IP Verse

Coelenterazine Analogues

Patent No. US11691976 (titled "Coelenterazine Analogues") was filed by Promega Corp on Aug 22, 2019.

What is this patent about?

’976 is related to the field of bioluminescent assays, specifically to novel coelenterazine analogues and their use as substrates for luciferase enzymes. Luciferases are widely used as reporter enzymes in biological research, enabling the study of gene expression, protein-protein interactions, and cell viability. Existing coelenterazine analogues suffer from limitations such as cell toxicity, light sensitivity, and poor solubility, hindering their effectiveness in various applications.

The underlying idea behind ’976 is to create new coelenterazine analogues with improved properties for use as luciferase substrates. The key inventive insight involves modifying the chemical structure of coelenterazine to enhance its aqueous solubility , bioluminescence signal kinetics , and overall performance in luciferase-based assays. This is achieved through specific substitutions on the phenyl group at the 6-position of the imidazopyrazinone core structure.

The claims of ’976 focus on novel compounds of formula (II), which are coelenterazine analogues with specific substitutions at various positions. The core structure is an imidazo[1,2-a]pyrazin-3(7H)-one, with substituents Y, R3, and R4 attached to the phenyl ring. The claims define the allowed chemical groups for Y (nitro, or —NRy1Ry2), R3, and R4, along with the number of such substituents (p and q).

In practice, the claimed compounds function as substrates for luciferase enzymes. When the luciferase enzyme interacts with the coelenterazine analogue, a chemical reaction occurs that produces light. The intensity of the light emitted is directly proportional to the amount of luciferase present, allowing for quantitative measurements. The specific substitutions defined in the claims are designed to improve the compound's interaction with the luciferase enzyme, leading to a brighter and more stable bioluminescent signal.

The advantage of these new coelenterazine analogues over prior solutions lies in their improved characteristics. The patent highlights enhanced aqueous solubility compared to existing substrates like furimazine, which is crucial for in vivo applications. Furthermore, the compounds exhibit improved bioluminescence signal kinetics, meaning a faster and more intense light output, making them more suitable for real-time monitoring of biological processes. The new compounds are designed to be less toxic and more biocompatible, expanding their utility in cellular assays and in vivo imaging.

How does this patent fit in bigger picture?

Technical landscape at the time

In the late 2010s when ’976 was filed, bioluminescent assays were used extensively at a time when luciferase reporter enzymes were valuable tools in the investigation of cellular physiology. At a time when protein engineering was intense to obtain small and environmentally insensitive luciferases, systems commonly relied on coelenterazine and coelenterazine analogues as substrates due to their brightness and acceptance in whole cell applications.

Novelty and Inventive Step

The application was filed in 2019. In 2022, a final rejection was issued for claims pertaining to certain coelenterazine analogues, based on anticipation by prior art. The examiner withdrew a species requirement, but indicated that further species election may be required if claims are amended to include structures outside the scope of formula II. The applicant's arguments were considered but not persuasive.

Claims

This patent contains 20 claims, with independent claims numbered 1, 11, 17, and 17. The independent claims generally focus on chemical compounds with specific formulas. The dependent claims generally narrow the scope of the independent claims by specifying particular substituents, quantities, or uses of the claimed compounds.

Key Claim Terms New

Definitions of key terms used in the patent claims.

Term (Source)Support for SpecificationInterpretation
Formula (II)
(Claim 1)		“In another aspect, disclosed are compounds of formula (II), or a tautomer or a salt thereof, wherein: Y is C 1-10 alkyl, halogen, CN, nitro, C 1-10 haloalkyl, C 1-4 haloalkylene-OC 1-4 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, —C(O)OR y1 , —CONR y1 R y2 , —OC(O)NR y1 R y2 , —NR y3 C(O)OR y1 , —OR y1 , —C 1-10 alkylene-OR y1 , —OC(O)R y1 , —NR y1 R y2 , —C 1-10 alkylene-NR y1 R y2 ; —NR y3 C(O)R y1 , —NR y3 C(O)NR y1 R y2 , —SO 2 R y1 , —SO 2 NR y1 R y2 , —NR y3 SO 2 R y1 , —SO 2 OR y1 , —OSO 2 R y1 , —OSO 3 R y1 , —OP(O)(OH)OR y1 , —OSi(C 1-10 alkyl) 3 , —OR y0 , —OG E , or —NR y1 G E ; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, 4, or 5; R 3 , at each occurrence, is independently halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A3 , —NR B3 R C3 , —OG E , or —NR B3 G E ; wherein optionally two adjacent R 3 groups, or Y and an adjacent R 3 group, together with the carbon atoms to which they are attached form a fused ring selected from a 5- to 7-membered cycloalkyl, a 5- to 7-membered cycloalkenyl, a phenyl, a 5- to 6-membered heteroaryl, or a 5- to 7-membered heterocyclyl, the optional fused ring being optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A3 , and —NR B3 R C3 ; G E , at each occurrence, independently comprises an enzyme substrate, wherein biotransformation by an enzyme converts G E to H; R 4 , at each occurrence, is independently halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A4 , or —NR B4 R C4 ; wherein two adjacent R 4 groups together with the carbon atoms to which they are attached optionally form a fused ring selected from a 5- to 7-membered cycloalkyl, a 5- to 7-membered cycloalkenyl, a phenyl, a 5- to 6-membered heteroaryl, or a 5- to 7-membered heterocyclyl, the optional fused ring being optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A4 , and —NR B4 R C4 ; R A3 and R A4 , at each occurrence, are independently H, C 1-10 alkyl, C 1-10 haloalkyl, C 3-12 cycloalkyl, —C(O)C 1-10 alkyl, —C(O)C 3-12 cycloalkyl, or —C(O)C 1-3 alkylene-C 3-12 cycloalkyl, wherein each cycloalkyl is optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 haloalkyl; R B3 , R B4 , R C3 , and R C4 , at each occurrence, are independently H, C 1-10 alkyl, C 1-10 haloalkyl, C 3-12 cycloalkyl, —C(O)C 1-10 alkyl, —C(O)C 3-12 cycloalkyl, —C(O)C 1-3 alkylene-C 3-12 cycloalkyl, —SO 2 C 1-10 alkyl, —SO 2 C 3-12 cycloalkyl, or —SO 2 C 1-3 alkylene-C 3-12 cycloalkyl, wherein each cycloalkyl is optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 haloalkyl; alternatively, R B3 and R C3 and/or R B4 and R C4 , together with the nitrogen atom to which each attaches form a 4- to 8-membered saturated or partially unsaturated heterocyclic ring, optionally substituted with 1-4 substituents independently selected from the group consisting of C 1-4 alkyl, C 1-4 haloalkyl, oxo, —OH, and —OC 1-4 alkyl; R y1 , R y2 , and R y3 , at each occurrence, are independently H, C 1-10 alkyl, C 1-10 haloalkyl, C 3-12 cycloalkyl, or C 1-3 alkylene-C 3-12 cycloalkyl, wherein each cycloalkyl is optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 haloalkyl; alternatively, R y1 and R y2 together with the nitrogen atom to which each attaches form a 4- to 8-membered saturated or partially unsaturated heterocyclic ring, optionally substituted with 1-4 substituents independently selected from the group consisting of C 1-4 alkyl, C 1-4 haloalkyl, oxo, —OH, and —OC 1-4 alkyl; and R y0 is a sugar moiety”A chemical structure as shown in the patent description, representing a class of coelenterazine analogues.
R3
(Claim 1)		“R 3 , at each occurrence, is independently halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A3 , —NR B3 R C3 , —OG E , or —NR B3 G E ; wherein optionally two adjacent R 3 groups, or Y and an adjacent R 3 group, together with the carbon atoms to which they are attached form a fused ring selected from a 5- to 7-membered cycloalkyl, a 5- to 7-membered cycloalkenyl, a phenyl, a 5- to 6-membered heteroaryl, or a 5- to 7-membered heterocyclyl, the optional fused ring being optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A3 , and —NR B3 R C3”A substituent on the compound of formula (II), which can be halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A3 , or —NR B3 R C3 .
R4
(Claim 1)		“R 4 , at each occurrence, is independently halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A4 , or —NR B4 R C4 ; wherein two adjacent R 4 groups together with the carbon atoms to which they are attached optionally form a fused ring selected from a 5- to 7-membered cycloalkyl, a 5- to 7-membered cycloalkenyl, a phenyl, a 5- to 6-membered heteroaryl, or a 5- to 7-membered heterocyclyl, the optional fused ring being optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A4 , and —NR B4 R C4”A substituent on the compound of formula (II), which can be halogen, CN, nitro, C 1-10 alkyl, C 1-10 haloalkyl, —OR A4 , or —NR B4 R C4 .
Ry1
(Claim 1)		“R y1 and R y2 , at each occurrence, are independently H, C 1-10 alkyl, C 1-10 haloalkyl, C 3-12 cycloalkyl, or C 1-3 alkylene-C 3-12 cycloalkyl, wherein each cycloalkyl is optionally substituted with 1-4 substituents independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 haloalkyl; and alternatively, R y1 and R y2 together with the nitrogen atom to which each attaches form a 4- to 8-membered saturated or partially unsaturated heterocyclic ring, optionally substituted with 1-4 substituents independently selected from the group consisting of C 1-4 alkyl, C 1-4 haloalkyl, oxo, —OH, and —OC 1-4 alkyl.”A substituent on the compound of formula (II), which can be H, C 1-10 alkyl, C 1-10 haloalkyl, C 3-12 cycloalkyl, or —C 1-3 alkylene-C 3-12 cycloalkyl.

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US11691976

PROMEGA CORP
Application Number
US16548214
Filing Date
Aug 22, 2019
Status
Granted
Expiry Date
Jul 21, 2040
External Links
Slate, USPTO, Google Patents