Cymanquine – A New Organometallic Anticancer Agent

Cymanquine – A New Organometallic Anticancer Agent

Experimental Procedures

1. Syntheses of Cymanquine

A dried round bottomed flask was charged with dry cymantrene (0.20 g, 1.0 mmol) dissolved in THF (2.5 mL) was reacted with n-BuLi (0.55 mL,1.20 mmol) at -50°C. After 1 hour, the solution was cooled down to -78°C and DMF (0.15 mL, 1.5 mmol) was added to the reaction mixture which was left at -78°C for 1h before warms it up slowly to room temperature for 2 hours. Hydrolysis was performed by addition of a satured solution of NH₄Cl solution. The aqueous layers were extracted twice with Diethyl Ether and the combined organic layers were washed with water, dried over Na₂SO₄, concentrated under vacuo.

Formylcymantrene was obtain as a yellow oil (93 %). The product is engaged directly in the next step without further purification. The product is degradable after 1-2 days without the following precautions: Keep away from light and keep on the freezer.

¹H NMR (500 MHz, DMSO-d₆) δ_H 9.62 (s, 1H), 5.82 – 5.80 (t, J= 2.3 Hz, 2H), 5.28 (t, J = 2.3 Hz, 2H)

¹³C{1H} NMR (126 MHz, CDCl₃): δ_C 189.05 (s, C=O), 88.40 (s, Cp), 86.20 (s, Cp).

Formylcymantrene (0.23 g, 1.0 mmol) dissolved in DMF was stirred and heated at reflux for 4 hours. Once the reflux over, the reaction mixture was cooled down at room temperature and treated with a solution of HCl 3M. Hence, the layers were separated, the aqueous layers were treated with NaOH 3M to pH=13, then extracted twice with Ethyl Acetate and the combined organic layers were washed with water solution, dried over Na₂SO₄, concentrated under vacuum.

(N,N-Dimethylaminomethyl)cymantrene was obtain as a yellow oil (78 %). The product was engaged without further purification.

¹H NMR (500 MHz, DMSO-d₆) δ 5.02 (t, J = 2.1 Hz, 1H), 4.96 (t, J = 2.1 Hz, 1H), 3.04 (s, 1H), 2.14 (s, 3H).

¹³C{1H} NMR (126 MHz, DMSO-d₆): δ_C 101.90 (s, Cp), 85.50 (s, Cp), 85.50 (s, Cp), 56.20 (s, CH₂), 44.85 (s, CH₃).

A dried round bottomed flask was charged with dry (N,N-dimethylaminemethyl)cymantrene (0.26 g, 1.0 mmol) dissolved in THF was reacted with n-BuLi (1.50 mmol, 1.50 eq) at -50°C. After 1 hour, the solution was cooled down to -78°C and DMF (2.5 mmol) was added to the reaction mixture which is left at -78°C for 1h before warms it up slowly to room temperature for 2 hours. Hydrolysis was performed by addition of the solution of HCl 3N solution. Hence, the layers were separated, the aqueous layers were treated with NaOH 3M to pH=13, then extracted twice with ethyl Acetate and the combined organic layers were washed with water solution, dried over Na₂SO₄, concentrated under vacuo.

(N,N-Dimethylaminomethyl)formylcymantrene was obtain as a brown oil (75 %). The product was engaged without further purification.

(N,N-dimethylaminomethyl)formylcymantrene (1.00 mmol, 1.00 eq) was dissolved in 10 mL of dry methanol at 273 K. Sodium borohydride (0.080 g, 2.0 mmol) was added by portion over 15 minutes and the solution was allowed to come back to room temperature. After 2 h, most of the solvent was removed under reduced pressure and the mixture was dissolved in 30 mL ethyl acetate. The solution was washed twice with 30 mL water and brine, the organic layer was isolated, dried over MgSO₄, filtered, and the solvent removed under reduced pressure.

α-hydroxymethyl-(N,N,-dimethylaminomethyl)cymantrene was obtained as a dark brown oil (68 %) sufficiently pure to be used in the next step.

¹H NMR (500 MHz, CDCl₃) δ 4.77 (ddd, J = 2.6, 1.7, 0.6 Hz, 1H), 4.68 (dd, J = 2.7, 1.7 Hz, 1H), 4.53 (t, J = 2.7 Hz, 1H), 4.41 (d, J = 12.7 Hz, 1H), 4.03 (d, J = 12.6 Hz, 1H), 3.60 (d, J = 12.9 Hz, 1H), 2.71 (dd, J = 12.9, 0.7 Hz, 1H), 2.27 (s, 6H).

¹³C{1H} NMR (126 MHz, CDCl₃): δ_C 103.80 (s, Cp), 100.25 (s, Cp), 84.95 (s, Cp), 84.10 (s, Cp), 78.25 (s, CH₂), 57.65 (s, CH₂), 44.55 (s, CH₃).

To a solution of α-hydroxymethyl-(N,N,-dimethylaminomethyl)cymantrene (50 mg, 0.17 mmol) of this compound dissolved in 5 mL of dry THF were added phthalimide (32 mg, 0.22 mmol) and triphenylphosphine (57 mg, 0.22 mmol). Then diisopropyl diazene-1,2 dicarboxylate (DIAD) (47 uL, 0.22 mmol) was slowly added. After 16 h, the solvent was removed and MeOH was added (5 mL). The product was engaged without further work-up or purification to the next step

Hydrazine hydrate (0.51 mmol) was added to a solution of α-phthalimidomethyl-(N,N,-dimethylaminomethyl)cymantrene (0.17 mmol) dissolved in 5 mL of dry MeOH, which was stirred at room temperature for 4 h. After concentrated the reaction mixture, the solution was quickly extracted three times with ethyl acetate. The pH of the aqueous layer was increased to 13 by addition of a solution of 1N NaOH and twice extracted with dichloromethane. The organic layers were combined, dried over MgSO₄, and the solvent removed under reduced pressure to α-aminomethyl-(N,N,dimethylaminomethyl)cymantrene, which was used directly in the next step

The α-aminomethyl-(N,N,-dimethylaminomethyl)cymantrene (1.0 mmol) was dissolved in a 2 mL dry NMP solution containing 4,7-dichloroquinoline (1.05 mmol) and refluxed overnight, after which the reaction mixture was cooled down and poured into water. A solution of NaOH 3N was added and the precipitate was filtered, the aqueous phase was extracted twice with Ethyl acetate. The precipitate was washed with MeOH. The combined organics layers and the washings of the precipitate were concentrated, and the residue was dissolved in THF. KOtBu was added and the compound purified by column chromatography over silica gel using AcOEt/Hexane/Et3N (10:10: 1) as the eluent. A light brown powder was obtained for CMQ (16% yield over the three steps from the hydroxyl compound), the solid did not seem to be overly air sensitive. It was stored in the dark owing to the light sensitivity common among cymantrene derivatives.

¹H NMR (400 MHz, CDCl₃–d₆) δ 7.83 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 6.38 (s, 1H), 5.06 (dd, J = 2.9, 1.8 Hz, 1H), 4.67 (d, J = 15.0 Hz, 1H), 4.60 (t, J = 2.3 Hz, 1H), 4.53 (t, J = 2.8 Hz, 1H), 4.31 (d, J = 15.0 Hz, 1H), 3.49 (d, J = 13.1 Hz, 1H), 2.77 (d, J = 13.1 Hz, 1H), 2.04 (s, 6H).

¹³C{1H} NMR (126 MHz, CDCl₃): δ_C 150.0 (s, Ar), 134.90 (s, Ar), 131.90 (s, Ar), 128.50 (s, Ar), 125.20 (s, Ar), 121.75 (s, Ar), 100.02 (s, Cp), 90.90 (s, Cp), 85.45 (Cp), 84.85 (Cp), 78.90 (Cp), 55.73 (s, CH₂), 45.02 (s, CH₂), 40.00 (s, CH₃).-