The Natural Products Journal, 2012, 2, 000-000
Antimicrobial and Radical Scavenging Activities of N-Hydroxycinnamoyl – L-Cysteine and - L-Proline Ethyl Esters Maya G. Chochkovaa,*, Elitsa Y. Chorbadzhiyska, Galya I. Ivanovab, Hristo Najdenskic, Mariana Ninovac and senka S. Milkova a
South-West University “Neofit Rilski”, Faculty of Mathematics and Natural Sciences, Department of Chemistry, 66, Ivan Mihailov Str., Blagoevgrad 2700, Bulgaria; bRequimte, Polo da Universidade do Porto, Departamento de Química, Rua do Campo Alegre, Porto 4169-007, Portugal; cThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. Bl. 26, Sofia 1113, Bulgaria Abstract: Hydroxycinnamic acids are widely distributed in the plant kingdom secondary metabolites, found also as simple derivatives including amides, esters, and glycosides. These acids and their derivatives are known to possess antibacterial, antiviral, anti-inflammatory, antioxidative, antiproliferative, immunostimulatory and neuroprotective properties. The aim of the present work was the synthesis of new hydroxycinnamoyl amides of the (L)- cysteine and (L)- proline and evaluation of their radical scavenging and antimicrobial activity. The structures of the synthesized analogues were characterized by UV, 1H NMR, 13C NMR, ESI-MS. The compounds were screened for their antibacterial (against Staphylococcus aureus 209, Streptococcus pyogenes 10535, Bacillus subtilis 1A95, Listeria monocytogenes C12) and antifungal (against Candida albicans 62) activities. All amides demonstrated the most potent activity against Streptococcus pyogenes, even higher in comparison with the free hydroxycinnamic acids. The ability of hydroxycinnamoyl amides to interact with 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical in vitro was also evaluated. The results obtained showed that the radical scavenging activity of the sinapoyl- and caffeoyl amides of L-cysteine is superior to the standards ferulic acid, eugenol and isoeugenol.
Keywords: Antimicrobial activity, DPPH radical, L-cysteine and - L-proline ethyl esters, N-hydroxycinnamoyl amides. 1. INTRODUCTION Substituted forms of cinnamic acid (CA), such as caffeic acid (CafA), sinapic acid (SA), ferulic acid (FA), p-coumaric acid (CoA) and etc. are the major subgroup of phenolic compounds. They are secondary metabolites, widely distributed in plant kingdom, found also as simple derivatives including amides (conjugated with mono- or polyamines; amino acids or peptides), esters, and glycosides. Hydroxycinnamic acids and their derivatives are known to possess antibacterial, antiviral, anti-inflammatory, antioxidative, antiproliferative, immunostimulatory and neuroprotective properties [1-9]. It is thought that the therapeutical effects on humans is due to the antioxidant and free radical scavenging properties of these compounds.
strains to traditional antibiotics stimulates the investigators on search of new and more active antimicrobials. Herein, we present results on examination of antimicrobial and radical scavenging activity against DPPH: of the obtained hydroxycinnamoyl amides of the (L)-cysteine and (L)-proline. 2. MATERIALS AND METHODS 2.1. Chemistry 2.1.1. Chemistry General
Recently, the antibacterial properties of cinnamic acids and their derivatives against Bacillus subtilis , as well as the antifungal effect of L-cysteine and its esters against fungi from Zygomycetes species, are reported in the literature [7, 8, 11]. The appearance of resistant pathogenic bacterial
(E)-3-Phenylacrylic(cinnamic), (E)-4-hydroxy-3methoxycinnamic-(ferulic), (E)-3,4-dihydroxycinnamic(caffeic), (E)-3,5-dimethoxy-4-hydroxycinnamic (sinapic) acids; 1- [3-(di-methylamino) propyl]-3-ethyl carbodiimide hydrochloride (EDC), trifluoroacetic acid (TFA), N-methyl morpholine (NMM), DPPH (1,1-diphenyl-2-picrylhydrazyl) radical were purchased from Sigma-Aldrich and amino acid derivatives were from Bachem.
*Address correspondence to this author at the South-West University “Neofit Rilski”, Faculty of Mathematics and Natural Sciences, Department of Chemistry, 66, Ivan Mihailov Str., Blagoevgrad 2700, Bulgaria; Fax: +359 73 88 55 16; E-mail: [email protected]
The synthesized amides were indentified by UV, 1H NMR (nuclear magnetic resonance), 13C NMR, 1H,1HCOSY (two dimensional (1H/1H) correlated spectroscopy ) and ESI-MS (electrospray ionization mass spectrometry). NMR spectra were acquired on a Bruker Avance II+ 600
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spectrometer operating at 600.13 MHz for protons, equipped with pulse gradient units, capable of producing magnetic field pulsed gradients in the z-direction of 56.0 G/cm. The measurements in CDCl3 solutions were carried out at ambient temperature (27º C) and tetramethylsilane (TMS) was used as an internal standard. 1H,1H- COSY spectra were recorded using the standard Bruker software. The UV spectra of the compounds and the reduction of DPPH. absorbance at 516 nm in C2H5OH solutions were measured using an “Agilent 8453” UV-VIS spectrophotometer. Mass spectra were measured by Esquire 3000 plus mass spectrometer.
(m, 1H, CHCH2), 6.26 (d, J=15.6 Hz, 1H, =CH), 6.32 (d, J=6.8 Hz, 1H, NH), 6.91 (d, J=8.0 Hz, 2H, m-ArH), 7.01 (d, J=1.6 Hz, 1H, o-ArH),7.06 (dd, J=8.0, 1.6 Hz, 1H, oArH), 7.28-7.34 (5H, ArH), 7.56 (d, J=15.6 Hz, 1H, =CH); 13 C NMR (600MHz, CDCl3) 14.13 (CH2CH3), 33.8 (CH2), 36.8 (CH2), 51.9 (CH), 55.9 (CH3), 62.0 (CH2CH3), 109.4 (CH/Ar), 114.7 (CH/Ar), 117.3 (=CH), 122.6 (CH/Ar), 127130 (CH/Ar), 137.8 (Cq/Ar), 142.1 (=CH), 146.7 (Cq/Ar), 147.6 (Cq/Ar), 165.8 (-CONH-), 170.9 (-COO CH2CH3); ESI-MS: 416.1 ([M + H]+), 438.3 ([M + Na]+).
Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck, Germany). The spots were visualized by spraying with ceric sulphate solution or by UV absorption at 254 nm.
UV (C2H5OH) max = 203,220,324 nm; 1H NMR (600MHz, CDCl3) 1.22 (t, J=7.3 Hz, 3H, CH2CH3), 2.89 (br. dd, J=13.8, 5.1 Hz, 2H, CHCH2a), 2.95 (br. dd, J=13.8, 4.4 Hz, 2H, CHCH2b), 3.66 (d, J=2.7 Hz, 2H, -S-CH2-Ph), 3.86 (s, 6H, OCH3), 4.16 (q, J=7.3 Hz, 2H, CH2CH3), 4.88 (m, 1H, CHCH2), 6.21 (d, J=15.5 Hz, 1H, =CH), 6.26 (d, J=7.4 Hz, 1H, NH), 6.69 (s, 2H, o-ArH), 7.15-7.27 (5H, ArH), 7.94 (d, J=15.5 Hz, 1H, =CH); 13C NMR (600MHz, CDCl3) 14.13 (CH2CH3), 33.8 (CH2), 36.8 (CH2), 51.9 (CH), 55.9 (CH3), 57.3 (CH3), 62.0 (CH2CH3), 104.8 (CH/Ar), 117.7 (=CH), 126-130 (CH/Ar), 142.2 (=CH), 147.1 (Cq/Ar), 147.3 (Cq/Ar), 165.7 (-CONH-), 170.9 (-COO CH2 CH3); ESI-MS: 446.3 ([M + H]+).
2.1.2. General Synthetic Procedure of Esters The ethyl esters of H-Cys(Bzl)-OH and Boc-Pro-OH were synthesized according to Brenner et al.,  and Dhaon et al., . Removal of the t-Boc (t-butyloxycarbonyl) group in Boc-Pro-OEt was accomplished with 50% TFA (trifluoroacetic acid) in CH2Cl2. 2.1.3. Typical Procedure for Synthesis of Hydroxycinnamic Amino Acid Amides  Substituted cinnamic acid (1 mM), 0.113g (1 mM) HOBt and 0.160 g (1 mM) EDC were dissolved in 5 ml CH2Cl2 . The resulting solution was cooled in an ice water bath and after 10 min a solution of (0.84 mM) HCl.H-Cys(Bzl)OC2H5 (or TFA.H-Pro- OC2H5 ) in 3 ml CH2Cl2 and 0.9 ml NMM was added. The mixture was stirred at 0 °C for 30 min and then at room temperature overnight. The mixture was diluted with additional CH2Cl2 and washed successively with 5% NaHSO4 and brine.The organic layer was dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was chromatographed on a silica gel column using CH2Cl2/ CH3 COCH3. (1) N-trans-Cinnamoyl-L-cysteine(Bzl)-ethylester, Yield 43.4% UV (C2H5OH) max = 204,223,297nm;1H NMR (600MHz, CDCl3) 1.22 (t, J=7.2 Hz, 3H, CH2CH3), 2.89 (dd, J=14.2, 5.2 Hz, 2H, CHCH2a), 2.94 (dd, J=14.2, 4.6 Hz, 2H, CHCH2b), 3.66 (d, J=5.6 Hz, 2H, -S-CH2-Ph), 4.16 (q, J=7.2 Hz, 2H, CH2 CH3), 4.87 (ddd, J= 7.0, 5.2, 4.6 Hz, 1H, CHCH2), 6.28 (d, J=7.0 Hz, 1H, NH), 6.32 (d, J=15.6 Hz, 1H, =CH), 7.57 (d, J=15.6 Hz, 1H, =CH), 7.24 (m, 5H, Ar-H), 7.31 (br t, J=7.9 Hz, 3H, m,p-ArH), 7.45 (dd, J=7.9, 2.0 Hz, 2H, o-Ar-H), 7.57 (d, J=15.6 Hz, 1H, =CH); 13 C NMR (600MHz, CDCl3) 14.18 (CH2CH3), 33.71 (CH-CH2), 36.8 (S-CH2), 51.9 (CH), 56.3 (CH3), 61.96 (CH2CH3), 119.78 (=CH), 126.93-131.70 (CH/Ar), 134.54 (Cq/Ar), 137.77 (Cq/Ar), 142.01 (=CH),165.47 (-CONH-), 170.80 (-COO CH2CH3); ESI-MS: 370.4 ([M + H]+), 492.5 ([M + Na]+), 739.2 ([2M + H ]+), 761.6 ([2M + Na]+).
(3) N-trans-Sinapoyl-L-cysteine(Bzl)-ethylester, Yield 49,3 %
(4) N-trans-Caffeoyl-L-cysteine(Bzl)-ethylester, Yield 19,8 % UV (C2H5OH) max = 204,326 nm; 1H NMR (600MHz, CDCl3) 1.20 (t, J=7.5 Hz, 3H, CH2CH3), 2.87 (br. dd, J=14.1, 5.5 Hz, 2H, CHCH2a), 2.91 (br. dd, J=14.1, 4.6 Hz, 2H, CHCH2b), 3.65 (d, J=3.9 Hz, 2H, -S-CH2-Ph), 4.15 (q, J=7.5 Hz, 2H, CH2 CH3), 4.83 (m, 1H, CHCH2), 6.15 (d, J=15.6 Hz, 1H, =CH), 6.54 (br.s, 1H, NH), 6.78 (d, J=8.1 Hz, 1H, m-ArH), 6.85 (d, J=8.1 Hz, 1H, o-ArH), 7.01 (s, 1H, o-ArH), 7.10-7.25 (m, 5H, ArH), 7.33 (2H, OH), 7.43 (d, J=15.6 Hz, 1H, =CH). 13C NMR (600MHz, CDCl3) 13.8 (CH2CH3), 33.7 (CH2), 37.2 (CH2), 62.5 (CH2CH3), 52.03 (CH); 114.28 (o-CHC-OH/Ar), 116.63 (=CH), 115.46 (m-CH/Ar), 121.66 (o-CH-C-/Ar), 126-130 (5CH/Ar), 142.66 (=CH), 162.3 (-CONH-), 178.4 (-COO CH2CH3); ESI-MS: 402.1([M + H]+), 424.5 ([M + Na]+). (5) N-trans-Feruloyl-proline-ethylester, Yield 24.8% UV (C2H5OH) max = 218, 237, 329 nm; 1H-NMR (CDCl3) /600 MHz/ = 1.26 (t, 3H, CH3), 1.88-2.06 (m, 4H, 2 x CH2), 3.78 (m, 2H, N-CH2), 3.92 (s, 3H, OCH3), 4.21 (q, 2H, -OCH2-), 4.62 (m, 1H, CH), 6.59 (d, 1H, J = 15.4 Hz, CH=), 6.91 (d, 1H, J = 8.2 Hz, Ar-H(m)), 6.99 (d, 1H, J = 1.9 Hz, Ar-H(o), 7.09 (dd, 1H, J = 8.2, 1.9 Hz), 7.53 (d, 1H, J = 15.4 Hz, CH=); 13C NMR (600MHz, CDCl3) 14.0 (CH2CH3), 23.4 (CH2), 28.7 (CH2), 47.8 (CH2), 55.7 (CH3), 59.1 (CH), 61.3 (CH2 CH3), 114.7 (CH/Ar), 116.7 (=CH), 121.4 (CH/Ar), 129.7 (CH/Ar), 142.1 (=CH), 148.4 (CH/Ar), 158.1 (-CON500
Tobramycin Cinnamic acid
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DPPH Radical Scavenging Activity of N-hydroxycinnamoyl Amino Acid Amides. The Values are Given as the Mean ± Standard Error ( % ) RSA 0.9 mM
Reaction time ( min ) AH
O R` OH
R`` R``` R` = R `` = R ``` = H, cinnamic acid ( CA); R` = H, R `` = OH, R ``` = OCH 3, ferulic acid ( FA); R` = R ``` = OCH 3, R `` = OH, sinapic acid ( SA); R` = H, R `` =R ``` = OH, caffeic acid ( CafA).
(1) CA-Cys(Bzl)-OEt; (2) FA-Cys(Bzl)-OEt; (3) SA-Cys(Bzl)-OEt; (4) CafA-Cys(Bzl)-OEt; (5) FA-Pro-OEt; (6) SA-Pro-OEt.
Scheme 1. Reaction and conditions: (a) HCl.H-AA-OEt(AA=Proor Cys(Bzl)), EDC/HOBt, NMM, CH2Cl2.
All pathogens are causal agents of medically important infections like listeriosis in animals and humans, with growing importance as a food-borne pathogen. Staphylococcus aureus is one of the causative agents of skin infections, pimples, boils, etc. Many different diseases can be caused by Streptococcus pyogenes ranging from mild, like strep throat and impetigo, to severe, like necrotizing fasciitis and streptococcal toxic shock syndrome. The last two pathogens are commonly implicated in respiratory tract infections varying from acute bacterial sinusitis to community acquired pneumonia. Bacillus subtilis is not considered a human pathogen, however it was included because is a widely accepted model organism for testing antibacterial activity of many naturally or artificially designed compounds.
Among Candida spp., Candida albicans was chosen because of its high incidence in candidiasis in the world . The screening results on the activity of the tested amides against the bacteria strains and fungi, expressed as minimal inhibitory concentration (MIC) in g/ml, are summarized on Table 1. The values of the estimated MIC of the amides varied from 62.5 to 500g/ml. Tobramycin as a conventional antibacterial agent, and ketoconazole as an antifungal agent, were included in the assay for comparison. Among the used strains, Gram positive S. pyogenes was the most sensitive strain to all of the tested analogues. Although the activity of the control tobramicyn is not reached, all of the tested cinnamoyl amides were promising
Biological Activities of N-hydroxycinnamoyl - Cysteine and - Proline
against one and the same strain (S. pyogenes) with MIC 62.5, followed by free hydroxycinnamic acids (MIC 125).
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3.3. Radical Scavenging Activity Against DPPH Radical As by this test the H radical/electron transferring ability of the stable DPPH radical is measured, the results obtained provided a rough index of the scavenging activity of the studied amides [16, 20]. The scavenging abilities of the synthesized compounds (1-6) and reference standards (quercetin, eugenol, isoeugenol, caffeic-, sinapic-, ferulic acids and D,L -tocopherol) are summarized on Table 2. The DPPH scavenging activity of cinnamoyl- and hydroxycinnamoyl amino acid amides and standards decreases as follows: quercetin > caffeic acid (CafA) > sinapic acid (SA) > tocopherol > 3 > 4 > ferulic acid (FA) > 6 > 2 > eugenol > isoeugenol > 5 > 1 > cinnamic acid (CA). It is worth to note that all tested hydroxycinnamoylamides (with the only exception of cinnamoyl amide (1)) have good antiradical activities. Among the tested amides, caffeoyl- and sinapoyl L-cysteine amides (3 and 4) have shown the highest %RSA, also higher than the standards as: FA> eugenol>isoeugenol. The absence of a phenolic group in compound (1) decreases RSA. Conversely, the presence of phenolic and methoxyl groups in the phenylpropenoyl part of the molecules increases significantly RSA. Although this study did not show correlation between antimicrobial and radical scavenging activity, the observed effects of the examined amides could serve as a basis for future planned synthesis of new active derivatives.
CONFLICT OF INTEREST 
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We acknowledge the financial support of South-West University "Neofit Rilski"(Project SRP-A7), Bulgaria and the National Science Fund for the purchase of Bruker Avance II+ 600 NMR spectrometer in the framework of the Program “Promotion of the Research Potential through Unique Scientific Equipment” (Project UNA-17/2005).
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Received: June 26, 2011
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Revised: December 21, 2011
Accepted: January 09, 2012