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1. WO2020115286 - CELL-PERMEABLE FLUOROGENIC FLUOROPHORES

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

Claims

1 . A compound of the general formula (10) or the general formula (1 O’)


wherein

R1 is selected from H and F, particularly R1 is H;

R2 and R3 can be any moiety, particularly R2 and R3 are independently selected from H, substituted or unsubstituted C to C4 alkyl optionally forming a bridge to the substituent designated Y, and a moiety having a molecular weight between 15 and 250 u (g/mol);

- X is selected from O, S, Se, TeO, PORx, POORx, S02, NRX, CRX2, SiRx2, GeRx2, and SnRx2, with each Rx being independently selected from H and an unsubstituted or substituted (particularly unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted) moiety selected from C C12 alkyl, C3-C8 cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, C7-Ci2 alkylaryl, phenyl and 5- or 6-membered heteroaryl, or two Rx moieties form a four-, five-, six- or seven-membered unsubstituted or amino-, hydroxy- and/or halogen substituted alkyl ring;

- Y is OH or NRY1 RY2, with

• RY1 , and RY2 each independently selected from H, an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted hydrocarbon moiety selected from C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, an unsubstituted phenyl or a phenyl substituted by any one or several of the following substituents:

unsubstituted C C4 alkyl, halogen, C C4 oxyalkyl, COOH, COORYC, CONRyc2, with RYC being selected from H and unsubstituted or amino- or hydroxy-substituted C C8 alkyl; or

• RY1 and RY2 together are a C3-C6 unsubstituted or hydroxy-, amino-, halogen-, alkoxy- and/or carboxy-substituted alkyl forming a 4-7-membered ring structure with Y; or

• one of RY1 and RY2, or both RY1 and RY2, together with R2 and/or R3, respectively, form an unsubstituted or hydroxy-, amino-, halogen-, carboxy- and/or aryl- substituted 4-7-membered alkyl or alkylene ring;

a) A and B together with the atoms that they are covalently coupled to form a phenyl ring or 5- or 6-membered heteroaryl ring, wherein the ring is unsubstituted or substituted by one of NRN1 RN2- and OH

and wherein the ring is optionally further by R4, R5 and/or R6, wherein

RN1 and RN2 have the same meanings as RY1 , and RY2 above,

R4 has the same meaning as R3, R5 has the same meaning as R1 , and R6 has the same meaning as R2

particularly A and B are a moiety of the general formula (50)


wherein

R4 and G are independently selected from H, substituted or unsubstituted Ci to C4 alkyl optionally forming a bridge to the substituent designated E, and a moiety having a molecular weight between 15 and 250 u (g/mol);

R5 is selected from H and F, particularly R5 is H;

E is OH or NREG1 REG2, wherein REG1 and REG2 have the same meanings as RY1 , and RY2 above, respectively, wherein optionally one of REG1 and REG2, or both REG1 and REG2, together with R4 and/or G, respectively, form an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted 4-7-membered alkyl ring;

b) A is an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted conjugated carbocycle or heterocycle having one, two or three rings, particularly A is an aromatic or heteroaromatic ring system, more particularly A is selected from an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted aryl, and heteroaryl, most particularly A is a substituted (particularly hydroxy-, amino-, halogen-, and/or carboxy-substituted) coumarin; and

B is selected from H (or D, F) and a moiety having a molecular weight between 15 and 250 u (g/mol);

U and V

• together with the atoms they are covalently coupled to form an unsubstituted or substituted 5-7 membered ring,

wherein the substitution is selected from one or several substituents independently selected from OH, SH, amine [particularly NRUV2] halogen, CN, NC, CNO, NCO, CNS, NCS, unsubstituted C C4 O-alkyl, C C4 S-alkyl, O- aryl, S-aryl, N02, CHO, CONRuv2, COORuv, COO-aryl, COO-alkylaryl (particularly benzyl), P03H, P03Ruv, S03H, S03Ruv and S02Ruv, with Ruv being selected from H, and C C4 unsubstituted alkyl

and the 5-7 membered ring is selected from

o cycloalkyl,

o aryl,

o heteroaryl,

particularly U and V form an unsubstituted or substituted 5-membered heteroaryl or phenyl, or

• U is selected from H, D and F and V is selected from H, D, F, and C to C4 unsubstituted or amino-, hydroxy- or halogen substituted alkyl,

Z is selected from

• CN, CHO, CORz, COORz, and CONRz2, wherein each Rz is independently selected from H, unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C C4 alkyl, halogen, C C4 oxyalkyl, COORc, CONRc2, with Rc being selected from H, C C8 alkyl; particularly Rz is selected from H, unsubstituted C C4 alkyl, and alkylaryl (particularly benzyl); or

• S02RS with Rs being selected from

o NH2, NHRsn, NRsn2, with each RSN independently selected from a

unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted moiety selected from C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, or from unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C C4 alkyl, halogen, O- C C4 alkyl, COOH, COORc, CONRc2, with Rc being selected from H and C C8 alkyl;

o an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted moiety selected from C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C C4 alkyl, halogen, O- C C4 alkyl, COOH, COORsc, CONRsc2, with Rsc being selected from H and C C8 alkyl; particularly Rsc is unsubstituted or fluorinated alkyl;

o ORso, with Rso selected from a unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted moiety selected from C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, or from unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C C4 alkyl, halogen, O- C C4 alkyl, COOH, COORc, CONRC2, with Rc being selected from H and C C8 alkyl;

• CH2Rf, CHRF2, and CRF3, with RF selected from CH2F, CHF2, CF3, CH2CI, and CHCI2, CCI3, particularly with RF being CF3 ;

• H

• CH2COORK1 or CH2CONHRK1 with RK1 selected from H, and unsubstituted or halogen-substituted Ci to C4 alkyl;

wherein optionally, the compound is covalently linked to a binding moiety M capable of attaching the compound to a biomolecule,

with the proviso that the following compounds are not claimed:


- R1 , R2, R3, R4, R5 and G are H, and

Y and E are NEt2 and

o Z is CONH2 or

o Z is CONHC6H5; or

o Z is CN or

o Z is selected from CH2CF3, CH2CHF2, and CH2CH2F, or

o Z is selected from CH2COOCH3 and CH2COOH, o Z is H,

or

Y and E are NMe2 and Z is selected from CH2COOCH3 and CH2COOH or

Y and E are NH2 and Z is selected from CH2COOH and CH2COOCH3 or

Y is OH and E is =0 (for formula 10) or E is OH (for formula 10’), and Z is C(=0)(CH3)C6H5;

or

R1 , R3, R4, and R5 are H, R2 and G are methyl, Y and E are NHEt and Z is CH2COOH or CH2COOCH3

or the compound is of formula (301), (302), (303), or (304)


2. The compound according to claim 1 , wherein the compound is covalently linked (particularly through any one of substituents A, B, R2, R3, R4, REG1 , REG2, REG, G, Rx, RY1 , RY2, RY, U, V, Rz, RPh, Rs, RK1) to M, and M is selected selected from:

a. a moiety selectively attachable by a covalent or quasi-covalent bond to a protein or a nucleic acid under conditions prevailing in cell culture or inside of a living cell, particularly a moiety able to form an ester bond, an ether bond, an amide bond, a disulfide bond, a Schiff base, or a moiety able to react in a click-

chemistry reaction, more particularly a moiety selected from -COCHCH2, -CO- NHS, biotin, an azide or ethyne moiety, a tetrazine moiety, a (bicyclo[6.1.0]nonyne) moiety, a cyclooctyne moiety, a transcyclooctene moiety and a maleimide, or a moiety employed for specific protein labelling,

b. a substrate of an 06-alkylguanine-DNA-alkyltransferase or a functional variant thereof,

particularly a 6-[(4-methylenephenyl)methoxy]-9H-purin-2-amine moiety of formula (71), or a pyrimidine derivative thereof, particularly a moiety of formula (72), or a moiety of formula (73),


c. a substrate of a haloalkane halotransferase,

particularly a 1 -chlorohexyl moiety as exemplarily shown below;


(74); or from

d. a substrate of dihydrofolate reductase, particularly the moiety:


e. a moiety capable of selectively interacting non-covalently with a biomolecule (particularly a protein or nucleic acid) under conditions prevailing in a living cell, wherein said moiety and said biomolecule form a complex having a dissociation constant kD of 10 4 mol/l or less, particularly wherein

- M has a molecular mass of more than 160 u but less than 1000 u,

particularly less than 700 u, more particularly less than 500 u, and M comprises up to five hydrogen bond donators, up to ten hydrogen bond acceptors; more particularly, M is selected from taxol, jasplaklinolide, a bis-benzimide DNA stain, pepstatin A and triphenylphosphonium; or

- M is an oligonucleotide having a sequence length of 10 to 40 nucleotides. f. a lipid, particularly a lipid selected from a ceramide derivative, a glyceride, or a fatty acid.

3. The compound according to claim 2, wherein the compound is connected to said binding moiety M through a covalent bond or a linker moiety L consisting of 1 to 50 atoms having an atomic weight of 12 or higher, particularly wherein L is a moiety described by a formula

- lL A1 n- lL J1 n s -L A2 m- ,L J2 m s -L A3 i J3

p-L p -L q-L q -, wherein

LA1 , LA2, LAS and l_A4 independently of each other are selected from
to C12 unsubstituted or amino-, hydroxyl-, carboxyl- or fluoro-substituted alkyl or C3 to C7 cycloalkyl, (CH2-CH2-0)r or (CH2-CH(0H)-CH2-0)r with r being an integer from 1 to 20, alkylaryl, alkylaryl-alkyl, and unsubstituted or C C4 alkyl-, halogen-, amino-, C C4 alkylamino-, imido-, nitro-, hydroxyl-, C C4 oxyalkyl-, carbonyl-, carboxyl-, sulfuryl- and/or sulfoxyl-substituted aryl or heteroaryl,

LJ1 , LJ2, LJ3 and LJ4 independently of each other are selected from -NRN5C(0)-, -C(0)N(RN5)-, -CN-, -NC-, -CO-, -OC(O)-, -C(0)0-,-NRN5-, -0-, -P(OOH)-, -OP(OOH)-, -R(00H)0-, -0P(00H)0-, -0P(00H)0-, -S-, -SO-, S02-,

with RN5 selected from H and unsubstituted or amino-, hydroxyl-, carboxyl or fluoro-substituted C to C6 alkyl, particularly RN5 is selected from H and unsubstituted C to C3 alkyl;

n, n’, m, m’, p, p’, q, q' and s independently from each other are selected from 0 and 1.

4. The compound according to claim 3, wherein L is -LA1-LJ1-LA2m-LJ2m’-LA3p, wherein

LA1 , LA2 and l_A3 are independently selected from unsubstituted, amino-, hydroxyl-, carboxyl- or fluoro substituted OΪ to C6 alkyl or C3 to C6 cycloalkyl, (CH2-CH2-0)r or (CH2-CH(0H)-CH2-0)r with r being an integer from 1 to 4, and

- LJ1 and LJ2 are selected independently from -NRN5C(0)-, -C(0)N(RN5)-, -CN-, -NC-, -CO-, -OC(O)-, -C(0)0-, NRN5-, -0-, and -S-, and

m, m’ and p independently from each other are selected from 0 and 1 .

5. The compound according to any one of the preceding claims, wherein the compound is connected to said binding moiety M through one of substituents A, R2, R3, R4, G, or a phenyl moiety formed by U and V.

6. The compound according to any one of the preceding claims, wherein any one of substituents R2, R3, R4, G and Rx independently of any other is selected from H and unsubstituted or fluoro-, amino-, hydroxyl-, S03H- and/or carboxyl substituted C to C4 alkyl, Ci to C4 alkenyl or Ci to C4 alkynyl, wherein optionally one of R2, R3, R4, G and Rx is linked to a moiety M, particularly wherein M is defined by claim 2, more particularly wherein M is linked to the compound by a moiety L as defined in claim 3 or 4.

7. The compound according to any one of the preceding claims, wherein A is a moiety of the general formula (60)


wherein RA is selected from H, OH, NRY1 RY2, with RY1 , and RY2 as defined in claim 1 , or one of RY1 , and RY2, or RA itself, is L-M, with L as defined in claim 3 or 4, and M as defined in claim 2.

8. The compound according to any one of the preceding claims, wherein

B is selected from H, D and F.

9. The compound according to any one of the preceding claims, wherein Z is selected from

• CN and CONRz2 wherein each Rz is independently selected from H, unsubstituted C C4 alkyl and alkylaryl (particularly benzyl); or

• S02RS with Rs being selected from

o NRsn2, with each RSN being independently selected from H,

unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted moiety selected from C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C C4 alkyl, halogen, O- C C4 alkyl, COOH, COORc, CONRc2, with Rc being selected from H and C C8 alkyl;

o unsubstituted or perfluorinated alkyl;

particularly Z is S02Rs with Rs being selected from

o NRsn2, with each RSN being independently selected from H,

unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy- substituted C C8 alkyl, C3-C8 cycloalkyl, C C4 acyl, C7-C12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C C4 alkyl, halogen, O- C C4 alkyl, COOH, COORc, CONRc2, with Rc being selected from H and C C8 alkyl;

o unsubstituted or perfluorinated alkyl.

10. The compound according to any one of the preceding claims, wherein U and V form an unsubstituted or substituted phenyl, wherein one or several substituents of phenyl are independently selected from OH, SH, amine [particularly NRUV2], halogen, CN, NC, CNO, NCO, CNS, NCS, unsubstituted C C4 O-alkyl, C C4 S-alkyl, O-aryl, S-aryl, N02, CHO, CONRuv2, COORuv, COO-aryl, COO-alkylaryl (particularly benzyl), P03H, P03Ruv, S03H, S03RUV and S02Ruv, with Ruv being selected from H, and C C4 unsubstituted alkyl, or wherein U and V form a phenyl substituted with a moiety L-M, wherein M has the meaning as specified in claim 2, and L has the meaning as specified in claim 3 or 4.

1 1. The compound according to any one of the preceding claims, wherein each Rx is independently selected from

unsubstituted or hydroxyl-, amino- or halogen-substituted OΪ to C4 alkyl, C2 to C4 alkenyl or C2 to C4 alkynyl,

unsubstituted or hydroxyl-, amino- or halogen-substituted C4 to C6 cycloalkyl or unsubstituted or hydroxyl-, alkyoxy-, amino- or halogen-substituted phenyl, particularly wherein Rx is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and phenyl,

more particularly wherein both Rx are the same for X selected from C, Si, Ge, and Sn.

12. The compound according to any one of the preceding claims 1 to 6 and 9 to 1 1 , wherein Y is NRY1 RY2 and E is NREG1 REG2 and

a. RY1 and RY2, and/or REG1 and REG2, are independently selected from H, unsubstituted and amino-, hydroxy-, carboxy- and/or fluoro-substituted C C6 alkyl, C! - C4 acyl, and C3 - C6 cycloalkyl,

particularly RY1 and RY2, and/or REG1 and REG2, are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and CH2CF3, more particularly wherein RY1 , RY2, REG1 and REG2 are the same and are selected from H or unsubstituted C C4 alkyl and C3 - C6 cycloalkyl;

b. RY1 together with RY2, and/or REG1 together with REG2 form a ring and are an unsubstituted or alkyl-, amino-, hydroxy-, carboxy- and/or fluoro-substituted C3 - C6 alkyl,

particularly -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)20(CH2)2- or -(CH2)2NRNN(CH2)2- with RNN being selected from H and unsubstituted C! to C4 alkyl;

c. RY1 and/or REG1 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C! -C6 alkyl, C! - C4 acyl, and C3 -C6 cycloalkyl, and RY2 together with R2 or R3, and/or REG2 together with G or R4, is an alkyl or heteroalkyl bridge selected from -(CH2)2-, -(CH2)3-, -CH2CH=CH- or -(CH2)4- or -CH2-0-, -CH2-NR5-, -CH2-S-, -CH2-Se-, -

(CH2)20-, -(CH2)2NRn-, -(CH2)2S-, -(CH2)2Se-, -CH2-0-CH2-, -CH2NR5-, -CH2S- CH2-, -CH2-Se-CH2-, -CH2-(1 ,2)phenyl-, and a mono- or dimethyl substituted derivative of any one of the foregoing alkyl or heteroalkyl bridge moieties;

d. RY1 and/or REG1 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted
-C6 alkyl,
- C4 acyl, and C3 -C6 cycloalkyl, and RY2 together with R2, and/or REG2 together with G, form an cyclic structure according to any one of substructures (41 ) to (44) or (51) to (54):


(44) (54),

wherein R11 , R12, R13, R14, R15 and R16 are selected from H, unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted Ci to C4 alkyl, halogen, S03R’, COOR’, CONR’2 with R’ selected from H and unsubstituted to C4 alkyl;

and R17 is selected from H unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C to C4 alkyl, halogen, N02, CN, S03R’, COOR’, CONR’2 with R’ selected from H and unsubstituted Ci to C4 alkyl;

particularly wherein R11 , R12, R13, R14, R15 and R16 are selected from H, methyl, CH2-S03H, Cl and F,

and R1 and R3 can have any of the meanings given herein; or

e. RY1 together with R3, and RY2 together with R2, and/or REG1 together with R4, and REG2 together with G, form a bi-cyclic structure according to any one of substructures (45) to (47) or (55) to (57):


wherein R11 , R12, R13, and R15 are selected from H, unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted Ci to C4 alkyl, halogen, S03R’, COOR’, CONR’2 with R’ selected from H and unsubstituted
to C4 alkyl;

particularly wherein R11 , R12, R13, and R15 are selected from H, methyl, CH2- SO3H , Cl and F,

and R1 and R5 can have any of the meanings given herein; or

f. RY2 and/or REG2 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted ^ -C6 alkyl, Ci - C4 acyl, and C3 -C6 cycloalkyl, and RY1 together with R3, and/or REG1 together with R4, form an cyclic structure according to any one of substructures (48) to (49) or (58) to (59):


wherein R1 and R5 can have any of the meanings given herein.

13. The compound according to claim 12, wherein RY1 together with RY2, and/or REG1 together with REG2 are -(CH2)3-, -CH2CHFCH2-, -CH2CF2CH2-, -CH2CH(CH3)CH2-, - CH2C(CH3)2CH2-, CH2CH(CN)CH2-, CH2CH(COOH)CH2-, CH2CH(CH2COOH)CH2-, - CH2CH(OCH3)CH2- and -CH2CH(N(CH3) 2)CH2-, particularly wherein the substituent is the same for RY1 with RY2, and REG1 with REG2.

14. The compound according to claim 12, wherein

RY1 together with R3, and RY2 together with R2, and/or REG1 together with R4, and REG2 together with G, form a bi-cyclic structure according to any one of substructures (45) to (47) or (55) to (57), and

Z is selected from

• CH2Rf, CHRf2, and CRF3, with RF selected from CH2F, CHF2, CF3, CH2CI, and CHCI2, CCI3, particularly with RF being CF3;

• H

• CH2COORK1 or CH2CONHRK1 with RK1 selected from H, and

unsubstituted or halogen-substituted to C4 alkyl

particularly wherein X is O.

15. The compound according to any one of the preceding claims, wherein

- Y is NRY1 RY2 and E is NREG1 REG2 ,

RY1 , RY2, REG1 and REG2 are unsubstituted or halogen-substituted C4 alkyl, particularly methyl, ethyl or CH2CF3;

optionally, R2, R3, R4 and G can be halogen (particularly F or Cl); and Z is selected from

• CH2Rf, CHRf2, and CRF3, with RF selected from CH2F, CHF2, CF3, CH2CI, and CHCI2, CCI3, particularly with RF being CF3;

• H

• CH2COORK1 with RK1 selected from H, and unsubstituted or halogen- substituted Ci to C4 alkyl;

particularly wherein X is O.

16. The compound according to any one of claims 1 to 5 or 7 to 1 1 , wherein the compound is of the general formula (30) or (30’)



wherein

Ra, B, U and V have the meanings as defined in claim 1 ,

- Y is NRY1 RY2,

RY1 and RY2 are unsubstituted or halogen-substituted - C4 alkyl, particularly methyl, ethyl or CH2CF3;

optionally, R2 and R3, can be halogen (particularly F or Cl); and

Z is selected from

• CH2Rf, CHRf2, and CRF3, with RF selected from CH2F, CHF2, CF3, CH2CI, and CHCI2, CCI3, particularly with RF being CF3;

• H

• CH2COORK1 with RK1 selected from H, and unsubstituted or halogen- substituted Ci to C4 alkyl;

particularly wherein X is O.

17. The compound according to any one of the preceding claims 1-6 and 9-15, wherein the compound is of the general formula (20) or (20’)


- Y is NRY1 RY2 and E is NREG1 REG2, wherein RY1 , RY2, REG1 and REG2 are individually unsubstituted or amino-, hydroxyl- or halogen-substituted
to C4 alkyl or C3 to C6 cycloalkyl, or RY1 together with RY2, and REG1 together with REG2 together with the N form an unsubstituted or methyl-, ethyl- propyl-, or halogen-substituted aziridine, pyrrolidine, piperidine, piperazine or morpholine, and/or

R1 and R5 are H, and/or

R2, R3, R4 and G are independently selected from

o H, halogen, S03H, and unsubstituted and amino-, hydroxy-, carboxy-, S03H-, and/or halogen-substituted C-\ -C4 alkyl, C02H, C02R, S02R with R being selected from to C4 unsubstituted alkyl,

and/or

X is selected from O, CRX2, SiRx2, particularly wherein X is O, and/or

each Rx is independently selected from unsubstituted or halogen-substituted
to C4 alkyl or C3 to C6 cycloalkyl and phenyl, and/or

U and V have the meaning defined in claim 9, and/or

Z has the meaning as defined in claim 1 , particularly the meaning as defined in claim 8, and

wherein optionally, one of G, R2, R3, R4, or a phenyl linking U and V bears a moiety M as defined in claim 2, linked to the compound by a covalent bond or a linker L, with L as defined as in claim 3 or 4.

18. The compound according to any one of claims 1 to 5 or 7 to 1 1 or 16, wherein the compound is of the general formula (30) or (30’)



wherein

RA has the meaning as defined in claim 7 and B is H, and/or

Y is NRY1 RY2, wherein RY1 and RY2 are individually unsubstituted or amino-, hydroxyl- or halogen-substituted
to C4 alkyl or C3 to C6 cycloalkyl, or RY1 together with RY2, and REG1 together with REG2 together with the N form an unsubstituted or methyl-, ethyl- propyl-, or halogen-substituted aziridine, pyrrolidine, piperidine, piperazine or morpholine, and/or

R1 is H, and/or

R2 and R3 are independently selected from

o H, halogen, S03H, and unsubstituted and amino-, hydroxy-, carboxy-, S03H-, and/or halogen-substituted
-C4 alkyl, C02H, C02R, S02R with R being selected from Ci to C4 unsubstituted alkyl,

and/or

X is selected from O, CRX2, SiRx2, particularly wherein X is O, and/or

each Rx is independently selected from unsubstituted or halogen-substituted
to C4 alkyl or C3 to C6 cycloalkyl and phenyl, and/or

U and V have the meaning defined in claim 10, and/or

Z has the meaning as defined in claim 1 , particularly the meaning as defined in claim 9, and

wherein optionally, one of R2, R3, RA, or a phenyl linking U and V bears a moiety M as defined in claim 2, linked to the compound by a covalent bond or a linker L, with L as defined as in claim 3 or 4.

19. The compound according to any one of the preceding claims 17 or 18, wherein R2, R3, R4, and G, are

• H, or

n1 2 n3 n4 ^ n5 ; _ A1 J1 3 A2 J2 3 A3 J3 3 A4 J4 3 one Of R , R , R , R , G, and R IS L n-L n -L m-L m -L p-L p -L q-L q -Ms, wherein |_A1 4, LJ1 4 ,n, n’ ...q’, s and M have the definitions recited above each
r phenyl,

- Y is NRY1 RY2 and E is NREG1 REG2; and

RY1 , RY2, REG1 and REG2 are individually unsubstituted or amino-, hydroxyl- or fluoro substituted Ci to C4 alkyl, or RY1 together with RY2, and REG1 together with REG2 form a ring and are -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)20(CH2)2- or -(CH2)2NH(CH2)2-.

20. The compound according to any one of the preceding claims 1 to 18, wherein R2 and G are F or Cl.

21. Use of a compound according to any one of the preceding claims or being characterized by one of the following combinations


- R1 , R2, R3, R4, R5 and G are H, and

Y and E are NEt2 and

o Z is CONH2 or

o Z is CONHC6H5; or

o Z is CN or

o Z is selected from CH2CF3, CH2CHF2, and CH2CH2F, or o Z is selected from CH2COOCH3 and CH2COOH,

o Z is H,

or

Y and E are NMe2 and Z is selected from CH2COOCH3 and CH2COOH or

Y and E are NH2 and Z is selected from CH2COOH and CH2COOCH3 or

Y is OH and E is =0 (for formula 10) or E is OH (for formula 10’), and Z is C(=0)(CH3)C6H5;

or

R1 , R3, R4, and R5 are H, R2 and G are methyl, Y and E are NHEt and Z is CH2COOH or CH2COOCH3

or the compound is of formula (301), (302), (303) or (304)


in a method of staining a biological sample.

22. A method to stain a sample, said method comprising the steps of:

a. contacting the sample with a compound according to any one of the preceding claims or being characterized by one of the following combinations


- R1 , R2, R3, R4, R5 and G are H, and

Y and E are NEt2 and

o Z is CONH2 or

o Z is CONHC6H5; or

o Z is CN or

o Z is selected from CH2CF3, CH2CHF2, and CH2CH2F, or o Z is selected from CH2COOCH3 and CH2COOH,

o Z is H,

or

Y and E are NMe2 and Z is selected from CH2COOCH3 and CH2COOH or

Y and E are NH2 and Z is selected from CH2COOH and CH2COOCH3 or

Y is OH and E is =0 (for formula 10) or E is OH (for formula 10’), and Z is C(=0)(CH3)C6H5;

or

R1 , R3, R4, and R5 are H, R2 and G are methyl, Y and E are NHEt and Z is CH2COOH or CH2COOCH3

or the compound is of formula (301), (302), (303) or (304)


b. illuminating the sample with light of a wavelength ranging from 400-800 nm; c. recording the presence and location of said compound in said sample by illuminating the sample with light of an appropriate excitation wavelength and recording light emitted from said sample, particularly at an appropriate emission wavelength l, more particularly a l close to the maximum of the emission spectrum of 400-800 nm.