Macrocyclic immunomodulators
Compounds are provided that are useful as immunomodulators. The compounds have the following Formula (I) or (II): including stereoisomers and pharmaceutically acceptable salts thereof, wherein R, R 1 , R 2a , R 2b , R 2c , R 3 , R 4 , R 5 , R 6a , R 6b , R 6c , m and n are as defined herein. Methods associated with preparation and use of such compounds, as well as pharmaceutical compositions comprising such compounds, are also disclosed.
1. A method of assaying a compound for PD-1/PD-L1 interaction activity, said method comprising
(a) affixing human PD-L1 to a solid surface;
(b) contacting the compound with the affixed human PD-L1 to form an interaction mixture;
(c) contacting the interaction mixture with human PD-1 complexed with biotin to form a competition mixture;
(d) washing the competition mixture with a wash buffer to form an incubation product;
(e) contacting the incubation product with Streptavidin-HRP to form a signal mixture;
(f) washing the signal mixture with a wash buffer to form an detection product;
(g) contacting the detection product with 3,3′,5,5′-Tetramethylbenzidine (TMB) to form a signal product; and
(h) measuring the absorbance of the signal product at 450 nm,
wherein said method comprises performing steps (a)-(h) with a positive control sample having a formula represented by the structure
or a pharmaceutically acceptable salt thereof, wherein:
W is N or C(R 9 );
X, Y, and V are each independently selected from the group consisting of a bond, O, NH, N(CH 3 ), C(O), methylene, and ethylene, wherein the methylene and ethylene are optionally substituted with one or two R 7a ;
R is selected from the group consisting of H, halogen, CN, C 1-3 haloalkyl, C 1-3 alkyl, and C 1-3 alkoxy;
R 1 is phenyl, optionally substituted with 1 to 3 R 1a substituents;
each R 1a is independently selected from the group consisting of halogen, —CN, —R c , —CO 2 R a , —CONR a R b , —C(O)R a , —OC(O)NR a R b , —NR b C(O)R a , —NR b C(O) 2 R c , —NR a —C(O)NR a R b , —NR a R b , —OR a , —O—X 1 —OR a , —O—X 1 —CO 2 R a , —O—X 1 —CONR a R b , —X 1 —OR a , —X 1 —NR a R b , —X 1 —CO 2 R a , —X 1 —CONR a R b , —SF 5 , and —S(O) 2 NR a R b , wherein each X 1 is a C 1-4 alkylene; each R a and R b is independently selected from hydrogen, C 1-8 alkyl, and C 1-8 haloalkyl, or when attached to the same nitrogen atom can be combined with the nitrogen atom to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O, and S, wherein the five or six-membered ring is optionally substituted with oxo; each R c is independently selected from the group consisting of C 1-8 alkyl, C 2-8 alkenyl, C 2-8 alkynyl, and C 1-8 haloalkyl; and optionally when two R 1a substituents are on adjacent atoms, they are combined to form a fused five, six, or seven-membered carbocyclic or heterocyclic ring optionally substituted with from 1 to 3 substituents independently selected from halogen, oxo, C 1-8 haloalkyl, and C 1-8 alkyl;
each of R 1b , R 1c , R 1d , and R 1e is independently selected from the group consisting of H, halogen, CF 3 , CN, C 1-4 alkyl, and —O—C 1-4 alkyl, wherein the C 1-4 alkyl and —O—C 1-4 alkyl are optionally further substituted with halogen, hydroxyl, methoxy, or ethoxy;
L is a linking group selected from the group consisting of:
wherein each of the subscripts q is independently 1, 2, 3, or 4, and L is optionally further substituted with one or two members selected from the group consisting of halogen, hydroxy, C 1-3 alkyl, —O—C 1-3 alkyl, C 1-3 hydroxyalkyl, C 1-3 haloalkyl, and —CO 2 H;
Z is selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyridyl, pyrimidinyl, guanidinyl, quinuclidine, and 8-azabicyclo[3.2.1]octane, each of which is optionally substituted with from 1 to 3 groups independently selected from halogen, hydroxy, C 1-3 alkyl, —NH 2 , —NHC 1-3 alkyl, —N(C 1-3 alkyl) 2 , —O—C 1-3 alkyl, C 1-3 hydroxyalkyl, C 1-3 haloalkyl, and —CO 2 H;
each R 2a , R 2b , and R 2c is independently selected from the group consisting of H, halogen, —CN, —R d , —CO 2 R e , —CONR e R f , —OC(O)NR e R f , —NR f C(O)R e , —NR f C(O) 2 R d , —NR e —C(O)NR e R f , —NR e R f , —OR e , —X 2 —OR e , —X 2 —NR e R f , —X 2 —CO 2 R e , —SF 5 , and —S(O) 2 NR e R f , wherein each X 2 is a C 1-4 alkylene; each R e and R f is independently selected from hydrogen, C 1-8 alkyl, and C 1-8 haloalkyl, or when attached to the same nitrogen atom can be combined with the nitrogen atom to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O, and S, and optionally substituted with oxo; and each R d is independently selected from the group consisting of C 1-8 alkyl, C 2-8 alkenyl, and C 1-8 haloalkyl;
each R 5 is independently selected from the group consisting of halogen, —CN, —R q , —CO 2 R r , —CONR r R s , —C(O)R r , —OC(O)NR r R s , —NR r C(O)R s , —NR r C(O) 2 R q , —NR r —C(O)NR r R s , —NR r R s , —OR r , —O—X 5 —OR r , —O—X 5 —NR r R s , —O—X 5 —CO 2 R r , —O—X 5 —CONR r R s , —X 5 —OR r , —X 5 —NR r R s , —X 5 —CO 2 R r , —X 5 —CONR r R s , —SF 5 , and —S(O) 2 NR r R s , wherein each X 5 is a C 1-4 alkylene; each R r and R s is independently selected from hydrogen, C 1-8 alkyl, and C 1-8 haloalkyl, or when attached to the same nitrogen atom can be combined with the nitrogen atom to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O, and S, and optionally substituted with oxo; and each R q is independently selected from the group consisting of C 1-8 alkyl and C 1-8 haloalkyl;
R 6a and R 6c are each independently selected from the group consisting of H, C 1-4 alkyl, and C 1-4 haloalkyl;
each R 7a and R 7b is independently selected from the group consisting of H, C 1-6 alkyl, CO 2 H, CH 2 OH, —CO 2 —(C 1-6 alkyl), and PO 3 H 2 , wherein C 1-6 alkyl is optionally substituted with one or two members selected from halogen, OH, NH 2 , CN, and CO 2 H;
each R 8a and R 8b is independently selected from the group consisting of H and C 1-6 alkyl, optionally substituted with halogen, OH, NH 2 , CN, and CO 2 H;
R 9 is selected from the group consisting of H, halogen, CN, C 1-6 alkyl, —O—C 1-6 alkyl, —SO 2 (C 1-6 alkyl), —C 1-6 alkyl-CO 2 H, —C 1-6 alkyl-CO 2 —C 1-6 alkyl, —C 1-6 alkyl-C(O)NH 2 , —C 1-6 alkyl-C(O)NHC 1-6 alkyl, and —C 1-6 alkyl-C(O)N(C 1-6 alkyl) 2 ; and
n is 0, 1, 2, or 3.
2. The method of claim 1 , further comprising step a-1) contacting the affixed human PD-L1 with a blocking solution, where step a-1) is performed after step a) but before step b).
3. The method of claim 2 , further comprising step a-2) washing the affixed human PD-L1 and blocking solution with a wash buffer, where step a-2) is performed after step a-1) but before step b).
4. The method of claim 3 , wherein step a-2) performed 3 times.
5. The method of claim 1 , further comprising step g-1) contacting the signal product with a blocking solution, where step g-1) is performed after step g) but before step h).
6. The method of claim 5 , further comprising step g-2) washing the signal product and blocking solution with a wash buffer, where step g-2) is performed after step g-1) but before step h).
7. The method of claim 1 , wherein said wash buffer comprises PBS/0.05% TWEEN-20.
8. The method of claim 2 , wherein said blocking solution comprises 2% BSA (w/v) in PBS with 0.05% TWEEN-20.
9. The method of claim 1 , wherein the positive control sample has the formula
or a pharmaceutically acceptable salt thereof.
10. The method of claim 1 , wherein the positive control sample has the formula
or a pharmaceutically acceptable salt thereof.
11. The method of claim 1 , wherein the positive control sample has the formula
or a pharmaceutically acceptable salt thereof.
12. The method of claim 1 , wherein the positive control sample has the formula
or a pharmaceutically acceptable salt thereof.
13. A kit comprising a compound of Formula (IIa) or Formula (IIb) and a label indicating the intended use, wherein Formula (IIa) and Formula (IIb) have the structure structures
or a pharmaceutically acceptable salt thereof, wherein:
W is N or C(R 9 );
X, Y, and V are each independently selected from the group consisting of a bond, O, NH, N(CH 3 ), C(O), methylene, and ethylene, wherein the methylene and ethylene are optionally substituted with one or two R 7a ;
R is selected from the group consisting of H, halogen, CN, C 1-3 haloalkyl, C 1-3 alkyl, and C 1-3 alkoxy;
R 1 is phenyl, optionally substituted with 1 to 3 R 1a substituents;
each R 1a is independently selected from the group consisting of halogen, —CN, —R c , —CO 2 R a , —CONR a R b , —C(O)R a , —OC(O)NR a R b , —NR b C(O)R a , —NR b C(O) 2 R c , —NR a —C(O)NR a R b , —NR a R b , —OR a , —O—X 1 —OR a , —O—X 1 —CO 2 R a , —O—X 1 —CONR a R b , —X 1 —NR a R b , —X 1 —CO 2 R a , —X 1 —CONR a R b , —SF 5 , and —S(O) 2 NR a R b , wherein each X 1 is a C 1-4 alkylene; each R a and R b is independently selected from hydrogen, C 1-8 alkyl, and C 1-8 haloalkyl, or when attached to the same nitrogen atom can be combined with the nitrogen atom to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O and S, wherein the five or six-membered ring is optionally substituted with oxo; each R c is independently selected from the group consisting of C 1-8 alkyl, C 2-8 alkenyl, C 2-8 alkynyl, and C 1-8 haloalkyl; and optionally when two R 1a substituents are on adjacent atoms, they are combined to form a fused five, six, or seven-membered carbocyclic or heterocyclic ring optionally substituted with from 1 to 3 substituents independently selected from halogen, oxo, C 1-8 haloalkyl, and C 1-8 alkyl;
each of R 1b , R 1c , R 1d , and R 1e is independently selected from the group consisting of H, halogen, CF 3 , CN, C 1-4 alkyl, and —O—C 1-4 alkyl, wherein the C 1-4 alkyl and —O—C 1-4 alkyl are optionally further substituted with halogen, hydroxyl, methoxy, or ethoxy;
L is a linking group selected from the group consisting of:
wherein each of the subscripts q is independently 1, 2, 3, or 4, and L is optionally further substituted with one or two members selected from the group consisting of halogen, hydroxy, C 1-3 alkyl, —O—C 1-3 alkyl, C 1-3 hydroxyalkyl, C 1-3 haloalkyl, and —CO 2 H;
Z is selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyridyl, pyrimidinyl, guanidinyl, quinuclidine, and 8-azabicyclo[3.2.1]octane, each of which is optionally substituted with from 1 to 3 groups independently selected from halogen, hydroxy, C 1-3 alkyl, —NH 2 , —NHC 1-3 alkyl, —N(C 1-3 alkyl) 2 , —O—C 1-3 alkyl, C 1-3 hydroxyalkyl, C 1-3 haloalkyl, and —CO 2 H;
each R 2a , R 2b , and R 2c is independently selected from the group consisting of H, halogen, —CN, —R d , —CO 2 R e , —CONR e R f , —OC(O)NR e R f , —NR f C(O)R e , —NR f C(O) 2 R d , —NR e —C(O)NR e R f , —NR e R f , —OR e , —X 2 —OR e , —X 2 —NR e R f , —X 2 —CO 2 R e , —SF 5 , and —S(O) 2 NR e R f , wherein each X 2 is a C 1-4 alkylene; each R e and R f is independently selected from hydrogen, C 1-8 alkyl, and C 1-8 haloalkyl, or when attached to the same nitrogen atom can be combined with the nitrogen atom to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O, and S, and optionally substituted with oxo; and each R d is independently selected from the group consisting of C 1-8 alkyl, C 2-8 alkenyl, and C 1-8 haloalkyl;
each R 5 is independently selected from the group consisting of halogen, —CN, —R q , —CO 2 R r , —CONR r R s , —C(O)R r , —OC(O)NR r R s , —NR r C(O)R s , —NR r C(O) 2 R q , —NR r —C(O)NR r R s , —NR r R s , —OR r , —O—X 5 —OR r , —O—X 5 —NR r R s , —O—X 5 —CO 2 R r , —O—X 5 —CONR r R s , —X 5 —OR r , —X 5 —NR r R s , —X 5 —CO 2 R r , —X 5 —CONR r R s , —SF 5 , and —S(O) 2 NR r R s , wherein each X 5 is a C 1-4 alkylene; each R r and R s is independently selected from hydrogen, C 1-8 alkyl, and C 1-8 haloalkyl, or when attached to the same nitrogen atom can be combined with the nitrogen atom to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O and S, and optionally substituted with oxo; and each R q is independently selected from the group consisting of C 1-8 alkyl and C 1-8 haloalkyl;
R 6a and R 6c are each independently selected from the group consisting of H, C 1-4 alkyl, and C 1-4 haloalkyl;
each R 7a and R 7b is independently selected from the group consisting of H, C 1-6 alkyl, CO 2 H, CH 2 OH, —CO 2 —(C 1-6 alkyl), and PO 3 H 2 , wherein C 1-6 alkyl is optionally substituted with one or two members selected from halogen, OH, NH 2 , CN, and CO 2 H;
each R 8a and R 8b is independently selected from the group consisting of H and C 1-6 alkyl, optionally substituted with halogen, OH, NH 2 , CN, and CO 2 H;
R 9 is selected from the group consisting of H, halogen, CN, C 1-6 alkyl, —O—C 1-6 alkyl, —SO 2 (C 1-6 alkyl), —C 1-6 alkyl-CO 2 H, —C 1-6 alkyl-CO 2 —C 1-6 alkyl, —C 1-6 alkyl-C(O)NH 2 , —C 1-6 alkyl-C(O)NHC 1-6 alkyl, and —C 1-6 alkyl-C(O)N(C 1-6 alkyl) 2 ; and
n is 0, 1, 2, or 3.
14. A kit of claim 13 , wherein the compound has the formula
or a pharmaceutically acceptable salt thereof.
15. A kit of claim 13 , wherein the compound has the formula
or a pharmaceutically acceptable salt thereof.
16. A method of claim 13 , wherein the compound has the formula
or a pharmaceutically acceptable salt thereof.
17. A kit of claim 13 , wherein the compound has the formula
or a pharmaceutically acceptable salt thereof.
18. The method of claim 1 , wherein step d) is performed 3 times.
19. The method of claim 1 , further comprising step d-1) contacting the incubation product with a blocking solution, where step d-1) is performed after step d) but before step e).
20. The method of claim 19 , further comprising step d-2) washing the incubation product and blocking solution with a wash buffer, where step d-2) is performed after step d-1) but before step e).