Mild, Organo-Catalysed Borono-Deamination as a Key to Late-Stage Pharmaceutical Precursors and 18F-Labelled Radiotracers

A tris(pentafluorophenyl)borane catalysed method for the synthesis of boronic acid esters from aromatic amines in yields of up to 93% was devised. Mild conditions, benign reagents, short reaction times, low temperatures and a wide substrate scope characterize the method. The reaction was found applicable to the synthesis of boronic acid ester derivatives of complex drug molecules in up to 86% isolated yield and high purity suitable for labelling. These boronates were subsequently labelled with [18F]fluoride ion in radiochemical yields of up to 55% with and even without isolation of the boronate-intermediate.


Supplementary Data Experimental Procedures
General Solvents and reagents used in the experiments described herein were procured from Sigma-Aldrich (Sigma-Aldrich AS, Norway) or Fluorochem (Fluorochem Ltd., UK), in highest available quality unless specified otherwise. Starting materials were obtained from commercial suppliers.
Intermediates and references were either obtained commercially or produced via standard methods from commercially available starting materials. The identity of intermediates and references was confirmed via comparison to literature reports. Solid phase extraction (SPE) cartridges were purchased from VWR (VWR International, Darmstadt, Germany) and Sigma-Aldrich (Sigma-Aldrich AS, Norway). TLC was conducted on Silica gel 60 F254 coated aluminium TLC plates (Merck KGaA, Darmstadt, Germany), and developed using mixtures of ethyl acetate:hexanes (v:v) unless otherwise stated. Compounds on TLC plates were visualized under UV light (254 or 356 nm) and by staining with iodine or potassium permanganate. Silica gel 60Å (40-63 µm, 230-400 mesh) (Merck-Millipore) was used as the solid phase for flash column chromatography, unless otherwise stated. Nuclear magnetic resonance spectra were recorded on a Bruker AVII 400 NMR instrument (Bruker ASX Nordic AB). Chemical shifts (δ) for 1 H (400 MHz), 13 C (100 MHz) and 19 F (376 MHz) resonances are reported in parts per million (ppm), relative to the solvent signal (CDCl3 δ = 7.223 ppm), downfield from a theoretical tetramethylsilane signal (TMS, δ = 0 ppm). Mass spectrometry was conducted on a Q-Tof-2 mass analyser (Micromass, Q-Tof-2TM) using ESI ion source in positive mode. HPLC analysis of compound purity and quality control was conducted on a Hewlett-Packard 1100 HPLC system (Matriks AS, Oslo, Norway) consisting of a quaternary pump, variable wavelength diode array detector and a Raytest Gina star radioactivity detector (Raytest GmBH, Straubenhardt, Germany) using GABI-star software (Raytest) for instrument control, data acquisition and processing. Three HPLC methods were developed. For determination of the identity and purity of radiotracers, a Luna PFP column (Phenomenex; 5 µm, 100 Å, 250 mm × 4.6 mm) with an isocratic mixture of MeCN-water; 55:45 was used at a flow rate of 1 mL/min (System A) or an isocratic mixture of MeCN-water 40:60 at 1.0 mL/min flow rate (System B). Alternatively, a Kinetex EVO (Phenomenex; 5u, C18, 100Å, 250 x 4.6 mm) with an isocratic mixture of ammonium formate buffer (25 mM, pH=9.2)-MeOH-MeCN; 60:30:10 at a 1.0 mL/min was used (System C). UV signals were detected at a wavelength of 254 nm. Radioactivity measurements during labelling experiments and radiotracer productions were performed using an Atomlab 300 dose calibrator (Biodex Medical Systems).

Recovery of B2pin2
Recovery of B2pin2 was achieved by dilution of the reaction mixture with 5 volumes of Et2O followed by cooling to 4 °C over night. The reagent was recovered as colourless crystals after decanting or filtration of the mother liquor. Following concentration, the product is obtained via purification on silica gel. Residual B2pin2 was recovered in the fractions eluting before the desired products. Recovery was calculated to be around 46% ( The recovered B2pin2 had higher purity (by 1 H-NMR spectroscopy) than the material obtained from commercial sources ( Figure S1).
In efforts to obtain a radiotracer for poly-ADP ribose polymerase (PARP) from the PARP-inhibitor rucaparib (1), we designed a total synthesis to introduce a leaving group to be substituted for [ 18 F]fluoride ion ( Figure S2). We then devised the following synthetic route ( Figure S3), to the boronic acid ester precursor synthesized by this method.
Synthetic procedures.
methyl 6-((tert-butoxycarbonyl)amino)-1H-indole-4-carboxylate (S1). Methyl-6-amino-1Hindole-4-carboxylate (3.5 g, 18.4 mmol) was dissolved in THF (50 mL). To the stirring solution was added di-tert-Butyl dicarbonate (4.42 g, 20.2 mmol) in one portion at room temperature. The solution was then heated to 50 °C, and gas evolution was visible in the form of bubbles after 5-10 min. After 2 h at this temperature gas evolution had ended and TLC showed consumption of all starting materials. The solvent was evaporated under reduced pressure, and the beige residue was recrystallized from EtOH/H2O to afford the product as small colourless/pink crystals in 96% yield ( methyl 6-((tert-butoxycarbonyl)amino)-3-formyl-1H-indole-4-carboxylate (S2). To a round bottomed flask which had been air dried under argon, was added DMF (5 mL) and cooled on an ice bath. POCl3 (3.2 mL, 34.4 mmol) was then added to the stirring solvent dropwise, being careful to maintain the temperature below 5 °C. Methyl 6-((tert-butoxycarbonyl)amino)-1H-indole-4carboxylate (5 g, 17.2 mmol) was dissolved in anhydrous DMF (10 mL), and added to the stirring DMF/POCl3 mixture over the course of 15 min. The solution was stirred on the ice bath for an additional 45 min, and then allowed to warm up to room temperature and stirred for an additional h. The solution was transferred into an Erlenmeyer flask, with crushed ice (400 mL) and stirred vigorously. A yellow paste immediately appeared. This suspension then neutralized by dropwise addition of NaOH (3 M) with vigorous stirring, until the pH was neutral (pH paper indicator) and then basified with saturated K2CO3, while maintaining the solution cooled. The solution was allowed to decant in the refrigerator overnight. The precipitate was filtered off, washed with water, and dried under high vacuum to leave a beige solid which was the product in 92% yield (5.04 g, 15.8 mmol), and which was pure enough to be used in the next step without further purification.  29, 168.95, 153.40, 138.91, 137.03, 135.47, 125.13, 118.63, 116.98, 115.27, 105.25, 79.75, 52.44, 28.61

H-NMR Experiments for the screening of reaction conditions
Spectra of samples from the screening of reaction conditions (General procedure for NMR experiments) were taken, using 4-nitroaniline as a model compound. Peaks for aromatic product and starting materials were identified by adding an external standard of 4-nitrophenyl boronate or 4nitroaniline to reaction samples. The chemical shifts of the different intermediates were accounted for (Table S1; Figures S4, S5, S6, S7 and S8), and were subsequently used to calculate the NMR yields. For the neopentyl derivatives, there was no change in the chemical shift for the aromatic signals for the products. (Table S1).

NMR yield calculation
Yield for individual reactions were calculated by integrating the proton signals originating from the products and any other intermediates present in the reaction mixture in the range between 13-6 ppm. The total integral from the product was then divided by the total integral of all the signals in the aromatic region and multiplied by 100 to give the yield (Equation 1).

= • 100 ( 1)
There is no need to account for the number of protons from which the signals originate, since the only source of aromatic protons in all reactions mixtures is the starting material.
Alternatively, the signal originating from the product (IP) either in the aliphatic region (5-0 ppm), and the sum of the integrals from all boron associated protons (IBtot) were normalized for the number of protons (N =12). The theoretical yield integral (IyTh) was found by dividing the normalized IBtot by the ratio between the amount of aniline to boron (monomer) in the mixture (Equation 2).
IP was then divided by IyTh multiplied by 100 to obtain the yield (Equation 3).

Experimental procedures for the Cu(II) mediated radiofluorination of 23a and 23b
Due to the poor solubility of 23a and 23b in conventional solvents and the need for an additional deprotection step to remove the PMB protective group from the benzylic amine, standard fluorination was performed slightly different. This is summarized in scheme 2.
Scheme S1 Reaction scheme for the copper mediated radiofluorination of 23a and 23b to obtain [ 18 F]28.

Preparation of the reagents and reaction procedure
Solution for elution of [ 18 F]fluoride: Was prepared in the same manner as previously described in materials and methods.

Labeling Procedure
Tetrakis(pyridine)copper(II) triflate solution (0.1 mL) is added to the potassium-15-C-5 [ 18 F]fluoride complex, followed by addition of radiolabeling precursor solution (0.8 mL) in a reactivial with a conical bottom The reactivial is counted, placed on the hotplate, and left to react for 20 min at 120 °C. After the time has elapsed the mixture is removed from the hotplate and placed in an ice bath for 30 seconds. CAN solution is added to the mixture and heated to 85 °C for 5 min. The reaction is quenched by cooling in an ice bath for 2 min. The mixture is diluted 1:1 with either ammonium formate buffer (25 mM, pH = 9.2) or phosphate buffered saline before injection into the HPLC, for which HPLC system C was used.

One-pot borono-deamination radiofluorination
We attempted the sequential borylation followed by direct copper mediated radiofluorination with [ 18 F]Fby using our optimized conditions. The following general protocol was used to label drug compounds from their amine starting materials: Aromatic amine (30 µmol) was dissolved in MeCN (150 µL) in a reactivial with a conical bottom. To the solution was added in succession B2pin2 or B2neop2 (15 µmol), B(C6F5)3 (2.5 mol%) and AmylONO (45 µmol). The vial was heated to 40 °C for 15 min. At the same time [ 18 F]F -(50-200 MBq) was eluted from the target water from the cyclotron and azeotropically dried as described in the article text. After 15 minutes had elapsed the reaction mixture was added to the dried fluoride, followed by a solution of tetrakis(pyridine)copper(II) triflate (30 µmol) and pyridine (120 µmol) in DMA (850 µL). The homogeneous solution was heated to 120 °C for 20 min, and cooled on an ice water. The solution was diluted 1:10 with MeCN and injected into an HPLC system.

Calculation of radiochemical yields (RCY) and quality control
Radiochemical conversions (RCC) were determined by injecting aliquots of diluted reaction mixtures into the analytical HPLC system A, B or C.
For calculation of radiochemical yields (RCY), an activity balance over the reaction was used, i.e. the percentage of activity in the product fraction at the end of syntheses was divided by the starting activity and the fraction was multiplied with 100%. For ease of screening, unreacted fluoride was separated from labelled products via liquid-liquid extraction with Et2O (1 mL).
A quality control by HPLC was conducted using systems confirm identity and determine radiochemical purity of the products. Product remaining in the aqueous phase after extraction was omitted. The activity corresponding to the product was divided by the total activity at this timepoint and multiplied with 100% to give the RCY.
Alternatively, for [ 18 F]23d the area under the peak of the radioactive product was divided by the remaining are of the chromatogram and multiplied by 100% to give the RCY.
Molar activity of the product was calculated from the concentration of the labelled compound in the final formulation as determined by HPLC. In brief, UV calibration curves were recorded using 5 different concentrations of the reference compound. The calibration curves were used to calculate the amount of substance in the isolated product. Total activity of the product was divided by the total amount of substance to obtain molar activities in MBq/nmol.

ICP-MS analysis
Labeled compounds were formulated in 10% EtOH in phosphate buffered saline (PBS) after purification. A sample of the formulated radiotracer was prepared for Cu-determination by ICP-MS by 1:9 dilution in 2% (V/V) ultra-pure HNO3, and injected to the ICP-MS system for copper determination. Samples were analyzed on an Agilent 8900 #100 ICP-MS instrument, in HE-KED MS-MS mode for 63 Cu and 65 Cu. The instrument was calibrated by measurement of standards containing 2.26, 20.2 and 200 µg/mL Cu, which were prepared identically to formulated samples.