Synthesis and biological evaluation of some new 2-oxazoline and salicylic acid derivatives

Starting from methyl salicylate and 2-amino-2-(hydroxymethyl)propane-1,3-diol 1a , or 2-amino- 2-methylpropane-1-ol 1b , the 2-oxazoline derivatives 2a , 2b or 3, as well as mono- 4a and 4b and bis- 5a and 5b derivatives of salicylic acid were synthesized. Reactions were performed by microwave irradiation in the presence of tetrabutylammonium bromide or metallic sodium as catalyst, as well as by conventional heating. Microwave-induced reaction of some diols, diamines and amino alcohols with methyl salicylate gave mono- and/or bis- derivatives of salicylic acid 4c, 5c, 5d, 6c, 8c, 7a, 7b, 8a and 8b . The mono- and bis-salicyloyl derivatives 4c , 5c and 5d were transformed to the corresponding phenyl-azo derivatives 9, 10c and 10d . The structure of compound 3 was proved by the X-ray analysis and the R-configuration on its stereocenter was confirmed. The antioxidant and cytotoxic activities of the synthesized derivatives were evaluated in a series of in vitro tests. Compounds 5d , 8b and 8c exhibited very strong activity against hydroxyl radical. Six 4c , 5d , 8a-c , 10c of 16 tested compounds inhibited growth of MDA-MB-231 cells at a nanomolar concentration. Compounds 8c and 10c showed high cytotoxicity against MCF7 cells, whereas compounds 4c , 5d , 8a-c and 10d showed high activity against K562 cells.


Introduction
The complex sequence of cellular and molecular changes that take place during cancer formation are mediated by the different endogenous and exogenous stimuli. 1 Among endogenous stimuli are intermediates of oxygen reduction, i.e. oxygen free radicals (OFR), or more generally, reactive oxygen species (ROS), which interact with DNA, forming various aducts.2a-c OFRs are important in the pathogenesis of many different diseases.2a-d ROS are also involved in the processes of aging.Because of that, the recent research activities have been directed to discovering new efficient antiradical and antioxidant compounds.[5][6][7][8][9][10][11][12][13][14] Bearing the above in mind, one part of the present research was directed to the microwaveinduced synthesis of some new derivatives of salicylic acid, starting from different amino alcohols, diamines and diols, as well as to the determination of their antioxidant and antiproliferative activities.Microvawe irradiation is an alternative to conventional heating for introducing energy into reactions.One of the main advantages of microwave-assisted organic synthesis is the drastic decrease of reaction times.There is a considerable interest in the rapid synthesis of pharmacologically active compounds, such as 2-oxazolines.Namely, 2-oxazolines represent a very interesting class of heterocyclic compounds that is widely used in synthetic organic chemistry.6][17] Recently, it has been reported on the one-pot synthesis of 2-hydroxyphenyl-4-methyloxazoline-4-carboxamide substructures, found also in some natural cytotoxic agents such as brasilibactin A. 18 Because of that, the other part of the present research was directed towards finding a suitable way for the microwave-catalyzed synthesis of some new 2-(2-hydroxyphenyl)-4-substituted derivatives of 2-oxazoline.Since the structures of these compounds are similar to those of some bioactive natural products such as the brasilibactin A, it was interesting to investigate their antioxidant and cytotoxic activities against selected human cancer cell lines. 19Some azo-salicylic acids show biological activity and also azo derivatives of some phenols are useful precursors for the synthesis of anticarcinogenic, antiviral, antimicrobial and antimalaric agents. 20Hence, our research was also concerned with the synthesis of phenyl-azo-salicyloyl derivatives of diamines and amino alcohols and with testing of their biological activity.

Starting compounds
Synthesized compounds When the microwave-assisted reaction of compounds 1a and 1b with methyl salicylate was carried out in the presence of sodium as catalyst, the products were also oxazolines 2a, 2b and 3 (Table 3).Oxazolines 2a, 2b and 3 were also formed at conventional heating of methyl salicylate and amino alcohol 1a and 1b in the presence of sodium as catalyst at 150 o C during 2h (Table 3).However, under these reaction conditions, 2-amino-2-(hydroxymethyl)propane-1,3-diol 1a, apart from compounds 2a and 3, afforded also the mono 4a 25 and bis 5a derivatives of salicylic acid, whereas 2-amino-2-methylpropane-1-ol 1b, in addition to the oxazoline derivative 2b, gave also the mono 4b and bis 5b derivatives of salicylic acid.It can be supposed that the oxazoline derivatives 2a and 2b are formed by cyclization of the amides 4a 25 and 4b, 26 generated primarly in the reaction.Salicyloyl-oxazoline 3 is probably formed in a similar way from the salicyloyl-amide 5a, or by transesterification of methyl salicylate with oxazoline 2a.Cyclization of compounds 4a and 4b into a heterocyclic ring proceeds probably according to the mechanism presented in Scheme 1. Microwave-assisted reactions of 2-aminoethanol 1c, ethane-1,2-diamine 1d, ethane-1,2-diol 6a, octane-1,8-diol 6b and propane-1,3-diamine 6c with methyl salicylate were also carried out in present work; the microwave power, temperature, and reaction time being the same as above (Table 3).Under these conditions, amino alcohol 1c as well as diols 6a and 6b gave mono 4c, 7a, 27 7b and bis 5c, 25 8a, 28 8b 29 derivatives of salicylic acid, whereas the diamines 1d and 6c gave only the respective bis derivatives 5d 28 and 8c 30 .
In our previous works, compounds 4c, 25 5c, 25 5d, 28 as well as 8a-8c [28][29][30] were prepared from the corresponding starting substrates in the presence of sodium as catalyst by conventional heating at 150 o C, for 2 h, the corresponding yields are given in Table 3.In the present work, at the same mole ratio of the reactants as in the above works 25,[28][29][30] the reaction time under microwave irradition was shortened to 15 min (Table 3).The microwave irradiation gave significantly higher yield of compound 4c compared to that obtained by conventional heating.The yield of compound 5c was somewhat lower, but the overall yield of 4c and 5c in the microwave-assisted reaction was significantly higher.It can be noticed that 2-aminoethanol 1c did not produce oxazoline derivative, either at conventional heating or microwave irradiation.
Coupling reactions of compunds 4c, 5c and 5d with benzenediazonium chloride at 0 o C yielded azo derivatives 9, 10c and 10d (Scheme 2).Molecular and crystal structure of compound 3 was solved using X-ray structural analysis (Figure 1), 31 showing the R-configuration at the stereocenter (C4) of the molecule.

Table 4. Intramolecular and intermolecular O−H…O hydrogen-bond parameters
Biological properties Antioxidant activity.The antioxidant activities of the synthesized derivatives of salicylic acid were evaluated in a series of in vitro tests.In the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, the ability of tested compounds to act as donors of hydrogen atoms or electrons in transforming DPPH• into its reduced form, DPPH-H, was measured by spectrophotometric method. 32All tested compounds were able to reduce the stable, purple-colored radical DPPH• into yellowcolored DPPH-H form (Table 5).Commercial synthetic antioxidants, 3,5-di-tert-butyl-4hydroxytoluene (BHT) and 3-tert-butyl-4-hydroxyanisole (BHA) were used as positive controls.
The strongest DPPH-scavenging activity showed the phenylazo derivatives 9 and 10c, which exhibited much stronger activity than their precursors 4c and 5c, which indicates a positive influence of the introduced phenylazo groups.By comparing the activities of mono derivatives 4a and 4c with the corresponding bis derivatives 5a and 5c it can be seen that the latter are more active, indicating that the introduction of the new salicyloyl group increased the DPPHscavenging effect.This could be observed with oxazoline derivatives, too.Namely, by introducing the salicyloyl group into the oxazoline 2a, the more active salicyloyl oxazoline 3 is obtained.Also, it can be noted that the length of methylene chain in the esters 8a and 8b and amides 5d and 8c does not influence essentially the IC 50 values.Commercial synthetic antioxidants BHT and BHA exhibited higher DPPH scavenging properties than those observed for the examined compounds.The hydroxyl radical scavenging activity of the tested compounds (Table 5) was measured by the deoxyribose assay. 32Protective effect of the tested compounds was followed as their ability to remove hydroxyl radicals from the test solution and prevent the degradation.High inhibition of degradation was observed for all tested compounds, especially for compounds 8c, 8b and 5d.Comparison of the IC 50 values for the tested compounds with the IC 50 values for BHT and BHA indicates that salicylic acid derivatives are very strong scavengers of OH radicals (generated in Fenton`s reaction).It is evident that the activity is essentially influenced by the length of methylene chain in the ester and amide bis-derivatives.Namely, the bis-ester 8b and bis-amide 8c are more active than the corresponding compounds with smaller number of methylene groups, 8a and 5d.Of the 2-oxazoline derivatives 2a, 2b and 3 the most active is oxazoline 3, which has a salicyloyl group at C-4 position.
The inhibition of lipid peroxidation (LP) was determined by measuring the formation of MDA, using liposomes as an oxidizable substrate. 32Some of the examined compounds showed notable inhibition of the Fe 2+ /ascorbate induced LP in liposomes, compound 10c expressing the highest inhibition.Comparison of the IC 50 values for tested compounds with the IC 50 values for BHT and BHA shows that azo-derivatives 9 and 10c could be classified as promising inhibitors of LP.
The bis derivatives appeared to be very active antiproliferative agents.Thus, bis-salicyloyl derivative with eight methylene groups, 8b, exhibited the most potent activity of all the tested compounds in the case of MDA-MB-231 cells.Its analog with two methylene groups, compound 8a, showed three times weaker activity against the mentioned cells, but still at the nanomolar level.Of the bis-amides 5d and 8c higher activity showed compound 8c, having three methylene groups.The bis-derivatives with phenylazo groups, 10c and 10d, showed also strong cytotoxicity, especially against three cell lines: MCF7, MDA-MB-231 and PC3.By examining the differences of the following pairs of compounds 2a and 3, 4a and 5a, 4b and 5b, 4c and 5c, 9 and 10c, 7a and 8a, as well as 7b and 8b, it can be noticed that the introduction of salicyloyl or phenylazo-salicyloyl group into the corresponding starting compound changed significantly cytotoxicity against all of examined cell lines of human tumors.In the majority of cases, these groups increased the cytotoxicity of the synthesized compounds.
The established antitumor drug doxorubicin (Dox) was used as reference, and its activity was in the range of IC 50 from 0.12 to 95.61 µM.The synthesized salicyloyl derivatives 4c, 5c, 5d, 8a-8c, 9, 10c and 10d were significantly more active against particular cell lines compared to Dox.A markedly higher cytotoxicity compared to Dox was observed with compounds 8a-8c.Namely, compound 8a was by 100 and compound 8b by 300 times more active against MDA-MB-231 cells than Dox.The most pronounced increase in the antiproliferative activity compared to Dox (i.e. by 385 times) showed compound 8c against Hs 294T cells.It should be noticed that compounds 5d, 8c, 9, 10c, and 10d exhibited strong cytotoxicity against PC3, in contrast to Dox, which was not practically toxic against these cells.Only compounds 2a, 3, 5b, 5c, and 8b were slightly toxic to normal fetal lung fibroblasts cells, MRC-5, unlike doxorubicin which was very toxic.
By comparing the antioxidant and antiproliferative activities it can be concluded that bisester 8b, which showed the highest activity as a scavenger of OH radicals (IC 50 0.012 mM), exhibited also the highest antiproliferative activity (MDA-MB-231 cell lines, IC 50 0.0004 µM).Similarly, the most potent LP inhibitor, bis-azo derivative 10c (IC 50 0.02 mM), showed cytotoxicity at a nanomolar concentration against three cell lines (MCF7, MDA-MB-231 and PC3).
It can be concluded that mono and bis derivatives of salicylic acid, with salicyloyl-and phenylazo-salicyloyl groups, as well as some new oxazoline derivatives, showed very potent cytotoxicity against K562, Hs 294T, HeLa S3, MCF7, PC3 and MDA-MB-231 cell lines, and strong antioxidant activity as scavengers of OH radicals and inhibitors of LP.

Experimental Section
General.Melting points were determined using a Büchi SMP 20 apparatus and are uncorrected.The infrared spectra (wave numbers in cm -1 ) were taken on a Nexus 670 FT-IR spectrometer.Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AC 250 apparatus operating at 250 MHz (proton) and 62.9 MHz (carbon), using standard Bruker software, with tetramethylsilane as the internal standard.Chemical shifts are given in ppm (δ-scale); coupling constants (J) are given in Hz.High resolution mass spectra (TOF) were recorded on a 6210 Time-of-Flight LC/MS Agilent Technologies (ESI+) instrument.GC/MS analyses were performed on an Agilent Technologies GC 7890A instrument with Mass Selective Detector 5975C.Absorbances of the reaction mixtures in free radical scavenging tests were recorded on a CECIL CE2021 spectrophotometer.The microwave reactor was a monomode system (Microwave Synthesis Sistem -Discover Bench Mate from CEM) with focused waves.Organic solutions were dried over Na 2 SO 4 and evaporated on a rotary evaporator under reduced pressure.Column chromatography was performed on Merck grade 60 silica gel (0.063-0.2 mm).

General procedure for preparation of azo-derivatives (9, 10c and 10d)
To NaOH solution (10%; 10 mmol for 4c; 20 mmol for 5c and 5d) the compounds 4c, 5c or 5d (5 mmol) were added and the solution was cooled to 0 -5 o C.After that the cold freshly prepared benzenediazonium chloride solution 31 (5 mL for 4c, 12.5 mL for 5c and 5d) was added very slowly (the pH 8-9).When all the diazonium salt solution was added, the mixture was left in an ice bath with stirring for 30 min.After that, distilled water and HCl (1:1 to make the pH 5-6) were added, and the content was stirred at the same temperature for 1 h.After the reaction was finished, the reaction mixture was filtered and crude products were recrystallyzed to obtained pure compounds 9, 10c or 10d.IC 50 values (the concentration of the tested compound in the reaction mixture which causes 50% of RSC) were determined by linear regression analysis from the obtained RSC values.Hydroxyl-radical scavenging assay.Hydroxyl-radicals scavenging capacity (HO • RSC) of the tested compounds was evaluated by measuring the degradation of 2-deoxy-D-ribose (Aldrich; Taufkirchen, Germany) in the reaction with OH radicals, generated in situ in Fenton's reaction. 32hese radicals attack the sugar 2-deoxy-D-ribose and degrade it into a series of fragments, some or all of which react on heating with 2-thiobarbituric acid (TBA) (Sigma; St. Louis, MO) at low pH to give a pink chromogen, which can be determined spectrophotometrically at 532 nm.Different aliquots (0.005-0.5 mL) of sample solution in methanol were added to test tubes (final concentration ranged from 0.01 to 8 mM), each containing 0.1 mL of 5 mM H 2 O 2 , 0.1 mL of 10 mM FeSO 4 and 0.1 mL of 0.05 M 2-deoxy-D-ribose and 0.067 M KH 2 PO 4 -K 2 HPO 4 buffer pH 7.4 to a final volume of 3 mL.The same reaction mixture without sample was used as the control.After an incubation period of 1 h at 37 o C, 2 mL of TBA reagent (10.4 mL of 60 % (v/v) HClO 4 , 3 g TBA and 120 g of trichloroacetic acid (Sigma; St. Louis, MO)) and 0.2 mL of 0.1 M EDTA (Sigma; St. Louis, MO) were added to the reaction mixture, and the tubes were heated at 100 o C for 20 min.After cooling, absorbance of the reaction mixtures and control were recorded at 532 nm.Percentage of HO • RSC was calculated using the following equation: RSC (%) = (A blank -A sample / A blank ) × 100.Three replicates were recorded for each sample; BHT and BHA were used as reference compounds.Determination of lipid peroxidation (LP).The extent of LP was determined by measuring the absorbance of adduct produced in the reaction between TBA and malondialdehyde (MDA) as an oxidation product in the peroxidation of phospholipids from liposomes, by the TBA assay. 32he commercial preparation of liposomes "PRO-LIPO S" (Lucas-Meyer, Hamburg, Germany), pH 5-7, was used as a model system of biological membranes.The liposomes, 225-250 nm in diameter, were obtained by dispersing the commercial preparation in demineralized water (1:10) in an ultrasonic bath.In the Fe 2+ /ascorbate-induced lipids peroxidation, a 60 µL suspension of liposomes was incubated with 20 µL of 0.01 M FeSO 4 , 20 µL of 0.01 M ascorbic acid, and 10 µL of tested compound and filled with 0.05 M KH 2 PO 4 -K 2 HPO 4 buffer, pH 7.4 to a final volume of 3 mL.Samples were incubated at 37 o C for 1 h, and LP was terminated by adding 2 mL of TBA reagent and 0.2 mL of EDTA, and heating the test tubes at 100 o C for 20 min.After centrifugation (4000 rpm for 10 min), the content of the MDA (TBARS) was determined by measuring the absorbance of the product at 532 nm.Analyses were compared with the commercial synthetic antioxidants BHT and BHA as a positive control.All reactions were carried out in triplicate.The percentage of LP inhibition was calculated by the following equation: where A o was the absorbance of the control (without the test compound) and A 1 was the absorbance in the presence of the inhibitor.Antiproliferative activity Cell lines.Six human tumor cell lines and one human non-tumor cell line were used in the study: human breast adenocarcinoma ER+, MCF7, human breast adenocarcinoma ER-, MDA-MB-231, prostate cancer PC3, cervix epithelioid carcinoma, HeLa S3, human melanoma, Hs 294T, chronic myelogenous leukemia, K562, and normal fetal lung fibroblasts, MRC-5.The cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5% of glucose (MCF7, MDA-MB-231, PC3, HeLa S3, Hs 294T and MRC-5) or in RPMI 1640 (K562).Media were supplemented with 10% of fetal calf serum (FCS, NIVNS) and antibiotics: 100 IU/mL of penicillin and 100 µg/mL of streptomycin (ICN Galenika).All cell lines were cultured in flasks (Costar, 25 cm 2 ) at 37 o C in the 100% humidity atmosphere and 5% of CO 2 .Only viable cells were used in the assay.Viability was determined by dye exclusion assay with trypan blue.SRB assay.Cytotoxicity was evaluated by colorimetric sulforhodamine B (SRB) assay. 33riefly, single cell suspension was plated into 96-well microtiter plates (Costar, flat bottom): 5 × 10 3 cells (MCF7, MDA-MB-231, PC3, HeLa S3, Hs 294T and MRC-5) or 10 4 (K562) cells per 180 mL of medium.Plates were pre-incubated 24 h at 37 o C, 5% CO 2 .Tested substances at concentrations ranging from 10 -8 to 10 -4 M were added to all wells except for the control ones.After incubation period (48 h /37 o C /5% CO 2 ) SRB assay was carried out as follows: 50 µL of 80% trichloroacetic acid was added to all wells; an hour later the plates were washed with distilled water, and 75 µL of 0.4% SRB was added to all wells; half an hour later the plates were washed with citric acid (1%) and dried at room temperature.Finally, 200 µL of 10 mmol Tris (pH 10.5) was added to all wells.Absorbance (A) was measured on the microplate reader (Multiscan MCC340, Labsystems) at 540/690 nm.The wells without cells, containing complete medium only, served as the blank.Cytotoxicity was calculated according to the formula: CI (%) = (1 -A sample / A control ) × 100.Data analysis.Two independent experiments were set out in quadruplicate for each concentration of the compound.IC 50 value defines the dose of compound that inhibits cell growth by 50%.The IC 50 of compounds was determined by Median effect analysis.Crystal structure determination A single crystal of compound 3 was mounted on a glass fiber and measured on an Oxford Diffraction Gemini S system.The diffraction data for compound 3 were collected at the temperature of 200 K with graphite-monochromated MoKα radiation (λ = 0.7107 Å).The data reduction was performed with the program package CrysAlis RED. 35The space group determination was based on an analysis of the Laue class and the systematically absent reflections.The structure of compound 3 was solved by direct methods using SIR92 36 and refined using full-matrix least-squares.Non-hydrogen atoms were refined anisotropically; the C-H hydrogen atoms were included at calculated positions riding on their attached atoms with fixed distances of 0.93 (CH) or 0.97 Å (CH 2 ) and all O-H hydrogen atoms were identified on difference electron density maps and isotropically refined.The data were subjected to the removal of the scattering contribution from heavily disordered solvent molecules, which must be n-hexane molecules, using the Squeeze routine as implemented in the program PLATON. 37ll calculations were performed using SHELXL97, 38 PARST 39 and PLATON, 37 as implemented in the WINGX 40 system of programs.The crystal data and refinement parameters are summarized in Table 7.

or 4b 2 Scheme 1 .
Scheme 1. Probable mechanism of cyclization of 4a and 4b to a heterocyclic ring.

Figure 1 .
Figure 1.ORTEP presentation of structure 3 with the labeling of non-H atoms.Displacement ellipsoids are shown at the 50% probability level and H atoms are drawn as spheres of arbitrary radii.Intramolecular hydrogen bonds are shown as dashed lines.

Figure 2 .
Figure 2. PLATON drawing showing the crystal packing of compound 3; Intermolecular hydrogen bond O2-H2...O2 is shown as dashed line (atoms that are not involved in O2-H2...O2 intermolecular hydrogen bond are not presented because of clarity).

Table 2 .
Microwave-assisted synthesis catalyzed by Bu 4 NBr in basic conditions (K 2 CO 3 )

Table 3 .
Comparison of the results of the synthesis of salicylic acid derivatives obtained by conventional heating and microwave irradiation, in the presence of sodium as catalyst a Temperature 150 o C, time 2 h.b Power applied 170W (except for 1a, 150 W), temperature 100 -160 o C, time 15 min (except for 1b, 20 min.).c The microwave-assisted synthesis was performed in the absence of sodium.

Table 5 .
IC 50 Values for radical scavenging activities of the tested compounds and commercial antioxidants BHA and BHT a 50% inhibition not achieved.

Table 6 .
IC 50 Values for in vitro cytotoxicity of the tested compounds and Dox C 18 H 17 NO 6 [M+H] + calculated: 343.11286, found: 344.11403.