Photoswitchable Azo- and Diazocine-Functionalized Derivatives of the VEGFR-2 Inhibitor Axitinib

In this study, we aimed at the application of the concept of photopharmacology to the approved vascular endothelial growth factor receptor (VEGFR)-2 kinase inhibitor axitinib. In a previous study, we found out that the photoisomerization of axitinib’s stilbene-like double bond is unidirectional in aqueous solution due to a competing irreversible [2+2]-cycloaddition. Therefore, we next set out to azologize axitinib by means of incorporating azobenzenes as well as diazocine moieties as photoresponsive elements. Conceptually, diazocines (bridged azobenzenes) show favorable photoswitching properties compared to standard azobenzenes because the thermodynamically stable Z-isomer usually is bioinactive, and back isomerization from the bioactive E-isomer occurs thermally. Here, we report on the development of different sulfur–diazocines and carbon–diazocines attached to the axitinib pharmacophore that allow switching the VEGFR-2 activity reversibly. For the best sulfur–diazocine, we could verify in a VEGFR-2 kinase assay that the Z-isomer is biologically inactive (IC50 >> 10,000 nM), while significant VEGFR-2 inhibition can be observed after irradiation with blue light (405 nm), resulting in an IC50 value of 214 nM. In summary, we could successfully develop reversibly photoswitchable kinase inhibitors that exhibit more than 40-fold differences in biological activities upon irradiation. Moreover, we demonstrate the potential advantage of diazocine photoswitches over standard azobenzenes.


Molecular Modeling
Molecular modeling of diazocine-functionalized axitinib derivative 7 in the ATP binding pocket of VEGFR-2 a b Supplementary figure 1. a) Calculated binding mode of C-diazocine-functionalized axitinib derivative E-7 (chair conformation) in the ATP binding pocket of VEGFR-2 (pdb: 4AG8) [29]. b) Superposition of Z-7 and VEGFR-2. While retaining the hydrogen bonds of the pharmacophore, the diazocine moiety clashes with the protein. Protein surface displayed in gray. Yellow dotted lines: hydrogen bonds; light blue dotted lines: π-π-interactions.

Synthetic route of azoaxitinib (2)
The synthesis of azoaxitinib (2) was performed once and neither reaction yields, nor the synthetic route were further optimized. The key step was the formation of the azo bond which finally succeeded in a Baeyer-Mills reaction (step iii). First, 2-fluoro-4-iodobenzonitrile (8) was cyclized to 6-iodo-1H-indazol-3-amine (9). Amine 9 was then treated with nitroso compound 11. Interestingly, we suppose the isolated product was the 2H/Z tautomer 12. Which could explain the broadness of the NH signal in the 1 H NMR spectra, as well as its poor solubility. In order to achieve higher yields in the last step, the Migita coupling should be carried out with a DHP protecting group on the azo compound 12. Instead of protection, however, the reaction iv) led to the isomerized product 13, which was indicated by a shift in the response time (tR) in HPLC analysis. However, 1 H NMR of compound 12 is quite similar to compound 13 except the NH signal tends to be sharper. A similar acidic modulation of isomers was found for azobis imidazoles [18]. This crude product was finally deployed in the Migita coupling with compound 15 to form azoaxitinib (2) in a yield of 21 %.

Synthesis of 6-iodo-1H-indazol-3-amine (9)
In a nitrogen-rinsed apparatus, 2-fluoro-4-iodobenzonitrile (8, 984 mg, 3.98 mmol) and 590 μL hydrazine monohydrate (3 eq, 12.2 mmol) were added to 25 mL of n-butanol and the reaction mixture was heated to reflux for 20 h. After cooling, 30 mL of deionized water was added, resulting in the precipitation of a white solid, which was filtered off and washed with deionized water. The organic phase was separated, and the aqueous phase was extracted three times with 30 mL of ethyl acetate. The combined organic phases were dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was recrystallized from diethyl ether and combined with the previously obtained solid.

Synthesis of 2-nitrosopyridine (11)
Pyridin-2-amine (10, 2.35 g, 25.0 mmol) was dissolved in 25 mL dichloromethane, and 2.00 mL dimethyl sulfide (27.5 mmol) was added. The reaction mixture was cooled to 0 °C and within 40 min a solution of N-chlorosuccinimide (3.33 g, 25.0 mmol) in 60 mL dichloromethane was added. After addition, the solution was stirred for 1 h at 0 °C and then for 1 h at room temperature. A solution of 1.03 g sodium methylate in 18 mL MeOH was added to the reaction mixture and stirred for 10 min, and stirred for another 2 h after the addition of 37 mL deionized water. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL dichloromethane each. The combined organic phases were washed with 50 mL deionized water, dried over sodium sulfate and the solvent was removed under reduced pressure. The isolated yellow oil was converted without further purification (crude yield: 926 mg; 6.00 mmol; 24 %). m-Chloroperbenzoic acid (1.71 g, 7.60 mmol) was dissolved in 43 mL dry dichloromethane and cooled to 0 °C. The raw product (926 mg, 6.00 mmol) was dissolved in 12 mL dichloromethane and added to the reaction mixture. After stirring for 90 min at 0 °C, 298 μL dimethyl sulfide (4.03 mmol) was added and the mixture was stirred for another 30 min. 30 mL of a saturated sodium bicarbonate solution was added and the phases were separated. The light green organic phase was washed once with 20 mL deionized water, dried over sodium sulfate, and the solvent was evaporated. No further purification took place.

Synthesis of (E)-1-(3-iodophenyl)-2-phenyldiazene (20)
Aniline (16) (1.0 g, 10.7 mmol) was dissolved in dichloromethane (50 mL) and potassium peroxomonosulfate (Oxone®) (4.9 g, 32.2 mmol) dissolved in deionized water (100 mL) was added. The reaction mixture was stirred for 6 h at room temperature and the solution turned green. The phases were separated, and the aqueous solution was added. The phase was extracted with dichloromethane (3 x 50 mL). The combined organic extracts were washed with 1 M hydrochloric acid, a saturated solution of sodium hydrogen carbonate and water (1 x 35 mL respectively) and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was absorbed in glacial acetic acid (100 mL). 3-Iodoaniline (18) (2.35 g, 10.7 mmol) was added to this solution and the reaction mixture was stirred for 24 h at room temperature. The solvent was evaporated and the residue purified by column chromatography on silica gel (120 g column, PE/EA gradient, 0 % EA → 20 % EA, 20 min, then another 20 with 20 % EA, 50 mL/min). An orange solid was obtained.

Synthesis of (E)-1-(4-iodophenyl)-2-phenyldiazene (21)
Aniline (16) (2.0 g, 10.7 mmol) was dissolved in dichloromethane (50 mL) and potassium peroxomonosulfate (Oxone®) (6.54 g, 43.0 mmol) dissolved in deionized water (100 mL) was added. The reaction mixture was stirred for 2 h at room temperature and the solution turned green. The phases were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The combined organic extracts were washed with 1 M hydrochloric acid, saturated sodium hydrogen carbonate solution and water (1 x 50 mL respectively) and dried over sodium sulfate. The solvent was evaporated, and the residue was dissolved in glacial acetic acid (250 mL). 4-Iodoaniline (19) (4.23 g, 19.3 mmol) was added to this solution and the reaction mixture was stirred at room temperature for 96 h. The solvent was removed under reduced pressure and the raw product was purified by column chromatography on silica gel (120 g column, PE/EA gradient, 0 % EA → 20 % EA, 20 min, then another 20 min 20 % EA, 50 mL/min). An orange solid was obtained.

Sulfur-diazocine derivates
Synthesis of 4-iodo-2-methyl-1-nitrobenzene (27) 3-Methyl-4-nitroaniline (25) (5.27 g, 34.6 mmol) was suspended in a mixture of deionized water (125 mL) and concentrated sulphuric acid (10.6 mL) and stirred with an overhead stirrer at the highest speed. The suspension was cooled to 0 °C and sodium nitrite (2.63 g, 38.1 mmol) dissolved in deionized water (12 mL) was added dropwise. The reaction mixture was stirred for 30 min and finally potassium iodide (8.05 g, 48.5 mmol) dissolved in deionized water (25 mL) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 16 h. The aqueous solution was extracted with ethyl acetate (3 x 100 mL) and the combined organic phases were washed with sodium thiosulfate solution (3 x 100 mL), water (1 x 100 mL) and saturated sodium chloride solution (1 x 50 mL). The organic phase was dried with sodium sulfate, the solvent was removed under reduced pressure and the residue was purified in portions by column chromatography on silica gel (120 g column, PE/EA gradient, 0 % EA → 10 % EA, 10 min, then another 10 min 10 % EA, 50 mL/min). A yellow solid was obtained.

Synthesis of 2-((4-iodo-2-nitrobenzyl)thio)aniline (31)
2,2'-Disulfanediyldianiline (30) (2.61 g, 7.64 mmol) was dissolved in dry THF (30 mL) under a nitrogen atmosphere and sodium borohydride (469 mg, 12.4 mmol) was added. The reaction mixture was heated for 4 h under reflux, whereby the solution gradually became opaque. The reaction temperature was reduced to 40 °C and then a solution of 1-(bromomethyl)-4-iodo-2-nitrobenzene (28) (2.61 g, 7.64 mmol) in THF (10 mL) was added. The solution was stirred at 40 °C for 3 h and was then added to ice water (200 mL). After no further gas evolution was observed, it was extracted with ethyl acetate (3 x 50 mL) and the organic phase was washed with saturated sodium hydrogen carbonate solution and saturated sodium chloride solution (1 x 75 mL each). The solvent was removed in vacuo and the residue purified in two portions by column chromatography on silica gel (120 g column, PE/EA gradient, 5 % EA → 40 % EA over 30 min, 50 mL/min). An orange oil was obtained.

Synthesis of (Z)-2-iodo-12H-dibenzo[b,f][1,4,5]thiadiazocine (36)
2-((5-Iodo-2-nitrobenzyl)thio)aniline (32) (917 mg, 2.37 mmol) was dissolved in ethanol (45 mL) and ammonium chloride (381 mg, 7.12 mmol) dissolved in deionized water (10 mL) was added. The reaction mixture was heated to 65 °C and zinc powder (466 mg, 7.12 mmol) was added. The progress of the reaction was continuously monitored by DC, and the reaction was stopped after 30 min due to increasing formation of the amino by-product. The reaction mixture was filtered hot and then cooled down to 0 °C. Iron(III) chloride hexahydrate (1.10 g, 4.04 mmol) dissolved in ice-cold deionized water (5 mL) was added dropwise to the cold solution. The reaction mixture was slowly brought to room temperature and the conversion of the nitroso intermediate 2-((5-iodo-2nitrosobenzyl)thio)aniline (34) was checked by DC. After 30 min glacial acetic acid (30 mL) was added and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under vacuum and diluted with dichloromethane (200 mL). The organic phase was washed with 1 M sodium hydroxide solution (3 x 70 mL) and saturated sodium chloride solution (1 x 50 mL) and dried over sodium sulfate. The solvent was evaporated, and the residue was purified by column chromatography on silica gel (120 g column, PE/EA gradient, 10 % EA → 40 % EA, 30 min, 50 mL/min). A yellow solid was obtained.
The remaining product mixture was used in the next stage without further purification. For this purpose, the mixture (3.49 g) of 1,2-bis(2-nitrophenyl)ethane (41) and 4-iodo-2-nitro-1-(2nitrophenethyl)benzene (40) was dissolved in ethyl acetate (300 mL) and tin(II) chloride dihydrate (19.8 g, 87.7 mmol) was added in portions. The reaction mixture was heated for 4 h under reflux and diluted with ethyl acetate (300 mL) after cooling to room temperature. To this solution 5 M sodium hydroxide solution was added dropwise (3 x 150 mL) and the aqueous phase was separated without shaking in the separating funnel to avoid emulsion formation by colloidal tin hydroxides. After separation of most of the tin hydroxides, the organic phase was washed with 5 M sodium hydroxide solution (3 x 150 mL), saturated sodium hydrogen carbonate solution (3 x 150 mL) and saturated sodium chloride solution (1 x 150 mL). The organic phase was dried over sodium sulfate, and the solvent was evaporated. The residue was purified in three portions by column chromatography on silica gel (120 g column, PE/EA gradient, 25 % EA → 65 % EA, 30 min, 50 mL/min). A colorless solid was obtained.
Stirring was continued at room temperature for further 3 h. The reaction was stopped afterwards due to increasing formation of the azoxy by-product. The reaction mixture was concentrated in vacuo and the residue was diluted with ethyl acetate (100 mL). The organic phase was washed with 1 M sodium hydroxide solution (3 x 50 mL) and saturated sodium chloride solution (1 x 50 mL) and dried over sodium sulfate. The solvent was evaporated, and the residue was purified by column chromatography on silica gel (40 g column, PE/EA gradient, 0 % EA → 70 % EA, 30 min, 25 mL/min). A yellow solid was obtained.
The solution was washed with saturated sodium hydrogen carbonate solution (3 x 25 mL), deionized water (1 x 25 mL) and saturated sodium chloride solution (1 x 25 mL) and dried over sodium sulfate. The solvent was removed under pressure and the residue purified by column chromatography on silica gel (12 g column, DCM (0.

Synthesis of 6-iodo-1H-indazole (46)
1H-Indazol-6-amine (45) (7.97 mL, 77.3 mmol) was suspended in a mixture of deionized water (5 mL) and acetone (2 mL). The suspension was cooled to 10 °C and concentrated sulfuric acid (0.4 mL, 6.76 mmol) was added dropwise. An aqueous solution of sodium nitrite (2.5 M, 2.7 mL, 6.76 mmol) was added dropwise and the reaction mixture was stirred for 1 h at 10 °C. This mixture was then added dropwise to an aqueous solution of potassium iodide (1.87 g, 11.3 mmol, dissolved in 3 mL of deionized water). The reaction mixture was stirred for 18 h at room temperature. The aqueous solution was extracted with ethyl acetate (3 x 30 mL) and the combined organic phases were washed with 30 % sodium sulfite solution (2 x 20 mL). The organic phase was dried over sodium sulfate and the solvent was removed under pressure. The residue was purified by column chromatography on silica gel (40 g column, PE/EA gradient, 10 % EA → 40 % EA, 11 min, 25 mL/min). An orange solid was obtained. 2-((1H-Indazol-6-yl)thio)-N-methylbenzamide (47) (400 mg, 1.41 mmol) was dissolved in dry DMF (12 mL) under nitrogen atmosphere and potassium hydroxide (780 mg, 5.65 mmol) was added. The reaction mixture was stirred for 30 min at room temperature, and iodine (717 g, 2.82 mmol) was added in portions. After 2.5 h the suspension was diluted with ethyl acetate (200 mL) and the organic phase was washed with sodium pyrosulfite solution (2 x 50 mL) as well as deionized water (1 x 50 mL). The organic phase was dried over sodium sulfate, the solvent was removed under reduced pressure and the raw product was recrystallized from methanol. A colorless crystalline solid was obtained.

Quantum chemical Calculations
Cartesian coordinates of the B3LYP/631-G*-optimized structures of sulfur-and carbon-bridged diazocines (figure 5 main text)