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1. AU2018351881 - Secondary amine-substituted coumarin compounds and their uses as fluorescent labels

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]
WHAT IS CLAIMED IS:
1.   A compound of Formula (I) or a salt thereof:
wherein:
X is 0, S, Se, or NR, where R" is H or C16alkyl;
R and R1 are each independently H, halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido, -NO 2, -S0 3H, -S0 2NH 2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
R2 and R4 are each independently H, halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido,
-NO2, -SO 3H, -SO 2NH2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; or one of R2 and R4 is linked to R3 to form an optionally substituted heterocyclic ring;
R3 is H, C16alkyl, substituted C2-6alkyl, optionally substituted C2-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted carbocyclyl, heterocyclyl, aryl, or heteroaryl, or R3 is linked to R2 or R4 to form an optionally substituted ring;
wherein when R is -CN, R3 is not C16alkyl;
each R 5 is independently halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido, -NO 2 , -S0 3H, -S0 2NH 2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and
m is 0, 1, 2, 3, or 4.
2.   The compound of claim 1, wherein R is H, halo, -CO 2H, amino, -OH, C-amido, N-amido, -NO2, -S0 3H, -S0 2NH2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.
3.   The compound of claim 1 or claim 2, wherein R2 and R4 are each independently H, halo, CN, -CO 2H, amino, -OH, C-amido, N-amido, -NO 2, -SO 3 H, -S0 2NH2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and neither of R2 and R4 is linked to R3 to form an optionally substituted heterocyclic ring.
4.   The compound of claim 2, wherein R2 is -SO 3H; and/or R4 is -SO 3H.
5.   The compound of any one of claims 1-4, wherein X is 0 or S.
6.   The compound of any one of claims 1-4, wherein X is NR", where R" is H or C16alkyl.
7.   The compound of any one of claims 1-6, wherein (a) R3 is H; or (b) R3 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl; or (c) R3 is ethyl.
8.   The compound of any one of claims 1-6, wherein R3 is substituted C2-6alkyl.
9.   The compound of claim 8, wherein R3 is C2-6alkyl substituted with -CO 2H.
10.   The compound of claim 8, wherein R3 is -(CH 2).COOH, where n is 2, 3, 4, 5 or 6.
11.    The compound of claim 8, wherein R3 is -(CH 2).SO 3H where n is 2, 3, 4, 5 or 6.
12.   The compound of any one of claims 1-11, wherein m is 0, 1, or 2.
13.   The compound of any one of claims 1-12, wherein each R 5 is independently optionally substituted C1.6alkyl, halo, -CN, -CO 2H, amino, -OH, -S 3H, or -S 2NH 2.
14.   The compound of claim 13, wherein each R 5 is halo, -CN, -CO 2 H, -S 3H, -S 2NH2 , or optionally substituted C16alkyl.
15.   The compound of claim 14, wherein R 5 is C 26- alkyl substituted with -CO 2H, -SO 3H, or -S0 2NH 2.
16.   The compound of claim 15, wherein R 5 is -(CH2)xCOOH, where x is 2, 3, 4, 5 or 6.
17.   The compound of claim 4, wherein R5 is -SO 3H or -S 2NH 2.
18.   The compound of any one of claims 1-16, wherein R1 is H, halo, or C16alkyl.
19.   The compound of claim 18, wherein R 1 is H or Cl.
20.    The compound of any one of claims 1-19, wherein R is H, halo, or C16alkyl.
21.   The compound of claim 20, wherein R1 is H or Cl.
22.    The compound of any one of claims 1-21, wherein R is H, halo, -CO 2H, amino, -OH, C amido, N-amido, -NO 2 , -SO 3H, -S0 2NH 2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.
23.   The compound of claim 22, wherein R is H.
24.    The compound of any one of claims 1-23, wherein R2 is H, optionally substituted alkyl, C1 . 4alkyl optionally substituted with -CO 2H or -SO 3 H, or -SO 3H.
25.   The compound of claim 24, wherein R2 is H or -SO 3H.
26.    The compound of any one of claims 1-23, wherein R2 is linked to R3 to form an optionally substituted heterocyclic ring, preferably pyrrolidine or piperidine, optionally substituted with one or more alkyl groups.
27.    The compound of any one of claims 1-25, wherein R4 is H, optionally substituted alkyl, C1 . 4alkyl optionally substituted with -CO 2H or -SO 3 H, or -SO 3H.
28.  The compound of any one of claims 1-25, wherein R4 is linked to R3 to form an optionally substituted heterocyclic ring, preferably a pyrrolidine or piperidine, optionally substituted with one or more alkyl groups.
29.    The compound of claim 1, wherein X is 0 or S; R isH;Ris H; R3 is -(CH2)COOH where n is 2, 3, 4, 5, or 6; R 5 is H, -SO 3H, or -SO 2NH 2 ; R2 is H or -SO 3H; and R4 is H or -SO 3H.
30.   The compound of claim 29, wherein R3 is -(CH2) 2COOH.
31.   The compound of claim 29, wherein R3 is -(CH2)COOH.
32.   A compound of Formula (II) or a salt thereof:
wherein:
X' is selected from 0, S, and NRP, where RP is H or C16alkyl;
R6 is H or C1.4alkyl;
R7 is H, halo, -CN, -OH, optionally substituted C1.4alkyl, optionally substituted C1.4alkenyl, optionally substituted C2-4alkynyl, -CO 2H, -SO 3H, -SO 2NH 2, -SO 2NH(C1 .4alkyl), -SO 2N(C1 4alkyl)2, and optionally substituted C1.4alkoxy;
R8 and R10 are each independently H, halo, -CN, -CO 2H, amino, -OH, -SO 3H, -SO 2NH 2 ,
-SO 2NH(C 14alkyl), -SO 2N(C 14alkyl)2, optionally substituted C1 .6alkyl, optionally substituted C1.6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C1 . 6alkoxy; or
one of R' and R10 is H, halo, -CN, -CO 2H, amino, -OH, -SO 3H, -SO 2NH 2 , -SO2NH(C1.4alkyl), -SO 2N(C 14alkyl)2, optionally substituted C 1.6alkyl, optionally substituted C16alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C1.6alkoxy, and the other of R' and R10 is taken with R9 to form an optionally substituted 4- to 7-membered heterocyclic ring;
R9 is C2-6alkyl or C16alkyl substituted with -CO 2H, -CO2C1.4alkyl, -CONH2, -CONH(C1.4alkyl), -CON(C1.4alkyl)2, -CN, -SO 3H, -SO 2NH 2, -SO2NH(C1.4alkyl), or -SO2N(C1.4alkyl)2; each R" is independently halo, -CN, carboxy, amino, -OH, C-amido, N-amido, nitro, -SO 3H, -S0 2NH2, -SO2NH(C 14alkyl), -SO2N(C 14alkyl) 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted C16alkoxy; and q is 0, 1, or 2.
33.   The compound of claim 32, wherein X' is 0 or X' is S.
34.   The compound of claim 32, wherein X' is NRP, where RP is H or C16alkyl.
35.    The compound of any one of claims 32-34, wherein R6 is H or R6 is C1.4alkyl.
36.    The compound of any one of claims 32-35, wherein R7 is H or R7 is optionally substituted C1.4alkyl, -CO 2H, -SO 3H, -S0 2NH 2, -SO2NH(C1.4alkyl), or -SO2N(C1.4alkyl)2.
37.   The compound of claim 36, wherein R7 is C1.4alkyl optionally substituted with -CO 2H.
38.    The compound of any one of claims 32-37, wherein R8 is H or R is -CO 2H, -SO 3H, or -S0 2NH 2.
39.    The compound of any one of claims 32-38, wherein R10 is H or is -C 2H, -SO 3H, or -S0 2NH 2.
40.    The compound of any one of claims 32-37, wherein (a) R8 is H and R10 is -SO 3H or (b) R8 is -SO 3H and R10 is H.
41.    The compound of any one of claims 32-37, wherein one of R' and R10 is H, halo, -CN, -CO 2H, amino, -OH, -SO 3 H, -S0 2NH 2, -SO2NH(C1.4alkyl), -SO2N(C1.4alkyl)2, optionally substituted C1.6alkyl, optionally substituted C1.6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C1.6alkoxy, and the other of R' and R10 is taken with R9 to form an optionally substituted 4- to 7-membered heterocyclic ring.
42.    The compound of any one of claims 32-41, wherein R9 is C2-6alkyl or R9 is C16alkyl substituted with -CO 2H, -CO2C1.4alkyl, -CONH 2, -CONH(C1.4alkyl), -CON(C1.4alkyl)2, -CN, -SO 3H, -S0 2NH 2, -SO 2NH(C1.4alkyl), or -SO2N(C1.4alkyl)2.
43. The compound of claim 42, wherein R9 is C.6alkyl substituted with -CO 2H, preferably where R9 is -(CH 2)y-CO 2H, where y is 2, 3, 4, or 5.
44. The compound of any one of claims 32-43, wherein each R" is independently halo, -CO 2H, -SO 3H, -S0 2NH 2, -SO2NH(C 14alkyl), -SO 2N(C 14alkyl)2, or optionally substituted alkyl.
45. The compound of any one of claims 32-44, wherein q is 0, 1, or 2.
46. A compound selected from the group consisting of.
and salts thereof
47. The compound according to any preceding claim, wherein the compound is attached to a nucleotide or oligonucleotide.
48. A nucleotide or oligonucleotide labeled with a compound according to any one of claims 1 49. The labeled nucleotide or oligonucleotide according to claim 48 wherein the compound according to claims 1-11 is attached via R3 of Formula (I) or R9 of Formula (II).
50.    The labeled nucleotide or oligonucleotide of claim 49, wherein R3 of Formula (I) is -(CH 2).COOH where n is 2, 3, 4, 5, or 6, or R9 of Formula (II) is -(CH2)yCOOH where y is 2, 3, 4, 5, or 6, and the attachment forms an amide using the -COOH group.
51.    The labeled nucleotide or oligonucleotide of anyone of claims 48 to 50, wherein the label is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.
52.    The labeled nucleotide or oligonucleotide according to any one of claims 48 to 51, further comprising a 3' OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.
53.   A kit comprising a first labeled nucleotide according to any one of claims 49 to 52 and a second labeled nucleotide.
54.    The kit of claim 53, wherein the second labeled nucleotide is labeled with a different compound that the first labeled nucleotide or is unlabeled.
55.    The kit of claim 54, wherein the first and second labeled nucleotides are excitable using a single laser wavelength.
56.    The kit of claim 53 or 54, further comprising a third nucleotide and a fourth nucleotide, wherein each of the second, third, and fourth nucleotides is labeled with a different compound or is unlabeled, wherein each label has a distinct absorbance maximum that is distinguishable from the other labels.
57.    The kit of claim 53, wherein the kit comprises four nucleotides, wherein a first of the four nucleotides is a labeled nucleotide according to any one of claims 48 to 52, a second of the four nucleotides carries a second label, a third nucleotide carries a third label, and a fourth nucleotide is unlabeled (dark).
58.    The kit of claim 53, wherein the kit comprises four nucleotides, wherein a first of the four nucleotides is a labeled nucleotide according to any one of claims 48 to 52, a second of the four nucleotides carries a second label, a third nucleotide carries a mixture of two labels, and a fourth nucleotide is unlabeled (dark).
59.   Use of a nucleotide according to any one of claims 1 to 46, an oligonucleotide according to claims 48 to 52, or a kit according to any one of claims 53 to 58 in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or a protein binding assay.
60.   Use according to claim 59 on an automated sequencing instrument, wherein said automated sequencing instrument comprises two lasers operating at different wavelengths.
61.   A method of sequencing comprising incorporating a nucleotide according to any one of claims 48 to 52 in a sequencing assay.
62.    The method of claim 61, further comprising detecting the nucleotide.
63.    The method of claim 61 or 62, wherein the sequencing assay is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.
64.   A method of synthesizing a compound of Formula (I) or Formula (II) as described herein.