LIM JONGWON (US)
CUMMING JARED (US)
TROTTER BENJAMIN WESLEY (US)
WU WEN LIAN (US)
LIM JONGWON (US)
CUMMING JARED N (US)
TROTTER BENJAMIN WESLEY (US)
| WO2016120305A1 | 2016-08-04 | |||
| WO2017027645A1 | 2017-02-16 |
| US20150056224A1 | 2015-02-26 |
| WHAT IS CLAIMED IS: 1. A compound of formula (I): (I) armaceutically acceptable salt thereof, wherein 1 and Base2 are each independently selected from the group consisting of , where Base1 and Base2 each may be independently substituted by 0-3 substituents R10, where each R10 is independently selected from the group consisting of F, CI, I, Br, OH, SH, H2, Ci-3 alkyl, C3-6 cycloalkyl, 0(Ci-3 alkyl), 0(C3-6 cycloalkyl), S(Ci-3 alkyl), S(C3-6 cycloalkyl), H(Ci-3 alkyl), H(C3-6 cycloalkyl), N(Ci-3 alkyl)2, and N(C3-6 cycloalkyl)2; Y and Ya are each independently selected from the group consisting of -0-, -S-, -S02-, -CH2-, and -CF2-; Xa and Xal are each independently selected from the group consisting of -0-, -S-, and -CH2 X and X l are each independently selected from the group consisting of -0-, -S-, and -CH2 Xc and Xcl are each independently selected from the group consisting of SR9, OR9, and R9R9; Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R1 and Rla Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R2 and R2a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R2 and R2a Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R3 is selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R3 Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R4 and R4a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R4 and R4a Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R5 is selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R5 Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R6 and R6a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R6 and R6a Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R7 and R7a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R7 and R7a Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R and R a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl, where said R8 and R8a Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, -0-Ci-C6 alkyl, -0-C2-C6 alkenyl, and -0-C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; each R9 is independently selected from the group consisting of H, Ci-C2o alkyl, , where each R9 Ci-C2o alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, -O-C1-C20 alkyl,-S-C(0)Ci-C6 alkyl, and C(0)OCi-C6 alkyl; optionally Rla and R3 are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where Rla and R3 are connected to form -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, said O is bound at the R3 position; optionally R2a and R3 are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R2a and R3 are connected to form -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, said O is bound at the R3 position; optionally R3 and R6a are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R3 and R6a are connected to form -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, said O is bound at the R3 position; optionally R4 and R5 are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R4 and R5 are connected to form -O- Ci-C6 alkylene, or -0-C2-C6 alkenylene, said O is bound at the R5 position; optionally R5 and R6 are connected to form -0-Ci-C6 alkylene, -0-C2-C6 alkenylene, or -0-C2-C6 alkynylene, such that where R5 and R6 are connected to form -0-Ci-C6 alkylene, -0-C2-C6 alkenylene, or -0-C2-C6 alkynylene, said O is bound at the R5 position; optionally R7 and R8 are connected to form Ci-C6 alkylene or C2-C6 alkenylene; and optionally R7a and R8a are connected to form Ci-C6 alkylene or C2-C6 alkenylene; and provided that at least one of x° and x°1 is r9 . 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein 1 and Base2 are each independently selected from the group consisting of may be independently substituted by 0-3 substituents R , where each R is independently selected from the group consisting of F, CI, I, Br, OH, SH, NH2, C1-3 alkyl, C3-6 cycloalkyl, 0(Ci-3 alkyl), 0(C3-6 cycloalkyl), S(Ci-3 alkyl), S(C3-6 cycloalkyl), NH(Ci-3 alkyl), NH(C3-6 cycloalkyl), N(C1-3 alkyl)2, and N(C3-6 cycloalkyl )2; Y and Ya are each independently selected from the group consisting of -O- and -S-; Xa and Xal are each independently selected from the group consisting of O and S; X and X l are each independently selected from the group consisting of O and S; Xc and Xcl are each independently selected from the group consisting of SR9, OR9, and NR9R9; Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 and R2a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, and Ci-C6 haloalkyl, where said R2 and R2a Ci-C6 alkyl or Ci-C6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N3; R3 is selected from the group consisting of H, F, CI, Br, I, OH, CN, N3, Ci-C6 alkyl, and Ci-C6 haloalkyl, where said R3 Ci-C6 alkyl or Ci-C6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N3; R4 and R4a are each independently selected from the group consisting of H, F, CI, I, Br, CN, OH, N3, Ci-C6 alkyl, and Ci-C6 haloalkyl, where said R4 and R4a Ci-C6 alkyl or Ci-C6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N3; R5 is selected from the group consisting of H, F, CI, Br, I, OH, N3, Ci-C6 alkyl, and Ci-C6 haloalkyl, where said R5 Ci-C6 alkyl or Ci-C6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R6 and R6a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N3, Ci-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, where said R6 and R6a Ci-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N3; R7 and R7a are each independently selected from the group consisting of H, Ci-C6 alkyl, and Ci-C6 haloalkyl, where said R7 and R7a Ci-C6 alkyl or Ci-C6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N3; R8 and R8a are each independently selected from the group consisting of H, Ci-C6 alkyl, and Ci-C6 haloalkyl, where said R8 and R8a Ci-C6 alkyl or Ci-C6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N3; each R9 is independently selected from the group consisting of H, Ci-C6 alkyl, , where each R9 Ci-C6 alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, -O-Ci-C20 alkyl,-S-C(0)Ci-C6 alkyl, and -C(0)OCi-C6 alkyl; optionally R3 and R6a are connected to form C2-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, said O is bound at the R3 position; and optionally R5 and R6 are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R5 and R6 are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, said O is bound at the R5 position. 3. The compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein Base1 and Base2 are each independently selected from the group consisting of Y and Ya are each independently selected from the group consisting of -O- and -S-; Xa and Xal are each independently selected from the group consisting of O and S; X and Xbl are each independently selected from the group consisting of O and S; c and Xcl are each inde endently selected from the group consisting of -OH, X and X are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 and R2a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N3, CF3, CH3, CH2OH, and CH2CH3; R3 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N3, CF3, CH3, CH2OH, and CH2CH3; R4 and R4a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N3, CF3, CH3, CH2OH, and CH2CH3; R5 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N3, CF3, CH3, CH2OH, and CH2CH3; R6 and R6a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N3, CF3, CH3, CH2OH, CH2CH3, -CH=CH2, -C≡CH, and -C≡C-CH; R7 and R7a are each independently selected from the group consisting of H, CF3, CH3, and CH2CH3; R8 and R8a are each independently selected from the group consisting of H, CF3, CH3, and CH2CH3; optionally R3 and R6a are connected to C2-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, said O is bound at the R3 position; and optionally R5 and R6 are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R5 and R6 are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, said O is bound at the R5 position. 4. The compound according to any one of claims 1 to 3, or a phramaceutically acceptable salt thereof, wherein 1 and Base2 are each independently selected from the group consisting of Y and Ya are each independently selected from the group consisting of -O- and -S-; Xa and Xai are each O; X and X l are each O; Xc and Xcl are each independently selected from the group consisting of -OH and -SH; Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 and R2a are each independently selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R3 is selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R4 and R4a are each independently selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R5 is selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R6 and R6a are each independently selected from the group consisting of H, F, CN, N3, CH3, -CH=CH2, and -C≡CH; R7 and R7a are each H; R8 and R8a are each H; optionally R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, such that O is bound at the R3 position; and optionally R5 and R6 are connected to form Ci-C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R5 and R6 are connected to form -0-Ci-C6 alkylene or -O-C2-C6 alkenylene, said O is bound at the R5 position. 5. The compound according to any one of claims 1 to 4, or a phramaceutically acceptable salt thereof, wherein 1 and Base2 are each independently selected from the group consisting of Y and Ya are each independently selected from the group consisting of -O- and -S-; Xa and Xal are each O; X and X l are each O; Xc and Xcl are each independently selected from the group consisting of -OH and -SH; Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 and R2a are each independently selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R3 is selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R4 and R4a are each independently selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R5 is selected from the group consisting of H, F, CI, OH, CN, N3, and CH3; R6 and R6a are each independently selected from the group consisting of H, F, CN, N3, CH3, -CH=CH2, and -C≡CH; R7 and R7a are each H; R8 and R8a are each H; optionally R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, such that where R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-Cs alkenylene, said O is bound at the R3 position; and optionally R5 and R6 are connected to form C C6 alkylene, C2-C6 alkenylene, -0-Ci-C6 alkylene, or -0-C2-C6 alkenylene, such that where R5 and R6 are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, said O is bound at the R5 position. 6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein Base1 and Base2 are each independently selected from the group consistin Y and Ya are each independently selected from the group consisting of -O- and -S-; Xa and Xai are each O; X and X l are each O; Xc and Xcl are each independently selected from the group consisting of SH and OH; Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 and R2a are each independently selected from the group consisting of H, F, and OH; R3 is selected from the group consisting of H, F, and OH; R4 and R4a are each independently selected from the group consisting of H, F, and OH; R5 is selected from the group consisting of H, F, and OH; R6 and R6a are each H; R7 and R7a are each H; R8 and R8a are each H; and optionally R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, such that where R3 and R6a are connected to form -0-Ci-C6 alkylene or -0-C2-C6 alkenylene, said O is bound at the R position. 7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Base1 and Base2 are each independently selected from the group consisting of Y and Ya are each independently selected from the group consisting of -O- and -S-; Xa and Xal are each O; X and Xbl are each O; Xc and Xcl are each independently selected from the group consisting of -SH and -OH; Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 is H; R2a is selected from the group consisting of H, F, and OH; RJ is selected from the group consisting of H, F, and OH; R4 is selected from the group consisting of H, F, and OH; R4a is H; R5 is selected from the group consisting of H, F, and OH; R6 and R6a are each H; R7 and R7a are each H; and R8 and R8a are each H. 8. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Base1 and Base2 are each independently selected from the group consisting of X and X l are each O; Xc and Xcl are each independently selected from the group consisting of -SH and Xd and Xdl are each independently selected from the group consisting of O and S; R1 and Rla are each H; R2 is H; R2a is selected from the group consisting of H, F, and OH; R3 is selected from the group consisting of H, F, and OH; R4 is selected from the group consisting of H, F, and OH; R4a is H; R5 is selected from the group consisting of H, F, and OH; R6 and R6a are each H; R7 and R7a are each H; and R8 and R8a are each H. 10. A pharmaceutical composition, said pharmaceutical composition comprising: (a) a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier. 11. A method of inducing an immune response in a subject, said method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 9 to the subject. 12. A method of inducing an immune response in a subject, said method comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 10 to the subject. 13. A method of inducing a STING-dependent type I interferon production in a subject, said method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 9 to the subject. 14. A method of inducing a STING-dependent type I interferon production in a subject, said method comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 10 to the subject. 15. A method of treating a cell proliferation disorder in a subject, said method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 9 to the subject. 16. The method of claim 15, wherein the cell proliferation disorder is cancer. 17. A method of treating a cell proliferation disorder in a subject, said method comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 10 to the subject. 18. The method of claim 17, wherein the cell proliferation disorder is cancer. |
CYCLIC DI-NUCLEOTIDE COMPOUNDS AS STING AGONISTS
FIELD OF THE INVENTION
The present disclosure relates to cyclic di-nucleotide compounds and derivatives thereof that may be useful as STF G (Stimulator of Interferon Genes) agonists that activate the STING pathway. The present disclosure also relates to processes for the synthesis and to uses of such cyclic di-nucleotide compounds. BACKGROUND OF THE INVENTION
The immune system has evolved to recognize and neutralize different types of threats in order to maintain the homeostasis of the host, and it is generally broken down into two arms: adaptive and innate. The adaptive immune system is specialized to recognize as foreign those antigens not naturally expressed in the host and to mount an anti-antigen response through the coordinated actions of many leukocyte subsets. The hallmark of adaptive immune responses is their ability to provide "memory" or long-lasting immunity against the encountered antigen. While this specific and long-lasting effect is critical to host health and survival, the adaptive immune response requires time to generate a full-blown response.
The innate immune system compensates for this time delay and is specialized to act quickly against different insults or danger signals. It provides the first line of defense against bacteria, viruses, parasites and other infectious threats, but it also responds strongly to certain danger signals associated with cellular or tissue damage. The innate immune system has no antigen specificity but does respond to a variety of effector mechanisms. Opsonization, phagocytosis, activation of the complement system, and production of soluble bioactive molecules such as cytokines or chemokines are all mechanisms by which the innate immune system mediates its response. By responding to these damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) described above, the innate immune system is able to provide broad protection against a wide range of threats to the host.
Free cytosolic DNA and RNA are among these PAMPs and DAMPs. It has recently been demonstrated that the main sensor for cytosolic DNA is cGAS (cyclic GMP-AMP synthase). Upon recognition of cytosolic DNA, cGAS catalyzes the generation of the cyclic- dinucleotide 2'3'-cGAMP, an atypical second messenger that strongly binds to the ER- transmembrane adaptor protein STING. A conformational change is undergone by cGAMP- bound STING, which translocates to a perinuclear compartment and induces the activation of critical transcription factors IRF-3 and NF-κΒ. This leads to a strong induction of type I interferons and production of pro-inflammatory cytokines such as IL-6, TNF-a and IFN-γ.
The importance of type I interferons and pro-inflammatory cytokines on various cells of the immune system has been very well established. In particular, these molecules strongly potentiate T-cell activation by enhancing the ability of dendritic cells and macrophages to uptake, process, present and cross-present antigens to T-cells. The T-cell stimulatory capacity of these antigen-presenting cells is augmented by the up-regulation of critical co-stimulatory molecules, such as CD80 or CD86. Finally, type I interferons can rapidly engage their cognate receptors and trigger the activation of interferon-responsive genes that can significantly contribute to adaptive immune cell activation.
From a therapeutic perspective, type I interferons are shown to have antiviral activities by directly inhibiting human hepatitis B virus and hepatitis C virus replication, and by stimulating immune responses to virally infected cells. Compounds that can induce type I interferon production are used in vaccines, where they act as adjuvants, enhancing specific immune responses to antigens and minimizing side effects by reducing dosage and broadening the immune response.
In addition, interferons, and compounds that can induce interferon production, have potential use in the treatment of human cancers. Such molecules are potentially useful as anti-cancer agents with multiple pathways of activity. Interferons can inhibit human tumor cell proliferation directly and may be synergistic with various approved chemotherapeutic agents. Type I interferons can significantly enhance anti-tumor immune responses by inducing activation of both the adaptive and innate immune cells. Finally, tumor invasiveness may be inhibited by interferons by modulating enzyme expression related to tissue remodeling.
In view of the potential of type I interferons and type I interferon-inducing compounds as anti-viral and anti-cancer agents, there remains a need for new agents that can induce potent type I interferon production. With the growing body of data demonstrating that the cGAS-STING cytosolic DNA sensory pathway has a significant capacity to induce type I interferons, the development of STING activating agents is rapidly taking an important place in today's anti-tumor therapy landscape. SUMMARY OF THE INVENTION
The present disclosure relates to novel compounds of general formula (I). In particular, the present disclosure relates to compounds having the general structural formula (I):
(I)
or pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, as described herein. Embodiments of the disclosure include compounds of general formula (I), and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, as well as synthesis and isolation of compounds of general formula (I), and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. The compounds of general formula (I), and their pharmaceutically acceptable salts, hydrates, solvates, and/or prodrugs, may be useful as agents to induce immune responses, to induce STING-dependent type I interferon production, and/or to treat a cell proliferation disorders, such as cancers, in a subject. The compounds of general formula (I) could further be used in combination with other therapeutically effective agents, including but not limited to, other drugs useful for the treatment of cell proliferation disorders, such as cancers. The invention further relates to processes for preparing compounds of general formula (I), and pharmaceutical compositions that comprise compounds of general formula (I) and
pharmaceutically acceptable salts thereof.
Other embodiments, aspects and features of the present disclosure are either further described in or will be apparent from the ensuing description, examples and appended claims.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure includes compounds of general formula (I), and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. These compounds and their pharmaceutically acceptable salts, hydrates, solvates, and/or prodrugs may be useful as agents to induce immune responses, to induce STING-dependent type I interferon production, and/or to treat a cell proliferation disorder. Embodiments disclosed herein relate to compounds of general formula (I): (I)
or a pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, wherein Base 1 and
Base 2 are each independently selected from the group consisting of ?
R , where each R is independently selected from the group consisting of F, CI, I, Br, OH, SH, H 2 , Ci-3 alkyl, C 3-6 cycloalkyl, 0(Ci -3 alkyl), 0(C 3-6 cycloalkyl), S(Ci -3 alkyl), S(C 3-6 cycloalkyl), H(Ci -3 alkyl), H(C 3-6 cycloalkyl), N(Ci -3 alkyl) 2 , and N(C 3-6 cycloalkyl) 2 ; Y and Y a are each independently selected from the group consisting of -0-, -S-, -S0 2 -, -CH 2 -, and -CF 2 -; X a and X al are each independently selected from the group consisting of -0-, -S-, and -CH 2 -; X and X l are each independently selected from the group consisting of -0-, -S-, and -CH 2 -; X c and X cl are each independently selected from the group consisting of SR 9 , OR 9 , and R 9 R 9 ; X d and X dl are each independently selected from the group consisting of O and S; R 1 and R la are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 1 and R la Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 2 and R 2a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 2 and R 2a Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 3 is selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and
-0-C 2 -C 6 alkynyl, where said R 3 Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 4 and R 4a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 4 and R 4a Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 5 is selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 5 Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and
-0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 6 and R 6a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 6 and R 6a Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 7 and R 7a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 7 and R 7a Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 8 and R 8a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl, where said R 8 and R 8a Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Ci-C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl, -0-Ci-C 6 alkyl, -0-C 2 -C 6 alkenyl, and -0-C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, 9 is independently selected from the group consisting of H,
Ci-C 20 alkyl, s w here each R 9 Ci-C 2 o alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, -O-C 1 -C 2 0 alkyl,-S-C(0)Ci-C 6 alkyl, and C(0)OCi-C 6 alkyl; optionally R la and R 3 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R la and R 3 are connected to form -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position; optionally R 2a and R 3 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 2a and R 3 are connected to form -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position; optionally R 3 and R 6a are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position; optionally R 4 and R 5 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 4 and R 5 are connected to form -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position; optionally R 5 and R 6 are connected to form -0-Ci-C 6 alkylene, -0-C 2 -C 6 alkenylene, or -0-C 2 -C 6 alkynylene, such that where R 5 and R 6 are connected to form -0-Ci-C 6 alkylene, -0-C 2 -C 6 alkenylene, or -0-C 2 -C 6 alkynylene, said O is bound at the R 5 position; optionally R 7 and R 8 are connected to form Ci-C 6 alkylene or C 2 -C 6 alkenylene; and optionally R 7a and 8a are conne to form Ci-C 6 alkylene or C 2 -C 6 alkenylene; and provided
that at least one of and is . In embodiments, the disclosure relates to compounds of general structural formula (I), or pharmaceutically acceptable salts, hydrates, or solvates thereof. In specific embodiments, the disclosure relates to compounds of general structural formula (I), or pharmaceutically acceptable salts thereof. In a first embodiment, Base 1 and Base 2 are each independently selected from the
group consisting of
where
Base 1 and Base 2 each may be independently substituted by 0-3 substituents R 10 , where each R 10 is independently selected from the group consisting of F, CI, I, Br, OH, SH, H 2 , C 1-3 alkyl, C3 cycloalkyl, (d -3 alkyl), (C 3-6 cycloalkyl), S(Ci -3 alkyl), S(C 3-6 cycloalkyl), H(Ci -3 alkyl), H(C 3-6 cycloalkyl), N(C 1-3 alkyl) 2 , and N(C 3-6 cycloalkyl) 2 . In particular aspects, Base 1 and
Base 2 are each independently selected from the group consisting of , where Base 1 and Base 2 each may be independently substituted by 0-3 substituents R , where each R is independently selected from the group consisting of F, CI, I, Br, OH, SH, H 2 , C 1-3 alkyl, C 3-6 cycloalkyl, (Ci alkyl), (C 3-6 cycloalkyl), S(Ci -3 alkyl), S(C 3-6 cycloalkyl), H(Ci -3 alkyl), H(C 3-6 cycloalkyl), N(C 1-3 alkyl) 2 , and N(C 3-6 cycloalkyl) 2 . In all aspects of this embodiment, all other groups are as provided in the general formula (I) above.
In a second embodiment, Y and Y a are each independently selected from the group consisting of -O- and -S-. In aspects of this embodiment, Y is and Y a is In additional aspects of this embodiment, Y is and Y a is -S-. In further aspects of this embodiment, Y is -S-, and Y a is In still further aspects of this embodiment, Y is -S-, and Y a is -S-. In this embodiment, all other groups are as provided in the general formula (I) above or in the first embodiment described above.
In a third embodiment, X a and X a are each independently selected from the group consisting of -S-, and -CH 2 -. In aspects of this embodiment, X a and X al are each independendently selected from the group consisting of -O- and -S-. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through second embodiments described above.
In a fourth embodiment, X and X l are each independently selected from the group consisting of -0-, -S-, and -CH 2 -. In aspects of this embodiment, X and X l are each independendently selected from the group consisting of -O- and -S-. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through third embodiments described above.
In a fifth embodiment, X c and X cl are each independently selected from the group consisting of -OR 9 , -SR 9 , and - R 9 R 9 , where each R 9 is independently selected from the group
consisting of H, Ci-C 2 o alkyl, and where each
R Ci-C 2 o alkyl is optionally substituted by 0 to 3 substituents independently selected from the group consisting of OH, -O-Ci-C 20 alkyl,-S-C(0)Ci-C 6 alkyl, and C(0)OCi-C 6 alkyl. In
of -OH,
, and In more particular aspects, X c and X cl are each independently selected from the group consisting of -OH and -SH. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourth embodiment described above.
In a sixth embodiment, X d and X dl are each independently selected from the group consisting of O and S. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fifth embodiments described above.
In a seventh embodiment, R 1 and R la are each H. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through sixth embodiments described above.
ghth embodiment, R 2 and R 2a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 2 and R 2a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 . In particular aspects, R 2 and R 2a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and
CH 2 CH 3 . In more particular aspects, R 2 and R 2a are each independently selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 . In even more particular aspects, R 2 and R 2a are each independently selected from the group consisting of H, F, and OH. In even still more particular aspects, R 2 is H. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through seventh embodiments described above.
In a ninth embodiment, R 3 is selected from the group consisting H, F, CI, I, Br,
OH, CN, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 3 Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 . In particular aspects, R 3 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 . In more particular aspects, R 3 is selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 . In even more particular aspects, R 3 is selected from the group consisting of H, F, and OH. In even still more particular aspects, R 3 is H. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through eighth embodiments described above.
In a tenth embodiment, R 4 and R 4a are each independently selected from the group consisting of H, F, CI, I, Br, CN, OH, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 4 and R a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 . In particular aspects, R 4 and R 4a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 . In more particular aspects, R 4 and R 4a are each independently selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 . In even more particular aspects, R 4 and R 4a are each
independently selected from the group consisting of H, F, and OH. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through ninth embodiments described above.
In an eleventh embodiment, R 5 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 5 Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 . In particular aspects, R 5 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 . In more particular aspects, R 5 is selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 . In even more particular aspects, R 5 is selected from the group consisting of H, F, and OH. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through tenth embodiments described above.
In a twelfth embodiment, R 6 and R 6a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl, where said R 6 and R 6a Ci-C 6 alkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 . In particular aspects, R 6 and R 6a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, CH 2 CH 3 , -CH=CH 2 , -C≡CH, and -C≡C-CH. In more particular aspects, R 6 and R 6a are each independently selected from the group consisting of H, F, CN, N 3 , CH 3 , -CH=CH 2 , and -C≡CH. In even more particular aspects, R 6 and R 6a are each H. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through eleventh embodiments described above.
In a thirteenth embodiment, R 7 and R 7a are each independently selected from the group consisting of H, Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 7 and R 7a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 . In particular aspects, R 7 and R 7a are each independently selected from the group consisting of H, CF 3 , CH 3 , and CH 2 CH 3 . In more particular aspects, R 7 and R 7a are each H. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through twelfth embodiments described above.
In a fourteenth embodiment, R 8 and R 8a are each independently selected from the group consisting of H, Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 8 and R 8a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH,
CN, and N 3 . In particular aspects, R 8 and R 8a are each independently selected from the group consisting of H, CF 3 , CH 3 , and CH 2 CH 3 . In more particular aspects, R 8 and R 8a are each H. In all aspects of this embodiment, all other groups are as provided in the general formula (I) above or in the first through thirteenth embodiments described above.
In a fifteenth embodiment, R la and R 3 are connected to form Ci-C 6 alkyl ene,
C 2 -C 6 alkenylene, -0-Ci-C 6 alkyl ene, or -0-C 2 -C 6 alkenylene, such that where R la and R 3 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position.
In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a sixteenth embodiment, R 2a and R 3 are connected to form Ci-C 6 alkylene,
C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 2a and R 3 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position.
In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a seventeenth embodiment, R 3 and R 6a are connected to form C 2 -C 6 alkylene,
C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene,, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position.
In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In an eighteenth embodiment, R 4 and R 5 are connected to form Ci-C 6 alkylene,
C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 4 and R 5 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position.
In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a nineteenth embodiment, R 5 and R 6 are connected to form Ci-C 6 alkylene,
C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 5 and R 6 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twentieth embodiment, R and R are connected to form Ci-C 6 alkyl ene or
C2-C6 alkenylene. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty-first embodiment, R a and R a are connected to form Ci-C 6 alkylene or C2-C6 alkenylene. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty-second embodiment, Base 1 and Base 2 are each independently selected
where Base 1 and Base 2 each may be independently substituted by 0-3 substituents R 10 , where each R 10 is independently selected from the group consisting of F, CI, I, Br, OH, SH, NH 2 , Ci -3 alkyl, C 3-6 cycloalkyl, 0(Ci -3 alkyl), 0(C 3-6 cycloalkyl), S(Ci -3 alkyl), S(C 3-6 cycloalkyl), NH(Ci -3 alkyl), NH(C 3-6 cycloalkyl), N(Ci -3 alkyl) 2 , and N(C 3-6 cycloalkyl) 2 ; Y and Y a are each independently selected from the group consisting of -O- and -S-; X a and X al are each
independently selected from the group consisting of O and S; X and X l are each independently selected from the group consisting of O and S; X c and X cl are each independently selected from the group consisting of SR 9 , OR 9 , and NR 9 R 9 ; X 4 and X dl are each independently selected from the group consisting of O and S; R 1 and R la are each H; R 2 and R 2a are each independently selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 2 and R 2a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 ; R 3 is selected from the group consisting of H, F, CI, Br, I, OH, CN, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 3 Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 ; R 4 and R 4a are each independently selected from the group consisting of H, F, CI, I, Br, CN, OH, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 4 and R 4a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 .; R 5 is selected from the group consisting of H, F, CI, Br, I, OH, N 3 , Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 5 Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 6 and R 6a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl, where said R 6 and R 6a Ci-C 6 alkyl, C 2 -C 6 alkenyl, and C 2 -C 6 alkynyl are substituted by 0 to 3 substituents selected from the group consisting of F, CI, Br, I, OH, CN, and N 3 ; R 7 and R 7a are each independently selected from the group consisting of H, Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 7 and R 7a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 ; R 8 and R 8a are each independently selected from the group consisting of H, Ci-C 6 alkyl, and Ci-C 6 haloalkyl, where said R 8 and R 8a Ci-C 6 alkyl or Ci-C 6 haloalkyl are substituted by 0 to 3 substituents selected from the group consisting of OH, CN, and N 3 ; each R 9 is independently selected from the group consisting of H, Ci-C 6 alkyl, f ° ° ° , and
R 9 Ci-C 6 alkyl is optionally substituted by 1 to 2 substituents independently selected from the group consisting of OH, -O-Ci-C 20 alkyl, -S-C(0)Ci-C 6 alkyl, and -C(0)OCi-C 6 alkyl; optionally R 3 and R 6a are connected to form C 2 -C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position; and optionally R 5 and R 6 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 5 and R 6 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty -third embodiment, Base 1 and Base 2 are each inde endentl selected
from the group consisting of
and Y a are each independently selected from the group consisting of -O- and -S-; X a and X a are each independently selected from the group consisting of O and S; X and X l are each inde endentl selected from the rou consistin of O and S; X c and X cl are each independently
independently selected from the group consisting of O and S; R 1 and R la are each H; R 2 and R 2a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 ; R 3 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 ; R 4 and R 4a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 ; R 5 is selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, and CH 2 CH 3 ; R 6 and R 6a are each independently selected from the group consisting of H, F, CI, I, Br, OH, CN, N 3 , CF 3 , CH 3 , CH 2 OH, CH 2 CH 3 , -CH=CH 2 , -C≡CH, and -C≡C-CH; R and R are each independently selected from the group consisting of H, CF 3 , CH 3 , and CH 2 CH 3 ; R 8 and R 8a are each
independently selected from the group consisting of H, CF 3 , CH 3 , and CH 2 CH 3 ; optionally R 3 and R 6a are connected to C 2 -C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position; and optionally R 5 and R 6 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 5 and R 6 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty -fourth embodiment, Base 1 and Base 2 are each independently selected
from the group consisting of
and Y a are each independently selected from the group consisting of -O- and -S-; X a and X a are each O; X and X l are each O; X c and X c are each independently selected from the group consisting of -OH, and -SH; X d and X dl are each independently selected from the group consisting of O and S; R 1 and R la are each H; R 2 and R 2a are each independently selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 3 is selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 4 and R 4a are each independently selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 5 is selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 6 and R 6a are each independently selected from the group consisting of H, F, CN, N 3 , CH 3 , -CH=CH 2 , and -C≡CH; R 7 and R 7a are each H; R 8 and R 8a are each H; optionally R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, such that O is bound at the R 3 position; and optionally R 5 and R 6 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 5 and R 6 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above. In a twenty-fifth embodiment, Base 1 and Base 2 are each independently selected
f
and Y a are each independently selected from the group consisting of -O- and -S-; X a and X ai are each O; X and X l are each O; X c and X c are each independently selected from the group consisting of -OH and -SH; X d and X dl are each independently selected from the group consisting of O and S; R 1 and R la are each H; R 2 and R 2a are each independently selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 3 is selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 4 and R 4a are each independently selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 5 is selected from the group consisting of H, F, CI, OH, CN, N 3 , and CH 3 ; R 6 and R 6a are each independently selected from the group consisting of H, F, CN, N 3 , CH 3 , -CH=CH 2 , and -C≡CH; R 7 and R 7a are each H; R 8 and R 8a are each H; optionally R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position; and optionally R 5 and R 6 are connected to form Ci-C 6 alkylene, C 2 -C 6 alkenylene, -0-Ci-C 6 alkylene, or -0-C 2 -C 6 alkenylene, such that where R 5 and R 6 are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 5 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty-sixth embodiment, Base 1 and Base 2 are each independently selected
and Y a are each independently selected from the group consisting of -O- and -S-; X a and X a are each O; X and X l are each O; X c and X c are each independently selected from the group consisting of SH and OH; X d and X dl are each independently selected from the group consisting of O and S; R 1 and R la are each H; R 2 and R 2a are each independently selected from the group consisting of H, F, and OH; R 3 is selected from the group consisting of H, F, and OH; R 4 and R 4: are each independently selected from the group consisting of H, F, and OH; R 5 is selected from the group consisting of H, F, and OH; R 6 and R 6a are each H; R 7 and R 7a are each H; R 8 and R 8a are each H; and optionally R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -O-C 2 -C 6 alkenylene, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -O-C 2 -C 6 alkenylene, said O is bound at the R 3 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty-seventh embodiment, Base 1 and Base 2 are each independently
selected from the rou consisting
; Y and Y a are each independently selected from the group consisting of -O- and -S-; X a and X al are each independently O; X and X l are each
independently O; X c and X cl are each independently selected from the group consisting of -SH and -OH; X d and X dl are each independently selected from the group consisting of O and S; R and R la are each H; R 2 is H; R 2a is selected from the group consisting of H, F, and OH; R 3 is selected from the group consisting of H, F, and OH; R 4 is selected from the group consisting of H, F, and OH; R 4a is H; R 5 is selected from the group consisting of H, F, and OH; R 6 and R 6a are each H; R 1 and R Is. are each H; and R 8 and R 83. are each H. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
In a twenty-eighth embodiment, Base 1 and Base 2 are each independently selected
from the group consisting of ; Y and Y a are each independently selected from the group consisting of -O- and -S-; X a and X al are each independently O; X and X l are each independently O; X c and X cl are each independently selected from the group consisting of -SH and -OH; X d and X dl are each independently selected from the group consisting of O and S; R 1 and R la are each H; R 2 is H; R 2a is selected from the group consisting of H, F, and OH; R 3 is selected from the group consisting of H, F, and OH; R 4 is selected from the group consisting of H, F, and OH; R 4a is H; R 5 is selected from the group consisting of H, F, and OH; R 6 and R 6a are each H; R 7 and R 7a are each H; and R 8 and R 8a are each H; and R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, such that where R 3 and R 6a are connected to form -0-Ci-C 6 alkylene or -0-C 2 -C 6 alkenylene, said O is bound at the R 3 position. In this embodiment, all other groups are as provided in the general formula (I) above or in the first through fourteenth embodiments described above.
A twenty-ninth embodiment relates to a pharmaceutical composition, said pharmaceutical composition comprising (a) a compound according to any one of general formula (I) above or the first through twenty-eighth embodiments described above or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier.
A thirtieth embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I) above or the first through twenty-eighth embodiments described above or a pharmaceutically acceptable salt thereof to the subject.
A thirty-first embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-ninth embodiment described above to the subject.
A thirty-second embodiment relates to methods of inducing a STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I) above or the first through twenty-eighth embodiments described above or a pharmaceutically acceptable salt thereof to the subject.
A thrity-third embodiment relates to methods of inducing STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-ninth embodiment described above to the subject. A thirty-fourth embodiment relates to methods of inducing STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a compound according to any one of general formula (I) above or the first through twenty-eighth embodiments described above or a pharmaceutically acceptable salt thereof to the subject.
A thirty-fifth embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a composition according to the twenty-ninth embodiment described above to the subject.
In each embodiment described herein, variables Base Base 2 , Y, Y a , X a , X al , X , X l , X c , X cl , X d , X dl , R 1 , R la , R 2 , R 2a , R 3 , R 4 , R 4a , R 5 , R 6 , R 6a , R 7 , R 7a , R 8 , R 8a , and R 9 of general formula (I), and the various aspects thereof, are each selected independently from each other.
A thirty-sixth embodiment relates to a compound selected from the group
consisting of
, and ? anc j pharmaceutically acceptable salts, hydrates, solvates, and prodrugs thereof. In aspects, the disclosure relates to the compounds set forth above, or pharmaceutically acceptable salts, hydrates, or solvates thereof. In specific embodiments, the disclosure relates to the compounds set forth above or pharmaceutically acceptable salts thereof.
A thirty-seventh embodiment relates to a pharmaceutical composition, said pharmaceutical composition comprising (a) a compound according to the thirty-sixth embodiment above or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier. A thirty-eight embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a compound according to the thirty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the subject.
A thirty-ninth embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition according to the thirty-seventh embodiment above to the subject.
A fortieth embodiment relates to methods of inducing STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a compound according to the thirty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the subj ect.
A forty-first embodiment relates to methods of inducing STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a composition according to the thirty-seventh embodiment above to the subject.
A forty-second embodiment relates to methods of inducing STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a compound according to the thirty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the subject.
A forty-third embodiment relates to methods of inducing a STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a composition according to the thirty-seventh embodiment above to the subject.
A forty-fourth embodiment relates to methods of treating a cell proliferation disorder in a patient in need of therapy, comprising administering a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt thereof to the patient. In aspects of this embodiment, the cell proliferation disorder is cancer.
A forty-fifth embodiment relates to methods of treating a cell proliferation disorder in a patient in need of therapy, said method comprising administering a therapeutically effective amount of a composition according to the thirty-seventh embodiment described above to the patient. In aspects of this embodiment, the cell proliferation disorder is cancer.
A forty-sixth embodiment relates to a compound selected from the exemplary species depicted in Examples 1 through 27 shown below.
A forty-seventh embodiment relates to a a pharmaceutical composition, said pharmaceutical composition comprising (a) a compound according to the forty-sixth embodiment above or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier.
A forty-eighth embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a compound according to the forty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the subject.
A forty-ninth embodiment relates to methods of inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition according to the forty-seventh embodiment above to the subject.
A fiftieth embodiment relates to methods of inducing STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a compound according to the forty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the subject.
A fifty-first embodiment relates to methods of inducing STING-dependent type I interferon production in a subject, comprising administering a therapeutically effective amount of a composition according to the forty-seventh embodiment above to the subject.
A fifty-second embodiment relates to methods of inducing STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a compound according to the forty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the subject.
A fifty-third embodiment relates to methods of inducing STING-dependent cytokine production in a subject, comprising administering a therapeutically effective amount of a composition according to the forty-seventh embodiment above to the subject.
A fifty-fourth embodiment relates to methods of treating a cell proliferation disorder in a patient in need of therapy, comprising administering a therapeutically effective amount of according to the forty-sixth embodiment above or a pharmaceutically acceptable salt thereof to the patient. In aspects of this embodiment, the cell proliferation disorder is cancer.
A fifty-fifth embodiment relates to methods of treating a cell proliferation disorder in a patient in need of therapy, said method comprising administering a therapeutically effective amount of a composition according to the fifty-seventh embodiment described above to the patient. In aspects of this embodiment, the cell proliferation disorder is cancer.
Other embodiments of the present disclosure include the following: (a) A pharmaceutical composition comprising an effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
(b) The pharmaceutical composition of (a), further comprising an active agent selected from the group consisting of STING agonist compounds, anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti -cancer and chemotherapeutic agents.
(c) A pharmaceutical combination that is (i) a compound of general formula (I), or a pharmaceutically acceptable salt thereof, and (ii) an active agent selected from the group consisting of STING agonist compounds, anti -viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer and chemotherapeutic agents; wherein the compound of general formula (I), or pharmaceutically acceptable salt thereof, and the active agent are each employed in an amount that renders the combination effective for inducing an immune response in a patient.
(d) A method of inducing an immune response in a patient, which comprises administering to the subject a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt thereof.
(e) A method of inducing an immune response in a patient, which comprises administering to the subject a therapeutically effective amount of a composition of (a), a composition of (b), or a combination of (c).
(f) A method of inducing STING-dependent type I interferon production in a patient, which comprises administering to the subject a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt thereof.
(g) A method of inducing STING-dependent type I interferon production in a patient, which comprises administering to the subject a therapeutically effective amount of a composition of (a), a composition of (b), or a combination of (c).
(h) A method of inducing STING-dependent cytokine production in a patient, which comprises administering to the subject a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt thereof.
(i) A method of inducing STING-dependent cytokine production in a patient, which comprises administering to the subject a therapeutically effective amount of a composition of (a), a composition of (b), or a combination of (c). (j) A method of treating a cell proliferation disorder in a subject, said method comprising administering a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt thereof to the subject;
(k) The method of (j), wherein the cell proliferation disorder is cancer.
(1) A method of treating a cell proliferation disorder in a subject, said method comprising administering a therapeutically effective amount of a composition of (a), a composition of (b) ,or a combination of (c) to the subject.
(m) The method of (1), wherein the cell proliferation disorder is cancer.
The present disclosure also includes a compound of the present disclosure for use (i) in, (ii) as a medicament for, or (iii) in the preparation of a medicament for: (a) inducing an immune response in a patient, or (b) inducing STING-dependent cytokine production in a patient. In these uses, the compounds of the present disclosure can optionally be employed in combination with one or more active agents selected from STING agonist compounds, anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other
immunomodulatory agents, lipids, liposomes, peptides, anti -cancer agents, and chemotherapeutic agents.
Additional embodiments of the disclosure include the pharmaceutical
compositions, combinations and methods set forth in (a) through (m) above and the uses set forth in the preceding paragraph, wherein the compound of the present disclosure employed therein is a compound of one of the embodiments, aspects, instances, occurrences, or features of the compounds described above. In all of these embodiments, the compound may optionally be used in the form of a pharmaceutically acceptable salt, as appropriate.
In the embodiments of the compound provided above, it is to be understood that each embodiment may be combined with one or more other embodiments, to the extent that such a combination provides a stable compound and is consistent with the description of the embodiments. It is further to be understood that the embodiments of compositions and methods provided as (a) through (m) above are understood to include all embodiments of the compounds, including such embodiments as result from combinations of embodiments.
The term "subject" (alternatively "patient") as used herein refers to a mammal that has been the object of treatment, observation, or experiment. The mammal may be male or female. The mammal may be one or more selected from the group consisting of humans, bovine (e.g., cows), porcine (e.g., pigs), ovine (e.g., sheep), capra (e.g., goats), equine (e.g., horses), canine (e.g., domestic dogs), feline (e.g., house cats), Lagomorpha (rabbits), rodents (e.g., rats or mice), Procyon lotor (e.g., raccoons). In particular embodiments, the subject is human.
As used herein, the term "immune response" relates to any one or more of the following: specific immune response, non-specific immune response, both specific and non- specific response, innate response, primary immune response, adaptive immunity, secondary immune response, memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression. In certain embodiments, a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, is administered in conjunction with one or more additional therapeutic agents including anti-viral compounds, vaccines intended to stimulate an immune response to one or more predetermined antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer agents, and chemotherapeutic agents, etc. In certain
embodiments, a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, is administered in conjunction with one or more additional compositions including anti -viral compounds, vaccines intended to stimulate an immune response to one or more predetermined antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other
immunomodulatory agents, lipids, liposomes, peptides, anti -cancer agents, and chemotherapeutic agents, etc. Compounds
The term "alkyl" refers to a monovalent straight or branched chain, saturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range. Thus, for example, "Ci -6 alkyl" (or "Ci-C 6 alkyl") refers to any of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- and tert-butyl, n- and iso-propyl, ethyl, and methyl. As another example, "Ci-4 alkyl" refers to n-, iso-, sec- and tert-butyl, n- and isopropyl, ethyl, and methyl.
As used herein, the term "alkylene" refers to a bivalent straight chain, saturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range.
As used herein, the term "alkenyl" refers to a monovalent straight or branched chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more double bond.
As used herein, the term "alkenylene" refers to a bivalent straight chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more double bond. As used herein, the term "alkynyl" refers to a monovalent straight or branched chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more triple bond.
As used herein, the term "alkynylene" refers to a bivalent straight chain, unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range and including one or more triple bond.
The term "halogen" (or "halo") refers to fluorine, chlorine, bromine, and iodine (alternatively referred to as fluoro, chloro, bromo, and iodo or F, CI, Br, and I).
The term "haloalkyl" refers to an alkyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen. Thus, for example, "Ci -6 haloalkyl" (or "Ci-C 6 haloalkyl") refers to a Ci to C 6 linear or branched alkyl group as defined above with one or more halogen substituents. The term "fluoroalkyl" has an analogous meaning except the halogen substituents are restricted to fluoro. Suitable fluoroalkyls include the series (CH 2 )o- 4 CF 3 (i.e., trifluoromethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoro-n-propyl, etc.).
As used herein, the term "haloalkenyl" refers to an alkenyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen.
As used herein, the term "haloalkynyl" refers to an alkynyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen.
As used herein, the term "alkoxy" as used herein, alone or in combination, includes an alkyl group connected to the oxy connecting atom. The term "alkoxy" also includes alkyl ether groups, where the term "alkyl" is defined above, and "ether" means two alkyl groups with an oxygen atom between them. Examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, methoxymethane (also referred to as "dimethyl ether"), and methoxyethane (also referred to as "ethyl methyl ether").
As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon containing one ring having a specified number of carbon atoms. Examples of cycloalkyl include
cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
As used herein, the term "heterocycle", "heterocyclyl", or "heterocyclic", as used herein, represents a stable 3- to 6-membered monocyclic that is either saturated or unsaturated, and that consists of carbon atoms and from one to two heteroatoms selected from the group consisting of N, O, and S. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. The term includes heteroaryl moieties. Examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, 1,3-dioxolanyl, furyl,
imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl, thiamo holinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, triazolyl and thienyl.
As used herein, the term "spirocycle" or "spirocyclic ring" refers to a pendant cyclic group formed by substituents on a single atom.
The term "compound" refers to the compound and, in certain embodiments, to the extent they are stable, any hydrate or solvate thereof. A hydrate is the compound complexed with water, and a solvate is the compound complexed with a solvent, which may be an organic solvent or an inorganic solvent.
A "stable" compound is a compound that can be prepared and isolated and whose structure and properties remain or can be caused to remain essentially unchanged for a period of time sufficient to allow use of the compound for the purposes described herein (e.g., therapeutic administration to a subject). The compounds of the present invention are limited to stable compounds embraced by general formula (I), or pharmaceutically acceptable salts thereof.
Unless expressly stated to the contrary, all ranges cited herein are inclusive; i.e., the range includes the values for the upper and lower limits of the range as well as all values in between. As an example, temperature ranges, percentages, ranges of equivalents, and the like described herein include the upper and lower limits of the range and any value in the continuum there between. Numerical values provided herein, and the use of the term "about", may include variations of ± 1%, ± 2%, ±3%, ± 4%, ± 5%, ± 10%, ± 15%, and ± 20% and their numerical equivalents.
As used herein, the term "one or more" item includes a single item selected from the list as well as mixtures of two or more items selected from the list.
In the compounds of general formula (I), and pharmaceutically acceptable salts of the foregoing, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure is meant to include all suitable isotopic variations of the compounds of general formula (I), and pharmaceutically acceptable salts of the foregoing. For example, different isotopic forms of hydrogen (H) include protium (1H), deuterium ( 2 H), and tritium ( 3 H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for
characterization of biological samples. Isotopically-enriched compounds within general formula (I), and the pharmaceutically acceptable salts of the foregoing, can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
In particular embodiments of the compounds of general formula (I), and/or pharmaceutically acceptable salts of the foregoing, the compounds are isotopically enriched with deuterium. In aspects of these embodiments, one or more of Base 1 , Base 2 , Y, Y a , X a , X al , X , X l , X c , X cl , X d , X dl , R 1 , R la , R 2 , R 2a , R 3 , R 4 , R 4a , R 5 , R 6 , R 6a , R 7 , R 7a , R 8 , R 8a , and R 9 may include deuterium.
As shown in the general structural formulas and the structures of specific compounds as provided herein, a straight line at a chiral center includes both (R) and (S) stereoisomers and mixtures thereof. Also, unless otherwise specified {e.g., 100% purified compound), reference to a particular stereochemistry at a position provides a compound having the indicated stereochemistry but does not exclude the presence of stereoisomers having different stereochemistry at the indicated position.
Recitation or depiction of a specific compound in the claims {i.e., a species) without a specific stereoconfiguration designation, or with such a designation for less than all chiral centers, is intended to encompass, for such undesignated chiral centers, the racemate, racemic mixtures, each individual enantiomer, a diastereoisomeric mixture and each individual diastereomer of the compound where such forms are possible due to the presence of one or more asymmetric centers.
The invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios. Thus, enantiomers are a subject of the invention in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios. In the case of a cis/trans isomerism, the invention includes both the cis form and the trans form, as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally a derivatization can be carried out before a separation of stereoisomers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, or it can be done on a final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates that are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Unless a particular isomer, salt, solvate (including hydrates) or solvated salt of such racemate, enantiomer, or diastereomer is indicated, the present invention includes all such isomers, as well as salts, solvates (including hydrates), and solvated salts of such racemates, enantiomers, diastereomers, and mixtures thereof.
Those skilled in the art will recognize that chiral compounds, and in particular sugars, can be drawn in a number of different ways that are equivalent. Those skilled in the art will further recognize that the identity and regiochemical position of the substituents on ribose can vary widely and that the same prinicples of steroechemical equivalence apply regardless of substituent. -limiting examples of such equivalence include those exemplified below.
Salts
As indicated above, the compounds of the present invention can be employed in the form of pharmaceutically acceptable salts. Those skilled in the art will recognize those instances in which the compounds of the invention may form salts. Examples of such compounds are described herein by reference to possible salts. Such reference is for illustration only. Pharmaceutically acceptable salts can be used with compounds for treating patients. Non- pharmaceutical salts may, however, be useful in the preparation of intermediate compounds.
The term "pharmaceutically acceptable salt" refers to a salt (including an inner salt such as a zwitterion) that possesses effectiveness similar to the parent compound and that is not biologically or otherwise undesirable (e.g., is neither toxic nor otherwise deleterious to the recipient thereof). Thus, an embodiment of the invention provides pharmaceutically acceptable salts of the compounds of the invention. The term "salt(s)", as employed herein, denotes any of the following: acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. Salts of compounds of the invention may be formed by methods known to those of ordinary skill in the art, for example, by reacting a compound of the invention with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in aqueous medium followed by lyophilization.
Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates ("mesylates"), naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates) and the like. Suitable salts include acid addition salts that may, for example, be formed by mixing a solution of a compound with a solution of a pharmaceutically acceptable acid such as
hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. Additionally, acids that are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.), Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley- VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould,
International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug
Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference thereto.
Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamine, t-butyl amine, choline, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen- containing groups may be quarternized with agents such as lower alkyl halides {e.g., methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others. Compounds carrying an acidic moiety can be mixed with suitable pharmaceutically acceptable salts to provide, for example, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands such as quaternary ammonium salts. Also, in the case of an acid (such as -COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound.
All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.
In addition, when a compound of the invention contains both a basic moiety, such as, but not limited to an aliphatic primary, secondary, tertiary or cyclic amine, an aromatic or heteroaryl amine, pyridine or imidazole, and an acidic moiety, such as, but not limited to tetrazole or carboxylic acid, zwitterions ("inner salts") may be formed and are included within the terms "salt(s)" as used herein. It is understood that certain compounds of the invention may exist in zwitterionic form, having both anionic and cationic centers within the same compound and a net neutral charge. Such zwitterions are included within the invention.
Methods of Preparing Compounds
Several methods for preparing the compounds of general formula (I), and pharmaceutically acceptable salts of the foregoing, are described in the following Schemes and Examples. Starting materials and intermediates are purchased from commercial sources, made from known procedures, or are otherwise illustrated. In some cases the order of carrying out the steps of the reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products.
In the following Methods and Schemes, PG1 or PG2 represents a protecting group, selected from the options described below. All other variables have the same meaning as provided above.
Method 1
One method for the preparation of examples of the instant invention is detailed in Scheme 1. This procedure was adequately modified from the previously reported procedure (Zhao, Jianwei et al., Thiophosphate analogs of c-di-GMP: Impact on polymorphism,
Nucleosides, Nucleotides and Nucleic Acids, 2009, 28, 352-378). The sequence starts with modified ribo-nucleoside with a nucleobase of which amino group was appropriately protected with an alkyl or phenyl carbonyl group, and DMTr ether at 5'-0 position. It was treated with diphenyl phosphorochloridate and subsequently lithium sulfide to convert the 2' -OH to a hydrogen phosphonothioate. Then, DMTr ether was removed under acidic condition. The resulting 5'-hydroxyl group was reacted with 2' -0-phosphoramidites of fully protected second modified ribo-nucleoside to give a coupled compound. It was immediately either thioated or oxidized with elemental sulfur or t-butyl hydroperoxide. Then, the 5'-hydroxyl group of the second ribo-nucleoside was deprotected with dichloroacetic acid. Using diphenyl
phosphorochloridate as a coupling reagent, the hydrogen phosphonothioate at 3'-0 of the first ribo-nucleoside was reacted with 5' -OH of the second ribo-nucleoside to give a cyclic product. It was immediately thioated with 3H-benzo[c][l,2]dithiol-3-one or elemental sulfur. Treatment with methylamine plus fluoride anion in case of silyl protection was used provided the desired cyclic dinucleotide lla or lib.
SCHEME 1
SCHEME 1 (CONT.)
O* SH 1Fb ( x=o ) 1Gb (x=o) ors
Method 2
Another method for the preparation of examples of the instant invention is detailed in Scheme 2. This procedure was modified from Scheme 1. The sequence starts with modified ribo-nucleoside with a nucleobase of which amino group was appropriately protected with an alkyl or phenyl carbonyl group, and DMTr ether at 5'-0 position. It was treated with diphenyl phosphorochloridate and subsequently lithium sulfide (Li 2 S) to convert the 2' -OH to a hydrogen phosphonothioate. Then, DMTr ether was removed under acidic conditions. The resulting 5' -hydroxyl group was reacted with 3 ' -phosphoramidites of fully protected second modified ribo-nucleoside to give a coupled compound. It was immediately thioated with elemental sulfur. Then, the 5'-hydroxyl group of the second ribo-nucleoside was deprotected with dichloroacetic acid. Using diphenyl phosphorochloridate as a coupling reagent, the hydrogen phosphonothioate at 2'-0 of the first ribo-nucleoside was reacted with 5' -OH of the second ribo-nucleoside to give a cyclic product. It was immediately thioated with 3H- benzo[c][l,2]dithiol-3-one or elemental sulfur. Treatment with methylamine plus fluoride anion in case of silyl protection was used provided the desired cyclic dinucleotide 21. SCHEME 2
Method 3
Another method for the preparation of examples of the instant invention is detailed in Scheme 3. This procedure was modified from the previously reported procedure (Dahl, Bjarne H. et al., Synthesis of oligodeoxynucleoside phosphorodithioates by a phosphotriester method, Nucleosides & Nucleotides, 1991, 10, 553-554). The sequence starts with modified ribo-nucleoside with a nucleobase of which amino group was appropriately protected with an alkyl or phenyl carbonyl group, DMTr ether at 5'-0 position, and a H- phosphonate at 2'-0 position. It was treated with diphenyl phosphorochloridate and subsequently a fully protected second modified ribo-nucleoside with 5' -OH to give a coupled compound. It was immediately thioated or oxidized with 3H-benzo[c][l,2]dithiol-3-one or elemental sulfur or aqueous iodine. Then, DMTr ether of the first ribo-nucleoside and 3'-0- protecting group of the second ribo-nucleoside was removed under appropriate conditions. Using S-(2,4-dichlorobenzyl) 0,0-bis(4-oxobenzo[d][l,2,3]triazin-3(4H)-yl) phosphorodithioate as a coupling reagent, 5' -OH of the first ribo-nucleoside was reacted with 3' -OH of the second ribo-nucleoside to give a cyclic product. Treatment of 2-methylundecane-2-thiol, methylamine plus fluoride anion in case of silyl protection was used provided the desired cyclic dinucleotide 3Ga or 3Gb
SCHEME 3
3Fb ( x=o ) 3Gb (x=o) Method 4
Another method for the preparation of examples of the instant invention is detailed in Scheme 4. This procedure was modified from Scheme 3. The sequence starts with modified ribo-nucleoside with a nucleobase of which amino group was appropriately protected with an alkyl or phenyl carbonyl group, DMTr ether at 5'-0 position, and a H-phosphonate at 2'- O position. It was treated with 2,4-dichlorobenzyl phosphorodichloridodithioate, 3- hydroxybenzo[d][l,2,3]triazin-4(3H)-one, and subsequently a protected second modified ribo- nucleoside with 3' - and 5' -OH to give a coupled compound. Then, DMTr ether of the first ribo- nucleoside was removed under an acidic conditions. Using 3-(bis(diisopropylamino)
phosphanyl)oxy)propanenitrile as a coupling reagent, 5' -OH of the first ribo-nucleoside was reacted with 3' -OH of the second ribo-nucleoside to give a cyclic product. Treatment of 2- methylundecane-2-thiol, methylamine plus fluoride anion in case of silyl protection was used provided the desired cyclic dinucleotide 4E.
Methods of Use
Compounds described herein having therapeutic applications, such as the compounds of general formula (I), the compounds of the Examples 1 through 27, and
pharmaceutically acceptable salts of the foregoing, may be administered to a patient for the purpose of inducing an immune response, inducing STING-dependent cytokine production and/or inducing anti-tumor activity. The term "administration" and variants thereof (e.g., "administering" a compound) means providing the compound to the individual in need of treatment. When a compound is provided in combination with one or more additional active agents (e.g., antiviral agents useful for treating HCV infection or anti-tumor agents for treating cancers), "administration" and its variants are each understood to include concurrent and sequential provision of the compound or salt and other agents.
The compounds disclosed herein may be STING agonists. These compounds are potentially useful in treating diseases or disorders including, but not limited to, cell proliferation disorders. Cell-proliferation disorders include, but are not limited to, cancers, benign
papillomatosis, gestational trophoblastic diseases, and benign neoplastic diseases, such as skin papilloma (warts) and genital papilloma.
In specific embodiments, the disease or disorder to be treated is a cell proliferation disorder. In certain embodiments, the cell proliferation disorder is cancer. In particular embodiments, the cancer is selected from brain and spinal cancers, cancers of the head and neck, leukemia and cancers of the blood, skin cancers, cancers of the reproductive system, cancers of the gastrointestinal system, liver and bile duct cancers, kidney and bladder cancers, bone cancers, lung cancers, malignant mesothelioma, sarcomas, lymphomas, glandular cancers, thyroid cancers, heart tumors, germ cell tumors, malignant neuroendocrine (carcinoid) tumors, midline tract cancers, and cancers of unknown primary (i.e., cancers in which a metastasized cancer is found but the original cancer site is not known). In particular embodiments, the cancer is present in an adult patient; in additional embodiments, the cancer is present in a pediatric patient. In particular embodiments, the cancer is AIDS-related.
In specific embodiments, the cancer is selected from brain and spinal cancers. In particular embodiments, the cancer is selected from the group consisting of anaplastic astrocytomas, glioblastomas, astrocytomas, and estheosioneuroblastomas (also known as olfactory blastomas). In particular embodiments, the brain cancer is selected from the group consisting of astrocytic tumor (e.g., pilocytic astrocytoma, subependymal giant-cell astrocytoma, diffuse astrocytoma, pleomorphic xanthoastrocytoma, anaplastic astrocytoma, astrocytoma, giant cell glioblastoma, glioblastoma, secondary glioblastoma, primary adult glioblastoma, and primary pediatric glioblastoma), oligodendroglial tumor (e.g., oligodendroglioma, and anaplastic oligodendroglioma), oligoastrocytic tumor (e.g., oligoastrocytoma, and anaplastic
oligoastrocytoma), ependymoma (e.g., myxopapillary ependymoma, and anaplastic
ependymoma); medulloblastoma, primitive neuroectodermal tumor, schwannoma, meningioma, atypical meningioma, anaplastic meningioma, pituitary adenoma, brain stem glioma, cerebellar astrocytoma, cerebral astorcytoma/malignant glioma, visual pathway and hypothalmic glioma, and primary central nervous system lymphoma. In specific instances of these embodiments, the brain cancer is selected from the group consisting of glioma, glioblastoma multiforme, paraganglioma, and suprantentorial primordial neuroectodermal tumors (sP ET).
In specific embodiments, the cancer is selected from cancers of the head and neck, including nasopharyngeal cancers, nasal cavity and paranasal sinus cancers,
hypopharyngeal cancers, oral cavity cancers (e.g. , squamous cell carcinomas, lymphomas, and sarcomas), lip cancers, oropharyngeal cancers, salivary gland tumors, cancers of the larynx (e.g., laryngeal squamous cell carcinomas, rhabdomyosarcomas), and cancers of the eye or ocular cancers. In particular embodiments, the ocular cancer is selected from the group consisting of intraocular melanoma and retinoblastoma.
In specific embodiments, the cancer is selected from leukemia and cancers of the blood. In particular embodiments, the cancer is selected from the group consisting of myeloproliferative neoplasms, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), myeloproliferative neoplasm (MPN), post-MPN AML, post- MDS AML, del(5q)-associated high risk MDS or AML, blast-phase chronic myelogenous leukemia, angioimmunoblastic lymphoma, acute lymphoblastic leukemia, Langerans cell histiocytosis, hairy cell leukemia, and plasma cell neoplasms including plasmacytomas and multiple myelomas. Leukemias referenced herein may be acute or chronic.
In specific embodiments, the cancer is selected from skin cancers. In particular embodiments, the skin cancer is selected from the group consisting of melanoma, squamous cell cancers, and basal cell cancers.
In specific embodiments, the cancer is selected from cancers of the reproductive system. In particular embodiments, the cancer is selected from the group consisting of breast cancers, cervical cancers, vaginal cancers, ovarian cancers, prostate cancers, penile cancers, and testicular cancers. In specific instances of these embodiments, the cancer is a breast cancer selected from the group consisting of ductal carcinomas and phyllodes tumors. In specific instances of these embodiments, the breast cancer may be male breast cancer or female breast cancer. In specific instances of these embodiments, the cancer is a cervical cancer selected from the group consisting of squamous cell carcinomas and adenocarcinomas. In specific instances of these embodiments, the cancer is an ovarian cancer selected from the group consisting of epithelial cancers.
In specific embodiments, the cancer is selected from cancers of the gastrointestinal system. In particular embodiments, the cancer is selected from the group consisting of esophageal cancers, gastric cancers (also known as stomach cancers),
gastrointestinal carcinoid tumors, pancreatic cancers, gallbladder cancers, colorectal cancers, and anal cancer. In instances of these embodiments, the cancer is selected from the group consisting of esophageal squamous cell carcinomas, esophageal adenocarcinomas, gastric
adenocarcinomas, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gastric lymphomas, gastrointestinal lymphomas, solid pseudopapillary tumors of the pancreas, pancreatoblastoma, islet cell tumors, pancreatic carcinomas including acinar cell carcinomas and ductal adenocarcinomas, gallbladder adenocarcinomas, colorectal adenocarcinomas, and anal squamous cell carcinomas.
In specific embodiments, the cancer is selected from liver and bile duct cancers. In particular embodiments, the cancer is liver cancer (also known as hepatocellular carcinoma). In particular embodiments, the cancer is bile duct cancer (also known as cholangiocarcinoma); in instances of these embodiments, the bile duct cancer is selected from the group consisting of intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma.
In specific embodiments, the cancer is selected from kidney and bladder cancers. In particular embodiments, the cancer is a kidney cancer selected from the group consisting of renal cell cancer, Wilms tumors, and transitional cell cancers. In particular embodiments, the cancer is a bladder cancer selected from the group consisting of urethelial carcinoma (a transitional cell carcinoma), squamous cell carcinomas, and adenocarcinomas.
In specific embodiments, the cancer is selected from bone cancers. In particular embodiments, the bone cancer is selected from the group consisting of osteosarcoma, malignant fibrous histiocytoma of bone, Ewing sarcoma, chordoma (cancer of the bone along the spine).
In specific embodiments, the cancer is selected from lung cancers. In particular embodiments, the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancers, bronchial tumors, and pleuropulmonary blastomas. In specific embodiments, the cancer is selected from malignant mesothelioma. In particular embodiments, the cancer is selected from the group consisting of epithelial
mesothelioma and sarcomatoids.
In specific embodiments, the cancer is selected from sarcomas. In particular embodiments, the sarcoma is selected from the group consisting of central chondrosarcoma, central and periosteal chondroma, fibrosarcoma, clear cell sarcoma of tendon sheaths, and Kaposi's sarcoma.
In specific embodiments, the cancer is selected from lymphomas. In particular embodiments, the cancer is selected from the group consisting of Hodgkin lymphoma (e.g., Reed- Sternberg cells), non-Hodgkin lymphoma (e.g., diffuse large B-cell lymphoma, follicular lymphoma, mycosis fungoides, Sezary syndrome, primary central nervous system lymphoma), cutaneous T-cell lymphomas, primary central nervous system lymphomas.
In specific embodiments, the cancer is selected from glandular cancers. In particular embodiments, the cancer is selected from the group consisting of adrenocortical cancer (also known as adrenocortical carcinoma or adrenal cortical carcinoma), pheochromocytomas, paragangliomas, pituitary tumors, thymoma, and thymic carcinomas.
In specific embodiments, the cancer is selected from thyroid cancers. In particular embodiments, the thyroid cancer is selected from the group consisting of medullary thyroid carcinomas, papillary thyroid carcinomas, and follicular thyroid carcinomas.
In specific embodiments, the cancer is selected from germ cell tumors. In particular embodiments, the cancer is selected from the group consisting of malignant extracranial germ cell tumors and malignant extragonadal germ cell tumors. In specific instances of these embodiments, the malignant extragonadal germ cell tumors are selected from the group consisting of nonseminomas and seminomas.
In specific embodiments, the cancer is selected from heart tumors. In particular embodiments, the heart tumor is selected from the group consisting of malignant teratoma, lymphoma, rhabdomyosacroma, angiosarcoma, chondrosarcoma, infantile fibrosarcoma, and synovial sarcoma.
In specific embodiments, the cell-proliferation disorder is selected from benign papillomatosis, benign neoplastic diseases and gestational trophoblastic diseases. In particular embodiments, the benign neoplastic disease is selected from skin papilloma (warts) and genital papilloma. In particular embodiments, the gestational trophoblastic disease is selected from the group consisting of hydatidiform moles, and gestational trophoblastic neoplasia (e.g., invasive moles, choriocarcinomas, placental-site trophoblastic tumors, and epithelioid trophoblastic tumors).
As used herein, the terms "treatment" and "treating" refer to all processes in which there may be a slowing, interrupting, arresting, controlling, or stopping of the progression of a disease or disorder described herein. The terms do not necessarily indicate a total elimination of all disease or disorder symptoms.
The terms "administration of and or "administering" a compound should be understood to include providing a compound described herein, or a pharmaceutically acceptable salt thereof, and compositions of the foregoing to a subject.
The amount of a compound administered to a subject is an amount sufficient to induce an immune response and/or to induce STING-dependent type I interferon production in the subject. In an embodiment, the amount of a compound can be an "effective amount" or "therapeutically effective amount," such that the subject compound is administered in an amount that will elicit, respectively, a biological or medical (i.e., intended to treat) response of a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician. An effective amount does not necessarily include considerations of toxicity and safety related to the administration of a compound.
An effective amount of a compound will vary with the particular compound chosen (e.g., considering the potency, efficacy, and/or half-life of the compound); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the subject being treated; the medical history of the subject being treated; the duration of the treatment; the nature of a concurrent therapy; the desired therapeutic effect; and like factors and can be routinely determined by the skilled artisan.
The compounds disclosed herein may be administered by any suitable route including oral and parenteral administration. Parenteral administration is typically by injection or infusion and includes intravenous, intramuscular, intratumoral, and subcutaneous injection or infusion.
The compounds disclosed herein may be administered once or according to a dosing regimen where a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound disclosed herein depend on the pharmacokinetic properties of that compound, such as absorption, distribution and half-life, that can be determined by a skilled artisan. In addition, suitable dosing regimens, including the duration such regimens are administered, for a compound disclosed herein depend on the disease or condition being treated, the severity of the disease or condition, the age and physical condition of the subject being treated, the medical history of the subject being treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual subject's response to the dosing regimen or over time as the individual subject needs change. Typical daily dosages may vary depending upon the particular route of administration chosen.
One embodiment of the present disclosure provides for a method of treating a cell proliferation disorder comprising administration of a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, to a subject in need of treatment thereof. In embodiments, the disease or disorder to be treated is a cell proliferation disorder. In aspects of these embodiments, the cell proliferation disorder is cancer. In further aspects of these embodiments, the cancer is selected from brain and spinal cancers, cancers of the head and neck, leukemia and cancers of the blood, skin cancers, cancers of the reproductive system, cancers of the gastrointestinal system, liver and bile duct cancers, kidney and bladder cancers, bone cancers, lung cancers, malignant mesothelioma, sarcomas, lymphomas, glandular cancers, thyroid cancers, heart tumors, germ cell tumors, malignant neuroendocrine (carcinoid) tumors, midline tract cancers, and cancers of unknown primary.
In one embodiment, disclosed herein is the use of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, in a therapy. The compound may be useful in a method of inducing an immune response and/or inducing STING-dependent type I interferon production in a subject, such as a mammal in need of such inhibition, comprising administering an effective amount of the compound to the subject.
In one embodiment, disclosed herein is a pharmaceutical composition comprising at least one compound of general formula (I), or at least one pharmaceutically acceptable salt of the foregoing, for use in potential treatment to induce an immune response and/or to induce STING-dependent type I interferon production.
One embodiment disclosed herein is the use of a compound of general formula
(I), or a pharmaceutically acceptable salt of the foregoing, in the manufacture of a medicament to induce an immune response and/or to induce STING-dependent type I interferon production. In embodiments, the disease or disorder to be treated is a cell proliferation disorder. In aspects of these embodiments, the cell proliferation disorder is cancer. In further aspects of these embodiments, the cancer is selected from brain and spinal cancers, cancers of the head and neck, leukemia and cancers of the blood, skin cancers, cancers of the reproductive system, cancers of the gastrointestinal system, liver and bile duct cancers, kidney and bladder cancers, bone cancers, lung cancers, malignant mesothelioma, sarcomas, lymphomas, glandular cancers, thyroid cancers, heart tumors, germ cell tumors, malignant neuroendocrine (carcinoid) tumors, midline tract cancers, and cancers of unknown primary.
Compositions
The term "composition" as used herein is intended to encompass a dosage form comprising a specified compound in a specified amount, as well as any dosage form that results, directly or indirectly, from combination of a specified compound in a specified amount. Such term is intended to encompass a dosage form comprising a compound of general formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug of the foregoing, and one or more pharmaceutically acceptable carriers or excipients. In embodiments, the dosage form comprises compounds of general structural formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and one or more pharmaceutically acceptable carriers or excipients. In specific embodiments, the dosage form comprises compounds of general structural formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients. Accordingly, the compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and one or more pharmaceutically acceptable carrier or excipients. By "pharmaceutically acceptable", it is meant the carriers or excipients are compatible with the compound disclosed herein and with other ingredients of the composition.
For the purpose of inducing an immune response and/or inducing STING- dependent type I interferon production, the compounds of general formula (I), or
pharmaceutically acceptable salts of the foregoing, can be administered by means that produces contact of the active agent with the agent's site of action. The compounds can be administered by conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. The compounds can be administered alone, but typically are administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. In one embodiment, disclosed herein is a composition comprising a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, and one or more pharmaceutically acceptable carriers or excipients. The composition may be prepared and packaged in bulk form in which a therapeutically effective amount of a compound of the disclosure can be extracted and then given to a subject, such as with powders or syrups.
Alternatively, the composition may be prepared and packaged in unit dosage form in which each physically discrete unit contains a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing.
The compounds disclosed herein and a pharmaceutically acceptable carrier or excipient(s) will typically be formulated into a dosage form adapted for administration to a subject by a desired route of administration. For example, dosage forms include those adapted for (1) oral administration, such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; and (2) parenteral administration, such as sterile solutions, suspensions, and powders for reconstitution. Suitable pharmaceutically acceptable carriers or excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable carriers or excipients may be chosen for a particular function that they may serve in the composition. For example, certain
pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the carrying or transporting of a compound disclosed herein, once administered to the subject, from one organ or portion of the body to another organ or another portion of the body. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to enhance patient compliance.
Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, lubricants, binders, disintegrants, fillers, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anti -caking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.
A skilled artisan possesses the knowledge and skill in the art to select suitable pharmaceutically acceptable carriers and excipients in appropriate amounts for the use in the compositions of the disclosure. In addition, there are a number of resources available to the skilled artisan, which describe pharmaceutically acceptable carriers and excipients and may be useful in selecting suitable pharmaceutically acceptable carriers and excipients. Examples include REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company), THE
HANDBOOK OF PHARMACEUTICAL ADDITIVES (Gower Publishing Limited), and THE HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (the American Pharmaceutical Association and the
Pharmaceutical Press).
The compositions of the disclosure are prepared using techniques and methods known to those skilled in the art. Some methods commonly used in the art are described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company).
In one embodiment, the disclosure is directed to a solid oral dosage form such as a tablet or capsule comprising a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, and a diluent or filler.
Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g., corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives, (e.g., microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The solid oral dosage form may further comprise a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pre-gelatinized starch) gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). The solid oral dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The solid oral dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.
Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The composition can also be prepared to prolong or sustain the release as, for example, by coating or embedding particulate material in polymers, wax, or the like.
The compounds disclosed herein may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or
polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the disclosure may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanacrylates, and cross- linked or amphipathic block copolymers of hydrogels.
In one embodiment, the disclosure is directed to a liquid oral dosage form. Oral liquids such as solutions, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of a compound or a pharmaceutically acceptable salt thereof disclosed herein. Syrups can be prepared by dissolving the compound of the disclosure in a suitably flavored aqueous solution; elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing a compound disclosed herein in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additives such as peppermint oil, or other natural sweeteners or saccharin or other artificial sweeteners and the like can also be added.
In one embodiment, the disclosure is directed to compositions for parenteral administration. Compositions adapted for parenteral administration include aqueous and nonaqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
Combinations
The compounds of general formula (I), and/or pharmaceutically acceptable salts of the foregoing, may be administered in combination with one or more additional active agents. In embodiments, one or more compounds of general formula (I), or one or more
pharmaceutically acceptable salts of the foregoing, and the one or more additional active agents may be co-administered. The additional active agent(s) may be administered in a single dosage form with the compound of general formula (I), or pharmaceutically acceptable salt of the foregoing, or the additional active agent(s) may be administered in separate dosage form(s) from the dosage form containing the compound of general formula (I), or pharmaceutically acceptable salt of the foregoing. The additional active agent(s) may be one or more agents selected from the group consisting of STING agonist compounds, anti-viral compounds, antigens, adjuvants, anti- cancer agents, CTLA-4, LAG-3 and PD-1 pathway antagonists, lipids, liposomes, peptides, cytotoxic agents, chemotherapeutic agents, immunomodulatory cell lines, checkpoint inhibitors, vascular endothelial growth factor (VEGF) receptor inhibitors, topoisomerase II inhibitors, smoothen inhibitors, alkylating agents, anti-tumor antibiotics, anti-metabolites, retinoids, and immunomodulatory agents including but not limited to anti-cancer vaccines. It will be understood the descriptions of the above additional active agents may be overlapping. It will also be understood that the treatment combinations are subject to optimization, and it is understood that the best combination to use of the compounds of general formula (I), or pharmaceutically acceptable salts of the foregoing, and one or more additional active agents will be determined based on the individual patient needs.
A compound disclosed herein may be used in combination with one or more other active agents, including but not limited to, other anti-cancer agents that are used in the prevention, treatment, control, amelioration, or reduction of risk of a particular disease or condition (e.g., cell proliferation disorders). In one embodiment, a compound disclosed herein is combined with one or more other anti-cancer agents for use in the prevention, treatment, control amelioration, or reduction of risk of a particular disease or condition for which the compounds disclosed herein are useful. Such other active agents may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure.
When a compound disclosed herein is used contemporaneously with one or more other active agents, a composition containing such other active agents in addition to the compound disclosed herein is contemplated. Accordingly, the compositions of the present disclosure include those that also contain one or more other active ingredients, in addition to a compound disclosed herein. A compound disclosed herein may be administered either simultaneously with, or before or after, one or more other active agent(s). A compound disclosed herein may be administered separately, by the same or different route of
administration, or together in the same pharmaceutical composition as the other agent(s).
Products provided as combinations may include a composition comprising a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, and one or more other active agent(s) together in the same pharmaceutical composition, or may include a composition comprising a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, and a composition comprising one or more other active agent(s) in separate form, e.g. in the form of a kit or in any form designed to enable separate administration either concurrently or on separate dosing schedules.
The weight ratio of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, to a second active agent may be varied and will depend upon the therapeutically effective dose of each agent. Generally, a therapeutically effective dose of each will be used. Combinations of a compound disclosed herein and other active agents will generally also be within the aforementioned range, but in each case, a therapeutically effective dose of each active agent should be used. In such combinations, the compound disclosed herein and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).
In one embodiment, this disclosure provides a composition comprising a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, and at least one other active agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a cell proliferation disorder, such as cancer.
In one embodiment, the disclosure provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules, and the like.
A kit of this disclosure may be used for administration of different dosage forms, for example, oral and parenteral, for administration of the separate compositions at different dosage intervals, or for titration of the separate compositions against one another. To assist with compliance, a kit of the disclosure typically comprises directions for administration.
Disclosed herein is a use of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, for treating a cell proliferation disorder, where the medicament is prepared for administration with another active agent. The disclosure also provides the use of another active agent for treating a cell proliferation disorder, where the medicament is administered with a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing. The disclosure also provides the use of a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing, for treating a cell proliferation disorder, where the patient has previously (e.g., within 24 hours) been treated with another active agent. The disclosure also provides the use of another active agent for treating a cell proliferation disorder, where the patient has previously (e.g., within 24 hours) been treated with a compound of general formula (I), or a pharmaceutically acceptable salt of the foregoing. The second agent may be administered a week, several weeks, a month, or several months after the administration of a compound disclosed herein.
STING agonist compounds that may be used in combination with the compounds of general formula (I), or pharmaceutically acceptable salts of the foregoing, disclosed herein include but are not limited to cyclic di-nucleotide compounds.
Anti-viral compounds that may be used in combination with the compounds of general formula (I), or pharmaceutically acceptable salts of the foregoing, disclosed herein include hepatitis B virus (HBV) inhibitors, hepatitis C virus (HCV) protease inhibitors, HCV polymerase inhibitors, HCV NS4A inhibitors, HCV NS5 A inhibitors, HCV NS5b inhibitors, and human immunodeficiency virus (HIV) inhibitors.
Antigens and adjuvants that may be used in combination with the compounds of general formula (I), or the pharmaceutically acceptable salts of the foregoing, include B7 costimulatory molecule, interleukin-2, interferon-y, GM-CSF, CTLA-4 antagonists, OX-40/0X- 40 ligand, CD40/CD40 ligand, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions. Adjuvants, such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, a toll-like receptor (TLR) 9 agonist as well as additional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein, LPS, monophosphoryllipid A, lipoteichoic acid, imiquimod, resiquimod, and in addition retinoic acid- inducible gene I (RIG-I) agonists such as poly I:C, used separately or in combination with the described compositions are also potential adjuvants.
CLTA-4 and PD-1 pathways are important negative regulators of immune response. Activated T-cells up-regulate CTLA-4, which binds on antigen-presenting cells and inhibits T-cell stimulation, IL-2 gene expression, and T-cell proliferation; these anti-tumor effects have been observed in mouse models of colon carcinoma, metastatic prostate cancer, and metastatic melanoma. PD-1 binds to active T-cells and suppresses T-cell activation; PD-1 antagonists have demonstrated anti-tumor effects as well. CTLA-4 and PD-1 pathway antagonists that may be used in combination with the compounds of general formula (la), the compounds of general formula (lb), the compounds of general formula (I), or the
pharmaceutically acceptable salts of the foregoing, disclosed herein, include ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-011, AMP-224, and MDX-1106.
"PD-1 antagonist" or "PD-1 pathway antagonist" means any chemical compound or biological molecule that blocks binding of PD-Ll expressed on a cancer cell to PD-1 expressed on an immune cell (T-cell, B-cell, or NKT-cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279, and SLEB2 for PD-1;
PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-Ll; and PDCD1L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In any of the treatment method, medicaments and uses of the present disclosure in which a human individual is being treated, the PD-1 antagonist blocks binding of human PD-Ll to human PD-1, and preferably blocks binding of both human PD-Ll and PD-L2 to human PD-1. Human PD-1 amino acid sequences can be found in NCBI Locus No. : NP 005009. Human PD-Ll and PD-L2 amino acid sequences can be found in NCBI Locus No. : NP_054862 and NP_079515, respectively.
PD-1 antagonists useful in any of the treatment method, medicaments and uses of the present disclosure include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-Ll, and preferably specifically binds to human PD-1 or human PD-Ll . The mAb may be a human antibody, a humanized antibody, or a chimeric antibody and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgGl or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab') 2 , scFv, and Fv fragments.
Examples of mAbs that bind to human PD-1, and useful in the treatment method, medicaments and uses of the present disclosure, are described in U.S. Patent Nos. US7488802, US7521051, US8008449, US8354509, and US8168757, PCT International Patent Application Publication Nos. WO2004/004771, WO2004/072286, and WO2004/056875, and U.S. Patent Application Publication No. US2011/0271358. Examples of mAbs that bind to human PD-L1, and useful in the treatment method, medicaments and uses of the present disclosure, are described in PCT International Patent Application Nos. WO2013/019906 and WO2010/077634 Al and in U.S. Patent No.
US8383796. Specific anti-human PD-L1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present disclosure include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C, and an antibody that comprises the heavy chain and light chain variable regions of SEQ ID NO:24 and SEQ ID NO:21, respectively, of WO2013/019906.
Other PD-1 antagonists useful in any of the treatment method, medicaments, and uses of the present disclosure include an immune-adhesion that specifically binds to PD-1 or PD- LI, and preferably specifically binds to human PD-1 or human PD-L1, e.g., a fusion protein containing the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immune-adhesion molecules that specifically bind to PD-1 are described in PCT International Patent Application Publication Nos. WO2010/027827 and WO2011/066342. Specific fusion proteins useful as the PD-1 antagonist in the treatment method, medicaments, and uses of the present disclosure include AMP -224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1.
Examples of cytotoxic agents that may be used in combination with the compounds of general formula (I), or pharmaceutically acceptable salts of the foregoing, include, but are not limited to, arsenic trioxide (sold under the tradename TRISENOX ® ), asparaginase (also known as L-asparaginase, and Erwinia L-asparaginase, sold under the tradenames ELSPAR ® and
KlDROLASE®).
Chemotherapeutic agents that may be used in combination with the compounds of general formula (I), or pharmaceutically acceptable salts of the foregoing, disclosed herein include abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5, 6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl- 1-Lproline-t-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'deoxy-8'-norvin- caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea andtaxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, nivolumab, onapristone, paclitaxel, pembrolizumab, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine.
Examples of vascular endothelial growth factor (VEGF) receptor inhibitors include, but are not limited to, bevacizumab (sold under the trademark AVASTIN), axitinib
(described in PCT International Patent Publication No. WO01/002369), Brivanib Alaninate ((S)- ((R)- 1 -(4-(4-Fluoro-2-methyl - lH-indol-5-yloxy)-5-methylpyrrolo[2, 1 -f] [ 1 ,2,4]triazin-6- yloxy)propan-2-yl)2-aminopropanoate, also known as BMS-582664), motesanib (N-(2,3- dihydro-3,3-dimethyl-lH-indol-6-yl)-2-[(4-pyridinylmethyl)am ino]-3-pyridinecarboxamide. and described in PCT International Patent Application Publication No. WO02/068470), pasireotide (also known as SO 230, and described in PCT International Patent Publication No.
WO02/010192), and sorafenib (sold under the tradename NEXAVAR).
Examples of topoisomerase II inhibitors, include but are not limited to, etoposide (also known as VP-16 and Etoposide phosphate, sold under the tradenames TOPOSAR,
VEPESID, and ETOPOPHOS), and teniposide (also known as VM-26, sold under the tradename VUMON).
Examples of alkylating agents, include but are not limited to, 5-azacytidine (sold under the trade name VIDAZA), decitabine (sold under the trade name of DECOGEN), temozolomide (sold under the trade names TEMCAD, TEMODAR, and TEMODAL), dactinomycin (also known as actinomycin-D and sold under the tradename COSMEGEN), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, sold under the tradename ALKERAN), altretamine (also known as hexamethylmelamine (HMM), sold under the tradename HEXALEN), carmustine (sold under the tradename BCNU), bendamustine (sold under the tradename TREANDA), busulfan (sold under the tradenames BUSULFEX ® and
MYLERAN ® ), carboplatin (sold under the tradename PARAPLATIN ® ), lomustine (also known as CCNU, sold under the tradename CEENU ® ), cisplatin (also known as CDDP, sold under the tradenames PLATINOL ® and PLATINOL®-AQ), chlorambucil (sold under the tradename
LEUKERAN ® ), cyclophosphamide (sold under the tradenames CYTOXAN ® and NEOSAR®), dacarbazine (also known as DTIC, DIC and imidazole carboxamide, sold under the tradename DTIC -DOME ® ), altretamine (also known as hexamethylmelamine (HMM) sold under the tradename HEXALEN ® ), ifosfamide (sold under the tradename IFEX ® ), procarbazine (sold under the tradename MATULANE ® ), mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, sold under the tradename MUSTARGEN®), streptozocin (sold under the tradename ZANOSAR ), thiotepa (also known as thiophosphoamide, TESPA and TSPA, and sold under the tradename THIOPLEX®.
Examples of anti-tumor antibiotics include, but are not limited to, doxorubicin (sold under the tradenames ADRIAMYCIN ® and RUBEX ® ), bleomycin (sold under the tradename LENOXANE®), daunorubicin (also known as dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, sold under the tradename CERUBIDINE®), daunorubicin liposomal (daunorubicin citrate liposome, sold under the tradename DAUNOXOME ® ), mitoxantrone (also known as DHAD, sold under the tradename NOVANTRONE®), epirubicin (sold under the tradename ELLENCE™), idarubicin (sold under the tradenames IDAMYCIN®, IDAMYCIN PFS ® ), and mitomycin C (sold under the tradename MUTAMYCIN®).
Examples of anti-metabolites include, but are not limited to, claribine (2- chlorodeoxyadenosine, sold under the tradename LEUSTATIN®), 5-fluorouracil (sold under the tradename ADRUCIL®), 6-thioguanine (sold under the tradename PURINETHOL ® ), pemetrexed (sold under the tradename ALIMTA ® ), cytarabine (also known as arabinosylcytosine (Ara-C), sold under the tradename CYTOSAR-U ® ), cytarabine liposomal (also known as Liposomal Ara-C, sold under the tradename DEPOCYT™), decitabine (sold under the tradename DACOGEN®), hydroxyurea and (sold under the tradenames HYDREA®, DROXIA™ and MYLOCEL™), fludarabine (sold under the tradename FLUDARA ® ), floxuridine (sold under the tradename FUDR ® ), cladribine (also known as 2-chlorodeoxyadenosine (2-CdA) sold under the tradename LEUSTATIN™), methotrexate (also known as amethopterin, methotrexate sodium (MTX), sold under the tradenames RHEUMATREX ® and TREXALL™), and pentostatin (sold under the tradename NIPENT®).
Examples of retinoids include, but are not limited to, alitretinoin (sold under the tradename PANRETIN ® ), tretinoin (all-trans retinoic acid, also known as ATRA, sold under the tradename VESANOID ® ), Isotretinoin (13-c/s-retinoic acid, sold under the tradenames
ACCUTANE®, AMNESTEEM®, CLARAVIS®, CLARUS®, DECUTAN®, ISOTANE®, IZOTECH®,
ORATANE®, ISOTRET ® , and SOTRET ® ), and bexarotene (sold under the tradename TARGRETIN®).
Activity: STING Biochemical HIcGAMP Competition Assay
The individual compounds described in the Examples herein are defined as
STING agonists by (i) binding to the STING protein as evidenced by a reduction in binding of tritiated cGAMP ligand to the STING protein by at least 20% at 20uM (concentration of compound being tested) in a STING Biochemical [3H]eGAMP Competition Assay and/or (ii) demonstrating interferon production with a 6% or greater induction of IFN-β secretion at 30uM in the THP1 ceil assay (where induction caused by cGAMP at 30uM was set at 100%).
The ability of compounds to bind STING is quantified by the ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter- binding assay. The binding assay employs STING receptor obtained from Hi-Five cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.
The following experimental procedures detail the preparation of specific examples of the instant disclosure. The compounds of the examples are drawn in their neutral forms in the procedures and tables below. In some cases, the compounds were isolated as salts depending on the method used for their final purification and/or intrinsic molecular properties. The examples are for illustrative purposes only and are not intended to limit the scope of the instant disclosure in any way. EXAMPLES
ABBREVIATIONS
19 F-NMR 19 F nuclear magnetic resonance spectroscopy
1H -NMR Proton nuclear magnetic resonance spectroscopy
31 P-NMR 31 P nuclear magnetic resonance spectroscopy
A Angstrom
AcOH Acetic acid
aq Aqueous
ATP Adenosine 5 '-triphosphate
BF 3 -OEt 2 Boron trifuoride diethyl etherate
Bn Benzyl
Bz Benzoyl
BzCl Benzoyl chloride
CD 3 OD Deuterium-enriched methyl alcohol, deuterium-enriched methanol
Ci Curie, a non-standard unit of radioactivity; ICi = 3 x 10 10 Bq, where Bq is Becquerel, the SI unit of radioactivity, equivalent to 1 disintegration per second (dps)
d Doublet
D 2 0 Deuterium-enriched water DBU l,8-diazabicyclo(5.4.0)undec-7-ene
DCA Dichloroacetic acid
DCM, CH 2 C1 2 Dichloromethane
ddd Doublet of doublet of doublet
ddt Doublet of doublet of triplet
DIEA N,N-diisopropylethylamine
DMF N,N-di m ethyl form ami de
DMSO Dimethyl sulfoxide
DMTr 4,4'-dimethoxytrityl
DMTrCl 4,4'-dimethoxytrityl chloride
DPCP Diphenyl phosphorochloridate
dq Doublet of quartet
#e# Standard exponential notation for #xl0 # ; for example 2e5 = 2xl0 3
EC50 half maximal effective concentration, concentration of a drug, antibody or toxicant that induces a response halfway between the baseline and maximum after a specified exposure time
EDTA Ethylenediaminetetraacetic acid
eq Equivalents
ES Electron spray
Et Ethyl
Et 2 0 Diethyl ether
Et 3 N, TEA Tri ethyl amine
Et 3 N 3HF Triethylamine trihydrogen floride
EtsSiH, Si(C 2 H 5 ) 3 H Triethylsilane
EtOAc Ethyl acetate
EtOH Ethyl alcohol, ethanol
g Gram
GTP Guanosine 5 '-triphosphate
h Hour
HEPES 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid, a
zwitterionic organic chemical buffering agent
hept Heptet
Hex Hexanes HF-Pyr, HF-Py Hydrogen fluoride - pyridine complex
HPLC High performance liquid chromatography
Hz Hertz
iPrMgCl-LiCl Isopropylmagnesium chloride lithium chloride compled
J NMR Coupling constant
L Liter
LCMS Liquid chromatography - mass spectroscopy
Li 2 S Dilithium sulfide
m Multiplet
M Molar, moles per liter
mCi Millicurie
Me, CH 3 Methyl
MeCN, ACN, CH 3 CH Acetonitrile
MeMgBr, CH 3 MgBr Methylmagnesium bromide
Me H 2 , CH 3 H 2 Methylamine
MeOH Methanol
mg Milligram
MgCl 2 Magnesium chloride
MHz Megahertz
min Minute(s)
mL,ml Milliliter
mM Millimole per liter
mm Millimeter
mmol Millimole
MOI Multiplicity of infection
mol Mole
N Normal
ng Nanogram(s)
nL Nanoliter
nm Nanometer
nM Nanomolar
pfu Particle-forming units
Ph phenyl PSI, psi Pounds per square inch
Py Pyridine
q Quartet
RPM, rpm Revolutions per minute
RT, rt Room temperature, approximately 25°C
s Singlet
sat Saturated
t Triplet
tBuOOH t-Butylhydrogen peroxide
TEAA Triethylammonium acetate
THF Tetrahydrofuran
TLC Thin layer chromatography
TMSC1 Trimethylsilyl chloride, Si(CH 3 ) 3 Cl
T R Retention time
TrisCl Tris(hydroxymethyl)aminom ethane hydrochloride
v/v Volume/volume
xg Times gravity; the force applied to a spinning sample in terms of multiples of the gravitational force
Emission wavelength
Xex Excitation wavelength
μg Microgram
μί, uL Microliter
μΜ, uM Micromolar
μιη Micrometer, micron
μιηοΐ Micromole
Preparation 1 : N-( 9-(T2R,3S,4S,5RV5-( (bis( 4-methoxyphenylK phenvnmethoxy)methvn-4- fluoro-3-hvdroxytetrahvdrofuran lH-purin-2-yl)isobutyramide
Step 1: N-(9-( (2R, 3S, 4S, 5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrah^
6,9-dihydro-lH-purin-2-yl)isobutyramide
To a suspension of 2-amino-9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-l,9-dihydro-6H-purin -6-one (Carbosynth catalog #
D10826, 1.50g, 5.26mmol) in Py (30mL) at 0°C to 5°C was added TMSCI (2.86g, 26.3mmol), and the mixture was stirred at RT for 30min. Then, isobutyric anhydride (2.50g, 15.8mmol) was added dropwise, and it was stirred for an additional for 2h. Then, MeOH (5.3mL) was added. After 5min, H 4 OH (10.5mL) was added dropwise and stirring was continued for 30min. The reaction mixture was concentrated under reduced pressure, and MeOH (2mL) in CH 2 CI 2 (18mL) was added to the residue. Insolubles were filtered off, and the filtrate was concentrated and purified by flash column chromatography with 2-10% MeOH in CH 2 C1 2 to give the product. LCMS (ES, m/z): 356.1 [M + H] + . 1H- MR: (400MHz, DMSO-i¾): δ 12.11 (s, 1H), 11.68 (s, 1H), 8.28 (s, 1H), 5.98 (d, J = 6.1Hz, 1H), 5.85 (d, J = 8.0Hz, 1H), 5.24 (t, J = 5.4Hz, 1H), 5.14 (d, J = 4.1Hz, 0.5H), 5.01 (d, J = 4.2Hz, 0.5H), 4.87-4.69 (m, 1H), 4.26 (t, J= 4.4Hz, 0.5H), 4.19 (t, J = 4.4Hz, 0.5H), 3.61 (t, J = 4.9Hz, 2H), 2.77 (hept, J = 6.8Hz, 1H), 1.13 (d, J = 6.7Hz, 6H). 19 F- MR: (376MHz, DMSO-i¾): δ -197.5 (s).
Step 2: N-(9-( (2R, 3S, 4S, 5R)-5-( (bis(4-methoxyphenyl) (phenyl)methoxy)methyl)-4-fIuoro-3- hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-
N-(9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetr ahydrofuran-2- yl)-6-oxo-6,9-dihydro-lH-purin-2-yl)isobutyramide (1.30g, 3.66mmol) was co-evaporated with Py (3x1 OmL) and re-dissolved in Py (26mL). To the solution at 0°C to 5°C was added DMTrCI (1.36g, 4.02mmol). It was stirred at RT for 3h and then concentrated. CH 2 CI 2 (40mL, with 1% Et 3 N) was added, and it was washed with sat aq NaHC0 3 (15mL), H 2 0 (lOmL) and brine (lOmL). The organic solution was dried (Na 2 S0 4 ), concentrated and purified by silica gel column chromatography using 0-10% MeOH in CH 2 C1 2 (1% Et 3 N) to give the product. LCMS (ES, m/z): 656.2 [M - H] " . 1H- MR: (400MHz, DMSO-i¾): δ 12.10 (s, 1H), 11.61 (s, 1H), 8.14 (s, 1H), 7.40-7.31 (m, 2H), 7.31-7.19 (m, 7H), 6.89-6.78 (m, 4H), 6.08 (d, J = 6.1Hz, 1H), 5.87 (d, J = 7.3Hz, 1H), 5.23 (dd, J = 4.1, 1.8Hz, OH), 5.10 (d, J = 4.4Hz, OH), 4.96 (dq, J = 22.4, 5.9Hz, 1H), 4.30 (dt, 7= 26.1, 4.6Hz, 1H), 3.74 (d, 7= 1.1Hz, 6H), 3.39 (dd, 7= 10.6, 5.7Hz, 1H), 3.22 (dd, 7 = 10.6, 3.8Hz, 1H), 2.76 (p, 7= 6.8Hz, 1H), 1.13 (d, 7= 6.8Hz, 6H). 19 F-NMR: (376MHz, DMSO-i¾): δ -198.1 (s, IF). Preparation 2: N-f7-^2R,4S,5R)-5-^bisH-methoxyphenyl)fphenyl)methoxy)methyl )-4- hvdroxytetrahydrofuran-2-yl)imidazo[2,l- 1 [l,2,41triazin-4-yl)benzamide
Step 1: (3R R,5R)-2-(4-aminoimidazo[2 -fJ[l ltriazin-7-yl)-3 -bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahydrofuran-2-ol
To a stirring mixture of 7-bromoimidazo[2,l- J[l,2,4]triazin-4-amine (41g, 0.19mol) in THF (0.50L) at 0°C was added MeMgBr (3.0M in THF, 66mL, 0.19mol) dropwise to maintain the internal temperature below 10°C. Bis(chlorodimethylsilyl)ethane (41g, 190mmol) was added in one portion. MeMgBr (3.0M in Et 2 0, 66mL, 0.19mol) was then added dropwise to maintain the internal temperature below 10°C. / ' -PrMgCI-LiCI (1.3M in THF,
0.16L, 0.21mol) was added while maintaining the internal temperature below 10°C. A mixture of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofu ran-2(3H)-one (160g, 0.38mol) in THF was added dropwise at 0°C, and the mixture was then allowed to warm to RT and was stirred for 12h. The mixture was diluted with sat aq H 4 CI (lOOmL) and extracted with EtOAc (3xl000mL). The combined organic layers were dried over Na 2 S0 4 and concentrated under reduced pressure. The resulting residue was purified by column chromatography (column height: 2500mm, diameter: 1000mm, 25% to 75% EtOAc gradient in hex) to afford (3R,4R,5R)- 2-(4-aminoimidazo[2, l- J[l,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methy l)- tetrahydrofuran-2-ol .
Step 2: 7-((3SAR,5R)-3 -bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)imidazo[2, 1-f] [ 1, 2, 4]triazin-4-amine
To a stirring mixture of (3R,4R,5R)-2-(4-aminoimidazo[2,l- J[l,2,4]triazin-7-yl)-
3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2 -ol (64g, 0.12mmol) in DCM (1.3L) at 0°C was added Et 3 SiH (81g, 0.69mol), and then BF 3 -OEt 2 (21g, 0.15 mol). The mixture was then allowed to warm to 25°C, and the mixture was stirred for lh. More BF 3 -OEt 2 (57g,
0.40mol) was added, and the mixture was then heated to 35°C for 4h. Upon cooling to RT, the mixture was quenched with sat aq NaHC0 3 (200mL) and then extracted with EtOAc (3x300mL) The combined organic layers were dried over Na 2 S0 4 and concentrated under reduced pressure. The resulting residue was purified by column chromatography (15-75% EtOAc gradient in hex) to afford 7-((3,S',4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetr ahydrofuran-2- yl)imidazo[2,l- J[l,2,4]triazin-4-amine. MS (ES, m/z) = 538 [M + H]+.
Step 3: (3R,4S,5R)-2-(4-aminoimidazo[2,l-fl ' [ 1 ,2,41 triazin-7-yl)-5-(hvdroxymethyl)- tetrahydrofuran-3, 4-diol
To a stirring mixture of 7-((3,S',4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)- tetrahydrofuran-2-yl)imidazo[2, l- J[l,2,4]triazin-4-amine (12g, 22mmol) in DCM (850mL) at -78°C was added BC1 3 (18g, 0.16mol) dropwise. Upon completion, the mixture was stirred at -78°C for 3h. After 3h, the mixture was quenched with MeOH (50mL) at -78°C, and the mixture was allowed to warm to 25°C. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (9-25% MeOH gradient in DCM) to afford (3R,4^,5R)-2-(4-aminoimidazo[2,l- J[l,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4- diol.
Step 4: (6aR, 8S, 9S, 9aS)-8-(4-aminoimidazoi2, 1-flil, 2, 41triazin- 7-yl)-2.2.4.4- tetraisopropyltetrahydro-6H-furo[ 3, 2-fJf 1,3,5, 2, 4]trioxadisilocin-9-ol
To a stirred mixture of (2,S',3R,4 ) S',5R)-2-(4-aminoimidazo[2, l- J[l,2,4]triazin-7- yl)-5-(hydroxym ethyl )tetrahydrofuran-3,4-diol (4.0g, 15mmol) in Py (0.10 L) was added 1,3- dichloro-l,l,3,3-tetraisopropyldisiloxane (5.8mL, 18mmol). After 3h, the mixture was diluted with toluene (50mL) and then concentrated. The resulting mixture was taken up in DCM and MeOH, and then silica gel (40g) was added. The mixture was concentrated, placed under vacuum for lh and purified by column chromatography (0-80% EtOAc gradient in hex) to afford (6aR,8^,9^,9a,S)-8-(4-aminoimidazo[2,l-f][l,2,4]triazin-7-yl )-2,2,4,4-tetraisopropyltetrahy 6H-furo[3,2- J[l,3,5,2,4]trioxadisilocin-9-ol. MS (ES, m/z) = 510 [M + H]+.
Step 5: Q-((6aR, 8S.9S.9aR)-8-(4-aminoimidazo[2, l-flfl, 2, 41triazin- 7-yl)-2.2.4.4- tetraisopropyltetrahydro-6H-furo[ 3, 2-f/f 1,3,5, 2, 4]trioxadisilocin-9-yl) lH-imidazole-1- carbothioate
To a mixture of (6aR,8^,9^,9a5)-8-(4-aminoimidazo[2, l-f][l,2,4]triazin-7-yl)-
2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2- J[l,3,5,2,4]trioxadisilocin-9-ol (6.45g, 12.7mmol) in CH 3 CN (63.0mL) and Py (63.0mL) was added Ι,Γ-thiocarbonyldiimidazole (2.71g,
15.2mmol). After 90min, more Ι, Γ-thiocarbonyldiimidazole (2.71g, 15.2mmol) was added, and the mixture was stirred overnight. After stirring overnight, the mixture was concentrated and purified by column chromatography (0-100% EtOAc gradient in hex) to afford 0-((6aR,SS,9S, 9aR)-8-(4-aminoimidazo[2, l- J[l,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-fu ro J[l,3,5,2,4]trioxadisilocin-9-yl) lH-imidazole-l-carbothioate. MS (ES, m/z) = 620 [M + H]+. Step 6: 7-(( 6aR, 8R, 9aS)-2, 2, 4, 4-tetraisopropyltetrahydro-6H-furo[ 3, 2- f] [ 1,3,5, 2, 4]trioxadisilocin-8-yl)imidazo[ 2, 1-f] [ 1, 2, 4]triazin-4-amine
To a mixture of O-((6aR,8^,9^,9aR)-8-(4-aminoimidazo[2,l- J[l,2,4]triazin-7-yl)- 2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2- J[l,3,5,2,4]trioxadisilocin-9-yl) (5.65g, 9.1 lmmol) in toluene (91.0mL) was added 2,2'-azobis(2-methylpropionitrile (0.300g, 1.82mmol) and tris(trimethylsilyl)silane ((TMS) 3 SiH, 4.22mL, 13.7mmol). The mixture was heated to 85°C for 30min. After 30min, the mixture was allowed to cool to RT and placed directly on the column and purified (0-80% EtOAc gradient in hex) to afford 7-((6aR,8R,9a,S 2,2,4,4-tetraisopropyl- tetrahydro-6H-furo[3 ,2- J [ 1 ,3 , 5,2,4]trioxadisilocin-8-yl)imidazo[2, 1 - J [ 1 ,2,4]triazin-4-amine. MS (ES, m/z) = 494 [M + H]+ 494.
Step 7: N-benzoyl-N-(7-( ( 6aR 8R 9aS)-2, 2, 4, 4-tetraisopropyltetrahydro-6H-furo[ , 2- f] [ 1, 3, 5, 2, 4]trioxadisilocin-8-yl)imidazo[ 2, 1-f] [ 1, 2, 4]triazin-4-yl)benzamide
To a mixture of 7-((6aR,8R,9a,S)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3, 2- J[l,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,l- J[l,2,4]triazin-4-amine (15.7g, 31.8mmol) in Py (64.0mL) was added BzCl (1 l .OmL, 95.0mmol), and the mixture was heated to 50°C for 45min.
After 45min, the mixture was allowed to cool to RT. After cooling, a precipitate formed and was filtered off. The filtrate was diluted with DCM (50mL) and toluene (50mL). The mixture was concentrated to about 50mL. The mixture was filtered, and the solids were washed with DCM.
The filtrate and washes were combined, loaded onto a column, and purified (0-50% EtOAc gradient in hex) to afford N-benzoyl-N-(7-((6aR,8R,9a,S)-2,2,4,4-tetraisopropyltetrahyd ro-6H- furo[3,2- J[l,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,l- J[l,2,4]triazin-4-yl)benzamide. MS (ES, m/z) = 702 [M + H]+.
Step 8: N-(7-( (2R, 4S, 5R)-4-hvdroxy-5-(hvdroxymethyl)tetrahvdrofuran-2-yl)imidazo[ 2, 1- f] [ 1, 2, 4]triazin-4-yl)benzamide
2-yl)imidazo[2, l-f][l,2,4]triazin-4-yl)benzamide (6.1g, 17mmol) in Py (86mL) at 0°C was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (5.8g, 17mmol), and the mixture was allowed to warm to RT overnight. After stirring overnight, the mixture was diluted with toluene and then concentrated under reduced pressure to afford the crude product. The crude product was purified by silica gel chromatography (0-100%) EtOAc gradient in hex) to afford N-(7- ((2R,4,S',5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methy l)-4-hydroxytetrahydrofuran-2- yl)imidazo[2,l- J[l,2,4]triazin-4-yl)benzamide. MS (ES, m/z) = 658 [M + H]+. Preparation 3: Ammonium f2R,3 .,4S,5R)-5-^bisf4- methoxyphenyl)fphenyl)methoxy)methyl)-4-^tert-butyldimethyls ilyl)oxy)-2-f2- isobutyramido-6-oxo-l,6-dihvdro-9H-purin-9-yl)tetrahydrothio phen-3-yl phosphonate
Step 1: N-(9-( (2R 3R 4S, 5R)-3, 4-dihydroxy-5-(7rydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo -
6,9-dihvdro-lH-purin-2-yl)isobutyramide
2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrothiophen- 2-yl)-l,9-dihydro-6H-purin-6-one (1.7g, 5.7mmol) was co-evaporated with Py (3x5mL) and then, re-dissolved in Py (34mL). To the mixture at 0°C was added TMSC1 (4.32g, 39.8mmol) dropwise. It was stirred at RT for lh and then, cooled to 0°C again. Isobutyric anhydride (1.348g, 8.52mmol) was added dropwise, and it was stirred at RT for 3h. It was quenched by the addition of H 2 0 (8.5mL). After 5min, H 4 OH (ca. 29%, 17mL) was added, and the mixture was stirred for 30min. It was concentrated and purified by column chromatography eluted with 1 to 30% MeOH in CH 2 C1 2 to give the product. LCMS (ES, m/z): 396.9 [M + H] + . 1H- MR
(400MHz, DMSO-i¾): δ 9.52 (br s, 2 H), 8.39 (s, 1H), 5.79 (d, J = 7.1Hz, 1H), 5.59 (s, 1H), 5.40 (s, 1H), 5.22 (s, 1H), 4.55 (d, J= 6.7Hz, 1H), 4.21 (s, 1H), 3.77 (t, J= 9.3Hz, 1H), 3.61 (s, 1H), 3.30 (dt, J= 6.4, 3.3Hz, 1H), 2.78 (p, J= 6.9Hz, 1H), 1.13 (d, J = 6.8Hz, 6H).
Step 2: N-(9-( (2R 3R 4S, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3, 4- dihydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-
To a mixture of N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)- tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-lH-purin-2-yl)iso butyramide (480mg, 1.299mmol) in pyridine (lOmL) was added 4,4'-(chloro(phenyl)methylene)-bis(methoxybenzene) (484mg, 1.43mmol). It was stirred at RT for 16h and then, concentrated. The crude product was purified by column chromatography on silica gel eluted with 1 to 30% MeOH in CH 2 C1 2 (containing 1% Et 3 N) to give the product. LCMS (ES, m/z): 672.2 [M + H] + . 1H- MR (400MHz, OMSO-d 6 + D 2 0): δ 8.08 (s, 1H), 7.39 (d, J= 7.2Hz, 2H), 7.32 (t, J= 7.6Hz, 2H), 7.26 (dt, J= 9.1, 3.3Hz, 5H), 6.94-6.87 (m, 4H), 5.75 (d, J= 5.9Hz, 1H), 4.39 (dd, J= 5.9, 3.5Hz, 1H), 4.14 (t, J= 3.9Hz, 1H), 3.74 (s, 6H), 3.49-3.37 (m, 2H), 3.33 (dd, J= 14.5, 7.3Hz, 1H), 2.87-2.67 (m, 1H), 1.11 (dd, J= 6.8, 1.6Hz, 6H).
Step 3: N-(9-( (2R 3R 4S, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert - butyldimethylsilyl)oxy)-3-hydroxytetrahydrothiophen-2-yl)
vDisobutyramide and N-(9-( (2R, 3R, 4S, 5R)-5-( (bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3- ((tert-buMdimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-y l)-6-oxo-6,9-dihydr
vDisobutyramide
To a solution of N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)- methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-6-oxo- 6,9-dihydro-lH-purin-2- yl)isobutyramide (580mg, 0.863mmol) in DMF (5mL) at RT was added lH-imidazole (147mg, 2.16mmol) and tert-butylchlorodimethylsilane (156mg, 1.04mmol). After 6h, the mixture was diluted with EtOAc (50mL) and washed with sat aq NaHC0 3 (2x20mL) and brine (20mL). It was dried (Na 2 S0 4 ), concentrated, and purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in H 2 0 to give the products. LCMS (ES, m/z): 786.3 [M + H] + .
Step 4: (2R 3R 4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((t ert- bu1yldimethylsilyl)oxy)-2-(2 sobu1yramido-6-oxo-l,6-dihydro-9H^urin-9- yDtetrahydrothiophen-3-yl phenyl phosphonate and (2R,3S,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-buMdimethylsi lyl)oxy)-5-(2-isobutyramido-6- oxo-1 , 6-dihydro-9 -purin-9-yl)tetrahydrothiophen-3-yl phenyl phosphonate
To a solution of a mixture of N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl) (phenyl)-methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3- hydroxytetrahydrothiophen-2- 6-oxo-6,9-dihydro-lH-purin-2-yl)isobutyramide and N-(9-((2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethyl silyl)oxy)-4- hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro- lH-purin-2-yl)isobutyramide (220mg, 0.280mmol) in Py (2mL) at 0°C was added diphenyl phosphonate (98mg, 0.420mmol). The resulting mixture was stirred at RT for 20min. It was used in the next reaction step without purification. LCMS (ES, m/z): 926.2 [M + H] + .
Step 5: ammonium (2R,3R, 4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((t ert- butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-l, 6-dihvdro-9H-purin-9- yl)tetrahydrothiophen-3-yl phosphonate
To the reaction mixture from Step 4 at 0°C was added Et 3 N (0.28mL, 2.0mmol) and H 2 0 (0.28mL). It was stirred at RT for 30min. It was concentrated, and the residue was partitioned between CH 2 C1 2 (40mL) and aq NaHC0 3 (5%, 30mL). The organic layer was washed with aq NaHC0 3 (5%, 2x30mL), dried (Na 2 S0 4 ), concentrated and purified by silica gel column chromatography using 0-10% MeOH in CHC1 3 containing 1% Et 3 N to give a mixture. The mixture was further purified by Prep-HPLC (XB ridge Shield RP18 OBD Column,
19x 150mm) eluted with 46 to 79% ACN in aq NH 4 HC0 3 (lOmM) over 7min to give the product. LCMS (ES, m/z): 850.2 [M + H] + . 1H- MR (400MHz, CD 3 OD): δ 8.18 (s, 1H), 7.68 (s, 0.5H), 7.59-7.49 (m, 2H), 7.45-7.36 (m, 4H), 7.37-7.30 (m, 2H), 7.28-7.22 (m, 1H), 6.95-6.87 (m, 4H), 6.16-6.07 (m, 2H), 4.88-4.87 (m, 1H), 4.69 (dd, J = 7.3, 3.3Hz, 1H), 3.81 (s, 6H), 3.51 (dd, J = 4.9, 1.9Hz, 2H), 3.37 (s, 1H), 2.67 (p, J = 6.9Hz, 1H), 1.21 (dd, J = 6.9, 0.9Hz, 6H), 0.77 (s, 9H), 0.01 (s, 3H), -0.28 (s, 3H). 31 P- MR (162MHz, DMSO-i¾): δ -0.74 (s).
Preparation 4: f2R,3S,4S,5R)-5-^bisf4-methoxyphenyl)fphenyl)methoxy)methyl) -4-fluoro- 2-f2-isobutyramido-6-oxo-l,6-dihvdro-9H-purin-9-yl)tetrahydr ofuran-3-yl hydrogen phosphonate
Step 1: (2R S S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)m
isobut\ramido-6-ox -L 6-dihydro-9H-purin-9-yl)te^
To a solution of N-(9-((2R,3S,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy) methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9- dihydro-lH-purin-2-yl)isobutyramide (prepared according to published procedures: Tetrahedron Letters, 1989, 30, 3171-3174. 630mg, 0.96mmol) in Py (5mL) under Ar was added diphenyl phosphonate (1.07g, 4.56mmol), and the mixture was stirred at RT for 20min. It was used for the next reaction step without purification. LCMS (ES, m/z): 798.3 [M + H] + .
Step 2: (2R3S S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluo ro-2-(2- isobut ramido-6-oxo-l, 6-dihydro-9H^urin-9-yl)tetrahydrofuran-3-yl phos
To the reaction mixture from Step 1 at 0°C was added H 2 0 (lmL), Et 3 N (lmL). The resulting mixture was stirred at RT for 20min. Then, it was concentrated, and the residue was partitioned between CH 2 CI 2 (50mL) and aq NaHC0 3 (5%, 20mL). The organic layer was washed with aq NaHC0 3 (5%, 20mL), dried (Na 2 S 2 0 4 ), concentrated and purified by silica gel column chromatography using 0-7% MeOH in CH 2 C1 2 (1% Et 3 N) to give the product. LCMS (ES, m/z): 722.2 [M + H] + . 1H- MR (400MHz, CD 3 OD): δ 7.76 (s, 1H), 7.74 (s, 0.5H), 7.51 (d, J= 1.5Hz, 1H), 7.48 (q, J= 2.4, 1.9Hz, 1H), 7.41-7.34 (m, 4H), 7.34-7.27 (m, 2H), 7.26-7.21
(m, 1H), 6.92-6.85 (m, 4H), 6.22 (s, 1H), 6.15 (s, 0.5H), 5.37 (d, J= 2.7Hz, 0.5H), 5.28-5.19 (m, 1.5H), 4.73-4.69 (m, 0.5H), 4.66-4.62 (m, 1H), 3.80 (s, 6H), 3.65-3.55 (m, 1H), 3.53-3.44 (m, 1H), 3.12 (q, J = 7.3Hz, 8H), 2.75 (p, J= 6.8Hz, 1H), 1.33-1.22 (m, 18H). 31 P-NMR: (162MHz, CD 3 OD): δ 2.67 (s, IP). Examples 1 and 2: 2-amino-9-rf5R,7R,8S,12aR,14R,15S,15aR,16RV14-f6-amino-9H-pu rin- 9-vn-15,16-difluoro-10-hvdroxy-2-sulfanyl-2,10-disulfidoocta hvdro-12H-5,8- methanofuro[3,2-ll[l,3.,6,9,ll,2,101pentaoxadiphosphacvclote tradecin-7-yll-l,9-dihvdro- 6H-purin-6-one (Diastereomer 1) and 2-amino-9-rf5R,7R,8S,12aR,14R,15S,15aR,16RV14- f6-amino-9H-purin-9-yl)-15,16-difluoro-10-hvdroxy-2-sulfanyl -2,10-disulfidooctahvdro- 12H-5,8-methanofuro[3,2-ll[l,3.,6,9,ll,2,101pentaoxadiphosph acvclotetradecin-7-yll-l,9- dihydro-6H-purin-6-one (Diastereomer 2)
Step 1: (2R 3R.4S 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofura n- phenyl phosphonate
z
To a solution of N-(9-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-p urin-6-yl)benzamide (2.1g, 3.1mmol) in anhydrous Py (14mL) was added diphenyl phosphonate (7.28g, 31.1mmol). The mixture was stirred at RT for 20min and was used for the next reaction step without purification. LCMS (ES, m/z): 816.3 [M + H] + .
Step 2: ammonium Q-((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetra^
To the reaction mixture from Step 1 at 0°C was added Li 2 S (714mg, 15.5mmol), and the mixture was stirred at RT for 15h. The mixture was concentrated under reduced pressure. The residue was partitioned between CH 2 C1 2 (200mL) and aq NaHC0 3 (5%, lOOmL). The organic layer was washed with aq NaHC0 3 (5%, 2x50mL), dried (Na 2 S0 4 ) and concentrated to give a crude product. The residue was purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq H 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 756.2 [M + H] + . 1H- MR (400MHz, OMSO-d 6 ): δ 11.25 (s, 2H), 8.76 (d, J= 9.9Hz, 3H), 8.25 (d, J =
4.1Hz, 2H), 8.03 (d, J= 7.6Hz, 4H), 7.58 (dt, J= 29.5, 7.6Hz, 6H), 7.41 (d, J= 7.7Hz, 4H), 7.32 - 7.13 (m, 14H), 6.94 (d, J= 5.9Hz, 1H), 6.84 (d, J= 8.4Hz, 8H), 6.57 (d, J= 4.0Hz, 1H), 6.51 (s, 1H), 5.54 (s, 1H), 5.37 (s, 1H), 5.10 (d, J = 15.3Hz, 2H), 4.24 (d, J= 14.1Hz, 2H), 3.70 (s, 13H), 2.48 (d, J = 1.3Hz, 2H), 2.36 (s, 1H), 2.05 (s, 6H), 1.24 - 1.09 (m, 2H). 31 P-NMR:
(162MHz, DMSO-i¾): δ 48.82 (s).
Step 3: yridin-l-ium 0-((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-yl) phosphonothioate
z
To a solution of ammonium 0-((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrah ydrofuran-3-yl)
phosphonothioate (400mg, 0.467mmol) in CH 2 C1 2 (7mL) at RT was added H 2 0 (84mg,
4.9mmol) and 2,2-dichloroacetic acid in CH 2 C1 2 (6%, 7mL). The mixture was stirred at RT for 15min, and then Et 3 SiH (5mL) was added. After 40min, Py (660mg, 8.4mmol) was added, and the mixture was stirred for 5min. The mixture was concentrated, and the residue was used for the next reaction step without purification. LCMS (ES, m/z): 452.0 [M - H] " .
Step 4: pyridin-l-ium Q-((2R.3R.4S.5R)-5-(6-benzamido-9H-purin-9-\l)-2-((((((2R.3S .4R 5R)-5- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluo
dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy
fIuorotetrahydrofuran-3-yl) phosphonothioate
The crude product of Step 3 was co-evaporated with MeCN (3x4mL), re- dissolved in MeCN (2mL), and dried by adding activated 4A molecular sieve (50mg).
(2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)met hyl)-4-fluoro-2-(2- isobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl)tetrahydrofura n-3-yl (2-cyanoethyl) diisopropylphosphoramidite (0.48g, 0.56mmol) was co-evaporated with MeCN (3x4mL), re- dissolved in MeCN (2mL), and dried by adding activated 4A molecular sieve (50mg). After 30min, it was added to the previously prepared mixture containing pyridin-l-ium O- ((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hyd roxymethyl)tetrahydrofuran-3- yl) phosphonothioate. The resulting mixture was stirred at RT for 30min and was used in the next reaction step without purification. LCMS (ES, m/z): 1208.2 [M - H] " .
Step 5: pyridin-l-ium Q-((2R.3R.4S 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R.3S 4R 5R)-5- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2- isobu1 ramM
dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoet ho
4-fIuorotetrahydrofuran-3-yl) phosphonothioate
To the mixture from Step 4 was added sulfur (1.50g, 46.7mmol) in one portion and 2,6-dimethylpyridine (0.541ml, 4.67mmol). The resulting mixture was stirred at RT for 2h. Then, the mixture was concentrated, and the crude product was used for the next step without purification. LCMS (ES, m/z): 1240.2 [M - H] " .
Step 6: ammonium Q-((2R3R4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2- cyanoethoxy) ( ( (2R, 3S, 4R, 5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-l, 6- dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothio y
fIuorotetrahydrofuran-3-yl) phosphonothioate
To a solution of the crude product of Step 5 in CH 2 C1 2 (7mL) at RT was added H 2 0 (93mg, 5.2mmol) and 2,2-dichloroacetic acid in CH 2 C1 2 solution (6%, 7.7mL, 4.6mmol). After 15min, Et 3 SiH (15mL) was added, and the mixture was stirred for additional 40min. Then, Py (665mg, 8.5mmol) was added, and the mixture was concentrated. The residue was purified by reverse phase (C 18) chromatography eluted with 0 to 95% ACN in aq H 4 HC0 3 (0.04%) to give the product. LCMS (ES, m/z): 938.1 [M + H] + .
Step 7: pyridinium (5R, 7R, 8S, 12aR, 14R, 15S, 15aR, 16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2- [(2-methylpropanoyl)amino]-6-oxo-L6-dihydro-9H-purin-9-y ^
9H-purin-9-yftoctahydro-12H-5, 8-methanofurof 3, 2- I] [1,3, 6,9, 11,2, lOlpentaoxadiphosphacyclotetradecine-2-thiolate 10-sulfide
To Py (12mL) at -40°C under Ar was added diphenyl phosphorochloridate (657mg, 2.44mmol) and then, a solution of ammonium 0-((2R,3R,4S,5R)-5-(6-benzamido-9H- purin-9-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-( hydroxymethyl)-2-(2- isobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl)tetrahydrofura n-3-yl)oxy)phosphorothioyl) oxy)methyl)-4-fluorotetrahydrofuran-3-yl) phosphonothioate (150mg, 0.122mmol, co- evaporated with Py 3x5mL) in CH 2 C1 2 (30mL) over 20min. The resulting mixture was stirred at -40°C for 20min. It was used in the next step immediately without purification. LCMS (ES, m/z): 922.2 [M + H] + .
Step 8: ammonium (5R, 7R,8S, 12aR, 14R, 15S, 15aR, 16R)-10-(2-cyanoethoxy)-l 5, 16-di[luoro-7- {2-[(2-methylpropanoyl)aminol-6-oxo-l, 6-dihydro-9H-purin-9-yl}-14-{6- [ (phenylcarbonyl)aminoJ-9H-purin-9-yl}octahydro-12H-5, 8-methanofuro[ 3, 2- lj[ 1,3, 6,9, 11,2, 10Jpentaoxadiphosphacyclotetradecine-2-thiolate 2, 10-disulfide
To the solution from Step 7 at -40°C was added 3H-benzo[c][l,2]dithiol-3-one (0.03 lg, 0.18mmol) and H 2 0 (8.1g, 450mmol). The mixture was stirred at RT for 40min. Then, the mixture was concentrated, and the residue was purified by reverse phase (CI 8)
chromatography eluted with 0 to 95% ACN in aq NH 4 HC0 3 (0.04%) to give the product. LCMS (ES, m/z): 954.2 [M + H] + . 1H-NMR: (400MHz, CD 3 OD) δ 8.77 (d, J= 7.7Hz, 1H), 8.20-8.03 (m, 3H), 7.71-7.64 (m, 1H), 7.64-7.51 (m, 3H), 7.35-7.17 (m, 4H), 7.07 (t, J= 7.3Hz, 1H), 6.30 (dd, J= 15.9, 8.0Hz, 1H), 5.85-5.33 (m, 2H), 4.59-4.37 (m, 2H), 4.32 (t, J = 11.7Hz, 1H), 4.24- 3.99 (m, 2H), 3.86-3.65 (m, 3H), 2.90 (t, J= 6.9Hz, 1H), 2.82-2.69 (m, 1H), 2.63 (ddd, J = 21.0, 7.1, 5.1Hz, 1H), 1.43-1.28 (m, 7H). 31 P-NMR: (162MHz, CD 3 OD): δ 115.38 (s), 63.59 (s). Step 9: 2-amino-9-f(5R, 7R.8S.12aR, 14R, 15S.15aR.16R)-14-(6-amino-9H-purin-9-yl)-15.16- difluoro-10-hydroxy-2-sulfanyl-2, 10-disulfidooctahydro-12H-5, 8-methanofurof 3, 2- 11 [1,3, 6,9, 11,2, lOlpentaoxadiphosphacyclotetradecin- 7-yl 1-1, 9-dihydro-6H-purin-6-one (Piaster eomers 1— 2)
Ammonium (5R,7R,8S, 12aR, 14R, 15 S, 15aR, 16R)- 10-(2-cyanoethoxy)-l 5, 16- difluoro-7- { 2-[(2-methylpropanoyl)amino] -6-oxo- 1 ,6-dihydro-9H-purin-9-yl } - 14- { 6- [(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-meth anofuro[3,2-l][l,3,6,9,l 1,2,10] pentaoxadiphosphacyclotetradecine-2-thiolate 2,10-disulfide (40mg, 0.064mmol) was dissolved in a solution of MeNH 2 in EtOH (30%, 5mL). The resulting solution was stirred at RT for 3h, then the solution was concentrated. The residue was suspended in EtOAc (lOmL) and stirred for lh. The mixture was filtered and washed with cold EtOAc (2xl0mL). The solid was purified by prep-HPLC (Atlantis Prep RP CI 8 OBD Column, 19mmx250mm) eluted with 0 to 27% ACN in aq NH 4 HC0 3 (50mM) over 25min to afford two diastereomers of 2-amino-9- [(5R, 7R, 8 S, 12aR, 14R, 15 S, 15 aR, 16R)- 14-(6-amino-9H-purin-9-yl)- 15, 16-difluoro- 10-hydroxy- 2-sulfanyl-2, 10-disulfidooctahydro-12H-5,8-methanofuro[3,2-
1] [ 1 ,3 ,6,9, 1 1 ,2, 10]pentaoxadiphosphacyclotetradecin-7-yl]- 1 ,9-dihydro-6H-purin-6-one:
Example 1 (diastereomer 1) (T R : 13.58min): LCMS (ES, m/z): 727.3 [M + H] + . 1H- MR: (400MHz, D 2 0) δ 8.33 (s, 1H), 8.27 (d, J = 2.4Hz, 1H), 8.20 (s, 1H), 6.63-6.46 (m, 1H), 6.08 (d, J = 8.7Hz, 1H), 5.78-5.24 (m, 4H), 4.55 (s, 1H), 4.46-4.01 (m, 4H), 3.67-3.44 (m, 1H). 31 P-NMR: (162MHz, D 2 0) δ 1 13.21 (s), 54.13 (s).
Example 2 (diastereomer 2) (T R : 12.77min): LCMS (ES, m/z): 727.3 [M + H] + . 1H- MR: (400MHz, D 2 0) δ 8.54 (s, 1H), 8.28 (d, J = 2.9Hz, 1H), 8.20 (s, 1H), 6.53 (dd, J = 22.7, 2.6Hz, 1H), 6.10 (d, J = 8.5Hz, 1H), 5.74-5.49 (m, 2H), 5.42-5.13 (m, 2H), 4.49-4.27 (m, 3H), 4.07 (s, 2H). 31 P- MR: (162MHz, D 2 0) δ 1 13.30 (s), 60.84 (s).
Examples 3 through 9, as shown in Table 1 below, were prepared according to procedures analogous to those outlined in Examples 1 and 2 above using appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.
Table 1
purin-6-one
Examples 10 and 11: 2-amino-9-rf5S,7R,8R,12aR,14R,15aSV14-f6-amino-9H-purin-9-vn - 2-hvdroxy-10-sulfanyl-2,10-disulfidooctahydro-12H-5,8-methan ofuro[3,2- 11 [1,3.,6,9 ,ll,2,101pentaoxadiphosphacvclotetradecin-7-yll-l,9-dihydro- 6H-purin-6-one (Diastereomer 1) and 2-amino-9-rf5S,7R,8R,12aR,14R,15aSV14-f6-amino-9H-purin-9-vn - 2-hvdroxy-10-sulfanyl-2,10-disulfidooctahydro-12H-5,8-methan ofuro[3,2- ll[l,3.,6,9,ll,2,101pentaoxadiphosphacvclotetradecin-7-yll-l ,9-dihvdro-6H-purin-6-one (Diastereomer 2)
Step 1: Ammonium Q-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-2-(2- isobut ramido-6-oxo- -dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl) phosphonothw
To a solution of (2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-
2-(2-isobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl)tetrah ydrofuran-3-yl phenyl phosphonate (2.35 mmol) in Py (12ml) at 0°C was added Li 2 S (539mg, 11.7mmol). The mixture was stirred at RT for lh. Then, the mixture was concentrated. DCM (50 mL) was added, and the mixture was washed with sat aq NaHC0 3 (20 mL). The layers were separated, and the aq layer was extracted with DCM (3x80mL). The combined organic layers were washed with brine (30 mL), dried (Na 2 S0 4 ), concentrated, and purified by reverse phase (CI 8) chromatography eluted with 0-50% ACN in aq H 4 HC0 3 (0.4%) to give the product. LCMS (ES, m/z): 720.2 [M + H] + . 1H- MR: (400MHz, DMSO-i¾): δ 12.11 (s, 1H), 11.75-11.70 (m, 1H), 8.15 (s, 1H), 7.32-7.12 (m, 9H), 7.00 (s, 1H), 6.82-6.78 (m, 4H), 6.14 (s, 1H), 5.30 (d, J= 12Hz, 1H), 4.51-4.47 (m, 1H), 3.72 (s, 6H), 3.15-3.14 (m, 2H), 2.83-2.76 (m, 1H), 2.21-2.16 (m, 1H), 1.13 (d, J= 8Hz, 1H). 31 P-NMR: (162MHz, DMSO-i¾): δ 48.27 (s).
Step 2: Pyridin-l-ium Q-( (2R 3R 5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-l, 6-dihydro- 9H-purin-9-yl)tetrahydrofuran-3-yl) phosphonothioate
To a solution of ammonium 0-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-2-(2 sobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl)tetrahydrofuran -3-yl) phosphonothioate (400mg, 0.543mmol) in DCM (6ml) was added H 2 0 (98mg, 5.4mmol) and 2,2-dichloroacetic acid in DCM (6%, 6mL). The mixture was stirred at RT for 20min, and then Et 3 SiH (6mL) was added. After 2h, Py (773 mg, 9.77mmol) was added, and the mixture was stirred for lOmin. Then, the mixture was concentrated, and the residue was co-evaporated with MeCN and toluene (1 : 1, 3x4mL) to give the crude product, which was used for the next reaction step without purification. LCMS (ES, m/z): 418.1 [M + H] + .
Step 3: Pyridin-l-ium 0-((2P3P5S)-5-((((((2R.3S.5R)-5-(6-benzamido-9H-purin-9-yl)- 2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran -3-yl)oxy)(2- cyanoethoxy)phosphanyl) oxy)methyl)-2-(2-isobutyramido-6-oxo-l, 6-dihydro-9H-purin-9- yl)tetrahydrofuran-3- l hos honothioate
To the crude product from Step 3 in MeCN (4mL) under Ar was added activated
4A molecular sieve (lOOmg). (2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)
diisopropylphosphoramidite (0.466g, 0.543mmol) was co-evaporated with MeCN (3x4mL), re- dissolved in MeCN (4mL), and dried by adding 4A molecular sieve (lOOmg). After 30min, it was transferred into a solution containing pyridin-l-ium 0-((2R,3R,5S)-5-(hydroxymethyl)-2-(2- isobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl)tetrahydrofura n-3-yl) phosphonothioate, and the mixture was stirred at RT for lh. It was used for the next reaction step. LCMS (ES, m/z): 1174.5 [M + H] + .
Step 4: Pyridin-l-ium Q-((2R3R5S)-5-((((((2R.3S.5R)-5-(6-benzamido-9H-purin-9-yl)- 2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydro^ cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2 sobutyramido-6-oxo- 6-dihydro-9 yl)tetrahydrofuran-3-yl) phosphonothioate
To the reaction mixture from Step 3 at RT was added sulfur (0.174g, 5.43mmol), and the mixture was stirred at RT for 3h. The reaction mixture was filtered, and the filtrate was concentrated to give a residue. It was co-evaporated with toluene (3x5ml) and CH 3 CN (3x5ml), and the crude product was used for the next reaction step without purification. LCMS (ES, m/z): 1206.3 [M + H] + .
Step 5: Ammonium 0-((2R,3R,5S)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl )-2-
(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)ph o
isobutyramido-6-oxo- -dihydro-9H- urin-9-yl)tetrahydrofuran-3-yl^
To a solution of the crude product from Step 4 in DCM (6ml) at RT was added H 2 0 (98mg, 5.4mmol) and 2,2-dichloroacetic acid in DCM (6%, 6mL). After 20min, Et 3 SiH (6mL) was added, and the mixture was stirred for 1.5h. Then, Py (773mg, 9.77mmol) was added. The mixture was stirred for lOmin, then concentrated, and the residue was purified by reverse phase (CI 8) chromatography eluted with 0-35% ACN in aq H 4 HC0 3 (0.04%) to give the product. LCMS (ES, m/z): 904.2 [M + H] + . H- MR (400MHz, DMSO-i¾): δ 12.11 (s, 1H), 11.73 (s, 1H), 11.23 (s, 1H), 8.75-8.70 (m, 2H), 8.17-8.05 (m, 3H), 7.68-7.55 (m, 4H), 7.20-7.14 (m, 3H), 6.56-6.51 (m, 1H), 6.10-6.05 (m, 1H), 5.32-5.18 (m, 3H), 4.58 (s, 1H), 4.36-4.32 (m, 1H), 4.27-4.20 (m, 4H), 3.62-3.57 (m, 2H), 3.13-3.10 (m, 1H), 2.98-2.93 (m, 1H), 2.80-2.69 (m, 2H), 1.21 (d, J = 8Hz, 1H), 1.13-1.10 (m, 6H). P-NMR: (162MHz, DMSO-i¾): δ 66.02 (s, IP), 47.44-47.30 (m, IP). Step 6: Pyridinium (5S, 7R,8R, 12aR, 14R, 15aS)-2-(2-cyanoethoxy)-7-{2-f(2-methylpropanoyl) aminol-6-oxo-l, 6-dihydro-9H-purin-9-yll-14-{6-f(phenylcarbonyl)aminol-9H-pu rin-9- yl}octahydro-12H-5, 8-methanofurof 3, 2-1] [ 1,3,6,9, 11,2, 1 OJpentaoxadiphosphacyclotetradecme- 10-thiolate 2 -sulfide
To Py (8mL) at -40°C under Ar was added DPCP (467mg, 1.74mmol), and a solution of ammonium 0-((2R,3R,5S)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl )-2- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy) phosphorothioyl)oxy)methyl)-2-(2- isobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl) tetrahydrofuran-3-yl) phosphonothioate (80mg, 0.087mmol) (co-evaporated with Py 3x4mL) in DCM (8mL) dropwise over 5min. The resulting mixture was stirred at -40°C for lh. The reaction mixture was used for the next without purification. LCMS (ES, m/z): 886.2 [M + H] + .
Step 7: Ammonium (5S, 7R,8R, 12aR, 14R, 15aS)-2-(2-cyanoethoxy)-7-{2-f(2-methylpropanoyl) aminol-6-oxo-l, 6-dihydro-9H-purin-9-yll-14-{6-f(phenylcarbonyl)aminol-9H-pu rin-9- yl}octahydro-12H-5, 8-methanofurof 3, 2-1] [ 1,3,6,9, 11,2, 1 OJpentaoxadiphosphacyclotetradecine- 10-thiolate 2, 10-disulfide
To the solution at -40°C from Step 6 was added sulfur (27.9mg, 0.870mmol) in one portion. After stirring at RT for 3h, the reaction mixture was concentrated and purified by reverse phase (CI 8) chromatography eluted with 0-35% ACN in aq H 4 HCO 3 (0.4%) to give the product. LCMS (ES, m/z): 918.2 [M + H] + . Step 8: 2-amino-9-[(5S, 7R8R 12aR 14R 15aS)-l 4-(6-ammo-9H-purin-9-yl)-2-hvdroxy-l 0- sulfanyl-2, 10-disulfidooctahydro-12H-5, 8-methanofurof 3, 2-1] [1, 3, 6,9, 11,2, 101
pentaoxadiphosphacyclotetradecm-7-yl]-l,9-dihydro-6H-purm -6-one
Ammonium (5S,7R,8R, 12aR, 14R, 15aS)-2-(2-cyanoethoxy)-7-{2-[(2- methylpropanoyl)amino]-6-oxo-l,6-dihydro-9H-purin-9-yl}-14-{ 6-[(phenylcarbonyl)amino]- 9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][l,3,6,9, l 1,2, 10]
pentaoxadiphosphacyclotetradecine-10-thiolate 2, 10-disulfide (20mg, 0.020mmol) was dissolved in a solution of Με Η 2 in EtOH (30%, lmL). The mixture was stirred at RT for 2h, then concentrated. The residue was purified by prep-HPLC (XBridge Shield RP C 18 OBD Column, 19mmx250mm) eluted with 4-12% ACN in aq NH 4 HCO 3 (lOmM) over 26min to afford two diastereomers of 2-amino-9-[(5 S,7R,8R, 12aR, 14R, 15aS)- 14-(6-amino-9H-purin-9-yl)-2- hydroxy-10-sulfanyl-2, 10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][l,3,6,9, l 1,2, 10] pentaoxadiphosphacyclotetradecin-7-yl]-l,9-dihydro-6H-purin- 6-one:
Example 10 (diastereomer 1) (T R : 13.88min): LCMS (ES, m/z): 689.0 [M - H] " . 1H- MR (300MHz, D 2 0): δ 8.33 (s, 1H), 8.07 (s, 1H), 8.78 (s, 1H), 6.35-6.33 (m, 1H), 5.73- 5.69 (m, 1H), 5.57-5.56 (m, 1H), 5.30-5.27 (m, 1H), 4.49-4.69 (m, 1H), 4.25-4.08 (m, 3H), 3.98- 3.81 (m, 2H), 2.91-2.72 (m, 2H), 2.56-2.34 (m, 2H). 31 P- MR: (121MHz, D 2 0): δ 1 13.61 (s), 55.67-55.58 (m).
Example 1 1 (diastereomer 2) (T R : 20.67min): LCMS (ES, m/z): 689.0 [M - H] " . 1H- MR: (300MHz, D 2 0): δ 8.29 (s, 1H), 8.27 (s, 1H), 8.24 (s, 1H), 6.37-6.33 (m, 1H), 5.74 (d, J= 7.2Hz, 1H), 5.51-5.43 (m, 1H), 5.33-5.29 (m, 1H), 4.50-4.47 (m, 1H), 4.32-4.29 (m, 1H), 4.22-4.12 (m, 1H), 3.98-3.96 (m, 2H), 3.84-3.78 (m, 1H), 3.00-2.87 (m, 2H), 2.56-2.38 (m, 2H). 31 P- MR: (121MHz, D 2 0): δ 1 15.35 (s), 54.08 (s).
Examples 12 through 15, as shown in Table 2 below, were prepared according to procedures analogous to those outlined in Examples 10 and 1 1 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step. Table 2
Example 16: 2-amino-9-rf5R,7R,8R,12aR,14R,15S,15aR,16SV14-f6-amino-9H-pu rin-9-vn- 15-fluoro-10,16-dihvdroxy-2-sulfanyl-2,10-disulfidooctahydro -12H-5,8-methanofuro[3,2- ll[1 ,9,ll,6,2,101tetraoxathiadiphosphacvclotetradecin-7-yll-l,9- dihydro-6H-purin-6-one
Step 1: (2R 3R, 4S 5R)-5-(6-(N-benzoylbenzamido)-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofura n-3-yl benzoate
To a solution of N-(9-((2R,3 S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-p urin-6-yl)benzamide (2.00g, 2.96mmol) in Py (20ml) at 0°C was added BzCl (0.832g, 5.92mmol) dropwise, and the resulting mixture was stirred at RT for 3h. Then, the mixture was concentrated to give a crude product, which was used for next reaction step without purification. LCMS (ES, m/z): 884.2 [M + H] + . Step 2: (2R 3R.4S.5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydr benzoate
To a solution of the crude product from Step 1 in MeOH (20ml) and THF (10ml) was added H 4 OH (5ml) dropwise, and the resulting mixture was stirred at RT for 30min. Then, the mixture was concentrated to give a crude product, which was used for next reaction step without purification. LCMS (ES, m/z): 780.3 [M + H] + .
Step 3: (2R 3R, 4S, 5R)-5-( 6-benzamido-9H-purin-9-yl)-4-fIuoro-2- (hydroxymethyl)tetrahydrofuran-3-yl benzoate
To a solution of the crude product from Step 2 in DCM (45mL) was added H 2 0 (533mg, 29.6mmol) and 2,2-dichloroacetic acid in DCM (6%, 44.4mL, 26.6mmol). The mixture was stirred at RT for lOmin. Then, Et 3 SiH (70mL) was added to the reaction. After lh, the reaction mixture (at 0°C to 5°C) was treated with Py (4.2g, 53mmol) and stirred at RT for 5min. The resulting solution was concentrated, and EtOAc (300mL) was added. The solution was washed with H 2 0 (3x50mL) and brine (50mL), dried (Na 2 S0 4 ), and purified by chromatography on silica gel using 0-10 % MeOH in DCM to the product. LCMS (ES, m/z): 478.1 [M + H] + . 1H- MR: (400MHz, DMSO-i¾) δ 11.26 (s, 1H), 8.79 (s, 1H), 8.65 (d, J = 2.6Hz, 1H), 8.12-8.00 (m, 4H),7.76-7.69 (m, 1H), 7.67-7.61 (m, 1H), 7.60-7.53 (m, 4H), 6.72 (dd, J= 17.8, 3.9Hz, 1H), 5.79 (d, J= 4.0Hz, 2H), 5.74 (s, 0.5H), 5.66 (s, 0.5H), 5.23 (t, J= 5.9Hz, 1H), 4.32 (d, J = 4.6Hz, 1H), 3.79 (m, 2H).
Step 4: pyridin-l-ium Q-(((2R, 3R 4S,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(benzoyloxy)-4- fluorotetrahydrofuran-2-yl)methyl) Q-((2R, 3R, 4S, 5R)-5-( (bis(4-methoxyphenyl) (phenyl) methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(24sobu
purin-9-yl)tetrahydrothiophen-3-yl) phosphorothioate
To a solution of (2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-
(hydroxymethyl)tetrahydrofuran-3-yl benzoate (275mg, 0.577mmol) and ammonium (2R,3R,4S, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert -butyldimethylsilyl)oxy)-2-(2- isobutyramido-6-oxo-l,6-dihydro-9H-purin-9-yl)tetrahydrothio phen-3-yl phosphonate (500mg, 0.577mmol) in Py (7mL) and DCM (3mL) at -40°C under Ar was added DPCP (3098mg, 11.53mmol) over 5min. The resulting mixture was stirred at -20°C to -40°C for 40min. The reaction mixture was used for the next step without work-up. LCMS (ES, m/z): 1309.4 [M + H] + . Step 5: ammonium 0-(((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(benzoylox y)-4- fluorotetrahydrofuran-2-yl)methyl) Q-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)-2-(2 sobutyramido-6-oxo-L 6-dih^
phosphorothioate
To the reaction mixture from Step 4 at -20°C was added 3H-benzo[c][l,2]dithiol-
3-one (0.146g, 0.866mmol) and H 2 0 (250mg). The mixture was stirred at RT for lh. Then, the reaction mixture was concentrated to give a crude product, which was used for the next reaction step without purification. LCMS (ES, m/z): 1341.2 [M + H] + .
Step 6: ammonium 0-(((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(benzoylox y)-4- fluorotetrahydrofuran-2-yl)methyl) Q-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)-2-(24sobut ramido-6-oxo-L 6-dihydro-9H-purin-9-yl)tetra
phosphor othioate
To a solution of the crude product from Step 5 in DCM (9mL) was added H 2 0
(104mg, 5.77mmol) and 2,2-dichloroacetic acid in DCM (6%, lOmL, 5.2mmol). The mixture was stirred at RT for lOmin and then, Et 3 SiH (15mL) was added. After lh, the mixture was treated with Py (l .Og) and stirred for 5min. The resulting solution was concentrated and purified by reverse phase (C 18) chromatography eluted with 0-95% ACN in aq H 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 1039.3 [M + H] + . 1H- MR (400MHz, DMSO-i¾) δ 12.06-12.01 (m, 1H), 11.62-11.59 (m, 1H), 11.27-11.26 (m, 1H), 8.78-8.77 (m, 1H), 8.68-8.64 (m, 1H), 8.39 (s, 1H), 8.15-8.00 (m, 4H), 7.78-7.51 (m, 6H), 7.31-6.93 (m, 4H), 6.76-6.55 (m, 1H), 5.89-5.86 (m, 1H), 5.77-5.53 (m, 2H), 5.41- 5.22 (m, 2H), 4.67-4.64 (m, 1H), 4.42-4.24 (m, 1H), 4.12-4.09 (m, 1H), 3.89-3.74 (m, 1H), 3.69-3.50 (m, 1H), 3.22 (t, J= 6.8Hz, 1H), 2.85-2.73 (m, 1H), 1.16- 1.02 (m, 6H), 0.90-0.85 (m, 9H), 0.19-0.10 (m, 6H). 31 P- MR: (162MHz, DMSO-i¾): δ 54.64- 53.95 (m).
Step 7: ammonium 0-(((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)methyl) Q-((2R3R4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)-2-(2-isobut ramido-6-oxo-l, 6-dihydro-9H-purin-9-yl)tetrah^
phosphor othioate
To a solution of ammonium 0-(((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)- 3-(benzoyloxy)-4-fluorotetrahydrofuran-2-yl)methyl) 0-((2R,3R,4S,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido -6-oxo-l,6-dihydro-9H-purin-9- yl)tetrahydrothiophen-3-yl) phosphorothioate (400mg, 0.379mmol) in MeOH (15mL), THF (12mL), and H 2 0 (3mL) at 0°C was added aq NaOH (2M, 3mL, 6mmol) over 3min. After lOmin, the reaction mixture was neutralized with AcOH and then concentrated. The residue was purified by reverse phase (CI 8) chromatography eluted with 0-95% ACN in aq NH 4 HCO 3
(5mM) to give the product. LCMS (ES, m/z): 935.2 [M + H] + . 1H-NMR: (400MHz, DMSO-i¾) δ 12.08-12.06 (m, 1H), 1 1.61-11.59 (m, 1H), 11.23 (br s, 1H), 8.76-8.75 (m, 1H), 8.56-8.54 (m, 1H), 8.37-8.35 (m, 1H), 8.06 (d, J= 7.9Hz, 2H), 7.66 (t, J= 7.4Hz, 1H), 7.56 (t, J= 7.6Hz, 2H), 7.28 - 6.94 (m, 4H), 6.64 - 6.44 (m, 1H), 6.16 - 5.83 (m, 2H), 5.41 - 5.10 (m, 3H), 4.71-4.64 (m, 1H), 4.36-4.32 (m, 1H), 4.06 - 3.85 (m, 1H), 3.82 - 3.52 (m, 4H), 3.23-3.19 (m, 1H), 2.85 - 2.71 (m, 1H), 1.15 - 1.04 (m, 6H), 0.90-0.89 (m, 9H), 0.17-0.15 (m, 6H). 31 P-NMR: (162MHz,
Step 8: ammonium (5R, 7R,8R,12aR,14R, 15S,15aR, 16S)-16-{ftert-butyl(dimethyl)silylloxyl-2- f(2,4-dichlorobenzyl)sulfanyl) '-15-fluoro-7-{2-f(2-methylpropanoyl)aminoi '-6-oxo-l ,6-dihydro- 9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}o ctahydro
methanofurof 3, 2-1] [ 1,3,9, 11,6,2,1 O]tetraoxathiadiphosphacyclotetradecine-10-thiolate 10-oxide 2-sulfide
To a solution of ammonium 0-(((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl) 0-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl) oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-l,6-dihydro- 9H-purin-9-yl)
tetrahydrothiophen-3-yl) phosphorothioate (360mg, 0.378mmol, co-evaporated with Py 3x5mL) in CH 3 CN (36mL) and DIEA (4mL) was added a solution of ^-2,4-dichl orobenzyl 0,0-bis(4- oxobenzo[<i][l,2,3]triazin-3(4H)-yl) phosphorodithioate (548mg, 0.945mmol) in 1,4-dioxane (4mL) dropwise over 3min. The mixture was stirred at RT for 16h and then concentrated. The residue was purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq H 4 HCO 3 (5mM) to give a crude product. The crude product was further purified with prep- TLC (developed by 3% MeOH in DCM) to give the product. LCMS (ES, m/z): 1189.0 [M + H] + . 31 P- MR: (162MHz, CD 3 OD): δ 96.19-95.17 (m), 61.86-59.92 (m).
Step 9: diammonium (5R, 7R,8R, 12aR, 14R, 15S, 15aR, 16S)-16-{[tert-butyl(dimethyl)silyl] oxy}-l 5- fiuoro-7-{2-[(2-methylpropanoyl)aminol-6-oxo-l,6-dihydro-9H^ urin-9-yll-14-{6- [ (phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5, 8-methanofurof 3, 2- I] [1,3,9, 11, 6, 2, 1 OJtetraoxathiadiphosphacyclotetradecine-2, 10-bis( thiolate) 10-oxide 2 -sulfide
To a stirred solution of ammonium (5R,7R,8R,12aR, 14R, 15S,15aR,16S)-16- {[tert-butyl(dimethyl)silyl]oxy}-2-[(2,4-dichlorobenzyl)sulf anyl]-15-fluoro-7-{2-[(2- methylpropanoyl)amino]-6-oxo-l,6-dihydro-9H-purin-9-yl}-14-{ 6-[(phenylcarbonyl)amino]- 9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][l,3,9, l 1,6,2,10]
tetraoxathiadiphosphacyclotetradecine-10-thiolate 10-oxide 2-sulfide (lOOmg, 0.082mmol) (co- evaporated with CH3CN 3x5mL) in DMSO (15mL) were added 2-methylundecane-2-thiol (99mg, 0.49mmol) and 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (lOOmg, 0.65mmol). The resulting mixture was stirred at RT for 3h. The mixture was purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq NH 4 HCO 3 (5mM) to give the product. LCMS (ES, m/z): 1029.3 [M + H] + . 31 P-NMR: (162MHz, CD 3 OD): δ 113.80 (s), 61.35 (s).
Step 10: dimethanaminium (5R, 7R,8R, 12aR, 14R, 15S, 15aR, 16S)-7-(2-amino-6-oxo-l, 6-dihydro- 9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(^^ fluorooctahydro-12H-5, 8-methano[uro[ 3, 2-11 [1,3,9,11,6,2,101
tetraoxathiadiphosphacyclotetradecine-2,10-bis(thiolate) 10-oxide 2 -sulfide
Diammonium (5R,7R,8R, 12aR, 14R, 15 S, 15aR, 16S)- 16-{ [tert-butyl(dimethyl) silyl] oxy } - 15 -fluoro-7- { 2- [(2-methylpropanoyl)amino] -6-oxo- 1 , 6-dihydro-9H-purin-9-yl } - 14- {6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-m ethanofuro[3,2-l]
[1,3,9, 1 l,6,2,10]tetraoxathiadiphosphacyclotetradecine-2, 10-bis(thiolate) 10-oxide 2-sulfide (40mg, 0.037mmol) was dissolved in a solution of CH 3 H 2 in EtOH (33%, lOmL). The mixture was stirred at RT for 3h, then was concentrated to give a crude product, which was used for next step without purification. LCMS (ES, m/z): 855.2 [M + H] + .
Step 11: 2-amino-9-[(5R 7R.8R.12aR.14R.15S.15aR16S)-14-(6-amino-9H-vurin-9-yl)-15- fluoro-10, 16-dihvdroxy-2-sul[anyl-2, 10-disulfidooctahydro-12H-5, 8-methanofiirof 3, 2- l] [1,3, 9,11, 6, 2, 1 OJtetraoxathiadiphosphacyclotetradecin- 7- lJ-l, 9-dihydro-6H-purin-6-one
To a stirred solution of the crude product from Step 10 in Py (1.5mL) were added Et 3 N (0.37g, 3.7mmol) and Et 3 N 3HF (0.30g, 1.85mmol). The resulting mixture was heated at 50°C for 24h. The mixture then was concentrated and purified by prep-HPLC (Atlantis Prep T3 OBD Column, 19mmx250mm) eluted with 7-25% ACN in aq NH 4 HC0 3 (lOmM) over 14min to afford a crude product (T R : 11.58min). The crude product was further purified by prep-HPLC (XBridge Prep Phenyl OBD Column, 19mmx250mm) eluted with 6-8% ACN in aq NH 4 HC0 3 (20mM) over 15min to afford the product (T R : 13.5min). LCMS (ES, m/z): 741.2 [M + H] + . 1H- NMR: (400MHz, D 2 0) δ 8.61 (s, 1H), 8.25 (d, J = 3.0Hz, 1H), 8.19 (s, 1H), 6.52 (dd, J = 23.3, 2.6Hz, 1H), 5.96 (d, J = 8.7Hz, 1H), 5.73-5.62 (m, 1H), 5.59 (m, 0.5 H), 5.46 (m, 0.5H), 5.40 (m, 1H), 4.55 (d, J = 3.4Hz, 1H), 4.46-4.28 (m, 2H), 4.25 (m, 2H), 4.00 (m, 1H), 3.63 (m, 1H). ji P- MR: (162MHz, D 2 0) δ 112.91 (s), 60.45 (s).
Example 17: 2-amino-9-rf5R,7R,8S,12aR,14R,15R,15aS,18SV14-f6-amino-9H-pu rin-9-vn- 18-fluoro-10-hvdroxy-10-oxido-2-sulfanyl-2-sulfidohexahydro- 14H-15,12a- fepoxymethano)-5,8-methanofuro[3,2-ll[l,3.,6,9,ll,2,101penta oxadiphosphacvclotetradecin- 7a2HVyll-l,9-dihvdro-6H-purin- -one
Step 1: N-(9-((lR,3R,4R, 7S)-l-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-7-((tert - butyldimethylsilyl)oxy)-2 ', 5-dioxabicyclo[ 2.2.1 ]heptan-3-yl)-9H-purin-6-yl)benzamide
N-(9-((lR,3R,4R,7S)-l-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-7- hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purin-6-yl)be nzamide (800mg, 1.17mmol) was co-evaporated with Py (3x5mL) and then re-dissolved in DMF (5ml) under Ar. To the solution was added imidazole (397mg, 5.84mmol) and tert-butylchlorodimethylsilane (440mg,
2.92mmol), and the solution was stirred at RT for 5h. Then, sat aq NaHC0 3 (5mL), H 2 0 (30ml) and EtOAc (50mL) were added. The layers were separated, and the aq layer was extracted with EtOAc (2x50mL). The combined organic solution was washed with brine (3x60mL), concentrated, and purified by silica gel column chromatography eluted with 1 to 10% MeOH in DCM (0.3% Et 3 N) to give the product. LCMS (ES, m/z): 801.3 [M + H] + . 1H- MR: (400MHz, DMSO-i¾) δ 11.26 (s, 1H), 8.79 (s, 1H), 8.60 (s, 1H), 8.10-8.03 (m, 2H), 7.71-7.62 (m, 1H), 7.59-7.55 (m, 2H), 7.44-7.36 (m, 2H), 7.37-7.18 (m, 7H), 6.94-6.84 (m, 4H), 6.20 (s, 1H), 4.85 (s, 1H), 4.73 (s, 1H), 4.02 (d, J= 8.0Hz, 1H), 3.87 (d, J= 7.9Hz, 1H), 3.73 (s, 6H), 3.47 (d, J = 10.9Hz, 1H), 3.33-3.30 (m, 1H), 0.72 (s, 9H), -0.01 (s, 3H), -0.07 (s, 3H). Step 2: N-(9-((lS,3R,4R, 7S)-7-((tert-butyldimethylsilyl)oxy)-l-(hvdroxymethyl)-2,5- dioxabicycloi 2.2.1 ]heptan-3-yl)-9H-purin-6-yl)benzamide
D T
To a solution of N-(9-((lR,3R,4R,7S)-l-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-7-((tert-butyM
6-yl)benzamide (850mg, 0.956mmol) in DCM (5ml) was added H 2 0 (172mg, 9.56mmol) and 2,2-dichloroacetic acid in DCM (6%, 12ml, 7.20mmol). After 20min, Et 3 SiH (20mL, 124mmol) was added. The resulting solution was stirred at RT for lh. Then, Py (1.36g, 17.2mmol) was added to the mixture, which was stirred forlOmin. The mixture was concentrated, and the residue was purified by reverse phase (CI 8) chromatography eluted with 0-100% ACN in aq H 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 498.2 [M + H] + . 1H- MR: (400MHz, DMSO-i¾) δ 11.23 (s, 1H), 8.77 (s, 1H), 8.56 (s, 1H), 8.09-8.02 (m, 2H), 7.71-7.62 (m, 1H), 7.59-7.55 m, 2H), 6.09 (s, 1H), 5.10-5.07 (m, 1H), 4.68 (s, 1H), 4.59 (s, 1H), 3.96 (d, J= 7.9Hz, 1H), 3.88-3.73 (m, 3H), 0.87 (s, 9H), 0.10 (d, J= 1.3Hz, 6H).
Step 3: yridin-l-ium ((1R,3R,4R, 7S)-3-(6-benzamido-9H-purin-9-yl)-7-((tert- butyldimethylsilyl)oxy)-2 ', 5-dioxabicyclof 2.2. l]heptan-l-yl)methyl ((2R, 3S, 4S, 5R)-5-( fbis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2 sobut ramido-6-oxo
purm-9-yl)tetrahydrofuran-3-yl) hos hite
To a solution of DPCP (3.66g, 13.7mmol) in Py (10ml) at -40°C was under Ar added a solution of N-(9-((l S,3R,4R,7S)-7-((tert-butyldimethylsilyl)oxy)-l-(hydroxymethy l)- 2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purin-6-yl)benzamide (340mg, 0.684mmol, co- evaporated with Py 3xlml) and triethylammonium (2R,3S,4S,5R)-5-((bis(4-methoxyphenyl) (phenyl )methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-l,6-dihyd ro-9H-purin-9- yl)tetrahydrofuran-3-yl phosphonate (560mg, 0.684mmol, co-evaporated with Py 3xlml) in Py (3ml) and DCM (3ml). The mixture was stirred for 40min. The reaction mixture was used for the next step immediately without purification. LCMS (ES, m/z): 1201.4 [M + H] + .
Step 4: yridin-l-ium ((1R,3R,4R, 7S)-3-(6-benzamido-9H-purin-9-yl)-7-((tert- butyldimethylsilyl)oxy)-2 ', 5-dioxabicyclof 2.2. l]heptan-l-yl)methyl ((2R, 3S, 5R)-5-( (bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(24sobutyra m
purin-9-yl)tetrahydrofuran-3-yl) phosphate
To the reaction mixture from Step 3 at -40°C, was added 3% I 2 in Py/H 2 0 (9/1, 6mL). The mixture was stirred at RT for lh. Then, 1.6 g Na 2 S 2 0 3 · 5H 2 0 (1.6g) in H 2 0 (8ml) was added. After 5min, the mixture was concentrated to give a crude product, which was used in next reaction step directly. LCMS (ES, m/z): 1217.3 [M + H] + .
Step 5: ammonium (( 1R 3R,4R S)-3-(6-benzamido-9H-purin-9-yl)-7-((tert- butyldimethylsilyl)oxy)-2 ', 5-dioxabicyclof 2.2. l]heptan-l-yl)methyl ((2R, 3S, 4S, 5R)-4-fluoro-5- (hydroxymethyl)-2-(2 sobutyramido-6-oxo-L 6-dihydro-9H-p
phosphate
To a solution of the crude product from Step 4 in DCM (8mL) were added H 2 0 (90mg, 5.0mmol) and 2,2-dichloroacetic acid in DCM (0.6M, 30mL, 18mmol). The solution was stirred at RT for 30min. Et 3 SiH (16mL) was added, and the resulting solution was stirred for lh. Then, Py (2.85g, 36.0mmol) was added, and the solution was concentrated. The residue purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq H 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 915.2 [M + H] + . 1H MR: (400MHz, MeOH-i¾) δ 8.70 (s, 1H), 8.41 (s, 1H), 8.18-8.11 (m, 2H), 7.92 (s, 1H), 7.73-7.66 (m, 1H), 7.61 (dd, J= 8.3, 6.9Hz, 2H), 6.25 (s, 1H), 5.93 (s, 1H), 5.43-5.26 (m, 2H), 4.67 (s, 1H), 4.62 (s, 1H), 4.57-4.43 (m, 1H), 4.26 (t, J= 4.5Hz, 2H), 4.11 (d, J = 7.9Hz, 1H), 3.96-3.82 (m, 3H), 2.73 (p, J= 6.9Hz, 1H), 1.25 (dd, J = 9.3, 6.9Hz, 6H), 0.91 (s, 9H), 0.14 (d, J= 9.5Hz, 6H). 31 P-NMR: (162MHz,
Step 6: Ammonium ((1R,3R,4R, 7S)-3-(6-benzamido-9H-purin-9-yl)-7-hydroxy-2,5- dioxabicycloi 2.2.1 ]heptan-l-yl)methyl ((2R, 3S, 4S, 5R)-4-fluoro-5-(hydroxymethyl)-2-(2- isobutyramido-6-oxo-l, 6-dihydro-9H-purin-9-yl)tetrahydrofurm phosphate
To a solution of ammonium ((lR,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-7- ((tert-butyldimethylsilyl)oxy)-2,5-dioxabicyclo[2.2. l]heptan-l-yl)methyl ((2R,3S,4S,5R)-4- fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-l,6-dihydr o-9H-purin-9-yl)
tetrahydrofuran-3-yl) phosphate (320mg, 0.343mmol) in Py (5ml) were added Et 3 N 3HF (2768mg, 17.17mmol) and Et 3 N (1737mg, 17.17mmol). The resulting mixture was heated at 50°C for 15h. Then, the mixture was concentrated and purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq H 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 801.1 [M + H] + . 1H MR: (400MHz, MeOH-<¾) δ 8.69 (s, 1H), 8.42 (s, 1H), 8.13 (dd, J= 7.4, 1.8Hz, 2H), 7.93 (s, 1H), 7.74-7.65 (m, 1H), 7.60 (dd, J= 8.4, 7.0Hz, 2H), 6.26 (s, 1H), 5.98 (s, 1H), 5.48 - 5.26 (m, 2H), 4.54 (s, 1H), 4.47 (s, 1H), 4.30 (dd, J= 14.7, 5.5Hz, 2H), 4.11 (d, J= 8.0Hz, 1H), 3.97-3.85 (m, 3H), 2.74 (p, J= 6.9Hz, 1H), 1.25 (dd, J= 9.7, 6.9Hz, 6H). 31 P NMR: (162MHz, MeOH-<¾) δ -1.42 (s, 1).
Step 7: N-{9-f(5R, 7R.8S.12aR.14R.15R.15aS.18S)-2-i(2.4-dichlorobenzyl)sulfanyl l-18-fluoro- 10-hydroxy-7-{2-[(2-methylpropanoyl)aminol-6-oxo-l,6-dihydro -9H^urin-9-yll-10-oxido-2- sulfidohexahydro-14H-15, 12a-(epoxymethano)-5,8-methanofuro[3,2-ll[l,3, 6,9, 11,2, 10] pentaoxadiphosphacyclotetradecin-14(12H)-yl]-9H-purin-6-yl}b enzamide
To a solution of ammonium ((lR,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-7- hydroxy-2,5-dioxabicyclo[2.2.1]heptan-l-yl)methyl ((2R,3S,4S,5R)-4-fluoro-5- (hydroxymethyl)-2-(2-isobutyramido-6-oxo-l,6-dihydro-9H-puri n-9-yl)tetrahydrofuran-3-yl) phosphate (268mg, 0.328mmol, co-evaporated with MeCN 3x2mL) in MeCN (30mL) under Ar added DIEA (3.3mL) and a solution of S-2,4-dichlorobenzyl-0,O-bis(4-oxobenzo[d][l,2,3] triazin-3(4H)-yl) phosphorodithioate (613 mg, 1.06 mmol) in 1,4-dioxane (3.5mL). The mixture was stirred at RT for 18 h, then concentrated. The residue was purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq H 4 HCO 3 (5mM) to give the product. LCMS (ES, m/z): 1053.1 [M - H] " . 31 P MR: (162MHz, MeOH-<¾) δ 95.72(s), 95.02 (s); -1.96(s), -3.17 (s).
Step 8: dipyridinium (5R, 7R,8S,12aR,14R,15R,15aS, 18S)-7-(2-amino-6-oxo-l,6-dihydro-9H- purin-9-yl)-18-fiuoro-14-{6-[(phenylcarbonyl)amino]-9H^
15, 12a-(epoxymethano)-5,8-methanofuro[ 3,2-11 f 1, 3, 6, 9,11, 2,101
pentaoxadiphosphacyclotetradecin-10( 12H)-olate 10-oxide 2 -sulfide
To a solution of N-{9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-2-[(2,4- dichlorobenzyl)sulfanyl]-18-fluoro-10-hydroxy-7-{2-[(2-methy lpropanoyl)amino]-6-oxo-l,6- dihydro-9H-purin-9-yl}-10-oxido-2-sulfidohexahydro-14H-15,12 a-(epoxymethano)-5,8- methanofuro[3 ,2-1] [1,3,6,9, 11,2, 1 OJpentaoxadiphosphacyclotetradecin- 14(12H)-yl]-9H-purin-6- yl}benzamide (35mg, 0.033mmol, co-evaporated with MeCN 3x2 mL) in DMSO (6.3mL) were added 2-methylundecane-2 -thiol (26.7mg, 0.132mmol) and 2,3, 4,6,7,8, 9,10-octahydropyrimido [l,2-a]azepine (30. lmg, 0.198mmol). The mixture was stirred at RT for 3h. The mixture was used for the next step without purification. LCMS (ES, m/z): 895.2 [M + H] + . Step 9: 2-Amino-9-i(5R.7R8S.12aR.14R.15R.15aS.18S)-14-(6-amino-9H-pu rin-9-yl)-18-fluoro- 10-hydroxy-l 0-oxido-2-sulfanyl-2-sulfidohexahvdro-14H-15,12a-(epoxymetha no)-5,8- methanofurof 3, 2-1] [ 1,3,6,9,11,2,1 OJpentaoxadiphosphacyclotetradecin- 7(12H)-yl]-l, 9-dihydro- 6H-purin-6-one
To the reaction mixture from Step 8 was added a solution of Me H 2 in EtOH (30%, 8mL). The resulting solution was stirred at RT for 3h. The solution was concentrated, and the residue was purified prep-HPLC (XBridge Prep Phenyl OBD Column, 19mmx250mm) eluted with 2-14% ACN in aq H 4 HCO 3 (lOmM) over 15min to afford the product (T R :
9.13min). LCMS (ES, m/z): 721.2 [M + H] + . 1H- MR: (400MHz, D 2 0) δ 8.36 (d, J = 1.9Hz, 2H), 8.16 (s, 1H), 7.72 (s, 1H), 6.14 (s, 1H), 6.08 (s, 1H), 5.60-5.44 (m, 2H), 5.02 (d, J = 9.5Hz, 1H), 4.78 (d, J = 6.5Hz, 1H), 4.67-4.50 (m, 1H), 4.36-4.21 (m, 2H), 4.17-4.06 (m, 2H), 4.00 (d, J = 8.5Hz, 1H). 31 P- MR: (162MHz, D 2 0) δ 114.34 (s), -3.21 (s).
Examples 18 through 19, as shown in Table 3 below, were prepared according to procedures analogous to those outlined in Example 17 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.
Table 3
Example 20: 2-amino-9-rf5R,7R,8R,12aR,14R,15S,15aR,16SV14-f6-amino-9H-pu rin-9-vn- 15-fluoro-10,16-dihvdroxy-10-oxido-2-sulfanyl-2-sulfidooctah ydro-12H-5,8- methanofuro [3,2-11 [1 ,9,1 l,6,2,101tetraoxathiadiphosphacvclotetraderin-7-yll-l,9-dihv dro- 6H-purin-6-one
Step 1: ((2R, 3R, 4S,5R)-5-(6-benzamido-9H^urin-9-yl)-3-(benzoyloxy)-4-fluoro^
2-vDmethyl ((2R, 3R, 4S, 5R)-5-( (bis(4-methoxyphenyl) (phenyl)methoxy)methyl)-4-((tert- buMdimethylsilyl)oxy)-2-(2 sobut ramido-6-oxo-l,6-dihydro-9H-purin-9- yl)tetrahydrothiophen- -yl) phosphate
To a mixture containing crude pyridin-l-ium ((2R,3R,4S,5R)-5-(6-benzamido- 9H-purin-9-yl)-3-(benzoyloxy)-4-fluorotetrahydrofuran-2-yl)m ethyl ((2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethyl silyl)oxy)-2-(2-isobutyram 6-oxo-l,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-yl) phosphite (~0.607mmol) in DCM (4mL) and Py (4mL) was added 3% I 2 in Py (1.8ml) and H 2 0 (0.2ml) over 5min. The mixture was stirred at RT for 30min. A solution of Na 2 S 2 0 3 5H 2 0 (0.5g) in H 2 0 (5ml) was added. After 5min, the mixture was concentrated to give a crude product. LCMS (ES, m/z): 1325.3 [M + H] + . Step 2: ((2R, 3R 4S, 5R)-5-( 6-benzamido-9H-purin-9-yl)-3-(benzoyloxy)-4-fluorotetrahydro furan- 2-vDmethyl ((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2- isobutyramido-6-oxo-L 6-dihydro-9H-purin-9-yl)tetrahydroM phosphate
To a solution of the crude product from Step 1 in DCM (8ml) at RT were added
H 2 0 (1 lOmg, 6.0mmol) and 2,2-dichloroacetic acid in DCM (0.6M, 36ml, 22mmol). The solution was stirred at RT for 30min. Then, Et 3 SiH (16ml) was added. The resulting solution was stirred for lh, and then, Py (3.42g, 43.2mmol) was added. The solution was concentrated, and the residue was purified by reverse phase (CI 8) chromatography eluted with 0-95% ACN in aq H 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 1023.1 [M + H] + .
Step 3: ((2R 3R 4S, 5R)-5-( 6-benzamido-9H-purm-9-yl)-4-fIuoro-3-hydroxytetrahydrofuran- 2- yQmethyl ((2R3R S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-( 2 s
6-oxo-l,6-dihydro-9H-purm-9-yl)tetrahydrothiophen-3-yl) phosphate
To a solution of ((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(benzoyloxy)-
4-fluorotetrahydrofuran-2-yl)methyl ((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)-2-(2-isobutyramido-6-oxo-l,6-dihydro-9H-puri n-9-yl)tetrahydrothiophen-3-yl) phosphate (280mg, 0.193mmol) in MeOH (10ml), THF (8ml) and H 2 0 (2mL) at 0°C was added aq NaOH (2N, 2ml, 4mmol) over 3min. After lOmin, the reaction mixture was neutralized with AcOH and concentrated under vacuum. The residue was purified by reverse phase (CI 8) chromatography eluted with 0-95% ACN in aq NH 4 HC0 3 (5mM) to give the product. LCMS (ES, m/z): 919.1 [M + H] + . 1H-NMR: (400MHz, DMSO-i¾) δ 8.76 (s, 1H), 8.57 (d, J= 1.7Hz, 1H), 8.42 (s, 1H), 8.12-8.04 (m, 2H), 7.66 (t, J= 7.4Hz, 1H), 7.56 (t, J= 7.6Hz, 2H), 6.52 (dd, J = 13.2, 4.7Hz, 1H), 6.35 (s, 1H), 5.84 (d, J = 8.8Hz, 1H), 5.36-5.22 (m, 2H), 5.21-5.01 (m, 2H), 4.63 (d, J= 3.4Hz, 1H), 4.36 (d, J= 19.2Hz, 1H), 3.78 (d, J= 5.3Hz, 1H), 3.68 (d, J= 11.7Hz, 2H), 3.56 (d, J = 5.9Hz, 1H), 3.22 (t, J = 7.1Hz, 1H), 2.79 (p, J = 6.8Hz, 1H), 1.10 (dd, J= 6.8, 4.7Hz, 6H), 0.92 (s, 9H), 0.17 (d, J= 4.7Hz, 6H). 31 P-NMR: (162MHz, OMSO-d 6 ) δ -1.15 (s). Step 4: (5R 7R.8R.12aR.14R 15S.15aR.16S)-16-{[tert-butyl(dimethyl)silylloxy}-2-[q
dichlorobenzyDsulfanyl] -15-fluoro- 7-{2-[ (2-methylpropanoyl)amino) '-6-oxo-l, 6-dihydro-9H- purin-9-yft-14-{6-[(phenylcarbonyl)aminol-9H-purin-9-yl\octa ^
11 [1,3,9,11, 6,2, 101 tetraoxathiadiphosphacyclotetradecin-10-olate 10-oxide 2 -sulfide
To a solution of ((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)methyl ((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)-2-(2-isobutyramido-6-oxo-l,6-dihydro-9H-puri n-9-yl)tetrahydrothiophen-3-yl) phosphate (180mg, 0.196mmol, co-evaporated with Py 3x4ml) in MeCN (18ml) was added diisopropylamine (2ml) and S-2,4-dichlorobenzyl 0,0-bis(4-oxobenzo[d][l,2,3]triazin-3(4H)-yl) phosphorodithioate in 1,4-dioxane (213mg, 0.369mmol). The resulting mixture was stirred at RT for 16h. The mixture was concentrated, and the residue was purified by reverse phase (CI 8) chromatography eluted with 0 to 95% ACN in aq H 4 HCO 3 (5mM) to give a crude product. It was further purified by prep-TLC developed with 5% MeOH in DCM (0.2% Et 3 N) to give the product. LCMS (ES, m/z): 1173.0 [M + H] + .
Step 5: (5R 7R.8R.12aR.14R.15S.15aR.16S)-16-{[tert-butyl(dimethyl)silyll oxy}-15-fluoro-7-{2- i(2-methylpropanoyl)amino) -6-oxo-l, 6-dihydro-9H-purin-9-yll-14-{6-[(phenylcarbonyl)aminol- 9H-purin-9-yll-2-sulfidooctahydro-12H-5,8-methano[uro [3,2-11
[1,3,9, 11,6,2,1 OJtetraoxathiadiphosphacyclotetradecin-10-olate 10-oxide 2 -sulfide
To a stirred solution of (5R,7R,8R, 12aR, 14R, 15S, 15aR, 16S)-16-{[tert- butyl(dimethyl)silyl]oxy}-2-[(2,4-dichlorobenzyl)sulfanyl]-1 5-fluoro-7-{2-[(2-methylpropanoyl) amino]-6-oxo-l,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbon yl)amino]-9H-purin-9- yl } octahydro- 12H-5,8-methanofuro[3 ,2-1] [1,3,9,11,6,2,1 OJtetraoxathiadiphosphacyclotetradecin- 10-olate 10-oxide 2-sulfide triethylammonium salt (30mg, 0.025mmol, co-evaporated with MeCN 3x2mL) in DMSO (2.5mL) were added 2-methylundecane-2-thiol (30mg, 0.15mmol) and 2,3, 4,6,7,8, 9,10-octahydropyrimido[l,2-a]azepine (30mg, 0.20mmol). The resulted mixture was stirred at RT for 3h. The reaction mixture was used for next step without purification. LCMS (ES, m/z): 1013.2 [M + H] + .
Step 6: (5R 7R.8R.12aR.14R 15S.15aR.16S)-7-(2-amino-6-oxo-1.6-dihydro-9H-purin-9-yl)-14 - (6-amino-9H-purin-9-yl)-l6-{[tert-bu1yl(dimethyl)silyl]oxy}- l5-fiuom
5, 8-methanofurof 3, 2-1 If 1,3,9, 11, 6, 2, 101 tetraoxathiadiphosphacyclotetradecin-10-olate 10-oxide 2 -sulfide
To the reaction mixture from Step 5 was added Me H 2 in EtOH (33%, 3.65ml). The resulting solution was stirred at ET for 3h. The solution was concentrated, and the residue was purified by reverse phase (CI 8) chromatography eluted with 0-95% ACN in aq H 4 HCO 3 (5mM) to give the product. LCMS (ES, m/z): 839.2 [M + H] + .
Step 7: 2-amino-9-f(5R 7R.8R.12aR.14R.15S.15aR 16S)-14-(6-amino-9H-purin-9-yl)-15-fluoro-
10,16-dihydroxy-l 0-oxido-2-sulfanyl-2-sulfidooctahydro-12H-5, 8-methanofurof 3, 2-
II fl, 3, 9,11, 6,2, lOltetraoxathiadiphosphacyclotetradecin- 7-yl 1-1, 9-dihydro-6H-purin-6-one
(5R,7R,8R, 12aR, 14R, 15S, 15aR, 16S)-7-(2-amino-6-oxo-l,6-dihydro-9H-purin-9- yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)sily l]oxy}-15-fluoro-2- sulfidooctahydro- 12H-5, 8-methanofuro[3 ,2-1] [1,3,9, 11,6,2,10]
tetraoxathiadiphosphacyclotetradecin-10-olate 10-oxide 2-sulfide (12mg, 0.013mmol) was suspended in Py (1.5mL) under Ar, and Et 3 N (121mg, 1.20mmol) and Et 3 N 3HF (144mg, 0.896mmol) were added. The mixture was warmed at 50°C for 16h. Then, the mixture was concentrated and purified by prep-HPLC (Atlantis Prep T3 OBD Column, 19mmx250mm) eluted with 6-15% ACN in aq H 4 HCO 3 (lOmM) over 1 lmin to afford the product (T R :
8.87min). LCMS (ES, m/z): 725.1 [M + H] + . 1H- MR: (400MHz, D 2 0): δ 8.40 (s, 1H), 8.27 (d, J= 2.6Hz, 1H), 8.22 (s, 1H), 6.53 (dd, J = 19.9, 3.3Hz, 1H), 5.96 (d, J= 8.7Hz, 1H), 5.71- 5.59 (m, 1H), 5.44-5.27 (m, 2H), 4.64 (d, J= 3.4Hz, 1H), 4.42-4.38 (m, 2H), 4.33-4.25 (m, 1H), 4.09 (t, J= 6.2Hz, 2H), 3.67-3.65 (m, 1H). 31 P-NMR: (162MHz, D 2 0) δ 112.88 (s), 0.90 (s).
Example 21, as shown in Table 4 below, was prepared according to procedures analogous to those outlined in Example 20 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.
Table 4
Example 22: 2-amino-9-rf5R,7R,8S,12aR,14R,15S,15aR,16RV14-f6-amino-9H-pu rin-9-vn- 15,16-difluoro-2-hvdroxy-2-oxido-10-sulfanyl-10-sulfidooctah ydro-12H-5,8- methanofuro[3,2-ll[l,3.,6,9,ll,2,101pentaoxadiphosphacvclote tradecin-7-yll-l,9-dihvdro- 6H-purin-6-one
Step 1: Q-(((2R, 3R 4S,5R)-5-(6-benzamido-9H^urin-9-yl)-4-fluoro-3-hvdroxytetrah vdrofuran- 2-vDmethyl) 0-((2R3SAR5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4-fluoro-2 isobutyramido-6-oxo-lH-purin-9(6H)-yl)tetrahydrofuran-3-yl) S-2, 4-dichlorobenzyl phosphor odithioate
2,4-dichlorobenzyl phosphorodichloridodithioate (714mg, 2.19mmol) and 3- hydroxybenzo[d][l,2,3]triazin-4(3H)-one (734mg, 4.50mmol) were kept under high vacuum for 2h, then added 1,4-dioxane (4ml) and Py (0.492ml, 6.08mmol). The mixture was stirred at RT for 2h, and resulting solid was filtered off. The solid was washed with 1,4- dioxane (3ml), and the combined filtrate was concentrated. DMF (5ml), activated 4A molecular sieve (300mg) and Py (0.492ml, 6.08mmol) were added to the residue. The mixture was stirred for 20min, and N- (9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)me thyl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-lH-purin-2-yl )isobutyramide (800mg,
1.22mmol) was added. After 5h, N-(9-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (454mg, 1.22mmol), Py (1ml) and 1-methylimidazole (0.97ml, 12mmol) were added. The mixture was stirred at RT overnight. Then, EtOAc (30 ml) and H 2 0 (20ml) were added. The layers were separated, and the organic layer was dried (Na 2 S0 4 ) and purified by column chromatography on silica gel eluted with 0 to 4% MeOH in DCM with (1% Et 3 N) to give the product. LCMS (ES, m/z): 1285 [M + H] + . Step 2: Q-(((2R, 3R, 4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetra hydrofuran- 2-yl)methyl) S-2, 4-dichlorobenzyl Q-((2R, 3S, 4R 5R)-4-fluoro-5-(hydroxymethyl)-2-(2- isobutyramido-6-oxo-lH-purin-9(6H)-yl)tetrahydrofuran-3-yl) phosphorodithioate
To a solution of 0-(((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3- hydroxytetrahydrofuran-2-yl)methyl) 0-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-lH-purin-9 (6H)-yl)tetrahydrofuran-3-yl) S-2,4-dichlorobenzyl phosphorodithioate (1.2g, 0.37mmol) in MeCN (15ml) was added pyrrole (0.078ml, l . lmmol) and trifluoroacetic acid (0.288ml, 3.74mmol). The resulting mixture was stirred at RT for 30min. The mixture was concentrated, and the residue was purified by column chromatography on silica gel, eluted with 0-5% MeOH in DCM to give the product. LCMS (ES, m/z): 982 [M + H] + .
Step 3: N-{9-f(5R 7R.8S. J2aR.14R, 15S.15aR.16R)-2-(2-cyanoethoxy)-10-i(2.4- dichlorobenzyDsulfanyl) '-15, 16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-l,6-dihydr o- 9H-purin-9-yl}-2-oxido-10-sulfidooctahydro-12H-5, 8-methanofurof 3, 2- 11 [1,3, 6,9, 11,2, 101pentaoxadiphosphacyclotetradecin-14-yll-9H-purin-6-yllben zamide
Diisopropylammonium tetrazolide (59.3mg, 0.346mmol) and 0-(((2R,3R,4S,5R)- 5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofu ran-2-yl)methyl) S-2,4- dichlorobenzyl 0-((2R,3 S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo -lH- purin-9(6H)-yl)tetrahydrofuran-3-yl) phosphorodithioate (170 mg, 0.173 mmol) were co- evaporated with CH 3 CN (3x5ml) and kept under high vacuum for 30min. To the residue was added MeCN (10ml), DMF (lmL), and activated 4A molecular sieve (300mg), and the mixture was stirred at RT for 20min. Then, cyanoethyl Ν,Ν,Ν',Ν'-tetraisopropylphosphorodiamidite (67.8mg, 0.225mmol) in CH 3 CN (1ml) was added to the mixture. After 20min, lH-tetrazole (60.7mg, 0.866mmol) was added. The reaction was stirred at RT for 1.5h, and then, tert-butyl hydroperoxide (5.0M in decane, 0.104ml, 0.519mmol) was added. The mixture was stirred at RT for lh. Then, EtOAc (30 ml) and aq Na 2 S 2 0 3 (5%, 10ml) were added. The layers were separated, and the organic layer was washed with brine, dried (Na 2 S0 4 ) and purified by column chromatography on silica gel eluted with 0-10% MeOH in DCM to give the product. LCMS (ES, m/z): 1096 [M + H] + . Step 4: 2-Amino-9-i(5R.7R.8S.12aR.14R.15S.15aR.16R)-14-(6-amino-9H-v urin-9-yl)-15.16- difluoro-2-hvdroxy-2-oxido-10-sulfanyl-10-sulfidooctahydro-1 2H-5, 8-methanofurof 3, 2- IJf 1,3,6,9,11,2,1 OJpentaoxadiphosphacyclotetradecin- 7-ylJ-l, 9-dihydro-6H-purin-6-one
To a solution of N-{9-[(5R,7R,8S,12aR, 14R,15S,15aR, 16R)-2-(2-cyanoethoxy)-
10-[(2,4-dichlorobenzyl)sulfanyl]-15, 16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-l,6- dihydro-9H-purin-9-yl}-2-oxido-10-sulfidooctahydro-12H-5,8-m ethanofuro[3,2-l]
[1,3,6,9,11,2, 10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}ben zamide (70mg, 0.064mmol) in DMSO (1ml) was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (0.038ml, 0.25mmol) and 2-methylundecane-2-thiol (0.090ml, 0.38mmol). The resulting mixture was stirred at RT for 3h, and then, Me H 2 in EtOH (30%, 2ml) was added. It was stirred at RT for 2h. The reaction mixture was concentrated and purified by prep-HPLC (X- Bridge BEH 150 Prep C18) eluted with ACN in aq H 4 HCO 3 (lOOmM) to give the product. LCMS (ES, m/z): 709 [M - H] " . 1H- MR (500MHz, D 2 0): δ 8.36 (1H, s), 8.23 (1H, s), 8.10 (1H, s), 6.53 (1H, dd, J = 21.0, 2.9Hz), 6.10 (1H, d, J = 8.6Hz), 5.44 (1H, d, J = 49.5Hz), 5.33- 5.22 (2H, m), 4.65 (2H, m), 4.44 (1H, d, J = 9.9Hz), 4.38-4.26 (2H, m), 4.14 (1H, d, J = 12.1Hz), 4.06 (1H, d, J = 9.6Hz). 31 P- MR: (202MHz, D 2 0): δ 118, - 1.4.
Example 23, as shown in Table 5 below, was prepared according to procedures analogous to those outlined in Example 22 above using the appropriate monomers, described as Preparations or as obtained from commercial sources, in the coupling step.
Table 5
Example 24: 2-amino-9-rf5SJR,8Rq2aR,14R,15aS)-10-hvdroxy-14-f6-oxo-l,6-d ihvdro-9H- purin-9-vn-2-sulfanyl-2,10-disulfidooctahvdro-12H-5,8-methan ofuror3,2-/i ri,3,6,9,ll,2,101 pentaoxadiphosphacvclotetradecin-7-yll-l,9-dihydro-6H-purin- 6-one (Diastereomer 1)
To 2-amino-9-[(5 S, 7R, 8R, 12aR, 14R, 15 aS)- 14-(6-amino-9H-purin-9-yl)- 10- hydroxy-2-sulfanyl-2, 10-disulfi dooctahydro- 12H-5,8-methanofuro[3 ,2-1] [1,3,6,9, 11,2, 10] pentaoxadiphosphacyclotetradecin-7-yl]-l,9-dihydro-6H-purin- 6-one (Example 3) (4.2 mg, 6.0 μπιοΐ) were added NaP0 4 H 2 buffer (pH 6.8, 50mM, lmL) and adenosine monphosphate deaminase (5mg). The reaction mixture was left to stir overnight, filtered, and purified by reverse phase HPLC (eluting MeCN/H 2 0 gradient with lOOmM TEAA modifier, linear gradient) to afford the product. LCMS (ES, m/z): 690 [M - H] " . 1H MR (600MHz, D 2 0): δ 8.62 (s, 1H), 8.33 (s, 1H), 8.21 (s, 1H), 6.54 (m, 1H), 5.92 (d, J = 7.3Hz, 1H), 5.65 (m, 1H), 5.42 (m, 1H), 4.67 (d, J = 8.4Hz, 1H), 4.48 (m, 1H), 4.41 (m, 1H), 4.24 (m, 1H), 4.10 (m, 1H), 3.97 (m, 1H), 3.08 (m, 2H), 2.64 (m, 1H), 2.52 (m, 1H).
Examples 25 through 27, as shown in Table 6 below, were prepared according to procedures analogous to those outlined in Example 24 above using the appropriate monomers, described as Preparations or Examples, or as obtained from commercial sources, in the coupling step.
Table 6
BIOLOGICAL EVALUATION
The individual compounds described in the Examples herein are defined as STING agonists by (i) binding to the STING protein as evidenced by a reduction in binding of tritiated cGAMP ligand to the STING protein by at least 20% at 20uM (concentration of compound being tested) in a STING Biochemical [3H]cGAMP Competition Assay and/or (ii) demonstrating interferon production with a 6% or greater induction of IFN-β secretion at 30uM in the ΊΉΡ1 ceil assay (where induction caused by cGAMP at 30uM was set at 100%).
[ 3 H1-cGAMP Synthesis
2.3mL of buffer solution containing 80mM TrisCl, 200mM MgCl 2 , and 20mM NaCl followed by 0.32mL of a lOmM aq solution of GTP was added to a plastic 50mL AMICON tube. A solution of [ 3 H]ATP (21Ci/mmol, 45mCi) in 0.5mL H 2 0 was then added followed by lmL of a lmg/mL solution of DNA (Herring testes activator DNA, Sigma, #D6898) and 53uL of a 47mM solution of cGAS enzyme. Additional H 2 0 was added to bring the total volume to lOmL.
The reaction was stirred for 2h at 37°C and then added directly to an Amicon Ultra-15 10K centrifuge tube and spun for lh at 4,000g. The collected solution was then purified on a semi-prep Mono Q column using the following mobile phases:
A: 0.05M TrisCl pH 8.5 adjusted with 1M NaOH
B: 0.05M TrisCl, 0.5M NaCl pH 8.5 adjusted with 1M NaOH Gradient: 100% A for 5min followed by a linear gradient to 50:50 (A:B) over 25min, 3mL/min, 254nm.
The collected product fractions were pooled and adjusted to a total volume of 30mL with buffer A. A total yield of 15.5mCi of [ 3 H]cGAMP was isolated at a radiochemical purity of 98.0% at a specific activity of 21.5Ci/mmol. cGAS Enzyme
A recombinant DNA vector was chemically synthesized to express the truncated human cGAS enzyme (residues 161-522). To aid in expression and purification, the amino terminus contains a hexahistidine tag, SUMO tag and TEV cleavage site. The recombinant enzyme was overexpressed in ROSETTA™ 2(DE3) Single Competent Cells (Novagen). Affinity purification was carried out using HIS-Select HF Nickel Affinity Gel (Sigma) followed by size exclusion chromatography using a Hi-Load 26/60 SUPERDEX200 prep grade column (GE
Healthcare). Fractions were pooled, concentrated, flash-frozen in liquid nitrogen and stored at -80°C until needed.
Example 28: 3 H-cGAMP filtration binding assay (HAQ STING)
The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter- binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni cell membranes (T.ni; Expression Systems, cat # 94-002F, www.expressionsystems.com)
overexpressing full-length HAQ STING and tritiated cGAMP ligand.
The basic HAQ STING filtration assay protocol is as follows:
The compounds were serially titrated by the Hamilton STARPlus CORE in a 96- well plate (Greiner, #651201) using a 1 :3 ten-point dose response format. After compound preparation, a 2.2ug/ml working concentration of STING membrane (SEQ. ID. No. 1) was prepared by diluting concentrated membrane into assay buffer (lx PBS; Invitrogen
#SH30028.02) and douncing 7x using a manual tissue homogenizer (Wheaton, #357546). 148uL of prepared membrane was then manually added to each well of a 96-well deep-well
polypropylene plate (Fisher Scientific, #12-566-121). Following membrane addition, 2uL of either titrated test compound, DMSO control (Sigma #276855), or cold cGAMP control was added to the appropriate wells using a BIOMEK FX. Compound and membrane then preincubated for 60min at RT to allow compound binding to equilibrate. Following equilibration, 8nM of [ 3 H] c-GAMP ligand was prepared by diluting into assay buffer, and 50uL of this working stock was then manually added to each well of the assay plate. Plates were then incubated at RT for 60min, and the contents of each assay plate were then filtered through a 96-well GF/B filter plate (PerkinElmer, #6005250) using a TOMTEC MACH III Cell Harvester equipped with 20mM HEPES buffer (Fisher Scientific, #BP299500). The filter plates were then dried at 55°C for 30min using a pressurized oven before 30uL of ULTIMA GOLD F scintillate was added to each well. Tritium levels for each reaction well were then measured using a PerkinElmer TopCount plate reader.
After normalization to controls, the percent activity for each compound concentration was calculated by measuring the amount of remaining radioactivity. The plot of percent activity versus the log of compound concentration was fit with a 4-parameter dose response equation to calculate EC 50 values.
The final reaction conditions were:
Component Volume (uL) Final Concentration
STING membrane 148 1.5ug/ml
3 H-cGAMP 50 2.0nM
Low Control (cold cGAMP) 2 lOuM
Test compound/DMSO 2 lOuM
Compound concentrations tested were 20.000, 637.00, 2.200, 0.740, 0.247, 0.082, 0.027, 0.009, 0.003, and Ο.ΟΟΙμΜ with 1.0% residual DMSO.
Full-Length STING (ΉΑΟ) Virus Generation
STING virus was generated using an insect cell baculovirus system. Spodoptera frugiperda S/21 cells (Kempbio, Inc.) were diluted to 5e5 cells/ml in Sf-900II SFM media (LifeTechnologies #10902088) without antibiotics. The cell suspension was added to each well of a treated 6-well plate (2mL per well, le6 cells total), and the cells were allowed to adhere for at least 30min. Meanwhile, a lmL co-transfection mix was assembled by combining 500ng of HAQ STING [STING(l-379)R71H,G230A,H232R,R293Q-GG-AviTag-GS-HRV3C- HIS8/pBACl] DNA (Genewiz custom synthesis) with lmL Sf-900II SFM media containing ΙΟμί Cellfectin® II Reagent (Invitrogen #10362100) and lOOng viral backbone BestBac 2.0, v- cath/chiA Deleted Linearized Baculovirus DNA (Expression Systems #91-002). The
transfection mixtures were allowed to incubate for 30min. After incubation, media was gently removed from the adhered cells in the 6-well plate, the lmL transfection mixtures were added (lmL per well), and the plate was placed in a humidified incubator at 27°C. The following day, lmL Sf-900II SFM media (no antibiotics) was added to each well of the 6-well plate. After media addition, the cells were allowed to incubate with DNA (SEQ. ID. No. 2) at 27°C for 5-7 days to generate the P0 viral stock. To generate PI viral stocks, 0.5mL of P0 viral supernatant was added to 50mL uninfected S/21 cells (seeded the day prior to infection at a density of 5xl0 5 cells/mL to allow for one overnight doubling) in Sf-900II SFM media containing 5μg/mL gentamicin (Invitrogen #15710072). The infected cells were then incubated at 27°C for 3days while shaking at 1 lOrpm (ATR Biotech Multitron Infors HT #AJ118). On day 3, PI cultures were counted using a ViCell XR (Beckman Coulter Life Sciences #383556) to confirm infection had occurred (cell size >3μπι larger than uninfected cells and viability approximately 85-95%). Cultures were harvested in 50mL conical tubes and centrifuged at 2000xg for lOmin at 4°C. The PI viral supernatants were poured off into clean 50ml centrifuge tubes, and the remaining PI cell pellets were used to generate Baculovirus Infected Insect Cells (BIICs). Cryopreservation media containing Sf-900II SFM media with 10% heat inactivated FBS, 10% DMSO (Sigma #D2650), and 5μg/ml gentamicin was prepared and sterilized through 0.22μΜ filter immediately prior to use. PI cell pellets were resuspended to a density of 2e7 cells/ml and aliquoted into cryovials (lmL per vial). Cryovials were placed in MR. FROSTY™ cell freezers O/N at -80°C and transferred to liquid nitrogen for long term storage the following day. To generate P2 viral stock, 0.5mL of the PI viral supernatant was added to 50mL uninfected S/21 cells (seeded the day prior to infection at a density of 5xl0 5 cells/mL to allow for one overnight doubling) in Sf- 900II SFM media containing 5μg/mL gentamicin. These cells were incubated at 27°C for 3days while shaking at 1 lOrpm before harvesting P2 stock with centrifugation at 2000xg for lOmin at 4°C. The P2 viral supernatants were poured off and discarded, while the P2 cell pellets were used to generate P2 BIICs following the same protocol described above. The baculovirus generation protocol has been validated to consistently produce P1/P2 BIICs with titers of 2e9 pfu/mL (2e7 cells/mLxlOO pfu/cell).
Full-Length STING (HAQ) Expression
To generate STING membranes, P1/P2 BIICs were amplified overnight by adding thawed BIICs to S/21 cells seeded at a density of 1.0xl0 6 cells/mL. The volume of BIIC used to infect the culture was calculated using an assumed BIIC titer of 2e9 pfu/ml to achieve an MOI of 10 in the overnight amplification. After culturing overnight, the cells were counted on a ViCell XR to confirm infection had occurred (cell size >3μπι larger than uninfected cells and viability approximately 80-90%). The volume of infected Sf21 cells from the overnight amplification used to infect the large-scale expression of Trichoplusia ni (T.ni; Expression Systems, cat # 94- 002F, www. expressi onsystems. corn) seeded at a density of 1.0x10 6 in cell media (ESF921 SFM containing 5μg/mL gentamicin) at MOI = 2.0 was calculated based on (100 pfu/infected S/21 cell). The cells were allowed to express for 48h at 27°C before harvesting the cell pellet, by centrifugation at 3,400xg for lOmin at 4°C. T. ni cells were counted on a ViCell XR to confirm infection had occurred (cell size >3μπι larger than uninfected cells and viability approximately 80-90%) prior to harvest. Full-Length STING (HAQ) Membrane Generation
Buffer stock reagents:
1) 1M HEPES pH 7.5, Teknova, Cat#H1035
2) 5M NaCl, Sigma Aldrich, Cat#S5150-lL
3) KC1, Sigma Aldrich, Cat#319309-500ML
4) Complete EDTA-free protease inhibitor tablets, Roche Diagnostics, Cat#l 1873580001
5) Benzonase, Universal Nuclease, Pierce, Cat#88702
Lysis buffer [25mM HEPES pH 7.5, lOmM MgCl 2 , 20mM KC1, (Benzonase 1 :5000, Complete Protease Inhibitor tab/50mL)] was added to the pellet of cells expressing full- length STING (HAQ) prepared above at 5mL Lysis buffer per g of cell pellet. The pellet was resuspended and dounced twenty times using a Wheaton Dounce Homogenizer to disrupt the cell membrane. Homogenized lysate was then passed through the EMULSIFLEX-C5 microfiuidizer at a pressure close to 5000PSI. The resuspended pellet was centrifuged at 36,000rpm (100,000xg) in a 45Ti rotor ultra-high speed centrifuge for 45min, 4°C. The supernatant was removed. The pellet then was resuspended in wash buffer [(25mM HEPES pH7.5, ImM MgCl 2 , 20mM KC1, 1M NaCl (Complete Protease Inhibitor tab/50mL)] at a volume of 50mL pellet/centrifuge tube. The pellet/wash buffer mixture was then homogenized, using a glass homogenizer on ice (20 strokes), followed by centrifugation at 36,000rpm for 45min at 4°C. The supernatant was removed. The wash step was repeated once more. The resulting membrane was resuspended in 20mM HEPES pH 7.5, 500mM NaCl, 10% glycerol, EDTA-free Protease Inhibitors
(ltablet/50mL). The protein concentration was measured by Bradford assay (Bio-Rad Protein Assay, Cat#500-0006), and protein enrichment was determined by SDS-PAGE and confirmed by Western blot. The resuspended membranes were stored at -80°C. Full-Length HAQ STING [STING(l-379)R71H,G230A,H232R,R293Q-GG-AviTag-GS-HRV3C- HIS8] Amino Acid Sequence:
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLL LNGVC SLAEELHHIHSRYRGS YWRTVRACLGCPLRRGALLLLSIYF YYSLPNAVGPPFT WMLALLGLSQALNILLGLKGLAPAEISAVCEKG FNVAHGLAWSYYIGYLRLILPELQA RIRTYNQHYN LLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTADRAGIK DRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCQTL EDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTS T MSQEPELLISGMEKPLPLRTDFSGGGL DIFEAQKIEWHEGSLEVLFQGPHHHHHHHH
(SEQ. ID. No. 1)
Full-length HAQ [STING(l-379JR71H, G230A,H232R,R293Q-GG-AviTag-GS-HRV3C- HIS8/pBACl] PlasmidDNA Sequence:
GGAACGGCTCCGCCC ACTATTAATGAAATTAAAAATTCC AATTTTAAAAAACGCAG CAAGAGAAACATTTGTATGAAAGAATGCGTAGAAGGAAAGAAAAATGTCGTCGAC ATGCTGAACAACAAGATTAATATGCCTCCGTGTATAAAAAAAATATTGAACGATTTG AAAGAAAACAATGTACCGCGCGGCGGTATGTACAGGAAGAGGTTTATACTAAACTG TTACATTGCAAACGTGGTTTCGTGTGCCAAGTGTGAAAACCGATGTTTAATCAAGGC TCTGACGCATTTCTACAACCACGACTCCAAGTGTGTGGGTGAAGTCATGCATCTTTT A ATC A A ATC CC A AGATGTGT AT A A AC C AC C A A AC TGC C A A A A A ATGA A A AC TGTC G ACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTG AATAATAAAACAATTATAAATGCTAAATTTGTTTTTTATTAACGATACAAACCAAAC GCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTG AGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATT TTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTC ATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCG TATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAG TACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTC GGAGTGTGTTGCTTT AATTATTAAATTTAT AT AATC AATGAATTTGGGATCGTCGGTT TTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTT TTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAA ACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAAT AACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCA ATATATAGTTGCTGATCAGATCTGATCATGGAGATAATTAAAATGATAACCATCTCG CAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAA ATATAGGATCCATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTC ACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGG GGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCC TGCAGCTGGGACTGCTGTTAAACGGGGTCTGCAGCCTGGCTGAGGAGCTGCACCAC ATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGGGCCTGCCTGGGCTG CCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCCCA AATGCGGTCGGCCCGCCCTTC ACTTGGATGCTTGCCCTCCTGGGCCTCTCGC AGGC A CTGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGT GAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATGGTCATATTACATCGGATA TCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTCGAACTTACAATCAGCATTA CAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCCATTGGA CTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCC AACATTCGCTTCCTGGATAA ACTGCCCCAGCAGACCGCTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACA GCATCTATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTAC GCCACCCCCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTGGCTTTAGC CGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCAGACACTTGAGGACATCCT GGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGC AGATGACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGG AAAAGGAAGAGGTTACTGTGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCC ACGATGTCCCAAGAGCCTGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCTCTC CGCACGGATTTCTCTGGCGGTGGCCTGAACGACATCTTCGAAGCCCAGAAAATCGA ATGGCATGAAGGCAGCCTGGAAGTGCTGTTCCAGGGCCCACACCACCATCATCACC ATCACCATTAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACTAACCTAGG TAGCTGAGCGCATGCAAGCTGATCCGGGTTATTAGTACATTTATTAAGCGCTAGATT CTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTT ATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATA TCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCA AACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAAC GCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGT GTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTC TTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTT GGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCC AACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCC TATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAAT TATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAAT TCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAA ATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCG CAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGG CGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACT GGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTT CTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCC GTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGG AGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAA TTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTG GCTGCAC AACGGAAGGTCGTCTGCTTCGAGGC AGCGCTTGGGGTGGTGGC AATTCA ATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTA TCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGA GATTGTCTCAAGCTCGGATCGATCCCGCACGCCGATAACAAGCCTTTTCATTTTTACT ACAGCATTGTAGTGGCGAGACACTTCGCTGTCGTCGAGGTTTAAACGCTTCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTAC ATGATCCCCC ATGTTGTGC AAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC AGTT CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCG CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTT TCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACG GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAG GGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA ACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCCATTCGCCATTCAGGCTGC GCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCA
(SEQ. ID. No. 2) Certain compounds of the disclosure were evaluated in HAQ STING in vitro binding assay as described above. The following table tabulates the biological data for these compounds as EC50 values.
Table 7: 3 H-cGAMP filtration binding assay for HAQ STING
Example 29: 3 H-cGAMP filtration binding assay (WT STING)
The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter- binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni cell membranes T.ni; Expression Systems, cat #94-002F, ww w ex ressions stem con: )
overexpressing full-length WT STING and tritiated cGAMP ligand.
The basic WT STING filtration assay protocol is as follows:
16nM of [ 3 H] c-GAMP ligand was prepared by diluting into assay buffer, and 50uL of this working stock was manually added to each well of the assay plate. After ligand addition, 2uL of either titrated test compound, DMSO control (Sigma #276855), or cold cGAMP control was added to the appropriate wells using a BIOMEK FX. The serially titrated compound was prepared on a Hamilton STARPlus CORE in a 96-well plate (Greiner, #651201) using a 1 :3 ten-point dose response format. Following compound addition, a 2.2ug/ml working
concentration of STING membrane (SEQ. ID. No. 3) was prepared by diluting concentrated membrane into assay buffer (lx PBS; Invitrogen #SH30028.02) and douncing 7x using a manual tissue homogenizer (Wheaton, #357546). 148uL of this prepared membrane was then manually added to each well of a 96-well deep-well polypropylene plate (Fisher Scientific, #12-566-121). Compound, ligand, and membrane then incubated for 60min at RT before the contents of each assay plate were filtered through a 96-well GF/B filter plate (PerkinElmer, #6005250) using a
- I l l - TOMTEC MACH III Cell Harvester equipped with 20mM HEPES buffer (Fisher Scientific, #BP299500). The filter plates were then dried at 55°C for 30min using a pressurized VWR oven before 30uL of ULTIMA GOLD F scintillate was added to each well. Tritium levels for each reaction well were then measured using a PerkinElmer TopCount plate reader.
After normalization to controls, the percent activity for each compound concentration was calculated by measuring the amount of remaining radioactivity. The plot of percent activity versus the log of compound concentration was fit with a 4-parameter dose response equation to calculate EC 50 values.
The final reaction conditions were:
Component Volume (uL) Final Concentration
STING membrane 148 1.5ug/ml
3 H-cGAMP 50 4.0nM
Low Control (cold cGAMP) 2 lOuM
Test compound/DMSO 2 lOuM
Compound concentrations tested were 20.000, 637.00, 2.200, 0.740, 0.247, 0.082,
0.027, 0.009, 0.003, and Ο.ΟΟΙμΜ with 1.0% residual DMSO.
Full-Length STING (WT) Virus Generation
STING virus was generated using an insect cell baculovirus system. Spodoptera frugiperda S/21 cells (Kempbio, Inc.) were diluted to 5e5 cells/ml in Sf-900II SFM media
(LifeTechnologies #10902088) without antibiotics. The cell suspension was added to each well of a treated 6-well plate (2mL per well, le6 cells total), and the cells were allowed to adhere for at least 30min. Meanwhile, a lmL co-transfection mix was assembled by combining 500ng of WT STING[STrNG(l-379)H232R-gg-AviTag-gs-HRV3C-HIS8/pBACl] (Genewiz custom synthesis) with lmL Sf-900II SFM media containing ΙΟμΙ. CELLFECTIN® II Reagent (Invitrogen #10362100) and lOOng viral backbone BestBac 2.0, v-cath/chiA Deleted Linearized Baculovirus DNA (Expression Systems #91-002). The transfection mixtures were allowed to incubate for 30min. After incubation, media was gently removed from the adhered cells in the 6-well plate, the lmL transfection mixtures were added (lmL per well), and the plate was placed in a humidified incubator at 27°C. The following day, lmL Sf-900II SFM media (no antibiotics) was added to each well of the 6-well plate. After media addition, the cells were allowed to incubate with DNA [(SEQ. ID. No. 4) and linearized viral backbone BestBac 2.0] at 27°C for 5-7 days to generate the P0 viral stock. To generate PI viral stocks, 0.5mL of P0 viral supernatant was added to 50mL uninfected S/21 cells (seeded the day prior to infection at a density of 5xl0 5 cells/mL to allow for one overnight doubling) in Sf-900II SFM media containing 5μg/mL gentamicin (Invitrogen #15710072). The infected cells were then incubated at 27°C for 3days while shaking at 1 lOrpm (ATR Biotech Multitron Infors HT #AJ118). On day 3, PI cultures were counted using a ViCell XR (Beckman Coulter Life Sciences #383556) to confirm infection had occurred (cell size >3μπι larger than uninfected cells and viability approximately 85-95%). Cultures were harvested in 50mL conical tubes and centrifuged at 2000xg for lOmin at 4°C. The PI viral supernatants were poured off into clean 50ml centrifuge tubes, and the remaining PI cell pellets were used to generate Baculovirus Infected Insect Cells (BIICs). Cryopreservation media containing Sf-900II SFM media with 10% heat inactivated FBS, 10% DMSO (Sigma #D2650), and 5μg/ml gentamicin was prepared and sterilized through 0.22μΜ filter immediately prior to use. PI cell pellets were resuspended to a density of 2e7 cells/ml and aliquoted into cryovials (lmL per vial). Cryovials were placed in MR. FROSTY™ cell freezers O/N at -80°C and transferred to liquid nitrogen for long term storage the following day. To generate P2 viral stock, 0.5mL of the PI viral supernatant was added to 50mL uninfected S/21 cells (seeded the day prior to infection at a density of 5xl0 5 cells/mL to allow for one overnight doubling) in Sf- 900II SFM media containing 5μg/mL gentamicin. These cells were incubated at 27°C for 3days while shaking at 1 lOrpm before harvesting P2 stock with centrifugation at 2000xg for lOmin at 4°C. The P2 viral supernatants were poured off and discarded, while the P2 cell pellets were used to generate P2 BIICs following the same protocol described above. The baculovirus generation protocol has been validated to consistently produce P1/P2 BIICs with titers of 2e9 pfu/mL (2e7 cells/mLxlOOpfu/cell).
Full-Length STING WT) Expression
To generate STING membranes, P1/P2 BIICs were amplified overnight by adding thawed BIICs to S/21 cells seeded at a density of l .OxlO 6 cells/mL. The volume of BIIC used to infect the culture was calculated using an assumed BIIC titer of 2e9 pfu/ml to achieve an MOI of 10 in the overnight amplification. After culturing overnight, the cells were counted on a ViCell XR to confirm infection had occurred (cell size >3μπι larger than uninfected cells and viability approximately 80-90%). The volume of infected S/21 cells from the overnight amplification used to infect the large-scale expression of Trichoplusia ni (T.ni; Expression Systems, cat # 94- 002F, www. expressi onsystems. com) seeded at a density of 1.0x10 6 in cell media (ESF921 SFM containing 5μg/mL gentamicin) at MOI = 2.0 was calculated based on (lOOpfu/infected S/21 cell). The cells were allowed to express for 48h at 27°C before harvesting the cell pellet, by centrifugation at 3,400xg for lOmin at 4°C. T. ni cells were counted on a ViCell XR to confirm infection had occurred (cell size >3μπι larger than uninfected cells and viability approximately 80-90%) prior to harvest.
Full-Length STING (WT) Membrane Generation
Buffer stock reagents:
1) 1 M HEPES pH 7.5, Teknova, Cat#H1035
2) 5 M NaCl, Sigma Aldrich, Cat#S5150-lL
3) KC1, Sigma Aldrich, Cat#319309-500ML
4) Complete EDTA-free protease inhibitor tablets, Roche Diagnostics, Cat#l 1873580001
5) Benzonase, Universal Nuclease, Pierce, Cat#88702
Lysis buffer [25mM HEPES pH 7.5, lOmM MgCl 2 , 20mM KC1, (Benzonase 1 :5000, Complete Protease Inhibitor tab/50mL)] was added to the pellet of cells expressing full- length STING (WT) prepared above at 5mL Lysis buffer per g of cell pellet. The pellet was resuspended and dounced twenty times using a Wheaton Dounce Homogenizer to disrupt the cell membrane. Homogenized lysate was then passed through the emulsiflex-C5 microfluidizer at a pressure close to 5000PSI. The resuspended pellet was centrifuged at 36,000rpm (100,000xg) in a 45Ti rotor ultra-high speed centrifuge for 45min, 4°C. The supernatant was removed. The pellet then was resuspended in wash buffer [(25mM HEPES pH 7.5, ImM MgCl 2 , 20mM KC1, 1M NaCl (Complete Protease Inhibitor tab/50mL)] at a volume of 50mL/pellet/centrifuge tube. The pellet/wash buffer mixture was then homogenized, using a glass homogenizer on ice (20 strokes), followed by centrifugation at 36,000rpm for 45min at 4°C. The supernatant was removed. The wash step was repeated once more. The resulting membrane was resuspended in 20mM HEPES pH 7.5, 500mM NaCl, 10% glycerol, EDTA-free Protease Inhibitors
(ltablet/50mL). The protein concentration was measured by Bradford assay (Bio-Rad Protein Assay, Cat#500-0006), and protein enrichment was determined by SDS-PAGE and confirmed by Western blot. The resuspended membranes were stored at -80°C.
Full-Length STING WT [STING(l-379)H232R-gg-AviTag-gs-HRV3C-HIS8] Amino Acid Sequence: MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLL LNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFT WMLALLGLSQALNILLGLKGLAPAEISAVCEKG FNVAHGLAWSYYIGYLRLILPELQA RIRTYNQHYN LLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIK DRV YSNS IYELLENGQRAGTC VLE Y ATPLQ TLF AM S Q YS Q AGF SREDRLEQ AKLFCRTL EDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTS T MSQEPELLISGMEKPLPLRTDFSGGGL DIFEAQKIEWHEGSLEVLFQGPHHHHHHHH
(SEQ. ID. No. 3) Full-length WT STING [STING(l-379)H232R-gg-AviTag-gs-HRV3C-HIS8/pBACl] plasmid sequence:
GGAACGGCTCCGCCCACTATTAATGAAATTAAAAATTCCAATTTTAAAAAACGCAG CAAGAGAAACATTTGTATGAAAGAATGCGTAGAAGGAAAGAAAAATGTCGTCGAC ATGCTGAACAACAAGATTAATATGCCTCCGTGTATAAAAAAAATATTGAACGATTTG AAAGAAAAC AATGTACCGCGCGGCGGTATGTACAGGAAGAGGTTTATACTAAACTG TTACATTGCAAACGTGGTTTCGTGTGCCAAGTGTGAAAACCGATGTTTAATCAAGGC TCTGACGCATTTCTACAACCACGACTCCAAGTGTGTGGGTGAAGTCATGCATCTTTT A ATC A A ATC CC A AGATGTGT AT A A AC C AC C A A AC TGC C A A A A A ATGA A A AC TGTC G ACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTG AATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATACAAACCAAAC GCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTG AGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATT TTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTC ATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCG TATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAG TACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTC GGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTT TTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTT TTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAA ACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAAT AACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCA ATATATAGTTGCTGATCAGATCTGATCATGGAGATAATTAAAATGATAACCATCTCG CAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAA ATATAGGATCCATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTC ACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGG GGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCC TGCAGCTGGGACTGCTGTTAAACGGGGTCTGCAGCCTGGCTGAGGAGCTGCGCCAC ATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGGGCCTGCCTGGGCTG CCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCCCA AATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCA CTGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGT GAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATGGTCATATTACATCGGATA TCTGCGGCTGATCCTGCC AGAGCTCC AGGCCCGGATTCGAACTT AC AATC AGC ATT A CAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCCATTGGA CTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAA ACTGCCCCAGCAGACCGGTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACA GCATCTATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTAC GCC ACCCCCTTGCAGACTTTGTTTGCCATGTCAC AATAC AGTC AAGCTGGCTTTAGC CGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCGGACACTTGAGGACATCCT GGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGC AGATGACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGG AAAAGGAAGAGGTTACTGTGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCC ACGATGTCCCAAGAGCCTGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCTCTC CGCACGGATTTCTCTGGCGGTGGCCTGAACGACATCTTCGAAGCCCAGAAAATCGA ATGGCATGAAGGCAGCCTGGAAGTGCTGTTCCAGGGCCCACACCACCATCATCACC ATCACCATTAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACTAACCTAGG TAGCTGAGCGCATGCAAGCTGATCCGGGTTATTAGTACATTTATTAAGCGCTAGATT CTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTT ATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATA TCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCA AACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAAC GCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGT GTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTC TTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTT GGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCC AACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCC TATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAAT TATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAAT TCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAA ATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCG CAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGG CGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACT GGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTT CTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCC GTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGG AGGCGCTGGAATGTTAGGC ACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTAT AA TTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTG GCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCA ATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTA TCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGA GATTGTCTCAAGCTCGGATCGATCCCGC ACGCCGATAACAAGCCTTTTC ATTTTTACT ACAGCATTGTAGTGGCGAGACACTTCGCTGTCGTCGAGGTTTAAACGCTTCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTC AACC AAGTC ATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGG CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC ATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCG CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTT TCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACG GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAG GGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA ACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCCATTCGCCATTCAGGCTGC GCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCA
(SEQ. ID. No. 4)
Certain compounds of the disclosure were evaluated in WT STING in vitro binding assay as described above. The following table tabulates the biological data for these compounds as EC 50 values. Table 8: 3 H-cGAMP filtration binding assay for WT STING
The ability of compounds to stimulate the secretion of interferon-beta from THP1 cells was measured using a human IFN-β AlphaLISA kit (Perkin Elmer, Cat. No. AL265F). The basic protocol is as follows:
A Labcyte Echo 550 acoustic dispenser was used to transfer 120nL of compound dissolved in DMSO into the wells of an empty, sterile 384-well microplate, (Corning, Cat. No. 3712). THP1 cells (American Type Culture Collection, Cat. No. TIB202) previously frozen in Recovery Medium (Life Technologies, Cat. No. 12648-010) were thawed and immediately diluted 10-fold into 37°C assay medium (RPMI 1640 + L-Glutamine & phenol red, Life Technologies, Cat. No. 11875-085; 0.5% heat inactivated fetal bovine serum, Sigma Aldrich, Cat. No. F4135; lmM Sodium Pyruvate, Life Technologies, Cat. No. 11360-070; lx nonessential amino acids; Life Technologies, Cat. No. 11140-050). The cell viability and count was ascertained using a Beckman Coulter V-Cell XR cell counter. The cell suspension was centrifuged at 200xg for 5min at RT. Cells were resuspended to a density of 0.8xl0 6 /mL in 37°C assay medium. Subsequent liquid transfers were performed using either a Matrix electronic multichannel pipette or an Agilent Bravo Automated Liquid Handling Platform.
The assay was started by dispensing 40μΕ of the previously prepared cell suspension into the wells of the plate containing compounds. After 5h incubation at 37°C, 5% C0 2 in a humidified atmosphere, the plate of cells and compounds was centrifuged at 200xg for 5min at RT. From each well, 5μΕ of supernatant was transferred into corresponding wells of a white 384-well plate (Perkin Elmer, Cat. No. 6005620). To these supernatant-containing wells was added ΙΟμΙ. of 5x Anti-Analyte Acceptor beads (50μg/mL of AlphaLISA HiBlock Buffer) and incubated for 30min at RT while shaking on an orbital plate shaker. To each well was added ΙΟμΙ. of 5x Biotinylated Antibody Anti-analyte (5nM in AlphaLISA HiBlock Buffer) and incubated on an orbital plate shaker for 60min at RT or overnight at 4°C. To each well was added 25μΙ ^ of 2x SA-Donor beads (80μg/mL in AlphaLISA HiBlock Buffer) and incubated for 30-45min at RT in the dark while shaking on an orbital plate shaker. The plate was then read on a Perkin Elmer Envision (λ εχ = 680nm, sm = 570nm). The percent effect of the AlphaLISA signal at each compound concentration was calculated based on 30uM cGAMP positive controls and 0.3% DMSO negative controls. The plot of percent effect versus the log of compound concentration was fit with a 4-parameter concentration response equation to calculate EC 50 values. The test compounds were tested at concentrations 30000, 10000, 3333, 1 1 1 1, 370.4, 123.4, 41.2, 13.7, 4.6, and 1.5nM with 0.3% residual DMSO. The control compound, cGAMP was tested at concentrations 100000, 33333, 1 1 1 1 1, 3704, 1235, 412, 137, 46, and 15nM with 0.3% residual DMSO.
Compounds of the disclosure were evaluated for IFN-β secretion in THPl cell culture as described above. The following table tabulates the biological data for these compounds as percent activation relative to 2'3 '-cGAMP at the 30μΜ concentration.
Table 9: IFN-β secretion in THPl cell culture (5h)
It will be appreciated that various of the above-discussed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. It also will be appreciated that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art and are also intended to be encompassed by the following claims.
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