Synthesis of boronated phenylalanine analogues with a quaternary center for boron neutron capture therapy

2-Amino-2-methyl-3-(4-dihydroxyborylphenyl)propionic acid ( 3 , α -methyl-BPA) and 1-amino- 3-(4-dihydroxyborylbenzyl)cyclobutanecarboxylic acid 4 , which are (4-dihydroxyboryl-phenyl)alanine (BPA) analogues containing a quaternary center, have been synthesized from 4-allylbromobenzene. α -Methyl-BPA has also been prepared from D,L-alanine, and the route is suitable for the synthesis of α -alkyl-BPA. Both 3 and 4 exhibit very similar R f values indicating similar lipophilicities. The products have been prepared as potential boron carriers for Boron Neutron Capture Therapy.


Introduction
Boron Neutron Capture Therapy (BNCT) is a binary therapy requiring selective accumulation of boron-10 in a tumor cell.2][3][4][5][6] Early studies revealed that selective accumulation of boron in a tumor cell and low levels of boron in the blood are crucial factors for the therapy.Consequently, over the years several classes of compounds have been examinated as potential boron carriers for BNCT.2][13][14] Although it is a simple boronated amino acid, containing only one boron atom per molecule, it achieves concentrations of boron in a tumor cell sufficient for the therapy.However, BPA and BSH are not ideal, since higher concentrations of boron are desirable.6][17][18][19][20][21] Recently, it was shown that amino acids, such as 1-aminocycloalkanoic acids, cross the blood brain barrier and localize in Glioblastoma Multiforme and metastatic malignant melanoma more avidly than BPA. 228][19][20][21] Such boronated cyclic amino acids contain a quaternary carbon atom bonded to the amino acid functionality.It seemed interesting to compare the effect of such quaternary center incorporated in an acyclic moiety of BPA analogue with a representative cycloalkyl analogue as potential boron carriers for BNCT.Consequently, we decided to prepare 2-amino-2-methyl-3-(4-dihydroxyborylphenyl)propionic acid (3, α-methyl-BPA), the simplest BPA analogue containing a quaternary center, and for comparison its cycloalkyl analogue, 1-amino-3-(4-dihydroxyborylbenzyl)cyclobutanecarboxylic acid 4, (Figure 1).

Results and Discussion
α-Methyl-BPA was prepared following two routes, from 4-allylbromobenzene 6 via hydantoin, and from D,L-alanine by deprotonation-benzylation. 1-(4-Bromophenyl)propan-2-one 9, a convenient intermediate for the first route, was prepared starting from 1,4-dibromobenzene 5 in an overall 70% yield and >99% purity.Lower yield than might be expected results from competing formation of 1,4-diallylbenzene 7 in the first step, leading to a mixture of 5-7.To suppress the formation of 7, 4-bromophenylmagnesium bromide was prepared from 5 by the addition of reagents in a reversed order, and the amount of 7 decreased to 10-15%.Both products have very close boiling points and their separation by distillation is inconvenient.Consequently, a mixture of 6 and 7, 86:14, was used for the oxymercuration-demercuration reaction.The product alcohol 8 was cleanly separated from the product diol by distillation, and was oxidized with PCC to give 9 of >99% purity, (Scheme 1).Its ketalization followed by lithiation and transmetallation with triisopropoxyborane gave 1-(4-dihydroxyborylphenyl)propan-2-one 11.Hydantoin 12, readily formed under standard conditions by treatment of 11 with potassium cyanide and ammonium carbonate, crystallized with one molecule of water per two hydantoin molecules.Alkaline hydrolysis of 12 under carefully controlled conditions produced 3, isolated in 68% yield.The hydrolysis conditions must be controlled since at lower temperature the reaction is not completed, whereas at higher temperatures deboronated products are formed.

Scheme 1
The second route to 3 required 4-bromomethylphenylboronic acid and D,L-alanine properly protected for the deprotonation step.Based on the literature reports, 23,24 4-chlorobenz-aldehyde was used to protect the amino group of D,L-alanine, and the carboxylic group was protected as a methyl or ethyl ester.Free radical bromination of p-tolylboronic acid 13 produced 4chlorobenzaldehyde acid, however, dibromination was a competing reaction.The crude product was a mixture of 13, mono-and dibrominated acids, its composition depending on the bromine/13 ratio used.The starting acid could be separated by crystallization, however, the solubility of dibrominated acid in several solvents tried was lower than 4-bromomethylphenylboronic acid, and crystallization was not effective for purification.To circumvent the difficulty, 13 was esterified with ethylene glycol.Although bromination of the ester 14 gave also a mixture of the unreacted starting ester, mono-and dibrominated esters, the monobrominated ester 15 of ≥95% purity was separated by vacuum distillation, and was used for the reaction with the carbanion generated by deprotonation of protected D,L-alanine with potassium bis(trimethylsilyl)amide (KHMDS).Treatment of the product 16 with 1M hydrochloric acid deprotected the amino and boronic acid groups to give 17.Deprotection of the methyl ester required reflux with 6M hydrochloric acid.When ethyl ester was used deprotonation required refluxing for a longer time and the yield of 3 was lower.The cyclobutyl analogue 4 of BPA was prepared starting from 6 via hydantoin 22 (Scheme 3), following the route recently used for the synthesis of such cyclobutyl analogues. 17The addition of dichloroketene to 6 gave 18 which was reduced without isolation to 3-(4-bromobenzyl)cyclobutanone 19.Lithiation of its ketal 20 and transmetallation with triisopropoxyborane followed by hydrolysis afforded 3-(4-dihydroxyborylbenzyl)cyclobutanone 21.The ketone was transformed into the corresponding hydantoin 22 which crystallized with one molecule of water. 1 H and 13 C analysis showed a mixture of diastereomers.The mixture was hydrolyzed under carefully controlled conditions to give 4, isolated by crystallization as a mixture of diastereomers.

Scheme 3
The above described boronated amino acids 3 and 4 exhibited very similar R f values, 0.29 and 0.31, respectively, using as a mobile phase acetonitrile-methanol-water, 10:2:1.5.The result reflects a similar lipophilicity of 3 and 4, and indicates that the methyl substituted quaternary center of 3 and the cyclobutyl ring of 4 have a similar effect.The compounds will be tested for BNCT.Lipophilicity of α-alkyl-BPA can be controlled by the size of alkyl groups, and the compounds can be readily prepared by monoalkylation of glycine followed by the route shown in Scheme 2. The formation of diastereomers, observed for cycloalkyl analogues, is avoided since only one stereogenic center is present, and the synthesis can be modified to achieve enantioselectivity.Work on the synthesis of α-alkyl-BPA is in progress.

Experimental Section
General Procedures. 1 H and 13 C NMR spectra were recorded on a Varian Gemini 200 MHz, a Bruker AMX 300 MHz and a Varian Inova 500 MHz spectrometers. 11B NMR spectra were recorded on the Varian Gemini 200 instrument.IR spectra were recorded on a FT-IR Spektrum 2000 Perkin Elmer spectrometer.Microanalyses were performed by the Microanalyses Laboratory, Institute of Organic Chemistry PAN, Warsaw.GC analyses were performed on a Hewlett Packard 5890 chromatograph equipped with a 30 m × 0.32 mm SPB-5 column, a 50 m × 0.32 mm Carbowax 20M column, and a 5 m × 0.53 mm HP-1 column.All glassware used for reactions with air sensitive compounds was dried at 150 °C for several hours, assembled hot, and cooled in a stream of nitrogen.Melting points were measured on a Boetius ESP4/SI, Carl Zeiss Jena, apparatus and are uncorrected.R f values were determined using a horizontal TLC chamber and TLC plates Macherey-Nagel Polygram ® SIL G/UV 254 , 0.20 mm layer.

1-(4-Dihydroxyborylphenyl)propan-2-one (11).
A 2.36M solution of n-butyllithium in hexanes (23.3 mL, 55.0 mmol) was added dropwise with stirring to a solution of 10 (12.90 g, 50.0 mmol) in tetrahydrofuran (150 mL) at -78°C under argon atmosphere, and the mixture was stirred for 2 h at this temperature.Triisopropoxyborane (12 mL, 51.0 mmol) was added dropwise and the mixture was left for 24 h at room temperature.It was cooled to 0°C, 2M hydrochloric acid (75 mL, 150 mmol) was added, and the mixture was stirred for 24 h at room temperature.The organic layer was separated and the aqueous layer was extracted with diethyl ether (3 × 10 mL).Solvents were removed and the remaining oil was dissolved in 2M sodium hydroxide, the solution was extracted with diethyl ether (2 × 50 mL), acidified with 3M hydrochloric acid, extracted with diethyl ether (3 × 50 mL) and the extract was washed with saturated brine (20 mL).Ether was removed and the product was obtained as a grey-white solid which was crystallized from ethyl acetate, 6.

1-(4-Dihydroxyborylphenyl)propan-2-one hydantoin (12).
A mixture of 11 (2.67 g, 15.0 mmol), 50% aqueous ethanol (45 mL), potassium cyanide (1.95 g, 30.0 mmol), and ammonium carbonate (6.82 g, 80.0 mmol) was placed in an autoclave and kept at 60°C for 4 h.The mixture was acidified with 6M hydrochloric acid, and filtered through a pad of activated carbon.Solvents were removed by evaporation at room temperature, and the solid material which remained was extracted with diethyl ether in a Soxlet apparatus.A pale beige small crystals precipitated from the concentrated solution, 3.   (20 mL) was added to the mixture at -78°C, stirring was continued for 3 h, and the mixture was allowed to warm to -10°C.Hydrochloric acid (60 mL, 60.0 mmol) was added below 0°C and the mixture was stirred overnight at room temperature.The mixture was alkalized with 3M sodium hydroxide solution, and extracted with diethyl ether (2 × 50 mL).The aqueous layer was separated, acidified with 2M hydrochloric acid to pH 6.5-7.0,concentrated under vacuum, 6M hydrochloric acid (100 mL, 0.60 mol) was added, and the mixture was refluxed for 3 h.Hydrochloric acid was removed under vacuum at room temperature.The product was crystallized from water, 3.96 g, 90%, mp 302-305°C (decomposition). 1 H, 13 C, and 11 B spectra were identical with the spectra described above.

2-Amino-3-(4-dihydroxyborylphenyl)-2-methylpropionic acid (3) By hydrolysis of 12
. A mixture of 12 (0.80 g, 3.3 mmol) and a 2M sodium hydroxide solution (33 mL, 16.5 mmol) was placed in an autoclave and kept at 120°C for 2 h.After cooling, the mixture was treated with 4M hydrochloric acid to pH 6.5-7.The precipitated gelatinous solid was filtered off and washed with warm water (25 mL).The filtrate was concentrated under vacuum and the product crystallized out.