New Anilides As Potential Antimicrobial

FARMACIA, 2008, Vol.LVI, 5

NEW ANILIDES AS POTENTIAL ANTIMICROBIALAGENTS. NOTE 1

ILEANA CHIRIŢĂ1, AL. MISSIR1, CAMELIA ELENA STECOZA1, CARMEN LIMBAN1, G.M. NIŢULESCU1, DIANA NUŢĂ1, L. MORUŞCIAG1, CARMELLINA BĂDICEANU1, CORINA ILIE2, M.T. CĂPROIU3

1Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy “Carol Davila”, Bucharest, Traian Vuia 6, sect. 2, 020956, Romania

2 National Institute of Chemical and Pharmaceutical Research and Development, Bucharest, Calea Vitan 112, sect.3, Romania

3 The Organic Chemistry Center of Romanian Academy "Costin C.D. Nenitescu” Bucharest, Splaiul Independentei, 202B, 77208, Romania

Abstract

Based on our experience in the synthesis of N-(2-dialkylaminoethyl)-benzanilides with antimicrobial effect, we developed some new compounds, amides of 2-phenylthiomethyl-benzoic acid. These new substances were characterized by their physico-chemical properties and by elemental analysis and spectrometric measurements: IR, 1H-NMR and 13C-NMR.

Rezumat

Având în vedere experienţa noastră în sinteza N-(2-dialchilaminoetil) benzanilidelor cu efecte antimicrobiene, am obţinut noi compuşi, amide ale acidului 2-feniltiometil-benzoic. Noile amide au fost caracterizate prin proprietăţile lor fizico-chimice şi prin analiză elementală şi spectrală: IR, 1H-RMN şi 13C-RMN.

amides of 2-phenylthio-methyl-benzoic acid
antimicrobial activity

INTRODUCTION

In the therapeutic field there are many amidic substances used as germicides and fungicides. They have a higher skin retention time, even after several washes with water and soap. These substances can be incorporated in detergents or soaps, and also in plastic or rubber materials used for toys manufacturing, food packing, single use dishes, thus contributing to these products sterilization.

In previous papers [1- 3] we presented the synthesis of some benzanilides with similar structures, having powerful bactericidal effect against S. aureus and antifungal action against C. albicans.

We continued our researches and there were synthesized new amides of 2-phenylthiomethyl-benzoic acidwith the general formula:

MATERIALS AND METHODS

In order to obtain the new compounds (1a-e) we followed four steps. The reactions are presented in Scheme 1 and Scheme 2.

- synthesis of the 2-phenylthiomethyl-benzoic acids (2a-c)

- synthesis of the mentioned acid chlorides (3a-c)

- synthesis of the N-(2-dialkylaminoethyl)-anilines (4a, 4b)

- synthesis of the original amides(hydrochlorides) (1a-e)

Scheme 1

Synthesis of the the 2-phenylthiomethyl-benzoic acids (2a-c)

and their acid chlorides (3a-c)

Scheme 2

Synthesis of the N-(2-dialkylaminoethyl)-anilines (4a, 4b)

andof the original amides (1a-e)

According to the methods described in the literature, the phtalide was prepared by reducing the phtalimide with copper-activated zinc in alkaline medium, because the method has good yields and goes without secondary products and the resulted compounds have desired purity [4].

The acids 2a-c resulted by phtalide condensation with a suspension of potassium salt of the corresponding thiophenolate, that have a good solubility in water and can be easily separated from xylene used as reaction solvent. The acids precipitate after treating the reaction mixture with a diluted hydrochloric acid solution [5].

The acid chlorides 3a-c resulted by treating the mentioned acids 2a-c with thionyl chloride in excess.

The amines 4a-b were obtained by alkylation of corresponding anilines 5a-b with N-(2-chloroethyl)-N,N-diethylamine hydrochloride, in anhydrous toluene, using sodium carbonate as hydrochloric acid acceptor [1, 2, 6].

The new amides resulted by coupling the acid chlorides 3a-c with the anilines(4a, 4b) in anhydrous toluene in the presence of a base (triethylamine). These amides were convertedto the corresponding hydrochlorides (1a-e) by treating the etheric solution of amides with ethereal hydrochloric acid, using a low temperature, under 5oC [1, 2, 6].

The synthesized compounds were characterized to establish their structure and purity, using the FT-IR, 1H-NMR and 13C-NMR spectrometry. The 1H-NMR and 13C-NMR spectra were registered with a Varian-Gemini-300 BB spectrometer operating at 300.075 MHz for proton and 75.462 MHz for carbon and an UNITY 400 plus, operating at 400 MHz for proton and 100 MHz for carbon. As solvents were used:

-deuterochloroform with a min. 99% isotopic purity;

-perdeuterated dimethylsulfoxyde with a min. 98% isotopic purity;

-trifluoroacetic acid with 99% purity.

Chemical shifts were recorded as δ values in parts per million (ppm) relative to tetramethylsilane as internal standard. Multiplicities are given together with the coupling constants (in Hz). The spectra were recorded at 20oC ± 1 temperature. In order to perform the right attribution of the signals, we used different techniques to simplify the spectra, like the homonuclear decoupling and the specific deuteration. In some cases the APT and COSY spectra were needed. The spectral assignments were performed according to the general structure:

The FT-IR spectra were recorded with a BRUKER Vertex-70 apparatus with an optical system with diamond. The ATR technique in the solid phase was used and each spectrum is a result of 32 independent scans.

The melting points were measured in glass capillary tubes, using an Electrothermal 9100 digital apparatus and are uncorrected.

RESULTS AND DISCUSSION

The synthesis of phtalide was performed according to the method described in previous paper [4].

The synthesis of the 2-phenylthiomethyl-benzoic acid (2a), 2-(4-methoxy-phenylthiomethyl)-benzoic acid (2b) and 2-(4-methyl-phenylthiomethyl)-benzoic acid (2c) [5, 7].

2-Phenylthio-methyl-benzoic acid (2a)

In a round–bottom flask, equipped with water removing device isdissolved 0.05 mol thiophenol in 30 mL xylene and 0.055 mol of potassium hydroxide are added. The reaction mixture is refluxed until 0.9 mL resulting water is removed by azeotropic distillation, while potassium thiophenolate precipitates; 0.05 mol of phtalide are added and the mixture is refluxed until it solidifies. The precipitate is dissolved in 10% potassium hydroxideand than is diluted with 50 mL water.

The aqueous phase is separated and acidulated with 1M hydrochloric acid (pH=3), when 2-phenylthiomethyl-benzoic acid (2a) precipitates. The resulting product is purified from ethanol:water (3:1); acid 2a is obtained as crystalline powder, with m.p. 109-111oC (75% yield).

Similarly to the previous case, using 4-methoxy-thiophenol or 4-methyl-thiophenol, the followingacidswere obtained:

2-(4-Methoxy-phenylthiomethyl)-benzoic acid (2b), white crystals, m.p. 133-135oC (ethanol:water= 3:1) (81% yield).
2-(4-Methyl-phenylthiomethyl)-benzoic acid (2c), white crystals, m.p. 124-126oC (ethanol:water= 3:1) (71% yield).

The synthesis of chlorides of 2-phenylthiomethyl-benzoic acid (3a), 2-(4-methoxy-phenylthiomethyl)-benzoic acid (3b) and 2-(4-methyl-phenylthiomethyl)-benzoic acid (3c) [5]

In a round–bottom flask, equipped with condenser and drying tube is placed a mixture of the acid (2a-2c) and thionyl chloride, in molar ratio 1:10; the mixture was refluxed for 90 minutes, thenthe thionyl chloride was removed under reduced pressure and the crude chlorides 3a-3c were obtained and were used without any purification in the next step of synthesis.

The synthesis of N-(2-diethylaminoethyl)-anilines (4a, 4b).

These amines were obtained using the methods described in our previous paper [5].

The synthesis of the original amidic compouns (1a-e)

N-(2-diethylaminoethyl)-N-phenyl-2-(phenylthiomethyl)-benzamide hydrochloride (1a)

To a stirred solution of 1.45g (0.00633 mol) N-(2-diethylaminoethyl)-3-chloraniline (5b) and 1.11g (1.5 mL) (0.011 mol) triethylamine in 20 mL dry toluene is added 2.5g (0.0095 mol) 2-phenylthiomethyl-benzoic acid chloride (3a). The mixture is refluxed for 10h. After cooling at room temperature and filtering the triethylamine hydrochloride, the toluene is evaporated in vacuum with a rotary evaporator; the residue is taken up in 30 mL chloroform and it is washed with 10% sodium carbonate solution (3x50 mL) and then with water. The chloroform solution is dried on anhydrous sodium sulfate and then the solvent is evaporated under vacuum. The resulting basic amide is brown-yellow oil, which was transformed in the corresponding hydrochloride by treating the etheric solution of basic amide with a saturated solution of hydrochloric acid in anhydrous ether which is dropwise added. Thus was obtained a white crystaline compound, m.p. 118-120oC (acetone) (75% yield).

TLC: single spot Rf=0.75 (eluent chloroform:methanol=4:1, developing system:UV, iodine atmosphere)

Elemental analysis: N% theoretical/experimental 5.72/5.78

1H-RMN(dmso-d6, δ ppm, J Hz): 11.29(bs, 1H, HN+); 7.45-7.90(m, 14H, Harom); 4.41(s, 2H, H-20); 4.30(bs, 2H, H-8); 3.25(bs, 2H, H-9); 3.12(bs, 4H, H-11-11’); 1.20(bs, 6H, H-12-12’).

13C-RMN(dmso-d6, δ ppm): 169.19(C-13); 141.94(Cq); 136.01(Cq); 135.37(Cq); 134.80(Cq); 130.03(CH); 129.12(CH); 128.73(CH); 128.72(CH); 128.27(CH); 127.67(CH); 127.31(CH); 126.36(CH); 126.07(CH); 126.06(CH); 46.61(C-9); 46.50(C-11-11’); 43.78(C-8); 34.29(C-20); 8.38(C-12-12’).

IR(ATR in solid, cm1): 3422; 3051; 2947; 2881; 2648; 2494; 1745; 1718; 1631; 1588; 1486; 1439; 1382; 1320; 1282; 1224; 1229; 1090; 1025; 908; 775; 736; 698; 654; 607; 528; 478; 445; 420.

All the original amides (hydrochlorides) 1b-e were obtained using the same method of synthesis, and they are presented as it follows:

N-(2-diethylaminoethyl)-N-phenyl-2-(phenylthiomethyl)-benzamide hydrochloride (1b)

- white crystals, m.p. 88-91oC (ethanol abs.) (70% yield).

- TLC: single spot Rf=0.8 (the same visualisation)

- elemental analysis: N% t/e: 6.16/6.25

1H-RMN(dmso-d6, δ ppm, J Hz): 11.29(bs, 1H, HN+); 7.45-7.90(m, 14H, Harom); 4.41(s, 2H, H-20); 4.30(bs, 2H, H-8); 3.25(bs, 2H, H-9); 3.12(bs, 4H, H-11-11’); 1.20(bs, 6H, H-12-12’).

IR(ATR in solid, cm1): 3422; 3051; 2947; 2881; 2648; 2494; 1745; 1718; 1631; 1588; 1486; 1439; 1382; 1320; 1282; 1224; 1229; 1090; 1025; 908; 775; 736; 698; 654; 607; 528; 478; 445; 420.

N-(2-diethylaminoethyl)-N-(3-chlorophenyl)-2-(4-methoxy-phenylthiomethyl)-benzamide hydrochloride (1c)

- yellow crystals, m.p. 94-96oC (chromatografic purification, chloroform/methanol 4:1 eluent) (35% yield).

- TLC: single spot Rf=0.76 (the same visualisation)

- elemental analysis: N% t/e: 5.39/5.28

1H-RMN(dmso-d6, δ ppm, J Hz): 7.62(bs, 1H, H-27.32(d, 2H, H-22-26, 8.7); 6.95-7.30(m, 7H, Harom); 6.91(d, 2H, H-23-25, 8.7); 4.27(s, 2H, H-20); 4.27(s, 2H, H-8); 3.75(s, 3H, H-27); 3.29(bs, 2H, H-9); 3.15(bs, 4H, H-12-12’); 1.21(bs, 6H, H-12-12’).

13C-RMN(dmso-d6, δ ppm): 170.21(C-13); 158.63(C-24); 143.42(C-1); 137.35(Cq); 136.22(Cq); 134.18(Cq); 132.35(CH); 132.34(C-22-26); 130.59(CH); 130.21(CH); 129.27(CH); 127.71(CH); 127.40(CH); 126.52(CH); 125.54(CH); 125.27(Cq); 114.82(C-23-25); 55.25(C-27); 46.84(C-9); 46.46(C-11-11’); 44.08(C-8); 36.49(C-20); 8.45(C-12-12’).

IR(ATR in solid, cm1): 3411; 3052; 2977; 2944; 2905; 2833; 2354; 1626; 1586; 1472; 1387; 1311; 1289; 1245; 1170; 1123; 1099; 1027; 887; 861; 828; 784; 760; 719; 686; 628; 523; 495; 444.

N-(2-diethylaminoethyl)-N-(3-chlorophenyl)-2-(4-methyl-phenylthiomethyl)-benzamide hydrochloride (1d)

- white-yellow crystals, m.p. 114-116oC (ethyl ether) (80% yield).

- TLC: single spot Rf=0.72 (the same visualisation)

- elemental analysis: N% t/e: 5.56/5.64

1H-RMN(dmso-d6, δ ppm, J Hz): 11.15(bs, 1H, H-N+); 7.53(bs, 1H, H-2); 7.28(d, 2H, H-22-26, 8.1); 7.14(d, 2H, H-23-25, 8.1); 6.95-7.35(m, 7H, Harom); 4.34(s, 1H, H-20); 4.27(bs, 2H, H-8); 3.27(bs, 2H, H-9); 3.16(bs,4H, H-11-11’); 2.26(s, 3H, H-27); 1.22(bs, 6H, H-12-12’).

13C-RMN(dmso-d6, δ ppm): 169.12(C-13); 147.62(Cq); 139.56(Cq); 135.85(Cq); 133.18(Cq); 132.01(Cq); 130.55(CH); 130.16(CH); 129.77(C-22-26); 129.77(CH); 129.32(CH); 129.13(C-23-25); 128.63(CH); 127.70(CH); 127.38(CH); 126.52(CH); 126.42(Cq); 46.79(C-9); 46.45(C-11-11’); 44.05(C-8); 34.92(C-20); 20.56(C-27); 8.42(C-12-12’).

IR(ATR in solid, cm1): 3052; 2979; 2881; 2336; 1626; 1587; 1491; 1474; 1442; 1392; 1321; 1244; 1177; 1122; 1101; 1078; 1034; 1017; 890; 839; 803; 784; 763; 751; 721; 692; 675; 625; 491.

N-(2-diethylaminoethyl)-N-phenyl-2-(4-methyl-phenylthiomethyl)-benzamide hydrochloride (1e)

-white-yellow crystals, m.p. 94-97oC (anhydrous toluene) (60% yield).

- TLC: single spot Rf=0.78 (the same visualisation)

- elemental analysis: N% t/e: 5.97/6.10

1H-RMN(dmso-d6, δ ppm, J Hz): 7.32(d, 1H, H-19, 7.2); 7.27(d, 2H, H-22-26, 8.0); 7.14(d, 2H, H-23-25, 8.0); 7.01(dl, 1H, H-16, 6.8); 6.95(t, 1H, H-17, 6.8); 7.34-7.07(m, 6H, H-2÷6, H-18); 4.34(s, 2H, H-20); 4.22(tl, 2H, H-8, 5.8); 3.28(tl, 2H, H-9, 5.8); 3.16(ql, 4H, H-11-11’, 6.3); 2.26(s, 3H, H-27); 1.19(t, 6H, H-12-12’, 6.3).

13C-RMN(dmso-d6, δ ppm): 169.44(C-13); 142.07(Cq); 135.90(Cq); 135.77(Cq); 134.72(Cq); 132.23(Cq); 130.15(CH); 129.85(CH); 129.24(CH); 129.15(CH); 129.11(CH); 128.82(CH); 127.71(CH); 127.43(CH); 126.36(CH); 46.85(C-9); 46.70(C-11-11’); 43.95(C-8); 35.03(C-20); 20.64(C-24); 8.52(C-12-12’).

IR(ATR in solid, cm1): 3369; 2986; 2943; 2884; 2392; 1634; 1590; 1492; 1449; 1419; 1383; 1306; 1276; 1231; 1173; 1124; 1090; 1031; 896; 799; 769; 733; 697; 647; 612; 582; 486.

The antimicrobial activity [8] of these new benzamides showed a good action, all amides being active against the reference strains and also against gram-negative clinical strains; all of them exhibit antifungal activity.

CONCLUSIONS

In this paper we presented the synthesis of new amides of 2-phenylthiomethyl-benzoic acid, using the optimized synthesis methods. The compounds were physico-chemical characterized and their structure and purity were established using the elemental analysis, TLC method and 1H-NMR, 13C-NMR and IR spectral analysis. The antimicrobial tests confirmed that these amides have a significant bactericidal and antifungal action.

The present research was financed by research contract CEEX 85/2006 between University of Medicine and Pharmacy “Carol Davila” and The MedicalScienceAcademy, the authority of Programme “Excellence Research Projects”.

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