5’-C ALKYL SUBSTITUTED RNA CAPS AND METHODS OF USE
Provided herein are compounds according to formula (I), wherein each of R 7 and R 8 is H or C 1 -C 6 -alkyl and at least one of R 7 and R 8 is C 1 -C 6 -alkyl, that are cap analogs for polynucleotides, e.g., RNA molecules, such as mRNA molecules. Also provided are capped polynucleotides, e.g., capped RNA molecules, such as capped mRNA molecules, wherein the 5 ′ end of the RNA molecule comprises a cap analog disclosed herein, drug products comprising the capped RNA molecules, methods for making capped polynucleotides disclosed herein, and kits for making the capped polynucleotides.
1 . A compound of Formula (I), or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof:
wherein
ring G is guanine or a modified guanine;
each of R 1 , R 2 , R 4 , and R 6 is independently H, C 1-6 alkyl, halo, OH, CN, or OR x , where R x is C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl, and R x is optionally substituted with one or more substituents independently selected from halo, OH, C 1-6 alkoxy, and OC(O)—C 1-6 alkyl;
R 1A is H, or R 1A and R 1 together form —O—C 1-6 alkylene-;
R 4A is H, or R 4A and R 4 together form —O—C 1-6 alkylene-;
X is O, S, SO, SO 2 , or Se;
L is a linker;
R 3 is H, C 1-6 alkyl, C 1-6 alkoxy, OH, halo, or a polynucleotide;
R 5 is OH, SH, C 1-6 alkyl, C 1-6 alkoxy, or N(R N5 ) 2 and each R N5 is independently H or C 1-6 alkyl;
each of R 7 and R 8 is H or C 1-6 alkyl, and at least one of R 7 and R 8 is C 1-6 alkyl;
W is O, S, or NS(O) 2 C 1-6 alkyl; and
each of ring A and ring B is independently a nucleobase.
2 . The compound, stereoisomer, tautomer, or salt of claim 1 , wherein
L is —Y 0 —(P(O)(R P )Y 1 )—(P(O)(R P )Y 2 ) m —(P(O)(R P )Y 3 ) n —,
each R P is independently H, halo, C 1-6 alkyl, OH, SH, SeH, or BH 3− ;
each of Y 0 , Y 1 , Y 2 , and Y 3 is independently O, S, NH, N(C 1-6 alkyl), or CH 2 ; and
m+n is 1 to 5.
3 . The compound, stereoisomer, tautomer, or salt of claim 1 or 2 , wherein m+n is 1 or 2.
4 . The compound, stereoisomer, tautomer, or salt of claim 3 , wherein L is —Y 0 —(P(O)(R P )Y 1 )—(P(O)(R P )Y 2 )—(P(O)(R P )Y 3 )—.
5 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 4 , wherein each R P is OH.
6 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 4 , wherein at least one R P is SH.
7 . The compound, stereoisomer, tautomer, or salt of claim 6 , wherein each R P is SH.
8 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 7 , wherein Y 0 is O.
9 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is O.
10 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is CH 2 .
11 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is NH.
12 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 8 , wherein Y 1 is S.
13 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is O.
14 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is CH 2 .
15 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is NH.
16 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 12 , wherein Y 2 is S.
17 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 16 , wherein Y 3 is O.
18 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 16 , wherein Y 3 is CH 2 .
19 . The compound, stereoisomer, tautomer, or salt of any one of claims 3 to 16 , wherein Y 3 is NH.
20 . The compound, stereoisomer, tautomer, or salt of claim 4 , wherein L is —O—(P(O)(OH)O)—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)O)—(P(O)(OH)O)—(P(O)(SH)O)—, —O—(P(O)(OH)NH)—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)S)—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)CH 2 )—(P(O)(OH)O)—(P(O)(OH)O)—, —O—(P(O)(OH)S)—(P(O)(OH)S)—(P(O)(OH)O)—, or —O—(P(O)(SH)NH)—(P(O)(SH)O)—(P(O)(SH)O)—.
21 . The compound, stereoisomer, tautomer, or salt of claim 20 , wherein L is —O—(P(O)(OH)O)—(P(O)(OH)O)—(P(O)(OH)O—.
22 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 21 , wherein R 1A is H.
23 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 22 , wherein R 1 is OH.
24 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 21 , wherein R 1A and R 1 together form —O—C 1-6 alkylene-.
25 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 24 , wherein R 2 is OH.
26 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 25 , wherein R 4A is H.
27 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 26 , wherein R 4 is OH.
28 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 25 , wherein R 4A and R 4 together form —O—C 1-6 alkylene-.
29 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 28 , wherein R 6 is OR x .
30 . The compound, stereoisomer, tautomer, or salt of claim 29 , wherein R 6 is OC 1-6 alkyl.
31 . The compound, stereoisomer, tautomer, or salt of claim 29 or 30 , wherein R 6 is methoxy.
32 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 31 , wherein X is S.
33 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 31 , wherein X is O.
34 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 33 , wherein ring G is guanine.
35 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 33 , wherein ring G is modified guanine.
36 . The compound, stereoisomer, tautomer, or salt of claim 35 ,
wherein
ring G is
R N1 is C 1-6 alkyl, C 1-6 alkylene-aryl, or C 1-6 alklyene-O-aryl; and
each R N2 is independently H, C 1-6 alkyl, or benzyl.
37 . The compound, stereoisomer, tautomer, or salt of claim 36 , wherein R N1 is Me or Et.
38 . The compound, stereoisomer, tautomer, or salt of claim 36 , wherein R N1 is benzyl or benzyloxy.
39 . The compound, stereoisomer, tautomer, or salt of any one of claims 36 to 38 , wherein at least one R N2 is C 1-6 alkyl.
40 . The compound, stereoisomer, tautomer, or salt of any one of claims 36 to 39 , wherein at least one R N2 is H.
41 . The compound, stereoisomer, tautomer, or salt of claim 40 , wherein each R N2 is H.
42 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 41 , wherein W is O.
43 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 41 , wherein W is NS(O) 2 C 1-6 alkyl.
44 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 43 , wherein R 5 is OH.
45 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 43 , wherein R 5 is SH.
46 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 43 , wherein R 5 is C 1-6 alkyl.
47 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 46 , wherein R 7 is C 1-6 alkyl.
48 . The compound, stereoisomer, tautomer, or salt of claim 47 , wherein R 7 is methyl, ethyl, or isopropyl.
49 . The compound, stereoisomer, tautomer, or salt of claim 47 or 48 , wherein R 7 is methyl.
50 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 46 , wherein R 7 is H.
51 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 50 , wherein R 8 is C 1-6 alkyl.
52 . The compound, stereoisomer, tautomer, or salt of claim 51 , wherein R 8 is methyl, ethyl, or isopropyl.
53 . The compound, stereoisomer, tautomer, or salt of claim 51 or 52 , wherein R 8 is methyl.
54 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 49 , wherein R 8 is H.
55 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 54 , wherein R 3 is OH or C 1-6 alkoxy.
56 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 54 , wherein R 3 is H or halo.
57 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 54 , wherein R 3 is a polynucleotide.
58 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 57 , wherein ring A is adenine.
59 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 58 , wherein ring B is guanine.
60 . The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 58 , wherein ring B is uracil.
61 . A compound having a structure as shown in Table 1, or a stereoisomer, tautomer, or salt thereof.
62 . An RNA molecule, wherein the 5′ end of the RNA molecule comprises the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 61 .
63 . The RNA molecule of claim 62 , wherein the RNA molecule is mRNA.
64 . The RNA molecule of claim 63 , wherein the mRNA encodes for one or more immunomodulatory agents (e.g., a checkpoint inhibitor such as an anti-PD-1 antibody, anti-PDL-1 antibody, anti-CTLA4, etc., an immunosuppression antagonist, a pro-inflammatory agent/cytokine), therapeutic proteins, antibodies, or antigens (e.g., a tumor specific antigen).
65 . The RNA molecule of any one of claims 62 to 64 , wherein the RNA molecule has a half-life that is more than that of a corresponding natural RNA molecule in a cellular environment.
66 . A drug product comprising the RNA molecule of any one of claims 62 to 65 and one or more pharmaceutically acceptable excipients.
67 . A kit for capping an RNA molecule comprising the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 61 , and an RNA polymerase.
68 . The kit of claim 67 , wherein the RNA molecule is mRNA.
69 . A method for making a capped RNA molecule from a polynucleotide template by in vitro transcription, comprising:
(a) combining a plurality of nucleotides, the polynucleotide template and an RNA polymerase to produce a reaction mix;
(b) incubating the reaction mix; and
(c) adding the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 61 to the mix in order to produce the capped RNA molecule.
70 . The method of claim 69 , wherein the RNA molecule is mRNA.
71 . The method of claim 69 or claim 70 , wherein at least a portion of the plurality of nucleotides are unmodified.
72 . The method of any one of claims 69 to 71 , wherein the at least a portion of the plurality of nucleotides are modified.
73 . The method of any one of claims 69 to 72 , wherein the polynucleotide template is a DNA template.
74 . The method of any one of claims 69 to 73 , wherein the capping efficiency is greater than about 95%.
75 . The method of any one of claims 69 to 74 , wherein the translation yield is greater than about 75%.