Synthesis and in vitro Antiproliferative Activity of Flavone and 6 ‐ Hydroxyflavone Oxime Ethers Derivatives

Herein we report the synthesis of a series of O-alkyl oximes of flavone and 6-hydroxyflavone using a simple experimental protocol under solvent free conditions with yields up to 87%. Cytotoxicity of all compounds was evaluated against MDA-MB-231, PC-3, A-549 and MRC-5 cells. IC50 values for two compounds were determined to be in the range 28.7-47.8 μM against all tested cell lines. Oxime ethers derivatives showed IC50 values between 28.7 and 49.5 μM against MDA-MB-231, while the best activity was obtained for 6-hydroxyflavone with an IC50 of 3.4 μM against this cell line. Compounds containing the substituent hydroxyl at the position six of flavone system displayed the best antiproliferative activity over MDA-MB-231 cells, being necessary this group to improve the sensibility on this type of cells. The antiproliferative activity of 6-hydroxyflavone is drastically diminished when the carbonyl group of flavone is changed by an oxime ether.


Introduction
Oxime ethers are useful and versatile compounds in organic synthesis, they are commonly easily prepared and relatively stable to moisture; thus, they can be stored under air during long periods. 1 Oxime is a pharmacophore group present in a variety of compounds with diverse biological activities, for instance, some α,β-unsaturated oximes are known as anticancer, 2 immunosuppressive, 3 and antibacterial 4 agents.
On the other hand, flavone scaffold is an important core in many compounds displaying a variety of pharmacological properties that depend on their substitution patterns.The wide range of biological activities of flavones has attracted great interest in the synthesis of derivatives with the objective of developing new therapeutic agents. 5lthough oxime ether derivatives of flavones could display relevant biological activity, there are only a few reports about these kind of compounds, suggesting that these derivatives are not as promptly prepared.The first synthesis of a flavone oxime was reported in 1952 using hydroxylamine, and 4-thionflavone or 4-thionflavone methiodide as precursos. 6Previous attempts using directly hydroxylamine and a flavone conduced to 3-o-hydroxyphenyl-5-phenylisooxazole instead of the oxime. 7eshcheryakova et al. 8 reported the synthesis of some flavone oxime ether derivatives using 4,4-dichloroflavene or 4-thionflavone and O-alkyl hydroxylamines, and the evaluation of their pharmacological activity revealed that they act on the central nervous system.Green et al. 9 achieved the synthesis of 2-arylchromenone oximes using Ollis or Lawesson methods; and patented their use as inhibitors of protein kinases.
Schann et al. 10 synthesized 2-heteroarylchromenone oximes as allosteric modulators of metabotropic glutamate receptors, via the synthesis of O-t-butyl oxime ethers employing microwave (MW) irradiation in methanol, and further deprotection.They also prepared some of these compounds directly with hydroxylamine under MW, with poor yields.
Therefore, this work was undertaken with the aim of extending the knowledge about the synthesis, characterization and applications of flavone oxime ethers.
We prepared and evaluated the antiproliferative activity of twelve derivatives containing O-alkyl, O-benzyl and O-allyl moieties.

Chemistry
In order to obtain the target compounds by a previously optimized protocol, we carried out experiments using the model reaction between flavone (1a) and O-methylhydroxylamine hydrochloride (2a) in pyridine as a solvent and a base (Table 1, entry 1).Under these conditions, no evidence of the formation of compound 3a was observed after 18 h, and the flavone was mostly recovered.
Then, we adapted procedures reported in the literature for analog structures using microwave irradiation.The reaction with O-methylhydroxylamine hydrochloride in methanol under microwave for 30 minutes (entry 2) 10 did not give rise to compound 3a; thus, we decided to perform a new attempt using the former conditions, adding pyridine as the base (entry 3).This procedure allowed us obtaining the desired oxime ether 3a in a 19% according to 1 H nuclear magnetic resonance (NMR) spectra of the crude mixture, as a single isomer.
D u e t o t h e l ow r e a c t iv i t y d i s p l a y e d b y flavone, we employed the conditions reported by Meshcheryakova et al., 8 that includes the transformation of flavone in 4,4-dichloroflavene (entry 4).The reaction was monitored by gas chromatography-mass spectrometry (GC-MS) and the dichloride intermediate was detected after the first step (a), but no evidence of the formation of 3a was observed after the second one (b).
A fifth experiment (entry 5), employing 6 equivalents of both O-methylhydroxylamine hydrochloride and dry pyridine at 140 °C was carried out in order to displace the equilibrium towards the formation of oxime ether 3a.After 2 h under these conditions, the starting material was not completely consumed, but the desired oxime ether was observed in 75% by 1 H NMR.
Guided by the previous result, and with the aim of finding the molar ratio of the reagents that allowed to obtain the highest yield of 3a, we performed several experiments varying the equivalents of both 2a and pyridine.Finally we found that conditions shown in entry 6, afforded the maximum amount of product (75% calculated by 1 H NMR, 54% isolated), with the lowest equivalents of starting materials.
Based on the results shown above, we synthesized a series of oxime ethers of flavone and 6-hydroxyflavone (3b-l) using the experimental conditions of entry 6.In order to synthesize oxime ethers 3c-f,i-l, it was necessary to obtain the O-allyl and O-benzylhydroxylamines 5c-f no commercially available, via Gabriel synthesis, and their hydrochlorides 2c-f (Scheme 1).
The E/Z geometries of oxime ethers 3a-l, were determined by 2D NOESY (nuclear Overhauser effect spectroscopy) experiments, which allowed to evidence correlations between hydrogens of O-CH 2 group and the heterocycle ring, showing that the E isomer was the most favored product.

Antiproliferative activity
Cytotoxicity of all compounds was evaluated against MDA-MB-231, PC-3, A-549 and MRC-5 cells.Flavone (1a)   and its oxime derivatives (3a-f) showed no relevant activity at the evaluated concentrations.IC 50 values for compounds 3h,i presenting a hydroxyl and a benzyl group, were determined to be in the range of 28.7-47.8μM for all tested cell lines.Compounds 3g-l containing a hydroxyl group at sixth position, showed IC 50 between 28.7-49.5 μM against MDA-MB-231; however, the best activity was obtained for 6-hydroxyflavone (1b) with an IC 50 of 3.4 μM against the same cell line (Table 2).Previous studies have shown that 6-hydroxyflavone also exhibits activity over the leukemia cell lines HL-60 (IC 50 = 2.8 μM) and MOLT-4 (IC 50 = 6.3 μM). 11It is important to emphasize that no antiproliferative activity was displayed by compound 1b against healthy fibroblasts from lung (MRC-5).
On the other hand, the antitumor activity of baicalein, a naturally ocurring flavonoid used in Chinese herbal medicine, which contains hydroxyl groups at positions 5, 6 and 7, has motivated to develop studies aimed to verify the inhibitory effects and the mechanism involved in its antimetastatic effect against MDA-MB-231 cells.The IC 50 value of baicalein (59.5 μM), 12 higher than the IC 50 determined for 6-hydroxyflavone in this work over the same cell line, motivates our interest in continuing the study of this compound.
It is important to notice that despite the IC 50 values found in this work are not comparable with the reference drug vincristine (IC 50 = 0.008 μM against MDA-MB-231), the search for novel cytotoxic agents with selectivity for a particular cell line, and the comprenhension of mechanisms of action, are necessary tasks towards the development of new drugs that allow to improve the existing anticancer therapies.The IC 50 value was defined as the concentration of the compound which caused a 50% decrease of the cell viability.

Conclusions
The synthesis of twelve flavone and 6-hydroxyflavone oxime ether derivatives was achieved with reasonably good yields as a single isomer (E), using a simple experimental protocol.Changing the carbonyl group of flavone for an oxime ether did not increase the cytotoxic activity of the derivatives against the tested line cells.Hydroxyl group at the position six of flavone system is necessary to display activity against MDA-MB-231 since all compounds containing this substituent showed antiproliferative activity over this cell line.The antiproliferative activity of 6-hydroxyflavone is drastically diminished when the carbonyl group of flavone is changed by an oxime ether.

Experimental
Chemistry 1 H and 13 C NMR spectra were acquired on a Bruker Avance spectrometer (300 and 75 MHz, respectively) in CDCl 3 or CD 3 OD.Chemical shifts and configuration were assigned with the help of HSQC-edit (heteronuclear single quantum correlation), HMBC (heteronuclear multiple bond correlation), COSY (correlation spectroscopy) and NOESY experiments.High-resolution mass spectra (HRMS) were recorded on an Agilent 6520 q-TOF-MS instrument with orthogonal ESI (electrospray ionization).GC-MS analyses were performed on an Agilent 6850 series II gas chromatograph coupled to an Agilent 5975B VL mass spectrometer (electron ionization, 70 eV) equipped with split/splitess inlet (split relation 15:1, 260 °C), Agilent 6850 series automatic injector, and Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm); initial oven temperature 80 °C for 1 min, then a temperature ramp of 10 °C min -1 to 320 °C (hold 3 min); total run time 28 min.Melting points were determined on a Thermo Fisher Scientific Rochford 9100 apparatus.

Method A (entry 3)
A solution of flavone 1a (1.0 mmol), O-methyl hydroxylamine hydrochloride (2.0 mmol), and pyridine (2.0 mmol) in methanol (6.6 mL), was irradiated at 150 W for 30 minutes.The reaction mixture was treated with distilled water and extracted with dichloromethane.The organic phase was dried with Na 2 SO 4 , filtered and concentrated under reduced pressure.The crude mixture was purified by column chromatography using silica gel and hexane:CH 2 Cl 2 (4:1), yielding 5% of 3a.

Method B (entry 6)
A mixture of flavone or 6-hydroxyflavone 1a-b (1.0 mmol), an appropriate O-alkyl hydroxilamine hydrochloride (2a-e) (2.5 mmol), and dry pyridine (3.5 mmol), was stirred at 140 °C for 2-4 h.The reaction mixture was treated with distilled water and extracted with dichloromethane.The organic phase was dried with Na 2 SO 4 , filtered and concentrated under reduced pressure.The organic combined were purified by column chromatography (silica gel, hexane:CH 2 Cl 2 4:1 or CH 2 Cl 2 ) to provide products 3a-l.

Synthesis of O-alkylhydroxylamine hydrochlorides
To a stirred solution of hydrazine sulfate (1.2 mmol), K 2 CO 3 (1.2mmol) and water (2.0 mL) in THF (2.0 mL), was added the corresponding O-alkyl-N-hydroxyphthalimide (1.0 mmol), and the mixture was refluxed during 5 h, then distilled water (10 mL) was added.The resulting mixture was extracted with ethyl ether (3 × 10 mL), the organic phase was dried with sodium sulfate, filtrated and the solvent evaporated under reduced pressure.The residue was chromatographed by CC (column chromatography) using silica gel, Hex:AcOEt 7:3 to give the corresponding hydroxylamine.Subsequently the hydroxilamine was disolved in dried ethyl ether and HCl(g) was bubbled to obtain the salt (3c-f).

Table 1 .
Evaluation of reaction conditions to obtain compound 3a