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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 C1-6alkyl;
R is H, halo, -CN, amino, -OH, C-amido, N-amido, -N02,
-SO 3H, -SO 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;
R 1 is H, halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido, -N2, -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;
R2 and R4 are each independently H, halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido,
-NO2, -SO3H, -SO 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;
R3 is C2-6alkyl substituted with -CO 2H or -SO 3H, optionally substituted heterocyclyl, or R 3 is linked to R2 or R to form an optionally substituted ring;
each R' is independently halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido, -NO2, -SO 3H, -SO 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 R2 and R4 are each independently H, halo, -CN, -CO 2H, amino, -OH, C-amido, N-amido, -NO2, -SO 3H, -SO 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 neither of R2 and R4 is linked to R 3 to form an optionally substituted heterocyclic ring.
3.   The compound of claim 1 or claim 2, wherein X is 0 or S; or
wherein X is NR, where R" is H or CI-6alkyl.
4.   The compound of any one of claims 1 to 3, wherein R 3 is -(CH2)nCOOH, where n is 2, 3, 4, 5 or 6.
5.   The compound of any one of claims 1-4, wherein R3 is -(CH2)nSO 3H where n is 2, 3, 4, 5 or 6.
6.   The compound of any one of claims 1-5, wherein m is 0, 1, or 2.
7.   The compound of any one of claims 1-6, wherein each R5 is independently optionally substituted Ci-6alkyl, halo, -CN, -CO 2H, amino, -OH, -SO 3H, or -SO 2NH2; or
wherein each R 5 is halo, -CN, -CO2H, -SO 3H, -SO 2NH 2, or optionally substituted C1. 6alkyl; or
wherein R 5 is C2-6alkyl substituted with -CO 2 H, -SO 3H, or
-SO 2NH 2; or
wherein R5 is -(CH2)xCOOH, where x is 2, 3, 4, 5 or 6; or
wherein R5 is -SO 3H or -SO 2NH2.
8.   The compound of any one of claims 1-7, wherein R1 is H, halo, or Ci-6alkyl; or wherein R 1 is H or Cl.
9.   The compound of any one of claims 1-8, wherein R is H, halo, or C1-6alkyl; or
wherein R is H or Cl; or
wherein R is H.
10.   The compound of any one of claims 1-9, wherein R2 is H, optionally substituted alkyl, C1 . 4alkyl optionally substituted with -CO 2H or -SO 3H, or -SO 3H; or
wherein     R2  is  H or  -SO  3H. 
11.   The compound of any one of claims 1-9, wherein R2 or R4 is linked to R3 to form an optionally substituted heterocyclic ring, or a pyrrolidine or a piperidine, wherein the pyrrolidine or piperidine are optionally substituted with one or more alkyl groups.
12.   The compound of any one of claims 1-10, wherein R4 is H, optionally substituted alkyl, Ci 4alkyl optionally substituted with -CO 2H or -SO 3H, or -SO 3H.
13.   The compound of claim 1, wherein X is 0 or S; R is H; R is H; R3 is -(CH2)nCOOH where n is 2, 3, 4, 5, or 6; R5 is H, -SO 3H, or -SO2NH 2; R2 is H or -SO 3H; and R4 is H or -SO 3H; optionally
a) wherein R3 is -(CH 2)2COOH; or
b) wherein R3 is -(CH2)COOH.
14.   A compound of Formula (II) or a salt thereof:
wherein:
X' is selected from 0, S, and NRP, where RP is H or CI-6alkyl;
R6 is H or C1-4alkyl;
R7 is H, halo, -CN, -OH, optionally substituted C-4alkyl, optionally substituted Ci-4alkenyl, optionally substituted C2-4alkynyl, -CO 2H, -SO 3H, -SO 2NH 2, -SO2NH(C1-4alkyl), -SO2N(C1 4alkyl)2, and optionally substituted Ci4alkoxy;
R8 and R10 are each independently H, halo, -CN, -CO 2H, amino, -OH, -SO 3H, -SO 2NH2,
-SO 2NH(Ci-4alkyl), -SO2N(Ci-4alkyl)2, optionally substituted CI-6alkyl, optionally substituted CI-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted Ci 6alkoxy; or
one of R 8 and R 10 is H, halo, -CN, -CO 2 H, amino, -OH, -SO 3H, -SO 2NH 2, -SO2NH(C1-4alkyl), -SO2N(Ci-4alkyl)2, optionally substituted C1-6alkyl, optionally substituted C1-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C1-6alkoxy, and the other of R8 and R 10 is taken with R9 to form an optionally substituted 4- to 7-membered heterocyclic ring;
R9 is C2-6alkyl substituted with -CO 2H, -CO2C1-4alkyl, -CONH 2, -CONH(C1-4alkyl), -CON(C1-4alkyl)2, -CN, -SO 3H, -SO 2NH 2, -SO 2NH(C1-4alkyl), or -SO2N(C1-4alkyl)2; each R" is independently halo, -CN, carboxy, amino, -OH, C-amido, N-amido, nitro, -SO 3H, -SO 2NH 2, -SO2NH(Cl-4alkyl), -SO2N(C1-4alkyl)2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted CI-6alkoxy; and q is 0, 1, or 2.
15.   The compound of claim 14, wherein at least one of the following conditions i) to vi) applies to the compound of Formula (II);
i) X' is 0 or X' is S, or X' is NRP, where RP is H or C1-6alkyl,
ii) R6 is H or R6 is C1-4alkyl,
iii) R 7 is H or R7 is optionally substituted C1-4alkyl, -CO 2H, -SO 3H, -SO 2NH2 ,
SO2NH(C1-4alkyl), or -SO2N(C-4alkyl)2, optionally R' is C1-4alkyl optionally substituted with -CO 2H,
iv) R8 is H or R8 is -CO 2 H, -SO 3H, or -SO2NH 2,
v) R 10 is H or is -CO 2H, -SO 3H, or -SO 2NH 2, and
vi) (a) R 8 is H and R 10 is -SO 3H or (b) R 8 is -SO 3H and R 10 is H.
16.   The compound of any one of claims 14-15, wherein one of R 8 and R10 is H, halo, -CN, -CO2H, amino, -OH, -SO 3H, -SO 2NH 2, -SO2NH(C1-4alkyl), -SO2N(Cl-4alkyl)2, optionally substituted C1-6alkyl, optionally substituted Ci-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted Ci-alkoxy, and the other of R8 and R 10 is taken with R 9 to form an optionally substituted 4- to 7-membered heterocyclic ring.
17.   The compound of claim 14, wherein R9 is C2-6alkyl substituted with -CO 2H, or where R9 is -(CH 2)y-CO 2H, where y is 2, 3, 4, or 5.
18.   The compound of any one of claims 14-17, wherein each R 1 is independently halo, -CO 2H, -SO 3H, -SO 2NH 2, -SO2NH(C1-4alkyl), -SO2N(Ci-4alkyl)2, or optionally substituted alkyl.
19.   A compound of claim 1 selected from the group consisting of:
and salts thereof.
20.   A nucleotide or oligonucleotide labeled with a compound according to any one of claims 1 19.
21.   The labeled nucleotide or oligonucleotide according to claim 20 wherein;
(A) the compound is attached via R3 of Formula (I) or R9 of Formula (II), optionally further wherein R3 of Formula (I) is -(CH 2)nCOOH 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; and/or
(B) 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; and/or
(C) a 3' OH blocking group is covalently attached to the ribose or deoxyribose sugar of the nucleotide.
22.   A kit comprising a first labeled nucleotide according to claim 20 or claim 21 and a second labeled nucleotide, optionally;
i) wherein the second labeled nucleotide is labeled with a different compound that the first labeled nucleotide or is unlabeled, optionally wherein the first and second labeled nucleotides are excitable using a single laser wavelength; and/or ii) 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; or
iii) wherein the kit comprises four nucleotides, wherein a first of the four nucleotides is a labeled nucleotide according to claim 20 or claim 21, a second of the four nucleotides carries a second label, a third nucleotide carries a third label, and a fourth nucleotide is unlabeled (dark); or
iv) wherein the kit comprises four nucleotides, wherein a first of the four nucleotides is a labeled nucleotide according to claim 20 or claim 21, 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).
23.   Use of a compound according to any one of claims 1 to 19, a nucleotide or an oligonucleotide according to claim 20 or claim 21, or a kit according to claim 22 in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or a protein binding assay.
24.   Use according to claim 23 on an automated sequencing instrument, wherein said automated sequencing instrument comprises two lasers operating at different wavelengths.
25.   A method of sequencing comprising incorporating a nucleotide according to claim 20 or claim 21 in a sequencing assay, optionally further comprising detecting the nucleotide, optionally 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.