lake City, Utah). After 20 min at 37 0 C and the amount of inorganic phosphate released assessed by the addition of Malachite Green reagent and absorbance measurement at 650 nm.
Assay 2) Macrophage TNF-α production. J774.1a macrophage cells were treated with 10 μg/mL of test compound dissolved in solvent (e.g. cyclodextran) for 40 minutes prior to the addition of lOOng/mL LPS. Culture supernatants were collected after 2 hr and 5 hr for TNF-α determination by ELISA.
Assay 3) Macrophage TNF-α NO assay. J774.1a macrophage cells were treated with 10 μg/ml of test compound dissolved in solvent for 40 minutes prior to the addition of LPS. Culture supernatants were collected after 24 hr. for determination of NO concentration using the Griess reagent.
Assay 4) Stimulation of mast cells by FcεRI crosslinking. Mast cells were pre-loaded overnight in BMMC medium lacking IL-3 with 0.1 μg/ml anti-DNP IgE (SPE-7, Sigma, Oakville, Ont). For calcium flux measurements, cells were incubated with 2 μM fura 2-acetoxymethyl ester (Molecular Probes, Eugene, OR) in Tyrode's buffer at 23 0 C for 45 min. Cells were then washed and incubated in the presence of the test compound 30 min prior to stimulation with the indicated concentration of DNP-human serum albumin (DNP-HSA). Calcium influx was monitored by spectrofluorometry as described previously. For analysis of intracellular signaling, cells were pre-loaded with anti-DNP IgE as above, pre-treated with the test compound for 30 min at 37°C and stimulated with 20 ng/ml DNP-HSA for 5 min. Total
MAPK cell lysates were then prepared and analyzed for phospho-PKB, phospho-p38 phospho-MAPK, Grb-2 (Cell Signalling, Mississauga, Ont) and SHIP by immunoblot analysis.
Assay 5) Mouse acute cutaneous anaphylaxis model. 6-8 week old CDl mice (University of British Columbia Animal Facility, Vancouver, BC) were sensitized to the hapten DNP by cutaneous application of 25 μL of 0.5% dinitroflourobenzene (DNFB) (Sigma, Oakville, Ont) in acetone to the shaved abdomen of mice for two consecutive days. 24 hrs later, test substances (dissolved in 10 μL of 1:2 DMSO:MeOH) were painted on the right ear while the left ear received vehicle control. 30 min after drug application, DNFB was applied to both ears to induce mast cell degranulation. A 6 mm punch was taken from the ear and immediately frozen on dry ice for subsequent determination of neutrophil myeloperoxidase (MPO) activity.
Assay 6) Mouse endotoxemia model. 6-8 week old C57B16 mice (VCHRI Mammalian Model of Human Disease Core Facility, Vancouver, BC) were orally administered the test compound 30 min prior to an IP injection of 2 mg/kg of LPS (E. CoIi serotype 0111 :B4, Sigma, Oakville, Ont). Blood was drawn 2 hrs later for determination of plasma TNFα by εLISA.
Assay 7) In vitro Mulitple Myeloma (MM) assay. The ability of SHIP activators to reduce tumor cell survival was assessed in MM cell lines treated with the test compound. The lines OPMl, OPM2, MM. IS and RPMI 8226 were plated at a density of 1 x 10 5 cells/mL in
200 μL of medium with various concentrations of the test compound, and viable cell numbers were determined on day 3 and day 5 by trypan blue exclusion. The lines RPMI 8226 and U266 were plated at a density of 1 x 10 cells/mL in 250 μL of medium with various concentrations of the test compound. At day 4, the medium of each culture was replaced by fresh medium containing the same concentration of test compound. At day 7, the viable cell number of each culture was determined by trypan blue exclusion.
MM cell lines were cultured in 96 well plates seeded with 3x10 cells suspended in 200 μL of medium along with various concentrations of test compound (and associated cyclodextran vehicle control), with LY294002 serving as a positive control in the experiments. After 24-48 hrs of culture, 1 Ci of [3H]-thymidine (GE Healthcare, Baie D'Urfe, Canada) being added for the final 8 hours. Cells were harvested and DNA associated radioactivity was measured via liquid scintillation counting using a Wallac Microbeta counter (Perkin-Elmer; Boston, MA).
Assay 8) In vivo Multiple Myeloma (MM) assay. Mice were inoculated with at two sites each with 3 x 10 luciferase expressing OPM2 cells suspended in 50 μL of growth medium and 50 μL of Matrigel basement membrane matrix (Becton Dickenson; Bedford, MA). Tumors were injected subcutaneously in the upper and lower flanks of the mice and allowed to establish for 2 weeks. After 2 weeks, a test compound or control vehicle was administered in a subcutaneous oil depot at a dose of 50 mg/kg every 3 days.
Tumors were measured using bioluminescence imaging on the Xenogen IVIS 200. Mice received intra-peritoneal injections of 200 μL of D-luciferin at 3.75 mg/mL in sterile PBS. Mice were then anesthetized with isofluorane and imaged 15 minutes post- injection of luciferin. Quantification of tumor size was performed using the Living Image™ software.
Various different SHIP modulating compounds and prodrugs thereof were tested in assays as described above and the results are shown quantitatively in the table below, where a '+' indicates a positive result for desired activity, a '-' indicates a negative result for desired activity, and a 'NT' indicates no testing.
Example 14
The ability of SHIP activators to reduce tumor cell survival was assessed in multiple myeloma (MM) cell lines treated with Compound 100 or AQX-016 A. The lines OPMl, OPM2, MM. IS and RPMI 8226 were plated at a density of 1 x 10 cells/mL in 200 μL of medium with various
concentrations of Compound 100, and viable cell numbers were determined on day 3 and day 5 by trypan blue exclusion. The lines RPMI 8226 and U266 were plated at a density of 1 x 10 cells/mL in 250 μL of medium with various concentrations of AQX-016A. At day 4, the medium of each culture was replaced by fresh medium containing the same concentration of AQX-016A. At day 7, the viable cell number of each culture was determined by trypan blue exclusion. Experiments were performed in triplicate. Compound 100 inhibits MM proliferation at lower concentrations than AQX-016A and the results are illustrated graphically in Figures 5A, 5B and 5C.
Example 15
Proliferation (DNA synthesis) assays. Proliferation was measured by measuring incorporation of [ H]-thymidine into cells. MM cell lines were cultured in 96 well plates seeded with 3x10 cells suspended in 200 μL of medium along with various concentrations of Compound 100 or AQX-016A (and associated cyclodextran vehicle control), with LY294002 serving as a positive control in the indicated experiments. After 24-48 hrs of culture, 1 μCi of [ H]-thymidine (GE Healthcare, Baie D'Urfe, Canada) being added for the final 8 hours. Plates were frozen, which also aided in cell lysis, to terminate the experiments. Cells were then harvested onto glass fibre filters using an automatic cell harvester (TomTech; Orange, CT) and DNA associated radioactivity was measured via liquid scintillation counting using a Wallac Microbeta counter (Perkin-Elmer; Boston, MA). Wells were set up in triplicate and data is expressed as mean +/- SEM. The results are illustrated graphically in Figures 6A, 6B, 6C, 6D and 6E.
Example 16
Formulation of compounds
For in vitro testing in the SHIP enzyme assay, AQX-016A and Compound 100 were dissolved in EtOH and diluted into aqueous buffer (20 mM Tris HCl, pH 7.5 and 10 mM MgCl 2 ). The actual concentration of drug in solution was determined by optical density measurement at 280 nm (λ max for both compounds) after high speed centrifugation at 14 000 X g for 30 min to remove precipitated drug. For testing on cells, compounds were formulated in the carrier cyclodextrin (Cyclodex Technologies, High Springs, FL) at 6 mM (2 mg/mL). For oral administration to animals, compounds were dissolved in 100% cremophore EL (Sigma-Aldrich
Canada, Oakville, Ontario) at 150 mM (50 mg/mL) prior to dilution to 6 mM in phosphate buffer saline. However, while these compounds caged in cyclodextrin or formulated in cremophore EL micelles were very soluble in aqueous solution, they could not be used in the SHIP enzyme assays because of interference from both cyclodextrin and cremophore EL.
Production of recombinant SHIP enzyme and SHIP C2 domain
N-terminal Hisβ tagged SHIP enzyme was expressed in mammalian 293T cells by transient transfection with pME18S-His-SHIP plasmid and purified to >95% homogeneity by Ni-chelating bead chromatography (Qiagen, Mississauga, Ontario). Recombinant SHIP C2 domain (amino acid residues 725 to 863) was expressed in E. coli transformed with a pET28C expression vector constructed as described below. Recombinant protein purified from the cell lysates by Nichelating bead chromatography was >95% pure.
In vitro SHIP enzyme assay. SHIP enzyme assays were performed in 96-well microtitre plates with 10 ng of enzyme/well in a total volume of 25 μL of 20 mM Tris HCl, pH 7.5 and 10 Mm MgCl 2 . SHIP enzyme was incubated with test extracts (provided in DMSO) or vehicle for 15 min at 23°C before the addition of 100 μM inositol- 1, 3, 4,5-tetrakisphosphate (Echelon Biosciences Inc, Salt Lake City, Utah). After 20 min at 37°C and the amount of inorganic phosphate released assessed by the addition of Malachite Green reagent and absorbance measurement at 650 nm. SHIP2 enzyme was purchased from Echelon Biosciences (Salt Lake City, Utah) and an equivalent amount of inositol phosphatase activity was used in the in vitro enzyme assay. Enzyme data are expressed as the mean of triplicates +/- SEM. Experiments were performed at least 3 times. (Figures 7A and 7B).
Compound 100 is as biologically active as AQX-016A at lower concentratons
AQX-016A was substantially more active on SHIP + + than SHIP " " cells indicates that AQX-016A specifically targets SHIP. However, the presence of a catechol moiety within AQX-016 A (Figure 7A) was potentially problematic since catechols can exhibit activities independent of their specific protein pocket binding interaction For example, catechols can bind metals or be oxidized to an ortho-quinone which can lead to covalent modification of proteins through redox reactions. A non-catechol version of AQX-016A designated Compound 100 (Nodwell M. and Andersen RJ, manuscript in preparation). Analogous to AQX-016A, Compound 100 enhanced SHIP enzyme activity in vitro (Figure 7A and 7B).
Like AQX-016A, Compound 100 also selectively inhibited TNFα production from SHIP + + but not SHIP "7" macrophages (Figure 7C). The EC50 for this inhibition was 0.3 - 0.6 μM. Oral administration of Compound 100 also efficiently inhibited the LPS-induced elevation of plasma TNFα levels in the mouse endotoxemia model (Figure 7D).
Example 17
Production of SHIP+/+ and SHIP-/- Bone Marrow Derived Macrophages and Mast cells.
Bone marrow cells were aspirated from 4 to 8 week old C57B16 x 129Sv mixed background mice and SHIP + + and SHIP " " mast cells prepared as described previously. Bone marrow derived macrophages from SHIP and SHIP mice were obtained and maintained in IMDM supplemented with 10% FCS, 150 μM MTG, 2% C127 cell conditioned medium as a source of macrophage colony stimulating factor (M-CSF) (macrophage medium)
LPS stimulation of macrophages. For the analysis of LPS-stimulated TNFα production, 2 x 10 cells were plated the night before in 24 well plates in macrophage medium. The next day, the medium was changed and AQX-016A or carrier was added to cells at the indicated concentrations for 30 min prior to the addition of 10 ng/mL LPS. Supernatants were collected for TNFα determination by ELISA (BD Biosciences, Mississauga, ON, Canada). For analysis of intracellular signaling, 2 xlO cells were plated the night before in 6 cm tissue culture plates. The next day, the cells were cultured in macrophage medium without M-CSF for 1 hr at 37°C and then pretreated with AQX-016A or carrier for 30 min prior to the addition of 10 ng/mL LPS for 15 min. Cells were washed with 4°C PBS and resuspended in lysis buffer (50 mM Hepes, 2 mM EDTA, ImM NaVO 4 , 100 mM NaF, 50 mM NaPP; and 1%NP4O) supplemented with Complete Protease Inhibitor Cocktail (Roche, Montreal, Canada). Lysates were rocked at 4°C for 30 min and clarified by centrifuging 20 min at 12000 x g. Lysates were then made 1 x in Laemmli's buffer, boiled 2 min and loaded onto 7.5% SDS polyacrylamide cells. Immunoblot analysis for phospho PKB (Cell Signalling, Mississauga, Ont), SHIP and actin (Santa Cruz, Santa Cruz, CA) were carried out as described previously.
Stimulation of mast cells by FcεRI crosslinking. Mast cells were pre-loaded overnight in BMMC medium lacking IL-3 with 0.1 μg/ml anti-DNP IgE (SPE-7, Sigma, Oakville, Ont). For calcium flux measurements, cells were incubated with 2 μM fura 2-acetoxymethyl ester
(Molecular Probes, Eugene, OR) in Tyrode's buffer at 23 0 C for 45 min. Cells were then washed and incubated in the presence of vehicle control, LY294002 or AQX-016A 30 min prior to stimulation with the indicated concentration of DNP-human serum albumin (DNP-HSA). Calcium influx was monitored by spectrofluorometry. For analysis of intracellular signaling, cells were pre-loaded with anti-DNP IgE as above, pre-treated with AQX-016A or buffer control for 30 min at 37°C and stimulated with 20 ng/ml DNP-HSA for 5 min. Total cell lysates were then prepared and analyzed for phospho-PKB, phospho-p38 phospho-MAPK, Grb-2 (Cell Signalling, Mississauga, Ont) and SHIP by immunoblot analysis.
AQX-016A inhibits macrophage and mast cell activation
The target specificity and biological efficacy of AQX-016A were assessed by comparing AQX-016A' s effects on PBK-regulated processes in primary SHIP + + vs SHIP " " macrophages and mast cells. Both LPS-induced macrophage and IgE-induced mast cell activation involve activation of PI3K-dependent pathways which have previously been shown to be negatively regulated by SHIP. LPS stimulation of macrophages is associated with a PIP3 -dependent release of pro- inflammatory mediators such as TNFα. The action of AQX-016A on SHIP + + vs SHIP " " bone marrow derived macrophages was examined. Cells were pretreated for 30 min with AQX-016A prior to stimulation with 10 ng/mL of LPS for 2 h. AQX-016A was able to suppress TNFα production in SHIP +/+ cells by 30% at 3 μM and 50 % at 15 μM (Figure 8A). In contrast, SHIP " " cells, TNFα production was indistinguishable from non-AQX-016A treated cells 13 at 3 μM and was suppressed 15% at 15 μM. For comparison, the PI3K inhibitor LY294002 inhibited both SHIP +/+ and SHIP "7" macrophages to the same extent (up to -40% at 15 μM). Activation of mast cells via IgE + antigen crosslinking of their IgE receptors results in elevation of intracellular calcium levels. As shown in Figure 8B, AQX-016A selectively inhibited IgE + antigen-induced calcium entry to a substantially greater degree in SHIP +/+ than in SHIP "7" bone marrow derived mast cells whereas LY294002 inhibited both SHIP +/+ and SHIP mast cells to the same extent. These data were consistent with AQX-016A inhibiting PI3K-dependent macrophage and mast cell responses in a SHIP-dependent manner.
The ability of AQX-016A to inhibit activation of PIP 3 -dependent downstream signalling proteins in SHIP + + vs SHIP " " cells was assessed. LPS stimulation of macrophages results in PKB phosphorylation. AQX-016A preferentially inhibited, in a dose dependent manner, LPS -stimulated PKB phosphorylation in SHIP + + but not in SHIP " macrophages. Similarly, AQX-016A inhibited the phosphorylation of PKB, p38 MAPK and ERK in SHIP +/+ but not in SHIP " " mast cells. Similar protein loading was confirmed by reblotting with either antibodies to PKB or Grb2. We also examined the ability of AQX-016A to inhibit PKB activation in non-ematopoietic, prostate epithelial LNCaP cells, which do not express SHIP. The human prostate cancer cell line LNCaP exhibits constitutive activation of PKB due to the loss of PTEN expression. LY294002 efficiently suppressed PKB phosphorylation whereas AQX-016A had no effect at doses up to 60 μM. Thus, AQX-016A inhibits PIP 3 -regulated intracellular signal transduction events in SHIP expressing hematopoietic cells, but not in SHIP-deficient hematopoietic or non-hematopoietic cells.
Example 18
Mouse endotoxemia model. 6-8 week old C57B16 mice (VCHRI Mammalian Model of Human Disease Core Facility, Vancouver, BC) were orally administered the indicated dose of AQX-016A, Compound 100 or dexamethasone or carrier 30 min prior to an IP injection of 2 mg/kg of LPS (E. CoIi serotype 0111:B4, Sigma, Oakville, Ont). Blood was drawn 2 hrs later for determination of plasma TNFα by ELISA. Results are representative of 3 independent experiments. (Figures 7D and 9) AQX-016A inhibits inflammation in vivo
AQX-016 A' s ability to provide protection by inhibiting inflammatory reactions in vivo was assessed in mouse models. The mouse model of endotoxic shock involves intraperitoneal (IP) injection of bacterial LPS and measurement of serum TNFα levels 2 hrs later. We orally administered AQX-016 A or the steroidal drug dexamethasone to mice 30 min prior to the LPS challenge. AQX-016A reduced the level of serum TNFα and did so to the same extent as dexamethasone (Figure 9).
Example 19
Mouse acute cutaneous anaphylaxis model. 6-8 week old CDl mice (University of British
Columbia Animal Facility, Vancouver, BC) were sensitized to the hapten DNP by cutaneous
application of 25 μL of 0.5% dinitroflourobenzene (DNFB) (Sigma, Oakville, Ont) in acetone to the shaved abdomen of mice for two consecutive days. 24 hrs later, test substances (dissolved in 10 μL of 1:2 DMSO:MeOH) were painted on the right ear while the left ear received vehicle control. 30 min after drug application, DNFB was applied to both ears to induce mast cell degranulation. A 6 mm punch was taken from the ear and immediately frozen on dry ice for subsequent determination of neutrophil myeloperoxidase (MPO) activity. Compound 100's ability to inhibit cutaneous anaphylaxis was assessed.
Anaphylactic or allergic responses are mediated by allergen-induced degranulation of pre-sensitized mast cells. The mouse ear edema/cutaneous anaphylaxis model involves pre-sensitization of mice with the haptenizing agent dinitrofluorobenzene (DNFB). One week later the allergic reaction is elicited by painting DNFB onto the ears of the mice. The efficacy of potential anti- inflammatory compounds is tested by topical application of the test substance to one ear and comparing the resulting ear edema or inflammation of the two ears. As shown in Figure 1OA, topically applied Compound 100 dramatically inhibited allergen- induced inflammation compared to the vehicle control-treated ear. AQX-016A was also able to inhibit DNFB-induced inflammation in this model.
AQX-016A inhibited mast cell degranulation in CDl mice sensitized to hapten DNP by cutaneous application of 25 μL of 0.5% (DNFB) in acetone to the shaved abdomen of mice for two consecutive days was also shown (Figure 10B). 20 μCi of tritiated thymidine ([ H]-Tdr (GE Healthcare, Piscataway, NJ) was injected IP one week after the first DNFB application. [ H]-Tdr labels rapidly dividing cells of the mouse, including neutrophils (30). 24 hrs later, test substances (dissolved in 10 μL of 1:2 DMSO:MeOH) were painted on the right ear while the left ear received vehicle control. 30 min after drug application, DNFB was applied to both ears to induce mast cell degranulation. The resulting inflammatory cell infiltration was quantified by taking a 6mm diameter punch from the ear 1 hr later for dissolution in Solvable (Perkin Elmer-Packard, Woodbridge, Ont) and liquid scintillation counting as described. The ability of test substances to inhibit mast cell degranulation was then determined by calculating the ratio of [ H]-Tdr in the test (right) ear vs the control (left) ear as described (30). One group of mice had DNFB applied only to the left ear leaving the right ear noninflamed, in order to control for basal [ H]-Tdr incorporation into ear parenchymal cells.
I l l
Example 20
Construction of the SHIP δC2 mutant and isolated C2 domain
A His6 tagged SHIP δC2 domain deletion mutant (deleting residues 725 to 863) in the mammalian expression vector pME18S was generated by a standard PCR-based methodology. An N-terminal His6 C2 domain construct was also generated by PCR inserted into the pET28C bacterial expression vector using EcoRI and Ndel restriction sites.
Protein Lipid Overlay Assays
Protein lipid overlay (PLO) assays were performed essentially as described with minor modifications. Lyophilized phosphatidylinositol-3,4-bisphosphate diC16 (PIP 2 , Echelon Biosciences, Salt Lake City, UT) was reconstituted in a 2:1.8 solution of methanol and water. PVDF membranes (Millipore, Missisauga, Ont) were initially wetted in methanol for 1 minute, and washed 3 X 5 min with water, and gently agitated in TBST buffer (20 mM Tris pH 7.5, 0.15 M NaCl (TBS) with 0.05% Tween 20) at 23°C overnight. Treated membranes were air-dried and dilutions of reconstituted lipids were spotted in 1 μl aliquots to give the indicated amount of PIP2 per membrane spot. Membranes were dried completely and blocked with blocking buffer (3% BSA in TBS with 0.05% NaN3) for 1 h at 23°C. Purified, recombinant C2 domain was diluted into blocking buffer (5 μM final) and treated with 4 μM Compound 100 or EtOH control for 30 min at 23 0 C prior to overnight incubation with the PIP2 spotted membranes. Membranes were washed 10 times over 50 min in TBST buffer at 23°C and incubated with anti-Hisg mouse IgG (Qiagen, Missisauga, Ont) for 1 h at 23 0 C. Membranes were washed as above and incubated with Alexa Fluor 660 anti-mouse goat anti-mouse IgG (Invitrogen, Burlington, Ont) for 1 h at 23 0 C. After washing, bound proteins were detected and quantified on a Li-Cor Odyssey scanner (Lincoln, NE).
SHIP is an allosterically activated enzyme
The allosteric regulation of enzymes has remained under-appreciated primarily because allosteric effectors are not easy to find. The molecular mechanism by which Compound 100 activated SHIP was investigated, first by performing classical enzyme kinetic analysis of its phosphatase activity. Activity measurements were performed with substrate concentrations from 10 - 100 μM. Plots of the initial reaction velocity at each substrate concentration is predicted to exhibit a hyperbolic profile if SHIP obeys conventional Michaelis-Menten kinetics. However, SHIP displayed sigmoidal reaction kinetics suggesting allosteric activation
by its end-product (Figure 1 IA). Addition of the SHIP product PI-3,4-P 2 to the enzyme reaction activated wild-type SHIP enzyme to a similar extent as Compound 100 (Figure HB).
The SHIP protein contains a C2 domain located at the carboxyterminal end of its phosphatase domain. C2 domains were first described in the protein kinase C family where it serves to bind Ca 2+ , but C2 domains have since been identified in other proteins where they have been shown to bind to a variety of ligands including lipids. SHIP lacking its the C2 domain (δC2 SHIP) was prepared. As shown in Figure 1 IB, although δC2 SHIP was as active as the wild-type SHIP, its activity could not be enhanced by the addition of either PI-3,4-P 2 or Compound 100. This suggests that the C2 domain may be required for the allosteric activation of SHIP activity and that it may be the binding site for its allosteric activators such as PI-3,4-P 2 and Compound 100.
Example 21
Scintillation Proximity Assays
Compound 100 was radiolabeled with tritium by GE Healthcare (Piscataway, NJ) to a specific activity of 42 Ci/mmole. Copper chelate (His-Tag) YSi SPA Scintillation Beads (GE ealthcare, Piscataway, NJ) were diluted in 0.25% BSA/TBS to 1.5 mg/mL and recombinant, Hisg-tagged protein added at the indicated concentrations: wild-type (1 pM), δC2 SHIP enzyme (1 pM) or C2 domain (10 nM). Protein was allowed to bind 1 h at 23 °C, and 250 μg of beads were aliquoted per well of a 96-well plate. 5 μCi of [ H]-Compound 100 was added per well, the plate gently agitated for 30 min and the amount of bead associated radioactivity quantified by counting in a Wallac BetaPlate plate scintillation counter.
Isolated recombinant, His^-tagged C2 domain was expressed and its PI-3,4-P 2 binding ability was determined using protein lipid overlay assays. Purified C2 domain was incubated with membrane strips spotted with PI-3,4-P 2 and bound protein detected using an anti-Hisό antibody. As shown in Figure 11C the C2 domain bound PI-3,4-P 2 and this binding was inhibited by Compound 100, consistent with both Compound 100 and PI-3,4-P 2 interacting with the C2 domain at a common binding site. Compound 100 was verified to directly bind the C2 domain using scintillation proximity assays (SPAs) in which SPA beads were coated with
either the C2 domain or control protein (BSA) prior to incubation with [ H]-Compound 100. As shown in Figure HD, the C2 domain did interact with [ H]-Compound 100. In complementary studies, [ 3 H]-Compound 100 bound to wild-type SHIP but not to SHIP lacking its C2 domain (Figure HE). Together, these data are consistent with Compound 100 directly binding to SHIP'S C2 domain, resulting in allosteric activation of the enzyme.
Example 22
A novel paradigm for inhibiting PDK signaling through activation of the phosphatases that negatively regulate this pathway is provided. SHIP is a particularly good target for immune/hematopoietic disorders because of its restricted expression to hematopoietic cells. Because the relative activity of phosphatases present in a cell will influence the efficacy of kinase inhibitors, as discussed by Knight and Shokat, SHIP agonists may also be used to potentiate the activation of PI3K inhibitors and promote tissue targeting of PBK inhibitors to the ematopoietic/immune cell compartment. Initial toxicology studies suggest both AQX-016A and Compound 100 are well tolerated and do not significantly affect peripheral blood cell counts or bone marrow progenitor numbers (data not shown).
Compound 100 exhibits efficacy at a submicromolar EC50 (Figure 7C) and this suggests that it possesses a low likelihood of off-target effects based on calculations by Knight and Shokat. Compound 100 had minimal off- target effects on a screen of 100 other kinases and phosphatases (Figures 12A and 12B). Compound profiling activity was undertaken using 100 protein kinase and phosphatase targets by SignalChem (Richmond, BC, Canada. www.signalchem.com) against compound Compound 100 (2 μM final concentration). Protein kinase assays were performed in the presence of 50 μ M ATP at 3O 0 C for 15 min. Protein phosphatase activites were determined using pNPP as substrate and were also performed at 37°C for 15 min. The activity of the enzymes in the presence of Compound 100 was compared to that in the vehicle control and expressed as a % change in activity relative to that observed in the vehicle control. Changes in activity of <25% were not considered significant. Enzymes affected by Compound 100 are plotted in an expanded graph in Figure 12B.
Example 23
MM Xenograft murine model. Mice were inoculated with at two sites each with 3 x 10 6 luciferase expresseing 0PM2 cells suspended in 50 μL of growth medium and 50 μL of
Matrigel basement membrane matrix (Becton Dickenson; Bedford, MA). Tumors were injected subcutaneously in the upper and lower flanks of the mice and allowed to establish for 2 weeks. After 2 weeks, Compound 100 or control vehicle was administered in a subcutaneous oil depot at a dose of 50 mg/kg every 3 days.
Tumors were measured using bioluminescence imaging on the Xenogen IVIS 200. Mice received intra-peritoneal injections of 200 μL of D-luciferin at 3.75 mg/mL in sterile PBS. Mice were then anesthetized with isofluorane and imaged 15 minutes post- injection of luciferin. Quantification of tumor size was performed using the Living Image™ software. The results are illustrated graphically in Figures 13 and 14.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the appended claims. All patents, patent applications and publications referred to herein are hereby incorporated by reference.
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