Design, Synthesis, and Anti-Proliferative Evaluation of [1,1′-biphenyl]-4-ols as Inhibitor of HUVEC Migration and Tube Formation

Allylated biphenol neolignans contain a variety of chemopreventive entities that have been used as anti-tumor drug leads. Herein, 37 allylated biphenols were evaluated for anti-proliferative activity by the MTT assay and inhibitory effect on the migration and tube formation of HUVECs featuring anti-angiogenic properties. 3-(2-Methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (5c) exerted an inhibitory effect on HUVECs compared to honokiol (IC50 = 47.0 vs. 52.6 μM) and showed significant blocking effects on the proliferation of C26, Hela, K562, A549, and HepG2 (IC50 = 15.0, 25.0, 21.2, 29.5, and 13.0 μM, respectively), superior to those of honokiol (IC50 = 65.1, 62.0, 42.0, 75.0, and 55.4 μM, respectively). Importantly, compound 5c inhibited the migration and capillary-like tube formation of HUVECs in vitro.

newly-grown segmental vessels from the dorsal aorta of zebrafish, and inappropriate vascularisation in the transgenic zebrafish screening model.

Chemistry
As depicted in Scheme 1, 1-allyloxy-4-bromobenzene (2) was prepared in a satisfactory yield (95%) by a convenient procedure starting from commercially available p-bromophenol and employing 1.1 equiv of allyl bromide in the presence of anhydrous K 2 CO 3 (1.3 equiv) as base and acetone as solvent. The intermediates 3a-t were obtained through Suzuki-Miyaura reactions [17,18]. Two cross-coupling methods were developed for the condensation of appropriate arylboronic acids with 2 under palladium catalysis under a N 2 atmosphere. Method A involved the use of Pd(OAc) 2 as a cross-coupling catalyst, PPh 3 as a reducing agent and K 2 CO 3 (2.0 M) as a base in isopropanol as solvent. The intermediates 3a-i was obtained by Method A in 55-80% yields. Another coupling method (used for 3j-t) was carried out by using Pd(PPh 3 ) 4 as a catalyst, 2.0 M K 3 PO 4 ·3H 2 O as a base and DMF as solvent. The intermediates 3j-t were obtained by Method B in much better yields (75-90%). The O-allylation of 3 with 3-bromoprop-1-ene or 1-bromo-3-methylbut-2-ene was performed in a good yield in the presence of anhydrous K 2 CO 3 . Next, the corresponding compounds 4a-t and 5a-c were synthesized through the Claisen rearrangement, which belongs to the [3,3]-sigmatropic concerted rearrangement category, in N,N-diethylaniline (boiling point = 216 °C) as solvent [19,20]. Moreover, the other allylated biphenols 6a-j and 7a-d were prepared following similar synthetic methods (Scheme 2).

Anti-Proliferative Activity and SAR Study
In order to find new potential angiogenic inhibitors, 37 allylated biphenols were primarily investigated for anti-proliferative activity on C26 and Hela tumor cells by the MTT assay and these results are shown in Table 1. In addition, predicted octanol/water log P (Clog P, miLog P, and Xlog P) were calculated using the ChemDraw software, Molinspiration online service and XlogP3 online service, respectively, to provide a measure of lipophilicity. Honokiol, a potent anti-angiogenic and anti-tumor drug lead, was selected as a positive control.
A relevant strategy for anti-angiogenesis is effectively inhibition of the proliferation of ECs. Thus, the result of anti-proliferative activity against HUVECs was selected as the main index and the IC 50 values against A549 and HepG2 cells were also outlined in Table 2. With comparable or superior inhibitory potency against C26 and Hela cells compared to honokiol, nine allylated biphenols were next selected for biological evaluation, with 5c being the most efficient. Compound 5c exhibited a moderate inhibitory activity against the proliferation of HUVECs (IC 50 = 57.0 μM) in contrast to honokiol (IC 50 = 40.0 μM) and exerted remarkable cytotoxic activities against A549 and HepG2 cells (IC 50 = 29.5 and 13.0 μM, respectively). At the same time, 7c presented comparable inhibitory potencies. The anti-proliferative effects on HUVECs and the inhibitory activities against A549 and HepG2 of tested compounds showed consistent tendencies. In addition, there was a potential positive correlation between the lipophilicity (Log P and cLogP) and inhibitory potency.

Effects on HUVEC Migration
EC migration is a relevant process in chemotaxis and an indispensable step to form new blood vessels. Inhibition on the process could block the formation of new blood vessels and further suppress the development of cancer. Therefore, to characterize the effects of allylated biphenols 5c and 7c on HUVEC migration, an in vitro migration assay was performed by the application of a slightly modified Boyden chamber. As depicted in Figure 1I, the HUVECs actively migrated to the serum-containing lower chamber within 6 h under the untreated conditions (control). Compared to the control, the mean number of invaded HUVECs (% of control group) treated with 5c and 7c at the concentration of 40 μM were 7.1 and 14.2%, respectively, and the cell migration was not likely to occur [ Figure 1(II)]. After treatment with 5c and 7c, even at 20 μM, the inhibitory effects on cell migration were also effective and the mean invasion rates remained below 50.0%. However, at 10 μM, the two biphenols hardly inhibited HUVEC migration. As a consequence, 5c and 7c exerted the potent inhibitory activity on the migration of HUVECs in a concentration-dependent manner and 5c was more potent than 7c. In the later stages of angiogenesis, ECs will assemble into an interconnected tubular network which is similar to in vivo capillary vascular beds. Inhibition on this formation of capillary-like tube networks will block the formation of new blood vessels. A tube formation assay was performed by plating HUVECs on Matrigel. In the blank control, the cells exhibited high mobility on Matrigel and constructed an intact tube network in 24 hours [ Figure 2(I)]. Compared to the control, the mean number of tube formation (% of control) treated with 5c and 7c at the concentration of 40 μM were 3.7 and 4.7%, respectively, and the disrupted tubular structures were sparse and incomplete [ Figure 2(II)]. After the treatment with 5c and 7c, even at 20 μM, the inhibitory effects were also effective and the mean tube formation remained at 23.4 and 40.6%, respectively. However, served at the low concentration of 10 μM, the two compounds were totally unable to inhibit tube formation of HUVECs. Our observation indicated that allylated biphenols (5c and 7c) could effectively terminate the formation of capillary-like tube networks and the effect of 5c was superior to that of 7c at the same concentration.

Chemistry
Chemical reagents of analytical grade were purchased from Chengdu Changzheng Chemical Factory (Sichuan, China). 1 H-NMR spectra were recorded at 400 MHz on a Varian Gemini 400 spectrometer (Varian, Palo Alto, CA, USA) and are reported in parts per million. Chemical shifts (δ) are quoted in ppm relative to the internal standard tetramethylsilane (TMS), where (δ) TMS = 0.00 ppm. The multiplicity of the signals is indicated as s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet defined as all multipeak signals where overlap or complex coupling of signals makes definitive descriptions of peaks difficult. Mass spectra were measured by a Premier quadrupole-time of flight (Q-TOF) mass spectrometer (Micromass, Manchester, UK) utilizing electrospray ionization (ESI). The purity of compounds was determined to be ≥97% by HPLC analysis using a photodiode array detector (Waters, Milford, MA, USA) and the chromatographic column was an Atlantis C 18 (150 mm × 4.6 mm, i.d. 5 μm) (Waters, Milford, Ireland). All compounds were dissolved as 0.1 mg/mL solutions in HPLC quality methanol with 10 μL injected on a partial loop fill at a flow rate of 1 mL/min and the column chamber was kept at 20 °C for the analysis. The moble phases were 70% methanol and 30% water (0.1% formic acid).

General Procedure Step I for the Preparation of 1-Allyloxy-4-Bromobenzene (2)
Allyl bromide (1.9 mL, 22 mmol) was slowly added into a solution of p-bromophenol (3.46 g, 20 mmol) and anhydrous K 2 CO 3 (3.6 g, 26 mmol) in acetone (25 mL), and the mixture was refluxed for 5 hours (TLC monitoring). After completing and cooling, the mixture was filtered to remove the solid and the filtrate was evaporated to dryness. The residue was extracted with diethyl ether (20 mL × 3) and 10% NaOH (20 mL × 2) and then the organic layer was combined and washed by brine (20 mL × 2), dried over anhydrous MgSO 4 , and concentrated under reduced pressure to afford a colorless oil (4.05 g, 19.01 mmol, 95.0%).

Step II for the Preparation of Intermediates 3 by Suzuki-Coupling Reaction
Method A (for 3a-i): Compound 2 (1.0 mmol) and arylboronic acids (1.2 mmol) were dissolved in isopropanol (4 mL) at room temperature and stirred for 10 min. After a clear solution was formed, Pd(OAc) 2 (0.01 mmol), PPh 3 (0.03 mmol) and anhydrous K 2 CO 3 (2.0 mol/L, 1 mL) were quickly added under a N 2 atmosphere, and the resulting mixture was stirred for a further 18 hours at 90 °C (TLC monitoring). After completion of the reaction, the mixture was filtered, and extracted with ethyl acetate (10 mL × 3). The extracts were combined and washed with brine (10 mL × 2), dried over anhydrous MgSO 4 , and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1:10) to give the intermediates 3a-i in satisfactory yields (55-80%).
Method B (for 3j-t): Compound 2 (1.0 mmol) and arylboronic acids (1.2 mmol) were dissolved in DMF (4 mL) at room temperature and stirred for 5 min. After a clear solution was formed, Pd(PPh 3 ) 4 (0.01 mmol), and a solution of K 3 PO 4 ·3H 2 O (2.0 mmol) in water (1 mL) were quickly added under a N 2 atmosphere, and the mixture was heated for 18 h at 100 °C (TLC monitoring). After completion, the reaction mixture was filtered, and extracted with ethyl acetate (10 mL × 3). The extract was combined and washed with brine (10 mL × 2), dried over anhydrous MgSO 4 , and concentrated under reduced pressure. The residue was purified by gel chromatography (ethyl acetate/petroleum ether = 1:10) to give the intermediates 3j-t in satisfactory yields (75-90%).

Step III for the Preparation of 4a-t
Allyl bromide or 1-bromo-3-methylbut-2-ene (1.3 mmol) was added to the solution of 3 (1.0 mmol) and anhydrous K 2 CO 3 (2.0 mmol) and refluxed for 5 hours. After completion (TLC monitoring) and cooling, the mixture was filtered, and the solution was evaporated to dryness. Next, the crude product was dissolved in N,N-diethylaniline and refluxed for 10 hours under a N 2 atmosphere (TLC monitoring). After completion and cooling, the solution was adjusted to pH = 4 with 2.5 N HCl and extracted with ethyl acetate (10 mL × 2) and the organic layer was combined and washed with water (10 mL × 2) and brine (10 mL × 1), dried over anhydrous MgSO 4 , and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1:10) to give the targeted 4a-t. The yield represents the total yield of the above four steps. The chemical and structural elucidation of eighteen compounds (4a-g, 4j, 4t, 5a-b, 6a-b, 6d-e, 6i-j, and 7a-b) have been reported in our previous publication [17].

Conclusions
To date, suppression of angiogenesis-dependent tumor growth has been a widely accepted strategy for cancer therapy. Although acquired drug resistance remains an insurmountable obstacle of tumortargeting therapy, it is unlikely to occur or at least at a low rate if the genetically stable ECs are targeted. Thus, ECs have been proven to be an attractive and potent target for angiogenesis therapy [21]. In the present study, 37 allylated biphenols represented a novel biphenyl structural motif and possessed a unique mode of action in anti-angiogenic and anti-tumor activity. In detail, 3-(2-methylbut-3-en-2-yl)-3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-ol (5c) showed the strongest inhibitory effects on the proliferation, migration and tube formation of HUVECs featuring anti-angiogenic properties, and its average anti-proliferative activities against four tumor cell lines (C26, Hela, A549, and HepG2) demonstrated that the biologically active allylated biphenol molecule has dual functions and the inhibitory effect of 5c was specific on HUVEC migration and tube formation, rather than resulting from its cytotoxicity.