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CN-122029172-A - DNA-dependent protein kinase inhibitors and compositions based on 6, 6-core silacycle and their use in gene editing

CN122029172ACN 122029172 ACN122029172 ACN 122029172ACN-122029172-A

Abstract

The present disclosure relates to DNA-PK inhibitors having the formula (I), (I) Or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or tautomer thereof, methods of preparing the foregoing and compositions thereof, and methods of using the compound of formula (I) in combination with a DNA cleaving agent.

Inventors

  • J.X.Qiao
  • C. Clarat
  • A. siren
  • J. Fernandez

Assignees

  • 朱诺治疗学股份有限公司

Dates

Publication Date
20260512
Application Date
20240822
Priority Date
20230825

Claims (20)

  1. 1. A compound of formula I: (I), or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or tautomer thereof, Wherein: A is a 6-membered heteroaryl or heterocycloalkyl containing at least one heteroatom selected from N, O and S, wherein said heteroaryl or heterocycloalkyl is optionally substituted with one or more R 5 ; R 1 is aryl or heteroaryl containing at least one heteroatom selected from N, O and S, wherein the aryl or heteroaryl is optionally substituted with one or more R 6 ; R 2 is H, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, -CN, OH, CH 2 OH、NH 2 , or CH 2 NH 2 ; R 3 and R 4 are each independently selected from the group consisting of-OH, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 haloalkyl, C 1 -C 4 alkylaryl and aryl; each R 5 is independently selected from H, halogen, oxo, thiocarbonyl, C 1 -C 4 alkyl, CD 3 、CD 2 CD 3 、C 1 -C 4 alkoxy, C 1 -C 6 haloalkyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl is optionally substituted with one or more R 6 , or Two geminal R 5 together with the geminal carbon atom between them form a C 3 -C 6 cycloalkyl group; Each R 6 is independently selected from H, halogen, NH 2 、OH、-CN、C(O)NHR 7 、C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, CD 3 、CD 2 CD 3 、C 1 -C 6 alkoxy, C 1 -C 6 haloalkyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl is optionally substituted with one or more R 7 ; Each R 7 is independently selected from H, halogen, OH, NH 2 , CHO, oxo, thiocarbonyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 6 haloalkyl, and N is an integer from 1 to 3.
  2. 2. The compound of claim 1, wherein R 1 is , Wherein B is a 5 or 6 membered aryl or heteroaryl containing one or more heteroatoms selected from N, O, S and Se.
  3. 3. The compound of claim 1, wherein R 1 is selected from: 、 、 、 、 、 、 、 、 And Wherein: X 1 、X 2 、X 3 、X 4 and X 5 are each independently N or C (R 6 ), and M is an integer from 1 to 3.
  4. 4. The compound of any one of the preceding claims, wherein the compound has formula (Ia-1), (Ia-2), or (Ia-3): (Ia-1), (Ia-2), or (Ia-3), Or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or tautomer thereof.
  5. 5. The compound of any one of the preceding claims, wherein the compound has formula (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ib-7), (Ib-8) or (Ib-9): (Ib-1), Wherein m is an integer of 1 to 3, (Ib-2), Wherein m is an integer of 1 to 3, (Ib-3), Wherein m is an integer of 1 to 3, (Ib-4), Wherein the method comprises the steps of M is an integer of 1 to 3, (Ib-5), Wherein the method comprises the steps of X 1 、X 2 、X 3 and X 4 are each independently N or C (R 6 ), and M is an integer of 1 to 3, (Ib-6), Wherein X 5 is N or C (R 6 ), and M is an integer of 1 to 3, (Ib-7), Wherein X 5 is N or C (R 6 ), and M is an integer of 1 to 3, (Ib-8), Wherein X 5 is N or C (R 6 ), and M is an integer from 1 to 3, or (Ib-9), Wherein B is a 5-or 6-membered aryl or heteroaryl optionally substituted with one or more R 6 and m is an integer from 1 to 3, Or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or tautomer thereof.
  6. 6. The compound of claim 1, wherein the compound is selected from the compounds provided in table 1, or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug, or tautomer thereof.
  7. 7. The compound of claim 1, wherein the compound is selected from the compounds provided in table 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug, or tautomer thereof.
  8. 8. The compound of any one of the preceding claims, wherein the compound is selected from the group consisting of: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 And Or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or tautomer thereof.
  9. 9. A pharmaceutically acceptable composition comprising a compound according to any one of claims 1-8 and a pharmaceutically acceptable carrier.
  10. 10. A composition, the composition comprising: a) DNA protein kinase inhibitor (DNA-PKI), and B) A DNA cutting agent; wherein the DNA-PKI is a compound according to any one of claims 1-8.
  11. 11. The composition of claim 10, further comprising a cell.
  12. 12. The composition of claim 10 or 11, further comprising donor DNA.
  13. 13. The composition of any one of claims 10-12, wherein the DNA cleaving agent comprises a CRISPR/Cas nuclease component and optionally a guide RNA component.
  14. 14. The composition of any one of claims 10-enumerated embodiments 43, wherein the DNA cleaving agent comprises a CRISPR/Cas nuclease that produces double-stranded DNA breaks or single-stranded DNA breaks.
  15. 15. The composition of any one of claims 10-14, further comprising a carrier.
  16. 16. The composition of any one of claims 10-15, further comprising an inhibitor of a micro-homology mediated terminal connection (MMEJ) pathway.
  17. 17. A method for targeted genome editing in a cell, the method comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any of claims 1-8.
  18. 18. A method of repairing a double-stranded DNA break in the genome of a cell, the method comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of claims 1-8.
  19. 19. A method of inhibiting or suppressing repair of DNA breaks in a cell via a non-homologous end joining (NHEJ) pathway, the method comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is a compound of any one of claims 1-8.
  20. 20. A method of targeted insertion of donor DNA into the genome of a cell, the method comprising contacting the cell with a DNA cleaving agent, the donor DNA, and DNA-PKI, wherein the DNA-PKI is a compound according to any one of claims 1-8.

Description

DNA-dependent protein kinase inhibitors and compositions based on 6, 6-core silacycle and their use in gene editing Cross Reference to Related Applications The application claims the benefit and priority of U.S. provisional patent application No. 63/578,848 filed on 25/8/2023, the entire contents of which are hereby incorporated by reference. Technical Field The present disclosure relates generally to compounds, compositions, methods, and kits for increasing genome editing efficiency by administering a DNA protein kinase (DNA-PK) inhibitor of general formula (I) and a genome editing system to one or more eukaryotic cells. The disclosure also relates to compositions comprising DNA-PK inhibitors of general formula (I), methods of inserting a polynucleotide of interest into the genome of a eukaryotic cell, and kits for inserting a gene of interest into the genome of a eukaryotic cell. The methods and kits can improve the efficiency of CRISPR/Cas-mediated polynucleotide insertion in cells, particularly in CRISPR engineered CAR-T cells. Incorporated by reference into the sequence listing The present application contains a sequence listing submitted in XML format via EFS-WEB and hereby incorporated by reference in its entirety. The XML copy was created at month 8 of 2023, 24, and named 055920-610P01US_SeqList_ST26.XML, 75: 75 KB in size. Background It is a long-felt goal to develop cost-effective and reliable methods for precisely targeted alteration of living cell genomes. Genome editing has the potential to eliminate genes that cause specific disorders (i.e., gene "knockouts"), or alternatively to provide a means for gene manipulation or insertion to correct genetic defects or enhance biological processes by gene "knockins". Genome editing can be applied to the treatment of a variety of disorders, including genetic disorders, hematological disorders, and cancer, and can be applied in methods of immunotherapy. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related (Cas) systems are prokaryotic immune systems (Ishino et al Journal of Bacteriology 169:169:5429-54 (1987)), which provide immunity against viruses and plasmids by targeting their nucleic acids in a sequence-specific manner (Soret et al Nature Reviews Microbiology 6:181-186 (2008)). Since its initial discovery, a number of groups have been extensively studied around the potential applications of CRISPR systems in genetic engineering, including gene editing (Jinek et al, science 337 (6096): 816-821 (2012); cong et al, science 339 (6121): 819-823 (2013); and Mali et al, science 339 (6121): 823-826 (2013)). CRISPR-Cas9 gene editing systems have been successfully used in a variety of organisms and cell lines. Cas9 endonucleases create double-stranded DNA breaks at the target sequence upstream of the Protospacer Adjacent Motif (PAM). The target sequence may then be removed, or the sequence of interest may be inserted into the target sequence using the cell's endogenous repair pathway. Endogenous DNA repair pathways include the non-homology mediated end-joining (NHEJ) pathway, the micro-homology mediated end-joining (MMEJ) pathway, and the Homology Directed Repair (HDR) pathway. NHEJ, MMEJ and HDR pathways repair double-stranded DNA breaks, but repair of such double-stranded DNA breaks may result in insertions or deletions at the site of the double-stranded break. In NHEJ, no homology template is required to repair breaks in DNA. NHEJ repair may be prone to error, although errors are reduced when DNA breaks include compatible overhangs. NHEJ and MMEJ are mechanically distinct DNA repair pathways, where each of them involves a different subset of DNA repair enzymes. Unlike NHEJ (which may be precise in some cases, or prone to error in some cases), MMEJ is always prone to error and results in both deletions and insertions at the site of repair. MMEJ related deletions are due to the micro-homology (2-10 base pairs) on both sides of the double strand break. In contrast, HDR requires a homology template to guide repair, but HDR repair is typically high fidelity and less prone to error. Thus, HDR-driven repair of double-stranded DNA breaks is superior to NHEJ or MMEJ-mediated repair, however, in many cell types HDR is limited by NHEJ activity at all cell cycle phases and HDR is primarily used in the S/G2 phase of cell growth (Mao et al, CELL CYCLE, 7:2902-2906 (2008)). With recent findings and implementations of CRISPR/Cas9 editing techniques, the ability to modify the genome of any cell at a precise location has improved. However, the ability to introduce specific targeted changes at a given locus is hampered by the fact that the primary cellular repair pathway that occurs after Cas 9-mediated DNA cleavage is the wrong non-homologous end joining (NHEJ) pathway. Homologous Directed Recombination (HDR) is not as efficient as NHEJ, reducing editing efficiency in eukaryotic cells. Although in some reports the efficiency of implementation