new chiral 4-substituted 2-cyanoethyl-oxazolines: synthesis and assessment of some biological...

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Chemico-Biological Interactions xxx (2014) xxx–xxx

CBI 7024 No. of Pages 8, Model 5G

16 April 2014

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journal homepage: www.elsevier .com/locate /chembioint

New chiral 4-substituted 2-cyanoethyl-oxazolines: Synthesisand assessment of some biological activities

http://dx.doi.org/10.1016/j.cbi.2014.04.0030009-2797/� 2014 Published by Elsevier Ireland Ltd.

Abbreviations: ASL, lysine acetylsalicylate; COX, cyclooxygenases; DMSO,dimethyl sulfoxide; DNA, deoxyribonucleic acid; DPPH, 1,1-diphenyl-2-pic-rylhydrazyl radical; EDTA, ethylenediaminetetraacetic acid; EtOH, ethanol; HIV-1,Human immunodeficiency virus; 1H NMR, proton nuclear magnetic resonance; IC50,concentration inhibiting a biological response by 50%; Me3SiCl, trimethylsilylchlo-ride; MICs, Minimum Inhibitory Concentrations; MBCs, Minimum BactericidalConcentrations; NBT, nitroblue tetrazolium; NSADs, n-steroidal anti-inflammatorydrugs; PGE, prostaglandin E; TLC, thin layer chromatography; TOFMS ES+, time-of-flight mass spectrometry; Zn(OAc)2, zinc acetate.⇑ Corresponding author. Tel.: +216 73500279; fax: +216 73500278.

E-mail address: [email protected] (B. Ben Hassine).

Please cite this article in press as: R. Hassani et al., New chiral 4-substituted 2-cyanoethyl-oxazolines: Synthesis and assessment of some biologicaities, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.04.003

Rym Hassani a, Yakdhan Kacem a, Hedi Ben Mansour b, Hamed Ben Ammar a, Béchir Ben Hassine a,⇑a Laboratoire de Synthèse Organique Asymétrique et Catalyse Homogène (UR11ES56), Faculté des Sciences, Avenue de l’environnement, 5019 Monastir, Tunisiab Laboratoire de biotechnologie et Valorisation de Bio Géo Ressources (LBVBGR), Institut Supérieur de Biotechnologie, ISBST BioTechPole Sidi Thabet Université Manouba,Ariana 2020, Tunisia

a r t i c l e i n f o

293031323334353637383940

Article history:Received 15 January 2014Received in revised form 12 March 2014Accepted 2 April 2014Available online xxxx

Keywords:OxazolineAmino alcoholMonohydrochlorideIminoetherBiological activities

414243444546474849505152

a b s t r a c t

This paper describes the synthesis of new enantiomerically pure 2-cyanoethyl-oxazolines in one stepstarting from a wide range of amino alcohols and 4-ethoxy-4-iminobutanenitrile with high to good yields(73–96%) via an appropriate procedure which can be used for a selective synthesis of mono-oxazolines. Asimple operation as well as a practical separation is additional eco-friendly attributes of this method. Allthe synthesized compounds were identified and characterized with their physicochemical features andtheir spectral data (1H NMR, 13C NMR and TOFMS ES+). Among the prepared mono-oxazolines, themono-oxazoline (3a) [3-[(4S)-4-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile] was tested to detectsome biological activities. This compound was studied in vitro given the various types of pharmacologicalproperties characterizing these compounds such as antioxidant, antimicrobial and analgesic activities.The antioxidant activity and mechanism of (3a) were identified using various in vitro antioxidant assaysincluding 1,1-diphenyl-2-picryl-hydrazyl (DPPH�), and superoxide anion radicals (O2

��) scavenging activ-ity. In addition, compared to Quercetin, the tested synthetic product reveals a relatively-strong antirad-ical activity towards the DPPH (activity percentage of 81.22%) free radicals and significantly decreasedthe reactive oxygen species such as (O2

��) formation evaluated by the non-enzymatic (nitroblue tetrazo-lium/riboflavine) and the enzymatic (xanthine/xanthine oxidase) systems. Related activity values were,respectively, 66% and 60.30%. The oxazoline (3a) showed a high ability to reduce the O2

�� generationand proved to be a very potent radical scavenger. On the other hand, the analgesic property of the3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a) was demonstrated. The subcutaneousadministration of (3a) produced a significant reduction in the number of abdominal constrictionsamounting to 73.81% in the acetic acid writhing test in mice. In addition to these advances, the oxazoline(3a) has been investigated as an antimicrobial agent. Our results showed that this molecule exhibitedvarious levels of antibacterial effect against all the tested bacterial strains.

� 2014 Published by Elsevier Ireland Ltd.

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1. Introduction

Literature reveals that nitrogen containing heterocyclic mole-cules constitutes the largest portion of chemical entities whichare part of many natural products, fine chemicals, biologically-active pharmaceuticals and agrochemicals playing a vital role inenhancing the quality of life.

Among a large variety of nitrogen-containing heterocycliccompounds, the 2-oxazolines have impregnated numerous sub-disciplines in the field of synthetic organic chemistry over100 years since their discovery [1]. This versatile heterocycleshas served as a protecting group, a coordinating ligand, and anactivating moiety, often exhibiting all of these characteristics in asingle transformation. The well-defined reactivity of chiral

l activ-

http://dx.doi.org/10.1016/j.cbi.2014.04.003

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2 R. Hassani et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx


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oxazolines has given rise to numerous highly-efficient strategiesfor an asymmetric synthesis including their use as ligands in anasymmetric catalysis.

Various properties have imposed their use in a multitude of dis-similar applications: as monomers in polymer production, as mod-erators in analytical processes, and as conformationally rigidpeptide mimics in medicinal chemistry. Even natural systems havechosen to incorporate 2-oxazolines into their chemical arsenal, asevidenced by the rapidly growing number of identified naturalproducts and their attendant pharmacological properties (Fig. 1).This could be explained also by the various applications of theseproducts in the synthesis of different therapeutic and biologi-cally-active compounds [2] such as antidiabetic, antihypertensive,antidepressive, antihypercholesterolemic, anticancer, anti HIV-1,antitumor and antialzheimer agents [3]. Thanks to their structuralrelationship with procaine, 2-oxazolines derivatives are expectedto have local anaesthetic properties [4]. In addition to these seriousmatters, some 2-oxazoline derivatives act as enzyme inhibitors[5,6]. Furthermore, the 2-oxazoline moieties are often found in var-ious bioactive natural compounds and designed medicinal agents,such as the Brasilibactin A [7]. These compounds exist as the keyfragment in numerous marine organisms, such as epi-oxazolinehalipeptin D [8].

On the other hand, 2-oxazolines are considered an importantclass of heterocycles and are versatile intermediates in syntheticorganic chemistry [9,10]. Moreover, chiral oxazolines have beenwidely used in asymmetric synthesis both as building blocks[11], protecting groups for amino alcohols [12] and as auxiliaries,metal entrapment ligands and ligands [13]. In this setting, a num-ber of methods have been developed for the preparation of 2-oxaz-olines from carboxylic acids [14], carboxylic esters [15], nitriles[16], aldehydes [17], hydroxyamides [18] and olefins [19]. In spiteof the potential utility of aforementioned routes for the synthesisof oxazoline derivatives, many of these procedures have demon-strated various drawbacks including strong acidic conditions, longreaction times, tedious work-up, low yields, use of expensive, toxicor non-reusable catalysts, high temperatures, harsh reaction condi-tions and use of toxic solvents such as CCl4, hexachloroethane orchlorobenzene and/or co-occurrence of several side reactions. Insome cases, more than one step is required for the synthesis ofthese heterocycles. Therefore, to avoid these limitations, there isstill a need to search for an efficient method with regard to toxicity,solubility and reaction time.

In the present work, we describe the synthesis in one step ofsome new chiral 4-substituted 2-cyanoethyl-oxazolines using a-amino alcohols and the monohydrochloride of an iminoether asstarting materials. The results indicate that the present protocolis potentially applicable for the chemoselective conversion of a-amino alcohols to their corresponding 2-oxazolines in the presenceof iminoether. It is well known that the oxazolines’ derivatives

Epi-oxazoline halipeptin D

Fig. 1. Examples of several biologically-ac

Please cite this article in press as: R. Hassani et al., New chiral 4-substituted 2-ities, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.20

have different biological activities. Based on the literature, we haveassessed their pharmacological properties such as antioxidant,antimicrobial and analgesic activities.

2. Materials and methods

2.1. Analytical methods

All reactions were monitored on thin-layer chromatographic(TLC) Merck 60 F-254 silica-gel plates (0.25 mm layer thickness).Column chromatography was performed on silica gel (70–230mesh) using dichloromethane and methanol mixture as eluents.Melting points were determined on an Electrothermal 9002 appa-ratus and were uncorrected. The optical rotations were measuredby an Atago Polax-2L polarimeter. NMR spectra were recorded ona Bruker AC 300 spectrometer [300 MHz (1H) and 75 MHz (13C)].All chemical shifts were reported as d values (ppm) relative tointernal tetramethylsilane. Time-of-flight mass spectroscopy (TOF-MS ES+) was carried out on Micromass, UK and Manchester. Ultra-violet (UV) detector (Jasco 2075) and a data system processor wasused (Clarity Lite) to confirm the presence of the targeted products.Detection was performed at 254 nm and 365 nm.

2.2. Chemicals

(S)-2-Amino-3-methylbutanoic acid (L-valine), S-(+)-a-amino-phenylacetic acid ((L)-(+)-(a)-phenylglycine), (S)-2-amino-3-phenylpropanoic acid (L-phenylalanine), (S)-2-Amino-4-methyl-pentanoic acid (L-leucine), (S)-2-Aminopropanoic acid (L-alanine),(S)-2-amino-3-(1H-indol-3-yl)propanoic acid (L-tryptophan), suc-cinonitrile and trimethylsilylchloride were purchased from Sig-ma–Aldrich. The (R)-2-amino-1-butanol c was purchased fromSigma–Aldrich while the a-amino alcohols a, b, d, e, f, g and e wereobtained by the reduction of the corresponding amino acids usingthe method developed by Meyers [20]. 1,1-Diphenyl-2-pic-rylhydrazyl radical (DPPH), ethylenediaminetetraacetic acid(EDTA), potassium phosphate buffer, a-tocopherol, nitro bluetetrazolium (NBT) and riboflavin were purchased from Aldrich(St. Louis, MO). All the other chemicals and solvents used in thiswork were of an analytical quality and purchased from commercialspots.

2.3. Preparation of the synthetic compounds (general procedure)

The synthesis of the 4-ethoxy-4-iminobutanenitrile monohy-drochloride (2) was performed according to the modified methoddescribed by Ben Ammar et al. [21]. The synthesis of 3-[4-ben-zyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a–g) was syn-thesized as described by Meyers [22] (Scheme 1). The structures

Brasilibactin A

tive and pharmacological oxazolines.

cyanoethyl-oxazolines: Synthesis and assessment of some biological activ-14.04.003


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Scheme 1. Synthesis of new 3[4,5-dihydro-1,3-oxazol-2-yl] propanenitriles (3a–g).

R. Hassani et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx 3


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of the final products (3a–g), as well as the one of the intermediatecompounds were confirmed by 1H NMR, 13C NMR and TOFMS ES+.

2.3.1. Synthesis of the 4-ethoxy-4-iminobutanenitrilemonohydrochloride

Succinonitrile (3 g, 37.45 mmol), absolute ethanol (3.45 g,74.91 mmol) and trimethylsilylchloride (TMSiCl) (4.07 g,37.45 mmol) were respectively added in a Schlenk tube. The solu-tion was cooled to �30 �C for 72 h until the precipitation of the saltwas achieved. Subsequently, the resulting salt was washed withanhydrous ether and dried under vacuum and inert atmosphere.The imidate chloride was obtained in 68% yield. 1H NMR (DMSO,300 MHz): 1.01–1.09 (t, 2H), 2.38–2.42 (t, 2H), 4.06–4.11 (t, 2H),4.42–4.49 (q, 3H), 7.23 (s, 1H), 7.40 (s, 1H). 13C NMR (DMSO,75 MHz): 14.05 (–CH3), 28.64 (CH2), 30.50 (CH2), 60.85 (CH2),119.04 (–CN), 171.52 (C@NH2

+).

2.3.2. Synthesis of the 4-substituted 2-cyanoethyl-oxazolines (3a–g)The synthesis of 3-[(4S)-4-benzyl-4,5-dihydro-1,3-oxazol-2-yl]

propanenitrile (3a) is representative. In a Schlenk tube equippedwith a magnetic bar, the monohydrochloride of the 4-ethoxy-4-iminobutanenitrile (1.07 g, 6.61 mmol) and the (L)-phenylalaninol(1 g, 6.61 mmol) were introduced respectively in 50 mL of anhy-drous methylene chloride. Then, the mixture was heated at refluxfor 10 h under an inert atmosphere. After the removal of NH4Cl byfiltration, the organic phase was evaporated and the obtained res-idue was chromatographed on silica gel to afford 96% of the com-pound (3a). The yield, optical rotation, and data for individualcompounds are given in (Table 1).

2.3.2.1. 3-[(4S)-4-Benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile(3a). 96%; Yellow oil; [dichloromethane/methanol (99:01)];[a]D = [�25; c 1, CHCl3]. 1H NMR (CDCl3, 300 MHz): 2.66–2.72(m, 4H), 2.70–2.72 (dd, 1H; J 4.2 Hz, J 2.4 Hz), 3.05 (dd, 1H; J13.8 Hz, J 5.4 Hz), 4.01 (dd, 1H; J 8.4 Hz, J 7.2 Hz), 4.22 (dd, 1H; J9.3 Hz, J 5.7 Hz), 4.37–4.43 (m, 1H), 7.18–7.28 (m, 5H). 13C NMR

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Table 1Preparation and physicochemical properties of new 2-cyanoethyl-oxazolines (3a–g)according to Scheme 1.

Entrya Config. Yield%b [a]D Physical aspect

3a L 96 �25 (c 1, CHCl3) Yellow oil3b L 93 +15 (c 1, CHCl3) Yellow oil3c L 85 +15 (c 1, CHCl3) Yellow oil3d D 89 �25 (c 1, CHCl3) Yellow oil3e L 85 +20 (c 1, CHCl3) Yellow oil3f L 80 �23 (c 1, CHCl3) Yellow oil3g L 73 �17 (c 1, CHCl3) Yellow oil

a Compounds (3a–g) was build-to-build distilled.b Yields of pure, isolated products (characterized by 1H, 13C NMR, TOFMS ES+).


(CDCl3, 75 MHz): 14.15 (CH2), 24.20 (CH2), 41.58 (CH2), 67.26(CH), 72.22 (CH2), 118.70 (CN), 126.67 (CH), 128.59 (CH), 129.35(CH), 137.55 (C), 164.35 (C). TOFMS ES+ for C13H14N2O theoretical[M+H]+: 215.1184; measured [M+H]+: 215.1184.

2.3.2.2. 3-[(4S)-4,5-Dihydro-4-phenyl-1,3-oxazol-2-yl] propanenitrile(3b). 93%; Yellow oil; [dichloromethane/methanol (99:01)].[a]D = [+15; c 1, CHCl3]. 1H NMR (CDCl3, 300 MHz): 2.63–2.77 (m,4H), 4.14 (dd, 1H; J 16.8 Hz, J 8.4 Hz), 4.66 (dd, 1H; J 10.2 Hz, J8.4 Hz), 5.22 (dd, 1H; J 18. 3 Hz, J 9.9 Hz), 7.21–7.33 (m, 5H). 13CNMR (CDCl3, 75 MHz): 14.18 (CH2), 24.28 (CH2), 69.59 (CH),75.30 (CH2), 118.73 (CN), 126.59–127.83 (CH), 128.65 (CH),128.76–128.88 (CH), 141.73 (C), 165.31(C). TOFMS ES+ forC12H12N2O theoretical [M+H]+: 201.1028; measured [M+H]+:201.1030.

2.3.2.3. 3-[(4R)-4,5-Dihydro-4-ethyl-1,3-oxazol-2-yl] propanenitrile(3c). 85%; Yellow oil; [dichloromethane/methanol (99:01)].[a]D = [+15; c 1, CHCl3]. 1H NMR (CDCl3, 300 MHz): 0.86–1.06 (m,3H), 1.47–1.67 (m, 2H), 2.58–2.65 (t, 2H), 2.68–2.74 (t, 2H), 3.91(dd, 1H; J 15.6 Hz, J 8.1 Hz), 4.02–4.03 (m, 1H), 4.05 (dd, 1H; J1.8 Hz, J 1.2 Hz). 13C NMR (CDCl3, 75 MHz): 10.19 (CH2), 14.56(CH3), 24.55 (CH2), 28.80 (CH2), 67.84 (CH), 72.88 (CH2), 116.71(CN), 164.08 (C). TOFMS ES+ for C8H12N2O theoretical [M+H]+:153.1028; measured [M+H]+: 153.1015.

2.3.2.4. 3-[(4S)-4,5-Dihydro-4-isopropyl-1,3-oxazol-2-yl] propaneni-trile (3d). 89%; Yellow oil; [dichloromethane/methanol (99:01)].[a]D = [�20; c 1, CHCl3]. 1H NMR (CDCl3, 300 MHz): 0.78 (d, 3H; J6.8 Hz), 0.85 (d, 3H; J 7 Hz), 1.12–1.22 (m, 1H), 1.57–1.73 (m,1H), 2.47–2.68 (m, 4H), 3.75–3.94 (m, 1H), 4.04–4.22 (m, 1H) .13CNMR (CDCl3, 75 MHz): 14.23 (CH2), 18.10 (CH3), 18.67 (CH3),24.24 (CH2), 32.56 (CH), 70.63 (CH2), 72.13(CH), 118.68 (CN),163.66 (C). TOFMS ES+ for C9H14N2O theoretical [M+H]+:167.1184; measured [M+H]+: 167.1180.

2.3.2.5. 3-[(4S)-4,5-Diyhdro-4-isobutyl-1,3-oxazol-2-yl] propaneni-trile (3e). 85%; Yellow oil; [dichloromethane/methanol (99:01)].[a]D=[+ 20; c 1, CHCl3]. 1H NMR (CDCl3, 300 MHz): 0.84–0.88 (m,6H), 1.13–1.27 (m, 1H), 1.42–1.56 (m, 1H), 1.61–1.71 (m, 1H),2.48–2.70 (m, 4H), 3.72–3.79 (m, 1H), 3.80–4.14 (m, 1H), 4.23–4.33 (m, 1H). 13C NMR (CDCl3, 75 MHz): 13.33 (CH2), 14.55 (CH3),15.06 (CH3), 23.06 (CH), 29.58 (CH2), 45.82 (CH2), 61.03 (CH),72.34 (CH2), 118.17 (CN), 164.25 (C). TOFMS ES+ for C10H16N2O the-oretical [M+H]+: 181.1341; measured [M+H]+: 181.1340.

2.3.2.6. 3-[(S)-4,5-Dihydro-5-methyl-1,3-oxazol-2-yl] propanenitrile(3f). 80%; Yellow oil; [dichloromethane/methanol (99:01)].[a]D = [�23; c 1, CHCl3]. 1H NMR (CDCl3, 300 MHz): 1.23 (m, 3H),2.48–2.63 (m, 4H), 3.29 (dd, 1H; J 13.8 Hz, J 7.2 Hz), 3.83 (dd, 1H;J 14.1 Hz, J 9.3 Hz), 4.55–4.65 (m, 1H). 13C NMR (CDCl3, 75 MHz):14.04 (CH2), 20.41 (CH3), 30.82 (CH2), 60.40 (CH2), 66.11 (CH),118.12 (CN), 169.53 (C). TOFMS ES+ for C8H12N2O theoretical[M+H]+: 139.0827; measured [M+H]+: 139.0830.

2.3.2.7. 3-[(4S)-((1H-Indol-3-yl)-methyl)-4,5-Diyhdro-1,3-oxazol-2-yl] propanenitrile (3g). 73%; Yellow oil; [dichloromethane/metha-nol (99:01)]. [a]D = [�17; c 1, MeOH]. 1H NMR (CDCl3, 300 MHz):2.60–2.66 (m, 4H), 2.84 (dd, 1H; J 14.4 Hz, J 7.8 Hz), 3.15 (dd, 1H;J 14.7 Hz, J 5.1 Hz), 4.02 (dd, 1H; J 8.4 Hz, J 7.2 Hz), 4.18 (dd, 1H;J 8.7 Hz, J 7.2 Hz), 4.43–4.58 (m, 1H), 7.02 (s, 1H), 7.06–7.11(td,1H; J 7.5 Hz, J 1.2 Hz), 7.13–7.19 (td, 1H; J 7.5 Hz, J 1.2 Hz),7.33 (d, 1H; J 7.2 Hz), 7.59 (d, 1H; J 8.1 Hz), 8.35 (s, NH). 13CNMR (CDCl3, 75 MHz): 14.07 (CH2), 29.45 (CH2), 60.95 (CH2),65.95 (CH), 72.00 (CH2), 115.78 (CN), 110.75–135.79 (CIndol),



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169.59 (C). TOFMS ES+ for C10H16N2O theoretical [M+H]+:254.1293; measured [M+H]+: 254.1298.

2.4. Superoxide radical-scavenging activity

The ability of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl]propanenitrile (3a) to ensnare superoxide anions was carried outaccording two methods using the non-enzymatic and enzymaticantioxidants assays.

2.4.1. A non-enzymatic antioxidant assayThe inhibition of NBT reduction by the photochemically-gener-

ated O2�� was used to determine the superoxide anion scavenging

activity of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] pro-panenitrile (3a) according to the method described by Ben Man-sour et al. [23]. In fact, the reaction mixture contained 6.5 mMEDTA, 4 lM riboflavin, 96 lM NBT, and 51.5 mM potassium phos-phate buffer (pH 7.4). Superoxide anions were measured by the in-crease in the absorbance at 560 nm after 6 min of illumination atroom temperature. The synthetic product (3a) as well as the refer-ence substance (Quercetin) were checked at different concentra-tions with three repetitions. IC50 (concentration required toinhibit NBT reduction by 50%) values were calculated from dose-inhibition curves [24].

2.4.2. Inhibition of xanthine oxidase ActivityThe inhibition of XOD-generated superoxide formation was

evaluated by measuring the UV absorbance of uric acid at295 nm as proposed by Ben Mansour et al. [23b]. The assay mix-ture consisted of a 100 lL solution of test compound at each con-centrations (100 lg/mL; 200 lg/mL and 300 lg/mL), a 200 lLsolution of xanthine (X) (final concentration, 50 lM) and hydroxyl-amine (final concentration, 0.2 mM), 200 lL 0.1 mM EDTA, and300 lL distilled water. The reaction was initiated by adding200 lL XOD (final concentration, 11 mU/mL) dissolved in 0.2 Mphosphate buffer (pH 7.5). Following incubation at 37 �C for30 min, the reaction was stopped by adding 100 lL 0.58 M HCl.The absorbance was then measured against a blank solution pre-pared in the same way as described above, but replacing XOD bybuffer solution (no production of uric acid). A control solutionwithout the test compound was prepared in the same manner asthe assay mixture to measure the total uric acid production whichwas calculated from the differential absorbance.

2.5. DPPH radical-scavenging activity

The nitrogen centered stable free radical 1,1-diphenyl-2-pic-rylhydrazyl (DPPH) has often been used to characterize antioxi-dants. It is reversibly reduced, and the odd electron in the DPPHfree radical gives a strong absorption maximum at 517 nm, (purplein color). This property makes it suitable for spectrophotometricstudies. A radical scavenging antioxidant reacts with the DPPH sta-ble-free radical and converts into the 1,1-diphenyl-2-picrylhydr-azine. The resulting decolorization is steochiometric with respectto the number of the captured electrons. The change in the absor-bance produced in this reaction has been used to measure the anti-oxidant properties. The free-radical scavenging capacity of the newsynthetic oxazoline was determined with DPPH�. Ethanol solutionswere prepared containing 100, 30, 10, 3 and 1 lg/mL of the syn-thetic product (3a) and 23.6 lg/mL of DPPH. After incubation for30 min at ambient temperature, the absorbance of the remainingDPPH was determined colorimetrically at 517 nm. Radical scaveng-ing activity was measured as the decrease in the absorbance of thesamples versus a DPPH standard solution. Results were expressedas ‘‘percentage inhibition’’ (%) of the DPPH and the mean 50%inhibiting concentration (IC50). % is defined by the formula:


(%) = [(ODcontrol � ODsample)/ODcontrol] � 100, where ODcontrol is theinitial absorbance and ODsample is the value for the test sampleafter incubation. IC50 was defined as the concentration (in lg/mL) of the substrate that causes as 50% loss of DPPH activity (color)and was calculated by using the Litchfield and Wilcoxon test [25].The results are expressed as the mean of data from at least threeindependent experiments.

2.6. Antimicrobial testing

The antimicrobial activity of a synthetic molecule was tested onthe Gram-positive bacteria Staphylococcus aureus ATCC 25923 andEnterococcus faecalis ATCC 29212 as well as the Gram negative bac-teria Escherichia coli ATCC 25922 using the microdilution method[26]. Overnight grown microbial suspensions were standardizedto approximately 105 cells/mL [27]. The microdilution methodwas used to determine the Minimum Inhibitory Concentrations(MICs) of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propane-nitrile (3a). To do this, 100 lL of a microbial suspension containing,approximately 105 cells/mL, was added to 100 lL of the oxazoline(3a) dissolved in a minimum of EtOH (concentrations ranging from10 lg/mL to 500 lg/mL in water). A set of tubes containing onlythe microbial suspension served as the negative control. Theseserially-diluted cultures were, then, incubated at 37 �C for 24 h.Subsequently, 10 lL of each culture was placed on a substance-freeMueller–Hinton agar plates and, further, incubated at 37 �C for24 h. The MIC was defined as the lowest concentration of the testedproduct that completely suppresses cell growth. A minimal Bacte-ricidal Concentration (MBC) was defined as the lowest concentra-tion of the extract that kills 99.99% of the tested bacteria [28].

2.7. Analgesic testing

The analgesic activity can be performed according to the meth-od of Koster et al. [29]. Swiss mice (20–30 g) were selected 1 dayprior to each test and were divided into three groups of six miceeach. The first group served as control (saline 10 mL/kg bw). Thesecond group was given the lysine acetylsalicylate (ASL)(200 mg/kg bw) by the same route, as a reference drug. The thirdgroup was treated with the oxazoline (3a) (100, 200, 300 and400 mg/kg bw). All animals received 10 mL/kg (i.p.) of 1% aceticacid, 30 min after treatment. The number of writhes was recordedduring 30 min, starting 5 min after the acetic acid injection. Awrithe is shown by an abdominal constriction and a stretching ofat least one hind limb.

3. Results and discussion

The 2-oxazoline ring system has been familiar for more than acentury. A number of reliable preparative methods, developedbefore this heterocycles, knew a widespread utility and are stillvaluable and in use today. The direct synthesis of 2-cyanometh-yl-4-ethyl-oxazolines has been investigated by Elliot et al. [30],starting from malononitrile and a-amino alcohols under acidicconditions. Garcia et al. [31], has made possible the preparationof the related 2-cyanomethyl-oxazolines from 2,2-dimethylmalo-nitrile and a-amino alcohols ranging from high to good yieldsusing the Zn(OAc)2 as catalyst, although the formation of bis-oxazoline and methyl oxazoline that is not defined in this work,was observed. However, this reported method for the synthesisof 2-oxazolines suffer from some disadvantages such as long reac-tion times, low yields, high temperatures with elimination ofammonia, problems associated with the solubility of amino alcoholin toluene and the lower stability of the unsubstituted oxazolinering which led to difficulties in scaling up reactions using amino



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alcohol as the starting material. Consequently, the design of a sim-ple, inexpensive and environmentally-friendly method for the syn-thesis of 2-oxazolines is a worthwhile.

The synthetic method of chiral 2-cyanoethyl-oxazolines isbased on the cyclocondensation of some a-amino alcohols withan iminoether such as the 4-ethoxy-4-iminobutanenitrile hydro-chloride (2). It is worth noting that the reaction of succinonitrilewith ethanol in the presence of gaseous hydrogen chloride in anhy-drous medium (diethyl ether) represents the most widespread ac-cess to compound (2). Meanwhile, this method suffers from the useof hydrogen chloride and strong acids. However, a new approachbased on the transformation of malononitrile in the iminoethermonohydrochloride has been reported [21]. In the present work,the 4-ethoxy-4-iminobutanenitrile hydrochloride (2) was preparedfrom succinonitrile (1) and trimethylsilylchloride using absoluteethanol as a solvent at �30 �C for 72 h (Scheme 1).

Simple washing furnished the rather unstable crude 4-ethoxy-4-iminobutanenitrile monohydrochloride (2) as a solid. A spectrumof the crude free iminoether monohydrochloride (2) could also beobtained but it was not possible to reach an accurate elementalanalysis of the crude product. Furthermore, the previously notedinstability of this class of salt compounds was then observed whenattempts were made to purify. Recrystallization and silica/aluminachromatography all failed to improve the situation and actuallyyielded a material of lower quality. The instability of the iminoe-ther monohydrochloride (2) prompted us to form more stableand easily characterizable derivatives in which the free imino-group was protected. This simple method allowed us to obtainan unstable product which can be degraded easily in the presenceof air, but with minimal impurities and without using strong acids.However, despite this instability, the iminoether monohydrochlo-ride (2) is a synthetic intermediate in the pathway leading to con-densation systems and a precursor of the formation of theoxazoline.

2-Oxazolines proved to be very sensitive to acidic conditionsundergoing the rearrangement leading to the opening cycle almostquantitatively. Nevertheless, the rearrangement can be preventedfrom working in inert conditions and in the absence of Lewis acidsand proton sources. We have demonstrated that the cycloconden-sation proceeded smoothly at refluxing methylene chloride,obtaining the expected product with 96% yield. Further, no sideproducts were detected in the reaction mixture as determined byTLC analysis and 1H NMR spectra. This method avoids the multi-step preparation of starting materials or the requirement of specialreagents. Hence, we designed a new type of oxazoline with a cya-no-group to get some branched 2-cyanoethyl-oxazolines. In allcases, the reaction affords the new 2-cyanoethyl-oxazolines (3a–g) in high yields without any racemization of the asymmetric car-bon (Table 1).

Recently, we have developed a synthesis method for easy andefficient preparation of chiral 2-oxazolines based on cycloconden-sation. We estimated that our methodology would be ready for thepreparation of a series of oxazoline with cyano group. The reactionbetween the a-amino alcohol and the monohydrochloride of animinoether allowed us to reach an unstable intermediate. Intramo-lecular cyclization converts the final product after the release ofNH4Cl. It should be noted that the method is selective and the con-version is complete for aromatic amino alcohols as for aliphaticamino alcohols. It is also important to note that the synthesis ofmono-oxazoline is a chemoselective, but a time-dependent reac-tion. Mono-oxazolines (3a–f) were satisfactorily produced after10 h whereas the mono-oxazoline (3g) was exclusively obtainedfrom the reaction of (L)-tryptophanol with iminoether after 20 h.Our previous studies showed that the lowest yield of oxazoline isoften obtained with (L)-tryptophanol. However, in our original con-ditions, the synthesis of the oxazoline series was obtained in CH2-


Cl2 except for the compound (3g), the reaction was developed in asolvent mixture CH2Cl2/EtOH as the (L)-tryptophanol is insoluble inCH2Cl2.

In 13C NMR spectroscopy, these compounds showed character-istic signals at 115.6–118.7 ppm for the cyano carbon atom, and inthe range 164.0–169.6 ppm for the sp2 C@N carbon atom. TheOCH2 protons of 4-monosubstituted oxazoline rings are diastereo-topic. Consequently, each of the three hydrogens of the oxazolinering displayed a separate signal in the 1H NMR spectra of com-pounds (3a–g). The assignment of these signals was done by ana-lyzing 13C–[1H], DEPT135 and HMQC spectra. 1D NOESYexperiment was varied out to differentiate between the diastereo-topic OCH2 protons.

We hypothesize that the oxazoline group with another func-tional group (CN) could provide a strong chelating effect in orderto bind to the active site. We anticipated that the rigidity of themolecule may also offer some advantages for a good drug. On theother hand, the variation of the substituent 2-oxazoline has a sig-nificant effect on the activity. In addition, aromatic groups havingoxazoline in position (4) and the cyano-group in position (2) faredmuch better and more specifically increase the bioactive effect. Toprove our hypothesis, we have started by testing the antioxidantactivity.

Radical scavengers may directly react and quench the peroxideradicals to terminate the peroxidation chain reactions and improvethe quality and stability of food products. Assays based upon theuse of DPPH� and O2

�� radicals are among the most popular spectro-photometric methods for the determination of the antioxidantcapacity of foods, beverages, vegetable extracts and chemical prod-ucts. These chromogens and radical compounds can directly reactwith antioxidants. Additionally, DPPH� and O2

�� scavenging meth-ods have been used to evaluate the antioxidant activity of com-pounds due to the simple, rapid, sensitive, and reproducibleprocedures [33]. First, it has been reported that antioxidant prop-erties of some oxazolines are effective mainly via the scavengingof superoxide anion radicals. The superoxide anion is the precursorof hydrogen peroxide, hydroxyl radical, and singlet oxygen whichinduce oxidative damage in lipids, proteins and DNA [34]. Superox-ide radicals are normally formed first, and their effects can be mag-nified because they produce other kinds of free radicals andoxidizing agents. Superoxide anions derived from dissolved oxygenby the nitroblue tetrazolium/riboflavine system will reduce NBT inthis system. The NBT assay is based on the capacity of the extractsto inhibit the photochemical reduction of nitroblue tetrazolium(NBT) in the presence of riboflavin.

In the presence of an antioxidant that can donate an electron toNBT, the purple color typical of the formazan decays, a change thatcan be followed spectrophotometrically at 560 nm. The syntheticproduct (3a) and the reference substance (Quercetin) were assayedat different concentrations with three repetitions. The oxazoline(3a) seems to be a potent antioxidant with an activity percentageof 66% at the highest concentration (10 mg/mL) compared to thepositive control, Quercetin, and an IC50 of 4.31 mg/mL (Fig. 2).

This result confirms the ability of (3a) to decrease the XOD-generated superoxide radical in a concentration-dependentmanner with inhibition percentages up to 30.55%, 46.31%and 60.30% at the concentrations 100 lg/mL, 200 lg/mL and300 lg/mL, respectively (Fig. 3).

On the other hand, DPPH is a molecule containing a stable-freeradical. In the presence of an antioxidant that can donate an elec-tron to DPPH, the purple color typical of the free DPPH radical de-cays, a change that can be followed spectrophotometrically at517 nm. This method is based on the reduction of DPPH in an alco-holic solution in the presence of a hydrogen-donating antioxidantdue to the formation of the non-radical form of DPPH–H in thereaction. DPPH is usually used as a reagent to evaluate the free



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Fig. 2. Scavenging effects of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] pro-panenitrile (3a) against photochemically-generated superoxide free radicals (O2

��).

Fig. 3. Inhibition of xanthine oxidase activity of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a).

Fig. 4. DPPH free-radical scavenging activity of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a).



16 April 2014

radical scavenging activity of antioxidants. This simple test canprovide information on the ability of a compound to donate anelectron and the mechanism of antioxidant action. The 3[(4S)-ben-zyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a) was a verypotent radical scavenger, with a decrease percentage decrease vs.the absorbance of the DPPH standard solution of 81.22%, at a con-centration of 100 lg/mL, and an IC50 values of 3.11 lg/mL (Fig. 4).This value was slightly greater than that of the positive control,3 lg/mL a-tocopherol.

Two different reactive species were used to evaluate the antiox-idant activity of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl]propanenitrile (3a), the DPPH and superoxide radicals. Since theDPPH radical is not biologically relevant, the DPPH assay was per-formed as a preliminary study to estimate the direct free-radicalscavenging abilities of the test compound. The results obtainedwith the tested product (3a) revealed a relatively strong antiradicalactivity towards the DPPH free radical.

Results indicated that the 3[(4S)-benzyl-4,5-dihydro-1,3-oxa-zol-2-yl] propanenitrile (3a) decreased significantly the NBT/ribo-flavin generated superoxide radical in a concentration dependentmanner. The tested synthetic product shows a high ability to re-duce the O2

�� generation. This activity is dose-dependent and hasa similar profile to that of the Quercetin. In fact, no significant dif-ference was observed when compared the IC50 of the tested com-pound with Quercetin.


The activity of (3a) against the superoxide radical is a semi-quantitative test [35], and the enzymatic assay (X/XOD) has morerelevance to physiological conditions than the photochemical-NBTassay. The XOD catalyses the oxidation of hypoxanthine and xan-thine to uric acid. During the oxidation of xanthine, the superoxideradicals and the hydrogen peroxide are formed, this enzyme is con-sidered as an important biological source of superoxide radicals[36]. We can, then, conclude that (3a) is considered an antioxidantnot only because it acts as free-radical scavengers, but also becauseit inhibits the XOD.Our results are very encouraging as far as thesuperoxide radical (O2

��) is known to be a highly toxic species thatis generated by numerous biological and photochemical reactions.Indeed, it can generates the hydroxyl radical which reacts withDNA bases, amino acids, proteins, and polyunsaturated fatty acids,and produces toxic effects.

In order to expand the library of oxazolines as a drug, analgesicactivity is of key interest. Most antiepileptic drugs are known tohave strong analgesic effects [37]. So far, the available analgesicdrugs exert a wide range of side effects and are either too potentor too weak. The search for new analgesic compounds was a prior-ity for pharmacologists and pharmaceutical industries [38]. Amongthe several models of visceral pain, the writhing test has beenmostly used as a standard screening method [39]. This model in-volves different nociceptive mechanisms, such as the sympatheticsystem (Biogenic amines release), cyclooxygenases (COX) and theirmetabolites and opioid mechanisms. Acetic acid acts indirectly byinducing the release of an endogenous mediator which stimulatesthe nociceptive neurons sensitive to NSAIDs (non-steroidal anti-inflammatory drugs) and/or opioids. The subcutaneous administra-tion of (3a) (100, 200, 300 and 400 mg/kg) produced a significantreduction in the number of abdominal constrictions throughoutthe entire period of observation in a dose- related manner withrespectively 61.9%, 64.29%, 71.43% and 73.81% in the acetic acidwrithing test (Table 2). The standard drug (ASL, 200 mg/kg) de-creased the number of abdominal constrictions by 85.71% in theacetic acid writhing test in mice (Table 2).

Using the conventional pharmacological model, we have dem-onstrated the analgesic property of the product which can inducean antinociception by a mechanism similar to nonnarcotics and/or narcotic drugs, perhaps by blocking the receptor or the releaseof endogenous substances that excite pain nerve endings [40].NSAIDs such as ASL produce their antinociceptive and anti-inflam-matory action via inhibiting cyclooxygenases in peripheral tissues,



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Table 2Analgesic effect of the subcutaneous administration of 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a) in the acetic acid 1% writhing test in mice.

Treatment Dose(mg/kg)

Number ofwrithes ± s.e.m.

Inhibition ofwrithing (%)

Control (saline 10 mL/kg) � 42.50 ± 2.00 �Synthetic product 100 16.50 ± 2.50** 61.90

200 15.00 ± 1.50** 64.29300 12.00 ± 3.00** 71.43400 11.50 ± 3.00 73.81

Lysine acetylsalicylate(reference drug)

200 6.16 ± 0.37** 85.71

Values are expressed as mean ± s.e.m. n = 6 animals.** P < 0.001.

Table 3Antibacterial activity of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propaneni-trile, expressed as Minimum Inhibitory Concentration (MIC) and as MinimumBactericidal Concentration (MBC).

S. aureus E. faecalis E. coli

ATCC25923 ATCC25922 ATCC 25922

MIC MBC MIC MBC MIC MBC

3[(4S)-benzyl-oxazoline]propanenitrilea (lg/mL)

15 275 32.5 350 64 411

Ampicilinb (lg/mL) 1.5 225 2.5 125 6 275

a Values were expressed as means ± standard deviation of three experiments.b Positive control.



16 April 2014

thereby reducing the PGE2 (prostaglandin E2) synthesis and inter-fering with the mechanism of transduction in primary afferentnociceptors [41]. This particular activity of the crude extract ofthe defensive secretion and its semi-purified fractions is probablyrelated to their anti-inflammatory properties [42]. The analgesicproperty of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] pro-panenitrile (3a) was demonstrated. Evaluating analgesic propertiesin a single assay may not provide a full understanding of the ac-tions of the synthetic product (3a) or it’s utility. Further works toascertain its mechanism of action are necessary.

We show continued interest in the exploration of the variouspharmacological activities of oxazoline (3a) as an active sitessource. Besides, in this context, another important feature of thebiological properties of (3a) was demonstrated by testing the anti-microbial activity.

The antibacterial activity of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a) was evaluated on three pathogenicbacteria. Our results revealed that this molecule exhibited variouslevels of antibacterial effect against all the tested bacterial strains.Minimum Inhibitory Concentration (MICs) values ranging from 15to over 64 lg/mL, and Minimum Bactericidal Concentration(MBCs) values ranging from 275 to more than 411 lg/mL (Table 3).S. aureus was the most susceptible bacterial species, followed by E.faecalis and finally Escherichia coli, with MIC values of 15, 32.5 and64 lg/mL respectively. Compared to ampicillin, used as a positivecontrol against S. aureus (225 lg/mL), the tested oxazoline (3a)exhibited the same activity with an MBC value of 275 lg/mL.E. coli was found to be the least sensitive strain to the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile.

To the best of our knowledge, the antimicrobial effect of oxazo-line containing an aromatic ring and a nitrile function was neverinvestigated. Our results indicate that Gram-positive bacteria aremore sensitive to the antimicrobial effect of the synthetic oxazo-line than Gram-negative ones. It is interesting to note that (3a)exhibited an antimicrobial activity, particularly towards organismsof interest to the medical field such as Staphylococci and Entero-cocci. Likewise, foodborne illness resulting from the consumption


of food contaminated with pathogenic bacteria, has been a realconcern to public health. E. coli accounted for the largest numberof epidemic cases and deaths.

These observations show that the synthesized compounds arecapable of inhibiting the growth of Gram positive bacteria andGram negative bacteria. The compound (3a) possesses superioran antimicrobial activity against all microorganisms under investi-gation. There still exists a need for the development of new antimi-crobial agents having superior activity and less side effects toovercome resistant strains of microorganisms. Further studies topuzzle out the mechanism of these compounds’ action and deter-mine whether their activity is lethal or inhibiting to microorgan-isms are underway.

4. Conclusion

In this paper, we report an efficient one-pot synthesis of newchiral 4-substituted 2-cyanoethyl-oxazolines with high yields bycondensation of optically-pure a-amino alcohols and iminoethermonohydrochloride. The simple work-up, mild conditions and highyields are features of this approach. This work falls within theframework of valorization of these compounds as bioactive prod-ucts. In this context, the antioxidant, analgesic and antimicrobialactivities of these compounds were assessed. These experimentsestablished that the investigated oxazolines had an excellent anti-microbial, analgesic activity and a good antioxidant property. Thisfact may be evidence for the potential of this class of compounds inthe search and development of new bioactive compounds. In termsof their impacts, virtually all the activities studied revealed encour-aging results.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency document

The Transparency document associated with this article can befound in the online version.

6. Uncited reference

[32].

Acknowledgments

The authors are grateful to the DGRS (Direction Générale de laRecherche Scientifique) of the Tunisian Ministry of Higher Educa-tion and Scientific Research for the financial support.

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