CAGING-GROUP-FREE PHOTOACTIVATABLE FLUORESCENT DYES AND THEIR USE
20250188284 ยท 2025-06-12
Inventors
- Richard LINCOLN (Heidelberg, DE)
- Alexey N. BUTKEVICH (Heidelberg, DE)
- Mariano L. BOSSI (Heidelberg, DE)
Cpc classification
International classification
Abstract
The invention relates to novel caging-group-free photoactivatable fluorescent dyes having the structural formula I:
##STR00001##
as well as to the corresponding photoactivated fluorescent dyes having the structural formula II:
##STR00002##
The invention further relates to the use of the photoactivatable compounds as such or after photoactivation, in particular as fluorescent tags, analytical reagents and labels in optical microscopy, imaging techniques, protein tracking, nucleic acid labeling, glycan analysis, capillary electrophoresis, flow cytometry or as a component of biosensors, or as analytical tools or reporters in microfluidic devices or nanofluidic circuitry.
Claims
1. A compound having the structural formula I: ##STR00129## wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8, independently of each other are selected from H, halogen, SO.sub.3H, CO.sub.2H, CN, NO.sub.2, CO.sub.2R, SO.sub.2R (with R in CO.sub.2R or SO.sub.2R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl), and an unsubstituted or substituted, in particular a halogen-, amino-, hydroxyl-, SO.sub.3H and/or carboxyl-substituted 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 alkylene, C.sub.2-C.sub.20 alkylyne, C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; and where the substituents R.sup.6 and R.sup.7, taken together with the atoms to which they are bound, may form a 5-8 membered ring structure; and/or where the substituents R.sup.7 and R.sup.8, taken together with the atoms to which they are bound, may form a 5-8 membered ring structure; R.sup.9, R.sup.10, R.sup.11, R.sup.12 are: a. 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.8 acyl, C.sub.1-C.sub.8 alkoxycarbonyl, and C.sub.7-C.sub.12 alkylaryl, and unsubstituted phenyl or phenyl substituted by unsubstituted alkyl, halogen, alkoxy, NO.sub.2, CO.sub.2H, CO.sub.2R and/or CONR.sub.2 (with each R in CO.sub.2R or CONR.sub.2 being selected independently from C.sub.1 to C.sub.4 unsubstituted alkyl); or b. R.sup.9 together with R.sup.10 and a nitrogen atom to which they are bound, and/or R.sup.11 together with R.sup.12 and a nitrogen atom to which they are bound form a 3-7 membered ring structure; or c. R.sup.9 and/or R.sup.11 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.10 together with R.sup.2 or R.sup.3 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.12 together with R.sup.4 or R.sup.5 and the atoms to which they are bound form a 5-7 membered ring structure; d. R.sup.9 together with R.sup.2 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.10 together with R.sup.3 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.11 together with R.sup.4 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.12 together with R.sup.5 and the atoms to which they are bound form a 5-7 membered ring structure; X is independently selected from: a. SiR.sup.14R.sup.15 or GeR.sup.14R.sup.15 group, where R.sup.14 and R.sup.15 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where both substituents R.sup.14 and R.sup.15, taken together with the Si or Ge to which they are attached, form a 4-7 membered ring structure; b. CR.sup.16R.sup.17 group, where R.sup.16 and R.sup.17 are each independently selected from H, F, CF.sub.3, CN, OR.sup.18, COR.sup.18, CO.sub.2R.sup.18, SO.sub.2R.sup.18, CONR.sup.18R.sup.19 (where R.sup.18 and R.sup.19 in COR.sup.18, CO.sub.2R.sup.18, SO.sub.2R.sup.18, and CONR.sup.18R.sup.19 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof), unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where both substituents R.sup.16 and R.sup.17, taken together with the C atom to which they are attached, form a 4-7 membered ring structure; Y is independently selected from: a. CO (carbonyl), CS (thiocarbonyl) or CNR.sup.20 (iminocarbonyl) group, where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; b. SO.sub.2 (sulfone) or S(O)(NR.sup.20) (sulfoximine) group, where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; c. P(O)(OH), P(O)(OR.sup.20) or P(O)(R.sup.20), where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; W is independently selected from O, S or NR.sup.21, where R.sup.21 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; R.sup.13 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where the substituents R.sup.13 and R.sup.20 taken together form a 5-8 membered ring structure.
2. A compound, which has the structural formula II and is obtainable by irradiation with ultraviolet, visible or infrared light through a one-photon absorption process or a multiphoton absorption process from any of the compounds of general formula I of claim 1: ##STR00130## where 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.11, R.sup.12, R.sup.13, X, Y and W are defined as in claim 1.
3. 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.11, R.sup.12 and R.sup.13 or through any of the groups X, Y, W to a binding moiety M selected from: a. a moiety selectively attachable by covalent bond to a protein or nucleic acid, particularly a moiety able to form an ester bond, a thioester bond, an ether bond, an amide or thioamide bond, a sulfide or disulfide bond, a carbon-carbon bond, a carbon-nitrogen bond such as a Schiff base, or a moiety able to react in a click-chemistry reaction with a corresponding functional group of a protein or nucleic acid, more particularly a moiety selected from COCHCH.sub.2, SO.sub.2CHCH.sub.2, COCH.sub.2I, COCCH, NCS, CONHS or another active ester, biotin, an azide or a tetrazine, a diazoalkane or diazoketone, a diazirine, an alkyne, a strained alkyne such as bicyclo[6.1.0]nonyne or cyclooctyne, a strained alkene such as trans-cyclooctene or norbornene, a maleimide; or from b. a substrate of a haloalkane transferase, particularly a 1-chlorohexyl or a (3-chloropropyl)aryl moiety as exemplarily shown below: ##STR00131## c. a substrate of O.sup.6-alkylguanine-DNA-alkyltransferase, particularly a (substituted) O.sup.6-benzylguanine, O.sup.2-benzylcytosine or 4-benzyloxy-6-halo- or 4-benzyloxy-6-pseudohalo-pyrimidine-2-amine moiety (where halo group is preferably chloro, and pseudohalo group is selected preferably, but without limitation, from CN and CF.sub.3) as exemplarily shown below: ##STR00132## or from d. a substrate of dihydrofolate reductase, particularly a 4-demethyltrimethoprim moiety as exemplarily shown below: ##STR00133## or from e. a moiety capable of selectively interacting non-covalently with a biomolecule, particularly a protein or nucleic acid, wherein said moiety and said biomolecule form a complex having a dissociation constant k.sub.D of 10.sup.6 mol/L or less, more particularly, M is selected from de-N-Boc-docetaxel, de-N-Boc-cabazitaxel, de-N-Boc-larotaxel or another taxol derivative, a phalloidin derivative, a jasplakinolide derivative, a bis-benzimide DNA stain, pepstatin A or triphenylphosphonium, e.g. as shown below: ##STR00134## ##STR00135## f. or wherein M is an oligonucleotide having a sequence length between 10 and 40 nucleotides; g. or wherein M is a lipid, particularly a sphingosine derivative such as a ceramide, or a phospholipid such as dioleoylphosphatidylethanolamine (DOPE) or dipalmitoylphosphatidylethanolamine (DPPE), or a fatty acid.
4. The compound according to claim 3, having one of the structural formulas I-1-I-30 or II-1-II-30: ##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## wherein 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.11, R.sup.12 or any one of the substituents R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, R.sup.21, if present, independently of any other is H or a moiety having a molecular weight between 15 and 1500 Da; particularly wherein: a) the substituents R.sup.9, R.sup.10, R.sup.D, R.sup.12 are selected from H and methyl, or any of the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 represents an azetidine ring, and b) 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 or one of the R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, R.sup.21, if present, is H or a moiety M having a molecular weight between 15 and 1500 Da, and c) the other substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are selected from H and F, and d) the other substituents R.sup.6, R.sup.7, R.sup.8 are selected from H and methyl, and e) the other substituents R.sup.14, R.sup.15, if present, are selected from methyl, ethyl, isopropyl or phenyl, f) the other substituents R.sup.16, R.sup.17, if present, are methyl, g) the other substituents R.sup.20, R.sup.21, if present, are selected from H, OH, methoxy and methyl.
5. The compound according to claim 4, having a molecular weight between 15 and 1500 Da and is characterized by a general formula -L-M.sub.s, where -L- is a covalent bond or a linker consisting of 1 to 50 atoms having an atomic weight of 12 or higher (in addition to the number of hydrogen atoms required to satisfy the valence rules) covalently connecting the compound of structure I-1-I-30 or II-1-II-30 to the binding moiety M.sub.s, particularly wherein said moiety M having a molecular weight between 15 and 1500 Da is characterized by a general formula
-L.sup.A1.sub.m-L.sup.J1.sub.m-L.sup.A2.sub.n-L.sup.A2.sub.n-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.2CH.sub.2O).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-, sulfonyl- 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 NRC(O), C(O)N(R), NRC(O)O, OC(O)N(R), C(R)=N, NC(R), C(O), OC(O), C(O)O, N(R), O, P(O)(OR), P(O)(OR)O, OP(O)(OR), OP(O)(OR)O, S, SO, SO.sub.2, SO.sub.2N(R), N(R)SO.sub.2N(R), N(R)SO.sub.2 with R selected from H and unsubstituted or amino-, hydroxyl-, carboxyl, sulfonate or fluoro substituted C.sub.1 to C.sub.6 alkyl, particularly when R is selected from H and methyl; m, m, n, n, p, p, q, q and s independently from each other are selected from 0 and 1, and M.sub.s is defined identical to binding moiety M.
6. The compound according to claim 5, wherein said moiety M having a molecular weight between 15 and 1500 Da is represented by one of the following structures: ##STR00150## ##STR00151## ##STR00152## ##STR00153##
7. The compound according to claim 1, wherein a. R.sup.9 and R.sup.10, and/or R.sup.11 and R.sup.12, are independently selected from H, unsubstituted and amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, particularly R.sup.9 and R.sup.10, and/or R.sup.11 and R.sup.12, are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, allyl and CH.sub.2CF.sub.3, b. R.sup.9 together with R.sup.10, and/or R.sup.9 together with R.sup.10, are independently forming an unsubstituted or alkyl-, amino-, hydroxy-, carboxy-, sulfonate- 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, (CH.sub.2).sub.2SO.sub.2(CH.sub.2).sub.2 or (CH.sub.2).sub.2NR.sup.22(CH.sub.2).sub.2 with R.sup.22 being selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl, particularly methyl; c. R.sup.9 and/or R.sup.11 are independently selected from H, unsubstituted and alkyl-substituted, particularly methyl-substituted, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.10 together with R.sup.2 or R.sup.3, and/or R.sup.12 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.2CHCH or (CH.sub.2).sub.4 or CH.sub.2O, CH.sub.2NR, CH.sub.2S, CH.sub.2SO.sub.2, (CH.sub.2).sub.20, (CH.sub.2).sub.2NR, (CH.sub.2).sub.2S, (CH.sub.2).sub.2SO.sub.2, CH.sub.2OCH.sub.2, CH.sub.2NR, CH.sub.2SCH.sub.2, CH.sub.2SO.sub.2CH.sub.2, with R selected from H and unsubstituted or amino-, hydroxyl-, carboxyl, sulfonate- or fluoro-substituted C.sub.1 to C.sub.6 alkyl, particularly when R is selected from H and methyl-, and a mono- or dimethyl-substituted derivative of any one of the foregoing alkyl or heteroalkyl bridge moieties; d. R.sup.10 and/or R.sup.11 are independently selected from H, unsubstituted and alkyl-, substituted, particularly methyl-substituted-, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.9 together with R.sup.2, and/or R.sup.12 together with R.sup.5, form a fused annular structure according to any one of the following substructures: ##STR00154## e. R.sup.9 and/or R.sup.12 are independently selected from H, unsubstituted and alkyl-substituted, particularly methyl-substituted, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.10 together with R.sup.3, and/or R.sup.11 together with R.sup.4, form a fused annular structure according to any one of the following substructures: ##STR00155## f. R.sup.9 together with R.sup.2, and R.sup.10 together with R.sup.3, and/or R.sup.12 together with R.sup.5, and R.sup.11 together with R.sup.4, form a fused biannular structure according to any one of the following substructures: ##STR00156##
8. The compound according to claim 1, wherein R is structurally identical to the substituent CR.sup.6CR.sup.7R.sup.8, in particular when the substituents R.sup.2 and R.sup.5 are structurally identical, and/or the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 are structurally identical, and/or the substituents R.sup.3 and R.sup.4 are structurally identical.
9. The compound according to claim 5, wherein: R.sup.1 is H, and/or R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently selected from H, halogen, CN, and/or R.sup.9, R.sup.10, R.sup.11 and R.sup.12 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.9 together with R.sup.10 together with the N atom to which they are bound, and R.sup.11 together with R.sup.12 together with the N atom to which they are bound form an unsubstituted or methyl-, hydroxy-, methoxy-, or halogen-substituted aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine-S,S-dioxide, and/or R.sup.13, R.sup.14, R.sup.15, if present, are selected from methyl, ethyl, isopropyl or phenyl, R.sup.16, R.sup.17, if present, are methyl, one of the substituents R.sup.6, R.sup.7, R.sup.8 and R.sup.20, R.sup.21, if present, is selected from a) 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; or b) -L.sup.A1.sub.m-L.sup.J1.sub.m-L.sup.A2.sub.n-L.sup.J2.sub.n-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, L.sup.A4, L.sup.J1, L.sup.J2, L.sup.J3, L.sup.J4 m, m, n, n, p, p, q, q, s and M.sub.s have the definitions recited above in claim 5, and the other substituents R.sup.6, R.sup.7, R.sup.8 and R.sup.20, R.sup.21, if present, are selected from H or methyl.
10. The compound according to claim 1, wherein the substituents NR.sup.9R.sup.10 and/or NR.sup.11R.sup.12 are independently represented by one of the following structures, particularly when the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 are structurally identical: ##STR00157##
11. The compound according to claim 1, wherein the fragment CR.sup.6CR.sup.7R.sup.8 is represented by one of the following structures: ##STR00158## ##STR00159##
12. The compound according to claim 1, wherein the group YWR.sup.13 is represented by one of the following structures: ##STR00160##
13. The compound according claim 1, wherein the group X is represented by one of the following structures: ##STR00161##
14. The compound according to claim 1, which is selected from the group of compounds below: ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166##
Description
BRIEF DESCRIPTION OF THE FIGURES
[0154]
[0155]
[0156]
[0157]
[0158]
[0159]
[0160] The present invention is further illustrated by the following specific but non-limiting examples.
EXAMPLE 1
Synthesis of the Starting Materials, Photoactivatable Compounds and Photoactivatable Labels
##STR00048##
[0161] Compound 1. A solution of trifluoromethanesulfonic anhydride (Tf.sub.2O, 1 M in CH.sub.2Cl.sub.2; 0.68 mL, 0.68 mmol, 1.5 equiv.) was added to the stirred solution of A1 (known compound: R. Lincoln et al. Nat. Chem. 2022, 14, 1013-1020) (160 mg, 0.46 mmol) in dry CH.sub.2Cl.sub.2 (7 mL) under argon, and the resulting dark blue solution was stirred at rt for 20 min. It was then transferred dropwise into the stirred mixture of aqueous ammonia (28% aq., 3.5 mL) and 1,2-dimethoxyethane (DME, 6 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 C. for 1 h, diluted with brine (30 mL), the product was then extracted with CH.sub.2Cl.sub.2 (320 mL) and the combined extracts were dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (25 g Interchim SiHP 30 m cartridge, gradient 0% to 100% A/B, A=CH.sub.2Cl.sub.2 ethanol-25% aq. NH.sub.3 80:20:2, BCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 117 mg (73%) of 1 as brown-orange solid.
[0162] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.94 (dd, J=8.2, 1.0 Hz, 1H), 7.05 (dd, J=17.3, 10.8 Hz, 1H), 6.87-6.79 (m, 4H), 5.70 (dd, J=17.3, 1.5 Hz, 1H), 5.32 (dd, J=10.8, 1.5 Hz, 1H), 3.05 (s, 6H), 3.02 (s, 6H), 0.45 (s, 6H).
[0163] .sup.13C NMR (101 MHz, CDCl.sub.3): 173.1, 150.5, 149.9, 138.5, 138.2, 137.8, 136.5, 134.3, 131.7, 128.1, 115.5, 115.4, 114.7, 113.9, 112.9, 40.5, 40.4, 1.8.
[0164] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.21H.sub.27N.sub.3Si 350.2047; Found 350.2042.
##STR00049##
[0165] Compound 2. Methyl chloroformate (12 L, 0.15 mmol, 1.5 equiv) was added to the stirred solution of compound 1 (35 mg, 0.10 mmol) and N,N-diisopropylethylamine (DIPEA; 87 L, 0.50 mmol, 5 equiv) in dry CH.sub.2Cl.sub.2 (1 mL), and the resulting solution was stirred at rt for 1 h. The mixture was evaporated on Celite, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 5% to 60% EtOAc/hexane) and freeze-dried from 1,4-dioxane to yield 29 mg (71%) of 2 as bright yellow solid.
[0166] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.67 (d, J=8.8 Hz, 1H), 7.33 (dd, J=17.3, 10.8 Hz, 1H), 6.87 (d, J=2.7 Hz, 1H), 6.84 (d, J=2.7 Hz, 1H), 6.83 (d, J=2.7 Hz, 1H), 6.69 (dd, J=8.8, 2.7 Hz, 1H), 5.66 (dd, J=17.3, 1.5 Hz, 1H), 5.26 (dd, J=10.8, 1.5 Hz, 1H), 3.68 (s, 3H), 3.04 (s, 6H), 3.02 (s, 6H), 0.48 (s, 6H).
[0167] .sup.13C NMR (101 MHz, CDCl.sub.3): 169.1, 163.4, 150.4, 150.2, 138.4, 138.1, 137.9, 137.8, 132.3, 130.4, 128.0, 115.4, 114.9, 114.3, 112.8, 111.6, 53.0, 40.33, 40.25, 2.2.
[0168] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.29N.sub.3O.sub.2Si 408.2102; Found 408.2097.
##STR00050##
[0169] Compound 3. Propargyl chloroformate (28 L, 0.286 mmol, 4 equiv) was added to the mixture of compound 1 (25 mg, 71.4 mol) and N,N-diisopropylethylamine (DIPEA; 62 L, 0.357 mmol, 5 equiv) in dry acetonitrile (0.5 mL), and the resulting solution was stirred at rt for 3 h. The solvents were evaporated, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25020 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 9.6 mg (31%) of 3 as brown solid.
[0170] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.71 (d, J=8.7 Hz, 1H), 7.32 (dd, J=17.3, 10.8 Hz, 1H), 6.87 (d, J=2.7 Hz, 1H), 6.84 (d, J=2.8 Hz, 1H), 6.83 (d, J=2.7 Hz, 1H), 6.68 (dd, J=8.8, 2.8 Hz, 1H), 5.66 (dd, J=17.3, 1.5 Hz, 1H), 5.28 (dd, J=10.8, 1.5 Hz, 1H), 4.68 (d, J=2.4 Hz, 2H), 3.05 (s, 6H), 3.02 (s, 6H), 2.42 (t, J=2.4 Hz, 1H), 0.48 (s, 6H).
[0171] .sup.13C NMR (101 MHz, CDCl.sub.3): 170.2, 161.8, 150.5, 150.3, 138.5, 138.4, 138.0, 137.8, 132.0, 130.1, 128.5, 115.3, 115.0, 114.4, 112.8, 111.6, 78.4, 74.6, 53.3, 40.32, 40.26, 2.2.
[0172] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.25H.sub.29N.sub.3O.sub.2Si: 432.2102, found: 432.2100.
##STR00051##
[0173] Compound 4. Chloroformate ester B1 was prepared according to the procedure from K. M. Anderson et al. Crystal Growth & Design 2006, 6(9), 2109-2113. Ethyl 6-hydroxyhexanoate (160 mg, 1 mmol) and pyridine (81 L, 1 mmol, 1 equiv) were added to the stirred solution of triphosgene (99 mg, 0.33 mmol, 0.33 equiv) in dry diethyl ether (2 mL), cooled to 0 C. The reaction mixture was stirred at 0 C. for 5 h, the precipitate of pyridine hydrochloride was filtered off on a short plug of Celite, washed with dry diethyl ether (3 mL), the filtrate was evaporated and the residue was redissolved in dry CH.sub.2Cl.sub.2 (1 mL) and used directly in the next step.
[0174] The prepared solution of B1 in CH.sub.2Cl.sub.2 (0.3 mL, 0.3 mmol, 3 equiv) was added to the stirred solution of compound 1 (35 mg, 0.1 mmol) and DIPEA (87 L, 0.5 mmol, 5 equiv) in dry CH.sub.2Cl.sub.2 (0.5 mL), and the resulting solution was stirred at rt for 1 h. The crude reaction mixture was evaporated, the residue was dissolved in the mixture of THF (2 mL) and ethanol (0.5 mL), and lithium hydroxide solution (21 mg of LiOH H.sub.2O in 0.5 mL water, 0.5 mmol, 5 equiv) was added to the mixture, which was left stirring at rt for 24 h (the second portion of LiOH H.sub.2O (21 mg, 0.5 mmol, 5 equiv) was added after 8 h). Acetic acid (100 L) was then added, and the reaction mixture was evaporated to dryness. The product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 40% to 100% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 47 mg (93% over 2 steps) of 4 as yellow solid.
[0175] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.69 (d, J=8.7 Hz, 1H), 7.37 (dd, J=17.3, 10.8 Hz, 1H), 6.92 (br.s, 1H), 6.88 (br.s, 2H), 6.72 (br.s, 1H), 5.66 (dd, J=17.3, 1.5 Hz, 1H), 5.29 (dd, J=10.8, 1.5 Hz, 1H), 4.02 (t, J=6.2 Hz, 2H), 3.05 (s, 6H), 3.02 (s, 6H), 2.22 (t, J=7.5 Hz, 2H), 1.57-1.44 (m, 4H), 1.16-1.03 (m, 2H), 0.49 (s, 6H).
[0176] .sup.13C NMR (101 MHz, CDCl.sub.3): 178.5, 168.8, 162.8, 150.0, 138.2, 137.9, 137.3, 128.1, 115.2, 112.8, 111.6, 65.4, 40.4, 33.7, 28.3, 25.2, 24.2, 2.5.
[0177] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.28H.sub.37N.sub.3O.sub.4Si: 508.2626, found: 508.2640.
##STR00052##
[0178] Compound 4-Halo. PyBOP solution (benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate; 15.6 mg in 100 L DMF, 30.0 mol, 1.5 equiv) was added to the stirred solution of compound 4 (10 mg, 19.7 mol) and HaloTag(O2) amine (7.7 mg, 30.0 mol, 1.5 equiv) in DMF (200 L) and DIPEA (70 L), and the reaction mixture was stirred at rt for 1.5 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 50% to 100% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) to give 11 mg (78%) of 4-Halo as viscous yellow oil.
[0179] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.66 (d, J=8.8 Hz, 1H), 7.36 (dd, J=17.3, 10.8 Hz, 1H), 6.89 (d, J=2.7 Hz, 1H), 6.84 (d, J=2.7 Hz, 1H), 6.83 (d, J=2.7 Hz, 1H), 6.68 (dd, J=8.8, 2.7 Hz, 1H), 5.97 (t, J=5.6 Hz, 1H), 5.65 (dd, J=17.3, 1.5 Hz, 1H), 5.26 (dd, J=10.8, 1.5 Hz, 1H), 4.02 (t, J=6.4 Hz, 2H), 3.63-3.50 (m, 8H), 3.48-3.41 (m, 4H), 3.04 (s, 6H), 3.01 (s, 6H), 2.11-2.04 (m, 2H), 1.77 (dq, J=8.0, 6.7 Hz, 2H), 1.60 (dq, J=8.0, 6.8 Hz, 2H), 1.56-1.32 (m, 8H), 1.21-1.10 (m, 2H), 0.48 (s, 6H).
[0180] .sup.13C NMR (101 MHz, CDCl.sub.3): 173.0, 169.1, 163.0, 150.4, 150.2, 138.2, 137.9, 137.8, 137.6, 132.5, 130.7, 128.1, 115.4, 115.0, 114.3, 112.8, 111.5, 71.4, 70.4, 70.2, 70.1, 65.6, 45.2, 40.4, 40.3, 39.3, 36.7, 32.6, 29.6, 28.6, 26.8, 25.6, 25.54, 25.45, 2.3.
[0181] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.38H.sub.57ClN.sub.4O.sub.5Si: 713.3860, found: 713.3865.
##STR00053##
[0182] Compound 4-BG. PyBOP solution (benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate; 15.3 mg in 50 L DMSO, 29.5 mol, 1.5 equiv) was added to the stirred solution of 4 (10 mg, 19.7 mol) and BG-NH.sub.2 (8 mg, 29.5 mol, 1.5 equiv) in DMSO (100 L) and DIPEA (50 L), and the reaction mixture was stirred at rt for 2 h. The volatiles were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) to give 14 mg (94%) of 4-BG as greenish-yellow solid.
[0183] HRMS (ESI) m/z: [M+2H].sup.2+ Calcd for C.sub.41H.sub.49N.sub.9O.sub.4Si: 380.6911, found: 380.6905.
##STR00054##
[0184] Compound 4-maleimide. TSTU solution (N,N,N,N-tetramethyl-O(N-succinimidyl)uronium tetrafluoroborate; 9.0 mg in 50 L DMF, 30 mol, 1.5 equiv) was added to the stirred solution of 4 (10 mg, 19.7 mol) in DMF (100 L) and DIPEA (30 L), and the reaction mixture was stirred at rt for 1 h. A solution of 1-(2-aminoethyl)maleimide hydrochloride (5.3 mg, 30 mol, 1.5 equiv) was then added, followed by DIPEA (50 L) and the reaction mixture was stirred for further 1 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 100% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) to give 8.5 mg (68%) of 4-maleimide as yellow solid.
[0185] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.33H.sub.43N.sub.5O.sub.5Si: 630.3106, found: 630.3104.
##STR00055##
[0186] Compound 5. A mixture of A2 (known compound: R. Lincoln et al. Nat. Chem. 2022, 14, 1013-1020) (220 mg, 0.568 mmol), potassium vinyltrifluoroborate (229 mg, 1.70 mmol, 3 equiv), [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (33 mg, 45.4 mol, 8 mol %) and potassium carbonate (312 mg, 2.27 mmol, 4 equiv) in 1,4-dioxane (5 mL) and water (1 mL) was stirred at 80 C. for 18 h. Upon cooling, the reaction mixture was diluted with brine (50 mL) and extracted with CH.sub.2Cl.sub.2 (320 mL). The combined extracts were dried over Na.sub.2SO.sub.4, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 5% to 30% EtOAc/hexane+20% CH.sub.2Cl.sub.2 constant additive) and freeze-dried from 1,4-dioxane to yield 168 mg (89%) of 5 as yellow solid.
[0187] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.20 (d, J=8.4, 1H), 7.90 (dd, J=17.2, 10.8, 1H), 6.79 (d, J=2.7 Hz, 1H), 6.77-6.72 (m, 2H), 6.70 (d, J=2.7, 1H), 5.44 (dd, J=17.2, 2.0 Hz, 1H), 5.27 (dd, J=10.8, 2.0 Hz, 1H), 3.11 (s, 6H), 3.09 (s, 6H), 1.72 (s, 6H).
[0188] .sup.13C NMR (101 MHz, CDCl.sub.3): 182.9, 153.6, 153.0, 152.2, 151.3, 143.6, 141.8, 129.3, 121.5, 118.1, 113.2, 111.5, 111.1, 108.3, 107.3, 40.4, 40.3, 38.9, 34.3.
[0189] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.22H.sub.26N.sub.2O: 335.2118, found: 335.2118.
##STR00056##
[0190] Compound 6. A solution of trifluoromethanesulfonic anhydride (Tf.sub.2O, 1 M in CH.sub.2Cl.sub.2; 0.72 mL, 0.72 mmol, 1.5 equiv.) was added to the stirred solution of compound 5 (160 mg, 0.479 mmol) in dry CH.sub.2Cl.sub.2 (7 mL) under argon, and the resulting blue-violet solution was stirred at rt for 20 min. It was then transferred dropwise into the stirred mixture of aqueous ammonia (28% aq., 3.5 mL) and 1,2-dimethoxyethane (DME, 6 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 C. for 45 min, diluted with brine (30 mL), the product was then extracted with CH.sub.2Cl.sub.2 (420 mL) and the combined extracts were dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 100% A/B, A=CH.sub.2Cl.sub.2 ethanol-25% aq. NH.sub.3 80:20:2, BCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 83 mg (52%) of 6 as brown-orange solid.
[0191] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.9 (br.s, 1H), 7.96 (br. d, J=7.5 Hz, 1H), 7.26-7.14 (m, 1H), 6.86 (d, J=2.6 Hz, 1H), 6.82 (d, J=2.5 Hz, 1H), 6.75-6.68 (m, 2H), 5.70 (dd, J=17.3, 1.6 Hz, 1H), 5.35 (dd, J=10.8, 1.6 Hz, 1H), 3.06 (s, 6H), 3.04 (s, 6H), 1.65 (s, 6H).
[0192] .sup.13C NMR (101 MHz, CDCl.sub.3): 167.7, 151.8, 150.9, 149.1, 147.7, 139.0, 138.8, 126.6, 124.4, 121.2, 115.6, 110.9, 110.8, 107.8, 107.1, 40.6, 40.5, 39.8, 32.0.
[0193] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.22H.sub.27N.sub.3: 334.2278, found: 334.2277.
##STR00057##
[0194] Compound 7. Chloroformate ester B1 was prepared according to the procedure from K. M. Anderson et al. Crystal Growth & Design 2006, 6(9), 2109-2113. Ethyl 6-hydroxyhexanoate (160 mg, 1 mmol) and pyridine (81 L, 1 mmol, 1 equiv) were added to the stirred solution of triphosgene (99 mg, 0.33 mmol, 0.33 equiv) in dry diethyl ether (2 mL), cooled to 0 C. The reaction mixture was stirred at 0 C. for 5 h, the precipitate of pyridine hydrochloride was filtered off on a short plug of Celite, washed with dry diethyl ether (3 mL), the filtrate was evaporated and the residue was redissolved in dry CH.sub.2Cl.sub.2 (1 mL) and used directly in the next step.
[0195] The prepared solution of B1 in CH.sub.2Cl.sub.2 (0.36 mL, 0.36 mmol, 3 equiv) was added to the stirred solution of compound 6 (40 mg, 0.12 mmol) and DIPEA (104 L, 0.6 mmol, 5 equiv) in dry CH.sub.2Cl.sub.2 (1.2 mL), and the resulting solution was stirred at rt for 18 h. The crude reaction mixture was evaporated, the residue was dissolved in the mixture of THF (2 mL) and ethanol (0.5 mL), and lithium hydroxide solution (50 mg of LiOH H.sub.2O in 0.5 mL water, 1.2 mmol, 10 equiv) was added to the mixture, which was left stirring at rt for 8 h. Acetic acid (150 L) was then added, and the reaction mixture was evaporated to dryness. The product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 20% to 80% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 66 mg (95% over 2 steps) of 7 as green hygroscopic solid, containing 1 eq./eq. 1,4-dioxane.
[0196] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.67 (d, J=8.8 Hz, 1H), 7.55 (dd, J=17.3, 10.8 Hz, 1H), 6.83 (d, J=2.5 Hz, 1H), 6.81 (d, J=2.5 Hz, 1H), 6.75 (d, J=2.5 Hz, 1H), 6.57 (dd, J=8.8, 2.5 Hz, 1H), 5.63 (dd, J=17.3, 1.6 Hz, 1H), 5.25 (dd, J=10.8, 1.6 Hz, 1H), 4.10 (t, J=6.6 Hz, 2H), 3.06 (s, 6H), 3.03 (s, 6H), 2.22 (t, J=7.5 Hz, 2H), 1.66 (s, 6H), 1.62-1.36 (m, 4H), 1.27-1.09 (m, 2H).
[0197] .sup.13C NMR (101 MHz, CDCl.sub.3): 179.0, 163.7, 163.5, 151.7, 151.2, 150.0, 149.3, 139.1, 138.4, 127.7, 122.7, 120.8, 114.1, 109.9, 109.4, 107.1, 106.7, 65.5, 40.43, 40.37, 34.0, 31.5, 28.5, 25.5, 24.4.
[0198] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.29H.sub.37N.sub.3O.sub.4: 492.2857, found: 492.2859.
##STR00058##
[0199] Compound 7-Halo. PyBOP solution (benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate; 15.9 mg in 100 L DMF, 30.5 mol, 1.5 equiv) was added to the stirred solution of compound 7 (10 mg, 20.3 mol) and HaloTag(O2) amine (hydrochloride salt; 7.9 mg, 30.5 mol, 1.5 equiv) in DMF (200 L) and DIPEA (80 L), and the reaction mixture was stirred at rt for 1 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 12.8 mg (90%) of 7-Halo as viscous blue oil.
[0200] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.39H.sub.57ClN.sub.4O.sub.5: 697.4090, found: 697.4088.
##STR00059##
[0201] Compound 8. A mixture of A3 (known compound: A. N. Butkevich et al. Angew. Chem. Int. Ed., 2016, 55(10), 3290-3294) (167 mg, 0.51 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (34 mg, 0.051 mmol, 10 mol %), ligand L1 (known compound: B. Ghaffari et al. J. Am. Chem. Soc. 2014, 136, 14345-14348) (25 mg, 0.102 mol, 20 mol %) and 4,4,4,4,5,5,5,5-octaethyl-2,2-bi(1,3,2-dioxaborolane) (B.sub.2(Epin).sub.2; 205 mg, 0.561 mmol, 1.1 equiv) in degassed octane (6 mL) was stirred at 120 C. for 22 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 and evaporated on silica, and the product (the major regioisomer of aryl boronate ester) was separated by flash column chromatography (40 g RediSep Rf cartridge, gradient 0% to 30% EtOAc/hexane+20% CH.sub.2Cl.sub.2 constant additive) to give 123 mg of yellow foam, which was used in the next step without further characterization.
[0202] Anhydrous copper(II) bromide (162 mg, 0.726 mmol, 3 equiv) and potassium fluoride (56 mg, 0.97 mmol, 4 equiv) were added to the boronate ester (123 mg, 0.242 mmol) followed by DMSO (2.5 mL), pyridine (0.39 mL, 4.84 mmol, 20 equiv) and water (0.25 mL), and the mixture was stirred at 80 C. for 1 h. The reaction mixture was diluted with sat. aq. Na.sub.2SO.sub.4 solution and extracted with CH.sub.2Cl.sub.2 (325 mL), the combined extracts were dried over Na.sub.2SO.sub.4. The filtrate was evaporated on silica and the mixture was separated by flash column chromatography (25 g Interchim SiHP 30 m cartridge, gradient 0% to 30% EtOAc/hexane+20% CH.sub.2Cl.sub.2 constant additive) to yield 69 mg (33%) of 8 as yellow solid.
[0203] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.89 (d, J=14.6 Hz, 1H), 7.01 (d, J=2.6 Hz, 1H), 6.85 (d, J=8.3 Hz, 1H), 6.76 (d, J=2.6 Hz, 1H), 3.09 (s, 6H), 3.02 (d, J=1.5 Hz, 6H), 1.68 (s, 6H).
[0204] .sup.19F NMR (376 MHz, CDCl.sub.3): 125.06.
[0205] .sup.13C NMR (101 MHz, CDCl.sub.3): 179.8 (d, J=1.9 Hz), 154.3, 153.1 (d, J=245.1 Hz), 152.2, 145.6 (d, J=2.7 Hz), 144.2 (d, J=8.9 Hz), 124.34, 124.28 (d, J=6.5 Hz), 118.2, 117.0 (d, J=1.3 Hz), 114.5 (d, J=22.4 Hz), 113.4 (d, J=3.6 Hz), 108.2, 42.5 (d, J=5.5 Hz), 40.1, 38.8, 34.2.
[0206] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.20H.sub.22BrFN.sub.2O: 405.0972, found: 405.0972.
##STR00060##
[0207] Compound 9. A mixture of compound 8 (88 mg, 0.217 mmol), potassium vinyltrifluoroborate (58 mg, 0.435 mmol, 2 equiv), [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (8.9 mg, 11.0 mol, 5 mol %) and potassium carbonate (75 mg, 0.543 mmol, 2.5 equiv) in 1,5-dioxane (1.6 mL) and water (0.32 mL) was stirred at 80 C. for 6 h. Upon cooling, the reaction mixture was diluted with brine (30 mL) and extracted with CH.sub.2Cl.sub.2 (320 mL). The combined extracts were dried over Na.sub.2SO.sub.4, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% EtOAc/hexane+20% CH.sub.2Cl.sub.2 constant additive) and freeze-dried from 1,4-dioxane to yield 59 mg (77%) of 9 as light-yellow solid.
[0208] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.87 (d, J=14.7 Hz, 1H), 7.85 (dd, J=17.1, 10.8 Hz, 1H), 6.88 (d, J=8.3 Hz, 1H), 6.77 (d, J=2.7 Hz, 1H), 6.70 (dd, J=2.6, 0.7 Hz, 1H), 5.45 (dd, J=17.1, 1.9 Hz, 1H), 5.29 (dd, J=10.8, 1.9 Hz, 1H), 3.12 (s, 6H), 3.01 (d, J=1.3 Hz, 6H), 1.70 (s, 6H).
[0209] .sup.19F NMR (376 MHz, CDCl.sub.3): 125.26.
[0210] .sup.13C NMR (101 MHz, CDCl.sub.3): 182.0 (d, J=1.7 Hz), 153.7, 153.2 (d, J=244.8 Hz), 152.4, 146.4 (d, J=2.6 Hz), 144.0 (d, J=9.1 Hz), 143.9, 141.5, 124.9 (d, J=6.2 Hz), 117.5 (d, J=1.0 Hz), 114.1 (d, J=22.1 Hz), 113.8 (d, J=3.5 Hz), 113.7, 111.5, 108.2, 42.5 (d, J=5.3 Hz), 40.2, 38.6, 34.2.
[0211] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.22H.sub.25FN.sub.2O: 353.2024, found: 353.2024.
##STR00061##
[0212] Compound 11. A solution of trifluoromethanesulfonic anhydride (Tf.sub.2O, 1 M in CH.sub.2Cl.sub.2; 0.55 mL, 0.55 mmol, 1.5 equiv.) was added to the stirred solution of compound 9 (134 mg, 0.38 mmol) in dry CH.sub.2Cl.sub.2 (5 mL) under argon, and the resulting blue solution was stirred at rt for 20 min. It was then transferred dropwise into the stirred mixture of aqueous ammonia (28% aq., 3 mL) and 1,2-dimethoxyethane (DME, 5 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 C. for 45 min, diluted with brine (50 mL), the product was then extracted with CH.sub.2Cl.sub.2 (320 mL) and the combined extracts were dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 50% A/B, A=CH.sub.2Cl.sub.2 ethanol-25% aq. NH.sub.3 80:20:2, BCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 71 mg (0.20 mmol) of crude imine 10 as viscous orange oil, which was used directly in the next step.
[0213] Chloroformate ester B1 was prepared according to the procedure from K. M. Anderson et al. Crystal Growth & Design 2006, 6(9), 2109-2113. Ethyl 6-hydroxyhexanoate (640 mg, 4 mmol) and pyridine (0.32 mL, 1 mmol, 1 equiv) were added to the stirred solution of triphosgene (396 mg, 0.33 mmol, 0.33 equiv) in dry diethyl ether (8 mL), cooled to 0 C. The reaction mixture was stirred at 0 C. for 5 h, the precipitate of pyridine hydrochloride was filtered off on a short plug of Celite, washed with dry diethyl ether (3 mL), the filtrate was evaporated, redissolved in dry hexane, microfiltered through a 0.22 m PTFE membrane filter, evaporated to yellowish oil (561 mg, crude chloroformate ester containing alkyl chloride as an impurity), which was used directly in the next step.
[0214] The above prepared crude B1 (0.15 mL, 0.6 mmol, 3 equiv) was added to the stirred solution of 10 (71 mg, 0.2 mmol) and DIPEA (150 L, 0.66 mmol, 4.3 equiv) in dry CH.sub.2Cl.sub.2 (0.5 mL), and the resulting solution was stirred at rt for 3 h. The crude reaction mixture was evaporated, the residue was redissolved in minimal volume of CH.sub.2Cl.sub.2, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 10% to 60% EtOAc/hexane) to yield 47 mg (23%) of 11 as viscous green-brown oil.
[0215] .sup.1H NMR (400 MHz, CD.sub.3CN): 7.36 (dd, J=17.4, 10.9 Hz, 1H), 7.33 (d, J=14.5 Hz, 1H), 7.07 (d, J=8.9 Hz, 1H), 6.90 (d, J=2.5 Hz, 1H), 6.78 (d, J=2.5 Hz, 1H), 5.67 (dd, J=17.4, 1.5 Hz, 1H), 5.20 (dd, J=10.9, 1.5 Hz, 1H), 4.11-4.03 (m, 4H), 3.06 (s, 6H), 2.95 (d, J=1.4 Hz, 6H), 2.20 (t, J=7.5 Hz, 2H), 1.62 (s, 6H), 1.60-1.46 (m, 4H), 1.27-1.16 (m, 5H).
[0216] .sup.19F NMR (376 MHz, CD.sub.3CN): 126.98.
[0217] .sup.13C NMR (101 MHz, CD.sub.3CN): 174.1, 163.4, 162.5 (d, J=1.6 Hz), 152.9 (d, J=242.4 Hz), 152.6, 151.2, 145.7 (d, J=2.8 Hz), 143.0 (d, J=8.1 Hz), 139.8, 138.9, 125.9 (d, J=7.1 Hz), 119.9, 114.8, 114.3 (d, J=4.1 Hz), 114.1 (d, J=23.6 Hz), 109.8, 108.2, 66.6, 60.8, 42.6 (d, J=5.2 Hz), 40.9, 40.4, 34.7, 31.7, 29.2, 26.2, 25.3, 14.6.
[0218] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.31H.sub.40FN.sub.3O.sub.4: 538.3076, found: 538.3068.
##STR00062##
[0219] Compound 12. Compound 11 (47 mg, 87.4 mol) was dissolved in the mixture of THF (2 mL) and ethanol (0.5 mL), and lithium hydroxide solution (37 mg of LiOH H.sub.2O in 0.5 mL water, 0.874 mmol, 10 equiv) was added to the mixture, which was left stirring at rt overnight (18 h). Acetic acid (150 L) was then added, and the reaction mixture was evaporated to dryness. The product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 80% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 39 mg (87% over 2 steps) of 12 as green lustrous solid.
[0220] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.47 (d, J=14.0 Hz, 1H), 7.44 (dd, J=17.2, 10.8 Hz, 1H), 6.97 (d, J=8.6 Hz, 1H), 6.81 (d, J=2.5 Hz, 1H), 6.75 (d, J=2.5 Hz, 1H), 5.65 (dd, J=17.2, 1.6 Hz, 1H), 5.27 (dd, J=10.8, 1.6 Hz, 1H), 4.14 (t, J=6.4 Hz, 2H), 3.07 (s, 6H), 2.96 (d, J=1.2 Hz, 6H), 2.27 (t, J=7.5 Hz, 2H), 1.64 (s, 6H), 1.63-1.53 (m, 2H), 1.33-1.21 (m, 2H).
[0221] .sup.19F NMR (376 MHz, CDCl.sub.3): 125.49.
[0222] .sup.13C NMR (101 MHz, CDCl.sub.3): 178.4, 162.9, 152.5 (d, J=244.3 Hz), 151.5, 150.2, 144.5 (d, J=2.9 Hz), 141.9 (d, J=8.4 Hz), 139.4, 138.1, 126.1 (d, J=7.2 Hz), 119.9, 114.7, 113.9 (d, J=23.3 Hz), 112.9 (d, J=3.7 Hz), 109.3, 107.0, 42.5 (d, J=5.1 Hz), 40.4, 40.1, 33.9, 31.7, 28.5, 25.6, 24.5.
[0223] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.29H.sub.36FN.sub.3O.sub.4 510.2763; Found 510.2774.
##STR00063##
[0224] Compound 12-Halo. PyBOP solution (benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate; 15.3 mg in 100 L DMF, 29.4 mol, 1.5 equiv) was added to the stirred solution of compound 12 (10 mg, 19.6 mol) and HaloTag(O2) amine (hydrochloride salt; 7.6 mg, 29.4 mol, 1.5 equiv) in DMF (200 L) and DIPEA (80 L), and the reaction mixture was stirred at rt for 1 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 50% to 100% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 12 mg (85%) of 12-Halo as viscous brown-yellow oil.
[0225] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.39H.sub.56ClFN.sub.4O.sub.5: 715.3996, found: 715.4001.
##STR00064##
[0226] Compound 13. A mixture of A4 (known compound: S. Shen et al. RSC Adv., 2017, 7(18), 10922-10927) (150 mg, 0.28 mmol), 3,3-difluoroazetidine hydrochloride (110 mg, 0.84 mmol, 3 equiv), RuPhos Pd G4 precatalyst (35.7 mg, 42 mol, 15 mol %), RuPhos ligand (20 mg, 42 mol, 15 mol %) and cesium carbonate (320 mg, 0.98 mmol, 3.5 equiv) in 1,4-dioxane (1 mL) was stirred at 80 C. for 20 h. Upon cooling, the reaction mixture was diluted with brine (30 mL) and extracted with ethyl acetate (320 mL). The combined extracts were dried over Na.sub.2SO.sub.4, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 10% to 50% EtOAc/hexane) and freeze-dried from 1,4-dioxane to yield 118 mg (99%) of 13 as yellowish solid.
[0227] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.40 (d, J=8.7 Hz, 2H), 6.62 (dd, J=8.7, 2.6 Hz, 2H), 6.57 (d, J=2.5 Hz, 2H), 4.36 (t, J=11.7 Hz, 8H), 0.46 (s, 6H).
[0228] .sup.19F NMR (376 MHz, CDCl.sub.3): 99.53.
[0229] .sup.13C NMR (101 MHz, CDCl.sub.3): 185.4, 150.8, 150.8, 140.7, 132.1, 131.9, 115.8 (t, J=274.6 Hz), 114.7, 113.6, 63.2 (t, J=26.7 Hz), 1.1.
[0230] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.21H.sub.20F.sub.4N.sub.2OSi: 421.1354, found: 421.1341.
##STR00065##
[0231] Compound 14. A mixture of compound 13 (111 mg, 0.264 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (8.8 mg, 13.2 mol, 5 mol %), ligand L2 (known compound: M. E. Hoque et al. J. Am. Chem. Soc. 2021, 143, 5022-5037) (4.6 mg, 26.4 mol, 10 mol %) and bis(pinacolato)diboron (74 mg, 0.29 mmol, 1.1 equiv) in degassed THF (2.5 mL) was stirred at 80 C. overnight. The reaction mixture was evaporated on Celite, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 20% EtOAc/CH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 108 mg (75%) of 14 as yellow solid.
[0232] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.43 (dd, J=8.3, 0.8 Hz, 1H), 6.74 (d, J=2.3 Hz, 1H), 6.58-6.53 (m, 2H), 6.46 (d, J=2.3 Hz, 1H), 4.43 (t, J=11.6 Hz, 4H), 4.42 (t, J=11.7 Hz, 4H), 1.43 (s, 12H), 0.40 (s, 6H).
[0233] .sup.19F NMR (376 MHz, CDCl.sub.3): 99.73, 99.87.
[0234] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.5, 152.0, 145.9, 139.7, 134.0, 133.7, 125.2, 115.8 (t, J=274.5 Hz), 115.5, 115.4 (t, J=274.5 Hz), 114.91, 114.86, 112.6, 62.9 (t, J=27.3 Hz), 25.6, 1.4.
[0235] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.31BF.sub.4N.sub.2O.sub.3Si: 546.2242, found: 546.2243.
##STR00066##
[0236] Compound 15. Copper(II) bromide (132 mg, 0.593 mmol, 3 equiv) and potassium fluoride (46 mg, 0.792 mmol, 4 equiv) were added to a stirred solution of compound 14 (108 mg, 0.198 mmol) in DMSO (1.5 mL), followed by addition of pyridine (320 L, 3.96 mmol, 20 equiv) and water (150 L), and the reaction mixture was stirred at 80 C. for 30 min. On cooling, it was diluted with water (30 mL) and extracted with ethyl acetate (320 mL). The combined extracts were dried over Na.sub.2SO.sub.4, the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 10% to 50% EtOAc/hexane) and freeze-dried from 1,4-dioxane to yield 104 mg (quant.) of 15 as light-yellow solid, containing 25 mol % dioxane.
[0237] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.17 (d, J=8.6 Hz, 1H), 6.85 (d, J=2.4 Hz, 1H), 6.61 (dd, J=8.7, 2.6 Hz, 1H), 6.53 (d, J=2.5 Hz, 2H), 4.35 (t, J=11.6 Hz, 4H), 4.34 (t, J=11.7 Hz, 4H), 0.46 (s, 6H).
[0238] .sup.19F NMR (376 MHz, CDCl.sub.3): 99.49, 99.51.
[0239] .sup.13C NMR (101 MHz, CDCl.sub.3): 186.8, 150.5, 150.0, 142.5, 138.4, 134.6, 131.4, 130.7, 125.0, 120.3, 115.8 (t, J=274.5 Hz), 115.5 (t, J=274.7 Hz), 114.3, 114.0, 113.8, 63.2 (t, J=26.8 Hz), 1.3.
[0240] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.21H.sub.19BrF.sub.4N.sub.2OSi: 499.0459, found: 499.0453.
##STR00067##
[0241] Compound 16. A mixture of compound 15 (104 mg, 0.208 mmol), potassium vinyltrifluoroborate (50 mg, 0.375 mmol, 1.8 equiv), [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (8.5 mg, 10.0 mol, 5 mol %) and potassium carbonate (72 mg, 0.52 mmol, 2.5 equiv) in 1,5-dioxane (1 mL) and water (0.3 mL) was stirred at 80 C. for 6 h. Upon cooling, the reaction mixture was diluted with brine (30 mL) and extracted with ethyl acetate (320 mL). The combined extracts were dried over Na.sub.2SO.sub.4, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 5% to 50% EtOAc/hexane) and freeze-dried from 1,4-dioxane to yield 79 mg (89%) of 16 as light-yellow solid.
[0242] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.22 (d, J=8.7 Hz, 1H), 7.47 (dd, J=17.3, 10.8 Hz, 1H), 6.61 (dd, J=8.7, 2.6 Hz, 1H), 6.58 (d, J=2.7 Hz, 1H), 6.55 (d, J=2.2 Hz, 1H), 5.46 (dd, J=17.2, 1.6 Hz, 1H), 5.28 (dd, J=10.8, 1.6 Hz, 1H), 4.37 (t, J=11.6 Hz, 4H), 4.34 (t, J=11.6 Hz, 4H), 0.46 (s, 6H).
[0243] .sup.19F NMR (376 MHz, CDCl.sub.3): 99.50, 99.51.
[0244] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.2, 150.5 (t, J=3.0 Hz), 150.1 (t, J=3.0 Hz), 144.1, 141.4, 140.5, 139.2, 134.6, 131.4, 130.9, 115.82 (t, J=274.5 Hz), 115.78 (t, J=274.5 Hz), 114.7, 114.2, 114.0, 113.8, 113.7, 63.2 (t, J=26.2 Hz), 1.1.
[0245] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.22F.sub.4N.sub.2OSi: 447.1510, found: 447.1510.
##STR00068##
[0246] Compound 17. Chloroformate ester B1 was prepared according to the procedure from K. M. Anderson et al. Crystal Growth & Design 2006, 6(9), 2109-2113. Ethyl 6-hydroxyhexanoate (160 mg, 1 mmol) and pyridine (81 L, 1 mmol, 1 equiv) were added to the stirred solution of triphosgene (99 mg, 0.33 mmol, 0.33 equiv) in dry diethyl ether (2 mL), cooled to 0 C. The reaction mixture was stirred at 0 C. for 5 h, the precipitate of pyridine hydrochloride was filtered off on a short plug of Celite, washed with dry diethyl ether (3 mL), the filtrate was evaporated and the residue was redissolved in dry CH.sub.2Cl.sub.2 (1 mL) and used directly in the next step.
[0247] The above prepared crude B1 (0.1 mL, 0.4 mmol, 3 equiv) dissolved in dry CH.sub.2Cl.sub.2 (0.5 mL) was added to the stirred solution of 16 (54 mg, 0.121 mmol) and DIPEA (105 L, 0.605 mmol, 5 equiv) in dry CH.sub.2Cl.sub.2 (0.6 mL), and the resulting solution was stirred at rt for 3 h. The crude reaction mixture was evaporated, the residue was redissolved in minimal volume of CH.sub.2Cl.sub.2, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 50% EtOAc/hexane) to yield 93 mg of crude 17 as viscous yellow solid which was used directly in the hydrolysis step.
[0248] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.32H.sub.37F.sub.4N.sub.3O.sub.4Si: 632.2562, found: 632.2564.
##STR00069##
[0249] Compound 18. The entire amount of crude 17 was dissolved in the mixture of THF (3 mL) and ethanol (0.75 mL), and lithium hydroxide solution (51 mg of LiOH H.sub.2O in 0.75 mL water, 1.21 mmol, 10 equiv) was added to the mixture, which was left stirring at rt overnight (18 h). Acetic acid (200 L) was then added, and the reaction mixture was evaporated to dryness. The product was isolated by preparative HPLC (Interchim Uptisphere Strategy C18HQ 25030 mm 10 m, solvent flow rate 32 mL/min, gradient 50% to 100% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 65 mg (89% over 2 steps) of 18 as viscous yellow solid.
[0250] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.66 (d, J=8.5 Hz, 1H), 7.33 (dd, J=17.3, 10.9 Hz, 1H), 6.67 (d, J=2.4 Hz, 1H), 6.62 (d, J=2.5 Hz, 1H), 6.58 (d, J=2.5 Hz, 1H), 6.48 (dd, J=8.4, 2.6 Hz, 1H), 5.66 (dd, J=17.3, 1.3 Hz, 1H), 5.33 (dd, J=10.9, 1.2 Hz, 1H), 4.30 (t, J=11.8 Hz, 4H), 4.28 (t, J=11.7 Hz, 4H), 4.02 (t, J=6.3 Hz, 2H), 2.25 (t, J=7.6 Hz, 2H), 1.56-1.45 (m, 4H), 1.17-1.06 (m, 2H), 0.48 (s, 6H).
[0251] .sup.19F NMR (376 MHz, CDCl.sub.3): 99.43.
[0252] .sup.13C NMR (101 MHz, CDCl.sub.3): 178.6, 168.7, 162.6, 149.5 (t, J=2.8 Hz), 149.4 (t, J=2.7 Hz), 138.5, 138.1, 137.9, 136.3, 134.9, 133.0, 128.0, 115.9 (t, J=274.7 Hz), 115.8 (t, J=274.7 Hz), 115.6, 115.3, 115.2, 113.0, 111.4, 65.8, 63.4 (t, J=26.2 Hz), 63.3 (t, J=26.3 Hz), 33.8, 28.5, 25.3, 24.4, 2.7.
[0253] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.30H.sub.33F.sub.4N.sub.3O.sub.4Si: 604.2249, found: 604.2247.
##STR00070##
[0254] Compound 18-Halo. PyBOP solution (benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate; 15.5 mg in 100 L DMF, 29.8 mol, 1.5 equiv) was added to the stirred solution of 18 (12 mg, 19.9 mol) and HaloTag(O2) amine (hydrochloride salt; 7.7 mg, 29.8 mol, 1.5 equiv) in DMF (200 L) and DIPEA (80 L), and the reaction mixture was stirred at rt for 1 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 50% to 100% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 14 mg (87%) of 18-Halo as viscous yellow oil.
[0255] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.40H.sub.53ClF.sub.4N.sub.4O.sub.5Si: 809.3483, found: 809.3491.
##STR00071##
[0256] Compound 19. In a 25 mL round-bottom flask, a mixture of A5 (known compound: M. Remmel et al. Small Methods 2024, 2301497) (181 mg, 0.36 mmol), vinylboronic acid pinacol ester (336 mg, 2.18 mmol, 6 equiv), tris(dibenzylideneacetone)dipalladium(0) (33 mg, 36 mol, 10 mol %), XPhos (35 mg, 72 mol, 20 mol %) and cesium carbonate (474 mg, 1.44 mmol, 4 equiv) in degassed dry acetonitrile (6 mL) was stirred at 80 C. overnight (18 h). On cooling, the reaction mixture was diluted with CH.sub.2Cl.sub.2 and filtered through a plug of Celite, washing with CH.sub.2Cl.sub.2 (100 mL). The filtrate was washed with brine, dried over Na.sub.2SO.sub.4 and the product was isolated by flash column chromatography (25 g Interchim SiHP 30 m cartridge, gradient 5% to 60% EtOAc/hexane) and freeze-dried from 1,4-dioxane to yield 92 mg (67%) of 19 as yellow solid.
[0257] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.04 (t, J=1.2 Hz, 2H), 7.21 (dd, J=17.8, 11.3 Hz, 1H), 6.48 (d, J=2.1 Hz, 2H), 5.38 (dd, J=11.3, 1.9 Hz, 1H), 5.15 (dd, J=17.8, 1.9 Hz, 1H), 3.46-3.38 (m, 4H), 3.14-3.06 (m, 2H), 3.01 (td, J=8.4, 1.2 Hz, 2H), 2.878 (s, 3H), 2.876 (s, 3H), 0.44 (s, 6H).
[0258] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.0, 154.6, 154.5, 141.1, 138.9, 138.8, 138.4, 134.1, 132.3, 131.3, 131.0, 126.0, 113.9, 107.8, 107.6, 55.3, 55.1, 35.0, 34.9, 29.2, 28.3, 1.0.
[0259] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.26N.sub.2OSi: 375.1887, found: 375.1882.
##STR00072##
[0260] Compound 20. A solution of trifluoromethanesulfonic anhydride (Tf.sub.2O, 1 M in CH.sub.2Cl.sub.2; 0.37 mL, 0.37 mmol, 1.5 equiv.) was added under argon atmosphere to the stirred solution of compound 19 (92 mg, 0.25 mmol) in dry CH.sub.2Cl.sub.2 (3 mL), cooled in ice-water bath, and the resulting dark blue solution was stirred at 0 C. for 20 min. It was then transferred dropwise into the stirred mixture of aqueous ammonia (28% aq., 2.5 mL) and 1,2-dimethoxyethane (DME, 5 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 C. for 30 min, diluted with brine (230 mL), the product was then extracted with CH.sub.2Cl.sub.2 (320 mL) and the combined extracts were dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 100% A/B, A=CH.sub.2Cl.sub.2 ethanol-25% aq. NH.sub.3 80:20:2, BCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 19 mg (21%) of 20 as brown-orange solid.
[0261] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.78 (s, 1H), 6.80 (dd, J=17.9, 11.5 Hz, 1H), 6.54 (d, J=6.5 Hz, 2H), 5.52 (dd, J=11.5, 1.6 Hz, 1H), 5.45 (dd, J=17.9, 1.6 Hz, 1H), 3.40-3.32 (m, 4H), 3.07 (t, J=8.2 Hz, 2H), 3.00 (t, J=8.5 Hz, 1H), 2.83 (s, 6H), 0.43 (s, 6H).
[0262] .sup.13C NMR (101 MHz, CDCl.sub.3): 173.8, 153.8, 153.4, 137.1, 136.0, 135.6, 133.4, 132.5, 131.2, 123.6, 118.9, 108.73, 108.67, 55.8, 55.7, 35.7, 29.8, 29.3, 28.6, 1.8.
[0263] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.27N.sub.3Si: 374.2047, found: 374.2062.
##STR00073##
[0264] Compound 21. Chloroformate ester B1 was prepared according to the procedure from K. M. Anderson et al. Crystal Growth & Design 2006, 6(9), 2109-2113. Ethyl 6-hydroxyhexanoate (160 mg, 1 mmol) and pyridine (81 L, 1 mmol, 1 equiv) were added to the stirred solution of triphosgene (99 mg, 0.33 mmol, 0.33 equiv) in dry diethyl ether (2 mL), cooled to 0 C. The reaction mixture was stirred at 0 C. for 5 h, the precipitate of pyridine hydrochloride was filtered off on a short plug of Celite, washed with dry diethyl ether (3 mL), the filtrate was evaporated and the residue was redissolved in dry CH.sub.2Cl.sub.2 (1 mL) and used directly in the next step.
[0265] The prepared solution of B1 in CH.sub.2Cl.sub.2 (0.2 mL, 0.2 mmol, 4 equiv) was added to the stirred solution of compound 20 (19 mg, 50.8 mol) and DIPEA (50 L, 0.29 mmol, 6 equiv) in dry CH.sub.2Cl.sub.2 (0.5 mL), and the resulting solution was stirred at rt for 1 h. The crude reaction mixture was evaporated, the residue was dissolved in the mixture of THF (1 mL) and ethanol (0.25 mL), and lithium hydroxide solution (21 mg of LiOH H.sub.2O in 0.25 mL water, 0.5 mmol, 10 equiv) was added to the mixture, which was left stirring at rt for 18 h. Acetic acid (100 L) was then added, and the reaction mixture was evaporated to dryness. The product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 80% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 14 mg (52% over 2 steps) of 21 as brown solid.
[0266] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.55 (s, 1H), 7.05 (dd, J=17.8, 11.5 Hz, 1H), 6.62 (s, 1H), 6.57 (s, 1H), 5.47 (dd, J=11.5, 1.6 Hz, 1H), 5.34 (dd, J=17.8, 1.6 Hz, 1H), 4.00 (t, J=6.1 Hz, 2H), 3.47-3.34 (m, 4H), 3.06 (t, J=8.1 Hz, 2H), 2.99 (t, J=8.2 Hz, 2H), 2.85 (s, 6H), 2.26 (t, J=7.4 Hz, 2H), 1.56-1.44 (m, 4H), 1.16-1.05 (m, 2H).
[0267] .sup.13C NMR (101 MHz, CDCl.sub.3): 135.3, 123.7, 118.0, 110.0, 109.1, 65.5, 55.5, 35.5, 33.9, 29.0, 28.2, 28.1, 25.2, 24.5, 2.4 (indirect detection from a gHSQC experiment, only H-coupled carbons are resolved).
[0268] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.30H.sub.37N.sub.3O.sub.4Si: 532.2626, found: 532.2647.
##STR00074##
[0269] Compound 21-Halo. PyBOP solution (benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate; 10.4 mg in 100 L DMF, 20.0 mol, 1.5 equiv) was added to the stirred solution of compound 21 (7 mg, 13.2 mol) and HaloTag(O2) amine hydrochloride (5.2 mg, 20.0 mol, 1.5 equiv) in DMF (150 L) and DIPEA (50 L), and the reaction mixture was stirred at rt for 1.5 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) to give 8.3 mg (86%) of 21-Halo as viscous brown-yellow oil.
[0270] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.61 (br.s, 1H), 7.05 (dd, J=17.8, 11.5 Hz, 1H), 6.62 (s, 1H), 6.59 (s, 1H), 6.26 (br.s, 1H), 5.47 (dd, J=11.5, 1.5 Hz, 1H), 5.34 (dd, J=17.8, 1.5 Hz, 1H), 4.01 (t, J=6.3 Hz, 2H), 3.65-3.50 (m, 8H), 3.50-3.34 (m, 8H), 3.07 (t, J=8.1 Hz, 2H), 3.00 (t, J=8.2 Hz, 2H), 2.87 (s, 6H), 2.10 (t, J=7.7 Hz, 2H), 1.82-1.71 (m, 2H), 1.66-1.32 (m, 10H), 1.22-1.08 (m, 2H), 0.47 (s, 6H).
[0271] .sup.13C NMR (101 MHz, CDCl.sub.3): 135.2, 118.1, 109.9, 109.4, 71.4, 70.3, 70.1, 65.7, 55.6, 45.1, 39.3, 36.6, 35.6, 32.7, 29.6, 29.1, 28.5, 28.3, 26.7, 25.6, 25.4, 2.3 (indirect detection from a gHSQC experiment, only H-coupled carbons are resolved).
[0272] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.40H.sub.57ClN.sub.4O.sub.5Si: 737.3860, found: 737.3844.
##STR00075##
[0273] Compound 21-NHS. TSTU solution (N,N,N,N-tetramethyl-O(N-succinimidyl)uronium tetrafluoroborate; 5.2 mg in 50 L DMF, 17.2 mol, 1.5 equiv) was added to the stirred solution of compound 21 (6.1 mg, 11.5 mol) in DMF (150 L) and DIPEA (50 L), and the reaction mixture was stirred at rt for 1.5 h. The organic solvents were evaporated in vacuo, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 30% to 100% EtOAc/hexane) and freeze-dried from 1,4-dioxane to yield 6.4 mg (89%) of 21-NHS as yellow solid.
[0274] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.34H.sub.40N.sub.4O.sub.6Si: 629.2790, found: 629.2782.
##STR00076##
[0275] Compound 21-maleimide. A solution of compound 21-NHS (5.7 mg, 9.1 mol), 1-(2-aminoethyl)maleimide hydrochloride (2.4 mg, 13.6 mol, 1.5 equiv) in DMF (200 L) and DIPEA (50 L) was stirred at rt for 1.5 h. The organic solvents were evaporated in vacuo, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25021.2 mm m, solvent flow rate 18 mL/min, gradient 30% to 80% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) to give 2.1 mg (35%) of 21-maleimide as yellow solid.
[0276] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.36H.sub.43N.sub.5O.sub.5Si: 654.3106, found: 654.3101.
##STR00077##
[0277] Compound 22. Dimethylthiocarbamoyl chloride (16 mg in 0.5 mL dry acetonitrile, 0.128 mmol, 1.5 equiv) was added to the mixture of compound 1 (30 mg, 85.7 mol) and N,N-diisopropylethylamine (DIPEA; 74 L, 0.429 mmol, 5 equiv), and the resulting solution was stirred at 60 C. for 6 h. The solvents were evaporated, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25020 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 5 mg (13%) of 22 as brown-orange solid.
[0278] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.10 (d, J=8.8 Hz, 1H), 7.53 (dd, J=17.3, 10.9 Hz, 1H), 7.03-6.71 (m, 5H), 5.60 (dd, J=17.3, 1.4 Hz, 1H), 5.30 (dd, J=10.9, 1.4 Hz, 1H), 3.40 (s, 3H), 3.06 (s, 6H), 3.04 (s, 6H), 3.01 (s, 3H), 0.50 (s, 6H).
[0279] .sup.13C NMR (101 MHz, CDCl.sub.3) 138.7, 129.1, 116.4, 115.9, 113.7, 113.2, 41.9, 40.5, 40.1, 1.8 (indirect detection from a gHSQC experiment, only H-coupled carbons are resolved).
[0280] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.24H.sub.32N.sub.4SSi: 437.2190, found: 437.2199.
##STR00078##
[0281] Compound 23. Dimethylcarbamoyl chloride (16 L, 0.171 mmol, 2 equiv) was added to the mixture of compound 1 (30 mg, 85.7 mol) and N,N-diisopropylethylamine (DIPEA; 74 L, 0.429 mmol, 5 equiv) in dry acetonitrile (0.5 mL), and the resulting solution was stirred at 50 C. for 5 h. The solvents were evaporated, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25020 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 80% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 26 mg (72%) of 23 as light brown solid.
[0282] .sup.1H NMR (400 MHz, CDCl.sub.3): 7.81 (d, J=8.8 Hz, 1H), 7.44 (dd, J=17.3, 10.8 Hz, 1H), 6.91 (d, J=2.8 Hz, 1H), 6.84 (d, J=2.8 Hz, 1H), 6.82 (d, J=2.8 Hz, 1H), 6.69 (dd, J=8.8, 2.8 Hz, 1H), 5.63 (dd, J=17.3, 1.5 Hz, 1H), 5.26 (dd, J=10.8, 1.5 Hz, 1H), 3.04 (s, 6H), 3.01 (s, 6H), 2.91 (s, 3H), 2.72 (s, 3H), 0.47 (s, 6H).
[0283] .sup.13C NMR (101 MHz, CDCl.sub.3): 166.1, 164.5, 150.1, 150.0, 138.4, 138.3, 137.9, 137.2, 132.5, 131.5, 128.7, 115.3, 115.1, 113.6, 112.9, 112.1, 40.4, 40.2, 37.1, 35.7, 1.7.
[0284] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.24H.sub.32N.sub.4OSi 421.2418; Found 421.2428.
##STR00079##
[0285] Compound 24. Ethyl 6-isocyanatohexanoate (36 L, 0.19 mmol, 1.5 equiv) was added to the stirred mixture of compound 1 (45 mg, 0.13 mmol) and DIPEA (68 L, 0.39 mmol, 3 equiv) in dry CH.sub.2Cl.sub.2 (0.9 mL), and the resulting solution was stirred at rt for 1 h. The crude reaction mixture was evaporated on Celite, the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 0% to 50% EtOAc/CH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 52 mg (76%) of 24 as light tan solid.
[0286] .sup.1H NMR (400 MHz, CD.sub.3CN): 7.68 (d, J=8.8 Hz, 1H), 7.44-7.30 (m, 1H), 6.95-6.90 (m, 3H), 6.70 (dd, J=8.8, 2.8 Hz, 1H), 5.65 (dd, J=17.4, 1.5 Hz, 1H), 5.61 (t, J=5.2 Hz, 1H), 5.17 (dd, J=11.0, 1.5 Hz, 1H), 4.06 (q, J=7.1 Hz, 2H), 3.06 (q, J=6.5 Hz, 2H), 3.02 (s, 6H), 2.98 (s, 6H), 2.19 (t, J=7.6 Hz, 2H), 1.48 (p, J=7.6 Hz, 2H), 1.43-1.34 (m, 2H), 1.20 (t, J=7.1 Hz, 3H), 1.16-1.07 (m, 2H), 0.46 (s, 6H).
[0287] .sup.13C NMR (101 MHz, CD.sub.3CN): 174.3, 164.8, 151.2, 151.0, 138.9, 138.4, 138.3, 132.3, 129.1, 116.5, 116.0, 113.7, 113.1, 111.8, 60.8, 40.5, 40.4, 40.3, 34.7, 30.1, 26.9, 25.5, 14.6, 2.3.
[0288] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.30H.sub.42N.sub.4O.sub.3Si: 535.3099, found: 535.3092.
##STR00080##
[0289] Compound 25. Lithium hydroxide solution (12 mg of LiOH H.sub.2O in 0.5 mL water, 0.28 mmol, 5 equiv) was added to the stirred solution of compound 24 (30 mg, 56 mol) in THE (2 mL) and methanol (0.5 mL), and the reaction mixture was left stirring overnight (20 h). The organic solvents were evaporated, and the product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 70% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 26 mg (91%) of 25 as brown-yellow solid.
[0290] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.0 (br.s, 1H), 7.66 (d, J=8.8 Hz, 1H), 7.49-7.34 (m, 1H), 7.09 (t, J=5.8 Hz, 1H), 6.93-6.85 (m, 3H), 6.67 (dd, J=8.8, 2.8 Hz, 1H), 5.66 (dd, J=17.3, 1.5 Hz, 1H), 5.16 (dd, J=10.8, 1.5 Hz, 1H), 3.00 (s, 6H), 2.95 (s, 6H), 2.14 (t, J=7.4 Hz, 2H), 1.49-1.30 (m, 4H), 1.18-1.07 (m, 2H), 0.45 (s, 6H).
[0291] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 174.5, 163.4, 149.8, 149.5, 137.7, 137.5, 137.1, 136.6, 131.4, 128.0, 115.4, 114.9, 113.3, 112.0, 110.3, 33.7, 29.2, 26.0, 24.4, 2.4.
[0292] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.28H.sub.38N.sub.4O.sub.3Si 507.2786; Found 507.2778.
##STR00081##
[0293] Compound 25-NHS. TSTU solution (N,N,N,N-tetramethyl-O(N-succinimidyl)uronium tetrafluoroborate; 8.4 mg in 100 L DMF, 28 mol, 1.5 equiv) was added to the stirred solution of compound 25 (10 mg, 18.7 mol) in DMF (100 L) and DIPEA (50 L), and the reaction mixture was stirred at rt for 1 h. The organic solvents were evaporated in vacuo, and the product was isolated by flash column chromatography (12 g Interchim SiHP 30 m cartridge, gradient 5% to 70% EtOAc/CH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 11 mg (97%) of 25-NHS as yellow solid.
[0294] .sup.1H NMR (400 MHz, CD.sub.3CN): 7.69 (d, J=8.8 Hz, 1H), 7.35 (dd, J=17.3, 10.7 Hz, 1H), 6.96-6.90 (m, 3H), 6.71 (dd, J=8.8, 2.8 Hz, 1H), 5.65 (dd, J=17.3, 1.5 Hz, 1H), 5.61 (t, J=5.6 Hz, 1H), 5.18 (dd, J=10.7, 1.5 Hz, 1H), 3.08 (q, J=6.5 Hz, 2H), 3.02 (s, 6H), 2.99 (s, 6H), 2.76 (s, 4H), 2.53 (t, J=7.5 Hz, 2H), 1.61 (p, J=7.6 Hz, 2H), 1.41 (dt, J=14.6, 6.8 Hz, 2H), 1.23-1.14 (m, 2H), 0.46 (s, 6H).
[0295] .sup.13C NMR (101 MHz, CD.sub.3CN): 139.0, 129.3, 116.6, 116.1, 114.0, 113.2, 112.0, 40.48, 40.44, 40.40, 31.4, 30.1, 26.6, 26.4, 25.2, 2.1 (indirect detection from a gHSQC experiment, only H-coupled carbons are resolved).
[0296] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.32H.sub.41N.sub.5O.sub.5Si 604.2950; Found 604.2942.
##STR00082##
[0297] Compound 26. N,N-Dimethylsulfamoyl chloride (31 L, 0.286 mmol, 4 equiv) was added to the mixture of compound 1 (25 mg, 71.4 mol) and N,N-diisopropylethylamine (DIPEA; 62 L, 0.357 mmol, 5 equiv) in dry acetonitrile (0.4 mL), and the resulting solution was stirred at 50 C. for 4 h. The solvents were evaporated, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25020 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from dioxane to give 6 mg (18%) of 26 as orange-yellow solid.
[0298] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.02 (d, J=8.8 Hz, 1H), 7.36 (dd, J=17.2, 10.8 Hz, 1H), 6.86 (d, J=2.7 Hz, 1H), 6.82 (app. t, J=2.5 Hz, 2H), 6.74 (dd, J=8.8, 2.8 Hz, 1H), 5.58 (dd, J=17.2, 1.5 Hz, 1H), 5.22 (dd, J=10.8, 1.5 Hz, 1H), 3.06 (s, 6H), 3.04 (s, 6H), 2.72 (s, 6H), 0.48 (s, 6H).
[0299] .sup.13C NMR (101 MHz, CDCl.sub.3): 176.6, 150.8, 150.2, 139.1, 138.9, 138.4, 137.9, 131.9, 131.5, 130.7, 114.8, 114.5, 113.6, 111.8, 111.6, 40.3, 40.1, 38.8, 2.6.
[0300] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.32N.sub.4O.sub.2SSi 457.2088; Found 457.2097.
##STR00083##
[0301] Compound 27. Fluoro-N,N,N,N-tetramethylformamidinium hexafluorophosphate (TFFH; 38 mg, 0.143 mmol, 2 equiv) was added to the mixture of 1 (25 mg, 71.4 mol) and N,N-diisopropylethylamine (DIPEA; 62 L, 0.357 mmol, 5 equiv) in dry acetonitrile (0.4 mL), and the resulting solution was stirred at rt for 2 h. The solvents were evaporated, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25020 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 90% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from aq. dioxane to give 27 mg (64%) of 27 as orange solid.
[0302] .sup.1H NMR (400 MHz, CDCl.sub.3+10% (v/v) DMSO-d.sub.6): 7.28 (dd, J=17.3, 10.8 Hz, 1H), 7.22 (d, J=9.0 Hz, 1H), 6.96 (dd, J=9.0, 2.8 Hz, 1H), 6.86 (d, J=2.8 Hz, 1H), 6.82 (d, J=2.8 Hz, 1H), 6.79 (d, J=2.8 Hz, 1H), 5.51 (dd, J=17.3, 1.3 Hz, 1H), 5.21 (dd, J=10.8, 1.3 Hz, 1H), 3.15 (s, 6H), 3.13 (s, 6H), 2.95 (s, 12H), 0.49 (s, 6H).
[0303] .sup.13C NMR (101 MHz, CDCl.sub.3+10% (v/v) DMSO-d.sub.6): 176.1, 166.6, 151.6, 151.0, 142.6, 140.3, 140.2, 139.1, 129.7, 127.6, 127.0, 115.8, 115.0, 113.9, 113.71, 113.68, 41.0, 39.9, 39.8, 1.0.
[0304] .sup.19F NMR (376 MHz, CDCl.sub.3) 73.1 (d, J=712.0 Hz, PF.sub.6.sup.).
[0305] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.26H.sub.38N.sub.5Si 448.2891; Found 448.2886.
##STR00084##
[0306] Compound 28. 2-Fluoro-1,3-dimethylimidazolidinium hexafluorophosphate (68 mg, 0.258 mmol, 3 equiv) was added to the mixture of compound 1 (35 mg, 85.7 mol) and N,N-diisopropylethylamine (DIPEA; 74 L, 0.429 mmol, 5 equiv) in dry acetonitrile (0.5 mL), and the resulting solution was stirred at rt for 1 h. The solvents were evaporated, and the product was isolated by preparative HPLC (ThermoFisher Hypersil Gold C18 25020 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 80% A:B, A-acetonitrile+0.1% (v/v) HCO.sub.2H, B-water+0.1% (v/v) HCO.sub.2H) and freeze-dried from aq. dioxane to give 35 mg (69%) of 28 as orange solid.
[0307] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.55 (d, J=9.0 Hz, 1H), 6.99 (dd, J=17.4, 10.8 Hz, 1H), 6.92 (dd, J=9.0, 2.8 Hz, 1H), 6.84-6.78 (m, 3H), 5.65 (dd, J=17.4, 1.1 Hz, 1H), 5.24 (dd, J=10.8, 1.1 Hz, 1H), 3.86-3.79 (m, 2H), 3.77-3.67 (m, 2H), 3.14 (s, 6H), 3.12 (s, 6H), 2.72 (s, 6H), 0.47 (s, 6H).
[0308] .sup.13C NMR (101 MHz, CDCl.sub.3): 175.5, 162.5, 151.8, 151.2, 141.1, 139.9, 138.8, 138.7, 130.0, 128.9, 126.9, 115.3, 115.2, 115.0, 113.8, 113.4, 48.4, 40.1, 32.9, 1.4.
[0309] .sup.19F NMR (376 MHz, CDCl.sub.3) 73.4 (d, J=712.4 Hz, PF.sub.6.sup.).
[0310] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.26H.sub.36N.sub.5Si 446.2734; Found 446.2726.
EXAMPLE 2
Characterisation of Exemplary Compounds of the Present Invention
General Materials and Methods
[0311] All chemical reagents (TCI, Sigma-Aldrich, Alfa Aesar) and dry solvents for synthesis (over molecular sieves, AcroSeal package, Acros Organics) were purchased from reputable suppliers and were used as received without further purification. The products were lyophilized from a suitable solvent system using Alpha 2-4 LDplus freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH).
Thin Layer Chromatography
[0312] Normal phase TLC was performed on silica gel 60 F.sub.254 (Merck Millipore, Germany). For TLC on reversed phase silica gel 60 RP-18 F.sub.254s (Merck Millipore) was used. Compounds were detected by exposing TLC plates to UV-light (254 or 366 nm) or heating with vanillin stain (6 g vanillin and 1.5 mL conc. H.sub.2SO.sub.4 in 100 mL ethanol), unless indicated otherwise.
Flash Chromatography
[0313] Preparative flash chromatography was performed with an automated Isolera One system with Spektra package (Biotage AG) using commercially available cartridges of suitable size as indicated (RediSep Rf series from Teledyne ISCO, Puriflash Silica HP 30 m series from Interchim).
Nuclear Magnetic Resonance (NMR) NMR spectra (.sup.1H, .sup.13C{.sup.1H}, .sup.19F) were recorded on a Bruker DPX 400 spectrometer. All spectra are referenced to tetramethylsilane as an internal standard (=0.00 ppm). Multiplicities of the signals are described as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet or overlap of non-equivalent resonances; br=broad signal. Coupling constants .sup.nJ.sub.X-Y are given in Hz, where n is the number of bonds between the coupled nuclei X and Y (J.sub.HH are always listed as i without indices).
Mass-Spectrometry (MS)
[0314] Low resolution mass spectra (100-1500 m/z) with electro-spray ionization (ESI) were obtained on a Shimadzu LC-MS system described below. High resolution mass spectra (HRMS) were obtained on a maXis II ETD (Bruker) with electrospray ionization (ESI) at the Mass Spectrometry Core facility of the Max-Planck Institute for Medical Research (Heidelberg, Germany).
High-Performance Liquid Chromatography (HPLC)
[0315] Analytical liquid chromatography-mass spectrometry was performed on an LC-MS system (Shimadzu): 2 LC-20AD HPLC pumps with DGU-20A3R solvent degassing unit, SIL-20ACHT autosampler, CTO-20AC column oven, SPD-M30A diode array detector and CBM-20A communication bus module, integrated with CAMAG TLC-MS interface 2 and LCMS-2020 spectrometer with electrospray ionization (ESI, 100-1500 m/z). Analytical column: Hypersil GOLD 502.1 mm 1.9 m, standard conditions: sample volume 1-2 L, solvent flow rate 0.5 mL/min, column temperature 30 C. General method: isocratic 95:5 A:B over 2 min, then gradient 95:5.fwdarw.0:100 A:B over 5 min, then isocratic 0:100 A:B over 2 min; solvent A=water+0.1% v/v HCO.sub.2H, solvent B=acetonitrile+0.1% v/v HCO.sub.2H.
[0316] Preparative high-performance liquid chromatography was performed on a Buchi Reveleris Prep system using the suitable preparative columns and conditions as indicated for individual preparations. Method scouting was performed on a HPLC system (Shimadzu): 2LC-20AD HPLC pumps with DGU-20A3R solvent degassing unit, CTO-20AC column oven equipped with a manual injector with a 20 L sample loop, SPD-M20A diode array detector, RF-20A fluorescence detector and CBM-20A communication bus module; or on a Dionex Ultimate 3000 UPLC system: LPG-3400SD pump, WPS-3000SL autosampler, TCC-3000SD column compartment with 27-port 6-position valves and DAD-3000RS diode array detector. The test runs were performed on analytical columns with matching phases (HPLC: Interchim 2504.6 mm 10 m C18HQ, Interchim 2504.6 mm 5 m PhC4, solvent flow rate 1.2 mL/min; UPLC: Interchim C18HQ or PhC4 752.1 mm 2.2 m, ThermoFisher Hypersil GOLD 1002.1 mm 1.9 am, solvent flow rate 0.5 mL/min).
Confocal and STED (Stimulated Emission Depletion) Microscopy
[0317] Confocal and STED images were acquired using two Abberior Expert Line (Abberior Instruments GmbH, Gttingen, Germany) fluorescence microscopes built on a motorized inverted microscope IX83 (Olympus, Tokyo, Japan). Microscope 1 is equipped with pulsed STED lasers at 595 nm and 775 nm shaped by Spatial Light Modulators (SLMs), and with 355 nm, 405 nm, 485 nm, 561 nm, and 640 nm excitation lasers, and a 100/1.40 oil immersion objective lens (Olympus). Microscope 2 is equipped with pulsed STED lasers at 655 nm and 775 nm, and with 520 nm, 561 nm, 640 nm, and multiphoton (Chameleon Vision II, Coherent, Santa Clara, USA) excitation lasers, and a 60/1.42 oil immersion objective lens (Olympus). The multiphoton laser is tuneable in the 680 nm-1080 nm range. Spectral detection is performed in both cases with avalanche photodiodes at spectral windows adjusted for each particular fluorophore.
[0318] Imaging and image processing was done with ImSpector software (v. 16.3.13367; Abberior Instruments GmbH, Gttingen, Germany), and all images are displayed as raw data unless otherwise noted.
Superresolution Single Molecule Localization Microscopy (SMLM)
[0319] SMLM/PALM images were acquired using an ONI Nanoimager V3 (Oxford Nanoimaging, Oxford, UK). The 405 nm activation laser was applied as CW illumination. All images were analyzed and processed using the ThunderSTORM plugin [M. Ovesn et al. Bioinformatics, 2014, 30(16), 2389-2390] on Image J (version 1.52p) or using the ONI Nanoimager Software, Development build: Apr. 9 2023 22:54:56 Version: 1.19.7.20230409223555-28f00b5.
MINFLUX Imaging
[0320] For MINFLUX imaging an Abberior 3D MINFLUX microscope (Abberior Instruments GmbH, Gttingen, Germany) was used. Details of the instrument are described in [Schmidt et al. Nat. Comm., 2021, 12, 1478]. The microscope, built on an Olympus IX83 body with a 60UPLXAPO60XO oil objective, was equipped with a 640 nm excitation laser, a 405 nm activation laser, 488 nm and 560 nm confocal lasers, a 980 nm stabilization laser and an xyz piezo stage (Piezoconcept) for active sample stabilization.
[0321] For acquisition the pinhole was set to 0.83 AU and signal was detected on APDs in the spectral window of Cy5 (650-720 nm). The excitation power was set to initially 33 W in the sample position for 2D imaging and to 56 W for 3D imaging. The activation 405 nm laser was attenuated with an additional ND2 filter. Activation was switched on and the power was gradually increased up to 1.1 W, sustaining the frequency of detected events, until the events became sparse in time and the imaging was stopped.
[0322] MINFLUX imaging was performed with modified imaging sequences based on the standard imaging sequences provided by the manufacturer. Images were post-processed and analyzed with a custom-built MATLAB (2022a, MathWorks) routine according to [Remmel et al. Small Methods. 2024, 8, 2301497].
EXAMPLE 3
Photolysis of the Exemplary Novel Compounds of the Invention and Chemometric Analysis of the Photoactivation and Photobleaching Reaction Kinetics
[0323] Solutions in phosphate buffer (100 mM, pH=7.0, 5.0 M dye) were irradiated in a previously described home-built setup [K. Uno et al. Adv. Opt. Mater. 2019, 7, 1801746] with a 405 nm LED source (M405L3, Thorlabs Inc.) in combination with a 10 nm bandpass filter (FB405-10, Thorlabs Inc.). During the irradiation, samples were maintained at 20 C. and continuously stirred. The absorption and emission of irradiated solutions was monitored at desired irradiation intervals. For such purpose, excitation was performed with an LED emitting at a wavelength suitable for each compound (e.g. 625 nm). Photobleaching reactions were performed in the same setup using a 625 nm LED source (M625L4, Thorlabs Inc.). Photobleaching quantum yields were calculated as previously described in [R. Lincoln et al. Nat. Chem. 2022, 14, 1013-1020; R. Lincoln et al. WO2023/284968A1].
[0324]
[0325]
EXAMPLE 4
Live and Fixed Cell Optical Microscopy of Cells Using Exemplary Novel Dyes of the Invention with Self-Labelling Enzymes
[0326] The entire sample preparation procedure was conducted under red light (generic 12 V red LED strips, IP65 waterproof, 620-640 nm). Stocks solutions of SNAP-tag ligand (4-BG) or HaloTag ligand derivatives of the compounds (4-Halo, 21-Halo) were prepared in DMSO (500 M-5 mM). U-2 OS cells that stably expressed Vimentin-HaloTag [Ratz et al. Sci. Rep. 2015, 5, 9592; Butkevich et al. ACS Chem. Biol. 2018, 13(2), 475-480] or HeLa cells that stably expressed COX8A-SNAP-tag8 [Stephan et al. Sci. Rep. 2019, 9, 12419] were grown for 12-72 h on glass coverslips. Cells were incubated in the dark for 30 min to overnight (depending on the dye and experiment) with the respective fluorescent ligands diluted from DMSO stock solutions with culture medium (without phenol red) to a final concentration of 500 nm-1 M. After labeling with dyes, the samples were protected from the ambient light. Cells were washed twice with cell culture medium for ca. 15-30 minutes; then the medium was changed for fresh media for live-cell imaging or fixed as described below.
[0327] For live-cell imaging cells were mounted in a live-cell chamber (CM-B18-1, Live Cell Instrument Co.) with Fluorobrite (A1896701, Gibco) supplemented with 10% (v/v) FBS (10500064, ThermoFisher), 2% (v/v) GlutaMAX (35050061, Gibco) and 1% (v/v) penicillin/streptomycin.
[0328] PFA fixation for preservation of vimentin filaments was performed with a 4% formaldehyde solution in PBS (pH 7.4) at room temperature for 25 min, rinsed with a quenching solution (QS, 0.1 M NH.sub.4Cl and 0.1 M glycine in PBS) and then incubated with QS for 10 min at room temperature then washed with PBS.
[0329]
[0330]
EXAMPLE 5
Multiplexing of Two Photoactivatable Dyes in Single Molecule Localization Microscopy by Selective Photoactivation
[0331] HeLa cells that stably expressed COX8A-SNAP were labelled with 4-BG as described in Example 4 above. For preservation of mitochondria cristae, cells were fixed with warm 8% formaldehyde solution in PBS (pH 7.4) at 37 degrees for 7 minutes, and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 10 minutes. To reduce unspecific binding, blocking buffer (5% BSA in PBS) was added and incubated for 10 minutes at room temperature, then washed with PBS. The coverslips were overlaid with the primary antibody for TOMM20 (from rabbit) in blocking buffer and incubated in a humid chamber for 1 h at room temperature and then washed with blocking buffer (35 min). The coverslips were then incubated with the secondary anti-rabbit nanobody labelled with PaX.sub.560-Maleimide [R. Lincoln et al. Nat. Chem. 2022, 14, 1013-1020, R. Lincoln et al. WO2023/284968A1] in blocking buffer in a humid chamber for 1 h at room temperature, and then washed with blocking buffer (35 min), and with PBS (35 min).
[0332] As it was determined that 560 nm excitation was sufficient to photoactivate compound 4 (but not PaX.sub.560), sequential imaging independently selects the two fluorophores. First, compound 4 was imaged with 640 nm excitation (270 mW, detection range 662-710 nm) using the 560 nm laser as the photoactivation laser (<0.1 mW to 3 mW). Upon depleting the localizations of 4, PaX.sub.560 is imaged using the 560 nm laser for excitation (180 mW, detection range 570-620 nm) and a 405 nm laser for photoactivation (<0.1 mW).
[0333]
EXAMPLE 6
Two-Color MINFLUX Imaging of Photoactivatable Dyes by Spectral Classification
[0334] U-2 OS cells were seeded as described in Example 4 above. Fixation was performed with a 4% formaldehyde solution containing 0.2% glutaraldehyde in PBS (pH 7.4) at 37 degrees for 10 min. Samples were incubated with a quenching solution (0.1% sodium borohydride in PBS) for 7 min at room temperature then washed twice with PBS. To permeabilize and reduce unspecific binding, a blocking buffer (5% BSA in PBS with 0.1% Triton X-100) was added and incubated for 30 minutes at room temperature.
[0335] The coverslips were overlaid with the primary antibodies for TOMM20 (from rabbit) and TIMM23 (from mouse) in blocking buffer and incubated in a humid chamber for 1 h at room temperature and then washed with PBS (35 min). The coverslips were then incubated with the secondary nanobodies labelled with 4-Maleimide (anti-rabbit) and 21-Maleimide (anti-mouse) in blocking buffer, in a humid chamber for 1 h at room temperature, and then washed with PBS (35 min). Samples were post-fixed with a 4% PFA solution for 10 min at room temperature, then washed with PBS (35 min).
[0336]
ADDITIONAL NUMBERED EMBODIMENTS OF THE INVENTION
1. A compound having the structural formula I:
##STR00085## [0337] wherein: [0338] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.1, independently of each other are selected from H, halogen, SO.sub.3H, CO.sub.2H, CN, NO.sub.2, CO.sub.2R, SO.sub.2R (with R in CO.sub.2R or SO.sub.2R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl), and an unsubstituted or substituted, in particular a halogen-, amino-, hydroxyl-, SO.sub.3H and/or carboxyl-substituted 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 alkylene, C.sub.2-C.sub.20 alkylyne, C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; [0339] and where the substituents R.sup.6 and R.sup.7, taken together with the atoms to which they are bound, may form a 5-8 membered ring structure; and/or where the substituents R.sup.7 and R.sup.8, taken together with the atoms to which they are bound, may form a 5-8 membered ring structure; [0340] R.sup.9, R.sup.10, R.sup.11, R.sup.12 are: [0341] a. 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.8 acyl, C.sub.1-C.sub.8 alkoxycarbonyl, and C.sub.7-C.sub.12 alkylaryl, and unsubstituted phenyl or phenyl substituted by unsubstituted alkyl, halogen, alkoxy, NO.sub.2, CO.sub.2H, CO.sub.2R and/or CONR.sub.2 (with each R in CO.sub.2R or CONR.sub.2 being selected independently from C.sub.1 to C.sub.4 unsubstituted alkyl); or [0342] b. R.sup.9 together with R.sup.10 and a nitrogen atom to which they are bound, and/or R.sup.11 together with R.sup.12 and a nitrogen atom to which they are bound form a 3-7 membered ring structure; or [0343] c. R.sup.9 and/or R.sup.11 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.10 together with R.sup.2 or R.sup.3 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.12 together with R.sup.4 or R.sup.5 and the atoms to which they are bound form a 5-7 membered ring structure; [0344] d. R.sup.9 together with R.sup.2 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.10 together with R.sup.3 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.11 together with R.sup.4 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.12 together with R.sup.5 and the atoms to which they are bound form a 5-7 membered ring structure; [0345] X is independently selected from: [0346] a. SiR.sup.14R.sup.15 or GeR.sup.14R.sup.15 group, where R.sup.14 and R.sup.15 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where both substituents R.sup.14 and R.sup.15, taken together with the Si or Ge to which they are attached, form a 4-7 membered ring structure; [0347] b. CR.sup.16R.sup.17 group, where R.sup.16 and R.sup.17 are each independently selected from H, F, CF.sub.3, CN, OR.sup.18, COR.sup.18, CO.sub.2R.sup.18, SO.sub.2R.sup.18, CONR.sup.18R.sup.19 (where R.sup.18 and R.sup.19 in COR.sup.18, CO.sub.2R.sup.18, SO.sub.2R.sup.18, and CONR.sup.18R.sup.19 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof), unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, [0348] or where both substituents R.sup.16 and R.sup.17, taken together with the C atom to which they are attached, form a 4-7 membered ring structure; [0349] Y is independently selected from: [0350] a. CO (carbonyl), CS (thiocarbonyl) or CNR.sup.20 (iminocarbonyl) group, where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; [0351] b. SO.sub.2 (sulfone) or S(O)(NR.sup.20) (sulfoximine) group, where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; [0352] c. P(O)(OH), P(O)(OR.sup.20) or P(O)(R.sup.20), where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; [0353] W is independently selected from O, S or NR.sup.21, where R.sup.21 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; [0354] R.sup.13 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where the substituents R.sup.13 and R.sup.20 taken together form a 5-8 membered ring structure.
2. A compound, which has the structural formula II and is obtainable by irradiation with ultraviolet, visible or infrared light through a one-photon absorption process or a multiphoton absorption process from any of the compounds of general formula I of embodiment 1:
##STR00086## [0355] where 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.D, R.sup.12, R.sup.13, X, Y and W are defined as in embodiment 1.
3. The compound according to embodiment 1 or 2, 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.D, R.sup.12 and R.sup.13 or through any of the groups X, Y, W to a binding moiety M selected from: [0356] a. a moiety selectively attachable by covalent bond to a protein or nucleic acid, particularly a moiety able to form an ester bond, a thioester bond, an ether bond, an amide or thioamide bond, a sulfide or disulfide bond, a carbon-carbon bond, a carbon-nitrogen bond such as a Schiff base, or a moiety able to react in a click-chemistry reaction with a corresponding functional group of a protein or nucleic acid, [0357] more particularly a moiety selected from COCHCH.sub.2, SO.sub.2CHCH.sub.2, COCH.sub.2I, COCCH, NCS, CONHS or another active ester, biotin, an azide or a tetrazine, a diazoalkane or diazoketone, a diazirine, an alkyne, a strained alkyne such as bicyclo[6.1.0]nonyne or cyclooctyne, a strained alkene such as trans-cyclooctene or norbornene, a maleimide; or from [0358] b. a substrate of a haloalkane transferase, particularly a 1-chlorohexyl or a (3-chloropropyl)aryl moiety as exemplarily shown below:
##STR00087## [0359] c. a substrate of O.sup.6-alkylguanine-DNA-alkyltransferase, particularly a (substituted) O.sup.6-benzylguanine, O.sup.2-benzylcytosine or 4-benzyloxy-6-halo- or 4-benzyloxy-6-pseudohalo-pyrimidine-2-amine moiety (where halo group is preferably chloro, and pseudohalo group is selected preferably, but without limitation, from CN and CF.sub.3) as exemplarily shown below:
##STR00088## [0360] or from [0361] d. a substrate of dihydrofolate reductase, particularly a 4-demethyltrimethoprim moiety as exemplarily shown below:
##STR00089## [0362] or from [0363] e. a moiety capable of selectively interacting non-covalently with a biomolecule, particularly a protein or nucleic acid, wherein said moiety and said biomolecule form a complex having a dissociation constant k.sub.D of 10.sup.6 mol/L or less, [0364] more particularly, M is selected from de-N-Boc-docetaxel, de-N-Boc-cabazitaxel, de-N-Boc-larotaxel or another taxol derivative, a phalloidin derivative, a jasplakinolide derivative, a bis-benzimide DNA stain, pepstatin A or triphenylphosphonium, e.g. as shown below:
##STR00090## ##STR00091## [0365] f. or wherein M is an oligonucleotide having a sequence length between 10 and 40 nucleotides; [0366] g. or wherein M is a lipid, particularly a sphingosine derivative such as a ceramide, or a phospholipid such as dioleoylphosphatidylethanolamine (DOPE) or dipalmitoylphosphatidylethanolamine (DPPE), or a fatty acid.
4. The compound according to embodiments 1-3, having one of the structural formulas I-1-I-30 or II-1-II-30:
##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## [0367] wherein 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.11, R.sup.12 or any one of the substituents R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, R.sup.21, if present, independently of any other is H or a moiety having a molecular weight between 15 and 1500 Da; [0368] particularly wherein: [0369] a) the substituents R.sup.9, R.sup.10, R.sup.D, R.sup.12 are selected from H and methyl, or any of the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 represents an azetidine ring, and [0370] b) 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 or one of the R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, R.sup.21, if present, is H or a moiety M having a molecular weight between 15 and 1500 Da, and [0371] c) the other substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are selected from H and F, and [0372] d) the other substituents R.sup.6, R.sup.7, R.sup.8 are selected from H and methyl, and [0373] e) the other substituents R.sup.14, R.sup.15, if present, are selected from methyl, ethyl, isopropyl or phenyl, [0374] f) the other substituents R.sup.16, R.sup.17, if present, are methyl, [0375] g) the other substituents R.sup.20, R.sup.21, if present, are selected from H, OH, methoxy and methyl.
5. The compound according to embodiment 4, wherein said binding moiety M defined as in embodiment 3 and having a molecular weight between 15 and 1500 Da is characterized by a general formula -L-M.sub.s, where -L- is a covalent bond or a linker consisting of 1 to 50 atoms having an atomic weight of 12 or higher (in addition to the number of hydrogen atoms required to satisfy the valence rules) covalently connecting the compound of structure I-1-I-30 or II-1-II-30 to the binding moiety M.sub.s (which is defined identical to binding moiety M in embodiment 3), [0376] particularly wherein said moiety M having a molecular weight between 15 and 1500 Da is characterized by a general formula
-L.sup.A1.sub.m-L.sup.J1.sub.m-L.sup.A2.sub.n-L.sup.J2.sub.n-L.sup.A3.sub.p-L.sup.J3.sub.p-L.sup.A4.sub.q-L.sup.J4.sub.q-M.sub.s, wherein [0377] 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.2CH.sub.2-0).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-, sulfonyl- and/or sulfoxyl substituted aryl or heteroaryl; [0378] L.sup.J1, L.sup.J2, L and L.sup.J4 independently of each other are selected from NRC(O), C(O)N(R), NRC(O)O, OC(O)N(R), C(R)N, NC(R), C(O), OC(O), [0379] C(O)O, N(R), O, P(O)(OR), P(O)(OR)O, OP(O)(OR), OP(O)(OR)O, S, SO, SO.sub.2, SO.sub.2N(R), N(R)SO.sub.2N(R), N(R)SO.sub.2 with R selected from H and unsubstituted or amino-, hydroxyl-, carboxyl, sulfonate or fluoro substituted C.sub.1 to C.sub.6 alkyl, particularly when R is selected from H and methyl; [0380] m, m, n, n, p, p, q, q and s independently from each other are selected from 0 and 1, and [0381] M.sub.s is defined identical to binding moiety M in embodiment 3.
6. The compound according to embodiment 5, wherein said moiety M having a molecular weight between 15 and 1500 Da is represented by one of the following structures:
##STR00112## ##STR00113## ##STR00114## ##STR00115##
7. The compound according to any one of the preceding embodiments, wherein [0382] a. R.sup.9 and R.sup.10, and/or R.sup.11 and R.sup.12, are independently selected from H, unsubstituted and amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, [0383] particularly R.sup.9 and R.sup.10, and/or R.sup.11 and R.sup.12, are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, allyl and CH.sub.2CF.sub.3, [0384] b. R.sup.9 together with R.sup.10, and/or R.sup.9 together with R.sup.10, are independently forming an unsubstituted or alkyl-, amino-, hydroxy-, carboxy-, sulfonate- 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, (CH.sub.2).sub.2SO.sub.2(CH.sub.2).sub.2 or (CH.sub.2).sub.2NR.sup.22(CH.sub.2).sub.2 with R.sup.22 being selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl, particularly methyl; [0385] c. R.sup.9 and/or R.sup.11 are independently selected from H, unsubstituted and alkyl-substituted, particularly methyl-substituted, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.10 together with R.sup.2 or R.sup.3, and/or R.sup.12 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.2CHCH or (CH.sub.2).sub.4 or CH.sub.2O, CH.sub.2NR, CH.sub.2S, CH.sub.2SO.sub.2, (CH.sub.2).sub.20, (CH.sub.2).sub.2NR, (CH.sub.2).sub.2S, (CH.sub.2).sub.2SO.sub.2, CH.sub.2OCH.sub.2, CH.sub.2NR, CH.sub.2SCH.sub.2, CH.sub.2SO.sub.2CH.sub.2, with R selected from H and unsubstituted or amino-, hydroxyl-, carboxyl, sulfonate- or fluoro-substituted C.sub.1 to C.sub.6 alkyl, particularly when R is selected from H and methyl-, and a mono- or dimethyl-substituted derivative of any one of the foregoing alkyl or heteroalkyl bridge moieties; [0386] d. R.sup.10 and/or R.sup.11 are independently selected from H, unsubstituted and alkyl-, substituted, particularly methyl-substituted-, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.9 together with R.sup.2, and/or R.sup.12 together with R.sup.5, form a fused annular structure according to any one of the following substructures:
##STR00116## [0387] e. R.sup.9 and/or R.sup.12 are independently selected from H, unsubstituted and alkyl-substituted, particularly methyl-substituted, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.10 together with R.sup.3, and/or R.sup.11 together with R.sup.4, form a fused annular structure according to any one of the following substructures:
##STR00117## [0388] f. R.sup.9 together with R.sup.2, and R.sup.10 together with R.sup.3, and/or R.sup.12 together with R.sup.5, and R.sup.11 together with R.sup.4, form a fused biannular structure according to any one of the following substructures:
##STR00118##
8. The compound according to any of the preceding embodiments, wherein R.sup.1 is structurally identical to the substituent CR.sup.6CR.sup.7R.sup.8, in particular when the substituents R.sup.2 and R.sup.5 are structurally identical, and/or the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 are structurally identical, and/or the substituents R.sup.3 and R.sup.4 are structurally identical.
9. The compound according to any of the preceding embodiments, wherein: [0389] R.sup.1 is H, and/or [0390] R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently selected from H, halogen, CN, and/or [0391] R.sup.9, R.sup.10, R.sup.11 and R.sup.12 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.9 together with R.sup.10 together with the N atom to which they are bound, and R.sup.11 together with R.sup.12 together with the N atom to which they are bound form an unsubstituted or methyl-, hydroxy-, methoxy-, or halogen-substituted aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine-S,S-dioxide, and/or [0392] R.sup.13, R.sup.14, R.sup.15, if present, are selected from methyl, ethyl, isopropyl or phenyl, [0393] R.sup.16, R.sup.17, if present, are methyl, [0394] one of the substituents R.sup.6, R.sup.7, R.sup.8 and R.sup.20, R.sup.21, if present, is selected from a) 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; or b) -L.sup.A1.sub.m-L.sup.J1.sub.m-L.sup.A2.sub.n-L.sup.J2.sub.n-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, L.sup.A4, L.sup.J1, L.sup.J2, L.sup.J3, L.sup.J4 m, m, n, n, p, p, q, q, s and M.sub.s have the definitions recited above in embodiment 5, and [0395] the other substituents R.sup.6, R.sup.7, R.sup.8 and R.sup.20, R.sup.21, if present, are selected from H or methyl.
10. The compound according to any one of the preceding embodiments, wherein the substituents NR.sup.9R.sup.10 and/or NR.sup.11R.sup.12 are independently represented by one of the following structures, particularly when the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 are structurally identical:
##STR00119##
11. The compound according to any one of the preceding embodiments, wherein the fragment [0396] CR.sup.6CR.sup.7R.sup.8 is represented by one of the following structures:
##STR00120## ##STR00121##
12. The compound according to any one of the preceding embodiments, wherein the group YWR.sup.13 is represented by one of the following structures:
##STR00122##
13. The compound according to any one of the preceding embodiments, wherein the group X is represented by one of the following structures:
##STR00123##
14. The compound according to any one of embodiments 1-4 which is selected from the group of compounds below:
##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128##