RO134432A0 - O-aryl-carbamoyl-oximino-fluorene derivative, pharmaceutical composition containing the same and their use - Google Patents

Abstract

The invention relates to a process for preparing O-aryl-carbamoyl-oximino-fluorene derivative compounds to be used in pharmaceutical compositions. According to the invention, the process consists in refluxing 9-fluorenone with hydroxylamine hydrochloride in a molar ratio of 1: 1.3 in methanol reaction medium, resulting in fluoren-9-one-oxime which is refluxed with aryl isocyanates in a ratio of 1: 1 in anhydrous tetrahydrofuran reaction medium, resulting in derivatives of the formula 1a-d, wherein R = mono or disubstituted phenyl, the compounds having low toxicity, being intended for antibacterial, antifungal and antibiofilm treatment.

Description

O-aryl-carbamoyl-oxymino-fluorene derivatives, a pharmaceutical composition containing them and their use

The invention relates to novel compounds of class O-aryl-carbamoyl-oxymino-fluoren and to a process for their preparation, compounds with bactericidal, fungicidal and antibiofilm effect.

In recent years, the emergence of resistance and multidrug resistance to known antimicrobial substances and the emergence of new etiological agents have led to increasing concerns about finding new antimicrobial agents, which is also a challenge to identify new strategies for treating infectious diseases, such as would be inhibiting the expression of virulence factors.

Antibiotic resistance cannot be eliminated by placing new analogues of existing antibiotics on the market, but it is necessary to find antibiotics with new structures. As a result, the development of prototype molecules, with original structure, which contributes to reducing the permanent risk of microbial resistance, is, worldwide, a priority direction of research of new antimicrobial chemotherapeutics with maximum efficacy and low toxicity, which represent solutions. effective treatments for infections with microorganisms MDR (multidrug-resistant), XDR (extensively drug-resistant) and PDR (pandrug-resistant) and, at the same time, prevent the permanent risk of emergence of new resistance mechanisms.

The urgent need to discover new compounds with high antimicrobial efficacy also stems from the fact that recent studies estimate that humanity may soon be left without effective antibiotics, as they are not used judiciously.

The encouraging results of research on fluorenic nucleus compounds, as well as the fact that carbamoyl and oximinic pharmacophore groups are presented in the literature, as having certain pharmacological qualities, led us to combine these biologically active fragments into a single original molecule.

Fluoren, a tricyclic aromatic hydrocarbon, is found in the structure of some drugs such as lumefantrine (antimalarial), imirestate (aldose reductase inhibitor), cycloprofen (analgesic, anti-inflammatory), renitolin (analgesic, anti-inflammatory), aminocar, ), hexafluronium bromide (muscle relaxant).

Worldwide, studies are known in the field of obtaining new antimicrobial agents from the class of fluorenic nucleus derivatives.

A number of new fluorine derivatives were obtained using Noctadecyl-9-oxo-9H-fluoren-4-carboxamide as the basic intermediate. The synthesized derivatives react with propylene oxide resulting in compounds with good surface properties. Biological degradation of these a2019 00319

30/05/2019 fluorine-based surfactants was higher than 96% after 7 days. Most compounds have variable inhibitory effects on Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa, Escherichia coli) bacterial strains, as well as against fungal strains (Aspergillus niger, Candida albicans and Curvularia sp.). Therefore, these compounds can be considered as multifunctional agents, with potential applications as wetting agents, paints, drugs, mild emulsifiers, household detergents, in the cosmetics and textile industry [El-Sayed R., Althagafi I. I., Ahmed S.A. Fluorene derivatives with multi-addressable properties: synthesis, characterization, and reactiviLy. J Surfact Deterg., 2017, 20 (3), DOI J 0.1007 / sl 1743-017-1958-4].

Derivatives of (fluoren-2-yl) (3-phenylbicyclo [2.2.1] hept-5-en-2-yl) methanone, which have the phenyl radical substituted with various atoms or functional groups, were obtained by the Diels-Alder reaction and exhibited in vitro antimicrobial and antioxidant properties [Thirunarayanan G. The in-vitro antimicrobial and antioxidant activities of some Diels-Alder diaryl methanone adducts. J. Pharm. Appl. Chem., 2017, 3 (1), 19-26],

Of the 3- (2-chloroquinolin-3-yl) -isoxazol-5-yl-methyl 2- (9-fluoro-9-yl-methoxycarbonylamino) -propionate derivatives having the 2-substituted quinoline ring with various radicals, compounds with -SH and -SeH groups showed good antibacterial activity against Gram-positive and Gram-negative bacteria (Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtillis), compared to the standard ciprofloxacin [Sharath N., Hale S. Bhojya Naik, Vinay Kumar B., Hoskeri J. Synthesis, antibacterial, molecular docking, DNA binding and photonuclease activity of quinoline isoxazoles. Der Pharmacia Sinica, 2012, 3 (2), 254-265],

Fluorenones have various therapeutic applications. Thus, tilorone and fluorenal have antiviral properties, benfluron is antineoplastic, and fluodipine is cardiodepressant. The 9fluorenone class contains many potentially active substances, which can be studied for the optimization of some leading molecules.

The study of the influence of tilorone (2,7-bis (2-diethylaminoethoxy) fluoren-9-one) on the DnaG activity of Staphylococcus aureus primase showed that it is inhibited by tilorone, although no inhibition of the growth of the pathogen mentioned in the presence of this compound was observed. Structural modulations were performed in order to facilitate the diffusion of the compound into the bacterial cell, to inhibit bacterial growth, and also to identify a promising leading compound. Thus, different 9-fluorenone derivatives were synthesized, using tilorone as a structural model, adding hydrocarbon chains with different lengths and end groups. These compounds were tested against Gram-positive and negative bacteria, and some of them showed a relatively low minimum inhibitory concentration on Bacillus anthracis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Burkholderia 2019 00319

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thailandensis and Francisella tularensis. The results also show that the distribution of tilorone can be improved by further modifying the length of the chain, the resulting compounds can be used as prototype molecules to design and develop in the future, stronger and more effective inhibitors of these pathogens [Choi S., Larson MA , Hinrichs SH, Narayanasamy P. Development of potential broad spectrum antimicrobials using C2-symmetric 9-lluorenone alkyl amine. Bioorg. Med. Chem. Lett., 2016, 26, 1997-1999],

Starting from 9-fluorenone, Schiff bases were synthesized which were investigated for their biological activity and molecular silica docking studies were performed with the target protein, represented by the catalase from Proteus mirabilis. Of the compounds, N, N'-bis-fluoro-9-ylidene-ethan-1,2-diamine has the highest docking score (81,947). Some of the compounds showed antimicrobial activity against the strains of Escherichia coli, Staphylococcus aureus, Psendomonas aeroginosa, Proteus mirabilis and Klebsiella pneumoniae comparable to the standard antibiotic, streptomycin, at a concentration of 100 pg / mL [Venkivathi S., D 1., Narasimhan S. Preparation of various SchifPs bases of 9-fluorenone and its biologica! application. Chem. Pharm.Res., 2012, 4 (10), 4477-4483],

A new series of 2,7-diamidofluorenones was synthesized, the study showing that some compounds have good antiproliferative activity, being type I topoisomerase inhibitors. It was found that the introduction of the linear alkyl group in the side chains resulted in better antiproliferative activity, compared to the introduction of a branched alkyl group or a bulky group. Compared to tertiary amino groups, the presence of secondary amino groups in the side chains increased antiproliferative activity. The results indicated that the fluorenone fragment could be a potential pharmacophore for the design of antitumor compounds in the class of topoisomerase inhibitors IB [Lee C.-C., Chang D.-M., Huang Κ.-R, Chen C. -L., Chen T.-C., Lo Y., Guh J.-H., Huang H.-S. Design, synthesis and antiproliferative evaluation of fluorenone analogs with DNA topoisomerase I inhibitory properties. Bioorg. Med. Chem., 2013, 21, 7125-7133].

The choice of the carbamoyloximinic moiety in the structure of the compounds covered by this patent is also based on the fact that this moiety is found in the structure of other compounds with antibacterial and antifungal action, such as NR-carbamoyl-2-adamantanoxime [Georgiev VS, Saeva GA 2- Adamantanone oxide carbamate derivatives; patent SHA 4652680, 1987, March 24] or antimycobacterial, Ν, Ν-dimethylcarbamoyloxyma 2-bromo-6-R-indeno [2, lc] quinolin-7-one [Upadhayaya RS, Lahore SV, Sayyed AY, Dixit SS, Shinde PD, Chattopadhyay J. Conformationallyconstrained indeno [2, lc] quinolines - a new class of anti-mycobacterial agents. Org. Biomol. Chem., 8 (2010), 2180-2977] or is also found in the structure of well-known compounds such as acaricides, insecticides and / or nematocides (alanicarb, aldicarb, aldoxicarb, tirpate, oxamil), as well as pesticides, of 2019 00319

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such as the derivatives of N-methyl-N - [(^ r / -butylphenyl) sulfenyl] carbamoyloxime [Durden Jr. J. A., Sousa A. A. Tertiary butyl substituted carbamoyl oxime pesticides, U.S. Patent 3998963, 1976, December 2].

This structural feature of carbamate oxime, present in the molecule of some compounds, contributes to the improvement of their pharmacological and pharmacokinetic properties [Ray S., Pathak S. R., Chaturvedi D. Organic carbamates in drug development. Part II: Antimicrobial agents - recent reports. Drugs. Fut. 2005, 30, 161].

The problem that the present invention solves consists in the synthesis of new compounds of class O-aryl-carbamoyl-oxymino-fluoren with bactericidal, fungicidal and antibiofilm effect.

The present invention relates to O-aryl-carbamoyl-oxymino-fluorene derivatives with the following general formula, selected from la-d, in which several pharmacophore fragments are associated in a single molecule (tricyclic fluorenic system, carbamoyl and oximinic groups). ):

la (R = -CuHs): 9- (phenylcarbamoyloxymin) fluorine

1b (R = -CftHCHCHsXS)): 9 - ((3-methyl-phenyl) carbamoyloximino) fluorine

Ic (R = -C ₆ H ₄ (C 1) (3)): 9 - ((3-chloro-phenyl) carbamoylooximino) fluorine

Id (R = -C ₆ H ₄ (C 1) 2 (3,4)): 9 - ((3,4-dichloro-phenyl) carbamoyloxymin) fluorine

Another object of the invention consists in a process for obtaining the O-arylcarbamoyl-oxymino-fluoren derivatives defined as above, which comprises the following steps:

a. Preparation of fluoren-9-on-oxime, by refluxing 9-fluorenone with hydroxylamine hydrochloride, in a molar ratio of 1: 1,3, the reaction medium being methanol:

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b. Preparation of the new O-aryl-carbamoyl-oxymino-fluoren derivatives by refluxing fluoren-9-on-oxime with arylisocyanates, in a molar ratio of 1: 1, the reaction medium being anhydrous tetrahydrofuran:

1a-d

R = -C ₆ H ₅ (la), -C ₆ H ₄ (CH ₃ ) (3) (1b), R = -C ₆ H ₄ (Cl) (3) (lc), -C ₆ H ₄ ( Cl) ₂ (3.4) (ld)

The advantage of the compounds according to the invention is that most of them exert an antibiofilm activity on bacteria and yeasts, having a value of the minimum eradication concentration of the biofilm (CMEB) between 0.09-5 mg / mL.

Optimal reaction conditions were established to obtain the new compounds with high purity and good yields.

The new compounds at -d were characterized by their physical constants (melting temperature, solubility).

Melting points were determined on an Electrothermal 9100 (Bibby Scientific Ltd., Stone, UK), in open capillaries.

The structures of the original compounds and intermediate compounds were determined by IR, H-NMR and ¹³ C-NMR spectral analyzes. Chemical shifts for hydrogen and carbon atoms have also been confirmed by 2D-NMR experiments.

IR spectra were recorded on a Bruker Vertex 70 FT-IR spectrometer (Bruker Corporation, Billerica, MA, USA).

H-NMR and ¹³ C-NMR spectra were recorded in deuterated chloroform (CDC1 ₃ ) or DMSO-d6, on a Bruker FOURIER 300 MHz instrument (Bruker Corporation, Billerica, MA, USA), operating at 300.0 MHz for 'H-NMR and at 75.0 MHz for ^l3 C-NMR and on a Bruker AvancelII 500 MHz instrument (Bruker Corporation, Billerica, MA, USA), operating at 500 MHz for proton and 125 MHz for carbon).

In NMR spectra, chemical shifts were recorded as δ, in parts per million (ppm), relative to tetramethylsilane as internal standard, and coupling constants (J) in Hertz. Standard abbreviations indicating the multiplicity of signals are used, as follows: s (singlet), d (doublet), t (triplet), m (multiple), dd (double doublet), td (triple doublet) and 1 (2019 signal 00319

30/05/2019 widened). H-NMR data are reported in the following order: chemical displacements, multiplicity, coupling constants, number of protons and signal / atom assignment.

For ^1S C-NMR data the order is as follows: chemical displacements and signal / atom assignment.

The results obtained with the help of spectral analysis confirm both the structure of the new compounds and intermediates used and the syntheses performed.

In the following, 8 embodiments of the invention will be presented.

EXAMPLE 1

Fluoren-9-on-oxime (N-fluoren-9-ylidenhydroxylamine)

5 g of 9-fluorenone (Mr 180,19) (0,028 mol) dissolved in 20 mL of methanol are introduced into a N-OH flask with a round bottom and 4 necks, fitted with a shaker, thermometer, rising refrigerant and drip funnel. over which 5.5 g of sodium hydroxide is gradually added with stirring (Mr 39.99). Gradually add 2.5 g of hydroxylamine hydrochloride (Mr 69,49) (0.036 mol), dissolved in 10 mL of methanol, over the mixture, gradually, with stirring and at 75-80 ° C. After hydroxylamine in methanol was added, the reaction mixture was refluxed for 5 hours. After cooling to room temperature, a mixture of 12.5 mL hydrochloric acid and 27 mL of water is gradually added to pH 8 as the oxime precipitates. Stir at room temperature for one hour, then filter the oxime under low pressure and wash thoroughly on the water filter. After isolating the precipitate, it is dried and then purified from xylene.

The result is 4.80 g of compound (Mr 195.22), crystallized, yellow, with a yield of 88.5% compared to 9fluorenone, T.t. 194.3-195.2 ° C, cold soluble in ethyl acetate, DMF, DMSO, pyridine and hot in methanol, ethanol, isopropanol, isobutanol, chloroform and xylene, insoluble in hexane and water.

Proof of the structure of the compound according to the invention H-NMR (DMSO-d 6, δ ppm, J Hz): 12.58 (s, N-OH); 8.37 (d, J = 7.3 Hz, 1H, H1); 7.90 (d, J = 7.6 Hz, 1H, H-4); 7.85 (d, J = 7.6, Hz, 1H, H-5 or H-8); 7.73 (d, J = 7.3 Hz, 1H, H-5 or H-8); 7.32-7.54 (m, 4H, H-2, H-3, H-6, H-7) ¹³ C-NMR (DMSO-d 6, δ ppm): 150.96 (C-9); 140.13 (C-4a); 139.25 (C-5a); 135.28 (C-8a); 130.73 (C-1); 129.65 (C-6 or C-7); 129.60 (C-6 or C-7); 128.48 (Cl); 128.35 (C-2); 127.98 (C-3); 120.80 (C-4); 120.38 (C-5 or C-8); 120.31 (C-5 or C-8) »2019 00319

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FT-IR (solid in ATR, v cm ' ¹ ): 3166w; 3039w; 2617m; 1642m; 1603m; 1588vs; 1485s; 1433m; 1319m; 1167s; 1090m; lOOls; 947s; 873s; 737w; 676vs; 621vs.

EXAMPLE 2

9- (phenylcarbamoyloxymin) fluoren (la)

0.59 g of fluoren-9-on-oxime (Mr 195.22) (0.003 mol) solubilized in 10 mL of anhydrous tetrahydrofuran is introduced into a round-bottomed flask. To this solution is added a solution of 0.36 g of phenyl isocyanate (Mr 119.12) (0.003 mol) in 10 mL of anhydrous tetrahydrofuran. The reaction mixture was refluxed on the water bath for 52 hours. After the time has elapsed, cool and evaporate the solvent under low pressure. The product obtained is purified from ethyl acetate 1.

The result is 0.60 g of compound (Mr 314.33) crystallized, yellow, with a yield of 63.2% over the oxime, T.t. 156.9-159 ° C, cold soluble in pyridine, DMF, DMSO, chloroform and hot in methanol, ethanol, isopropanol, isobutanol, ethyl acetate, xylene, insoluble in hexane and water.

Proof of the structure of the novel compound according to the invention ‘H-NMR (CDCl 3, δ ppm, ./hz): 8.56 (sl, 1H, NH); 8.46 (d,. = 7.5 Hz, 1H, H-1); 7.79 (d,. = 7.5 Hz, 1H, H-4); 7.61-7.56 (m, 4H, H-5, H-8, H-12, H-16); 7.45 (td,. / = 7.5 Hz,. / = 1.0 Hz, 1H, H-6 or H-7); 7.44 (td,. / = 7.5 Hz,. / = 1.1 Hz, 1H, H-7 or H-6); 7.41 (t,. = 7.8 Hz, 2H, H-13, H-15); 7.32 (t,. = 7.5 Hz, 1H, H-2); 7.30 (t,. = 7.5 Hz, 1H, H-3); 7.16 (t.

¹³ C-NMR (CDCl ₃ , δ ppm): 155.97 (C-9); 151.75 (C-10); 142.31 (C-4a); 141.47 (C-5a); 136.80 (C-8a); 133.93 (C-la); 132.84 (C-6 or C-7); 131.82 (C-6 or C-7); 130.99 (C1); 129.72 (C1); 129.17 (C-13, C-15); 128.83 (C-2); 128.23 (C-3); 124.48 (C-14); 122.55 (C-4); 120.33 (C-5 or C-8); 120.16 (C-5 or C-8); 119.79 (C-12, C-16).

FT-IR (solid in ATR, v cm ' ¹ ): 3267m; 3138w; 3064w; 2962w; 1724vs; 1603s; 1543vs; 1501m; 1446s; 1315m; 1208vs; 1017m; 952vs; 837w; 809w; 788w; 749m; 729m; 688m; 646w.

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9 - ((3-methyl-phenyl) carbamoyloxymin) fluorene (1b)

The compound was prepared by the above method, requiring 0.399 g of 3-methylphenyl isocyanate (Mr 133.15, d ₄ ²⁰ = 1.033; 0.003 mol).

0.56 g of compound (Mr 328.36) were obtained after recrystallization from ethyl acetate, yielding 56.4% of the oxime, T.t. 171.2- 174.3 ° C, cold soluble in pyridine, DMF, DMSO, chloroform and hot in methanol, ethanol, isopropanol, isobutanol, ethyl acetate, xylene, insoluble in hexane and water.

Proof of the structure of the novel compound according to the invention ¹ H-NMR (CDCl 3, δ ppm, J Hz): 8.51 (sl, 1H, NH); 8.48 (d, J = 7.5 Hz, 1H, H1); 7.81 (d,. = 7.5 Hz, 1H, H-4); 7.60 (d, J = 7.5 Hz, 1H, H-5 or H-8); 7.59 (d, J = 7.5 Hz, 1H, H-5 or H8); 7.48 (t, J = 7.5 Hz, 1H, H-6 or H-7); 7.45 (t, J = 7.5 Hz, 1H, H-6 or H-7); 7.41 (sl, 1H, H12); 7.40 (d, 1H, H-16); 7.34 (t, J = 7.5 Hz, 1H, H-2 or H-3); 7.31 (t, J = 7.5 Hz, 1H, H-2 or H-3); 7.28 (t,. = 8.2 Hz, 1H, H-15); 6.99 (d, J = 8.2 Hz, 1H, H-14); 2.40 (s, 3H, H-13 ').

¹³ C-NMR (CDCl ₃ , δ ppm): 155.92 (C-9); 151.72 (C-10); 142.34 (C-4a); 141.51 (C-5a); 139.14 (C-13); 136.71 (C-8a); 134.00 (C-1); 132.85 (C-6 or C-7); 131.92 (C-11); 131.80 (C-6 or C-7); 131.08 (Cl); 129.00 (C-2); 128.86 (C-15); 128.25 (C-3); 125.32 (C-14); 122.58 (C-4); 120.43 (C-16); 120.34 (C-5 or C-8); 120.17 (C-5 or C-8); 116.91 (C-12); 21.50 (C-13 ').

FT-IR (solid ATR, v cm ' ¹ ): 3259m; 3149w; 3084w; 3054w; 3022w; 2920w; 1729vs; 1613s; 1555s; 1491m; 1449m; 1316m; 1211vs; 1169m; 1155m; 1028m; 968s; 900s; 780m; 728m; 690m.

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EXAMPLE 4

9 - ((3-chloro-phenyl) carbamoyloxymin) fluorene (Ic)

The synthesis follows the procedure described for the preparation of the compound of Example 2, using 0.46 g of 3-chlorophenyl isocyanate (Mr = 153.57, d ₄ ²⁰ = 1.269; 0.003 mol).

The result was 0.7 g of compound (Mr 348.77), after recrystallization from ethyl acetate, with a yield of 66.4% over hydrazide, T.t. 155.7-158.3 ° C, soluble cold in pyridine, DMF, DMSO, chloroform and hot in methanol, ethanol, isopropanol, isobutanol, ethyl acetate, xylene, insoluble in hexane and water.

Proof of the structure of the novel compound according to the invention ‘H-NMR (CDCl 3, δ ppm, J Hz): 8.59 (sl, 1H, NH, deuterable); 8.60 (d, J = 7.5 Hz, 1H, H-1); 7.82 (d, J = 7.5 Hz, 1H, H-4); 7.66 (t, J = 1.9 Hz, 1H, H-12); 7.59 (d, J = 7.5 Hz, 1H, H-5 or H-8); 7.58 (d, J = 7.5 Hz, 1H, H-5 or H-8); 7.56 (t, J = 7.5 Hz, 1H, H-6 or H-7); 7.54 (m, 1H, H-14); 7.50 (t, J = 7.5 Hz, 1H, H-6 or H-7); 7.40 (t, J = 7.5 Hz, 1H, H-2 or H-3); 7.37 (t, J = 7.5 Hz, 1H, H-2 or H-3); 7.36 (t, J = 8.1 Hz, 1H, H-15); 7.19 (dd, J = 8.1 Hz, J = 1.9 Hz, 1H, H-16).

¹³ C-NMR (CDCl 3, δ ppm): 156.31 (C-9); 151.16 (C-10); 142.45 (C-4a); 141.60 (C-5a); 138.02 (C-8a); 134.88 (C-13); 133.83 (C-1); 133.08 (C-6 or C-7); 132.04 (C-6 or C-7); 131.13 (Cl); 130.30 (C-15); 129.73 (C-11); 128.96 (C-2); 128.34 (C-3); 124.57 (C-16); 122.64 (C-4); 120.45 (C-5 or C-8); 120.28 (C-5 or C-8); 119.80 (C-12); 117.74 (C-14).

FT-IR (solid ATR, v cm ' ¹ ): 3251w; 3189w; 3125w; 3081w; 2962w; 1728vs; 1596vs; 1545s; 1481m; 1450m; 1415w; 1306m; 1204vs; 1022m; 968vs; 874s; 812w; 779m; 726m; 682m.

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EXAMPLE 5

9 - ((3,4-dichloro-phenyl) carbamoyloxymin) fluorine (Id)

The same synthetic procedure was followed as for the compound of Example 2, using 0.564 g of 3,4-dichlorophenyl isocyanate (Mr 188.01; 0.003 mol).

0.75 g of compound (Mr 383.23) is obtained after purification from ethyl acetate, with a yield of 64.8% over the oxime, T.t. 157.9-161.2 ° C, cold soluble in pyridine, DMF, DMSO, chloroform and hot in methanol, ethanol, isopropanol, isobutanol, ethyl acetate, xylene, insoluble in hexane and water.

Proof of the structure of the novel compound according to the invention 'H-NMR (CDCl ₃ , δ ppm, J Hz): 8.60 (sl, 1H, NH, deuterable); 8.53 (d,. = 7.7 Hz, 1H, H1); 7.80 (d, J = 7.7 Hz, 1H, H-4); 7.78 (d, J = 2.3 Hz, 1H, H-12); 7.62 (d, J = 7.2 Hz, 1H, H-5 or H-8); 7.61 (d, J = 7.2 Hz, 1H, H-5 or H-8); 7.43-7.51 (m, 4H, H-6, H-7, H-15, H-16); 7.35 (t,. = 7.7 Hz, 1H, H-2); 7.32 (t, J = 7.7 Hz, 1H, H-3).

^{, 3} C-NMR (CDCl ₃ , δ ppm): 156.53 (C-9); 151.45 (C-10); 142.48 (C-4a); 141.63 (C-5a); 136.37 (C-8a); 133.74 (C-1); 133.05 (C-14); 133.16 (C-6 or C-7); 132.13 (C-6 or C-7); 131.11 (Cl); 130.73 (C-16); 129.69 (C-11); 128.97 (C-2); 128.35 (C-3); 127.86 (C-13); 122.66 (C-4); 121.41 (C-12); 120.48 (C-5 or C-8); 128.32 (C-5 or C-8); 118.37 (C-15).

FT-IR (solid ATR, v cm ' ¹ ): 3366m; 1768m; 1597w; 1574w; 1504vs; 1477m; 1450w; 1380w; 1320w; 1287w; 1235w; 1185w; 1025m; 956m; 920w; 848w; 781w; 724m; 685w.

EXAMPLE 6

The antimicrobial activity of the newly synthesized compounds was evaluated against 5 microbial strains, respectively: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Candida albicans subCCie 900 biofilms developed on an inert substrate.

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a. Determination of the minimum inhibitory concentration (MIC)

The determination of MIC was performed by the method of serial binary microdilutions in Mueller Hinton broth, distributed in 96-well microplates. 10 concentrations of each compound (with values between 5-0.009 mg / mL) were obtained after performing serial binary dilutions in a final volume of 100 pL of medium, from a stock solution of 10 mg / mL, made in DMSO (dimethyl sulfoxide). Subsequently, the wells were seeded with 20 pL of 0.5 MacFarland microbial suspension. The positive control was represented by the microbial culture cultured in the absence of the test compound, and the negative one by the sterile culture medium. The microplates were incubated at 37 ° C for 24 hours, the MIC value being set at the level of the last well with a clear, transparent content, similar in appearance to the negative control.

The tested compounds showed antimicrobial activity with MIC values between 0.156 and 10 mg / mL (Table 1), the most sensitive strains being the S. aureus strain, followed by the P. aeruginosa strain. The increased efficacy of compound Id against the 5. aureus strain (MIC 0.156 mg / mL) is noted.

Table 1. Results of quantitative testing of antimicrobial activity and corresponding MIC values (mg / mL)

Chemical compound bMicrobial strain to 1b wedge id Enterococcus faecalis CVTCC 29212 2.5 10 5 5 Staphylococcus aureus ⁱⁿ KYCC 25923 2.5 2.5 2.5 0.156 gPseudomonas aeruginosa ATCC 27853 2.5 2.5 2.5 2.5 fEscherichia coli ATCC 25922 2.5 5 2.5 5 / Candida albicans ATCC 90029 2.5 5 5 5

b. Determination of the minimum bactericidal concentration (CMB)

After reading the MIC values, 10 pL volumes of the liquid culture developed in the well corresponding to the MIC value and from all previous wells were seeded on solid PCA (plate count agar) medium, in order to determine the CMB value (corresponding to the concentration at which total inhibition was obtained of microbial growth on solid medium).

The obtained results showed CMB values between 0.312 and 10 mg / mL. It is noted that the CMB values were similar or twice higher than the CMI values (Table 2).

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Table 2. Results of quantitative testing of antimicrobial activity and corresponding CMB values (mg / mL)

Chemical compound Microbial strain to 1b wedge id Enterococcus faecalis ATCC 29212 5 10 5 5 Staphylococcus aureus ATCC 25923 5 5 5 0,312 Pseudomonas aeruginosa ATCC 27853 2.5 2.5 2.5 2.5 Escherichia coli ATCC 25922 2.5 5 5 5 Candida albicans ATCC 90029 5 10 10 10

c. Determination of the anti-biofilm activity of the tested compounds

It was performed by the microtiter method, using the microplates used to determine the CMI and CMB values. For this purpose, the wells were washed two to three times with sterile saline, after which the biofilms adhered to the walls of the wells were fixed with 100pL of cold methanol for 5 minutes, stained with 1% alkaline solution of purple crystal, for 15 minutes, and then resuspended with 33% acetic acid. The minimum eradication concentration of biofilms (CMEB) was established as the last concentration of the compound at which the absorbance value was observed to decrease to 490 nm, compared to the positive control.

All compounds tested inhibited the development of bacterial and fungal biofilms tested, CMEB values being between 0.009 and 1.25 mg / mL, up to hundreds of times lower than the corresponding CMI and CMB values (Table 3).

It is worth noting the very high sensitivity of the biofilm of E. faecalis to compound Ic, of aureus to compound Id, of P. aeruginosa in and 1b and of C. aobicans in all compounds tested, but especially in 1b.

Table 3. Antibiofilm activity test results and corresponding CMEB values (mg / mL)

----- Chemical compound Microbial strain ~ to 1b wedge id Enterococcus faecalis ATCC 29212 1.25 1.25 0,312 1.25 Staphylococcus aureus ATCC 25923 5 2.5 5 0.019 Pseudomonas aeruginosa ATCC 27853 0.009 0.156 1.25 1.25 Escherichia coli ATCC 25922 0.625 0.078 1.25 0.625 Candida albicans ATCC 90029 0,312 0.078 0,312 0,312

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d. Investigation of potential mechanisms of antimicrobial action by flow cytometry

Flow cytometry (CF) was performed on microbial cultures obtained after 18-24 hours of incubation of microbial cells in the presence of subinhibitory concentrations of the analyzed compounds. Thus, microbial suspensions with a density of approximately IO ⁶ CFU / mL were prepared in sterile saline phosphate buffer from exponentially growing microbial cultures, obtained on agarized medium. Subinhibitory concentrations of the compounds made in Muller-Hinton liquid culture medium were inoculated with an equal amount of microbial suspension and incubated for 18-24 hours at 37 ° C. Microbial suspensions inoculated in liquid medium were used as growth control. After incubation, fluorochrome DÎBAC4 solution (3) [bis- (1,3-dibutylbarbituric acid) trimethinoxonol] Invitrogen / Life Technologies, Carlsbad, was added to obtain a final concentration of 0.5 pg / mL (Nuding S., Zabel LT Deiection, Identification, and susceptibilily lesting of bacteria by flow cytometry. J. Bacteriol. Parasitol. S5-005, 2013, doi: 10.4172 / 21559597.S5-005). Fluorescence intensity (IF) was measured with an Accuri C6 plus flow cytometer in the FITC fluorescence channel. Growth control was used to locate the microbial cell population and to exclude cell debris. Fluorescence measurements were performed only for cells in this gate. DÎBAC4 dye (3) was used to detect changes in microbial membrane potential produced by treatment with the analyzed compounds. The fraction of microbial cells in the analyzed population that showed increased green fluorescence (corresponding to membrane depolarization) was calculated based on the exclusion of fluorescence events corresponding to untreated growth control. An increased fluorescence intensity (median fluorescence intensity = MIF) of at least twice was considered to correspond to depolarized microbial cells. For each subinhibitory concentration, a staining index (CI) was calculated to represent the ratio of fluorescence intensity (IF) of treated microbial cells to untreated cells.

Analysis of the effects of the compounds on the membrane potential of the tested microbial strains showed that at subinhibitory concentrations, they produce a depolarization of the plasma membrane. suggesting that the plasma membrane is one of the targets of the antimicrobial activity of these compounds. Analyzes showed that compound Ic did not cause changes in membrane potential in strains of £. faecalis ATCC 292Î2 and 5. aureus ATCC 25923.

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Table 4. Staining index values at subinhibitory concentrations of the analyzed compounds

——Chemical compound Microbial strains __ to 1b wedge id E. coli ATCC 25922 Subinhibitory concentrations tested 1.25 mg / mL 2.5 mg / mL 1.25 mg / mL 1.25 mg / mL Coloring index value 80.7 10.2 3.2 173.3 E. faecalis ATCC 29212 Subinhibitory concentrations tested 2.5 mg / mL 2.5 mg / mL 1.25 mg / mL 1.25 mg / mL Coloring index value 2.21 3.99 0.32 44.54 P. aeruginosa ATCC 27892 Subinhibitory concentrations tested 2.5 mg / mL 2.5 mg / mL 2.5 mg / mL 2.5 mg / mL Coloring index value 6.68 6.5 2.38 20.45 S. aureus ATCC 25923 Subinhibitory concentrations tested 2.5 mg / mL 2.5 mg / mL 2.5 mg / mL 1.25 mg / mL Coloring index value 3.5 8.17 0.19 4.2 C. albicans ATCC 90029 Subinhibitory concentrations tested 2.5 mg / mL 2.5 mg / mL - 2.5 mg / mL Coloring index value 4.94 2.82 - 20,21

EXAMPLE 7

Crustacean toxicity Artemia franciscana Kellog, was evaluated based on the methods of B. M. Meyer et al. (Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med., 1982, 45 (5), 31-4) and T. W, Sam (Sam TW Toxicity testing using the brine shrimp Artemia salina. In: Bioactive natural products detection, isolation, and structural determination. Boca Raton (FL) ,: CRC Press; 1993. p. 441-56), with small adaptations suggested by more recent sources (Cock IE, Kalt FR Toxicity evaluation of Xanthorrhoea johnsonii leaf methanolic extract using the Artemia franciscana bioassay. Pharmacogn Mag. 2010, 6 (23), 166- 171) (Cock IE, Van Vuuren SF A comparison of the antimicrobial activity and toxicity of six combretum and two terminalia species from Southern Africa. Pharmacogn. Mag. 2015, 11 (41), 208-218) (Artoxkit M. Artemia toxicity screening test for estuarine and marine waters. Standard Operational Procedure. Microbiotests, Mariakerke- Gent., 2014).

Materials and methods

The oocysts were obtained from a commercial source (S.K. Trading), with the origin declared as 100% from Great Salt Lake (USA). As an average, artificial seawater obtained from a mixture of commercially available salts (Coral Marine, Grotech) dissolved in distilled water, with a few minutes of sonication, at a concentration of 30 g / L was used, according to the recommendations of the oocyst supplier. Hatching was performed at an average temperature of 26 ° C, using an air pump to ensure adequate aeration of the environment and was initiated 48 hours before testing. The test was performed in a plate of 24 wells (6 x 4), in triplicate (three wells for each concentration evaluated). Due to the limited solubility, the substances la-d were suspended in artificial seawater using sodium alginate (0.045%), the test being performed at the solubility limit. Concentrations 14 of 2019 00319

30/05/2019 used were 100, 50, 25, 12.5 and 6.2 pg / mL for each substance, being obtained by successive dilutions from the initial suspension. A solution of sodium alginate in artificial seawater (0.045%) was used as a negative control. The hatched nauplii were separated from the remains of oocysts and transferred to the wells using a micropipette, after prior concentration in a well using artificial light. Between 10 and 15 nauplii per well were collected, which were placed in contact with the test suspensions (1.5 mL of test suspension per well) (Libralato G., Prato E., Migliore L., Cicero AM, Manfra L. A review of toxicity testing protocols and endpoints with Artemia spp. Ecol Indic., 2016, 69, 35- 49). All nauplii, dead or alive, were counted and recorded within 24 hours of being placed in contact with the test suspensions. The nonlinear modeling of the concentration-lethality relationship was performed by a four-parameter logistics model (4PL), implemented in several robust variants for estimating the parameters in the R ..dr4pl 'package (Landis JT, An H., Bailey AG, Dittmer DP, Brown JS dr4pl: Dose Response Data Analysis using the 4 Parameter Logistic (4pl) Model [Internet]. 2019. Available from: https://CRAN.Rproject.org/package=dr4pl).

Results and discussions

Of the four substances tested, three (la, 1b and Ic) did not show any toxicity at the evaluated concentrations (in suspension, therefore at the solubility limit), all nauplii being alive and showing normal movements. Substance Id, however, showed a pronounced toxicity, highlighted on the lethality curve (concentration-response) and in the IC50 value (14.63 pg / mL, 95% CI 11.80 - 18.15 pg / mL) (Figure 1) . The use of other robust methods for modeling the concentration-response relationship led to very similar results, both in terms of the IC50 value and its 95% CI. In the literature it has been suggested that an IC50 value in the toxicity test on Artemia sp. between 10 and 30 pg / mL corresponds to a moderate toxicity, similar to that of cyclophosphamide, for which the IC50 reported in the literature for Artemia sp. is 16.3 pg / mL (Moshi MJ, Innocent E., Magadula JJ, Otieno DF, Weisheit A., Mbabazi PK, et al. Brine shrimp toxicity of some plants used as traditional medicines in Kagera Region, north western Tanzania. Tanzan J. Health Res., 2010, 12 (1), 63-67). The acute toxicity of compound ld is close to that of cyclophosphamide. The other compounds evaluated (at, 1b and Ic) did not show any toxicity at concentrations up to 100 pg / mL, so their toxicity can be considered modest (but the solubility limits that did not allow the evaluation should be taken into account). toxicity when applied as a solution).

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Figure 1. Concentration-response curve for the lethality of substance Id on Artemia franciscana Kellog nauplii, constructed on the basis of a four-parameter logistic model (x-axis corresponds to a logarithmic scale).

EXAMPLE 8

Research on the antioxidant activity of compounds in .1b and silica has included:

a. The scavenger capacity of the DPPH radical

The principle of the method

The violet free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) is reduced in the presence of natural / synthetic antioxidants to the corresponding yellow hydrazine. This change is accompanied by a decrease in absorbance values [Dudonne S., Vitrac X., Coutierre P., Woillez M., Merillon J.M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD and ORAC assays. J. Agric. Food. Chem., 2009, 57 (5), 1764-1778] [Brand - Williams W., Cuvelier ME., Berset C. Use of a free radical method to evaluate antioxidant activity, LWT-Food Sci. Technol., 1995, 28 (1), 25-30].

From the point of view of the mechanism of action, the method is mainly based on electron transfer and is widely used due to its simplicity and correlation with other methods [Dudonne S., Vitrac X., Coutierre P., Woillez M., Merillon J.M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS. FRAP, SOD and ORAC assays. J. Agric. Food. Chem., 2009, 57 (5), 1764-1778] [Deepshikha G, Methods for determination ol ’antioxidant capacity: a review. 1JPSR, 2015, 6 (2), 546-566] [Prior RL, Wu X, Schaich K, Standardized methods for the determination of antioxidant capacity and phenolics in food and dietary supplements. .1. Agric. Food. Chem., 2005, 53, 4290-4302], 2019 00319

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The method is not standardized so far, so in the literature there are various variants of work. Absolute methanol or hydroalcoholic mixtures (> 50%) can be used for free radical solubilization [Sharma O.P., Bhat T.K. DPPH antioxidant assay revisited, Food Chem., 2009, 113 (4), 1202-1205]; the contact time between free radical and substrate varies between 5 90 min. or until reaching the plateau state [Robu S., Aprotosoaie AC, Miron A., Cioancă O., Stănescu U., Hăncianu M. In vitro antioxidant activity of ethanolic extracts from some Lavandula species, cultivated in Romania, Farmacia, 2012, 60 (3), 394-401] [Yuan YV, Bone DE, Carrington MF Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro, Food Chem., 2005, 91 (3), 485-494] [Brand Williams W., Cuvelier ME., Berset C. Use of a free radical method to evaluate antioxidant activity, LWT-Food Sci. Technol., 1995, 28 (1), 25-30] [Molyneux P. The use of the stable free radical diphenylpicryl-hydrazyl (DPPH) for estimating antioxidant activity, Songklanakarin J. Sci. Technol., 2004, 26 (2), 212-219], and the determinations are performed at a wavelength between 515-520 nm [Molyneux P. The use of the stable free radical diphenylpicryl-hydrazyl (DPPH) for estimating antioxidant activity, Songklanakarin J. Sci. Technol., 2004, 26 (2), 212-219] [Molyneux P., 2004],

Our determinations were based on the method described by Ohnishi M. et al. (1994) [Ohnishi M., Morishita H., Toda S., Shirataki Y., Kimura M. Inhibitory effects of chlorogenic acids on linoleic acid peroxidation and haemolysis, Phytochemistry, 1994, 36 (3), 579-583], cited of German MP et al. [Germano MP, Cacciola F., Donato P., Dugo P., Certo G., D'Angelo V., Mondello L., Rapisarda A. Betula pendula leaves: polyphenolic characterization and potential innovative use in skin whitening products, Fitoterapia, 2012, 83 (5), 877-882],

Necessary reagents and solvents

DPPH (Sigma-Aldrich, Germany), ascorbic acid (Roth, Germany), ethanol, dimethylsulfoxide (DMSO) were used.

Preparation of the DPPH solution

0.0039 g DPPH free radicals were solubilized in a flask rated at 100 mL absolute ethanol to give a 0.1 mM solution. For determinations, freshly prepared, light-protected solution was always used. Absolute ethanol was chosen for free radical solubilization, since the tested compounds, respectively the reference substance (ascorbic acid), were subsequently solubilized in a mixture of absolute ethanol: DMSO = 99: 1 (v / v), respectively absolute ethanol.

Preparation of solutions to be analyzed

The compounds were solubilized in a 25 mL volumetric flask in a mixture of absolute ethanol: DMSO = 99: 1 (v / v), obtaining a stock solution with a concentration of 1000 μΜ. By dilution with the solvent mixture, solutions of concentration 500 μΜ, 250 μΜ, 100 μΜ, 75 μΜ, 50 μΜ and 25 μ conc were obtained.

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Preparation of the solution to obtain the ascorbic acid standard curve

0.1 g Ascorbic acid was solubilized in a 100 mL volumetric flask in absolute ethanol. The curve was constructed on the concentration range 0.005- 0.04 mg / mL.

Working technique

0.5 mL of the 25-1000 μΜ solution was treated with 3 mL of 0.1 mM DPPH solution. The samples were kept at rest, in the dark for 30 minutes [Costea T., Lupu A-R, Vlase L., Nencu I., Gîrd C.E. Phenolic content and antioxidant activity of a raspberry leaf dry extract. Rom. Biotech. Lett., 2016, 21 (2), 11345-11356] [Ohnishi M., Morishita H., Toda S., Shirataki Y., Kimura M. Inhibitory effects of chlorogenic acids on linoleic acid peroxidation and haemolysis, Phytochemistry, 1994, 36 (3), 579-583]. Subsequently, the absorbances were measured at λ = 515 nm, on a Jasco V-530 spectrophotometer (Jasco, Japan), against absolute ethanol, used as a blank.

Inhibition (%) of DPPH radical activity was calculated according to the formula [Brand Williams W., Cuvelier ME., Berset C. Use of a free radical method to evaluate antioxidant activity, LWT-Food Sci. Technol., 1995, 28 (1), 25-30]:

Acontrol - Approves „1% = --------------- x 100 where:

Acontrol

A comroi ⁼ absorbance of the 0.1 mM DPPH solution in the absence of compounds (1,000 ± 0.02).

Sample ⁼ absorbance of the DPPH solution in the presence of compounds after 30 min.

Antioxidant activity was expressed in ascorbic acid equivalents (mM ascorbic acid / g substance). These were calculated by interpolating the absorbance values of the DPPH solution in the presence of compounds, in the equation of the straight line of the standard curve (concentration vs. absorbance). The standard curve was previously constructed, under the same conditions, in the range 0.005-0.04 mg / mL (n = 5, R ² = 0.997).

The ascorbic acid standard curve is shown in Table 5 and Figure 2.

Table 5. Ascorbic acid standard curve (DPPH method)

Concentration (mg / mL) absorbance 0,005 .8276 0.01 .7533 0.02 .5511 0.03 .3332 0.04 .1094

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0.9 ............................................... .....................

0.8 ......... y = 40.73x * 0.950 _n7 A, R ² - 0.997

.................................................. ..................................... X 1

I 5 0.2 ............................................. .......................................... X!

<xi | QJ ................................................. .................... ♦ j | o ................. - |

O 0.01 0.02 0.03 0.040.05

Acorbic acid mg / mL

Figure 2. Ascorbic acid standard curve

Statistical analysis

For each concentration tested, the determinations were performed in triplicate and the results represent the mean of three independent determinations ± standard deviation (SD). Microsoft Office (Excell 2007) and GraphPad Prism v.5 software (GraphPad, USA) were used for statistical interpretation of the data. The comparison of the antioxidant activity of the analyzed compounds was performed using the ANOVA test followed by the Tukey post-test. The results were considered statistically significant if p <0.05.

Results and discussions

The results regarding the scavenger capacity of the DPPH radical are presented in Tables 6-8 and Figure 3.

The results obtained (Tables 6, 7) showed that the absorbance values decrease, with increasing concentration, regardless of the compound analyzed. DPPH free radical inhibition varies between 12.66% (at a concentration of 25 μΜ) and 24.64% (Ic at a concentration of 1000 μΜ). The compound la also inhibited by 19.81% the activity of the free radical DPPH, at a maximum concentration of 1000 μΜ. The lowest value of DPPH radical inhibition, at the maximum concentration, was recorded for compound 1b (16.29%).

In general, the scavenger capacity of the DPPH radical on the concentration range used is low (below 30%), probably due to the apolar nature of the compounds tested and the mechanism of action of the method used, based mainly on electron transfer. Ascorbic acid equivalents could not be determined for any of the compounds analyzed (Table 8) because the absorbance values did not fit into the ascorbic acid standard curve.

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Table 6. Absorbance values of the solutions to be analyzed in the presence of the DPPH radical

Compound Concentration (μΜ) 25μΜ 75 μΜ 100 μΜ 250 μΜ 500 μΜ 1000 μΜ to 0.9310 ± 0.0078 0.9252 ± 0.0061 0.9234 + 0.0048 0.9171 ± 0.00014 0.9117 + 0.0033 0.9102 + 0.0051 0.8547 ± 0.059. 1b 0.9269 + 0.0016 0.9260 + 0.00084 0.9205 ± 0.0028 0.9175 ± 0.0029 0.9161 + 0.0016 0.9134 + 0.0009 0.8923 ± 0.000. wedge 0.8770 ± 0.0031 0.8698 + 0.0006 0.8624 + 0.0042 0.8584 ± 0.00063 0.8552 ± 0.00084 0.8486 + 0.0065 0.8026 ± 0.002

Results are mean ± SD (n = 3)

Table 7. Inhibition (%) of the DPPH radical by the analyzed compounds

Compound Concentration (μΜ) 25μΜ 50 μΜ 75 μΜ 100 μΜ 250 μΜ 500 μΜ 1000 μΜ to 12.66 ± 0.7353 13.20 ± 0.5798 13.36 ± 0.4596 13.96 ± 0.0141 14.46 ± 0.3181 14.61 ± 0.4808 19.81 ± 5.6144 1b 13.04 ± 0.1626 13.13 + 0.0848 13.64 + 0.2687 13.93 + 0.2828 14.05 + 0.1626 14.31 ± 0.0989 16.29 ± 0.0494 wedge 17.72 + 0.2969 18.39 + 0.063 19.09 ± 0.4030 19.47 + 0.0565 20.17 ± 0.6434 20.39 ± 0.6081 24.64 ± 0.2192

Results are mean ± SD (n = 3)

Table 8. Ascorbic acid equivalents - DPPH method

Compound Ascorbic acid equivalents (mM ascorbic acid / g substance) to when 1b when wedge when

nd - indeterminate

Figure 3. Graphical representation of DPPH free radical inhibition by the analyzed compounds

conclusions

The tested compounds show a modest capacity (<30%) of scavenger of the DPPH radical on the analyzed concentration range.

The highest values of inhibition (%), at a maximum concentration of 1000 μΜ, were recorded for compounds la and Ic. Ascorbic acid equivalents were not determined because the absorbances did not fit the ascorbic acid standard curve.

b. The scavenger capacity of the ABTS radical

The ABTS method uses the free radical ABTS ' ⁺ as the reaction agent.

The radical can be obtained by reacting the ammonium salt of 2,2-azino-bis (3-ethyl-benzothiazolin-6-sulfonic) acid with potassium persulfate [Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med., 1999, 26 (9-10): 1231-1237], manganese dioxide [Miller J.N., Sampson J., Candeias L.P., Bramley P.M., Rice-Evans C.A. Antioxidant activities of carotens and xantophylls, FEBS Letters, 1996, 384 (3), 240-242], 2,2'-azobis- (2-amidinopropane) dichloride [Magalhăes L.M., Segundo M.A., Reis S., Lima J.L.F.C. Methodological aspects about in vitro evaluation of antioxidant properties, Anal. Chem. Acta, 2008, 613 (1), 1-19] or CU enzymes (peroxidase) [Prior R.L., Wu X., Schaich K., Slandardized a 2019 00319

30/05/2019 methods for the determination of antioxidant capacity and phenolics in food and dietary supplemenls. J. Agric. Food. Chem., 2005, 53, 4290-4302],

The principle of the method

The radical ABTS ' ⁺ (blue) in the presence of a natural / synthetic antioxidant fades; color changes being accompanied by decreased absorbance values [Dudonne S., Vitrac X., Coutierre P., Woillez M., Merillon JM Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial inlerest using DPPH, ABTS, FRAP , SOD and ORAC assays. J. Agric. Food. Chem., 2009, 57 (5), 1764-1778].

The method has multiple advantages: it is simple, reproducible, independent of pH and can be applied to both lipophilic and hydrophilic antioxidants. From the point of view of the mechanism of action, the method is mainly based on proton transfer [Prior R.L., Wu X., Schaich K., Standardized methods for the determination of antioxidant capacity and phenolics in food and dietary supplements. J. Agric. Food. Chem., 2005; 53: 4290-4302].

Similar to the DPPH method, the method is not standardized; In the literature there are many working variants, and the reaction time between radical and substrate varies from 4-6 min. up to 60 min. [Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Rădic. Biol. Med., 1999, 26 (9-10): 1231-1237] [Thaipong K., Boonprakob U., Crosby K., Cisneros-Zevallos L., Byrne D.H. Comparison of ABTS, DPPH, FRAP and ORAC assays for estimating activity front guava fruits, J. Food Compost. Anal., 2006, 19 (67), 669-675],

To evaluate the free radical scavenger capacity, the method described by Re R. et al. [Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Rădic. Biol. Med., 1999, 26 (9-10): 1231-1237].

Reagents and solvents used

The ammonium salt of ABTS (Sigma-Aldrich, Germany), potassium persulfate (Merck, Germany), ascorbic acid (Roth, Germany), dimethylsulfoxide (DMSO), ethanol were used.

Reagent preparation

ABTS ' ⁺ radical was obtained by the reaction between the ammonium salt of 2,2-azino-bis (3-ethyl-benzothiazolin-6-sulfonic acid) (7.4 mM solution) and potassium persulphate (2.6 mM solution). [Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Rădic. Biol. Med., 1999, 26 (9-10): 1231-1237].

- Obtaining the solution of the ammonium salt of 2,2-azino-bis (3-ethyl-benzothiazolin-6-sulfonic): 0.3808 g of ammonium salt were solubilized in 100 mL of distilled water (in a volumetric flask). A solution of concentration 7.4 mM (solution A) was obtained.

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- Obtaining potassium persulphate solution: 0.0707 g of potassium persulphate were solubilized in 100 mL of distilled water (100 mL volumetric flask) to give a solution of 2.6 mM concentration (solution B).

The free radical ABTS ' ⁺ was obtained by mixing solutions A and B in a ratio of 1: 1 (v / v) and keeping in contact for 16 hours.

The solution obtained was kept away from light. 1 mL of the solution obtained was diluted with 50 mL of absolute ethanol so that the absorbance at λ = 734 nm was 0.700 ± 0.02.

Preparation of solutions to be analyzed

The compounds were solubilized in a 25 mL volumetric flask in a mixture of absolute ethanol: DMSO = 99: 1 (v / v), obtaining a stock solution with a concentration of 1000 pM. By dilution with the solvent mixture, solutions of concentration 500 pM, 250 pM, 100 pM, 75 pM, 50 pM and 25 pM were obtained.

Preparation of the solution to obtain the ascorbic acid standard curve

0.1 g of ascorbic acid was solubilized in 100 mL of absolute ethanol. The curve was constructed on the concentration range of 0.01-0.1 mg / mL.

Working technique

0.5 mL of the 25-1000 pM concentration solutions were treated with 3 mL of ABTS ' ⁺ ethanolic solution, stirring and keeping in the dark for 6 minutes. The absorbance of the samples was measured with a Jasco V-530 spectrophotometer at λ = 734 nm, relative to absolute ethanol, used as a blank. The reduction of the absorbance values represents the inhibition of the ABTS ⁺ solution and is calculated according to the equation:

η / τ 1 -1 AbS (i = 0 min) - AbS (i = ft min)% Inhibition = ----------------------- xlOO, where :

Abs \ i = o)

Abs _{t =} omin ⁼ value of extinction of the ABTS ' ⁺ solution in the absence of test compounds (0,700 ± 0,02),

Abs _{t = 6 min} = value of extinction of the ABTS ' ⁺ solution after 6 minutes of incubation with the compounds to be analyzed.

Antioxidant activity was expressed in ascorbic acid equivalents (mM ascorbic acid / g substance). These were calculated by interpolating the absorbance values of the ABTS * ⁺ solution in the presence of compounds, in the equation of the ascorbic acid standard curve (concentration vs. absorbance), the standard curve was previously constructed, under the same conditions, in the range 0.01- 0.01 mg / mL (n = 7, R ² = 0.9912).

The ascorbic acid standard curve is shown in Table 9 and Figure 4.

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Table 9. Ascorbic acid standard curve (ABTS ' ⁺ method)

Ascorbic acid concentration (mg / mL) absorbance 0.01 .6151 0.02 .5425 0.04 .3989 0.05 .3528 0.07 .2176 0.1 .0844

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.02 0.04 0.06 0.08 0.1 0.12

Ascorbic acid mg / mL

Figure 4. Ascorbic acid standard curve

Statistical analysis

For each concentration tested, the determinations were performed in triplicate and the results represent the mean of three independent determinations ± standard deviation (SD). Microsoft Office (Excell 2007) and GraphPad Prism v.5 software (GraphPad, USA) were used for statistical interpretation of the data. The comparison of the antioxidant activity of the analyzed compounds was performed using the ANOVA test followed by the post-Tukey test. The results were considered statistically significant if p <0.05.

Results and discussions

The results on the scavenger capacity of the ABTS ' ⁺ radical are presented in Tables 10-12 and Figure 5.

Our results showed that the scavenger activity of the free radical ABTS ' ⁺ increases with concentration, regardless of the compound analyzed (Table 10).

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Table 10. Absorbance values of the solutions to be analyzed in the presence of the radical ABTS ' ⁺

Compound Concentration (μΜ) _25μΜ:: 50 μΜ 75 μΜ 100 μΜ 500 μΜ 1000 μΜ to 0.5002 ± 0.007 0.5780 ± 0.0094 0.5773 + 0.0099 0.5690 + 0.0055 0.5663 + 0.0033 0.5626 ± 0.0014 0.5546 ± 0.0074 1b 0.5322 ± 0.01364 0.5278 ± 0.0071 0.5256 ± 0.0049 0.5203 + 0.0018 0.5172 ± 0.0007 0.5161 ± 0.0068 0.5109 + 0.0063 wedge 0.5848 ± 0.0040 0.5780 ± 0.0081 0.5741 ± 0.0067 0.5717 + 0.0065 0.5705 ± 0.0063 0.5685 + 0.0080 0.5658 ± 0.0091

Results are mean ± SD (n = 3)

Table 11. Inhibition (%) of the ABTS ' ⁺ radical by the analyzed compounds

Compound Concentration (μΜ) 25μΜ 50 μΜ ₇₅ 100 μΜ 1 250 μΜ 500 μΜ 1000 μΜ IF1 16.65 + 1.0040 16.99 ± 1.3364 17.07 ± l, 4283 18.26 ± 0.8061 18.65 ± 0.4879 19.18 ± 0.2121 20.33 + 1.0818 1F4 23.50 + 1.9586 24.17 ± l, 025 24.25 ± 0.3535 25.26 + 0.2687 25.71 ± 0.098 25.85 + 0.9828 26.60 ± 0.091 IF5 12.53 + 0.6010 13.55 ± 1.2162 14.14 + 1.011 14.49 ± 0.9828 14.67 ± 0.9405 14.98 ± l, 2020 15.38 ± 1.4354

Results are mean ± SD (n = 3)

Table 12. Ascorbic acid equivalents - ABTS ' ⁺ method

Compound Ascorbic acid equivalents (mM ascorbic acid / g substance) to 3.0624 ± 3.0629 1b 4.7816 ± 4.8765 wedge 2.7217 ± 2.7262

The results represent the mean ± SD (n = 3) of 2019 00319

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Figure 5. Graphical representation of ABTS ' ⁺ free radical inhibition by the analyzed compounds

Inhibition values vary between 12.53% (Ic at 25 μΜ) and 26.60% (1b at 1000 μΜ). Regardless of the concentration used, the highest values of ABTS ' ⁺ radical inhibition were obtained for compound 1b, while compound Ic showed the weakest free radical scavenger capacity.

Compared to the DPPH method, the inhibition values on the concentration range used were higher in the case of the ABTS method, for compounds at and 1b, while for compound Ic the percentages were lower. We consider that the observed differences are due to the different mechanism of action of the radicals and the polarity of the compounds.

The antioxidant activity of the compounds increases in the order: Ic <la <1b (Table 12); the results being closely correlated with the percentage of free radical inhibition, on the concentration range used.

The ANOVA test did not show statistically significant differences for the tested compounds (p> 0.05).

conclusions

The tested compounds show ABTS ' ⁺ free radical scavenger capacity on the analyzed concentration range. The highest values of free radical inhibition were obtained for compound 1b, in close correlation with the values of ascorbic acid equivalents. Antioxidant activity increases in the order of Ic <to <1b. The results obtained did not show statistically significant differences between the analyzed compounds.

Claims (5)

1. O-aryl-carbamoyl-oxymino-fluorene compounds, characterized in that they have the following general formula: la (R = -CâHs): 9- (phenylcarbamoylooximino) fluorine 1b (R = -C ₆ H ₄ (CH 3) (3)): 9 - ((3-methyl-phenyl) carbamoylooximino) fluorine Ic (R = -C ₆ H ₄ (C 1) (3)): 9 - ((3-chloro-phenyl) carbamoylooximino) fluorine Id (R = -C ₆ H ₄ (C 1) 2 (3,4)): 9 - ((3,4-dichloro-phenyl) carbamoyloxymin) fluorine Process for obtaining the O-aryl-carbamoyl-oxymino-fluoren derivatives, la-d, according to claim 1, characterized in that it takes place by refluxing fluoren-9-on-oxime with arylisocyanates, the reaction medium being anhydrous tetrahydrofuran. 3. Pharmaceutical compositions, characterized in that they contain as the only active principle bactericidal, fungicidal and antibiofilm derivatives of O-aryl-carbamoyl-oxymino-fluorene, as defined in claim 1. 4. Pharmaceutical compositions exert their bactericidal and fungicidal effect by depolarizing the plasma membrane, suggesting that the plasma membrane is one of the targets of the antimicrobial activity of these compounds. Use of O-aryl-carbamoyl-oxymino-fluorene derivatives, as defined in claim 1, for the preparation of low toxicity pharmaceutical compositions for bactericidal, fungicidal and antibiofilm treatment.