Preparation of metal-pyridine derivative complexes for use in medical imaging

Abstract

The present invention relates to methods of forming metal-pyridine derivative complexes using pericyclic reactions with metal-1,2,4-triazine derivative complexes and a dienophile as the reactants. The reactants are bioorthogonal and the methods are particularly useful in preparing imaging agents.

Claims

1. A method of forming a pyridine; the method comprising: contacting a compound comprising a 1,2,4-triazine with a compound comprising a dienophile to form a compound comprising a pyridine, wherein the 1,2,4-triazine is complexed to a metal.

2. A method of claim 1, wherein the compound comprising the 1,2,4-triazine is a compound of formula (I): ##STR00052## wherein M is the metal, which may be bonded to other ligands; R.sup.1, R.sup.2 and R.sup.3 are each independently selected from H, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6 alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sub.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, C(O)NR.sup.4R.sup.4, S(O)R.sup.4, S(O).sub.2R.sup.4, C.sub.3-C.sub.6-cycloalkyl, 3-18-membered heterocycloalkyl, 5-, 6-, 9-, 10- or 14-membered heteroaryl, phenyl, naphthyl, pyrenyl and fluorophore; or R.sup.1 and R.sup.3, together with the atom to which they are attached, form a monocyclic or polycyclic aryl or heteroaryl; wherein if any of R.sup.1, R.sup.2 and R.sup.3 are C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.6-cycloalkyl, 3-6-membered heterocycloalkyl, 5-, 6-, 9-, 10- or 14-membered heteroaryl, phenyl, naphthyl or pyrenyl, that group can be substituted with from 1 to 5 R.sup.6 groups; R.sup.4 is independently at each occurrence selected from H and C.sub.1-C.sub.4-alkyl; and R.sup.5 is independently at each occurrence selected from H, C.sub.1-C.sub.4-alkyl, C(O)—C.sub.1-C.sub.4-alkyl and S(O).sub.2—C.sub.1-C.sub.4-alkyl; R.sup.6 is selected from 3-18-membered heterocycloalkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, fluorophore, phenyl, naphthyl, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sup.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, N((CH.sub.2).sub.nCO.sub.2R.sub.4).sub.2, C(O)NR.sup.4R.sup.4, S(O)R.sup.4 and S(O).sub.2R.sup.4; wherein if R.sup.6 is 3-18-membered heterocycloalkyl, C.sub.1-C.sub.6-alkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, phenyl, or naphthyl, R.sup.6 can be substituted with from 1 to 5 R.sup.7 groups; R.sup.7 is selected from 3-18-membered heterocycloalkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, fluorophore, phenyl, naphthyl, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sup.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, N((CH.sub.2).sub.nCO.sub.2R.sub.4).sub.2, C(O)NR.sup.4R.sup.4, S(O)R.sup.4 and S(O).sub.2R.sup.4; wherein if R.sup.7 is 3-18-membered heterocycloalkyl, C.sub.1-C.sub.6-alkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, phenyl, or naphthyl then R.sup.7 can be substituted with from 1 to 5 R.sup.8 groups; R.sup.8 is selected from C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sup.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, C(O)NR.sup.4R.sup.4, S(O)R.sup.4 and S(O).sub.2R.sup.4; wherein the compound of formula (I) optionally comprises a biological targeting moiety that is attached via one of the alkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, C.sub.3-C.sub.6-cycloalkyl, 3-6-membered heterocycloalkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, phenyl, naphthyl groups of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 or R.sup.8; n is independently at each occurrence an integer selected from 0, 1 and 2; where any of R.sup.6, R.sup.7 or R.sup.8 are phenyl, said phenyl group may be bonded to the metal; where any of R.sup.2, R.sup.3, R.sup.6, R.sup.7 or R.sup.8 are SR.sup.4, NR.sup.4R.sup.5, (CH.sub.2).sub.nCOOR.sup.4, OR.sup.4 or N((CH.sub.2).sub.nCO.sub.2R.sub.4).sub.2, R.sup.4 may be absent and the S, N or O may be bonded to the metal.

3. A method of claim 2, wherein R.sup.3 is a 5-, 6-, 9-, 10- or 14-membered heteroaryl group.

4. A method of claim 2, wherein R.sup.3 is a 2-pyridyl group.

5. A method of claim 2, wherein R.sup.2 is H.

6. A method of claim 2, wherein R.sup.1 is H.

7. The method of claim 2, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.6 and R.sup.7 are selected such that the compound comprising the 1,2,4-triazine further comprises a fluorophore.

8. The method of claim 1, wherein the dienophile is a C.sub.3-C.sub.10-cycloalkene or C.sub.7-C.sub.10-cycloalkyne.

9. The method of claim 1, wherein the metal is a transition metal, actinide or lanthanide.

10. The method of claim 1, wherein the metal is selected from Ru, Ir, Eu, Gd, Tb, Pt, Re, Tc, and Zn.

11. The method of claim 1, wherein the metal is complexed to one or more ligands other than the 1,2,4-triazine.

12. The method of claim 1, wherein the compound comprising the 1,2,4-triazine further comprises a biological targeting moiety.

13. The method of claim 12, wherein the method is a method of medical imaging or imaging a biological sample and wherein the compound comprising the 1,2,4-triazine further comprises a biological targeting moiety and the method comprises: administering the compound comprising the 1,2,4-triazine to a subject or a biological sample; and administering the compound comprising the dienophile to the subject or a biological sample, in order to contact the compound comprising the dienophile with the compound comprising the 1,2,4-triazine to form the compound comprising the pyridine; and detecting the image.

14. The method of claim 1, wherein the compound comprising the dienophile further comprises a biological targeting moiety.

15. The method of claim 14, wherein the method is a method of medical imaging or imaging a biological sample and wherein the compound comprising the dienophile further comprises a biological targeting moiety and the method comprises: administering the compound comprising the dienophile to a subject or a biological sample; and administering the compound comprising the 1,2,4-triazine to the subject or a biological sample, in order to contact the compound comprising the 1,2,4-triazine with the compound comprising the dienophile to form the compound comprising the pyridine; and detecting the image.

16. A kit for medical imaging or for imaging a biological sample, the kit comprising: a compound comprising a 1,2,4-triazine; and a compound comprising a dienophile; wherein the 1,2,4-triazine is complexed to a metal; and wherein either: A) the compound comprising the 1,2,4-triazine further comprises a biological targeting moiety; or B) the compound comprising the dienophile further comprises a biological targeting moiety.

17. The kit of claim 16, wherein the compound comprising the 1,2,4-triazine is a compound of formula (I): ##STR00053## wherein M is the metal, which may be bonded to other ligands; R.sup.1, R.sup.2 and R.sup.3 are each independently selected from H, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6 alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sub.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, C(O)NR.sup.4R.sup.4, S(O)R.sup.4, S(O).sub.2R.sup.4, C.sub.3-C.sub.6-cycloalkyl, 3-18-membered heterocycloalkyl, 5-, 6-, 9-,10 or 14-membered heteroaryl, phenyl, naphthyl, pyrenyl and fluorophore; or R.sup.1 and R.sup.3, together with the atom to which they are attached, form a monocyclic or polycyclic aryl or heteroaryl; wherein if any of R.sup.1, R.sup.2 and R.sup.3 are C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.6-cycloalkyl, 3-6-membered heterocycloalkyl, 5-, 6-, 9-, 10- or 14-membered heteroaryl, phenyl, naphthyl or pyrenyl that group can be substituted with from 1 to 5 R.sup.6 groups; R.sup.4 is independently at each occurrence selected from H and C.sub.1-C.sub.4-alkyl; and R.sup.5 is independently at each occurrence selected from H, C.sub.1-C.sub.4-alkyl, C(O)—C.sub.1-C.sub.4-alkyl and S(O).sub.2—C.sub.1-C.sub.4-alkyl; R.sup.6 is selected from 3-18-membered heterocycloalkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, fluorophore, phenyl, naphthyl, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sup.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, N((CH.sub.2).sub.nCO.sub.2R.sub.4).sub.2, C(O)NR.sup.4R.sup.4, S(O)R.sup.4 and S(O).sub.2R.sup.4; wherein if R.sup.6 is 3-18-membered heterocycloalkyl, C.sub.1-C.sub.6-alkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, phenyl, or naphthyl, R.sup.6 can be substituted with from 1 to 5 R.sup.7 groups; R.sup.7 is selected from 3-18-membered heterocycloalkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, fluorophore, phenyl, naphthyl, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sup.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, N((CH.sub.2).sub.nCO.sub.2R.sub.4).sub.2, C(O)NR.sup.4R.sup.4, S(O)R.sup.4 and S(O).sub.2R.sup.4; wherein if R.sup.7 is 3-18-membered heterocycloalkyl, C.sub.1-C.sub.6-alkyl, 5-, 6-, 9-,10- or 14-membered heteroaryl, phenyl, or naphthyl then R.sup.7 can be substituted with from 1 to 5 R.sup.8 groups; R.sup.8 is selected from C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, halo, nitro, cyano, C(O)R.sup.4, (CH.sub.2).sub.nCO.sub.2R.sup.4, OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, C(O)NR.sup.4R.sup.4, S(O)R.sup.4 and S(O).sub.2R.sup.4; wherein the compound of formula (I) optionally comprises a biological targeting moiety that is attached via one of the alkyl, C.sub.3-C.sub.6-alkenyl, C.sub.1-C.sub.6-alkynyl, C.sub.3-C.sub.6-cycloalkyl, 3-6-membered heterocycloalkyl, 5-, 6-, 9-, 10- or 14-membered heteroaryl, phenyl, naphthyl groups of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 or R.sup.8; n is independently at each occurrence an integer selected from 0, 1 and 2; where any of R.sup.6, R.sup.7 or R.sup.8 are phenyl, said phenyl group may be bonded to the metal; where any of R.sup.2, R.sup.3, R.sup.6, R.sup.7 or R.sup.8 are SR.sup.4, NR.sup.4R.sup.5, (CH.sub.2).sub.nCOOR.sup.4, OR.sup.4 or N((CH.sub.2).sub.nCO.sub.2R.sub.4).sub.2, R.sup.4 may be absent and the S, N or O may be bonded to the metal.

18. A kit of claim 17, wherein R.sup.3 is a 5-, 6-, 9-, 10- or 14-membered heteroaryl group.

19. A kit of claim 17, wherein R.sup.3 is a 2-pyridyl group.

20. A kit of claim 17, wherein R.sup.2 is H.

21. A kit of claim 17, wherein R.sup.1 is H.

22. A kit of claim 17, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.6 and R.sup.7 are selected such that the compound comprising the 1,2,4-triazine further comprises a fluorophore.

23. A kit of claim 16, wherein the dienophile is a C.sub.3-C.sub.10-cycloalkene or C.sub.5-C.sub.10-cycloalkyne.

24. A kit of claim 16, wherein the metal is a transition metal, actinide or lanthanide.

25. A kit of claim 16, wherein the metal is selected from Ru, Ir, Eu, Gd, Tb, Pt, Re, Tc, and Zn.

26. A kit of claim 16, wherein the metal is complexed to one or more ligands other than the 1,2,4-triazine.

27. A kit of claim 16 wherein the compound comprising the 1,2,4-triazine further comprises a biological targeting moiety.

28. A kit of claim 16, wherein the compound comprising the dienophile further comprises a biological targeting moiety.

Description

EXAMPLES

5-(furan-2-yl)-3-(pyridine-2-yl)-1,2,4-triazine (DVS-35)

(1) ##STR00028##

(2) A mixture of 2-(furan-2-yl)-2-oxoacetaldehyde (15 mmol) and (Z)-picolinohydrazonamide (2.0 g, 15 mmol) in ethanol (100 mL) was heated under reflux for 3 hours. All volatiles were removed by rotary evaporation under reduced pressure. The residue was then triturated with water and the formed solid was filtered off, washed with water and dried. Yield 2.3 g (70%). The product was used without further purification. M.p. 92° C. .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 9.56 (s, 1H), 8.89 (d, 1H, J=4.7 Hz), 8.63 (t, 1H, J=8.3 Hz), 7.97-7.87 (m, 1H), 7.78-7.70 (m, 1H), 7.67 (d, 1H, J=3.5 Hz), 7.53-7.43 (m, 1H), 6.74-6.64 (m, 1H).

3-(pyridin-2-yl)-1,2,4-triazine-5-carboxylic acid (DVS-42)

(3) ##STR00029##

(4) The triazine DVS-35 (4.88 g, 21.7 mmol) was dissolved in pyridine (20 mL) and then water (200 mL) was added. To this solution, at stirring at room temperature, potassium permanganate (13.8 g, 87 mmol) was added in small (0.5 g) portions over a period of 2 hours. The temperature of the reaction mixture was kept below 55° C. The mixture was filtered and the filtrate was evaporated to dryness. The product was then recrystallized from a small amount of water and dried at room temperature overnight. (It should be noted that the filtrate contains a big amount of the product). Yield 0.97 g (19%). .sup.1H NMR (D.sub.2O, 400 MHz): δ 9.55 (s, 1H), 8.62 (d, 1H, J=4.6 Hz), 8.39 (d, 1H, J=7.8 Hz), 7.98 (t, 1H, J=7.8, 7.2 Hz), 7.54 (t, 1H, J=4.6, 7.2 Hz); IR, v, cm.sup.−1: 1627 (C═O).

VNK-246

(5) ##STR00030##

(6) DVS-35 (1.25 g, 5.6 mmol) was dissolved in a mixture of water/pyridine (10/1v/v) at 50° C. At this solution, at stirring, potassium permanganate (3.7 g, 23.4 mmol, 4.18 eq.) was added in portions of approx. 0.3 g over a period of 30 min. The mixture was then stirred at 50-60° C. for 30 min and filtered while still hot. The dark solid from the filter (MnO.sub.2) was placed in a beaker and water (100 mL) was added. The suspension was stirred and heated to 90° C. and then filtered. All filtrates were combined and all volatiles were removed by rotary evaporation under reduced pressure. The residue was dissolved in 20 mL of water and 3M HCl was added dropwise (gas evolution) till slightly acidic pH. After that, a solution of zinc sulphate in water (excess) was added, the mixture was heated to 70° C. and then left overnight. Precipitated solid was filtered off, washed with water (3×10 mL) and acetone (3×5 mL) to give VNK-246 as yellow solid. Yield 570 mg (44%). .sup.1H NMR (DMSO-D.sub.6, 400 MHz): δ.sub.H 10.21 (s, 1H), 8.89 (d, 1H, J=7.6 Hz), 8.42 (d, 1H, J=4.6 Hz), 8.27 (t, 1H, J=7.2, 8.0 Hz), 7.68 (t, 1H, J=4.6, 7.2 Hz).

(7) ##STR00031##

(8) A solution of terbium chloride (1 eq) or europium chloride in water was added to a solution of the starting potassium salt of the triazine ligand (3 eq) in water. The mixture was stirred at 55° C. for 2 days. The mixture was allowed to cool to room temperature and the solid was filtered off to give the target complex.

Preparation of VNK-405, VNK-406, VNK-408, VNK-421, VNK-423, and VNK-548

(9) ##STR00032##

VNK-405

(10) To a solution of 2-oxo-2-(pyridin-2-yl)acetaldehyde (100 mmol) in a mixture of DMSO (100 mL) and water (200 mL), potassium carbonate (200 mmol) and thiosemicarbazide (9.1 g, 100 mmol) were added and the reaction mixture was stirred at RT for 30 minutes. The mixture was then warmed up to 50-60° C. and stirred at this temperature for 1 hour. The mixture was then neutralised by addition of an excess of acetic acid casing a formation of the solid. The solid was filtered off, washed with water and dried in oven. Yield 11.3 g (59%). .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ0 9.08 (s, 1H), 8.81 (br.d, J=4.6 Hz, 1H), 8.34 (d, J=7.7 Hz, 1H), 8.06 (br.t, J=7.7 Hz, 1H) 7.68 (dd, J=7.7, 4.6 Hz, 1 H).

VNK-406

(11) A mixture of VNK-405 (11.1 g, 58.4 mmol) and hydrazine hydrate (14.6 g, 292 mmol, 5 eq) was heated under reflux for 15 minutes (solid formed). Methanol (20 mL), water (20 mL) and acetic acid (10 mL) were added. The solid was filtered off, washed with water (30 mL) and methanol (30 mL). Yield 5.96 g (54%). .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ 9.36 (s, 1H), 8.88 (br.s, 1H), 8.74 (br.d, J=4.6 Hz, 1H), 8.40 (br.s, 1H), 8.02 (ddd, J=7.8, 7.8, 1.8, 1H), 7.59 (m, 1H), 4.45 (s, 2H).

VNK-408

(12) A mixture of VNK-406 (940 mg, 5 mmol), methanol (100 mL) and sodium methoxide (2.7 g, 50 mmol) was stirred at 75° C. (bath temperature) for 14 hours. Acetic acid (5 mL) and water (20 mL) were added and the mixture was evaporated to a volume of approximately 15 mL. The mixture was basified by addition of 33% aqueous ammonia (5 mL). Precipitated solid was filtered off, washed with water (20 mL) and dried. Yield 526 mg (67%). .sup.1H NMR (400 Hz, CDCl.sub.3): δ 10.30 (d, J=1.8 Hz 1H), 9.71 (d, J=1.8 Hz 1H), 8.79 (br.d, J=4.6 Hz, 1H), 8.54 (d, J=7.8 Hz, 1H), 7.92 (ddd, J=7.8, 7.8, 1.8, 1H), 7.49 (dd, J=7.4, 4.6 Hz, 1 H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ 157.3, 154.0, 151.7, 150.1, 147.4, 137.5, 126.7, 122.9.

VNK-421 Chloride

(13) A mixture of [(tpy).sub.2Ir(μ-Cl)].sub.2 VNK-411 (226 mg, 0.4 mmol for Ir), VNK-408 (90 mg, 0.6 mmol, 1.5 eq), chloroform (10 mL) and methanol (10 mL) was heated under reflux for 2 hours. The solvent was evaporated. The product was purified by column chromatography using silica gel as a stationary phase and a mixture of DCM/methanol mixture 5/1, v/v as an eluent. A green fraction was collected. The solvent was evaporated and the residue was dissolved in DCM (2 mL). To this solution, ether (20 mL) was added causing the formation of a solid. The solid was filtered off, washed with ether to give VNK-421RR chloride. Yield 202 mg (70%). .sup.13C NMR (100 MHz, CDCl.sub.3): δ 167.7, 167.2, 155.3, 154.9, 151.7, 151.3, 149.7, 149.6, 149.5, 148.8, 146.9, 141.6, 141.4, 141.1, 140.6, 140.5, 138.7, 138.6, 132.6, 132.1, 131.3, 125.1, 124.9, 124.7, 124.6, 123.5, 123.4, 119.7, 119.5, 22.0

VNK-527

(14) A mixture of [Ir(pqi)2]Cl2 VNK-525 (221 mg, 0.35 mmol for Ir) and VNK-502B (64 mg, 0.41 mmol, 1.15 eq) in chloroform (10 mL) and methanol (10 mL) was heated under reflux for 14 hours. The solvent was evaporated by rotary evaporation to a volume of approximately 2 mL. Diethyl ether (25 mL) was added causing formation of a solid. The solid was filtered off, washed with ether. The product was purified by column chromatography using silica gel as stationary phase and a mixture of DCM/MeOH 20/1 as a mobile phase. Yield 75 mg (27%).

VNK-423

(15) A mixture of bis(1,10-phenanthroline)dichlororuthenium(II) (212 mg, 0.4 mmol), VNK-408 (80 mg, 0.51 mmol, 1.27 eq) in 6 mL of water was heated in a microwave oven at 120° C. (150 W max.), for 70 min. The aqueous mixture was washed with DCM in a separating funnel (3×15 ml). To the aqueous layer, saturated solution of NaPF.sub.6 (6 mL) was added causing the formation of amorphous precipitate. DCM (50 mL) was added, but it does not dissolve the solid completely. The DCM and organic layers were drained. The residue was treated with water and mechanically removed from the sides of the separating funnel. The solid was filtered off, washed with water and little amount of ethanol. Yield 84 mg (17%). .sup.1H NMR (400 Hz, acetine-D.sub.6): δ 10.48 (br.s, 1H), 9.35 (br.s, 1H), 9.21 (d, J=8.0 Hz, 1H), 8.87 (d, J=8.0 Hz, 2H), 8.71-8.81 (m, 4H), 8.18-8.43 (m, 10H), 7.91-7.98 (m, 2H), 7.71-7.80 (m, 2 H), 7.65 (t, J=6.8 Hz, 1H).

VNK-548

(16) To a solution of the triazine VNK-408 (158 mg, 1 mmol) in toluene (15 mL), Re(CO).sub.5Cl (360 mg, 1 mmol) was added and the mixture was heated under reflux for 2 hours. The mixture was allowed to cool to RT. The solid was filtered off, washed with toluene and petrol ether to give VNK-548. Yield 406 mg (87%) .sup.1H NMR (400 Hz, DMSO-D.sub.6): δ 10.70 (s, 1H), 10.22 (s, 1H), 9.20 (d, J=4.6 Hz, 1H), 8.51 (d, J=7.8 Hz, 1H), 8.26 (t, J=7.8, 1H), 7.81 (dd, J=7.4, 4.6 Hz, 1 H). .sup.13C NMR (100 MHz, DMSO-D.sub.6): δ 197.7, 196.4, 188.2, 157.8, 155.1, 154.7, 152.2, 149.0, 141.1, 131.5, 128.2.

Preparation of VNK-479, VNK-480, VNK-481, VNK-482, VNK-483R, VNK-514, and VNK-515

(17) ##STR00033## ##STR00034##

3-mercapto-5-(6-methylpyrinin-2-yl)-1,2,4-triazine VNK-479

(18) To a solution of the dicarbonyl XX (74 mmol) in DMSO (74 mL) and water (150 mL), thiosemicarbazide (6.73 g, 74 mmol) and Na.sub.2CO.sub.3 (7.7 g, 74 mmol) were added and the mixture was stirred at RT for 10 minutes. The mixture was then warmed up to 50-60° C. and stirred at this temperature for 1 hour. The mixture was then filtered and the filtrate was neutralised by careful addition of acetic acid. The formed red solid was filtered off, washed with water and dried at room temperature for 14 hours and then at 100° C. for 30 min. Yield 7.6 g (50%). This product was used without further purification. Small amount of the product was recrystallised from ethanol to prepare analytically pure sample. .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ 9.06 (s, 1H), 8.14 (d, J=7.7 Hz, 1H), 7.93 (t, J=7.7 Hz, 1H), 7.54 (d, J=7.7, 1 H), 2.57 (s, 3H). .sup.13C NMR (400 MHz, DMSO-D.sub.6): δ 181.6, 159.3, 156.8, 150.2, 138.7, 135.7, 127.8, 121.3, 24.6.

3-hydrazino-5-(6-methylpyrinin-2-yl)-1,2,4-triazine VNK-480

(19) To a suspension of VNK-479 (6.12 g, 30 mmol) in ethanol (30 mL), hydrazine hydrate (3 g, 60 mmol, 2 eq) was added. The mixture was heated under reflux for 1 hour. More of hydrazine (2.3 g, 46 mmol) was added. Ethanol was distilled off, the temperature of the reaction mixture was around 100° C. Water (30 mL) was added. The formed solid was filtered off and washed with water. Yield 2.6 g (42%). .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ 9.34 (s, 1H), 8.87 (br.s, 1H, NH), 8.21 (br. d, J=7.7 Hz, 1H), 7.90 (t, J=7.7 Hz, 1H), 7.44 (d, J=7.7, 1 H), 4.56 (br.s, 2H, NH.sub.2), 2.55 (s, 3H). .sup.13C NMR (400 MHz, DMSO-D.sub.6): δ 164.0, 158.9, 154.0, 151.9, 138.4, 137.6, 126.6, 120.0, 24.6.

5-(6-methylpyrinin-2-yl)-1,2,4-triazine VNK-481

(20) A mixture of VNK-480 (1020 mg, 5 mmol), methanol (100 mL) and sodium methoxide (2.7 g, 50 mmol) was stirred at 75° C. (bath temperature) for 14 hours. Acetic acid (5 mL) and water (100 mL) were added and the mixture was extracted with DCM (3×30 mL). Organic layer was washed with saturated solution of sodium bicarbonate, dried over MgSO.sub.4. This solution (approx. 90 mL) was applied to a column of silica gel (50 g). The column was then eluted with a DCM/ethyl acetate mixture (5/1, v/v) collecting the first yellow fraction. The solvent was removed by rotary evaporation, the residue was suspended in petrol ether (5 mL) and filtered to give after drying the desired product. Yield 440 mg (51%). .sup.1H NMR (400 Hz, CDCl.sub.3): δ 10.29 (d, J=2.0 Hz, 1H), 9.68 (d, J=2.0 Hz, 1H), 8.31 (d, J=7.7 Hz, 1H), 7.77 (t, J=7.7 Hz, 1H), 7.33 (d, J=7.7 Hz, 1H), 2.66 (s, 3H). .sup.13C NMR (400 MHz, CDCl.sub.3): δ 159.2, 157.3, 154.2, 151.0, 147.5, 137.6, 126.4, 120.0, 24.6.

5-(6-(Bromomethyl)pyridin-2-yl)-1,2,4-triazine VNK-482

(21) N-Bromosuccinimide (161 mg, 0.91 mmol) and AIBN (17 mg, 0.1 mmol) were added to a solution of VNK-481 (172 mg, 1 mmol) in CCl.sub.4 (15 mL) under argon. The reaction mixture was stirred under reflux and irradiation with a (20 W) halogen lamp for 2 hours. The mixture was then applied to a column of dry silica gel and eluded first with DCM, followed by a mixture of DCM/ethyl acetate 5/1 v/v. The first yellow fraction contained the product of dibromination. The second yellow fraction was the desired product. Yield 61 mg (24%). The third fraction is unreacted starting material. .sup.1H NMR (400 Hz, CDCl.sub.3): δ 10.32 (d, J=2.0 Hz, 1H), 9.72 (d, J=2.0 Hz, 1H), 8.46 (d, J=7.7 Hz, 1H), 7.93 (t, J=7.7 Hz, 1H), 7.65 (d, J=7.7 Hz, 1H), 4.65 (s, 2H). .sup.13C NMR (400 MHz, CDCl.sub.3): δ 157.6, 157.3, 153.5, 151.5, 147.5, 138.7, 126.5, 122.1, 33.2.

VNK-483R

(22) ##STR00035##

(23) A mixture of VNK-482 (200 mg, 0.8 mmol) and tri-tert-butyl ester (475 mg, 0.8 mmol), acetonitrile (50 mL) and potassium carbonate (552 mg, 4 mmol) heated under reflux for 14 hours. The mixture was filtered by suction filtration. The filtrate was applied to a short (10 cm, diameter 2.5 cm) column of silica gel, without evaporation of the solvent. The column was firstly eluted with acetonitrile (50 mL) followed by a mixture of acetonitrile/MeOH, 5/1, v/v. The yellow fraction was collected and evaporated to dryness to give VNK-483R (250 mg) as an amorphous solid. The product was used in the next step without further purification.

VNK-514

(24) ##STR00036##

(25) VNK-483R (250 mg) was dissolved in 10 mL of 6M HCl. The solution was stirred overnight and then evaporated to dryness. The residue was triturated with acetone and the solid was filtered off, washed with diethyl ether to give VNK-514. .sup.1H NMR (400 Hz, D2O): δ 10.27 (d, J=2.0 Hz, 1H), 9.91 (d, J=2.0 Hz, 1H), 8.64 (d, J=7.7 Hz, 1H), 8.30 (t, J=7.7 Hz, 1H), 7.99 (d, J=7.7 Hz, 1H), 4.93-5.01 (m, 8H), 3.1-3.9 (m, 16H).

VNK-516

(26) ##STR00037##

(27) VNK-514 (81 mg, 0.1 mmol) was dissolved in 7 mL of methanol. 0.4 mL of 0.34 M solution of LaCl.sub.3*7H.sub.2O (0.136 mmol) in methanol was added, followed by 0.4 mL (0.726 g/mL, 2.88 mmol) of triethylamine. The mixture was heated under reflux for 4 hours, after that evaporated to dryness. 2-Propanol (7 mL) was added and the suspension was heated under reflux for 10 minutes and then filtered (quite slow filtration, very fine precipitate). The solid on filter was washed with 2-propanol and dried in oven at 100° C. for 10 min. Yield 56 mg. (86%)

VNK-517

(28) VNK-514 (130 mg, 0.16 mmol) was dissolved in 10 ml of methanol. 0.4 mL of 0.38 M solution of TbCl.sub.3*6H.sub.2O (0.18 mmol) in methanol was added, followed by 0.22 mL (0.726 g/mL, 1.6 mmol) of TEA. The mixture was heated under reflux for 24 hours, after that evaporated to dryness to give the final complex.

VNK-609

(29) ##STR00038##

(30) RED-283 (1.675 g, 6.6 mmol) was dissolved in dioxane (25 mL). Selenium dioxide (0.735 g, 6.6 mmol) was dissolved in a mixture of dioxane (5 ml) and water (0.2 mL) at heating. The SeO.sub.2 solution was added to the solution of RED-283 and the mixture was heated under reflux for 24 hours. The mixture was filtered while boiling hot. The filtrate was evaporated to dryness to give yellow oil. This product was used in the next step without any further purification.

(31) The crude product from previous step was dissolved in ethanol (10 mL). Thiosemicarbazide (590 mg, 6.6 mmol) was added, followed by water (15 mL) and K.sub.2CO.sub.3 (1.38 g, 10 mmol, 1.3 eq). The mixture was stirred at RT for 5 minutes. Ethanol was evaporated by rotary evaporation (bath temperature 94° C.). The aqueous solution was stirred for 15 minutes at 94° C., then allowed to cool to RT. The mixture was transferred into a beaker (250 mL) and acetic acid (6 mL) was added causing formation of a solid. The solid was filtered off, washed with water to give VNK-601. This product was used in the next step without any further purification.

(32) A mixture of crude mercaptotriazine VNK-601 from previous step, ethanol (25 mL) and hydrazine (0.63 mL, approx. 3 eq) was heated under reflux for 14 hours. Water was added and the mixture was extracted with DCM. Organic layer was dried over MgSO.sub.4, filtered and evaporated to dryness to give VNK-608 crude. This product was used in the next step without any further purification.

(33) A mixture of VNK-608 crude, methanol (100 mL) and sodium methoxide (2.48 g, 50 mmol) was stirred at 75° C. (bath temperature) for 14 hours. Acetic acid (5 mL) was added and the mixture was evaporated to a volume of approximately 5 mL. Water and conc. ammonia (2 mL) were added causing formation of an oily precipitate. Water was decanted. The residue was dried on rotary evaporator under vacuum at 90° C. The product VNK-609 was purified by using Isolera machine (25 g cartridge and a mixture of DCM (70%) and EA (30%)). Yield 270 mg (14% overall after 4 steps). .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ 9.81 (d, J=2.4 Hz, 1H), 9.69 (d, J=2.4 Hz, 1H), 8.75 (m, 1H), 8.59 (t, J=1.6 Hz, 1H), 8.28 (m, 1H), 7.82 (m, 1H), 7.31 (m, 1H), 1.44 (s, 9H).

VNK-612

(34) ##STR00039##

(35) The starting ligand VNK-609 (170 mg, 0.586 mmol) was dissolved in acetic acid (25 mL). To this solution, potassium tetrachloroplatinate (240 mg, 0.58 mmol) was added and the mixture was heated under reflux under argon atmosphere for 18 hours. The mixture was allowed to cool to RT and filtered. The dark solid on the filter was washed with methanol, water and again methanol. (234 mg). NMR showed mixture of products. The solid was placed into a test tube, 4 mL of DMSO was added and the mixture was heated until boiling begins. After that the mixture was allowed to cool to RT, methanol (8 mL) was added and the mixture was filtered to give red solid on filter. Yield 75 mg (25%).

VNK-614

(36) ##STR00040##

(37) In an NMR tube VNK-612 (35 mg, 0.067 mmol) was suspended in DMSO-D.sub.6 (0.6 mL). BCN-C10 (27 mg, 0.08 mmol, 1.2 eq) was added. The mixture was heated to boiling point. Evaluation of nitrogen was apparent. NMR showed 100% conversion to the product. .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ 9.35 (d, J=5.2 Hz Pt satellites, 1H), 8.91 (s, Pt satellites, 1H), 7.92 (br.t, J=7.2 Hz, 1H), 7.70 9br.d, J=7.2 Hz, 1H), 7.47 (s, 2H), 7.41 (s, Pr satellites, 1H), 7.25 (m, 1H), 4.6 (br.s, 1H), 3.82 (m, 2H), 2.5-3.2 (m, 8H), 1.27 (m, 16H), 0.8 (m, 12H).

VNK-450

(38) ##STR00041##

(39) A mixture of the hydrazine (24.2 g, 100 mmol) and triethylorthoformyate (18 mL, 16 g, 108 mmol) was heated until all the starting material dissolved. Catalytic amount of para toluene sulphonic acid was added and the mixture was heated under reflux for 1 hour (during this time quite a lot of precipitate formed). The mixture was allowed to cool to RT and the solid was filtered off, washed with methanol and dried. Yield 13 g (52%).

VNK-457

(40) ##STR00042##

(41) 1,2,4-triazine-4-oxide (5.04 g, 20 mmol) was suspended in DCM (40 mL). Trimethylsilyl cyanide (2.4 g, 24 mmol, 1.2 eq) was added, followed by trimethylamine (2.4 g, 24 mmol). The mixture was heated under reflux for 15 minutes and then filtered through a pad of silica gel collecting yellow fraction. The solvent was evaporated to give a pure product. Yield 2.2 g (42%).

VNK-458

(42) ##STR00043##

(43) 5-cyano-1,2,4-triazine (2.1 g, 8 mmol) was dissolved in DMF (10 mL). To this solution, hydrazine hydrate (1.33 g, 26.6, 3.3 eq) was added. The mixture was stirred at RT for 15 minutes then at 60-70° C. for 10 minutes. The mixture was allowed to cool to RT and water 50 mL was added. Precipitated solid was filtered off, washed with water and dried to give VNK-458 as yellow solid. Yield 2.0 g (94%).

VNK-459

(44) ##STR00044##

(45) To a stirred suspension 3-hydrazino-1,2,4-triazine (517 mg, 1.94 mmol) in ethanol (15 mL) acetylacetone (213 mg, 2.13 mmol) was added. The mixture was heated under reflux for 30 minutes. The mixture was allowed to cool to RT and the solid was filtered off, washed with ethanol to give VNK-459. Yield 527 mg (82%).

VNK-461

(46) ##STR00045##

VNK-421 Chloride

(47) A mixture of [Ir(tpy)2]Cl2 VNK-411 (226 mg, 0.4 mmol for Ir), VNK-459 (145 mg, 0.44 mmol, 1.1 eq), chloroform (10 mL) and methanol (10 mL) was heated under reflux for 2 hours. The solvent was evaporated to a volume of approximately 2 mL. Diethyl ether (10 mL) was added. Precipitated solid was filtered off, washed with ether to give VNK-461 (230 mg). (64%)

VNK-234

(48) ##STR00046##

(49) Sodium (600 mg, 26 mmol, 6 eq.) was dissolved in methanol (50 mL). To this solution, 3-hydrazino-1,2,4-triazine (800 mg, 4.27 mmol) was added (dark solution formed immediately) and the mixture has stirred at 80° C. for 15 hours. Excess of acetic acid (5-8 mL) was added and the mixture was evaporated. Water (50 mL) was added followed by a little amount of ammonia solution (to neutralise remaining AcOH). Precipitated solid was filtered off, washed with water to give the crude product. After drying the solid was purified by column chromatography (silica gel, ethylacetate). Yield 330 mg (49%).

VNK-472

(50) ##STR00047##

(51) A mixture of the hydrazine triazine (2 g, 10.4 mmol), methanol (100 mL) and sodium methoxide (5.9 g, 109 mmol) was stirred at 75° C. (bath temperature) for 14 hours. Acetic acid (5 mL) and water (20 mL) were added and the mixture was evaporated to a volume of approximately 15 mL. The mixture was basified by addition of 33% aqueous ammonia (5 mL). Precipitated solid was filtered off, washed with water (20 mL) and dried. The product was then purified by column chromatography.

VNK-194

(52) ##STR00048##

(53) 3-Hydrazino-5-(4-methoxyphenyl)-1,2,4-triazine (8.8 g, 40 mmol) was dissolved in acetic acid (220 mL) at slight heating, the mixture was allowed to cool to RT. Cu.sub.2SO.sub.4 (17.6 g) and NaOAc (17.6 g) were dissolved in water (220 mL) and this solution was added to the first one. The mixture was stirred at RT for 14 hours and then evaporated to dryness by rotary evaporation under reduced pressure. The residue was triturated with ethyl acetate (100 ml) and DCM (100 mL) and filtered. The solid on filter was washed with DCM and discarded. The filtrate was evaporated to dryness, dissolved in DCM and filtered through short pad of silica gel. The solvent was removed to give the pure product.

VNK-469

(54) ##STR00049##

(55) The diamino derivative (750 mg) was suspended in DMF (20 mL). Butyl nitrite (4 mL) was added and the mixture was heated under reflux for 14 hours. All volatiles were removed by rotary evaporation. The product was purified by column chromatography. Yield 5%.

(56) Kinetics

(57) ##STR00050##
IEDDA Reaction Kinetics

(58) The reaction between the tetrazine and triazine iridium complexes (the substrates) and the alkyne BCN were carried out in methanol at 25° C. under pseudo-first order conditions, with BCN in at least 10-fold excess. Reactions were followed by monitoring the loss of the substrate absorbance spectrophotometrically at a suitable wavelength. Observed pseudo-first-order rate constants, k.sub.obs, were obtained using nonlinear regression of the monoexponential loss of absorbance with time. Second order rate constants, k.sub.2, were obtained from linear regression of plots of k.sub.obs against BCN concentration. Pseudo-first-order and second order plots are shown below. The data is summarised in Table 1.

(59) The alkyne BCN has the following structure

(60) ##STR00051##

(61) TABLE-US-00001 TABLE 1 Second order rate constants, k.sub.2, for the reaction between triazine iridium complexes and BCN. Except where stated, reactions were carried out spectrophotometrically in methanol at 25° C. Compound k.sub.2 / M.sup.−1 s.sup.−1 VNK408 0.059 ± 0.001 VNK477 (Cl.sup.− salt) 7.87 ± 0.32 VNK477 (PF.sub.6.sup.− salt) 8.23 ± 0.31 VNK421 (Cl.sup.− salt) 7.745 ± 0.131

(62) This data demonstrates that N4-complexation of the triazine to Ir(III) centre increases the rate constant by more than two orders of magnitude.