STABILITY-ENHANCING COMPOSITIONS AND METHODS OF PREPARING COMPOUNDS
20250085300 ยท 2025-03-13
Inventors
- Liyue HUANG (Sharon, MA, US)
- David B. TERRY (Watertown, MA, US)
- Joshua D. WAETZIG (Waltham, MA, US)
- Chong-Hui Gu (Waban, MA)
Cpc classification
G01N33/94
PHYSICS
G01N33/6851
PHYSICS
C07D519/00
CHEMISTRY; METALLURGY
International classification
G01N33/94
PHYSICS
C07D519/00
CHEMISTRY; METALLURGY
Abstract
Chiral glutarimide stereochemical identity-preserving methods and compositions are disclosed. Also disclosed are methods of preparing chiral glutarimides stereoretentively.
Claims
1. A method of determining the level of a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring in a subject, the method comprising: collecting a body fluid from the subject into a container comprising a solution comprising citric acid to form a composition; and determining the level of the chiral glutarimide in the composition, thereby determining the level of a chiral glutarimide in a subject.
2. A method of preparing a composition comprising a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring and a body fluid, the method comprising collecting the body fluid from a subject into a container comprising a solution comprising citric acid, the body fluid comprising the chiral glutarimide.
3. A composition comprising a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring, a body fluid, and citrate buffer, wherein the composition is enriched for one of the stereoisomers of the epimerizable stereogenic center.
4. The method or composition of any one of claims 1 to 3, wherein the body fluid is blood.
5. The method or composition of any one of claims 1 to 4, wherein the solution comprising citric acid is 0.5M to 5M.
6. The method or composition of any one of claims 1 to 5, wherein the solution comprising citric acid is 3M citric acid.
7. The method or composition of any one of claims 1 to 6, wherein the ratio of body fluid to the solution of citric acid in the composition is 99 to 1.
8. A method of determining the level of a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring in a subject, the method comprising: collecting a body fluid from the subject; separating the body fluid into two or more components; combining one of the components and citric acid to form a composition; and determining the level of the chiral glutarimide in the composition, thereby determining the level of a chiral glutarimide in a subject.
9. A method of preparing a composition comprising a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring and a component of a body fluid, the method comprising combining citric acid and the component of the body fluid collected from a subject.
10. A composition comprising a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring, a component of a body fluid, and citrate buffer, wherein the composition is enriched for one of the stereoisomers of the epimerizable stereogenic center.
11. The method or composition of any one of claims 8 to 10, wherein the component of a body fluid is blood serum or blood plasma.
12. The method or composition of claim 11, wherein the component of a body fluid is blood serum.
13. The method or composition of claim 11, wherein the component of a body fluid is blood plasma.
14. The method or composition of any one of claims 8 to 13, wherein the citric acid is in lyophilized form.
15. The method or composition of any one of claims 8 to 14, wherein the ratio of the component of body fluid to citric acid in the composition is 99 to 1.
16. The method of any one of claims 8, 9, or 11 to 15, wherein the body fluid is stored at 0 C. to 4 C. between the step of collecting the body fluid and the step of preparing the component of the body fluid.
17. The method or composition of any one of claims 1 to 16, wherein the chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring is a compound of formula l:
A-L-B Formula I, wherein L is a linker; B is a degradation moiety having the structure: ##STR00083## wherein *designates the stereoenriched epimerizable stereogenic center at the -carbon of the glutarimide ring; Y.sup.1 is ##STR00084## R.sup.3 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; q is 0, 1, 2, 3, or 4; each R.sup.2 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; and Z is a substituent; and A is a protein binding moiety, or a pharmaceutically acceptable salt thereof.
18. The method or composition of claim 17, wherein the protein binding moiety has the structure of Formula E-3, Formula E-4, Formula G-2, Formula G-3, or Formula E-5: ##STR00085## ##STR00086## wherein Y.sup.2 is N or CR.sup.23; R.sup.22 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; R.sup.23 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl; s is 0, 1, 2, 3, or 4; each R.sup.25 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; R.sup.53 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl; R.sup.54 is H or optionally substituted C.sub.2-C.sub.9 heteroaryl; R.sup.55 is H or N(R.sup.a).sub.2, wherein each R.sup.a is independently H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; each of X.sup.5, X.sup.6, X.sup.7, and X.sup.8 is, independently, N or CR.sup.56; each R.sup.56 is, independently, H or N(R.sup.a).sub.2, wherein R.sup.a is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; R.sup.57 is optionally substituted C.sub.2-C.sub.10 heterocyclyl; each of Y.sup.2 and Y.sup.3 is, independently, N or CR.sup.58; and each R.sup.58 is, independently, H or optionally substituted C.sub.1-C.sub.6 alkyl, wherein if R.sup.53 is H and R.sup.54 is H, then R.sup.55 is NR.sup.a; if R.sup.54 is H and R.sup.55 is H, then R.sup.53 is optionally substituted C.sub.3-C.sub.10 carbocyclyl; and if R.sup.53 is H and R.sup.55 is H, then R.sup.54 is optionally substituted C.sub.2-C.sub.9 heteroaryl, or a pharmaceutically acceptable salt thereof.
19. The method or composition of claim 17 or 18, wherein A has the structure of Formula E-3.
20. The method or composition of claim 17 or 18, wherein A has the structure of Formula E-4.
21. The method or composition of claim 17 or 18, wherein A has the structure of Formula G-2.
22. The method or composition of claim 17 or 18, wherein A has the structure of Formula G-3.
23. The method or composition of claim 17 or 18, wherein A has the structure of Formula E-5.
24. The method or composition of any one of claims 17 to 23, wherein s is 0, 1, or 2.
25. The method or composition of any one of claims 17 to 24, wherein the degradation moiety has the structure of Formula A-1: ##STR00087## wherein Y.sup.1 is ##STR00088## R.sup.3 and R.sup.4 are, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; q is 0, 1, 2, 3, or 4; and each R.sup.2 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino, or a pharmaceutically acceptable salt thereof.
26. The method or composition of claim 25, wherein R.sup.3 is H or optionally substituted C.sub.1-C.sub.6 alkyl.
27. The method or composition of claim 26, wherein R.sup.3 is H or CH.sub.3.
28. The method or composition of claim 27, wherein R.sup.3 is H.
29. The method or composition of claim 27, wherein R.sup.3 is CH.sub.3.
30. The method or composition of any one of claims 25 to 29, wherein Y.sup.1 is ##STR00089##
31. The method or composition of claim 30, wherein Y.sup.1 is ##STR00090##
32. The method or composition of any one of claims 25 to 31, wherein each R.sup.2 is, independently, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, hydroxyl, or optionally substituted amino.
33. The method or composition of any one of claims 25 to 34, wherein q is 0 or 1.
34. The method or composition of claim 33, wherein q is 0.
35. The method or composition of any one of claims 25 to 34, wherein the degradation moiety has the structure of Formula A-1a: ##STR00091##
36. The method or composition of any one of claims 25 to 34, wherein the degradation moiety has the structure of Formula A-1b: ##STR00092##
37. The method or composition of any one of claims 25 to 34, wherein the degradation moiety has the structure of Formula A-1c: ##STR00093##
38. The method or composition of any one of claims 25 to 34, wherein the degradation moiety has the structure of Formula A-1d: ##STR00094##
39. The method or composition of any one of claims 25 to 34, wherein the degradation moiety has the structure: ##STR00095##
40. The method or composition of any one of claims 17 to 39, wherein the linker has the structure of Formula II:
A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-(D)-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2 Formula II wherein A.sup.1 is a bond between the linker and A; A.sup.2 is a bond between B and the linker; each of B.sup.1, B.sup.2, B.sup.3, and B.sup.4 is, independently, optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, optionally substituted C.sub.2-9 heterocyclyl, O, S, S(O).sub.2, or NR.sup.N; R.sup.N is H, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; each of C.sub.1 and C.sub.2 is, independently, carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; f, g, h, I, j, and k are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-to-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2.
41. The method or composition of claim 40, wherein each of B.sup.1, B.sup.2, B.sup.3, and B.sup.4 is, independently, optionally substituted C.sub.1-C.sub.4 alkyl, optionally substituted C.sub.1-C.sub.4 heteroalkyl, or NR.sup.N.
42. The method or composition of claim 40 or 41, wherein R.sup.N is H or optionally substituted C.sub.1-4 alkyl.
43. The method or composition of claim 42, wherein R.sup.N is H or CH.sub.3.
44. The method or composition of any one of claims 40 to 43, wherein each of B.sup.1 and B.sup.4 is, independently, ##STR00096##
45. The method or composition of claim 44, wherein B.sup.1 is ##STR00097##
46. The method or composition of any one of claims 40 to 45, wherein each of C.sup.1 and C.sup.2 is, independently, ##STR00098##
47. The method or composition of claim 46, wherein C.sup.1 is ##STR00099##
48. The method or composition of any one of claims 1 to 47, wherein the chiral glutarimide has the structure: ##STR00100##
49. The method or composition of any one of claims 1 to 48, wherein the chiral glutarimide has the structure: ##STR00101##
50. The method or composition of any one of claims 1 to 49, wherein the chiral glutarimide is enriched for one of the stereoisomers at the epimerizable stereogenic center.
51. A method of preparing a chiral glutarimide or a salt thereof comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring, the epimerizable stereogenic center being enriched for one of the stereoisomers, and the method comprising reacting a stereoenriched aminoglutarimide with a carboxybenzaldehyde, wherein the chiral glutarimide is of the following structure: ##STR00102## wherein *designates the stereoenriched epimerizable stereogenic center at the -carbon of the glutarimide ring; Y.sup.1 is ##STR00103## R.sup.3 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; q is 0, 1, 2, 3, or 4; each R.sup.2 is, independently, halogen, optionally substituted C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; and Z is L-A; wherein L is a linker; and A is a protein binding moiety; wherein the enantioenriched aminoglutarimide is of the following structure: ##STR00104## or a salt thereof, wherein all variables are same as those in the chiral glutarimide; and wherein the carboxybenzaldehyde is of the following structure: ##STR00105## wherein PG is an O-protecting group, and all remaining variables are same as those in the chiral glutarimide.
52. The method of claim 51, wherein R.sup.3 is H or optionally substituted C.sub.1-C.sub.6 alkyl.
53. The method of claim 52, wherein R.sup.3 is H or CH.sub.3.
54. The method of claim 53, wherein R.sup.3 is H.
55. The method of claim 53, wherein R.sup.3 is CH.sub.3.
56. The method of any one of claims 51 to 55, wherein Y.sup.1 is ##STR00106##
57. The method of claim 56, wherein Y.sup.1 is ##STR00107##
58. The method of any one of claims 51 to 57, wherein each R.sup.2 is, independently, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, hydroxyl, or optionally substituted amino.
59. The method of any one of claims 51 to 58, wherein q is 0 or 1.
60. The method of claim 59, wherein q is 0.
61. The method of any one of claims 51 to 60, wherein the enantioenriched aminoglutarimide is reacted with the carboxybenzaldehyde under the reductive amination conditions.
62. The method of any one of claims 51 to 61, wherein A has the structure of Formula E-3, Formula E-4, Formula G-2, Formula G-3, or Formula E-5: ##STR00108## wherein Y.sup.2 is N or CR.sup.23; R.sup.22 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; R.sup.23 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl; s is 0, 1, 2, 3, or 4; each R.sup.25 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.5-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; R.sup.53 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl; R.sup.54 is H or optionally substituted C.sub.2-C.sub.9 heteroaryl; R.sup.55 is H or N(R.sup.a).sub.2, wherein each R.sup.a is independently H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; each of X.sup.5, X.sup.6, X.sup.7, and X.sup.8 is, independently, N or CR.sup.56; each R.sup.56 is, independently, H or N(R.sup.a).sub.2, wherein R.sup.a is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; R.sup.57 is optionally substituted C.sub.2-C.sub.10 heterocyclyl; each of Y.sup.2 and Y.sup.3 is, independently, N or CR.sup.58; and each R.sup.58 is, independently, H or optionally substituted C.sub.1-C.sub.6 alkyl, wherein if R.sup.53 is H and R.sup.54 is H, then R.sup.55 is NR.sup.a; if R.sup.54 is H and R.sup.55 is H, then R.sup.53 is optionally substituted C.sub.3-C.sub.10 carbocyclyl; and if R.sup.53 is H and R.sup.55 is H, then R.sup.54 is optionally substituted C.sub.2-C.sub.9 heteroaryl.
63. The method of claim 62, wherein A has the structure of Formula E-3.
64. The method of claim 62, wherein A has the structure of Formula E-4.
65. The method of claim 62, wherein A has the structure of Formula G-2.
66. The method of claim 62, wherein A has the structure of Formula G-3.
67. The method of claim 62, wherein A has the structure of Formula E-5.
68. The method of any one of claims 62 to 67, wherein s is 0, 1, or 2.
69. The method of any one of claims 51 to 68, wherein the linker has the structure of Formula II:
A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-(D)-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2 Formula II wherein A.sup.1 is a bond between the linker and A; A.sup.2 is the valency of Z; each of B.sup.1, B.sup.2, B.sup.3, and B.sup.4 is, independently, optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, optionally substituted C.sub.2-9 heterocyclyl, O, S, S(O).sub.2, or NR.sup.N; R.sup.N is H, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; each of C.sup.1 and C.sup.2 is, independently, carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; f, g, h, I, j, and k are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-to-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2.
70. The method of claim 69, wherein each of B.sup.1, B.sup.2, B.sup.3, and B.sup.4 is, independently, optionally substituted C.sub.1-C.sub.4 alkyl, optionally substituted C.sub.1-C.sub.4 heteroalkyl, or NR.sup.N.
71. The method of claim 69 or 70, wherein R.sup.N is H or optionally substituted C.sub.1-4 alkyl.
72. The method of any one of claims 69 to 71, wherein R.sup.N is H or CH.sub.3.
73. The method of any one of claims 69 to 72, wherein each of B.sup.1 and B.sup.4 is, independently, ##STR00109##
74. The method of claim 73, wherein B.sup.1 is ##STR00110##
75. The method of any one of claims 69 to 74, wherein each of C.sup.1 and C.sup.2 is, independently, ##STR00111##
76. The method of claim 75, wherein C.sup.1 is ##STR00112##
77. The method of any one of claims 51 to 76, wherein the carboxybenzaldehyde is of the following structure: ##STR00113##
78. The method of claim 77, further comprising the step of preparing the carboxybenzaldehyde from a first reactant and a second reactant, wherein the first reactant is of the following structure: ##STR00114## and wherein the second reactant is of the following structure: ##STR00115##
79. The method of claim 78, wherein the step of preparing the carboxybenzaldehyde is performed under the nucleophilic aromatic substitution reaction conditions.
80. The method of claim 78 or 79, further comprising the step of preparing the first reactant from a third reactant and a fourth reactant, wherein the third reactant is a compound of the following structure: ##STR00116## and wherein the fourth reactant is a compound of the following structure: ##STR00117## wherein PG.sup.N is an N-protecting group.
81. The method of claim 80, wherein the step of preparing the first reactant comprises reacting the third reactant and the fourth reactant under reductive amination reaction conditions and removing the N-protecting group.
82. The method of any one of claims 51 to 81, further comprising the step of preparing the salt of the chiral glutarimide, wherein the step comprises reacting a free-base form of the chiral glutarimide with an acid to produce the salt of the chiral glutarimide.
83. The method of claim 82, wherein the acid is citric acid, and the salt of the chiral glutarimide is a citrate salt of the chiral glutarimide.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0198] The invention provides methods and compositions useful for preparing or storing a chiral glutarimide having an epimerizable stereogenic center at the -carbon of the glutarimide ring, while reducing or eliminating epimerization of the epimerizable stereogenic center. Thus, methods and compositions disclosed herein may reduce or eliminate erosion of the stereochemical enrichment of the chiral glutarimide at the epimerizable stereogenic center at the -carbon of the glutarimide ring.
[0199] The methods and compositions described herein take advantage of the stabilizing effect of citric acid upon the stereochemistry of the epimerizable stereogenic center at the -carbon of the glutarimide ring in the chiral glutarimide. Thus, the methods and compositions may be used to slow down significantly epimerization at the -carbon of the glutarimide ring in chiral glutarimides, e.g., in certain media (e.g., blood or a component thereof, such as blood plasma or blood serum) otherwise capable of promoting such epimerization and concomitant erosion of the stereochemical enrichment. The methods described herein include methods of determining the level of a chiral glutarimide including an epimerizable stereogenic center at the -carbon of the glutarimide ring in a subject; methods of preparing a composition including a chiral glutarimide including an epimerizable stereogenic center at the -carbon of the glutarimide ring and a body fluid; and methods of preparing a composition including a chiral glutarimide including an epimerizable stereogenic center at the -carbon of the glutarimide ring and a component of a body fluid.
[0200] The invention also provides a stereoretentive synthesis of a chiral glutarimide. Advantageously, the synthesis approach described herein introduces the glutarimide ring at the end of the synthesis and reduces exposure of the epimerizable stereogenic center at the -carbon of the glutarimide ring to various reaction, work up, and purification conditions, which could promote epimerization.
Stability-Enhancing Compositions and Methods
[0201] The compositions and methods disclosed herein typically combine a chiral glutarimide, a body fluid (e.g., blood) or a component thereof (e.g., blood plasma or blood serum), and citric acid. A chiral glutarimide includes an epimerizable stereogenic center at the -carbon of the glutarimide ring that is typically stereochemically enriched for one of the stereoisomeric forms of the stereogenic center (e.g., S or R). For example, in compositions and methods disclosed herein, the epimerizable stereogenic center at the -carbon of the glutarimide may be stereochemically enriched by at least 20% in favor of the stereochemical orientation (e.g., S) that is stereochemically enriched (e.g., stereochemical excess of at least 10%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%; up to stereochemically pure). Thus, compositions and method disclosed herein may be substantially stereochemical enrichment-preserving for an epimerizable stereogenic center at the a-carbon of the glutarimide ring.
[0202] Preparation of a composition containing a body fluid (e.g., blood) and the chiral glutarimide (e.g., as a subject's blood sample, e.g., the subject having been administered the chiral glutarimide) typically includes the step of collecting the body fluid from a subject into a container including a solution (e.g., an aqueous solution) containing citric acid (e.g., 0.5 M to 5 M citric acid or 3 M citric acid).
[0203] Preparation of a composition containing a body fluid component (e.g., blood plasma or blood serum) and the chiral glutarimide (e.g., as a subject's blood plasma or serum sample, e.g., the subject having been administered the chiral glutarimide) typically includes the step of combining citric acid and the component of the body fluid collected from a subject. For the combining step, citric acid may be, e.g., in lyophilized form. The methods described herein for the preparation of a composition containing a body fluid component may also include the step of preparing the body fluid component from the body fluid collected from the subject. The component of the body fluid may be prepared from the body fluid using methods known in the art. For example, blood plasma may be prepared by centrifugation of blood; blood serum may be prepared by clotting and subsequent centrifugation of blood. The methods described herein for the preparation of a composition containing a body fluid component may further include the step of collecting the body fluid from the subject (e.g., the subject having been administered the chiral glutarimide). The body fluid collections methods are known in the art and, e.g., for blood, typically involve arterial sampling, venipuncture sampling, or fingerstick sampling. The body fluid may be stored at 0 C. to 4 C. (e.g., in wet-ice bath) between the step of collecting the body fluid and the step of preparing the component of the body fluid (e.g., for up to 2 hours). Typically, the body fluid component is combined with citric acid immediately after the component's preparation.
[0204] In the compositions and methods described herein, the concentration of citric acid may be, e.g., 0.005M to 0.05M (e.g., 0.03M) after the citric acid is combined with blood or a component thereof.
Chiral Glutarimides
[0205] The compositions and methods described herein typically contain a chiral glutarimide having an epimerizable stereogenic center at the -carbon of the glutarimide ring. Such chiral glutarimides are typically of the following structure:
##STR00035##
where [0206] *designates the stereoenriched epimerizable stereogenic center at the -carbon of the glutarimide ring; [0207] A is a non-hydrogen group; and [0208] R.sup.3 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl. [0209] The chiral glutarimides can often be used as a Cereblon ligand, e.g., to be used as degraders for targeted protein degradation. Accordingly, the chiral glutarimide may be, e.g., a compound of formula I:
A-L-B Formula I, [0210] where [0211] L is a linker; [0212] B is a degradation moiety including a Cereblon ligand including an epimerizable stereogenic center at the -carbon of the glutarimide ring in the Cereblon ligand; and
[0213] A has the structure of Formula E-3, Formula E-4, Formula G-2, Formula G-3, or Formula E-5:
##STR00036## [0214] where [0215] Y.sup.2 is N or CR.sup.23; [0216] R.sup.22 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; [0217] R.sup.23 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl; [0218] s is 0, 1, 2, 3, or 4; [0219] each R.sup.25 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; [0220] R.sup.53 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl; [0221] R.sup.54 is H or optionally substituted C.sub.2-C.sub.9 heteroaryl; [0222] R.sup.55 is H or N(R.sup.a).sub.2, wherein each R.sup.a is independently H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal Ra, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; [0223] each of X.sup.5, X.sup.6, X.sup.7, and X.sup.8 is, independently, N or CR.sup.56; [0224] each R.sup.56 is, independently, H or N(R.sup.a).sub.2, wherein R.sup.a is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; [0225] R.sup.57 is optionally substituted C.sub.2-C.sub.10 heterocyclyl; [0226] each of Y.sup.2 and Y.sup.3 is, independently, N or CR.sup.58; and [0227] each R.sup.58 is, independently, H or optionally substituted C.sub.1-C.sub.6 alkyl, [0228] wherein if R.sup.53 is H and R.sup.54 is H, then R.sup.55 is NR.sup.a; if R.sup.54 is H and R.sup.55 is H, then R.sup.53 is optionally substituted C.sub.3-C.sub.10 carbocyclyl; and if R.sup.53 is H and R.sup.55 is H, then R.sup.54 is optionally substituted C.sub.2-C.sub.9 heteroaryl, [0229] or a pharmaceutically acceptable salt thereof.
[0230] In some embodiments, A has the structure of Formula E-3. In some embodiments, A has the structure of Formula E-4. In some embodiments, A has the structure of Formula G-2. In some embodiments, A has the structure of Formula G-3. In some embodiments, A has the structure of Formula E-5. In some embodiments, s is 0, 1, or 2.
[0231] In some embodiments, the degradation moiety has the structure of Formula A-1:
##STR00037## [0232] wherein
##STR00038## [0233] Y.sup.1 is [0234] R.sup.3 and R.sup.4 are, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; [0235] q is 0, 1, 2, 3, or 4; and [0236] each R.sub.2 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino, [0237] or a pharmaceutically acceptable salt thereof.
[0238] In some embodiments, the degradation moiety has the structure of Formula A-1a:
##STR00039##
[0239] In some embodiments, the degradation moiety has the structure of Formula A-1b:
##STR00040##
[0240] In some embodiments, the degradation moiety has the structure of Formula A-1c:
##STR00041##
[0241] In some embodiments, the degradation moiety has the structure of Formula A-1d:
##STR00042##
[0242] In some embodiments, the degradation moiety has the structure:
##STR00043##
[0243] In some embodiments, the linker has the structure of Formula II:
A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-(D)-(B.sup.3).sub.i-(C.sub.2).sub.j-(B.sup.4).sub.k-A.sup.2 Formula II [0244] wherein [0245] A.sup.1 is a bond between the linker and A; [0246] A.sup.2 is a bond between B and the linker; [0247] each of B.sup.1, B.sup.2, B.sup.3, and B.sup.4 is, independently, optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, optionally substituted C.sub.2-9 heterocyclyl, O, S, S(O).sub.2, or NR.sup.N; [0248] R.sup.N is H, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; [0249] each of C.sup.1 and C.sup.2 is, independently, carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; [0250] f, g, h, I, j, and k are each, independently, 0 or 1; and [0251] D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-to-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2.
[0252] In some embodiments, the chiral glutarimide is compound S-D1:
##STR00044##
Stereoretentive Synthesis of Chiral Glutarimides
[0253] The invention further provides stereoretentive approaches for the synthesis of chiral glutarimides. The methods disclosed herein thus may be used to prepare a chiral glutarimide comprising an epimerizable stereogenic center at the -carbon of the glutarimide ring, the epimerizable stereogenic center being enriched for one of the stereoisomers. The method typically include the step of reacting an stereoenriched aminoglutarimide with a carboxybenzaldehyde, wherein the chiral glutarimide is of the following structure:
##STR00045## [0254] wherein [0255] *designates the stereoenriched epimerizable stereogenic center at the -carbon of the glutarimide ring; [0256] Y.sup.1 is
##STR00046## [0257] R.sup.3 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; [0258] q is 0, 1, 2, 3, or 4; [0259] each R.sub.2 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; and [0260] Z is a substituent;
wherein the enantioenriched aminoglutarimide is of the following structure:
##STR00047## [0261] or a salt thereof, [0262] wherein all variables are same as those in the chiral glutarimide;
and
wherein the carboxybenzaldehyde is of the following structure:
##STR00048## [0263] wherein [0264] PG is an O-protecting group (e.g., alkyl, such as methyl), and [0265] all remaining variables are same as those in the chiral glutarimide.
[0266] In some embodiments, the enantioenriched aminoglutarimide is reacted with the carboxybenzaldehyde under the reductive amination conditions. Reductive amination reaction conditions are known in the art. Typically, reductive amination involves a reaction between a carbonyl functional group in an aldehyde or ketone with a primary or secondary amine in the presence of a 1,2-reducing agent (e.g., NaBH.sub.3CN, NaBH(OAc).sub.3, or NaBH.sub.4/acetic acid) to produce a secondary or tertiary amine, respectively.
[0267] In some embodiments, Z is -L-A,
where [0268] L is a linker, and [0269] A has the structure of Formula E-3, Formula E-4, Formula G-2, Formula G-3, or Formula E-5:
##STR00049## [0270] wherein [0271] Y.sup.2 is N or CR.sup.23; [0272] R.sup.22 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; [0273] R.sup.23 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl; [0274] s is 0, 1, 2, 3, or 4; [0275] each R.sup.25 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxyl, thiol, or optionally substituted amino; [0276] R.sup.53 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl; [0277] R.sup.54 is H or optionally substituted C.sub.2-C.sub.9 heteroaryl; [0278] R.sup.55 is H or N(R.sup.a).sub.2, wherein each R.sup.a is independently H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; [0279] each of X.sup.5, X.sup.8, X.sup.7, and X.sup.8 is, independently, N or CR.sup.56; [0280] each R.sup.56 is, independently, H or N(R.sup.a).sub.2, wherein R.sup.a is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl, or two geminal R.sup.a, together with the nitrogen atom to which they are attached, combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl; [0281] R.sup.57 is optionally substituted C.sub.2-C.sub.10 heterocyclyl; [0282] each of Y.sup.2 and Y.sup.3 is, independently, N or CR.sup.58; and [0283] each R.sup.58 is, independently, H or optionally substituted C.sub.1-C.sub.6 alkyl, [0284] wherein if R.sup.53 is H and R.sup.54 is H, then R.sup.55 is NR.sup.a; if R.sup.54 is H and R.sup.55 is H, then R.sup.53 is optionally substituted C.sub.3-C.sub.10 carbocyclyl; and if R.sup.53 is H and R.sup.55 is H, then R.sup.54 is optionally substituted C.sub.2-C.sub.9 heteroaryl.
[0285] In some embodiments, the linker has the structure of Formula II:
A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.h-(D)-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2 Formula II [0286] wherein [0287] A.sup.1 is a bond between the linker and A; [0288] A.sup.2 is the valency of Z; [0289] each of B.sup.1, B.sup.2, B.sup.3, and B.sup.4 is, independently, optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, optionally substituted C.sub.2-9 heterocyclyl, O, S, S(O).sub.2, or NR.sup.N; [0290] R.sup.N is H, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; [0291] each of C.sup.1 and C.sup.2 is, independently, carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; [0292] f, g, h, I, j, and k are each, independently, 0 or 1; and [0293] D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.f-(C.sup.1).sub.g-(B.sup.2).sub.n-to-(B.sup.3).sub.i-(C.sup.2).sub.j-(B.sup.4).sub.k-A.sup.2.
[0294] In some embodiments, the carboxybenzaldehyde is of the following structure:
##STR00050##
[0295] In some embodiments, the method further comprises the step of preparing the carboxybenzaldehyde from a first reactant and a second reactant, wherein the first reactant is of the following structure:
##STR00051##
and
wherein the second reactant is of the following structure:
##STR00052##
[0296] In some embodiments, the step of preparing the carboxybenzaldehyde is performed under the nucleophilic aromatic substitution reaction conditions.
[0297] In some embodiments, the method further comprises the step of preparing the first reactant from a third reactant and a fourth reactant,
wherein the third reactant is a compound of the following structure:
##STR00053##
and
wherein the fourth reactant is a compound of the following structure:
##STR00054##
wherein PG.sup.N is an N-protecting group (e.g., Boc).
[0298] In some embodiments, the step of preparing the first reactant comprises reacting the third reactant and the fourth reactant under reductive amination reaction conditions and removing the N-protecting group. N-protecting groups can be removed methods known in the art.
[0299] In some embodiments, the method further comprises the step of preparing the salt of the chiral glutarimide, wherein the step comprises reacting a free-base form of the chiral glutarimide with an acid to produce the salt of the chiral glutarimide. In some embodiments, the acid is citric acid, and the salt of the chiral glutarimide is a citrate salt of the chiral glutarimide.
EXAMPLES
[0300] Throughout the Examples, compound numbers are as shown in Table 1.
TABLE-US-00001 TABLE 1 Compounds Compound No. Structure D1
[0301] Examples 6-8 illustrate the preparation of compounds D1, as well as compounds S-D1 and R-D1 through resolution of racemate D1. Example
Example 1-BRD9 Degrader Depletes BRD9 Protein
[0302] The following example demonstrates the depletion of the BRD9 protein in synovial sarcoma cells treated with a BRD9 degrader.
[0303] Procedure: Cells were treated with DMSO or the BRD9 degrader, Compound 1 (also known as dBRD9, see Remillard et al, Angew. Chem. Int. Ed. Engl. 56 (21): 5738-5743 (2017); see structure of compound 1 below), for indicated doses and timepoints.
##STR00058##
[0304] Whole cell extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer's protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was incubated with antibodies against BRD9 (1:1,000, Bethyl laboratory A303-781A), GAPDH (1:5,000, Cell Signaling Technology), and/or MBP (1:1,000, BioRad) overnight at 4 C. Membranes were washed three times for 10 min and incubated with anti-mouse or anti-rabbit antibodies conjugated with either horseradish peroxidase (HRP,
[0305] Results: Treatment of SYO1 synovial sarcoma cells with the BRD9 degrader Compound 1 results in dose dependent (
Example 2Inhibition of Growth of Synovial Cell Lines by BRD9 Inhibitors and BRD9 Degraders
[0306] The following example demonstrates that BRD9 degraders and inhibitors selectively inhibit growth of synovial sarcoma cells.
Procedures:
[0307] Cells were treated with DMSO or the BRD9 degrader, Compound 1, at indicated concentrations, and proliferation was monitored from day 7 to day 14 by measuring confluency over time using an IncuCyte live cell analysis system (
[0308] Cells were seeded into 12-well plates and treated with DMSO, 1 M BRD9 inhibitor, Compound 2 (also known as BI-7273, see Martin et al, J Med Chem. 59 (10): 4462-4475 (2016); see structure of compound 2 below), or 1 M BRD9 degrader, Compound 1.
##STR00059##
[0309] The number of cells was optimized for each cell line. Growth medium and compounds were refreshed every 3-5 days. SYO1, Yamato, A549, 293T and HS-SY-II cells were fixed and stained at day 11. ASKA cells were fixed and stained at day 23. Staining was done by incubation with crystal violet solution (0.5 g Crystal Violet, 27 ml 37% Formaldehyde, 100 mL 10PBS, 10 mL Methanol, 863 dH20 to 1 L) for 30 min followed by 3washes with water and drying the plates for at least 24 h at room temperature. Subsequently plates were scanned on an Odyssey CLx Imaging system (
[0310] Cells were seeded into 96-well ultra-low cluster plate (Costar, #7007) in 200 L complete media and treated at day 2 with DMSO, Staurosporin, or BRD9 degrader, Compound 1, at indicated doses (
[0311] Results: As shown in
Example 3Selective Inhibition of Growth of Synovial Cell Lines by BRD9 Degraders and BRD9 Binders
[0312] The following example demonstrates that BRD9 degraders and binders selectively inhibit growth of synovial sarcoma cells.
[0313] Procedure: Cells were seeded into 6-well or 12-well plates and were treated daily with a BRD9 degrader (Compound 1), a bromo-domain BRD9 binder (Compound 2), E3 ligase binder (lenalidomide), DMSO, or staurosporin (positive control for cell killing), at indicated concentrations. The number of cells was optimized for each cell line. Growth media was refreshed every 5 days. By day 14, medium was removed, cells were washed with PBS, and stained using 500 L of 0.005% (w/v) crystal violet solution in 25% (v/v) methanol for at least 1 hour at room temperature. Subsequently plates were scanned on an Odyssey CLx Imaging system.
[0314] Results: As shown in
Example 4Inhibition of Cell Growth in Synovial Sarcoma Cells
[0315] The following example shows that BRD9 degraders inhibit cell growth and induce apoptosis in synovial sarcoma cells.
[0316] Procedure: SYO1 cells were treated for 8 or 13 days with DMSO, a BRD9 degrader (Compound 1) at 200 nM or 1 M, or an E3 ligase binder (lenalidomide) at 200 nM. Compounds were refreshed every 5 days. Cell cycle analysis was performed using the Click-iT Plus EdU Flow Cytometry Assay (Invitrogen). The apoptosis assay was performed using the Annexin V-FITC Apoptosis Detection Kit (Sigma A9210). Assays were performed according to the manufacturer's protocol.
[0317] Results: As shown in
Example 5Composition for SS18-SSX1-BAF
[0318] The following example shows the identification of BRD9 as a component of SS18-SSX containing BAF complexes.
[0319] Procedure: A stable 293T cell line expressing HA-SS18SSX1 was generated using lentiviral integration. SS18-SSX1 containing BAF complexes were subject to affinity purification and subsequent mass spectrometry analysis revealed SS18-SSX1 interacting proteins.
[0320] Results: As shown in
Example 6Preparation of 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (Intermediate H)
##STR00060## ##STR00061##
Step 1: Preparation of 6-chloro-4-methylpyridine-3-carboxamide (Intermediate B)
##STR00062##
[0321] To a stirred mixture of 6-chloro-4-methylpyridine-3-carboxylic acid (20.00 g, 116.564 mmol, 1.00 equiv) and NH.sub.4Cl (62.35 g, 1.17 mol, 10.00 equiv) in dichloromethane (DCM; 400 mL) was added DIEA (22.60 g, 174.846 mmol, 3.00 equiv). After stirring for 5 minutes, HATU (66.48 g, 174.846 mmol, 1.50 equiv) was added in portions. The resulting mixture was stirred for 3 hours at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (PE/EtOAc) from 1/1 to 3/2 to afford 6-chloro-4-methylpyridine-3-carboxamide (18.30 g, 61.3%) as a yellow solid. LCMS (ESI) m/z: [M+H].sup.+=171.
Step 2: Preparation of 6-chloro-N-[(1E)-(dimethylamino) methylidene]-4-methylpyridine-3-carboxamide (Intermediate C)
##STR00063##
[0322] To a stirred mixture of 6-chloro-4-methylpyridine-3-carboxamide (18.30 g, 107.268 mmol, 1.00 equiv) and in 2-methyltetrahydrofuran (100 mL) was added DMF-DMA (19.17 g, 160.903 mmol, 1.50 equiv) at 80 C. under nitrogen atmosphere, and stirred for additional 1 hour. Then the mixture was cooled and concentrated to afford 6-chloro-N-[(1E)-(dimethylamino)methylidene]-4-methylpyridine-3-carboxamide (26.3 g, 91.3%) as a yellow crude solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=226.
Step 3: Preparation of 6-chloro-2H-2,7-naphthyridin-1-one (Intermediate D)
##STR00064##
[0323] To a stirred mixture of 6-chloro-N-[(1E)-(dimethylamino) methylidene]-4-methylpyridine-3-carboxamide (26.30 g) in THF (170.00 mL) was added t-BuOK (174.00 mL, 1 mol/L in THF). The resulting solution was stirred at 60 C. under nitrogen atmosphere for 30 minutes. Then the mixture was cooled and concentrated under reduced pressure. The crude solid was washed with saturated NaHCO.sub.3 solution (100 mL) and collected to give 6-chloro-2H-2,7-naphthyridin-1-one (14.1 g, 67.0%) as a pink solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=181.
Step 4: Preparation of 6-chloro-2-methyl-2,7-naphthyridin-1-one (Intermediate E)
##STR00065##
[0324] To a stirred mixture of 6-chloro-2H-2, 7-naphthyridin-1-one (14.10 g, 78.077 mmol, 1.00 equiv) in anhydrous THF (280.00 mL) was added NaH (9.37 g, 234.232 mmol, 3.00 equiv, 60%) in portions at 0 C. After 10 minutes, Mel (33.25 g, 234.232 mmol, 3.00 equiv) was added at 0 C., and the mixture was allowed to stir for 10 minutes at 0 C., and then the mixture was allowed to stir for 12 hours at room temperature. The resulting mixture was concentrated under reduced pressure. The crude solid was slurried with water (100 mL), and the solid was filtered and collected to give the 6-chloro-2-methyl-2,7-naphthyridin-1-one (14.6 g, 94.1%) as a yellow solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=195.
Step 5: Preparation of 4-bromo-6-chloro-2-methyl-2,7-naphthyridin-1-one (Intermediate F)
##STR00066##
[0325] To a stirred mixture of 6-chloro-2-methyl-2,7-naphthyridin-1-one (8.00 g, 41.106 mmol, 1.00 equiv) in DMF (160.00 mL) was added NBS (8.78 g, 49.327 mmol, 1.20 equiv), and the resulting mixture was stirred for 2 hours at 90 C. The reaction mixture was cooled and diluted with DCM (150 mL) and washed with water (3100 mL). The organic layers were dried and concentrated. Then the residue was slurried with EtOAc (20 mL), and the slurry was filtered. The filter cake was washed with EtOAc (20 mL) to give 4-bromo-6-chloro-2-methyl-2,7-naphthyridin-1-one (6.32 g, 55.7%) as a white solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=273.
Step 6: Preparation of 6-(azetidin-1-yl)-4-bromo-2-methyl-2, 7-naphthyridin-1-one (Intermediate G)
##STR00067##
[0326] To a solution of 4-bromo-6-chloro-2-methyl-2,7-naphthyridin-1-one (5.00 g, 18.281 mmol, 1.00 equiv) and azetidine hydrochloride (3.2 g, 54.843 mmol, 3 equiv) in DMSO (50.00 mL) was added K.sub.2CO.sub.3 (12.6 g, 91.404 mmol, 5 equiv). The resulting solution was stirred at 130 C. for 2 hours. The resulting mixture was cooled and diluted with water (100 mL), and then extracted with EtOAc (3100 mL). The combined organic layers were washed with saturated NaCl solution (350 mL), dried over anhydrous Na.sub.2SO.sub.4, and concentrated under reduced pressure to afford 6-(azetidin-1-yl)-4-bromo-2-methyl-2,7-naphthyridin-1-one (3.7 g, 68.8%) as a grey solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=294.
Step 7: Preparation of 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (Intermediate H)
##STR00068##
[0327] To a solution of 6-(azetidin-1-yl)-4-bromo-2-methyl-2,7-naphthyridin-1-one (1.42 g, 4.827 mmol, 1.00 equiv) and 4-formyl-3,5-dimethoxyphenylboronic acid (1.52 g, 7.241 mmol, 1.5 equiv) in dioxane (16.00 mL) and H.sub.2O (4.00 mL) was added Pd(dppf)Cl.sub.2 (353.2 mg, 0.483 mmol, 0.1 equiv) and Cs.sub.2CO.sub.3 (3.15 g, 9.655 mmol, 2 equiv), and the resulting solution was stirred at 70 C. for 2 hours. The resulting mixture was cooled and concentrated under reduced pressure. The residue was slurried with water (30 mL) and filtered, and the filter cake was collected. This solid was further slurried with MeOH (30 mL) and filtered. The solid was collected to afford 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (1.42 g, 77.5%) as a grey solid. LCMS (ESI) m/z: [M+H].sup.+=380.
Example 7Preparation of 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (Intermediate P)
##STR00069##
Step 1: Preparation of methyl 5-bromo-2-(bromomethyl)benzoate (Intermediate J)
##STR00070##
[0328] To a stirred mixture of methyl 5-bromo-2-methylbenzoate (50.00 g, 218.271 mmol, 1.00 equiv) in CCl.sub.4 (500.00 mL) was added NBS (38.85 g, 218.271 mmol, 1.00 equiv) and BPO (5.59 g, 21.827 mmol, 0.10 equiv). After stirring for overnight at 80 C., the mixture was purified by silica gel column chromatography, eluted with PE/EtOAc (50:1) to afford methyl 5-bromo-2-(bromomethyl)benzoate (67 g, 74.75%) as a yellow oil. LCMS (ESI) m/z: [M+H].sup.+=307.
Step 2: Preparation of tert-butyl 4-(6-bromo-1-oxo-3H-isoindol-2-yl)-4-carbamoylbutanoate (Intermediate K)
##STR00071##
[0329] To a stirred mixture of methyl 5-bromo-2-(bromomethyl)benzoate (67.00 g, 217.554 mmol, 1.00 equiv) and tert-butyl (4S)-4-amino-4-carbamoylbutanoate hydrochloride (62.32 g, 261.070 mmol, 1.20 equiv) in DMF (100.00 mL) was added DIEA (112.47 g, 870.217 mmol, 4 equiv). After stirring for overnight at 80 C., the mixture was concentrated under reduced pressure. The residue was added water (200 mL) and stirred for 1 h at room temperature. The resulting mixture was filtered, the filter cake was added water (100 mL) and stirred. The precipitated solids were collected by filtration and washed with water (330 mL). This resulted in tert-butyl 4-(6-bromo-1-oxo-3H-isoindol-2-yl)-4-carbamoylbutanoate (55 g, 60.46%) as an off-white solid. LCMS (ESI) m/z: [M+H].sup.+=397.
Step 3: Preparation of tert-butyl 2-[2-[4-(tert-butoxy)-1-carbamoyl-4-oxobutyl]-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (Intermediate L)
##STR00072##
[0330] To a stirred solution of tert-butyl 4-(6-bromo-1-oxo-3H-isoindol-2-yl)-4-carbamoylbutanoate (10.00 g, 25.172 mmol, 1.00 equiv) and tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate hydrochloride (8.60 g, 32.723 mmol, 1.30 equiv) in dioxane (200.00 mL) was added Cs.sub.2CO.sub.3 (24.60 g, 75.516 mmol, 3.00 equiv) and RuPhos Palladacycle Gen.3 (2.11 g, 2.517 mmol, 0.10 equiv). After stirring for overnight at 100 C. under nitrogen atmosphere, the resulting mixture was filtered while hot, and the filter cake was washed with 1,4-dioxane (350 mL). The filtrate was concentrated under reduced pressure to afford tert-butyl 2-[2-[4-(tert-butoxy)-1-carbamoyl-4-oxobutyl]-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro [3.5]nonane-7-carboxylate (21 g, crude) as a black solid. LCMS (ESI) m/z: [M+H].sup.+=543.
Step 4: Preparation of tert-butyl 2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (Intermediate M)
##STR00073##
[0331] To a stirred mixture of tert-butyl 2-[2-[(1S)-4-(tert-butoxy)-1-carbamoyl-4-oxobutyl]-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (13.68 g, 25.208 mmol, 1.00 equiv) in THF (100.00 mL) was added t-BuOK in THF (25.00 mL, 25.000 mmol, 0.99 equiv). The resulting mixture was stirred for 2 hours at room temperature. The mixture was acidified to pH 6 with 1 M HCl (aq.) and then neutralized to pH 7 with saturated NaHCO.sub.3(aq.). The resulting mixture was extracted with EtOAc (3200 mL). The combined organic layers were concentrated under reduced pressure. This resulted in tert-butyl 2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (7.8 g, 59.43%) as a yellow solid. LCMS (ESI) m/z: [M+H].sup.+=469.
Step 5: Preparation of 3-(6-[2,7-diazaspiro[3.5]nonan-2-yl]-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione (Intermediate N)
##STR00074##
[0332] To a stirred mixture of tert-butyl 2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (7.80 g, 16.647 mmol, 1.00 equiv) in DCM (10.00 mL) was added trifluoroacetic acid (TFA; 5.00 mL). After stirring for 2 hours at room temperature, the resulting mixture was concentrated under vacuum. This resulted in 3-(6-[2,7-diazaspiro[3.5]nonan-2-yl]-1-oxo-3H-isoindol-2-yl) piperidine-2,6-dione (6 g, 92.93%) as a light yellow solid. LCMS (ESI) m/z: [M+H].sup.+=369.
Step 6: Preparation of tert-butyl 4-([2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonan-7-yl]methyl)piperidine-1-carboxylate (Intermediate O)
##STR00075##
[0333] To a stirred solution of 3-(6-[2,7-diazaspiro[3.5]nonan-2-yl]-1-oxo-3H-isoindol-2-yl) piperidine-2,6-dione (4.00 g, 8.685 mmol, 1.00 equiv, 80%) and tert-butyl 4-formylpiperidine-1-carboxylate (1.48 g, 6.939 mmol, 0.80 equiv) in DMF (20.00 mL) was added NaBH(OAc).sub.3 (3.68 g, 17.363 mmol, 2.00 equiv) at room temperature. The resulting mixture was stirred for 2 hours at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was purified by reverse flash chromatography with the following conditions (column, C18 silica gel; mobile phase, CH.sub.3CN in water (0.1% FA), 0 to 100% gradient in 40 minutes; detector, UV 254 nm). This resulted in tert-butyl 4-([2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonan-7-yl]methyl)piperidine-1-carboxylate (2.8 g, 51.29%) as a dark yellow solid. LCMS (ESI) m/z: [M+H].sup.+=566.
Step 7: Preparation of 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (Intermediate P)
##STR00076##
[0334] To a stirred mixture of tert-butyl 4-([2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonan-7-yl]methyl)piperidine-1-carboxylate (2.80 g, 4.949 mmol, 1.00 equiv) in DCM (5.00 mL) was added TFA (2.00 mL). The mixture was stirred for 2 hours at room temperature. The resulting mixture was concentrated under reduced pressure to afford 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (3.9 g, crude) as a yellow solid. LCMS (ESI) m/z: [M+H].sup.+=466.
Example 8Preparation of 3-[6-(7-[[1-([4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethox yphenyl]methyl)piperidin-4-yl]methyl]-2,7-diazaspiro[3.5]nonan-2-yl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione TFA Salt (Compound D1 TFA Salt)
##STR00077##
[0335] A solution of 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (4.5 g, 10.52 mmol, 1.00 equiv) and 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (4.0 g, 10.52 mmol, 1.00 equiv) and titanium (IV) isopropoxide (3.0 g, 10.52 mmol, 1.00 equiv) in DMSO (100 mL) was stirred at room temperature for 3 hours. Then NaBH(OAc).sub.3 (8.92 g, 42.08 mmol, 4.00 equiv) was added in batches to the above resulting solution, and the resulting mixture was stirred at room temperature overnight. The reaction was quenched by the addition of water (30 mL), and then the solution was filtered. The filter cake was wash by water and acetonitrile. Then the filtrate was concentrated in vacuo. The crude product was purified by reverse phase flash chromatography with the following conditions (Column: AQ C.sub.18 Column, 50250 mm 10 um; Mobile Phase A: Water (TFA 0.1%), Mobile Phase B: ACN; Flow rate: 100 mL/minute; Gradient: 5 B to 25 B in 35 minutes; 254/220 nm). Pure fractions were evaporated to dryness to afford 3-[6-(7-[[1-([4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxyphenyl]methyl)piperidin-4-yl]methyl]-2,7-diazaspiro[3.5]nonan-2-yl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione TFA salt (3.2 g, 32.3%) as a white solid. .sup.1H NMR (400 MHZ, DMSO-d6) 10.96 (s, 1H), 9.01 (s, 1H), 7.59 (s, 1H), 7.36 (d, J=8.0 Hz, 1H), 6.72 (s, 2H), 6.68 (d, J=8.1 Hz, 2H), 6.20 (s, 1H), 5.07 (dd, J=13.3, 5.1 Hz, 1H), 4.35-4.13 (m, 2H), 4.06-3.95 (m, 4H), 3.80 (s, 6H), 3.57 (s, 4H), 3.47 (s, 5H), 2.97-2.75 (m, 3H), 2.70-2.55 (m, 1H), 2.44-2.16 (m, 7H), 2.13-1.88 (m, 5H), 1.80-1.67 (m, 4H), 1.61 (d, J=12.4 Hz, 2H), 1.53-1.33 (m, 1H), 1.13-0.94 (m, 2H). LCMS (ESI) m/z: [M+H].sup.+=829.55.
[0336] Enantiomers of compound D1 were separated by supercritical fluid chromatography on chiral support to produce compound S-D1 and compound R-D1.
[0337] Compound D1 is of the following structure:
##STR00078##
[0338] Compound S-D1 is of the following structure:
##STR00079##
[0339] Compound R-D1 is of the following structure:
##STR00080##
Example 9SYO1 BRD9 NanoLuc Degradation Assay
[0340] This example demonstrates the ability of the compounds of the disclosure to degrade a Nanoluciferase-BRD9 fusion protein in a cell-based degradation assay.
[0341] Procedure: A stable SYO-1 cell line expressing 3FLAG-NLuc-BRD9 was generated. On day 0 cells were seeded in 30 L media into each well of 384-well cell culture plates. The seeding density was 8000 cells/well. On day 1, cells were treated with 30 nL DMSO or 30 nL of 3-fold serially DMSO-diluted compounds (10 points in duplicates with 1 M as final top dose). Subsequently plates were incubated for 6 hours in a standard tissue culture incubator and equilibrated at room temperature for 15 minutes. Nanoluciferase activity was measured by adding 15 L of freshly prepared Nano-Glo Luciferase Assay Reagent (Promega N1130), shaking the plates for 10 minutes and reading the bioluminescence using an EnVision reader.
[0342] Results: The Inhibition % was calculated using the following formula: % Inhibition=100(Lum.sub.HCLum.sub.Sample)/(Lum.sub.HCLum.sub.LC). DMSO treated cells are employed as High Control (HC) and 1 M of a known BRD9 degrader standard treated cells are employed as Low Control (LC). The data was fit to a four parameter, non-linear curve fit to calculate IC.sub.50 (UM) values as shown in Table 2. As shown by the results in Table 2, a number of compounds of the present disclosure exhibit an IC.sub.50 value of <1 M for the degradation of BRD9, indicating their use as compounds for reducing the levels and/or activity of BRD9 and their potential for treating BRD9-related disorders.
TABLE-US-00002 TABLE 2 SYO1 BRD9-NanoLuc Degradation Compound No. SYO1 BRD9-NanoLuc degradation IC.sub.50 (nM) D1 0.13 D2 0.18
Example 10Degradation of BRD9 Inhibits the Growth of Synovial Sarcoma Tumor In Vivo
[0343] Method: NOD SCID mice (Beijing Anikeeper Biotech, Beijing) were inoculated subcutaneously on the right flank with the single cell suspension of SYO-1 human biphasic synovial sarcoma tumor cells (5106) in 100 L Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). The mice were randomized into either control group [10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol (PEG400) and 50% water], or treatment group D1 when the mean tumor size reached about 117 mm.sup.3. Mice were dosed daily through intraperitoneal (i.p.) route over the course of 3 weeks. All dose volumes were adjusted by body weights in terms of mg/kg.
[0344] Results: As shown in
Example 11Compound D1 Causes Degradation of BRD9 in Synovial Sarcoma Tumor In Vivo
[0345] Method: Mice were treated with D1, 1 mg/kg, i.p. for 4 weeks. Mice were then euthanized, and tumors were collected at 8 hours, 72 hours, and 168 hours post last dose. Tumors were lysed with 1x RIPA lysis buffer (Boston BioProducts, BP-115D) with protease and phosphatase protein inhibitor (Roche Applied Science #04906837001 & 05892791001). Equal amounts of lysate (30 g) were loaded in in 4-12% Bis-Tris Midi Protein Gels in 1MOPS buffer; samples ran at 120 V for 120 minutes. Protein was transferred to membrane with TransBlot at 250 mA for 150 minutes, and then membranes were blocked with Odyssey blocking buffer for 1 hour at room temperature. Membranes were hybridized overnight in cold room with primary antibodies. Images acquired using Li-COR imaging system (Li-COR Biotechnology, Lincoln, Nebraska).
[0346] Table 3 shows detection antibody information.
TABLE-US-00003 TABLE 3 Antibody Vendor Cat# Species Dilution BRD9 Bethyl, (Montgomery, TX) A303-781A Rabbit 1:1000 GAPDH CST, (Danvers, MA) 97166 Mouse 1:2000
[0347] Results: As shown in
Example 12the Effect of Compounds S-D1 and R-D1 on Synovial Sarcoma Cells
[0348] Method. Synoial sarcoma cells were plated in 6-well plate at 500-100 k cells/well and treated with serial concentrations of BRD9 degrader (10 nM top concentration, diluted 1:3) the next day for two time points at 37 C. Cells were then harvested, washed with cold PBS, and frozen in cell pellets at 80 C. Lysates were prepared by resuspending thawed pellets in 1x RIPA Lysis and Extraction buffer (Thermo Fisher, Cat #89900) with 1Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (Thermo Fisher, Cat #78441) and 1:1000 dilution Pierce Universal Nuclease for Cell Lysis 25 ku (Thermo Fisher, Cat #88700). Lysates were incubated on ice for 10 minutes and then centrifuged in 4 C. at maximum speed (15,000 rpm) for 10 minutes. Samples were then analyzed for total protein using BCA protein quantification assay and diluted to 1 g/L with lysis buffer and 1NuPAGE LDS Sample Buffer (4) (Thermo Fisher, Cat #NP0007) and 1DTT from 30 stock (Cell Signaling Technologies, Cat #14265S). Samples with 20-25 ug of total protein were loaded into 4-12% Bis-Tris Mini-Gel with 1MES Running buffer and run at 150V for 45 minutes. Gels were transferred using Trans-Blot Turbo Transfer System (semi-dry) at 25V for 10 minutes (High MW setting) on nitrocellulose blots. Blots were blocked in 5% milk in TBST for 1 hour and probed with BRD9 antibody (Bethyl Labs, Cat #A303-781A, 1:750 for SYO1, and Cell Signaling Technologies, Cat #71232S for ASKA) and beta-Actin antibody (Cell Signaling Technologies, Cat #3700, 1:2000) overnight at 4 C. The next day, blots were washed in TBST 3x and probed with 1:5000 IRDye 680LT Goat anti-Rabbit IgG Secondary Antibody (LICOR, Cat #926-68021) and 1:10000 IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LiCOR, Cat #926-32210) in LiCOR Odyssey Blocking Buffer (TBS) for 1 hour at room temperature. Blots were washed in TBST 3x and scanned at 700 nM and 800 nM wavelength using LiCOR Odyssey CLx Imaging System. Western blot signal was quantified using same analyses program included in the same machine. BRD9 signal was quantified by normalizing to beta-actin signal and all samples were normalized to DMSO, set as 100% signal.
[0349] For the assessment of interconversion between Enantiomer 1 and Enantiomer 2 in cell medium, the following test was performed. Enantiomer 1 and Enantiomer 2 (each was 40 M in DMSO) was spiked into cell medium (DMEM+Glutamax+10% FBS) at a final concentration of 0.2 M and incubated at 37 C. and 5% CO.sub.2 in duplicate. At designated time point, aliquot (50 L) was taken and processed by the addition of 150 L of acetonitrile containing 0.1% formic acid and internal standard for LC/MS-MS analysis. Peak areas of both Enantiomer 1 and Enantiomer 2 were determined for each sample using a chiral specific analytical method. The results are summarized in Table 5 below.
[0350] Results. To assess BRD9 degradation activity of two enantiomers, degrader treatment and subsequent western-blot experiments were carried out using two synovial sarcoma cell lines (SYO-1 and ASKA). Significant more potent BRD9 degradation activity was observed with Enantiomer 2, with a fitted DC.sub.50 value of 0.092 nM, comparing to 2.8 nM for Enantiomer 1 in SYO-1 with 1 h treatment time (
TABLE-US-00004 TABLE 4 Cell Fitted Enantiomer Enantiomer Line DC.sub.50 (nM) 1 2 ASKA 0.5 h >10 0.34 SYO-1 1 h 2.8 0.092 ASKA 2 h 0.38 0.012 SYO-1 6 h 0.066 0.023
[0351] Epimerization of the chiral center in thalidomide or other IMiD drugs and their derivatives is reported. The acidic hydrogen in the chiral center can be scrambled under physical or neutral pH conditions. To investigate the chiral stability under cell assay conditions for these degraders, we performed a time course study for Enantiomer 1 and Enantiomer 2 in cell culture medium at 37 C. There is no detectable Enantiomer 2 in Enantiomer 1 samples at time 0 or 0.5 h. But substantial Enantiomer 2 was detected at later time points, accounting for 12% and 30% of the total at 2 h and 6 h, respectively (Table 5). Similarly, Enantiomer 2 is converted to Enantiomer 1 over time and its effective concentration was reduced to 63% at 6h (Table 5). These data indicate that epimerization rate is relatively fast under the cell assay conditions, and suggest that the time-dependent BRD9 degradation activity for Enantiomer 1 is likely due to the converted Enantiomer 2. Overall, these data indicate that Enantiomer 2 is the active enantiomer in degrading BRD9 in cells.
TABLE-US-00005 TABLE 5 Enantiomer 1 Dosing Enantiomer 2 Dosing Mean peak Mean peak area ratio area ratio of Enantiomer of Enantiomer 2 over % 1 over % Time Enantiomer 1 Enantiomer Enantiomer 2 Enantiomer (h) peak area ratio 2 peak area ratio 2 0 0.0 0.0 0.01 99 0.5 0.0 0.0 0.06 95 2 0.13 12 0.22 82 6 0.43 30 0.60 63
Example 13The Effect of Compounds S-D1 and R-D1 on Synovial Sacroma Cells
[0352] Method. The SYO-1 tumor cells were maintained in vitro as adherent cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 C. in an atmosphere of 5% CO2 in air. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. BALB/c Nude mice (Shanghai Lingchang biological science) were inoculated subcutaneously on the right flank with (510.sup.6) in 0.1 mL of phosphate buffered saline (PBS). The treatment(described in the table below)was started on day 19 after tumor inoculation, when the average tumor size reached 499 mm.sup.3.
TABLE-US-00006 TABLE 6 Treatment information Antibody Number of mice Vehicle control, sulfobutylether-- 3 cyclodextrin (SBECD), Single Dose, 10 L/g Racemic D1, 0.5 mg/kg i.v., Single 12 Dose, 10 L/g (20% SBECD) Enantiomer 1, 0.25 mg/kg i.v., Single 18 Dose, 10 L/g (20% SBECD) Enantiomer 2, 0.25 mg/kg i.v., Single 18 Dose, 10 L/g (20% SBECD) Enantiomer 2, 1 mg/kg i.v., Single 18 Dose, 10 L/g (20% SBECD)
[0353] Mice were treated with racemic D1, 1 mg/kg, i.p. for 4 weeks, mice were euthanized, and tumors collected 1, 4, 8, 24, 48 and 72-hour post last dose. Tumors were lysed with 1RIPA lysis buffer (Boston BioProducts, BP-115D) with protease and phosphatase protein inhibitor (Roche Applied Science #04906837001 & 05892791001). Equal amount of lysate (30 ug) were loaded in in 4-12% Bis-Tris Midi Protein Gels in 1MOPS buffer; samples ran at 120 V for 120 min. Proteins was transferred to membrane (NC) with TransBlot at 250 mA for 150 minute, then membranes were blocked with Odyssey Blocking buffer for 1 hour at room temperature. Membranes were hybridized overnight in cold room with primary antibodies. Images acquired using Li-COR imaging system (Li-COR Biotechnology, Lincoln, Nebraska)
TABLE-US-00007 TABLE 7 Detection antibody information Antibody Vendor Cat# Species Dilution BRD9 Bethyl, (Montgomery, TX) A303-781A Rabbit 1:1000 GAPDH CST, (Danvers, MA) 97166 Mouse 1:2000
[0354] Results. Pharmacodynamic activities of Enantiomer 1, Enantiomer 2, and racemic compound D1 were evaluated in SYO-1 Xenograft model. Enantiomer 2 demonstrated significant activity which was assessed by BRD9 protein level using western blot assay
Example 14Stereoretentive Preparation of Compound S-D1
##STR00081## ##STR00082##
[0355] Preparation of Compound X. To V and Win MeOH were added NaOAc (2.0 eq) and sodium triacetoxyborohydride (3.0 eq), and the reaction mixture was stirred. Upon completion of the reaction, the reaction was quenched with water, followed by further dilution with NaOH (7%). The mixture was seeded with compound X. To the reaction mixture was added more NaOH (7%) until the pH of the solution was 8-9. The slurry was filtered and washed to obtain compound X in >90% yield.
[0356] Hydrogenation of Compound X, Formation of Compound Q. To a mixture of compound X in MeOH was added 1% w/w Pd/C. This mixture was put under 40 psi hydrogen pressure and stirred for 16 h. The reaction mixture was then filtered and washed with MeOH. The resulting solution was concentrated, and then IPAc was added. This mixture was concentrated to remove MeOH, and further IPAc was added. The IPAc solution was then concentrated, and to the mixture was added n-heptane to allow for precipitation of the product. This mixture was cooled and stirred overnight. The solid was filtered and dried to provide Compound Q in 75% yield.
[0357] Reductive Amination to Compound R: Compound Q and Compound H Coupling. To Compound Q and Compound H in NMP, were added NaOAc (4.0 eq) and sodium triacetoxyborohydride (3.0 eq), and the reaction mixture is stirred. Upon completion of the reaction, the reaction was quenched NH.sub.4Cl (10% in water) the solution is filtered and washed with water. To the solution was added MeTHF, and the resulting mixture was basified with NaOH (15%) to pH 8-10. The layers were separated, and the aqueous layer was further extracted with MeTHF. The MeTHF layers were combined and washed with water to remove residual NMP. The organic layer was concentrated and seeded with compound R. To this mixture was added methylcyclohexane, and the mixture was cooled. The slurry was filtered and washed to obtain compound R in >80% yield.
[0358] Deprotection of Compound R: Formation of Compound S. To Compound R was added 5% H.sub.2SO.sub.4, and mixture is heated and stirred. The reaction was quenched by the addition of 16% NaOH solution to basify to pH 6-8. The solution was washed with DCM and 20% K.sub.2CO.sub.3 solution was added to adjust the pH to 9-11. The layers were separated, and the aqueous layer was further extracted with DCM. The organic layers were combined, and the solution was solvent swapped from DCM to THF. As a solution in THF, compound PB was isolated by heating and then cooling to RT followed by the addition of MBTE to allow for further precipitation. The slurry was stirred overnight. The slurry was filtered and washed to obtain compound S in >70% yield.
[0359] SNAR: Synthesis of Compound U. To a solution of compound S, compound T, and DMAc was added Na.sub.2CO.sub.3. The mixture was then stirred and heated overnight. The reaction was cooled and filtered to remove inorganics. The reaction was then quenched with NH.sub.4Cl, and DCM was added. The layers were separated, and the aqueous layer was washed with DCM once again, and then organic layers were combined. The combined organic layers were washed with NH.sub.4HCO.sub.3. The layers were separated, and the NH.sub.4HCO.sub.3 process was repeated. The layers were separated, and the organic layer was concentrated. MeCN was added, and the mixture was concentrated to remove DCM. The mixture was then seeded with compound U, cooled, and stirred overnight. The slurry was then filtered and dried to provide compound U in >60% yield.
[0360] Synthesis of Compound S-D1. To compound U and compound V was added a THF/MeOH mixture. This solution was cooled and stirred, and AcOH was added. NaCNBH.sub.3 was then added, while the mixture continued cooling. The reaction mixture was then allowed to stir overnight at RT until reaction completion. The reaction was quenched by the addition of a mixture of DCM/DMSO and Na.sub.2CO.sub.3. The layers were separated, and the organic layer was washed with Na.sub.2CO.sub.3 and then brine. The organic layer were separated and concentrated. To the concentrated organic layer was added MeCN, and the mixture was filtered. The filtrate was then further concentrated, and DMF was added. To the DMF solution was added water at RT, and the mixture was stirred for 10 h. The slurry was filtered, and drying the filter cake afforded compound S-D1 in 60% yield.
[0361] Recrystallization of Compound S-D1. Compound S-D1 was dissolved in DMSO (alternatively, NMP or DMF may be used), and a mixture of THF (or acetone)/water was then added. The mixture was seeded, and then further THF (or acetone)/water was added. The slurry was then filtered and dried to give compound S-D1 in 75-100% yield.
Example 15Forced Degradation Study of Compound D1
Solution Preparation
[0362] Diluent 1: H.sub.2O [0363] Diluent 2: dimethylsulfoxide (DMSO) [0364] Diluent 3: [0.05% formic acid (FA) in acetonitrile (MeCN)]: H.sub.2O=1:1 (v/v) [0365] 2 N HCl was prepared by weighing 19.7076 g concentrated aq. HCl (37%) into a 100 ml volumetric flask containing approximately 20 mL diluent 1, diluting to volume with diluent 1, and mixing well. [0366] 0.002 N HCl was prepared as follows. 1 mL of 2 N HCl into was transferred into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluted to volume with diluent 1, and mixed well. Then, 1 mL of this solution was pipetted into a 10 mL volumetric flask, diluted to volume with diluent 1, and mixed well. [0367] Diluent 4: 0.001 N HCl was prepared as follows. 1 mL of the 2 N HCl was transferred into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluted to volume with diluent 1, and mixed well. Then, 5 mL of this solution were pipetted into a 100 mL volumetric flask, made up to volume with diluent 1 and mixed well. [0368] 2 N NaOH was prepared as follows. 8.0248 g of NaOH were weighed into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well. [0369] 0.1 N NaOH was prepared as follows. 5 mL of the 2 N NaOH were transferred into a 100 mL volumetric flask containing approximately 5 mL diluent 1, diluted to volume with diluent 1, and mixed well. [0370] 0.002 N NaOH was prepared as follows. 1 mL of the 2 N NaOH was transferred into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluted to volume with diluent 1, and mixed well. Then, 10 mL of this solution were pipetted into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well. [0371] 6% Hydrogen peroxide was prepared as follows. 20 mL of concentrated H.sub.2O.sub.2 (30%) were transferred to a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well. [0372] 92% relative humidity control was achieved using a dryer containing a saturated potassium nitrate solution. [0373] 1 N Citric acid was prepared as follows. 19.2032 g of citric acid were weighed into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well. [0374] pH=4.0 50 mM Sodium citrate buffer was prepared as follows. 1.4814 g of sodium citrate were weighed into a 100 mL volumetric flask, diluted up to volume with diluent 1, and mixed well. pH was adjusted to 4.0 by 1 N citric acid. [0375] pH=5.0 50 mM Sodium citrate buffer was prepared as follows. 1.4815 g of sodium citrate were weighed into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well. pH was adjusted to 5.0 by 1 N citric acid. [0376] pH=7.0 Phosphate buffer was prepared as follows. 0.6963 g of KH.sub.2PO.sub.4 were weighed into a 100 mL volumetric flask, 29.1 mL of the 0.1 N NaOH were transferred into the same volumetric flask, diluted to volume with diluent 1, and mixed well.
Sample Preparation
[0377] Sample stock solution was prepared by weighing 60.74 mg of compound D1 to a 100 mL volumetric flask, dissolving by ultrasonic in 50 mL of diluent 2, diluting to volume with diluent 2, and mixing well.
[0378] Unstressed sample solution was prepared by weighing 21.32 mg of compound D1 into a 100 ml volumetric flask, dissolving by ultrasonic in 50 mL of diluent 3, diluting to volume with diluent 3, and mixing well.
[0379] Sensitivity solution (LOQ) was prepared as follows. 1.0 mL of unstressed sample solution was pipetted into a 100 mL volumetric flask, diluted to volume with diluent 3, and mixed well. Then, 5 mL of this sample solution were pipetted into a 100 mL volumetric flask, diluted to volume with diluent 3, and mixed well.
[0380] Sample solution for pH=4.0 solution stability was prepared as follows. 20.96 mg of compound D1 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 ml of pH=4.0 50 mM sodium citrate buffer, diluted to volume with pH=4.0 50 mM sodium citrate buffer, and mixed well.
[0381] Sample solution for pH=5.0 solution stability was prepared as follows. 20.75 mg of compound D1 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 ml of pH=5.0 50 mM sodium citrate buffer, diluted to volume with pH=5.0 50 mM sodium citrate buffer, and mixed well.
[0382] Sample solution for pH=7.0 solution stability was prepared as follows. 19.91 mg of compound D1 and 684.71 mg KH.sub.2PO.sub.4 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 1, transferred 29.1 mL of 0.1 N NaOH into the same volumetric flask, diluted to volume with diluent 1, and mixed well.
Forced Degradation
Stress Conditions
TABLE-US-00008 TABLE 8 Stress Conditions and Sampling time points Degradation type Stress Solvent/Conditions Time Point Acid 1N HCl at RT 0 h, 1 d, 2 d, 3 d Hydrolysis Basic 0.001N NaOH at RT 0 h, 1 h Hydrolysis Oxidation 3% Hydrogen Peroxide at RT 0 h, 8 h Photolysis 1.10 W/m.sup.2/25 C. for solid 13 h, 26 h, 39 h (1.10 W/m.sup.2/25 C. with 13 hours equal to visible 1.2M lux hrs and UV 647 Wh/m.sup.2) Thermal 80 C. for solid 1 d, 3 d, 7 d 80 C. for solution 1 d, 7 d Humidity 92% RH 1 d, 3 d, 7 d Solution pH = 4.0, 50 mM Sodium 0 h, 1 d, 3 d, 7 d Stability citrate buffer at RT pH = 5.0, 50 mM Sodium 0 h, 1 d, 3 d, 7 d citrate buffer at RT pH = 7.0 Phosphate 0 h, 1 d, 3 d, 7 d buffer at RT
[0383] Notes: 1. The target endpoint of a stress study was to form approximately 5-15% of total degradation product. 2. Based on actual degradation of the sample, the stress conditions including concentration of sample and reagent, and temperature, humidity, light may be adjusted. 3. After stressing samples in acid and base, neutralize them before placing into freezer. 4. All degradable samples before analysis must be placed into the 2 C. to 8 C. condition.
[0384] Acid degradation (1 N HCl at RT). 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial, 3 mL of 2 N HCl solution were added, and the resulting mixture was mixed well. Samples were prepared in quadruplicate and kept at RT. At the sampling point, 2 mL of the sample were transferred into an 8 mL vial and neutralized with 1 mL of 2 N NaOH.
[0385] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
[0386] Basic degradation (0.001 N NaOH at RT). 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial, 3 mL of 0.002 N NaOH solution were added, and the resulting mixture was mixed well. Samples were prepared in triplicate and kept at RT. At the sampling point, 2 mL of the solution were transferred into an 8 mL vial and neutralized with 1 mL of 0.002 N HCl.
[0387] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
[0388] Oxidation degradation (3% H.sub.2O.sub.2 at RT). 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial, 3 mL of 6% H.sub.2O.sub.2 solution were added, and the resulting mixture was mixed well. Samples were prepared in quadruplicate and kept at RT. At the sampling time point, 2 mL of the solution were transferred into an 8 mL vial, neutralized with 1 mL of diluent 3, and mixed well.
[0389] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
[0390] Photolysis degradation (solid). About 60 mg of compound D1 were placed onto a watch glass. Samples were prepared in triplicate and placed in a photo chamber (see Table 8). At the sampling time point, the sample was placed in a vial for analysis. About 20.02.0 mg of the sample into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 3, diluted to volume with diluent 3, and mixed well.
TABLE-US-00009 TABLE 9 Photolysis degradation of samples' mass weight Sampling Name* Weight (mg) PSD-13 h 20.44 PSD-26 h 19.93 PSD-39 h 19.43 *PSD-13 h, PSD-26 h and PSD-39 h in category mean the compound is degradation product under photolysis solid stress condition in 13 h, 26 h, and 39 h, respectively.
[0391] Dark control sample: one sample was prepared as described above for this test, but the watch glass was covered with aluminum foil, and processed as described above.
Thermal Stress Degradation (80 C. for Solid and Solution).
[0392] Solid: about 60 mg of compound D1 were weighted into an 8 mL vial. Samples were prepared in triplicate and placed in an oven at 80 C. At the sampling time point, about 20.02.0 mg of the sample were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 ml of diluent 3, diluted to volume with diluent 3, and mixed well.
TABLE-US-00010 TABLE 10 Thermal degradation of samples' mass weight Sample Name* Weight (mg) T-1 d 19.82 T-3 d 22.07 T-7 d 21.68 *T-1 d, T-3 d and T-7 d in category mean the compound is degradation product under thermal solid stress condition in 1 d, 3 d and 7 d, respectively.
[0393] Solution: 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial. Samples were prepared in triplicate and placed them in an oven at 80 C. At the sampling time point, 1 mL of the solution was transferred into an 8 mL vial, neutralized with 2 mL of diluent 3, and mixed well.
[0394] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
[0395] Humidity degradation (92% RH for solid). About 60 mg of compound D1 were transferred into a 20 mL vial (without cap). Samples were prepared in triplicate and placed in a desiccator at 92% RH. At the sampling time point, about 20.02.0 mg of the sample were transferred into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 3, diluted to volume with diluent 3, and mixed well.
TABLE-US-00011 TABLE 11 Humidity degradation of samples' mass weight Sample Name* Weight (mg) H-1 d 21.02 H-3 d 21.41 H-7 d 20.11 *H-1 d, H-3 d and H-7 d in category mean the compound is degradation product under humidity solid stress condition in 1 d, 3 d and 7 d, respectively.
[0396] Solution Stability. Samples were prepared tested under the solution stability conditions noted in Table 8. The sampling time points were as noted in Table 8.
[0397] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
Sample Analysis
TABLE-US-00012 TABLE 12 HPLC Method 1 Mobile Phase A H.sub.2O + 0.1% TFA Mobile Phase B MeCN + 0.1% TFA Column Poroshell 120 SB-AQ (3.0*150 mm, 2.7 m) Needle Wash MeCN: H.sub.2O = 1:1 Injection volume 3 L Column Temperature 40 C. Flow Rate 0.8 mL/min Detection 210 nm Gradient Time (min) % A % B 0.0 198 2 7.0 70 30 12.0 50 40 15.0 5 195 17.0 5 195 Post time 5 min Run time 17 min
TABLE-US-00013 TABLE 13 Summary % Degradation Stress Solvent/ Sample Sample % Mass Type Conditions Name Number Time Area % Degradation Balance Unstressed N/A N/A 0 N/A 94.172 N/A N/A Acid 1N HCl RT A 0 1 0 h 93.734 0.438 108.2 Hydrolysis A 1 2 1 d 89.391 4.781 106.5 A 2 3 2 d 85.180 8.992 107.6 A 3 4 3 d 82.047 12.125 106.9 Base 1N NaOH RT B 0 5 0 h 68.962 25.210 104.0 Hydrolysis B 1 6 1 h 17.331 76.841 98.0 Oxidation 3% H.sub.2O.sub.2 RT O 0 7 0 h 77.988 16.184 106.8 O 8 8 8 h 33.755 60.417 105.2 Photolysis 1.10 W/m.sup.2/25 C. for P 1 9 13 h 91.084 3.088 101.0 solid with P 2 10 26 h 90.916 3.256 92.1 13 hours equal to P 3 11 39 h 90.736 3.436 105.7 visible 1.2M lux hrs and UV 647 Wh/m.sup.2 Thermal 80 C. Solid TSD 1 12 1 d 93.448 0.724 105.7 TSD 3 13 3 d 93.194 0.978 97.6 TSD 7 14 7 d 82.192 11.980 92.7 80 C. Solution TSN 1 15 1 d 94.053 0.119 100.5 TSN 7 16 7 d 31.937 62.235 104.2 Humidity 92% RH H 1 17 1 d 92.927 1.245 98.8 H 3 18 3 d 92.081 2.091 99.5 H 7 19 7 d 90.890 3.282 99.8 Solution pH = 4.0, 50 mM pH 4 0 20 0 h 93.516 0.656 90.1 Stability Sodium citrate pH 4 1 21 1 d 93.331 0.841 93.7 buffer at RT pH 4 3 22 3 d 92.873 1.299 93.9 pH 4 7 23 7 d 92.130 2.042 93.7 pH = 5.0 50 mM pH 5 0 24 0 h 92.498 1.674 95.3 Sodium citrate pH 5 1 25 1 d 92.250 1.922 103.1 buffer at RT pH 5 3 26 3 d 92.039 2.133 100.0 pH 5 7 27 7 d 89.514 4.658 100.8 pH = 7.0 Phosphate pH 7 0 28 0 h 92.638 1.534 97.1 buffer at RT pH 7 1 29 1 d 85.252 8.920 102.6 pH 7 2 30 2 d 79.002 15.170 100.6
TABLE-US-00014 TABLE 14 Summary result of mass balance, resolution and purity factor Mass Balance *Resolution Purity Factor Sample Pass/ Pass/ Pass/ Number Purity % Result Criteria Fail Result Criteria Fail Result Criteria Fail 4 82.047 106.9 90%-110% Pass 2.0 1.2 Pass 999.943 >990 Pass 6 17.331 98.0 1.8 996.409 8 33.755 105.2 1.0 Fail 998.510 11 90.736 105.7 1.8 Pass 999.780 14 82.192 92.7 1.8 999.941 16 31.937 104.2 1.8 999.708 19 90.890 99.8 1.9 999.438 23 92.130 93.7 2.0 999.830 27 89.514 100.8 2.0 999.639 30 79.002 100.6 2.0 998.160 *Resolution was the adjacent impurity with the main peak. According to the result above, samples 6 and 8 were used to method optimized.
Example 16Forced Degradation Study of Compound S-D1
Solutions Preparation
[0398] Mobile phase: methanol (MeOH):acetonitrile (MeCN)=3:7 (v/v)+25 mM formic acid (FA)+25 mM NH.sub.3. Prepared by transferring 300 mL MeOH, 700 mL MeCN, 970 L FA, and 12.5 mL Ammonia in to 1 L bottle, mixed well.
[0399] Needle wash solution: MeOH [0400] Diluent 1: H.sub.2O [0401] Diluent 2: 0.05% FA in MeCN:THF=1:1 (v/V) [0402] 2 N HCl was prepared by weighing 19709.02 mg concentrated aq. HCl (37%) into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluting to volume with diluent 1, and mixing well. [0403] 0.3 N HCl was prepared by transferring 15 mL of the 2 N HCl (see above) into a 100 ml volumetric flask containing approximately 20 mL diluent 1, diluting to volume with diluent 1, and mixing well. [0404] 2 N NaOH was prepared by weighing 7996.77 mg NaOH into a 100 mL volumetric flask, made up to volume with diluent 1 and mixed well. [0405] 0.3 N NaOH was prepared by transferring 15 mL of the 2 N NaOH (see above) into a 100 ml volumetric flask containing approximately 5 mL diluent 1, diluting to volume with diluent 1, and mixing well. [0406] 0.1 N NaOH was prepared by transferring 5 mL of the 2 N NaOH (see above) into a 100 mL volumetric flask containing approximately 5 mL diluent 1, diluting to volume with diluent 1, and mixing well. [0407] 1 N Citric acid was prepared by weighing 19193.47 mg citric acid into a 100 mL volumetric flask, diluting to volume with diluent 1, and mixing well. [0408] pH=4.0 50 mM Sodium citrate buffer was prepared by weighing 1474.25 mg sodium citrate into a 100 mL volumetric flask, diluting to volume with diluent 1, and mixing well. pH was adjusted pH to 4.0 by 1 N citric acid. [0409] pH=5.0 50 mM Sodium citrate buffer was prepared by weighing 1474.11 mg sodium citrate into a 100 mL volumetric flask, diluting to volume with diluent 1, and mixing well. pH was adjusted to 5.0 by 1 N citric acid.
[0410] Sample solution stock was prepared by weighing 501.53 mg of compound S-D1 to a 100 ml volumetric flask, dissolving by ultrasonic in 50 mL of diluent 2, diluting to volume with diluent 2, and mixing well.
[0411] Unstressed sample solution was prepared by weighing 50.83 mg of compound S-D1 into a 100 mL volumetric flask, dissolving by ultrasonic in 50 mL of diluent 2, diluting to volume with diluent 2, and mixing well.
[0412] Sensitivity solution (LOQ) was prepared as follows. 1.0 mL of unstressed sample solution were pipetted into a 100 mL volumetric flask, diluted to volume with diluent 2, and mixed well. Then 2 mL of this sample solution were pipetted into a 10 mL volumetric flask, diluted to volume with diluent 2, and mixed well.
[0413] Sample solution for pH=4.0 solution stability was prepared as follows. 50.79 mg of compound S-D1 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of pH=4.0 50 mM sodium citrate buffer (see above), diluted to volume with pH=4.0 50 mM sodium citrate buffer, and mixed well.
[0414] Sample solution for pH=5.0 solution stability was prepared as follows. 50.66 mg of compound S-D1 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 ml of pH=5.0 50 mM sodium citrate buffer (see above), diluted to volume with 50 ml of pH=5.0 50 mM sodium citrate buffer and mixed well.
[0415] Sample solution for pH=7.0 solution stability was prepared as follows. 50.78 mg of compound S-D1 and 680.42 mg KH.sub.2PO.sub.4 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 1, transferred 29.1 mL of the 0.1 N NaOH (see above), diluted to volume with diluent 1, and mixed well.
Forced Degradation
TABLE-US-00015 TABLE 15 Stress Conditions and Sampling Time Points Degradation Stress Sampling type Solutions/Conditions Time Point Acid Hydrolysis 1N HCl at RT 0 h, 1 d, 2 d, 3 d Basic Hydrolysis 0.15N NaOH at RT 0 h, 1 h Thermal 50 C. for solid 1 d, 3 d, 7 d Solution pH = 4.0, 50 mM Sodium 0 h, 1 d, 2 d, 3 d Stability citrate buffer at RT pH = 5.0, 50 mM Sodium 0 h, 1 d, 2 d, 3 d citrate buffer at RT pH = 7.0 Phosphate 0 h, 1 d, 2 d, 3 d buffer at RT Note: After stressing samples in acid or base, the samples were neutralized before placing into freezer.
[0416] Acid degradation (1 N HCl at RT). 3 mL of the sample stock solution (see above) into an 8 mL vial, added 3 mL of 2 N HCl solution, and mixed well. Samples were prepared in quadruplicate and kept at room temperature (RT). At the sampling time point, 1 mL of the sample was transferred into a 5 mL volumetric flask, neutralized with 0.5 mL of 2 N NaOH, diluted to volume with diluent 2, and mixed well.
[0417] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound S-D1.
[0418] Basic degradation (0.15 N NaOH at RT). 3 mL of the sample stock solution (see above) into an 8 mL vial, added 3 mL of 0.3 N NaOH solution, and mixed well. Samples were prepared in quadruplicate and kept at room temperature (RT). At the sampling time point, 1 mL of the sample was transferred into a 5 mL volumetric flask, neutralized with 0.5 mL of 2 N NaOH, diluted to volume with diluent 2, and mixed well.
[0419] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound S-D1.
[0420] Thermal stress degradation (50 C. for solid). Approximately 150 mg of compound S-D1 were weighed into an 8 mL vial. Multiple samples were prepared and placed in an oven at 50 C. At the sampling time points, each of the 50.57 mg, 50.84 mg, and 50.84 mg samples were transferred into their respective 100 mL volumetric flasks, dissolved by ultrasonic in 50 mL of diluent 2, diluted to volume with diluent 2, and mixed well.
[0421] Solution Stability. The study was performed according to the conditions outlined in Table 15 using sample solution for pH=4.0 solution stability, sample solution for pH=5.0 solution stability, and sample solution for pH=7.0 solution stability (see above).
[0422] Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of sample.
Sample Analysis
TABLE-US-00016 TABLE 16 HPLC Method Mobile Phase MeOH:MeCN = 3:7 (v/v) + 25 mM FA + 25 mM NH.sub.3 Column Cellulose SB, 100*4.6 mm, 3.0 m Needle Wash MeOH Injection volume 1 L Column Temperature 30 C. Flow Rate 0.8 mL/min Diluent 0.05% FA in MeCN:THF = 1:1 (v/V) Autosampler RT Temperature Detection 280 nm Isocratic Time (minute) Mobile Phase (%) 0.0 100 8.0 100 Data Record Time 8 minutes
TABLE-US-00017 TABLE 17 Summary Degradation Stress Sample Area % Area % % Type Solvent/Conditions Time Number (R-D1) (S-D1) Degradation Unstressed N/A N/A 0 0.823 99.177 N/A Acid 1N HCl RT 0 h 1 0.825 99.176 0.002 Hydrolysis 1 d 2 0.828 99.172 0.005 2 d 3 0.834 99.166 0.011 3 d 4 0.839 99.161 0.016 Base 0.15N NaOH RT 0 h 5 19.083 80.917 18.260 Hydrolysis 1 h 6 53.033 46.967 52.210 Thermal 50 C. 1 d 7 1.001 98.999 0.178 3 d 8 1.154 98.847 0.331 7 d 9 1.203 98.797 0.380 Solution pH = 4.0, 0 h 10 0.921 99.079 0.098 Stability 50 mM Sodium 1 d 11 0.962 99.038 0.139 citrate buffer at RT 2 d 12 1.124 98.876 0.301 3 d 13 1.305 98.695 0.482 pH = 5.0 0 h 14 1.471 98.529 0.648 50 mM Sodium 1 d 15 1.861 98.139 1.038 citrate buffer at RT 2 d 16 2.230 97.770 1.407 3 d 17 2.825 97.175 2.002 pH = 7.0 0 h 18 11.555 88.445 10.732 Phosphate buffer at 1 d 19 19.745 80.255 18.922 RT 2 d 20 28.374 71.626 27.551 3 d 21 35.926 64.074 35.103
TABLE-US-00018 TABLE 18 Summary result of resolution Resolution between Resolution between compound R-D1 peak compound S-D1 peak and its adjacent peak and its adjacent peak Purity Factor Sample Pass/ Pass/ Pass/ Number Result Criteria Fail Result Criteria Fail Result Criteria Fail 4 1.3 1.2 Pass 2.0 1.2 Pass 999.917 >990 Pass 6 1.8 4.4 996.140 9 1.8 2.3 999.152 13 1.7 3.7 999.947 17 1.9 4.1 999.616 21 2.1 3.9 999.728
Example 17Stability of Compound S-D1 in Blood and Blood Plasma
[0423] Compound S-D1 (1 M) was incubated at 37 C. in triplicates with plasma and blood of human, monkey, and rat, and samples were taken from each incubation at 0, 30, 60, 120 and 240 min. Propantheline (5 UM, human and monkey) or mevinolin (5 UM, rat) were used as a positive control for plasma and blood stability. Samples were analyzed by Ultra Performance Liquid Chromatography with Tandem Mass Spectrometric Detection (UPLC/MS-MS). Throughout the study, control compounds performed as expected.
TABLE-US-00019 LC conditions were as follows: Instrument: Shimadzu 30 AD Column: Waters XSelectHSS T3 2.5 m (2.1 50 mm) Column 40 C. temperature: Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile Injection volume: 1 L Time A B Elution rate (min) (%) (%) (mL/min) 0 95 5 0.7 0.2 95 5 0.7 0.7 2 98 0.7 1.2 2 98 0.7 1.25 95 5 0.7 1.5 95 5 0.7
TABLE-US-00020 Mass conditions were as follows: Instrument: Triple Quad 5500 (AB sciex, USA) Ion source: Turbo spray Ionization model: ESI Scan type: MRM Ionization mode: Positive Mass spectrometer conditions: Test Article Q1 (m/z) Q3 (m/z) DP (v) EP(v) CE(v) CXP(v) Alprazolam 309.1 281.1 80 10 36 10 Mevinolin 405.3 199 85 15 15 10 Propantheline 369.2 99.7 80 10 50 10
[0424] Plasma samples were prepared as follows. Plasma was acquired from suppliers and stored at 80 C. prior to use. A water bath was set to 37 C. Frozen plasma (stored at 80 C.) was thawed immediately prior to use in the 37 C. water bath. The plasma was centrifuged at 2,000 g for 5 minutes to remove clots and collect supernatant into a fresh tube. pH of the plasma was then checked. The present study only utilized plasma that was thawed once and was within the range of pH 7.2 to 8.0.
[0425] Ratios of compound R-D1 over compound S-D1 were calculated to assess the conversion from compound S-D1 to compound R-D1 in human, monkey, and rat plasma or blood incubated with compound S-D1 up to 4 h (Table 19). Under the experimental condition, ratios of compound R-D1 over compound S-D1 in plasma or blood increased over time and were generally similar among the three species evaluated. Ratios of compound R-D1 over compound S-D1 were 0.01 at time 0 and reached 0.6-0.7 in plasma or 0.1-0.2 in blood after 4 h of incubations.
TABLE-US-00021 TABLE 19 Ratios of compound R-D1 over compound S-D1 in human, monkey, and rat plasma or blood Matrix Time (min) Human Monkey Rat Plasma 0 0.00749 0.00524 0.0138 0.00170 0.0130 0.00603 30 0.0288 0.00791 0.0630 0.000692 0.0572 0.00774 60 0.0712 0.00964 0.123 0.00256 0.124 0.00512 120 0.268 0.0220 0.279 0.000599 0.300 0.0472 240 0.611 0.0473 0.570 0.0140 0.713 0.0539 Blood 0 0.0140 0.000573 0.0138 0.00122 0.0141 0.000410 30 0.0332 0.00319 0.0292 0.000973 0.0237 0.00113 60 0.0500 0.00223 0.0466 0.00747 0.0344 0.00133 120 0.0975 0.00138 0.0900 0.00142 0.0569 0.00227 240 0.180 0.00438 0.152 0.000572 0.101 0.00255 Values are means SD of triplicate determinations.
[0426] Percent remaining of total (compound R-D1 and compound S-D1) was calculated to assess degradation in human, monkey, and rat plasma or blood incubated with compound S-D1 to 4 h (Table 20). While degradation was minimal in human and rat blood, loss of total in human and rat plasma increased over time. After 4 h of incubations, 100% remaining in human and rat blood, and 60-70% remaining in human and rat plasma were observed. Degradation in monkey plasma and blood appeared to be similar with 80% remaining observed at 4 h.
TABLE-US-00022 TABLE 20 Percent Remaining of compounds R-D1 and S-D1 in human, monkey, and rat plasma or blood Time % remaining of total Matrix (min) Human Monkey Rat Plasma 0 100 100 100 30 94.5 7.02 88.9 3.81 90.4 4.01 60 96.1 0.757 96.3 2.41 88.3 7.05 120 84.2 3.80 94.3 6.75 78.0 1.51 240 72.6 4.21 83.4 4.58 63.0 6.48 Blood 0 100 100 100 30 100 4.91 96.4 5.46 115 4.38 60 101 2.59 98.1 5.70 112 5.97 120 98.5 6.24 94.2 7.93 95.1 11.7 240 99.9 7.21 80.0 4.56 115 5.86 Values are means SD of triplicate determinations
OTHER EMBODIMENTS
[0427] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
[0428] While the invention has been described in connection with specific embodiments thereof, it will be understood that invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
[0429] Other embodiments are in the claims.