Synthesis and biological evaluation of boronic acid-containing phenstatin analogues

A series of boronic acid-containing benzophenones was synthesized by introducing a boronic acid as an acceptor-type functional group into the aromatic ring B of the phenstatin skeleton. Among the compounds synthesized, 4c in which a hydroxyl group on the aromatic ring B of the phenstatin was replaced by a boronic acid, exhibited significant cell growth inhibition and the GI 50 values toward B-16 and 1-87 cell lines are 0.013  M and 0.087  M, respectively.


Introduction
Combretastatin A-4 (1a) was isolated by Pettit and coworkers from the stem wood of the South African tree Combretum caffrum in 1982. 1 This compound is one of the most potent antimitotic agents and exhibits potent cytotoxicity against a wide variety of human cancer cells including multidrug resistant cancer cell lines. 2Phenstatin (2a) is a synthetic tubulin assembly inhibitor developed by the same research group in 1998. 3This compound is a benzophenone-type molecule and the functional groups on the two phenyl rings are similar to combretastatin A-4. 4 Based on the structures of both lead compounds, various benzophenone derivatives have been developed including aminobenzophenones (2b-c) 5,6 and isocombretastatin A (3). 7 These analogues have a donor-type functional group, as a common feature, substituted on the aromatic ring B (Figure 1).We are interested in introducing boronic acid as an acceptor-type functional group into the aromatic ring B in the phenstatin skeleton.
A boron atom has a vacant orbital and interconverts with ease between the neutral sp 2 and the anionic sp 3 hybridization states, which generates a new stable interaction between a boron atom and a donor molecule through a covalent bond. 8Therefore, it is expected that the boron atoms introduced into biologically active molecular frameworks would interact with a target protein not only through hydrogen bonds but also through covalent bonds, and this interaction might produce a potent biological activity. 91][12][13][14][15] Previously, we reported the synthesis of boronic acid-containing combretastatin A-4 analogue 1d. 11,16Herein, we report the synthesis and biological evaluation of boronic acid-containing phenstatin analogues (Figure 2).

Results and Discussion
The boronic acid-containing phenstatin analogues 4a and 4b were synthesized from 3,4,5trimethoxybenzaldehyde and dihalobenzenes as shown in Scheme 1.The reaction of 1-bromo-4iodobenzene 5a with n-butyllithium in THF generated the 4-bromophenyllithium, which reacted with 3,4,5-trimethoxybenzaldehyde to give the corresponding carbinol 6a in 82% yield. 17In a similar manner, carbinol 6b was obtained from 1,3-diiodobenzene 5b in 65% yield.The carbinols 6a and 6b were converted into the corresponding ketones 7a 17 and 7b using pyridinium chlorochromate (PCC) as an oxidizing agent and the ketal protection of the carbonyl groups was performed by treating with triomethyl orthoformate in methanol under acidic conditions to give 8a and 8b in 86% and 81% yields, respectively.Treatment of the ketals 8a and 8b with nbutyllithium and triisopropyl borate at -78 o C, followed by acidic hydrolysis, afforded the boronic acid-containing phenstatin analogues 4a and 4b in 37% and 72% yields in two steps, respectively.Scheme 1. Synthesis of boronic acid-containing phenstatin analogues 4a and 4b.
We also synthesized boronic acid-containing phenstatin analogue 4c (Scheme 2).The reaction of 5-bromo-1,2,3-trimethoxybenzene with n-butyllithium in THF generated the corresponding aryllithium, which reacted with 3-bromo-4-methoxybenzaldehyde to give the carbinol 9 in 83% yield.The carbinol 9 underwent oxidation by treatment with PCC to give the corresponding ketone 10 in 94% yield.Protection of the carbonyl group in ketone 10 followed by introduction of boronic acid using n-butyllithium and triisopropyl borate at -78 o C and deprotection of the ketal group under acidic conditions gave phenstatin analogue 4c in 52% yield in three steps.
We next examined inhibition of cell growth by the boronic acid analogues using B16 and 1-87 cell lines.The results are summarized in Table 1.The benzophenones 4a and 4b exhibited 50% inhibition of cell growth at 0.85 M and 0.37 M concentrations toward B16 cells and at 3.1 M and 1.9 M concentrations toward 1-87 cells, respectively.The benzophenone 4c, which has a boronic acid group at the meta position and a methoxy group at the para position, exhibited higher inhibition of cell growth against both cell lines and the GI50 values of 4c toward B16 and 1-87 cells are 0.013 M and 0.087 M, respectively.The boronic acid-containing combretastatin A-4 analogue 1d, which was previously synthesized as a tubulin polymerization inhibitor in our group 11 as well as Brown's group, independently, 16 was more potent in cell growth inhibition.In all cases, B16 cells are more sensitive toward the synthesized boronic acids than 1-87 cells.ClogP (partition coefficient of a compound between octanol/water speculated by structure-based speculated program) values of each compound are also shown in Table 1.According to the ClogP values, one of the low inhibitory potency of 4a and 4b toward cell growth may be due to higher hydrophilicity, which could prevent the compounds from crossing the cell membrane.Scheme 2. Synthesis of boronic acid-containing phenstatin analogue 4c.We also examined the effects of the boronic acid-containing phenstatin analogues 4a-c on in vitro polymerization of tubulin.The tubulin was purified from porcine brains according to the Shelanski protocol 18 with modification. 19Although it was reported that phenstatin significantly inhibits tubulin polymerization in vitro with an IC50 value of 1.1 M, the boronic acid containing phenstatin analogues 4a-c did not exhibited inhibition potency even at 100 M.These results indicate that the current boronic acid containing phenstatin analogues, especially compound 4c, possess significant inhibitory activity toward cell growth without interacting microtubule system.

Conclusions
We succeeded in synthesizing the boronic acid-containing phenstatin analogues 4a-4c.The lithiation of the aromatic ring B of the phenstatin with n-butyllithium is essential for introduction of a boronic acid moiety in the molecules.Among the boronic acid-containing phenstatin analogues synthesized, compound 4c, in which a hydroxyl group on the aromatic ring B of the phenstatin was replaced by a boronic acid, exhibited significant cell growth inhibition.Since the current synthesized compounds 4a-4c did not show any inhibition potency toward tubulin polymerization process in vitro, they are considered to inhibit cell growth by different mechanisms other than inhibition of the microtubule system.

Experimental Section
General. 1 H NMR and 13 C NMR spectra were measured on a JEOL JNM-AL 300 (300 MHz) or VARIAN UNITY-INOVA 400 (400 MHz) spectrometers.Chemical shifts of 1 H NMR were expressed in parts per million downfield from CDCl3 as an internal standard ( = 7.24) in CDCl3 or from CD3OD as an internal standard ( = 3.35).Chemical shifts of 13 C NMR were expressed in parts per million downfield from CDCl3 as an internal standard ( = 77.0) in CDCl3 or from CD3OD as an internal standard ( = 49.3).Analytical thin layer chromatography (TLC) was performed on a glass plates (Merck Kieselgel 60 F254, layer thickness 0.2 mm or RP-18 F254s, layer thickness 0.2 mm).Visualization was accompanied by UV light (254 nm), I2 and KMnO4.Column chromatography was performed on silica gel (Merck Kieselgel 70-230 mesh).All reactions were carried out under an argon atmosphere using standard Schlenk techniques.Most chemicals and solvents were analytical grade and used without further purification.

Table 1 .
Cell growth inhibition of boronic acid containing phenstatin analogues a The compounds were assayed at least three times and the GI50 values reported here are mean of an average of three experiments.
a b Mouse B-16 melanoma cell line.c Human lung carcinoma 1-87 cell line.d Clog P values were speculated by using structure-based calculation program, ChemBioDraw Ultra ver.12, PerkinElmer Inc.