Function through bio-inspired, synthesis-informed design: step-economical syntheses of designed kinase inhibitors

We describe here step-economical, function-oriented strategies towards the syntheses of potent kinase inhibitors inspired by the natural product staurosporine.


General Methods
Unless otherwise noted, air-and moisture-sensitive reactions were carried out in oven-dried (>110 ºC) glassware capped with rubber septa under a positive pressure of dry nitrogen or argon from a manifold or balloon. Likewise, air-and moisture-sensitive reagents, solvents, and solutions were transferred via syringe or stainless steel cannula under a dry inert atmosphere. Stirring was achieved using oven-dried (>110 ºC) Teflon®coated magnetic stir bars cooled under a stream of dry nitrogen or argon.
Reactions were run at room temperature (rt, 20-25 °C) unless otherwise noted in the experimental procedure. Elevated temperatures were maintained through the use of a silicone oil bath, which was equilibrated under constant current through a wire-heating element. For reactions below room temperature, the term "-78 °C" refers to a bath of acetone and dry ice, "-40 °C" refers to a slurry of acetonitrile and dry ice, "-20 ºC" refers to a slurry of NaCl salt and ice, and "0 °C" refers to an ice-water bath. "Concentration" of solvent refers to removal of solvent using a Büchi rotary evaporator equipped with a portable vacuum pump. Removal of residual solvents was accomplished by evacuation of the vessel using a high vacuum line maintained at 0.1-1.0 torr.

Reagents and Solvents
Unless otherwise specified, all commercial reagents and solvents were purchased from Aldrich Chemical Company, TCI America, or Acros Organics and were used without further purification with the following exceptions. [(C 10 H 8 )Rh(COD)]SbF 6 catalyst was synthesized according to literature procedures. 1 Vinylcyclopropane 1 was purchased from Aldrich. Tetrahydrofuran, diethyl ether, dichloromethane, and toluene were purified via passage through an activated alumina column (Solv-Tek, Inc.).

Chromatographic and Spectroscopic Methods
Thin layer chromatography (TLC) was performed using 250 μm glass-backed, silica gel 60 F254 coated plates from Merck. The plates were visualized by ultraviolet light (254 nm) and treatment with acidic p-anisaldehyde stain or potassium permanganate stain followed by gentle heating on a hot plate. Proton and carbon NMR spectra were measured on a Varian Inova-300 ( 1 H at 300 MHz, 13  formic acid in water) ramped to 100% Solvent B (0.1% formic acid in acetonitrile) in 3 minutes, and held for 1 minute. The column was a 2.1x50mm Zorbax 300 SB C18 Rapid Resolution 1.8u and the flow rate was 0.2mL/min. Infrared (IR) spectra were recorded on a Perkin-Elmer TM 1600 series FTIR spectrometer. IR data is reported as frequencies in wavenumbers (cm -1 ). Samples were prepared as thin films on a NaCl salt plate.

5-chloro-2-iodo-1H-indole (2)
Following Bergman's method 2 , in a round-bottom flask, 5-chloroindole (5.30 g, 34.9 mmol) was dissolved in dry THF (70 mL) and cooled to -78 ºC under argon atmosphere. n-butyllithium (32.4 mL, 1.13 M solution in hexanes) was added via a syringe dropwise over 10 min at -78 °C. After the resulting white suspension was stirred for 30 min at -78 °C, CO 2 (g) was bubbled through the reaction mixture for 15 min, and the obtained clear solution was stirred at -78 °C for an additional 10 min. The solution was carefully warmed up to rt over 1 h while maintaining good ventilation to remove the CO 2 gas. The solvent was removed using a rotary evaporator to obtain a pale yellow foamy solid. After the solid residue was dissolved in dry THF (70 mL) at rt under an argon atmosphere, the clear solution was cooled down to -78 °C, and tert-butyllithium (27.5 mL, 1.33 M solution in pentane) was added via a syringe dropwise over 25 min. The resulting yellow-orange solution was stirred for 1 h at -78 °C. A solution of 1,2-diiodoethane (9.74 g, 34.9 mmol) in dry THF (15 mL) was added dropwise over 30 sec. On addition, the apricot reaction mixture turned colorless and turbid. After the mixture was stirred for 20 min at -78 °C, saturated aqueous NH 4 Cl (10 mL) was added, and it was allowed to warm up to rt.
Water (20 mL) and diethyl ether (50 mL) were added and the dark brown organic phase was collected, and the aqueous phase was extracted twice with diethyl ether (50 mL).
The combined organic phases was washed with saturated aqueous Na 2 S 2 O 3 and brine, dried over anhydrous MgSO 4 , and concentrated in vacuo to afford a yellow-brown syrup. The crude residue was purified by flash column chromatography (5% Et 2 O/petroleum ether) to give the product as a white solid (5.83 g, 73 %). flask equipped with a reflux condenser, diester 8 (174.5 mg, 0.313 mmol) was suspended in ethanol (6.0 mL). 1 N aqueous KOH (2.0 mL) was added in one portion via a syringe at rt and the suspension was heated to 70 ºC. The mixture turned clear within 5 min. After 12 h, KOH (70 mg) in water (1.0 mL) was added and the reaction mixture was refluxed for an additional 10 h until TLC indicated complete consumption of the starting material. The reaction mixture was cooled down to rt and diluted with water (10 mL). Dichloromethane (10 mL), and 20% citric acid (10 mL) were added and stirred for 10 min. The organic phase was collected and the aqueous phase was extracted with dichloromethane (4 Í 10 mL). The combined organic phases were washed with water (10 mL) and brine (2 Í10 mL), and concentrated in vacuo to afford a yellow oil. Absolute ethanol (5 mL) was added and evaporated to yield ~200 mg of a yellow powder.
In a round-bottom flask equipped with a reflux condenser, the crude product was dissolved in dry acetonitrile (30 mL) and heated to a gentle reflux at 80 ºC.       -6-(methylamino)-4,5,6,7,8,9-hexahydro-1H-   Dichloromethane (5 mL) and saturated aqueous NH 4 Cl (5 mL) were added, and the organic phase was collected. 3N aqueous NaOH (3 mL) was added to the aqueous phase that was subsequently extracted with dichloromethane (2 × 5 mL). The combined organic phases were washed with brine (10 mL) and water (10 mL), dried over MgSO 4 , filtered, and concentrated in vacuo to give a yellow powder.
In a round-bottom flask equipped with a magnetic stir bar, the crude residue was dissolved in dry MeOH (0.85 mL) and CH 2 Cl 2 (1.7 mL). Dimethylamine hydrochloride (61 mg, 0.75 mmol), 4Å molecular sieves (20 mg), and sodium cyanoborohydride (2. mg, 0.03 mmol) were added in this order, in on portion respectively. Triethylamine (0.10 µL, 0.075 mmol) was added and the yellow suspension was stirred at rt for 48 h until TLC analysis indicated complete consumption of the starting material. The mixture was diluted with CH 2 Cl 2 (5 mL) and aqueous NaHCO 3 (10 mL) was added.
The organic phase was isolated, and the aquoeous phase was extracted with CH 2 Cl 2 (3 x 5 mL). The combined organic phases were washed with brine (5 mL), dried over MgSO 4 , and concetrated in vacuo to afford a yellow powder. This material was directly used in the next step.
In a round-bottom flask equipped with a magnetic stir bar, the yellow powder was suspended in CH 2 Cl 2 (1.25 mL). Triisopropylsilane (51 µL, 0.25 mmol) was added in one portion, and TFA (192 µL, 2.5 mmol) was added via syringe dropwise over 30 sec.
On addition, the suspension turned into a bright yellow solution, which was stirred at rt for 30 min. The reaction mixture was diluted with CH 2 Cl 2 (5 mL) and concentrated in vacuo to yield a yellow-brown residue. The crude product was purified by preparative reverse-phase HPLC (Alltech Alltima C18 10u, 250 mm X ID 22 mm, 5 -95% acetonitrile/water gradient, flow rate = 20 mL/min) to afford the desired product as a bright yellow poder (6.8 mg, 43% yield) after lyopholization. intermediates had converged to a single orange spot on TLC (p-anisaldehyde stain), the reaction solution was passed through a short pad of silica gel (EtOAc eluent) and concentrated. The crude product was purified using flash column chromatography (40%  EtOAc/pentane) to give the desired product as a red amorphous solid (296 mg, 79%).
A solution of the amine (7.50 mg, 0.016 mmol) in anhydrous CH 2 Cl 2 (0.82 mL) under Ar atmosphere was treated with triisopropylsilane (34 µL, 0.16 mmol) in one portion, causing the reaction mixture to become a cloudy white suspension. TFA (0.13 mL, 1.65 mmol) was next added dropwise over 30 sec, which caused the solution to become colorless. After 30 min, the stir bar was removed and CH 2 Cl 2 (5.0 mL) was

PKC Inhibition Assay
A PKC inhibition assay was performed following the protocol developed by Kikkawa and coworkers. 5 The assay can be scaled as desired. Histone solution (0.33 mg/mL in 20 mM Tris buffer) was prepared from a stock solution (33 mg/mL, stored frozen). Phosphatidylserine (PS) vesicles were prepared by the addition of PS (10 mg/mL in chloroform, 240 µL) and dioleoylglycerol (10 mg/mL in ethanol, 40 µL) to a glass vial followed by removal of the chloroform under a stream of nitrogen. To the neat PS was added chilled 20 mM Tris assay buffer (2 mL) and the resulting mixture was sonicated To each eppendorf tube, 10 µL of PKC solution and10 µL of the inhibitor solution were added. After addition of the inhibitor, each tube was vortexed. For negative controls, 70 µL of cocktail 1 was added to tubes 1 and 2. To the rest of the tubes were added 60 µL of cocktail 2. All the tubes were then transferred to the hot hood, and 20 µL of "Autogo" solution was added to each tube and vortexed immediately. A start countdown of 7 min was initiated when "Autogo" was added to the first tube. Addition to all the tubes should be completed within 7 min. When the countdown is over, "Stop" solution was added to each tube and vortexed. The interval between the addition of "Autogo" and "Stop" solution should be 7 min for each tube.