NOVEL TUNABLE PHOTOACTIVATABLE SILICON RHODAMINE FLUOROPHORES

Abstract

The invention relates to a compound characterized by general formula (100), wherein R.sup.1 and R.sup.6 are H or F, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 can be any substituent, R.sup.7, R.sup.8, R.sup.N1, R.sup.N2, R.sup.N3 and R.sup.N4 are a hydrocarbon moiety, one of R.sup.9 and R.sup.10 is hydrogen and the other one is hydrogen or a saturated carbon atom connected to any substituent, and its use in staining and live cell fluorescence imaging.

##STR00001##

Claims

1. A compound characterized by general formula (100): ##STR00051## wherein R.sup.1 and R.sup.6 are independently selected from hydrogen and fluorine, particularly R.sup.1 and R.sup.6 are hydrogen; R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently of each other is selected from H, halogen, SO.sub.3H, CO.sub.2H, NO.sub.2, CO.sub.2R, SO.sub.2R (with R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl) and an unsubstituted or substituted (particularly unsubstituted or halogen-, amino-, hydroxyl-, SO.sub.3H- and/or carboxyl substituted) moiety selected from C.sub.1-C.sub.20 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; R.sup.7 and R.sup.8 are independently selected from: unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.12 alkylene, C.sub.2-C.sub.12 alkylyne and C.sub.7-C.sub.12 alkylaryl; unsubstituted or substituted 5- or 6-ring aryl, particularly phenyl, particularly wherein R.sup.7 is selected from H and a substituted or unsubstituted alkyl, alkenyl, alkenyl, alkylary or aryl having x carbon atoms and R.sup.8 is selected from H and a substituted or unsubstituted alkyl, alkenyl, alkenyl, alkylary or aryl having y carbon atoms and the sum of x and y is between 0 and 12, more particularly between 0 and 10, even more particularly between 0 and 8, even more particularly between 0 and 6; R.sup.N1, R.sup.N2, R.sup.N3 and R.sup.N4 are: independently selected from H, unsubstituted and substituted C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, and C.sub.7-C.sub.12 alkylaryl, and unsubstituted phenyl or phenyl substituted by COOH—, COOR, CONR.sub.2, unsubstituted alkyl, halogen, O-alkyl, and/or NO.sub.2; or R.sup.N1 together with R.sup.N2, and/or R.sup.N3 together with R.sup.N4 are a C.sub.3, C.sub.4, C.sub.6 unsubstituted or substituted alkyl forming a 3-7 sized ring structure; or R.sup.N1 and/or R.sup.N3 are independently selected from H and unsubstituted and substituted C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and C.sub.7-C.sub.12 alkylaryl, and R.sup.N2 together with R.sup.2 or R.sup.3, and/or R.sup.N4 together with R.sup.4 or R.sup.5, is an unsubstituted or substituted C.sub.2, C.sub.3 or C.sub.4 alkyl, or an unsubstituted or substituted C.sub.2, C.sub.3 or C.sub.4 N-, O-, S-, or Se-alkyl; one of R.sup.9 and R.sup.19 is hydrogen and the other one is hydrogen or a saturated carbon atom connected to a moiety selected from H, halogen, SO.sub.3H, CO.sub.2H, NO.sub.2, CO.sub.2R, SO.sub.2R (with R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl) and an unsubstituted or substituted (particularly unsubstituted or halogen-, amino-, hydroxyl-, SO.sub.3H— and/or carboxyl substituted) moiety selected from C.sub.1-C.sub.20 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof and wherein optionally the compound is covalently linked to a binding moiety M via any of the substituents.

2. The compound according to claim 1, wherein the compound is covalently linked (particularly through any one of substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.N1, R.sup.N2, R.sup.N3 and R.sup.N4) to a binding moiety M selected from: a. a moiety selectively attachable by covalent bond to a protein or 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 selected from —COCHCH.sub.2, —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 from b. a substrate of O.sup.6-alkylguanine-DNA-alkyltransferase, particularly a 6-[(4-methylenephenyl)methoxy]-9H-purin-2-amine moiety of formula (110), or a pyrimidine derivative thereof, particularly a moiety of formula (111) or (112), ##STR00052## c. a substrate of a haloalkane halotransferase, particularly a 1-chlorohexyl moiety as exemplarily shown below; ##STR00053## or from d. a substrate of dihydrofolate reductase, particularly the moiety: ##STR00054## e. a moiety capable of selectively interacting non-covalently with a biomolecule (particularly a protein or nucleic acid) under conditions prevailing in a live cell, wherein said moiety and said biomolecule form a complex having a dissociation constant k.sub.D of 10.sup.−6 mold 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 and is characterized by an octanol-water partition coefficient logP of below 5.6; more particularly, M is selected from taxol, jasplaklinolide, a bis-benzimide DNA stain, pepstatin A and triphenylphosphonium; or wherein 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 1, wherein any one of substituents R.sup.2, R.sup.3, R.sup.4, R.sup.5, and one of R.sup.9 and R.sup.10 independently of any other is H or a moiety having a molecular weight between 15 and 1500 u (g/mol); particularly wherein one of substituents R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.9 and R.sup.10 is a moiety having a molecular weight between 15 and 1500 u and the other ones are selected from H and unsubstituted or fluoro-, amino-, hydroxyl-, SO.sub.3H— and/or carboxyl substituted C.sub.1 to C.sub.4 alkyl, alkenyl or alkynyl; more particularly wherein one of substituents R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.9 and R.sup.19 is a moiety having a molecular weight between 15 and 1500 u and the other ones are H.

4. The compound according to claim 3, wherein said moiety having a molecular weight between 15 and 1500 u is characterized by a general formula -L-M, wherein L is a linker covalently connecting the compound of structure (1) to the binding moiety M as defined above, and L is a covalent bond or a linker consisting of 1 to 50 atoms having an atomic weight of 12 or higher, particularly wherein said moiety having a molecular weight between 15 and 1500 u is characterized by a general formula
L.sup.A1.sub.n-L.sup.J1.sub.n′-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.p-L.sup.J3.sub.p′-L.sup.A4.sub.q-L.sup.J4.sub.q′-M.sub.s, wherein L.sup.A1, L.sup.A2, L.sup.A3 and L.sup.A4 independently of each other are selected from C.sub.1 to C.sub.12 unsubstituted or amino-, hydroxyl-, carboxyl- or fluoro substituted alkyl or cycloalkyl, (CH.sub.2—CH.sub.2—O).sub.r or (CH.sub.2—CH(OH)—CH.sub.2—O).sub.r with r being an integer from 1 to 20, alkylaryl, alkylaryl-alkyl, and unsubstituted or alkyl-, halogen-, amino-, alkylamino-, imido-, nitro-, hydroxyl- oxyalkyl-, carbonyl-, carboxyl-, sulfuryl- and/or sulfoxyl substituted aryl or heteroaryl, L.sup.J1, L.sup.J2, L.sup.J3 and L.sup.J4 independently of each other are selected from —NR.sup.5C(O)—, —C(O)N(R.sup.5)—, —CN—, —NC—, —CO—, —OC(O)—, —C(O)O—, —NR.sup.N5—, —O—, —P(OOH)—, —OP(OOH)—, —P(OOH)O—, —OP(OOH)O—, —OP(OOH)O—, —S—, —SO—, SO.sub.2—, with R.sup.N5 selected from H and unsubstituted or amino-, hydroxyl-, carboxyl or fluoro substituted C.sub.1 to C.sub.6 alkyl, particularly R.sup.N5 is selected from H and unsubstituted C.sub.1 to C.sub.3 alkyl; n, n′, m, m′, p, p′, q, q′ and s independently from each other are selected from 0 and 1, and M has the meaning defined above.

5. The compound according to claim 4, wherein L is -L.sup.A1-L.sup.J1-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.p, wherein L.sup.A1, L.sup.A2 and L.sup.A3 are independently selected from C.sub.1 to C.sub.6 unsubstituted, amino-, hydroxyl-, carboxyl- or fluoro substituted alkyl or cycloalkyl, and (CH.sub.2—CH.sub.2—O).sub.r or (CH.sub.2—CH(OH)—CH.sub.2—O).sub.r with r being an integer from 1 to 4, and L.sup.J1 and L.sup.J2 are selected independently from —NR.sup.5C(O)—, —C(O)N(R.sup.5)—, —CN——NC—, —CO—, —OC(O)—, —C(O)O—, NR.sup.N5—, —O—, and —S—, and m, m′ and p independently from each other are selected from 0 and 1.

6. The compound according to claim 1, wherein R.sup.7 and R.sup.8 are independently selected from unsubstituted or hydroxyl-, amino- or halogen-substituted C.sub.1 to C.sub.4 alkyl, alkenyl or alkynyl, unsubstituted or hydroxyl-, amino- or halogen-substituted C.sub.3 to C.sub.6 cycloalkyl or unsubstituted or hydroxyl-, alkyoxy-, amino- or halogen-substituted phenyl, particularly methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and phenyl, more particularly wherein R.sup.7 and R.sup.8 are the same.

7. The compound according to claim 1, wherein a. R.sup.N1 and R.sup.N2, and/or R.sup.N3 and R.sup.N4, are independently selected from H, unsubstituted and amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, particularly R.sup.N1 and R.sup.N2, and/or R.sup.N3 and R.sup.N4, are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and CH.sub.2CF.sub.3, b. R.sup.N1 together with R.sup.N2, and/or R.sup.N3 together with R.sup.N4 together are an unsubstituted or alkyl-, amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.3-C.sub.6 alkyl, particularly —(CH.sub.2).sub.3—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —(CH.sub.2).sub.2O(CH.sub.2).sub.2— or —(CH.sub.2).sub.2NR.sup.NN(CH.sub.2).sub.2— with R.sup.NN being selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; c. R.sup.N1 and/or R.sup.N3 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.N2 together with R.sup.2 or R.sup.3, and/or R.sup.N4 together with R.sup.4 or R.sup.5, is an alkyl or heteroalkyl bridge selected from —(CH.sub.2).sub.2—, —(CH.sub.2).sub.3—, —CH.sub.2CH═CH— or —(CH.sub.2).sub.4— or —CH.sub.2—O—, —CH.sub.2—NR.sup.5—, —CH.sub.2—S—, —CH.sub.2—Se—, —(CH.sub.2).sub.2O—, —(CH.sub.2).sub.2NR.sup.N—, —(CH.sub.2).sub.2S—, —(CH.sub.2).sub.2Se—, —CH.sub.2—O—CH.sub.2—, —CH.sub.2NR.sup.5—, —CH.sub.2S—CH.sub.2—, —CH.sub.2—Se—CH.sub.2—, —CH.sub.2-(1,2)phenyl-, and a mono- or dimethyl substituted derivative of any one of the foregoing alkyl or heteroalkyl bridge moieties; d. R.sup.N1 and/or R.sup.N3 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.N2 together with R.sup.2, and/or R.sup.N4 together with R.sup.5, form an annular structure according to any one of substructures (101) to (104) or (101′) to (104′): ##STR00055## wherein R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are selected from H, unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl, halogen, SO.sub.3R, COOR′, CONR′.sub.2 with R selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; and R.sup.17 is selected from H unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl, halogen, NO.sub.2, CN, SO.sub.3R, COOR′, CONR+.sub.2 with R selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; particularly wherein R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are selected from H, methyl, CH.sub.2—SO.sub.3H, Cl and F, and R.sup.1, R.sup.3, R.sup.7 and R.sup.5 can have any of the meanings given herein; or e. R.sup.N1 together with R.sup.3, and R.sup.N2 together with R.sup.2, and/or R.sup.N3 together with R.sup.4, and R.sup.N4 together with R.sup.5, form a bi-annular structure according to any one of substructures (105) to (107) and/or (105′) to (107′): ##STR00056## wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.15 are selected from H, unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl, halogen, SO.sub.3R, COOR′, CONR′.sub.2 with R selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; particularly wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.15 are selected from H, methyl, CH.sub.2—SO.sub.3H, Cl and F, and R.sup.1, R.sup.3, R.sup.7 and R.sup.8 can have any of the meanings given herein; or f. R.sup.N2 and/or R.sup.N4 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.N1 together with R.sup.3, and/or R.sup.N3 together with R.sup.4, form an annular structure according to any one of substructures (108) to (109) and/or (108′) to (109′): ##STR00057## wherein R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.7 and R.sup.8 can have any of the meanings given herein.

8. The compound according to claim 7, wherein R.sup.N1 together with R.sup.N2, and/or R.sup.N3 together with R.sup.N4 together are —(CH.sub.2).sub.3—, —CH.sub.2CHFCH.sub.2—, —CH.sub.2CF.sub.2CH.sub.2—, —CH.sub.2CH(CH.sub.3)CH.sub.2—, —CH.sub.2C(CH.sub.3).sub.2CH.sub.2—, CH.sub.2CH(CN)CH.sub.2—, CH.sub.2CH(COOH)CH.sub.2—, CH.sub.2CH(CH.sub.2COOH)CH.sub.2—, —CH.sub.2CH(OCH.sub.3)CH.sub.2— and —CH.sub.2CH(N(CH.sub.3).sub.2)CH.sub.2—, particularly wherein the substituent is the same for R.sup.N1 with R.sup.N2, and R.sup.N3 with R.sup.N4.

9. The compound according to claim 1, wherein R.sup.1, R.sup.6 and R.sup.9 are H, and/or R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are selected from H, halogen, SO.sub.3H, and unsubstituted and amino-, hydroxy-, carboxy-, SO.sub.3H—, and/or halogen-substituted C.sub.1-C.sub.4 alkyl, CO.sub.2H, CO.sub.2R, SO.sub.2R with R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl, and/or R.sup.7 and R.sup.8 are independently selected from unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl or C.sub.3 to C.sub.6 cycloalkyl and phenyl, and/or R.sup.N1, R.sup.N2, R.sup.N3 and R.sup.N4 are individually unsubstituted or amino-, hydroxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl or C.sub.3 to C.sub.6 cycloalkyl, or R.sup.N1 together with R.sup.N2, and R.sup.N3 together with R.sup.N4 together with the N form an unsubstituted or methyl-, ethyl- propyl-, or halogen-substituted aziridine, pyrrolidine, piperidine, piperazine or morpholine and/or R.sup.10 is selected from unsubstituted or amino-, hydroxyl-, carboxyl- and/or halogen-substituted C.sub.2 to C.sub.12 alkyl or C.sub.3 to C.sub.7 cycloalkyl; -L.sup.A1.sub.n-L.sup.J1.sub.n′-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.p-L.sup.J3.sub.p′-L.sup.A4.sub.q-L.sup.J4.sub.q′-M.sub.s,wherein L.sup.A1 . . . 4, L.sup.J1 . . . 4, n, n′ . . . q′, s and M have the definitions recited above.

10. The compound according to claim 1, wherein R.sup.2 and R.sup.5 are F or Cl.

11. The compound according to claim 1, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.9 are H, R.sup.7 and R.sup.8 are C.sub.1 to C.sub.4 alkyl or phenyl, R.sup.N1, R.sup.N2, R.sup.N3 and R.sup.N4 are individually unsubstituted or amino-, hydroxyl- or fluoro substituted C.sub.1 to C.sub.4 alkyl, or R.sup.N1 together with R.sup.N2, and R.sup.N3 together with R.sup.N4 together are —(CH.sub.2).sub.3—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —(CH.sub.2).sub.2O(CH.sub.2).sub.2— or —(CH.sub.2).sub.2NH(CH.sub.2).sub.2— and R.sup.19 is selected from unsubstituted or amino-, hydroxyl-, carboxyl- and/or fluoro substituted C.sub.2 to C.sub.12 alkyl or C.sub.3 to C.sub.7 cycloalkyl; or R.sup.10 is -L.sup.A1.sub.n-L.sup.J1.sub.n′-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.p-L.sup.J3.sub.p′-L.sup.A4.sub.q-L.sup.J4.sub.q′-M.sub.s, wherein L.sup.A1 . . . 4, L.sup.J1 . . . 4, n, n′ . . . q, s and M have the definitions recited above.

12. A compound selected from: a. 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoic acid (2a); b. 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-3-methylbutanoic acid (3a); c. N.sup.3,N.sup.3,N.sup.7,N.sup.7,5,5-Hexamethyl-10-propylidene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine (4a); d. N.sup.3,N.sup.3,N.sup.7,N.sup.7,5,5-Hexamethyl-10-methylene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine (5a); e. 3-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoic acid (22); f. 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-2,2-dimethylbutanoic acid (28); g. 4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanoic acid (6a); h. 4-(3,7-Bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoic acid (36) i. 3-(3,7-Di(azetidin-1-yI)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoic acid (42); j. 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide (44); k. 2,5-Dioxopyrrolidin-1-yl-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoate (45); l. N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (47); m. N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (49); n. 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(4-(6-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)butyl)butanamide (51); o. (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-yl benzoate (53); p. 8-(4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanoic acid (55); q. (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(8-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-yl benzoate (56); r. 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)butanamide (58); s. 4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide (59); t. 4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)butanamide (64); u. 1-(4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide (65); v. N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (66); w. 3-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-2-(4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-3-oxopropane-1-sulfonic acid (69);

Description

BRIEF DESCRIPTION OF THE FIGURES

[0168] FIG. 1 shows (A) structures of PA-SiR 1a, the fluorescent cation 1b and the non-fluorescent compound 1c. (B) Top UV-Vis spectra of PA-SiR 2a (20 μm in PBS) before and after activation by UV irradiation (2 min). The spectra of the activated solution were measured every 1 min. Bottom Change of absorbance after activation at four different wavelengths over time. (C) Excitation and emission spectrum of the cation 2b.

[0169] FIG. 2 shows (A) structures of the measured PA-SiR's. (B) Decay of absorbance at 646 nm normalized to the initial value reached after photoactivation of each compound. Compounds were dissolved in PBS/ACN (10 μM, 7:3, F) except 2a in PBS pH=7.3, 11.1 or 3.1, where the pH was adjusted by addition of HCl and NaOH. (C) Different PA-SiR probes together with the measurement on their respective protein-tag (10 μM PBS, M).

[0170] FIG. 3 U2OS cells expressing HaloTag-H2B fusion stained with PA-SiR-Halo for 1 h (1 μM) and washed once for 3 min. Images were taken before and after irradiation with the 355 nm laser on a confocal microscope. Scale bar 30 μm.

EXAMPLES

Example 1

General Description of All Species Involved in this Study

[0171] Photoactivatable silicon rhodamines were synthesised in three steps from commercially available starting materials in analogy to a previously published synthesis (Grimm et al., ACS Cent. Sci. 2017, 3, 975-985).

[0172] Upon UV irradiation of an aqueous solution of PA-SiR 2a, the fluorescent and blue-coloured cation 2b is forming (FIG. 1A), as confirmed by LC-MS. Compound 2b has an excitation maximum at 646 nm and an emission maximum at 668 nm. The extinction coefficient is 25,000 M.sup.−1cm.sup.−1 (in PBS at pH 7.3) and the quantum yield in aqueous solution was measured to be 0.37, whereas 2a shows only absorbance in the UV-range (FIGS. 1B and C and Table 1). However, the blue solution of 2b undergoes discoloration over time (FIG. 1C). It was shown by .sup.1H NMR that the discoloration observed by UV-Vis spectroscopy, which takes place on a time scale of roughly 10 min, could be assigned to the formation of a new species. The new species corresponds to the hydroxylated form 2c (LC-MS) as the fluorescent cation 2b is attacked by nucleophiles at the electrophilic central carbon. Thus, 2b is in equilibrium with the hydroxylated form 2c in aqeous solution, which leads to the discoloration of the blue solution within 10 min. The thermodynamics and kinetics of these reactions are discussed in more detail below.

Example 2

Fine-Tuning of the Equilibria Between Fluorescent Cation and the Non-Fluorescent Compounds

[0173] In order to study the kinetic properties of the second equilibrium (FIG. 1A, k.sub.2 and k.sub.−2), UV-Vis spectroscopy was used. In order to achieve the highest possible time resolution, the photoactivation was carried out in the spectrometer. These measurements revealed a dependence of the photoactivation and the second equilibrium between 1b and 1c (both kinetics and thermodynamics) on pH and solvent polarity (FIG. 2B). Effects such as solubility, pH, nucleophilicity and electrophilicity of the involved species play an important role.

[0174] Moreover, similar UV-Vis analysis of several analogues illustrated the influence on this equilibrium of different substituents at various positions (FIG. 2). It was shown that the ratio of the fluorescent cation 1b to hydroxylated compound 1c could be tuned in both directions in comparison to the standard compound 2a by structural changes at the periphery.

[0175] Since both the kinetic properties and the equilibrium of the second step could be tuned by chemical modifications and changes in solvent, the inventors hypothesised that also the microenvironment of protein surfaces can influence the stability of the fluorescent cation 1 b. Therefore, SNAP-tag and HaloTag substrates were synthesised and reacted with their respective protein tags. It could be shown that the same fluorophore showed different behaviour on the two protein-tags (FIG. 2C). A stable signal was achieved on the surface of HaloTag. In contrast, with SNAP-tag a fast decay was seen. The difference in decay rates permits to distinguish different molecular species such as SNAP and HaloTag labelled with the same PA-SiR. The decay rate also reports on the environment of the activated fluorophore.

[0176] Motivated by the versatility of the compounds the inventors synthesised corresponding probes from PA-SIR and various ligands (PA-SiR-Halo, PA-SiR-SNAP, PA-SiR-Actin, PA-SiR-DNA, PA-SiR-Tubulin) and tested them for applications in live-cell imaging. The olefinic structure is not charged and therefore ideal to pass the plasma membrane. The compounds showed good cell-permeability and could be activated efficiently in live cells in both widefield (DAPI channel) and confocal microscopy (355 nm laser). PA-SiR-SNAP and PA-SiR-Halo probes were successfully localised in Hela cells expressing either SNAP-tag or HaloTag in the nucleus. PA-SiR-Halo was also successfully localised to H2B (FIG. 3). PA-SiR-Actin, PA-SiR-DNA and PA-SiR-Tubulin were localised to their targets.

Example 3

General Procedure A for the Silane Introduction

[0177] 3-Bromo-N,N-dimethylaniline (13) (3.20 g, 16.00 mmol, 2.0 eq.) was dissolved in dry Et.sub.2O (45 mL) and cooled down to −78° C. sec-BuLi (14.0 mL, 18.40 mmol, 2.3 eq., 1.3 M in cyclohexane) was added dropwise over 15 min and the mixture was stirred for 30 min at −78° C. Dichlorodimethylsilane (14) (1.0 mL, 8.00 mmol, 1.0 eq.) was added dropwise over 10 min at −78° C. The mixture was stirred for 10 min at −78° C. and then warmed up to room temperature and stirred for 1 h. The mixture was quenched with aqueous saturated NaHCO.sub.3 solution. The aqueous layer was extracted with Et.sub.2O (3×150 mL) and the combined organic layers were dried over MgSO.sub.4, filtered and evaporated to afford the crude product.

Example 4

General Procedure B for the Bromination

[0178] A solution of 15 (1.85 g, 6.18 mmol, 1.0 eq.) and ammonium acetate (95 mg, 1.24 mmol, 0.2 eq.) in ACN (30 mL) was cooled down to 0° C. NBS (2.3 g, 12.98 mmol, 2.1 eq.) was added portion wise over 10 min. The mixture was stirred at 0° C. for 30 min and then warmed up to room temperature and stirred for 2 h. A mixture of aqueous saturated NaHCO.sub.3 solution and water 1:1 was added. The aqueous layer was extracted with CH.sub.2Cl.sub.2 (3×100 mL) and the combined organic layers were dried over MgSO.sub.4, filtered and evaporated to afford the crude product.

Example 5

General Procedure C for the Ring Closure

[0179] A solution of 16 (365 mg, 0.8 mmol, 1.0 eq.) in dry THF (8 mL) was cooled down to −78° C. sec-BuLi (1.4 mL, 1.76 mmol, 2.2 eq., 1.3 M in cyclohexane) was added dropwise over 5 min and the mixture was stirred for 30 min at −78° C. A solution of glutaric anhydride (17) (100 mg, 0.88 mmol, 1.1 eq.) in dry THF (1.0 mL) was added to the mixture. The mixture was stirred at −78° C. for 15 min and then warmed up to room temperature and stirred for 30 min. Acetic acid (2 mL) was added to the mixture. The blue mixture was adsorbed on SiO.sub.2 (2 g).

Example 6

Materials and Methods

[0180] All chemical reagents and anhydrous solvents for synthesis were purchased from commercial suppliers (Acros, Apollo, Armar, Bachchem, Biomatrik, Fluka, Fluorochem, LC Laboratories, Merck, Reseachem, Roth, Sigma-Aldrich and TCI) and used without further purification. BG-NH.sub.2 48 and Halo-NHBoc 43 were available in the Johnsson group. Jasplakinolide-NHBoc 48 was obtained from a custom synthesis. Composition of mixed solvents is given by volume ratio (v/v). Reactions in the absence of air and moisture were performed in oven-dried glassware under Ar or N2 atmosphere. Flash column chromatography was performed using a CombiFlash Rf system (Teledyne ISCO) using SiO.sub.2 RediSep® Rf columns at 25° C. The used solvent compositions are reported individually in parentheses. Analytical thin layer chromatography was performed on glass plates coated with silica gel 60 F254 (Merck). Visualisation was achieved using UV light (254 nm). Evaporation in vacuo was performed at 25-60° C. and 900-10 mbar. .sup.1H, .sup.13C, and .sup.19F NMR spectra were recorded on AV 400 and AV 600 Bruker spectrometers at 400 MHz or 600 MHz (.sup.1H), 101 MHz or 151 MHz (.sup.13C), 377 MHz or 566 MHz (.sup.19F) respectively. All spectra were recorded at 298 K. Chemical shifts δ are reported in ppm downfield from tetramethylsilane using the residual deuterated solvent signals as an internal reference (CDCl.sub.3: δ.sub.H=7.26 ppm, δ.sub.C=77.16 ppm; CD.sub.3OD: δ.sub.H=3.31 ppm, δ.sub.C=49.00 ppm; DMSO-d.sub.6: δ.sub.H=2.50 ppm, δ.sub.C=39.52 ppm; ACN-d.sub.3: δ.sub.H=1.94 ppm, δ.sub.C=118.26 ppm). For .sup.1H, .sup.13C and .sup.19F NMR, coupling constants J are given in Hz and the resonance multiplicity is described as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sext (sextet), sept (septet), m (multiplet) and br. (broad). High-resolution mass spectrometry (HRMS) was performed by the MS-service of the EPF Lausanne (SSMI) on a Waters Xevo® G2-S Q-Tof spectrometer with electron spray ionisation (ESI). Liquid chromatography coupled to mass spectrometry (LC-MS) was performed on a Shimadzu MS2020 connected to a Nexera UHPLC system equipped with a Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1×50 mm) column. Buffer A: 0.05% HCOOH in H.sub.2O Buffer B: 0.05% HCOOH in ACN. Analytical gradient was from 10% to 90% B within 6 min with 0.5 mL/min flow unless otherwise stated. Preparative reverse phase high-performance liquid chromatography (RP-HPLC) was carried out on a Dionex system equipped with an UltiMate 3000 diode array detector for product visualisation on a Waters Symmetry C18 column (5 μm, 3.9×150 mm) or on a Waters SunFire™ Prep C18 OBD™ (5 μm, 10×150 mm) column. Buffer A: 0.1% TFA in H.sub.2O Buffer B: ACN. Typical gradient was from 10% to 90% B within 32 min with 3 or 4 mL/min flow.

Example 7

In Vitro Characterization and Microscopy

[0181] General Considerations

[0182] Three different UV light sources were used in the following experiments: a transilluminator (Biometra TI 1, 312 nm, high), a photography flash (Agfatronic 261B, plastic cover removed), a monochromator (Polychrome V, FEI, at 330 nm for 12 s) abbreviated as T, F and M. PBS (6.7 mm, Lonza) was used in all experiments.

[0183] .sup.1H NMR Analysis

[0184] PA-SiR 2a (1 mg, 2.5 pmol) was dissolved in PBS/D.sub.2O (1 mL, 90:10) and NaOH (1 μL, 5 M) was added to achieve better solubility as PA-SiR 2a was isolated as its TFA salt (pH=7-8, pH paper). .sup.1H NMR spectra were measured on a Bruker AV 600 spectrometer at 600 MHz and 298 K. Chemical shifts δ are reported in ppm downfield from tetramethylsilane using the DMSO signal (δ.sub.H=2.50 ppm) instead of the residual deuterated solvent signal as an internal reference. Spectra were measured with either NS =128 using a water suppression pre-saturation sequence. UV irradiation was performed outside of the spectrometer for the indicated times (T). After each irradiation step the NMR sample was transferred to the NMR spectrometer.

[0185] LC-MS Analysis

[0186] PA-SiR 2a was dissolved in water (50 pM). UV irradiation was performed in a quartz cuvette (Hellma Analytics) for the indicated times (T). Aliquots were taken to measure LC-MS at defined time points using an analytical gradient from 10% to 100% B within 6 min with 0.5 mL/min flow.

[0187] Fluorescence Measurements and Determination of Quantum Yield

[0188] Fluorescence spectra were measured on an Infinite M1000 (Tecan) plate reader. Quantum yields were determined using a Quantaurus QY (Hamamatsu).

[0189] UV-Vis Analysis

[0190] PA-SiRs were prepared as stock solutions in dry DMSO and diluted in PBS or PBS/ACN such that the final concentration of DMSO did not exceed 5% v/v. PBS solutions of different pH were adjusted by addition of HCI or NaOH solution using a pH meter.

[0191] Full Absorbance Spectra Measurements of PA-SiR 2a

[0192] Solutions were prepared in PBS (10 μm or 20 μM) at the indicated pH. UV irradiation was performed outside of the spectrometer for the indicated period (T). Absorbance spectra were recorded using a SHIMADZU UV spectrophotometer (UV-1800) and 1 cm fluorescence quartz cuvettes (Hellma Analytics). Spectra were recorded every minute.

[0193] pH Dependence of PA-SiR 2a

[0194] Solutions of 2a in PBS (10 μM) at the desired pH were prepared in 1 cm fluorescence quartz cuvette (Hellma Analytics). UV irradiation was performed directly inside the spectrophotometer during the running experiment at a fixed distance (F, single flash). Kinetic absorbance measurements were recorded on a Perkin-Elmer Lambda 950 spectrophotometer at 646 nm.

[0195] UV-Vis Analysis of Various PA-SiRs

[0196] Solutions were prepared in PBS/ACN (10 μM, 7:3) in 1 cm fluorescence quartz cuvettes (Hellma Analytics). UV irradiation was performed directly inside the spectrophotometer during the running experiment at a fixed distance (F, single flash). Kinetic absorbance measurements were recorded on a Perkin-Elmer Lambda 950 spectrophotometer at 646 nm.

[0197] UV-Vis Analysis of PA-SiR-Halo and PA-SiR-SNAP

[0198] SNAP-tag protein and HaloTag protein were available in the Johnsson lab as 5.3 and 3.6 mm solutions in HEPES-Glycerol (1:1). PA-SiR-Halo and PA-SiR-SNAP were dissolved in PBS (10 μM) and protein (20 μM, 2.0 equiv.) was added. The mixture was incubated for 1 h (HaloTag) or 2 h (SNAP-tag) before UV-Vis measurement. UV irradiation was performed directly inside the spectrophotometer during the running experiment at a fixed distance (M, 12 s). Kinetic absorbance measurements were recorded on a Jasco V770 spectrophotometer equipped with a Peltier cooling element (PAC743R) at 646 nm.

[0199] Plasmids

[0200] Mammalian expression vectors with a SNAP-tag-NLS or SNAP-tag-Halo-tag-NLS fusion construct were available in the Johnsson lab. H2B-Halo was constructed from a pEBTet plasmid and the commercially available pCLIPf-H2B plasmid (NEB).

[0201] Cell Culture and Transfection

[0202] HeLa or U2OS cells were cultured in high-glucose DMEM media with GlutaMAX-1 (Life Technologies) supplemented with 10% FBS (Life Technologies) in a humidified 5% CO.sub.2 incubator at 37° C. Cells were split every 3-4 days or at confluency.

[0203] Cells were seeded on glass bottom 35 mm dishes (Mattek) one day before imaging. Transient transfection of cells was performed using Lipofectamine™ 2000 reagent (Life Technologies) according to the manufacturer's recommendations: DNA (2.5 μg) was mixed with OptiMEM I (100 μL, Life Technologies) and Lipofectamine™ 2000 (6 μL) was mixed with OptiMEM I (100 μL). The solutions were incubated for 5 min at room temperature. They were mixed and incubated for 20 min at room temperature. Prepared DNA-Lipofectamine complex was added to a glass bottom 35 mm dish with cells at 50-70% confluency. After 6 h incubation in a humidified 5% CO.sub.2 incubator at 37° C. the medium was changed to a fresh high-glucose DMEM medium with GlutaMAX-1 supplemented with 10% FBS. The cells were incubated for 1-2 days before imaging.

[0204] Staining

[0205] Cells were stained with 1-3 μM PA-SiR (1-2 h, 37° C.), washed with phenol-red free DMEM medium (Life Technologies) or PBS (once for 3 min, 37° C.) and imaged in the same medium.

[0206] Widefield Microscopy

[0207] Imaging was performed using a Leica DMI6000B microscope equipped with a Hamamatsu-C9100 EM-CCD camera and a HCX PL APO 100.0×1.47 Oil objective and a standard Cy5 filter set was used. Activation of the fluorophores was achieved by irradiation with the DAPI-channel at 100% for 10-20 s. The same settings (exposure time=150 ms, gain=3, EM-gain=800, transmission=12.5%) were used for the images taken before and after UV irradiation in the SiR-channel.

[0208] Confocal Microscopy

[0209] Confocal imaging was performed on a Leica DMi8 microscope equipped with a Leica TCS SP8 X scanhead, a white light laser, a 355 nm laser (Coherent) and HC PL APO 63×1.47 Oil objective. The 355 nm laser was used to perform the photoactivation.

[0210] All images were processed with Fiji.

Example 9

Synthesis

3,3′-(Dimethylsilanediyl)bis(N,N-dimethylaniline) (15)

[0211] ##STR00010##

[0212] Following general procedure, A, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.70:30) gave 15 (1.598 g, 67%) as a colourless oil.

[0213] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.56 (s, 6H), 2.94 (s, 12H), 6.78 (ddd, J=8.3, 2.8, 1.0 Hz, 2H), 6.92-6.98 (m, 4H), 7.22-7.31 ppm (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−2.01, 40.82, 113.68, 117.28, 118.46, 121.49, 122.86, 128.60, 139.07, 150.05 ppm; HRMS (ESI): m/z calcd for C.sub.18H.sub.27N.sub.2Si.sup.+ [M+H].sup.+ 299.1938, found 299.1940; LCMS (LC, 10% to 90%): t.sub.R=3.31 min.

3,3′-(Dimethylsilanediyl)bis(4-bromo-N,N-dimethylaniline) (16)

[0214] ##STR00011##

[0215] Following general procedure B, flash column chromatography (SiO.sub.2, hexane/CH.sub.2Cl.sub.2 100:0.fwdarw.0:100) gave 16 (2.270 g, 80%) as a beige solid.

[0216] .sup.1H NMR (400 MHz, CDCl.sub.3) δ =0.75 (s, 6H), 2.88 (s, 12H), 6.60 (dd, J=8.7, 3.2 Hz, 2H), 6.84 (d, J=3.2 Hz, 2H), 7.35 ppm (d, J=8.7 Hz, 2H); .sup.13C NMR (101 MHz, CDC.sub.3) δ=−0.79, 40.71, 115.40, 116.94, 121.92, 133.10, 138.87, 149.03 ppm; HRMS (ESI): m/z calcd for C.sub.18H.sub.25Br.sub.2N.sub.2Si.sup.+ [M+H].sup.+ 455.0148, found 455.0145; LCMS (LC, 10% to 100%): t.sub.R=5.18 min.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoicacid (2a)

[0217] ##STR00012##

[0218] Following general procedure C, flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 100:0.fwdarw.90:10) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 2a (23 mg, 7%) as a green solid.

[0219] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.50 (s, 6H), 2.44 (t, J=7.2 Hz, 2H), 2.68 (d, J=7.4 Hz, 2H), 3.21 (s, 6H), 3.24 (s, 6H), 5.98 (t, J=7.3 Hz, 1H), 7.40 (ddd, J=39.8, 8.5, 2.7 Hz, 2H), 7.51-7.69 ppm (m, 4H); .sup.13C NMR (101 MHz, CD.sub.3OD) δ=−1.11, 26.63, 34.76, 45.68, 46.39, 120.02, 121.75, 123.67, 123.77, 128.57, 131.34, 134.12, 138.48, 140.26, 140.97, 143.25, 143.43, 144.46, 150.16, 176.34 ppm; HRMS (ESI): m/z calcd for C.sub.23H.sub.29N.sub.2O.sub.2Si.sup.−[M−H].sup.31 393.2004, found 393.1992; LCMS (LC, 10% to 100%): t.sub.R=2.58 min.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-3-methylbutanoic acid (3a)

[0220] ##STR00013##

[0221] Following general procedure C, flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 100:0.fwdarw.90:10) and RP-HPLC (3 mL/min, 10% to 65% B in 32 min) gave 3a (0.9 mg, 0.6%) as a light blue solid.

[0222] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=0.37 (s, 6H), 1.04 (d, J=7.0 Hz, 3H), 2.26 (br. s, 3H, overlaps with the residual solvent signal), 2.52 (s, 1H, overlaps with the residual solvent signal), 2.92-2.97 (m, 12H), 5.51 (d, J=10.3 Hz, 1H), 6.77-6.95 (m, 2H), 7.03 (s, 2H), 7.21-7.38 ppm (m, 2H); HRMS (ESI): m/z calcd for C.sub.24H.sub.31N.sub.2O.sub.2Si.sup.− [M−H].sup.− 407.2160, found 407.2155; LCMS (LC, 10% to 100%): t.sub.R=2.64 min.

N.SUP.3.,N.SUP.3.,N.SUP.7.,N.SUP.7.,5,5-Hexamethyl-10-propylidene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine (4a)

[0223] ##STR00014##

[0224] Following general procedure C, flash column chromatography (SiO.sub.2, hexane/EtOAc 90:10.fwdarw.70:30) and RP-HPLC (3 mL/min, 10% to 100% B in 32 min) gave 4a (61 mg, 35%) as a white solid.

[0225] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.42 (s, 6H), 1.02 (t, J=7.4 Hz, 3H), 2.40 (p, J=7.4 Hz, 2H), 2.94 (s, 6H), 2.97 (s, 6H), 5.73 (t, J=7.3 Hz, 1H), 6.74 (td, J=8.4, 2.8 Hz, 2H), 6.91 (d, J=2.9 Hz, 1H), 6.97 (d, J=2.8 Hz, 1H), 7.29 (d, J=8.5 Hz, 1H), 7.39 ppm (d, J=8.5 Hz, 1H).

[0226] N.sup.3,N.sup.3,N.sup.7,N.sup.7,5,5-Hexamethyl-10-methylene-5,10-dihydrodibenzo[b,e]siline-3,7-diamine (5a)

##STR00015##

[0227] Following general procedure C, washing of the crystals with MeOH gave 5a (195 mg, 76%) as a colourless solid.

[0228] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.44 (s, 6H), 2.99 (s, 12H), 5.41 (s, 2H), 6.79 (dd, J=8.7, 2.8 Hz, 2H), 6.93 (d, J=2.8 Hz, 2H), 7.59 ppm (d, J=8.7 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−2.14, 40.85, 111.33, 114.17, 115.88, 126.93, 134.83, 135.20, 147.69, 149.17 ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.27N.sub.2Si.sup.+ [M+H].sup.+ 323.1938, found 323.1936; LCMS (LC, 10% to 100%): t.sub.R=3.79 min.

3-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoic acid (22)

[0229] ##STR00016##

[0230] Following general procedure C, flash column chromatography (SiO.sub.2, DCM/MeOH 100:0.fwdarw.90:10) gave 22 (46 mg, 24%) as a green solid.

[0231] .sup.1H NMR (400 MHz, CD.sub.3CN) δ=0.36-0.50 (br. s, 6H), 3.09 (s, 6H), 3.12 (s, 6H), 3.34 (d, J=7.5 Hz, 2H), 6.06 (t, J=7.5 Hz, 1H), 7.25 (dd, J=8.5, 2.7 Hz, 1H), 7.41 (dd, J=8.5, 2.7 Hz, 1H), 7.44 (d, J=8.5 Hz, 1H), 7.50 (d, J=2.7 Hz, 1H), 7.55-7.62 ppm (m, 2H); .sup.13C NMR (101 MHz, CD.sub.3CN) δ=35.56, 44.04, 45.48, 121.04, 122.09, 123.23, 125.15, 127.88, 130.45, 137.61, 138.75, 139.07, 142.38, 144.34, 146.14, 147.53, 172.88 ppm (two signals are hidden by the residual solvent signal).

3,7-Bis(dimethylamino)-4′,4′,5,5-tetramethyl-3′,4′-dihydro-5H,5′H-spiro[dibenzo[b,e]siline-10,2′-furan]-5′-one (24)

[0232] ##STR00017##

[0233] Following general procedure C, flash column chromatography (SiO.sub.2, hexane/EtOAc 70:30.fwdarw.0:100) and RP-HPLC (3 mL/min, 10% to 100% B in 32 min) gave 24 (8.4 mg, 4%) as a white solid.

[0234] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=0.45 (s, 3H), 0.59 (s, 3H), 1.10 (s, 6H), 2.28 (s, 2H), 2.96 (s, 12H), 6.87-6.97 (m, 2H), 7.09-7.20 (m, 2H), 7.33 ppm (d, J=8.7 Hz, 2H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ=−2.83, 0.35, 26.86, NMe.sub.2 hidden by the residual solvent signal, 40.93, 56.27, 83.83, 114.08, 116.97, 122.44, 133.63, 141.72, 147.69, 182.28 ppm; HRMS (ESI): m/z calcd for C.sub.24H.sub.33N.sub.2O.sub.2Si.sup.+ [M+H].sup.+ 409.2306, found 409.2304; LCMS (LC, 10% to 100%): t.sub.R=3.95 min.

3,7-Bis(dimethylamino)-10-isopropyl-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silin-10-ol (26)

[0235] ##STR00018##

[0236] Following general procedure C, flash column chromatography (SiO.sub.2, hexane/EtOAc 90:10.fwdarw.50:50) gave 26 (68 mg, 38%) as a white solid.

[0237] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.45 (s, 3H), 0.48 (s, 3H), 0.70 (d, J=6.8 Hz, 6H), 2.97 (s, 12H), 6.82 (dd, J=8.8, 2.9 Hz, 2H), 6.93 (d, J=2.9 Hz, 2H), 7.75 ppm (d, J=8.8 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−0.62, 0.57, 18.19, 40.85, 43.42, 79.44, 113.52, 117.00, 127.22, 134.53, 141.39, 148.52 ppm.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-2,2-dimethylbutanoic acid (28)

[0238] ##STR00019##

[0239] Following general procedure C, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.0:100) gave 28 (12 mg, 6%) as a green solid.

[0240] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.41 (s, 6H), 1.16 (s, 6H), 2.71 (d, J=7.1 Hz, 2H), 2.94 (s, 6H), 2.98 (s, 6H), 5.73 (t, J=7.1 Hz, 1H), 6.74 (ddd, J=8.5, 6.7, 2.8 Hz, 2H), 6.91 (d, J=2.8 Hz, 1H), 6.96 (d, J=2.8 Hz, 1H), 7.26 (d, J=8.5 Hz, 1H), 7.38 ppm (d, J=8.5 Hz, 1H).

3,3′-(Diisopropylsilanediyl)bis(N,N-dimethylaniline) (30)

[0241] ##STR00020##

[0242] Following general procedure A, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.80:20) gave 30 (0.440 g, 31%) as a colourless oil.

[0243] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.99 (d, J=7.4 Hz, 12H), 1.56 (hept, J=7.3 Hz, 2H), 2.93 (s, 12H), 6.81 (ddd, J=8.3, 2.7, 1.0 Hz, 2H), 6.91-7.01 (m, 4H), 7.21-7.31 ppm (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=10.06, 17.82, 40.96, 113.65, 120.91, 125.16, 128.13, 133.91, 149.74 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.35N.sub.2Si.sup.+ [M+H].sup.+ 355.2564, found 355.2567; LCMS (LC, 10% to 100%): t.sub.R=4.87 min.

3,3′-(Diisopropylsilanediyl)bis(4-bromo-N,N-dimethylaniline) (31)

[0244] ##STR00021##

[0245] Following general procedure B, flash column chromatography (SiO.sub.2, hexane/CH.sub.2Cl.sub.2 100:0.fwdarw.0:100) gave 31 (1.087 g, 73%) as a white solid.

[0246] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=1.17 (d, J=7.5 Hz, 12H), 2.00 (p, J=7.4 Hz, 2H), 2.92 (s, 12H), 6.60 (dd, J=8.8, 3.3 Hz, 2H), 6.97 (d, J=3.2 Hz, 2H), 7.33 ppm (d, J=8.7 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=12.68, 19.12, 40.80, 115.10, 117.63, 122.75, 133.40, 136.75, 148.57 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.33Br.sub.2N.sub.2Si.sup.+ [M+H].sup.+ 511.0774, found 511.0779; LCMS (LC, 10% to 100%): t.sub.R=5.73 min.

4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanoic acid (6a)

[0247] ##STR00022##

[0248] Following general procedure C, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.50:50) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 6a (6.7 mg, 7%) as a light blue solid.

[0249] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=1.09 (dd J=7.7 Hz, 12H), 1.50-1.65 (m, 2H), 2.41 (t, J=7.4 Hz, 2H), 2.65 (q, J=7.6 Hz, 2H), 3.20 (s, 6H), 3.24 (s, 6H), 5.90 (t, J=7.2 Hz, 1H), 7.37 (dd, J=8.6, 2.7 Hz, 1H), 7.48-7.61 (m, 4H), 7.69 ppm (d, J=8.5 Hz, 1H); .sup.13C NMR (101 MHz, CD.sub.3OD) δ=12.87, 18.43, 26.94, 34.84, 44.48, 45.82, 118.48, 120.96, 122.99, 123.90, 128.71, 131.61, 133.71, 134.66, 135.99, 141.75, 142.16, 143.85, 145.71, 150.46, 176.48 ppm; HRMS (ESI): m/z calcd for C.sub.27H.sub.37N.sub.2O.sub.2Si.sup.− [M−H].sup.− 449.2630, found 449.2622; LCMS (LC, 10% to 90%): t.sub.R=3.92 min.

5-Bromo-2-fluoro-N,N-dimethylaniline (33)

[0250] ##STR00023##

[0251] A solution of 5-bromo-2-fluoroaniline (32) (5.0 g, 26.3 mmol, 1.0 eq.) in MeOH (30 mL) was treated with acetic acid (40 mL) and paraformaldehyde (3.9 g, 131.6 mmol, 5.0 eq.). The mixture was cooled down to 0° C. and stirred for 15 min. NaBH.sub.3CN (5.0 g, 78.9 mmol, 3.0 eq.) was added portion wise to the mixture over 10 min. The mixture was warmed up to room temperature and was stirred for 16 h. The mixture was evaporated and then neutralised with aqueous NaOH solution (4 mL, 5 M). The aqueous layer was extracted with CH.sub.2Cl.sub.2 (3×100 mL). The combined organic layers were dried over MgSO.sub.4, filtered and evaporated to afford the crude product. Flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.85:15) gave 33 (3.990 g, 70%) as a yellow oil.

[0252] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=2.85 (d, J=1.0 Hz, 6H), 6.86 (dd, J=12.6, 8.4 Hz, 1H), 6.90-6.99 ppm (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=42.68 (d, J=4.5 Hz), 116.87 (d, J=3.3 Hz), 117.58 (d, J=22.8 Hz), 121.18 (d, J=3.9 Hz), 123.28 (d, J=7.9 Hz), 142.10 (d, J=9.7 Hz), 154.07 ppm (d, J=245.3 Hz); .sup.19F NMR (376 MHz, CDCl.sub.3) δ=−124.69-−124.58 ppm (m); HRMS (ESI): m/z calcd for C.sub.8H.sub.10BrFN.sup.+ [M+H].sup.+ 217.9975, found 217.9978 ; LCMS (LC, 10% to 90%): t.sub.R=4.09 min.

5,5′-(Dimethylsilanediyl)bis(2-fluoro-N,N-dimethylaniline) (34)

[0253] ##STR00024##

[0254] Following general procedure A, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.85:15) gave 34 (2.110 g, 73%) as a yellow oil.

[0255] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.52 (s, 6H), 2.83 (d, J=0.9 Hz, 12H), 6.98-7.06 ppm (m, 6H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−1.86, 43.01 (d, J=3.9 Hz), 115.92 (d, J=19.8 Hz), 123.97 (d, J=3.4 Hz), 127.57 (d, J=7.5 Hz), 134.00 (d, J=4.3 Hz), 140.33 (d, J=8.0 Hz), 156.31 ppm (d, J=248.0 Hz); .sup.19F NMR (376 MHz, CDCl.sub.3) δ=−121.34-−121.21 ppm (m); HRMS (ESI): m/z calcd for C.sub.18H.sub.25F.sub.2N.sub.2Si.sup.+ [M+H].sup.+ 335.1750, found 335.1754; LCMS (LC, 10% to 90%): t.sub.R=4.89 min.

5,5′-(Dimethylsilanediyl)bis(4-bromo-2-fluoro-N,N-dimethylaniline) (35)

[0256] ##STR00025##

[0257] Following general procedure B, flash column chromatography (SiO.sub.2, hexane/CH.sub.2Cl.sub.2 100:0.fwdarw.0:100) gave 35 (2.329 g, 78%) as a beige solid.

[0258] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.74 (s, 6H), 2.80 (d, J=1.0 Hz, 12H), 6.94 (d, J=10.2 Hz, 2H), 7.20 ppm (d, J=12.5 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−0.80, 42.70 (d, J=4.0 Hz), 119.91 (d, J=8.6 Hz), 120.90 (d, J=23.2 Hz), 126.70 (d, J=4.0 Hz), 134.08 (d, J=4.0 Hz), 139.37 (d, J=7.5 Hz), 155.57 ppm (d, J=252.7 Hz); .sup.19F NMR (376 MHz, CDCl.sub.3) δ=−118.89 ppm (t, J=11.3 Hz); HRMS (ESI): m/z calcd for C.sub.18H.sub.23Br.sub.2F.sub.2N.sub.2Si.sup.+ [M+H].sup.+ 490.9960, found 490.9954; LCMS (LC, 10% to 100%): t.sub.R=5.50 min.

4-(3,7-Bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoic acid (36)

[0259] ##STR00026##

[0260] Following general procedure C, flash column chromatography (SiO.sub.2, hexane/EtOAc 80:20.fwdarw.0:100) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 36 (24 mg, 27%) as a white solid.

[0261] .sup.1H NMR (400 MHz, ACN-d.sub.3) δ=0.45 (s, 6H), 2.45 (t, J=7.1 Hz, 2H), 2.61-2.68 (m, 2H), 3.08 (d, J=12.9 Hz, 12H), 5.98 (t, J=7.2 Hz, 1H), 7.33 (ddd, J=13.5, 12.1, 1.1 Hz, 2H), 7.50-7.63 ppm (m, 2H); .sup.13C NMR (101 MHz, CD.sub.3OD) δ=−1.27, 26.48, 34.67, 44.58 (d, J=3.2 Hz), 45.20 (d, J=2.8 Hz), 115.24 (d, J=20.2 Hz), 117.95 (d, J=20.4 Hz), 124.85, 133.20 (d, J=4.2 Hz), 133.95, 134.29, 134.82 (d, J=4.3 Hz), 139.92, 140.07, 142.25 (d, J=6.8 Hz), 149.66 (d, J=8.1 Hz), 155.32 (d, J=87.3 Hz), 157.79 (d, J=87.8 Hz), 176.37 ppm; .sup.19F NMR (376 MHz, ACN-d.sub.3) δ=−122.40-−122.10 (m), −122.07-−120.43 ppm (m); HRMS (ESI): m/z calcd for C.sub.23H.sub.27F.sub.2N.sub.2O.sub.2Si.sup.− [M−H].sup.− 429.1815, found 429.1816; LCMS (LC, 10% to 90%): t.sub.R=4.22 min.

1-(3-Bromophenyl)azetidine (39)

[0262] ##STR00027##

[0263] A solution of 1,3-dibromobenzene (38) (2.3 g, 10.0 mmol, 1.5 eq.), Pd.sub.2dba.sub.3 (305 mg, 0.3 mmol, 0.05 eq.), xantphos (387 mg, 0.7 mmol, 0.1 eq.) and NaO.sup.tBu (1.9 g, 20.0 mmol, 3.0 eq.) in 1,4-dioxane (50 mL) was degassed with argon for 5 min. Azetidine (37) (452 μL, 6.7 mmol, 1.0 eq.) was added and the mixture was again degassed with argon for 10 min. The mixture was stirred at 100° C. for 2 h. The mixture was let cool down to room temperature and was diluted with EtOAc and aqueous saturated NaHCO.sub.3 solution. The organic layer was washed with aqueous saturated NaHCO.sub.3 (2×50 mL), brine, dried over MgSO.sub.4, filtered and evaporated to afford the crude product. Flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.80:20) gave 39 (1.224 g, 86%) as a yellow oil.

[0264] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=2.37 (p, J=7.3 Hz, 2H), 3.87 (t, J=7.3 Hz, 4H), 6.34 (ddd, J=8.1, 2.2, 0.9 Hz, 1H), 6.56 (t, J=2.1 Hz, 1H), 6.82 (ddd, J=7.9, 1.9, 0.9 Hz, 1H), 7.04 ppm (t, J=8.0 Hz, 1H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=16.99, 52.42, 109.93, 114.17, 120.00, 123.14, 130.29, 153.32 ppm; HRMS (ESI): m/z calcd for C.sub.9H.sub.11BrN.sup.+ [M+H].sup.+ 212.0069, found 221.0065; LCMS (LC, 10% to 90%): t.sub.R=4.17 min.

Bis(3-(azetidin-1-yl)phenyl)dimethylsilane (40)

[0265] ##STR00028##

[0266] Following general procedure A, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.60:40) gave 40 (0.473 g, 54%) as a colourless oil.

[0267] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.51 (s, 6H), 2.34 (p, J=7.2 Hz, 4H), 3.86 (t, J=7.2 Hz, 8H), 6.47 (ddd, J=8.1, 2.4, 0.8 Hz, 2H), 6.61 (d, J=2.1 Hz, 2H), 6.90 (dt, J=7.2, 1.1 Hz, 2H), 7.20 ppm (t, J=7.6 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−2.09, 17.17, 52.61, 112.27, 116.87, 123.43, 128.35, 138.92, 151.64 ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.27N.sub.2Si.sup.+ [M+H].sup.+ 323.1938, found 323.1944; LCMS (LC, 10% to 90%): t.sub.R=4.88 min.

Bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane (41)

[0268] ##STR00029##

[0269] Following general procedure B, flash column chromatography (SiO.sub.2, hexane/EtOAc 100:0.fwdarw.80:20) gave 41 (0.085 g, 38%) as a beige solid.

[0270] .sup.1H NMR (400 MHz, CDCl.sub.3) δ=0.71 (s, 6H), 2.33 (p, J=7.2 Hz, 4H), 3.81 (t, J=7.2 Hz, 8H), 6.31 (dd, J=8.5, 2.9 Hz, 2H), 6.51 (d, J=3.0 Hz, 2H), 7.31 ppm (d, J=8.5 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ=−0.89, 17.04, 52.59, 114.15, 117.54, 120.40, 132.95, 138.93, 150.62 ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.25Br.sub.2N.sub.2Si.sup.+ [M+H].sup.+ 479.0148, found 479.0146; LCMS (LC, 10% to 100%): t.sub.R=5.40 min.

3-(3,7-Di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)propanoic acid (42)

[0271] ##STR00030##

[0272] A solution of 41 (91 mg, 0.19 mmol, 1.0 eq.) in dry THF (3 mL) was cooled down to −78° C. sec-BuLi (0.4 mL, 0.56 mmol, 3.0 eq., 1.3 M in cyclohexane) was added dropwise over 5 min and the mixture was stirred for 30 min at −78° C. A solution of succinic anhydride (21) (21 mg, 0.21 mmol, 1.1 eq.) in dry THF (1.0 mL) was added to the mixture. The mixture was stirred at −78° C. for 15 min and then warmed up to room temperature and stirred for 30 min. Saturated ammonium chloride solution was added and extracted with EtOAc (2×25 mL). The combined organic layers were dried over MgSO.sub.4, filtered and evaporated to afford the crude product. Flash column chromatography (SiO.sub.2, hexane/EtOAc 90:10.fwdarw.70:30) and RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 42 (0.63 mg, 0.8%) as a light blue solid.

[0273] .sup.1H NMR (400 MHz, CD.sub.3CN) δ=0.33 (m, 6H), 2.34-2.37 (m, 4H, hidden by residual solvent signal), 3.28 (d, J=6.8 Hz, 2H), 3.89 (td, J=7.2, 3.3 Hz, 8H), 5.80 (t, J=7.6 Hz, 1H), 6.49 (ddd, J=11.0, 8.3, 2.6 Hz, 2H), 6.68 (d, J=2.6 Hz, 1H), 6.73 (d, J=2.5 Hz, 1H), 7.22 (d, J=8.3 Hz, 1H), 7.32 ppm (d, J=8.4 Hz, 1H).

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide (44, PA-SiR-Halo)

[0274] ##STR00031##

[0275] A solution of 2a (1.8 mg, 4.5 μmol, 1.0 eq.) in DMSO (250 μL) was treated with DIEA (2.2 μL, 13.5 μmol, 3.0 eq.) and TSTU (1.6 mg, 5.4 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of Halo-NHBoc 43 (2.0 mg, 6.2 μmol, 1.4 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 80 μL) was shaken for 5 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 44 (1.3 mg, 48%) as a light blue solid.

[0276] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.45 (s, 6H), 1.30-1.47 (m, 4H), 1.56 (p, J=6.8 Hz, 2H), 1.73 (p, J=6.9 Hz, 2H), 2.34 (t, J=7.3 Hz, 2H), 2.69 (q, J=7.3 Hz, 2H), 3.10 (s, 6H), 3.14 (s, 6H), 3.16-3.26 (m, 2H), 3.33 (d, J=5.7 Hz, 3H, overlaps with the residual solvent signal), 3.40-3.52 (m, 8H), 5.85 (t, J=7.2 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 7.28 (s, 1H), 7.35 (s, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 8.02-8.12 ppm (m, 1H); HRMS (ESI): m/z calcd for C.sub.63H.sub.51ClN.sub.3O.sub.3Si.sup.+ [M+H].sup.+ 600.3383, found 600.3386; LCMS (LC, 10% to 100%): t.sub.R=3.61 min.

2,5-Dioxopyrrolidin-1-yl-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanoate (45)

[0277] ##STR00032##

[0278] A solution of 2a (5.1 mg, 13.0 μmol, 1.0 eq.) in DMSO (300 μl) was treated with DIEA (6.0 μL, 39 μmol, 3.0 eq.) and TSTU (4.7 mg, 15.6 μmol, 1.2 eq.). The mixture was shaken for 30 min at room temperature and then acidified with TFA (2 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32 min) gave 45 (2.1 mg, 33%) as a light green solid.

[0279] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.51 (br. s, 6H), 2.77-2.89 (m, 8H), 3.21 (s, 6H), 3.26 (s, 6H), 5.98-6.09 (m, 1H), 7.31 (dd, J=8.4, 2.8 Hz, 1H), 7.46 (dd, J=8.4, 2.7 Hz, 1H), 7.51 (d, J=2.7 Hz, 1H), 7.55 (d, J=8.5 Hz, 1H), 7.59 (d, J=2.7 Hz, 1H), 7.68 ppm (d, J=8.5 Hz, 1H); .sup.13C NMR (101 MHz, CD.sub.3OD) δ=−1.78, 24.76, 25.08, 30.31, 42.65, 44.22, 116.61, 119.21, 120.15, 121.18, 127.08, 129.51, 133.26, 136.90, 138.08, 138.48, 139.02, 140.84, 141.30, 142.95, 168.29, 170.40 ppm; HRMS (ESI): m/z calcd for C.sub.27H.sub.34N.sub.3O.sub.4Si.sup.+ [M+H].sup.+ 492.2312, found 492.2311; LCMS (LC, 10% to 90%): t.sub.R=3.50 min.

N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (47, PA-SiR-SNAP)

[0280] ##STR00033##

[0281] A solution of 45 (1.2 mg, 3.0 μmol, 1.0 eq.) in DMSO (100 μl) was treated with DIEA (1.5 μL, 9 μmol, 3.0 eq.) and BG-NH.sub.2 46 (1.0 mg, 3.8 μmol, 1.3 eq.). The mixture was shaken for 10 min at room temperature and then acidified with TFA (2 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32 min) gave 47 (1.5 mg, 77%) as a green solid.

[0282] .sup.1H NMR (600 MHz, DMSO-d.sub.6) δ=0.35 (br. s, 8H (should be 6H)), 2.24 (t, J=7.4 Hz, 2H), 2.55-2.65 (m, 1H, overlaps with the residual solvent signal), 2.92 (s, 6H), 2.94 (s, 6H), 4.23 (d, J=5.9 Hz, 2H), 5.48 (s, 2H), 5.66 (t, J=7.0 Hz, 1H), 6.80-6.87 (m, 2H), 6.97-7.05 (m, 2H), 7.22 (d, J=7.9Hz, 2H), 7.26 (d, J=8.5 Hz, 1H), 7.29 (d, J=8.5 Hz, 1H), 7.39 (d, J=8.0 Hz, 2H), 8.35 (t, J=6.0 Hz, 1H), 8.94 ppm (s, 1H); HRMS (ESI): m/z calcd for C.sub.36H.sub.43N.sub.8O.sub.2Si.sup.+ [M+H].sup.+ 647.3278, found 647.3274; LCMS (LC, 10% to 100%): t.sub.R=2.41 min.

N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (49, PA-SiR-Actin)

[0283] ##STR00034##

[0284] A solution of 2a (1.8 mg, 4.5 μmol, 1.0 eq.) in DMSO (300 μL) was treated with DIEA (2.2 μL, 13.5 μmol, 3.0 eq.) and TSTU (1.6 mg, 5.4 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of jasplakinolide-NHBoc 48 (3.8 mg, 5.0 μmol, 1.1 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 80 μL) was shaken for 2 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 30 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 49 (1.3 mg, 27%) as a light blue solid.

[0285] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=0.37 (s, 6H), 0.73-0.83 (m, 4H), 0.89 (d, J=6.8 Hz, 3H), 1.06 (p, J=7.1, 6.7 Hz, 1H), 1.15 (d, J=6.3 Hz, 3H), 1.19-1.29 (m, 6H), 1.30-1.41 (m, 1H), 1.42-1.55 (m, 5H), 1.75-1.91 (m, 2H), 2.08-2.20 (m, 3H), 2.58-2.71 (m, 2H), 2.72-2.93 (m, 2H), 2.93-2.96 (m, 12H), 3.00 (s, 3H), 4.44-4.58 (m, 1H), 4.66 (h, J=6.3 Hz, 1H), 4.91 (t, J=7.1 Hz, 1H), 5.18 (ddd, J=11.6, 8.8, 3.1 Hz, 1H), 5.50 (dd, J=11.3, 5.2 Hz, 1H), 5.65 (t, J=7.1 Hz, 1H), 6.64-6.72 (m, 2H), 6.77-6.88 (m, 2H), 6.93 (t, J=7.4 Hz, 1H), 6.97-7.06 (m, 3H), 7.09-7.15 (m, 2H), 7.23-7.34 (m, 3H), 7.66 (t, J=7.7 Hz, 2H), 7.73 (t, J=5.6 Hz, 1H), 8.64 (d, J=8.8 Hz, 1H), 9.30 (br. s, 1H), 10.78 ppm (d, J=2.3 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.61H.sub.80N.sub.7O.sub.7Si.sup.+ [M+H].sup.+ 1050.5883, found 1050.5902; LCMS (LC, 10% to 90%): t.sub.R=3.91 min.

4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(4-(6-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)henoxy)butyl)butanamide (51, PA-SiR-DNA)

[0286] ##STR00035##

[0287] A solution of 2a (1.1 mg, 3.0 μmol, 1.0 eq.) in DMSO (150 μL) was treated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and HATU (1.4 mg, 3.6 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of 50 (2.1 mg, 3.6 μmol, 1.2 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 80 μL) was shaken for 5 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 15 min and then acidified with TFA (3 μL) and diluted with water (200 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32 min) gave 51 (1.4 mg, 53%) as a light green solid.

[0288] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.49 (d, J=7.8 Hz, 6H), 1.59-1.75 (m, 2H), 1.75-1.89 (m, 2H), 2.36 (t, J=7.3 Hz, 2H), 2.71 (q, J=7.3 Hz, 2H), 3.02 (s, 3H), 3.15 (s, 6H), 3.17 (s, 6H), 3.25 (t, J=6.9 Hz, 2H), 3.61-3.77 (m, 4H), 3.90-4.03 (m, 4H), 4.07 (t, J=6.2 Hz, 2H), 5.93 (t, J=7.2 Hz, 1H), 7.14 (d, J=8.7 Hz, 2H), 7.23 (dd, J=8.5, 2.7 Hz, 1H), 7.29-7.35 (m, 2H), 7.38-7.44 (m, 2H), 7.45-7.52 (m, 2H), 7.58 (d, J=8.5 Hz, 1H), 7.73 (d, J=9.0 Hz, 1H), 7.90 (d, J=8.6 Hz, 1H), 8.04 (dd, J=8.6, 1.7 Hz, 1H), 8.11 (d, J=8.6 Hz, 2H), 8.40 ppm (d, J=1.7 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.52H.sub.62N.sub.9O.sub.2Si.sup.+ [M+H].sup.+ 872.4790, found 872.4797; LCMS (LC, 10% to 100%): t.sub.R=2.42 min.

[0289] (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-yl benzoate (53)

##STR00036##

[0290] A solution of 2a (1.8 mg, 4.6 μmol, 1.0 eq.) in DMSO (130 μL) was treated with DIEA (2.3 μL, 13.8 μmol, 3.0 eq.) and TSTU (1.7 mg, 5.5 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of docetaxel (52) (20 mg, 25.2 μmol, 5.5 eq.) in formic acid (370 μL) was shaken for 45 min. The solution was evaporated, coevaporated with toluene (3×500 μL) and dried on the high vacuum for 1 h. The residue was taken up in DMSO (200 μL) and a quarter of this solution (50 μL, 6.3 μmol, 1.4 eq.) was added to the other mixture. The mixture was shaken for 1 h and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 53 (0.47 mg, 9%) as a light blue solid.

[0291] HRMS (ESI): m/z calcd for C.sub.61H.sub.74N.sub.3O.sub.13Si.sup.+ [M+H].sup.+ 1084.4985, found 1084.5005; LCMS (LC, 10% to 90%): t.sub.R=2.74 min.

8-(4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanoic acid (55)

[0292] ##STR00037##

[0293] A solution of 2a (1.6 mg, 4.0 μmol, 1.0 eq.) in DMSO (120 μL) was treated with DIEA (4.6 μL, 28.0 μmol, 7.0 eq.) and TSTU (1.4 mg, 4.8 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. Amino-octanoic acid (54) (1.5 mg, 9.2 μmol, 2.3 eq.) was added to the mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 55 (1.0 mg, 47%) as a blue solid.

[0294] HRMS (ESI): tn/z calcd for C.sub.31H.sub.46N.sub.3O.sub.3Si.sup.+ [M+H].sup.+ 536.3303, found 536.3293; LCMS (LC, 10% to 90%): t.sub.R=2.92 min.

(2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-Acetoxy-9-((3-(8-(4-(3,7-bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)octanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-β]oxet-12-yl benzoate (56, PA-SiR-Tubulin)

[0295] ##STR00038##

[0296] A solution of 55 (1.0 mg, 2.0 μmol, 1.0 eq.) in DMSO (120 ∞L) was treated with DIEA (1.0 μL, 6.0 μmol, 3.0 eq.) and TSTU (0.7 mg, 2.4 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of docetaxel (52) (20 mg, 25.2 μmol, 12.6 eq.) in formic acid (370 μL) was shaken for 45 min. The solution was evaporated, coevaporated with toluene (3×500 μL) and dried on the high vacuum for 1 h. The residue was taken up in DMSO (200 μL) and a tenth of this solution (20 μL, 2.5 μmol, 1.3 eq.) was added to the other mixture. The combined mixture was shaken for 1 h and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 56 (0.24 mg, 10%) as a blue solid.

[0297] HRMS (ESI): tn/z calcd for C.sub.69H.sub.89N.sub.4O.sub.14Si.sup.+ [M+H].sup.+ 1225.6139, found 1225.6103; LCMS (LC): t.sub.R=3.72 min, 10% to 90% gradient.

[0298] 4-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)butanamide (58)

##STR00039##

[0299] A solution of 2a (1.0 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μL) was treated with DIEA (2.1 μL, 12.5 μmol, 5.0 eq.) and TSTU (0.9 mg, 3.0 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. Me-Tetrazine-NH.sub.2.HCl (57) (0.7 mg, 3.0 μmol, 1.2 eq.) was added to the mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 58 (0.70 mg, 50%) as a purple solid.

[0300] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.41 (s, 6H), 2.46 (t, J=6.9 Hz, 2H), 2.78 (q, J=7.0 Hz, 2H), 3.04 (s, 3H), 3.13 (s, 6H), 3.22 (s, 6H), 4.42-4.50 (m, 2H), 5.96 (t, J=7.1 Hz, 1H), 7.17 (d, J=8.6 Hz, 1H), 7.35 (s, 2H), 7.39-7.44 (m, 2H), 7.42-7.50 (m, 2H), 7.56 (d, J=8.5 Hz, 1H), 8.28-8.34 (m, 2H), 8.60 ppm (t, J=6.0 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.33H.sub.40N.sub.7OSi.sup.+ [M+H].sup.+ 578.3058, found 578.3051; LCMS (LC, 10% to 90%): t.sub.R=3.31 min.

4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide (59)

[0301] ##STR00040##

[0302] A solution of 6a (1.3 mg, 3.0 μmol, 1.0 eq.) in DMSO (150 μL) was treated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and TSTU (1.1 mg, 3.6 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of Halo-NHBoc 43 (1.3 mg, 4.0 μmol, 1.3 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 80 μL) was shaken for 3 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 59 (1.02 mg, 55%) as a blue oil.

[0303] .sup.1H NMR (400 MHz, CD.sub.3CN) δ=1.03 (d, J=7.4 Hz, 12H), 1.29-1.44 (m, 4H), 1.48-1.57 (m, 4H), 1.69-1.76 (m, 2H), 2.19 (t, J=7.4 Hz, 2H), 2.51-2.63 (m, 2H), 3.03 (s, 6H), 3.04 (s, 6H), 3.23-3.31 (m, 2H), 3.37 (d, J=6.5 Hz, 2H), 3.42 (d, J=5.7 Hz, 2H), 3.48-3.53 (m, 4H), 3.56-3.61 (m, 2H), 5.72 (t, J=7.2 Hz, 1H), 6.43 (s, 1H), 7.04 (dd, J=8.5, 2.7 Hz, 1H), 7.14-7.22 (m, 1H), 7.26 (d, J=2.7 Hz, 1H), 7.33 (d, J=2.7 Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.51 ppm (d, J=8.6 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.37H.sub.59ClN.sub.3O.sub.3Si.sup.+ [M+H].sup.+ 656.4009, found 656.4009; LCMS (LC, 10% to 90%): t.sub.R=5.09 min.

(9H-Fluoren-9-yl)methyl(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)carbamate (61)

[0304] ##STR00041##

[0305] A solution of Fmoc-N-PEG2-AcOH 60 (21.2 mg, 55.0 μmol, 1.1 eq.) in DMSO (625 μL) was treated with DIEA (41 μL, 250.0 μmol, 5.0 eq.) and TSTU (18.1 mg, 60.0 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of Halo-NHBoc 43 (16.2 mg, 50.0 μmol, 1.0 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 750 μL) was shaken for 5 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (100 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (20 μL). RP-HPLC (4 mL/min, 10% to 90% B in 32 min) gave Halo-PEG2 61 (19 mg, 63%) as a colourless oil.

[0306] .sup.1H NMR (400 MHz, ACN-d.sub.3) δ=1.23-1.34 (m, 2H), 1.34-1.42 (m, 2H), 1.43-1.54 (m, 2H), 1.61-1.78 (m, 2H), 3.26 (q, J=5.5 Hz, 2H), 3.27-3.40 (m, 4H), 3.39-3.47 (m, 4H), 3.46-3.52 (m, 4H), 3.52-3.64 (m, 6H), 3.89 (s, 2H), 4.22 (t, J=6.7 Hz, 1H), 4.37 (d, J=6.6 Hz, 2H), 5.90 (s, 1H), 7.16 (s, 1H), 7.33 (t, J=7.5 Hz, 2H), 7.41 (t, J=7.4 Hz, 2H), 7.65 (d, J=7.4 Hz, 2H), 7.83 ppm (d, J=7.5 Hz, 2H); .sup.13C NMR (101 MHz, ACN-d.sub.3) δ=26.10, 27.34, 30.23, 33.27, 39.19, 41.54, 46.17, 48.12, 66.69, 70.33, 70.56, 70.61, 70.64, 70.88, 70.95, 71.53, 71.71, 120.93, 126.01, 128.05, 128.60, 142.13, 145.22, 157.34, 170.79 ppm; HRMS (ESI): m/z calcd for C.sub.31H.sub.44ClN.sub.2O.sub.7.sup.+ [M+H].sup.+ 591.2832, found 591.2831; LCMS (LC, 10% to 90%): t.sub.R=3.42 min.

(9H-Fluoren-9-yl)methyl(28-chloro-15-oxo-3,6,9,12,19,22-hexaoxa-16-azaoctacosyl)carbamate (63)

[0307] ##STR00042##

[0308] A solution of Fmoc-N-PEG4-CH.sub.2-AcOH 62 (27.0 mg, 55.0 μmol, 1.1 eq.) in DMSO (625 μL) was treated with DIEA (41 μL, 250.0 μmol, 5.0 eq.) and TSTU (18.1 mg, 60.0 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of Halo-NHBoc 43 (16.2 mg, 50.0 μmol, 1.0 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 750 μL) was shaken for 5 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (20 μL). RP-HPLC (4 mL/min, 10% to 90% B in 32 min) gave Halo-PEG4 63 (19 mg, 55%) as colourless oil.

[0309] .sup.1H NMR (400 MHz, ACN-d.sub.3) δ=1.27-1.45 (m, 4H), 1.46-1.58 (m, 2H), 1.68-1.80 (m, 2H), 2.34 (t, J=6.1 Hz, 2H), 3.23 (q, J=5.5 Hz, 2H), 3.29 (q, J=5.6 Hz, 2H), 3.39 (t, J=6.6 Hz, 2H), 3.44-3.58 (m, 22H), 3.63 (t, J=6.1 Hz, 2H), 4.23 (t, J=6.8 Hz, 1H), 4.35 (d, J=6.8 Hz, 2H), 5.80 (s, 1H), 6.69 (s, 1H), 7.34 (td, J=7.4, 1.1 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.66 (d, J=7.5 Hz, 2H), 7.83 ppm (d, J=7.5 Hz, 2H); .sup.13C NMR (101 MHz, ACN-d.sub.3) δ=26.13, 27.34, 30.27, 33.28, 37.32, 39.85, 41.52, 46.18, 48.12, 66.78, 67.82, 70.19, 70.37, 70.70, 70.91, 70.94, 70.97, 71.07, 71.13, 71.57, 120.93, 126.06, 128.06, 128.62, 142.12, 145.21, 157.29, 171.97 ppm; HRMS (ESI): m/z calcd for C.sub.36H.sub.54ClN.sub.2O.sub.9.sup.+ [M+H].sup.+ 693.3512, found 693.3514; LCMS (LC, 10% to 90%): t.sub.R=4.19 min.

4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)-N-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)butanamide (64)

[0310] ##STR00043##

[0311] A solution of 6a (1.1 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μL) was treated with DIEA (2 μL, 12.5 μmol, 5.0 eq.) and TSTU (0.9 mg, 3.0 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of Halo-PEG2 61 (1.8 mg, 3.0 μmol, 1.2 eq.) in ACN/piperidine (9:1, 100 μL) was shaken for 15 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 100% B in 32 min) gave 64 (1.29 mg, 64%) as a blue oil.

[0312] HRMS (ESI): m/z calcd for C.sub.43H.sub.70ClN.sub.4O.sub.6Si.sup.+ [M+H].sup.+ 801.4748, found 801.4742; LCMS (LC, 10% to 90%): t.sub.R=4.05 min.

1-(4-(3,7-Bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide (65)

[0313] ##STR00044##

[0314] A solution of 6a (1.1 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μL) was treated with DIEA (2 μL, 12.5 μmol, 5.0 eq.) and TSTU (0.9 mg, 3.0 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of Halo-PEG4 63 (1.8 mg, 3.0 μmol, 1.2 eq.) in ACN/piperidine (9:1, 100 μL) was shaken for 15 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 100% B in 32 min) gave 65 (0.48 mg, 21%) as a blue oil.

[0315] HRMS (ESI): m/z calcd for C.sub.48H.sub.80ClN.sub.4O.sub.8Si.sup.+ [M+H].sup.+ 903.5428, found 903.5424; LCMS (LC, 10% to 90%): t.sub.R=4.37 min.

N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (66)

[0316] ##STR00045##

[0317] A solution of 6a (1.5 mg, 3.3 μmol, 1.0 eq.) in DMSO (150 μl) was treated with DIEA (1.6 μL, 9.9 μmol, 3.0 eq.) and TSTU (1.2 mg, 4.0 μmol, 1.2 eq.) was shaken for 20 min. BG-NH.sub.2 46 (1.1 mg, 4.0 μmol, 1.2 eq.) was added. The mixture was shaken for 10 min at room temperature and acidified with TFA (2 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 66 (0.73 mg, 31%) as a light blue solid.

[0318] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=0.99 (d, J=7.4 Hz, 12H), 1.37-1.53 (m, 2H), 2.20 (t, J=7.5 Hz, 2H), 2.52-2.59 (m, 2H), 2.93 (s, 6H), 2.95 (s, 6H), 4.23 (d, J=5.7 Hz, 2H), 5.50 (s, 2H), 5.59 (t, J=7.0 Hz, 1H), 6.85 (d, J=8.6 Hz, 1H), 6.90 (d, J=8.2 Hz, 1H), 6.99 (s, 2H), 7.21-7.32 (m, 3H), 7.35 (d, J=8.6 Hz, 1H), 7.45 (d, J=7.8 Hz, 2H), 8.38 (t, J=5.9 Hz, 1H), 8.43-8.47 ppm (m, 1H); HRMS (ESI): m/z calcd for C.sub.40H.sub.51N.sub.8O.sub.2Si.sup.+ [M+H].sup.+ 703.3899, found 703.3901; LCMS (LC, 10% to 90%): t.sub.R=3.44 min.

2-Amino-3-((4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-3-oxopropane-1-sulfonic acid (68)

[0319] ##STR00046##

[0320] A solution of Fmoc-cysteic-acid (67) (8.6 mg, 22.0 μmol, 1.1 eq.) in DMSO (250 μl) was treated with DIEA (165 μL, 100.0 μmol, 5.0 eq.) and TSTU (7.2 mg, 24.0 μmol, 1.2 eq.) was shaken for 20 min. BG-NH.sub.2 46 (5.4 mg, 20.0 μmol, 1.0 eq.) was added and the mixture was shaken for 45 min at room temperature. The mixture was poured into Et.sub.2O (2 mL) and the precipitate was washed with Et.sub.2O (2×1 mL). The solid was redissolved in ACN/DBU 9:1 (500 μL) and shaken for 10 min. The mixture was acidified with TFA (2 μL) and diluted with water and DMSO. RP-HPLC (4 mL/min, 10% to 70% B in 32 min) gave 68 (5 mg, 62%) as a light blue solid.

[0321] HRMS (ESI): m/z calcd for C.sub.16H.sub.20N.sub.7O.sub.5S.sup.+ [M+H].sup.+ 422.1241, found 422.1241; LCMS (LC, 10% to 90%): t.sub.R=0.73 min.

3-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-2-(4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamido)-3-oxopropane-1-sulfonic acid (69)

[0322] ##STR00047##

[0323] A solution of 6a (1.1 mg, 2.5 μmol, 1.0 eq.) in DMSO (120 μl) was treated with DIEA (24 μL, 150.0 μmol, 60.0 eq.) and TSTU (0.9 mg, 3.0 μmol, 1.2 eq.) was shaken for 10 min. 68 (1.2 mg, 3.0 μmol, 1.2 eq.) was added. The mixture was shaken for 10 min at room temperature and then acidified with TFA (20 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 69 (0.27 mg, 13%) as a light blue solid.

[0324] HRMS (ESI): m/z calcd for C.sub.43H.sub.56N.sub.9O.sub.6SSi.sup.+ [M+H].sup.+ 854.3838, found 854.3823; LCMS (LC, 10% to 90%): t.sub.R=2.64 min.

N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-5,5-diisopropyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (70)

[0325] ##STR00048##

[0326] A solution of 6a (1.3 mg, 3.0 μmol, 1.0 eq.) in DMSO (150 μL) was treated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and TSTU (1.1 mg, 3.6 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of jasplakinolide-NHBoc 48 (2.8 mg, 3.6 μmol, 1.2 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 80 μL) was shaken for 3 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 0% to 90% B in 32 min) gave 70 (0.75 mg, 23%) as a light blue solid.

[0327] .sup.1H NMR (400 MHz, CD.sub.3CN) δ=0.95-1.02 (m, 4H), 1.00-1.09 (m, 13H), 1.11 (d, J=6.3 Hz, 4H), 1.27-1.39 (m, 4H), 1.48-1.62 (m, 2H), 1.78-1.89 (m, 5H), 2.20-2.34 (m, 4H), 2.40-2.52 (m, 2H), 2.62-2.71 (m, 3H), 2.71-2.88 (m, 2H), 2.95 (s, 3H), 3.04 (s, 6H), 3.09 (s, 6H), 3.20 (s, 3H), 4.69 (q, J=5.9 Hz, 1H), 4.74-4.80 (m, 1H), 4.96 (t, J=6.9 Hz, 1H), 5.10-5.19 (m, 1H), 5.54 (dt, J=10.1, 5.0 Hz, 1H), 5.82 (t, J=7.3 Hz, 1H), 6.59 (d, J=6.9 Hz, 2H), 6.76 (d, J=6.5 Hz, 2H), 6.98-7.05 (m, 4H), 7.09-7.11 (m, 2H), 7.28-7.40 (m, 4H), 7.44-7.54 (m, 2H), 7.59 (dd, J=13.1, 8.2 Hz, 2H), 8.35-8.53 (m, 1H), 10.11 ppm (s, 1H); HRMS (ESI): m/z calcd for C.sub.65H.sub.88N.sub.7O.sub.7Si.sup.+ [M+H].sup.+ 1106.6509, found 1106.6520; LCMS (LC, 10% to 90%): t.sub.R=4.86 min.

N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-(3,7-bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (71)

[0328] ##STR00049##

[0329] A solution of 36 (1.2 mg, 2.7 μmol, 1.0 eq.) in DMSO (120 μl) was treated with DIEA (1.3 μL, 8.1 pmol, 3.0 eq.) and TSTU (1.0 mg, 3.2 ∥mol, 1.2 eq.) was shaken for 20 min. BG-NH.sub.2 46 (0.9 mg, 3.2 μmol, 1.2 eq.) was added. The mixture was shaken for 10 min at room temperature and then acidified with TFA (2 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 71 (1.1 mg, 60%) as a white solid.

[0330] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.40 (s, 6H), 2.35-2.45 (m, 2H), 2.72 (t, J=7.1 Hz, 2H), 2.93 (s, 6H), 2.94 (s, 6H), 4.37 (d, J=4.3 Hz, 2H), 5.60 (s, 2H), 5.87 (t, J=7.2 Hz, 1H), 7.18 (d, J=9.8 Hz, 1H), 7.22 (d, J=10.0 Hz, 1H), 7.25-7.33 (m, 4H), 7.34-7.42 (m, 2H), 8.34 (s, 1H), 8.52 ppm (t, J=6.0 Hz, 1H); .sup.19F NMR (376 MHz, CD.sub.3OD) δ=−123.44-−123.20 (m), −123.13-−122.77 ppm (m); HRMS (ESI): m/z calcd for C.sub.36H.sub.41F.sub.2N.sub.8O.sub.2Si.sup.+ [M+H].sup.+ 683.3084, found 683.3082; LCMS (LC, 10% to 90%): t.sub.R=3.50 min.

N-(4-((4R,7R,10S,13S,19S,E)-7-((1H-Indol-2-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl)butyl)-4-(3,7-bis(dimethylamino)-2,8-difluoro-5,5-dimethyldibenzo[b,e]silin-10(5H)-ylidene)butanamide (72)

[0331] ##STR00050##

[0332] A solution of 36 (1.3 mg, 3.0 μmol, 1.0 eq.) in DMSO (120 μL) was treated with DIEA (1.5 μL, 9.0 μmol, 3.0 eq.) and TSTU (1.1 mg, 3.6 μmol, 1.2 eq.). The mixture was shaken for 20 min at room temperature. In a separate vial a solution of jasplakinolide-NHBoc 48 (2.8 mg, 3.6 μmol, 1.2 eq.) in CH.sub.2Cl.sub.2/TFA (8:2, 80 μL) was shaken for 2 min. The solution was evaporated and dried on the high vacuum for 1 h. The residue was taken up in DMSO (50 μL) and added to the other mixture. The mixture was shaken for 10 min and then acidified with TFA (3 μL). RP-HPLC (3 mL/min, 10% to 90% B in 32 min) gave 72 (0.22 mg, 6%) as a white solid.

[0333] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=0.46 (s, 6H), 0.73-1.01 (m, 4H), 1.05 (d, J=6.8 Hz, 3H), 1.16 (d, J=6.3 Hz, 3H), 1.18-1.26 (m, 1H), 1.29 (s, 3H), 1.29-1.47 (m, 2H), 1.50-1.71 (m, 5H), 1.81-1.94 (m, 3H), 2.34 (q, J=7.0 Hz, 2H), 2.59 (ddd, J=10.4, 6.9, 3.0 Hz, 1H), 2.70 (dd, J=8.9, 5.1 Hz, 3H), 2.80-3.01 (m, 2H), 3.03 (d, J=1.2 Hz, 9H), 3.08 (s, 6H), 4.70 (t, J=5.7 Hz, 1H), 4.78-4.84 (m, 1H), 5.02 (t, J=7.0 Hz, 1H), 5.24 (td, J=9.1, 4.0 Hz, 1H), 5.56-5.66 (m, 1H), 5.93 (t, J=7.3 Hz, 1H), 6.69-6.75 (m, 2H), 6.93-7.03 (m, 3H), 7.03-7.09 (m, 2H), 7.24-7.39 (m, 3H), 7.46 (dd, J=11.2, 9.3 Hz, 2H), 7.56-7.63 (m, 1H), 8.39 ppm (d, J=8.5 Hz, 1H); HRMS (ESI): m/z calcd for C.sub.61H.sub.78F.sub.2N.sub.7O.sub.7Si.sup.+ [M+H].sup.+ 1086.5695, found 1086.5704; LCMS (LC, 10% to 90%): t.sub.R=4.85 min.

Abbreviations

[0334] ACN, acetonitrile; NBS, N-bromosuccinimide; PBS, phosphate buffered saline; secBuLi secondary butyl lithium;