Bifunctional Phenol Quinone Methide Precursors : Synthesis and Biological Activity

New bifunctional quinone methide (QM) precursors, bisphenols 2a–2e, and monofunctional QM precursor 7 were synthesized. Upon treatment with fluoride, desilylation triggers formation of reactive intermediates, QMs, which was demonstrated by trapping QM with azide or methanol. The ability of QMs to alkylate and cross-link DNA was assayed by investigation of the effects of QMs to DNA denaturing, but without conclusive evidence. Furthermore, treatment of a plasmid DNA with compounds 2a–2e and KF, followed by the analysis by alkaline denaturing gel electrophoresis, did not provide evidence for the DNA cross-linking. MTT test performed on two human cancer cell lines (MCF7 breast adenocarcinoma and SUM159 pleomorphic breast carcinoma), with and without fluoride, indicated that 2a–2e or the corresponding QMs did not exhibit cytotoxic activity, in line with the lack of ability to cross-link DNA. The lack of reactivity with DNA and biological activity were explained by sequential formation of QMs where bifunctional cytotoxic reagent is probably never produced. Instead, the sequential generation of monofunctional QM followed by a faster hydrolysis leads to the destruction of biologically active reagent. The findings described here are particularly important for the rational design of new generation of QM precursor molecules that will attain desirable DNA reactivity and cytotoxicity.


INTRODUCTION
UINONE methides (QMs) are important intermediates in chemistry and photochemistry of phenols, [1] owing to their applications in organic synthesis, [2] and biological activity. [3]It has been demonstrated that QMs react with amino acids [4] and proteins, [5] and inhibition of some enzymes has been reported including hydroxylases, [6] β-lactamase, [7] β-glucosidases, [8] phosphatase [9] or ribonuclease-A. [10]However, biological action of QMs has mostly been related to their reactivity with nucleosides [11] and alkylation of DNA, [12] since some anticancer antibiotics such as mitomycin [13] exert their antiproliferative action on metabolic formation of QMs that alkylate DNA.In particular, S. Rokita et al. demonstrated reversible alkylation ability of QMs leading to "immortalization of QM" by DNA as a nucleophile, [14] whereas Freccero et al. investigated ability of QMs to alkylate G4 regions of DNA. [15]Ms are very reactive species that cannot be stored, they have to be generated in situ.12b,c] On the other hand, photochemical methods offer much milder approach to QMs, particularly for biological systems. [19]The most common reactions to generate QMs in photochemical reactions are photodehydration [20] and photodeamination from the suitably substituted phenols. [4,21]An on-going interest in our group is the photochemical generation of quinone methides (QM) from suitable precursors, and investigation of their biological effects. [22]In particular, we have recently demonstrated that photogenerated QM Q formed from anthrol 1 (Scheme 1) exhibit higher cytotoxicity on cancer stem cell lines then on normal cancer cells. [23]22d,23] However, all molecules that we studied to date were monofunctional, so in principle, they could only alkylate DNA.On the other hand, DNA cross-linking by bifunctional molecules is known to be the most cytotoxic event leading to the cell death. [24]erein we report an investigation of F − induced desilylation and generation of bi-functional QMs that are anticipated to enable DNA cross-linking (Figure 1).Bisphenol derivatives 2 are bi-functional derivatives of phenols that are substituted by TBDMS at the phenolic oxygen and by acetyl at the benzyl alcohol.12b,c] The QM precursors are separated by alkyl spacer of different lengths to probe for the effect of molecular structure to the efficiency of DNA crosslinking.
Formation of QMs was probed by F − induced reactions and trapping with nucleophiles.Antiproliferative activity of bisphenols was investigated in vitro against two human cancer cell lines: MCF-7 (breast adenocarcinoma) and SUM159 (pleomorphic breast carcinoma).The idea was to link the antiproliferative activity to the ability of QMs to cross-link DNA molecules.Interestingly, treatment of plasmid DNA in the presence of bisphenols and analysis by alkaline agarose electrophoresis, or measurement of DNA denaturation show that QMs do not cross-link plasmid DNA, and the reasons for the lack of reactivity were disclosed.carbaldehyde.Salicylaldehyde component was TBDMSprotected 3 [25] (for the preparation see the SI) that reacted with double Grignard reagent formed from 1,n-dibromoalkane.The reaction gave alkoxide intermediates that can be quenched by H2O and isolated as alcohols 4, or acetylated in one-pot (Scheme 2).An attempt to quench the alkoxide with acetic anhydride failed, but the treatment with acetyl chloride was successful giving acetyl esters.The one pot procedure gave better yields and one purification step less was needed.It should be noted that the double Grignard reaction created two stereogenic centers in 4 or 2, and therefore, a mixture of two enantiomers (RR, SS) and the meso-compound (SR) were obtained, which were inseparable by achiral chromatography.On attempts to prepare bisphenol containing alkyl spacer with less than 6 C-atoms, in the Grignard reaction with 1,4-dibromobutane or 1,3-dibromopropane, a reduction of the TBDMS-protected aldehyde took place giving benzyl alcohol 5.Such β-hydride transfer reactions are known to compete with Grignard reactions, [26] particularly with sterically congested carbonyl groups. [27]In our case, the di-Grignard reagent formed from 1,4-dibromobutane contained reactive H-atoms at the β-position (Figure 2), allowing the facile β-hydride transfer and preventing the addition to the carbonyl group.Furthermore, based on literature precedent, formation of di-Grignard reagent from 1,3-dibromopropane is proble-matic since the di-Grignard reagent is very unstable. [28]Therefore, only bisphenols containing more than 6 C atoms in the linker were synthesized.In addition to bisphenol derivatives, we have synthesized also model compounds 6 and 7 (Figure 3), that contain only one phenol moiety, and which were needed in the biological investigations.

QM Formation by F − Induced Desilylation
Formation of QM in the desilylation reaction was first probed on model compound 7.The compound was dissolved in DMSO and diluted with PBS buffer (pH 7.4).Formation of QM was initiated by addition of KF (incubation 1 h).The formation of QMs was proven by quenching with nucleophiles, NaN3, the ubiquitous QM quencher [19] whereupon adduct 8 was isolated by preparative HPLC (Scheme 3).Furthermore, we have shown that adducts were formed practically immediately after the addition of KF, whereas in the absence of KF adducts were not formed over 24 h.
To show that bisphenols can also undergo F − desilylation giving QMs we have treated 2c with KF in methanolic solution.The reaction gave two products 9 and 10 (Scheme 4) that were isolated and characterized.Formation of 10, where only one benzyl alcohol is substituted by methoxy group indicates that formation of QMs and methanolysis probably take place sequentially.That is, upon formation of a QM, the reaction of solvolysis probably takes place more rapidly than formation of the second QM in the same molecule.Thus, it is not probable that one molecule contains two QM centers at the same time.

Investigation of DNA Cross-linking Ability of Compounds
After demonstrating that F − induces formation of QMs, DNA cross-linking ability of bisphenols 2 was assayed.Plasmid pCMVbeta DNA (2 μg) in PBS buffer was mixed with 2a-2e and 7 (1 mM), and treated with KF (200 mM).After 18 h incubation, the alkaline agarose gel electrophoresis was performed, as described in the Experimental section.12d] Interstrand cross-linking activities of psoralen were evident as X-band of circular form (X-CC).However, no X-bands and no difference in migratory ability compared to control DNA were observed in the presence of 2 (Figure 4).

Investigation of the DNA Cross-linking by Thermal Denaturation
Compounds that bind to DNA cause stabilization of the duplex and increase of the DNA melting temperature (∆Tm).
Similarly, we anticipated that the covalent binding, and particularly, cross-linking of DNA should affect the ∆Tm.Therefore, the ability of compounds 2b-2e to form bifunctional QMs and cross-link DNA was assayed also by their effects on ∆Tm (Figure 5 and Figures S1 and S2 in the supporting information).The problem in the measurement represented poor solubility of 2 under these conditions.Therefore, the measurement was conducted in H2O-DMSO solvent mixture (9:1, or 8:2 for 2c), containing sodium cacodylate buffer (c = 0.05 M, pH = 7.0).Under such conditions, ct-DNA revealed Tm = 75.1±0.5 °C.Addition of compounds alone or in combination with KF did not affect the Tm significantly, changes being within the error of the method (±0.5 °C, Figure 5), suggesting that the eventual alkylation and cross-linking was either of very low efficiency (leaving large amount of free DNA available for denaturation) or did not take place at all.However, the thermal denaturation experiment could not give the unambiguous conclusion whether QMs from 2 were able to alkylate and cross-link at least some of DNA, since even low cross-linking efficiency could have measurable biological effect.Therefore, we decided to assay these systems on human cancer cell lines.

Antiproliferative Activity
Antiproliferative effect of the F − generated QMs on two cancer cell lines, MCF7 (breast adenocarcinoma) and SUM159 (pleomorphic breast carcinoma) was investigated.Cells were divided into two groups; cells treated with compounds and KF, and cells that were treated only with compounds.KF was added two hours after the compounds in order to allow bisphenols to permeate the cell's membrane.Cells were incubated with all of the compounds for three days.The activities expressed as IC50 (concentration that causes 50 % inhibition of the cell growth) are compiled in Table 1.In general, all of the compounds exhibited low antiproliferative activity.Compound 7 had moderate activity toward MCF7 cells.We observed enhancement of cytotoxicity in SUM159 cells upon activation of compound 7 with KF, but this effect could also be attributed to toxicity of KF toward SUM159 cell line (Figure S4).Small improvement of cytotoxicity was also observed in MCF7 cells after activation of compound 2d.

DISCUSSION
12b,c] Furthermore, KF induced methanolysis of bisphenol 2c, indicating that formation of QMs takes place upon treatment of 2a-2e with KF.However, we do not have any evidence if the formation of bis-QM takes place (such as 2a-bisQM, Scheme 5).Namely, it is plausible that F − induced formation of QM takes place stepwise giving 2a-QM.In principle, this monofunctional QM molecule can undergo two competitive reactions, hydrolysis to 11 and secondary elimination to Table 1.IC50 values (in µM) (a) induced with compounds 2 and 7 Pathways presented in Scheme 5 may account for the lack of DNA cross-linking with compounds 2. Namely, the cross-linking only feasible if is formed, or if 2a-QM reacts faster with DNA then with H2O.Although, the product 11 forms 11-QM, it can only alkylate DNA and cannot induce DNA cross-linking.This situation is different to bifunctional phenols bearing leaving groups at the position 2 and 6, such as 13.We have demonstrated by transient spectroscopy that 13 undergoes photodeamination giving 13-QMa.21b] This specific reactivity of bifunctional QMs leads to long-lived transient species and enable reversible reactions of the QM along a DNA chain as if the QM "walks on the DNA". [29]ttempts to induce DNA cross-linking by 2a-2e and 7, and probe the process by the alkaline agarose gel electrophoresis or thermal denaturation failed, or gave no unambiguous conclusion.Furthermore, we have shown that compounds in the presence of KF do not exhibit cytotoxicity.These findings indicate that bisphenols 2 probably do not form bifunctional QMs.Consequently, they cannot cross-link DNA and do not show significant cytotoxicity. of QMs to alkylate and cross-link DNA was assayed by investigation of the effects of QMs to DNA denaturing and the treatment of a plasmid DNA with compounds 2a-2e and 7, and KF, followed by the by alkaline denaturing electrophoresis.Both methods did not provide evidence for the DNA cross-linking, in agreement with the MTT test performed on two human cancer cell lines (MCF7 breast adenocarcinoma and SUM159 pleomorphic breast carcinoma), which indicated that or the corresponding QMs did not exhibit significant cytotoxic activity.The lack of DNA reactivity was rationalized by sequential formation of QMs where bifunctional cytotoxic reagent is probably never produced.Instead, the sequential generation of monofunctional QM followed by a faster hydrolysis leads to the destruction of biologically active reagent.The findings described here are particularly important for the rational design of new generation of QM precursor molecules that should have structures that enable long-lived QM species such as 2,6-bifunctional phenols.

General
Chemicals for the synthesis were purchased from the usual suppliers, whereas solvents for the synthesis and chromatographic separations were purified by distillation, or used as received (p.a.grade). 1 H and 13 C NMR spectra were recorded on a Bruker AV-300, 400 or 600 MHz.The NMR spectra were taken in CDCl3 or DMSO-d6 at rt using TMS as a reference.HRMS were obtained on an Applied Biosystems 4800 Plus MALDI TOF/TOF instrument (AB, Foster City, CA).For the sample analysis a Shimadzu HPLC equipped with a Diode-Array detector and a Phenomenex Luna 3u C18(2) column was used.Mobile phase was CH3OH-H2O (20 %).For the chromatographic separations silica gel (Merck 0.05-0.2mm)was used.Analytical thin layer chromatography was performed on Polygram® SILG/UV254 (Machery-Nagel) plates.In the irradiation experiments, CH3CN and MeOH were HPLC grade pure, and mQ-H2O (Millipore) was used.ct-DNA was purchased from Aldrich.Preparation of known precursor molecule 5 is fully described in the ESI.

One-pot Synthesis of 7
In a two neck flask (25 mL), equipped with a condenser and septum, under inert N2 atmosphere, Mg (0.060 g, 2.47 mmol), dry THF (2mL) and a crystal of iodine were added.A small quantity of the solution of bromoethane (0.18 mL, 2.41 mmol) in THF (5 mL) was added dropwise through the septum via a syringe.When the reaction was initiated by heating, the remaining bromoethane solution was added at rt.After the addition was complete, the reaction mixture was heated at the temperature of reflux for 1 h, cooled to rt, and a solution of the protected salicylaldehyde (3, 0.47 g, 2.00 mmol,) in THF (3 mL) was added dropwise.The reaction mixture was stirred at rt for 1 h, and then acetyl chloride (0.18 mL, 2.53 mmol) was added and the stirring was continued over 2 h.The solvent was removed on a rotary evaporator and the residue was purified on a column of silica gel using hexane/diethyl ether (9:1) as eluent to afford the pure product (0.36 g, 58 %) in the form of colorless viscous oil.

Preparation of Bisphenol Derivatives 4 -General
Procedure In a two neck flask (50 mL), equipped with a condenser and septum, under inert N 2 atmosphere, Mg (1.5 mmol), dry diethyl ether (1 mL / 1 mmol Mg) and a crystal of iodine were added.A small quantity of the solution of 1,ndibromoalkane (0.6 mmol) in ether (5 mL / 1 mmol) was added dropwise through the septum via a syringe.When the reaction was initiated by heating, the remaining dibromoalkane solution was added at rt or with a moderate heating.After the addition was complete, the reaction mixture was heated at the temperature of reflux for 1 h, cooled to rt, and a solution of the protected salicylaldehyde (3, 1 mmol) in dry ether (2.5 mL) was added dropwise.The reaction mixture was stirred at rt for 2 h, and then a saturated aqueous solution of NH4Cl (5 mL) was added and the mixture was stirred for 15 min.The mixture was transferred to a separatory funnel, the layers were separated and the aqueous layer was extracted with ethyl acetate (3×15 mL).The organic extracts were dried over anhydrous MgSO4, fil-tered, and the solvent was removed on a rotary evaporator.The residue was chromatographed on a column of silica gel using hexane/ethyl acetate (7:3) as eluent.

Acetylation of 4 -General Procedure
A two neck flask (20 mL), equipped with a condenser and septum, under N2 inert atmosphere, was charged with 4 (1 mmol), ether (10 mL) and pyridine (4.4 mmol).The reaction mixture was heated reflux for 5 and then cooled to rt.Acetyl chloride (4.5 mmol) was added and the mixture was heated at reflux for 30 min.The solvent was removed on a rotary evaporator and the residue was chromatographed on a column of silica gel using hexane/diethyl ether (9:1) as eluent to afford the pure product.

One-pot synthesis of 2 -general procedure
In a two neck flask (50 mL), equipped with a condenser and septum, under inert N2 atmosphere, Mg (1.3 mmol), dry ether (2mL) and a crystal of iodine were added.A small quantity of the solution of the 1,n-dibromoalkane (0.5 mmol) in ether (5 mL) was added dropwise through the septum via a syringe.When the reaction was initiated by heating, the remaining dibromoalkane solution was added at rt.After the addition was complete, the reaction mixture was heated at the temperature of reflux for 1 h, cooled to rt, and a solution of the protected salicylaldehyde (3, 1 mmol) in ether (3 mL) was added dropwise.The reaction mixture was stirred at rt for 2 h, and then acetyl chloride (2.2 mmol) was added and the stirring was continued over 2 h.The solvent was removed on a rotary evaporator and the residue was purified on a column of silica gel using hexane/diethyl ether (9:1) as eluent to afford the pure product in the form of colorless viscous oil.

Methanolysis of 2c
Bisphenol 2c (30 mg, 0.05 mmol) was dissolved in methanol (2.0 mL), and a solution of KF (19 mg, 0.33 mmol) in H2O (0.3 mL) was added dropwise.The mixture was stirred at rt for 1 h, transferred to a separatory funnel, diluted with H2O (10 mL) and extracted with diethyl ether (3 × 5 mL).The combined organic extracts were dried over anhydrous MgSO4, filtered and the solvent was removed on a rotary evaporator.The residue was purified on a column of silica gel using cyclohexane/EtOAc (7:1) as eluent to afford bisphenol 9 (8 mg, 49 %) and 10 (6 mg, 33 %) in the form of colorless viscous oils.

Alkaline Agarose Gel Assay
Plasmid CMVbeta DNA (2 μg) was mixed with bisphenols 2a-2e (1 mM) and KF (200 mM) in PBS buffer (pH = 7.4).After 18 h incubation, the samples were added to the alkaline agarose gel loading buffer [50 mM NaOH, 1 mM ethylenediaminetetraacetic acid (EDTA), 3 % Ficoll, and 0.02 % bromophenol blue] and loaded on a 1 % agarose gel.Prior to the loading, the gel was soaked in 50 mM NaOH and 1 mM EDTA for 1h, and the same solution was used as the running buffer.Gels were run at 30 V constant voltage in horizontal electrophoresis system (BIO-RAD, USA) for 5 h.After the run, gels were neutralized with 0.5 M Tris (pH 7) for 30 min, and stained with ethidium bromide (1 μg/mL) for 30 min.Resulting products were visualized and documented with UV light at 254 nm (Image Master VDS, Pharmacia Biotech, Sweden).As a positive control, instead of 2 and KF, the plasmid was treated with psoralene (20 µM) and irradiated in a Luzchem reactor 5 min with 6 lamps (1 lamp 8 W) at 300 nm.

Scheme 5 .
Scheme 5. Plausible pathways for the formation of QM and bisQM from 2a and their hydrolysis.

Figure 6 .
Figure 6.Structure of bifunctional QM precursor 13 and the corresponding QMs.