Food Chemistry 183 (2015) 8–17

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Analytical Methods

Semi-synthesis of new antimicrobial esters from the natural oleanolic and maslinic acids Karim Chouaïb a, Fayçal Hichri a, Asma Nguir a, Majda Daami-Remadi b, Nicolas Elie c, David Touboul c, Hichem Ben Jannet a,⇑, M’hamed Ali Hamza a a Laboratoire de Chimie Hétérocyclique, Produits Naturels et Réactivité. Equipe: Chimie Médicinale et Produits Naturels, Département de Chimie, Faculté des Sciences de Monastir, Université de Monastir, Avenue de l’Environnement, 5019 Monastir, Tunisia b UR13AGR09, Production Horticole Intégrée au Centre Est Tunisien, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Mariem, Université de Sousse, 4042 Chott-Mariem, Tunisia c Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France

a r t i c l e

i n f o

Article history: Received 14 November 2014 Received in revised form 6 March 2015 Accepted 8 March 2015 Available online 14 March 2015 Keywords: Olea europaea Oleanolic acid Maslinic acid Pentacyclic triterpenoids esters Antibacterial activity Antifungal activity

a b s t r a c t In this article, we report an effective procedure for the selective isolation of oleanolic acid 1 and maslinic acid 2 (3.4 and 8.5 mg/g DW, respectively) from pomace olive (Olea europaea L.) using an ultrasonic bath, and the synthesis of a series of new triterpenic acid esters. The compounds were characterized by their spectral data and were evaluated for their antimicrobial activity. Among the compounds tested, those having sulfur and chlorine atoms were found to be antibacterial. They showed activity against two Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis and two Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (MICs within a range of 5–25 lg/mL). The fungus Penicillium italicum was found to be the most sensitive to both sulfur derivatives: (3b)-3-((thiophene2-carbonyl)oxy)-olean-12-en-28-oic acid (1a) (IZ = 22 mm) and (2a,3b-2,3-bis((thiophene-2carbonyl)oxy)olean-12-en-28-oic acid (2a) (IZ = 24 mm). Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of new therapeutics and antimicrobial strategies (Wright, 2010). Some plants derived secondary metabolites, e.g., pentacyclic triterpenoids have a potential as a new class of antibacterial agents as they are active against many bacterial species, both Gram-positive and Gram-negative, and they specifically target the cell envelope (Kurek, Nadkowska, Pliszka, & Wolska, 2012; Walencka et al., 2007). Triterpenoids are a large and structurally diversed group of natural products (Hill & Connolly, 2012) that display nearly 200 distinct skeletons. These compounds are found in food, medicinal herbs and various other plants in free form or bound to glycosides. The latter and their derivatives have been studied for their antineoplastic, anti-inflammatory, antiulcerogenic, antimicrobial, anti-plasmodial, antiviral (anti-HIV) characteristics. They are hepato- and cardio-protective, analgesic, anti-mycotic, immunomodulatory and they have tonic effects (Akihisa & Yasukawa, 2006; Cassels & Asencio, 2011; Kuo, Qian, ⇑ Corresponding author. Tel.: +216 73500279; fax: +216 73500278. E-mail address: [email protected] (H. Ben Jannet). http://dx.doi.org/10.1016/j.foodchem.2015.03.018 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

Morris-Natschke, & Lee, 2009; Ríos, 2010; Shanmugam, Nguyen, Kumar, Tan, & Sethi, 2012). In our previous research, we reported the synthesis and the antibacterial and anti-acetylcholinesterase activity of various oleanolic acid congeners as well as the structure–activity relationship conclusions (Hichri, Ben Jannet, Cheriaa, Jegham, & Mighri, 2003). Oleanolic acid (3b-hydroxyolean-12-en-28-oic acid, 1) and maslinic acid (2a,3b-dihydroxyolean-12-en-28-oic acid, 2) are natural pentacyclic triterpenoid compounds (Fig. 1), widely distributed throughout the vegetable kingdom (Herrera, Rodríguez-Rodríguez, & Ruiz-Gutiérrez, 2006; Liu, 2005). These acids are present in olive-pomace oil (García-Granados, 1997), being the main components of the protective wax-like coating of the olive skin. Both triterpenic acids and some closely related compounds display remarkable pharmacological characteristics such as being antitumor, antibacterial, anti-HIV, anti-inflammatory, antioxidant, and hepatoprotective (Huang et al., 2011; Parra, Rivas, MartínFonseca, García-Granados, & Martínez, 2011; Pollier & Goossens, 2012; Wang et al., 2010). In this report, we describe the effective extraction of large amounts of these compounds from olive-pressing residues using an ultrasonic bath. To explore the roles of the introduced acyl

K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

COOH

HO

3

COOH

HO HO

Oleanolic acid (1)

2 3

Maslinic acid (2)

Fig. 1. Chemical structures of the natural triterpenic compounds 1 and 2.

substituent at C-3 position, we prepared a series of new triterpenic acid esters 1a–j from oleanolic acid 1 and 2a–o from maslinic acid 2 using appropriate cyclic anhydrides and acid chlorides involving N,N-dimethyl-4-aminopyridine (DMAP) as a catalyst. Compounds 1 and 2 and their acylated derivatives were screened for their antimicrobial activity towards two Grampositive and two Gram-negative bacteria as well as against five fungal plant pathogens. 2. Materials and methods 2.1. General experimental procedures Solvents were purified and dried using standard methods. Melting points were determined on a Büchi 510 apparatus using capillary tubes. Commercial TLC plates (Silica gel 60, F254, sds) were used to monitor the progress of the reaction. Column chromatography was performed with silica gel 60 (particle size 40–63 lm, sds). HRMS were acquired with a LCT Premier XE (Waters, ESI technique, positive mode) mass spectrometer. For ESI experiments, leucine-enkephalin peptide was employed as the LockSpray lockmass. 1H (300 MHz, 16–32 scans) and BB-decoupled 13C (75 MHz, 256–2048 scans) NMR spectra were recorded at room temperature (rt) on a Bruker AM-300 Fourier Transform spectrometer equipped with a 10 mm probe in deuterated chloroform, acetone and pyridine with all chemical shifts (d), reported in ppm, refereed to residual non deuterated solvent. Coupling constants were measured in Hz and signals are using the following abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet, etc.

9

eliminate triglycerides. Then, dried at 35 °C for 24 h in an electrical furnace, milled and immersed in 4 L of methanol. The mixture was kept at room temperature and occasionally agitated to facilitate the maceration process. Five days after, the sample was decanted and filtered through a wire funnel. The methanolic solution was evaporated in vacuo to dryness in a rotary evaporator at 50 °C; this yielded 120 g of extract. This pasty extract was treated with hexane, and three fractions were collected using an ultrasonic bath (0.5 h, 50 °C): fraction 1 is composed of the hexane extract, fraction 2 oily consisting mainly of triglycerides and the drying of fraction 3 provided 25.5 g of a white solid formed according to TLC analysis by of two major compounds. About 25 g of this solid was separated by silica gel column chromatography (petroleum ether:EtOAc 8:2, 7:3 then 1:1) to give 1 and 2 (Table 1). 2.3. Synthesis 2.3.1. General procedure for the synthesis of oleanolic acid esters using acid chlorides The appropriate acid chloride (1.1 equiv) was added to the mixture of 1 (0.01 g, 0.22 mmol) and N,N-dimethyl-4-aminopyridine (DMAP) (1 equiv), in refluxing anhydrous pyridine, and the mixture was refluxed for overnight and then concentrated in vacuum under reduced pressure. The resulting mixture was washed with water to remove salts then extracted with chloroform. The organic layer was dried over sodium sulfate. The solvent was removed under reduced pressure to give the esters 1a–e, which were chromatographed over a column of silica gel using petroleum ether– ethyl acetate (9:1, v/v) as an eluent in 91–98% yield (Table 2).

2.2. Collection, extraction and isolation of compounds 1 and 2

2.3.2. General procedure for the synthesis of oleanolic acid esters using cyclic anhydrides To a solution of 1 (0.01 g, 0.22 mmol) in 3 mL DMSO is added the appropriate cyclic anhydride (0.88 mmol) in 2 mL DMSO. The solution is thermostatted at 40 °C. N,N-Dimethyl-4-aminopyridine (DMAP) (0.22 mmol) is added to the solution while stirring. The reaction of the mixture is kept at 40 °C for 24 h. The reaction’s product was diluted in distilled water, washed with a solution of HCl (3 M), then extracted with ethyl acetate. The organic layer was dried over sodium sulfate. After the evaporation of the solvent, the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate 6:4) or recrystallization to give the esters 1f–j, in 81–92% yield (Table 2).

The chemlali pomace olive was collected from the factory of soap located in Sousse, Tunisia and kept at a temperature of 20 °C in the dark until its use. The solid olive oil waste (2 kg) resulting from olive fruit pressing was washed with hexane (1 mL/g) to

2.3.3. General procedure for the synthesis of maslinic acid esters using acid chlorides To a solution of 2 (0.01 g, 0.21 mmol) and of N,N-dimethyl-4aminopyridine (DMAP) (2 equiv) in refluxing anhydrous pyridine,

Table 1 Methods and extraction yield of oleanolic acid 1 and maslinic acid 2 from different pomace olive cultivars. Cultivar

Extraction methods

Yield of extraction (mg/g DW) Oleanolic acid 1

Maslinic acid 2

References

Picual Hojiblanca Arbequina Non indicated

Solid–liquid extraction (maceration)

0.500 0.500 0.400 0.015

1.200 1.300 1.500 0.034

Guinda, Rada, Delgado, Gutiérrez-Adánez, and Castellano (2010)

Manzanilla Hojiblanca Cacereña Kalamata

Solid–liquid extraction (centrifugation)

0.274 0.565 0.185 0.841

0.824 0.904 0.295 1.318

Romero, García, Medina, Ruíz-Méndez, Castro, and Brenes (2010)

Picual Kalamon

Ultrasonic assisted extraction

1.003 0.838

2.440 2.100

Goulas and Manganaris (2011)

Chemlali

Solid–liquid then ultrasonic assisted extractions

3.400

8.500

This work

Gil et al. (1997)

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K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

Table 2 Synthesis of oleanolic (1) and maslinic (2) acid derivatives

COOH

X

R1

A / DMAP

HO

R2O

COOH 2 3

1,2 1 : X=H

1a - j

2 : X=OH

2a - o

. Compound

R1

1a

H

1b

H

1c

H

1d

H

R2

O

A

Yield (%)a

R2Cl

98

R2Cl

94

R2Cl

91

R2Cl

95

R2Cl

94

S

O

Cl

Ph

O

Cl

O

Cl 1e

H

1f

H

1g

H

Cl O

O

O

O

O

92

HOOC

O

O

82

O O

COOH 1h

O

H

O

91

O

COOH 1i

H

1j

H

O

O

O

O

O

O

O

O

85

HOOC

O

81

HOOC

O

S 2b

O

O

2a

Cl

O O

2d

Cl

59

R2Cl

48

R2Cl

55

R2Cl

45

S Cl

O

Ph

Ph 2c

R2Cl

O O

Cl

O

11

K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17 Table 2 (continued) Compound

R1

R2

O

Cl

O

2e

Yield (%)a

R2Cl

52

O

Cl

Cl O

A

Cl O O

O

2f

O

H

Cl

O S 2g

S

O

Cl

H

O

O

Cl 2j

H

Cl O

Cl

O

2i

O

Cl

O

39

Cl Cl

Cl O

O

H

O

O HOOC

O

O

O

O

O

O

O

O

O

82

O

COOH

COOH O HOOC

O O

O

O

O

O

O

O

O

O

76

HOOC

O HOOC

88

O

COOH

O

2o

81

O

COOH

2n

O

HOOC

O

2l

28

Cl

2k

a

31

Cl

H

O

2m

32

Ph

O

Cl

O

Cl

Ph 2h

33

O

78

HOOC

Isolated yield of product after purification by column chromatography or recrystallization.

the appropriate acid chloride (2.2 equiv) was added and the mixture was refluxed. The reaction was monitored by TLC till its completion in around 4 h. The solvent was then removed under reduced pressure. The resulting mixture was washed with water to remove salts, then extracted with chloroform. The organic layer was dried over sodium sulfate and then concentrated in vacuum. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate 7:3) to give the C-2, C-3 diacylated 2a–e and the C-2 monoacylated 2f–j esters in 45–59%, 28–39% yield, respectively (Table 2). 2.3.4. General procedure for the synthesis of maslinic acid esters using cyclic anhydrides To a solution of maslinic acid (0.01 g, 0.21 mmol) in 3 mL DMSO is added the appropriate cyclic anhydride (1.7 mmol) in 2 mL DMSO. The solution is thermostatted at 40 °C and N,N-dimethyl4-aminopyridine (0.44 mmol) is added while stirring. The reaction’s mixture is kept at 40 °C for 24 h. The reaction’s product

was isolated and then diluted in distilled water, then extracted with ethyl acetate. After washing it with a solution of hydrochloric acid (3 M), the organic layer was dried over sodium sulfate. After the evaporation of the solvent, the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate 6:4) to give the esters 2k–o in 81–94% yield (Table 2). 2.4. Antimicrobial assay 2.4.1. Antibacterial activity 2.4.1.1. Bacterial strains. The antibacterial activity of compounds 1 and 2 as well as of their acylated derivatives was tested against five microorganisms, including reference strains consisting of Gram-negative rods: Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) and Gram-positive cocci: Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212). The bacterial strains were cultured over night at 37 °C in Muller Hinton agar.

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K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

2.4.1.2. Antibacterial assay. MIC and MBC values were determined by a microtiter plate dilution method (Jabrane, Ben Jannet, Mastouri, Mighri, & Casanova, 2010) after dissolving the sample in 10% DMSO solution. Sterile 10% DMSO solution (100 lL) was pipetted into all wells of the micro-titer plate before transferring 100 lL of stock solution to the microplate. Serial dilutions were made to obtain concentration ranging from 10 to 0.0775 mg/mL. Finally, 50 lL of 106 colony forming units (cfu/mL) (according to McFarland turbidity standards) of standards microorganism suspensions were inoculated on to microplates and incubated at 37 °C for 24 h. At the end of the incubation period, the plates were evaluated for the presence or absence of bacterial growth. All the samples were screened three times against each microorganism. Imipenem was employed as a positive control against Grampositive and Gram-negative bacteria. The final concentration of DMSO in the well had no effect on bacterial growth. 2.4.2. Antifungal activity 2.4.2.1. Test fungi. The antifungal activity of compounds 1 and 2 as well as of their acylated derivatives was tested against five fungal species namely: Aspergillus flavus, Aspergillus niger, Penicillium digitatum, Penicillium italicum and Trichoderma harzianum. These fungi were obtained from the Laboratory of Phytopathology of the Regional Center of Research on Horticulture and Organic Agriculture (CRRHAB) of Chott-Mariem, Tunisia. They were cultured at 25 °C on potato dextrose agar (PDA) medium 1 week before use. 2.4.2.2. Antifungal assay. The antifungal activity of the compounds tested was screened by using the disc diffusion method (Baker, Stocker, Culver, & Thornsberry, 1991). A conidial suspension of the tested fungi was prepared (104–105 conidia/mL) and added to PDA medium cooled at 45 °C and poured uniformly into Petri plates (diameter 90 mm). Sterilized paper discs (6 mm, Whatman No. 1 filter paper) were impregnated with 20 lL (1000 lg/mL) of the compound dissolved in DMSO and placed on the culture plates whereas the negative control plates had no product added to the filter paper. In the positive control plates, discs were imbibed with the same volume of a carbendazim suspension (0.5 mg/mL). The diameter of the inhibition zone (mm) around the disc was measured after incubation at 25 °C for 4 days and compared with the controls. The test was performed in triplicate. 3. Results and discussion 3.1. Chemistry 3.1.1. Isolation of compounds 1 and 2 Extraction of bioactive compounds from vegetable materials with a solvent is a classical operation applied in many industrial processes, particularly the pharmaceutical industry. It is obvious that medical interest in plants derived drugs has led to an increased need for ideal extraction methods, which could obtain the maximum of the bioactive constituents in a shortest processing time with a low cost. Many reports indicated that ultrasound was an important factor in the compounds extraction yield of natural material (Wang et al., 2013). Indeed, ultrasonic-assisted extraction has been proven to significantly decrease extraction time and increase extraction yields in many vegetable materials (Eh & Teoh, 2012; Khan, Abert-Vian, Fabiano-Tixier, Dangles, & Chemat, 2010; Londoño-Londoño et al., 2010; Vinatoru, 2001; Wang et al., 2015; Zhou, Fu, & Li, 2015). In this work, compounds 1 (6.8 g, 3.4 mg/g DW for pomace olive) and 2 (17 g, 8.5 mg/g DW for pomace olive) were obtained by the treatment of the methanolic extract of the defatted pomace

olive cultivar: chemlali with hexane in an ultrasonic bath (0.5 h, 50 °C). As shown in Table 1, whatever the cultivar used, our extraction way followed (solid–liquid maceration then ultrasonic assisted extraction) resulted the recovery of significantly higher yields in oleanolic acid 1 and maslinic acid 2 compared with those of previous works following conventional extraction methods or even using ultrasonic assisted extraction with different solvents (ethanol, methanol, ethanol:methanol (1:1, v/v) and ethyl acetate). The spectroscopic data of 1 and 2 are in agreement with those reported by Gil, Haïdour, and Ramos (1997). 3.1.2. Synthesis Compound 1 was heated to reflux overnight with 1.1 equiv of the appropriate acid chloride in anhydrous pyridine in the presence of N,N-dimethyl-4-aminopyridine (DMAP) in catalytic amount. The desired new compounds 1a–e were obtained in yields ranging from 91% to 98% (Table 2). Similarly, compound 2 was refluxed with 2.2 equiv of the appropriate acid chloride in anhydrous pyridine for only 4 h to give the C2-acyl substituent (2f–j) and C2 and C3-di-acyl substituents (2a–e). On the other hand, compounds 1f–j and 2k–o were prepared by heating compounds 1 and 2 with 2 and 4 equiv of the appropriate anhydride, respectively, in DMSO in the presence of N,N-dimethyl-4-aminopyridine (catalyst) overnight. The structures were further supported by 1H NMR, 13C NMR and DEPT, which showed all the expected carbon signals corresponding to triterpenic acid derivatives. HRMS of all the derivatives were also in agreement with their molecular formula. The reagents and yields are reported in Table 2. The structure of compounds (1a–e, 2a–j) has been characterized by 1H NMR showing new signals at the region 4.78–5.79 and 6.87– 8.14 ppm attributed to protons of the introduced acyl group(s). Further, the 13C NMR spectra of (1a–e, 2a–j) exhibited the presence of signals at 162.8–172.6 ppm attributable to the carbonyl of acyl group(s). Then, the structures of compounds (1f–j, 2k–o) can be easily deduced from their 1H and 13C NMR data. In fact, the 13C NMR spectra showed essentially the appearance of signals at 166.5–173.9 and 170.8–183.5 ppm due to carbonyl of acyl group and carboxylic acid, respectively. The 1H NMR spectra of compounds (1g–j, 2l–o) showed signals in the region 6.15–8.05 ppm attributed to ethylenic and aromatic protons of the introduced acyl group(s). 3.1.2.1. (3b)-3-((Thiophene-2-carbonyl)oxy)-olean-12-en-28-oic acid (1a). Dark red solid; mp: 286–287 °C; 1H NMR (300 MHz, C5D5N): d 7.99 (1H, dd, J = 4.8; 2.4 Hz), 7.77 (1H, dd, J = 6.3; 3.9 Hz), 7.16 (1H, dd, J = 8.7; 1.2 Hz), 5.50 (1H, t, J = 3.3 Hz), 4.88 (1H, dd, J = 11.1; 4.5 Hz), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.14 (4H, m), 1.74 (2H, m), 1.54–120 (12H, m), 1.24 (4H, m), 1.11 (3H, s), 1.05 (3H, s), 1.01 (3H, s), 0.97 (3H, s), 0.90 (3H, s), 0.87 (3H, s), 0.83 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.5, 169.8, 146.4, 138.1, 135.5, 135.1, 130.4, 124.3, 82.7, 57.4, 48.6, 48.2, 48.0, 48.6, 44.1, 43.7, 41.6, 40.9, 40.5, 39.1, 35.8, 34.9, 34.7, 32.5, 31.6, 30.4, 29.9, 29.7, 27.7, 25.4, 25.3, 25.2, 20.4, 19.0, 18.1; HRMS (ESI+): calcd. for (C35H51O4S)+ [M+H]+ 567.3430, found 567.3491 . 3.1.2.2. (3b)-3-((2S)-2-chloro-2-phenylacetoxy)-olean-12-en-28-oic acid (1b). White solid; mp: 291–293 °C; 1H NMR (300 MHz, C5D5N): d 7.47 (5H, m), 6.02 (1H, d, J = 4.3 Hz), 5.46 (1H, s), 4.94 (1H, dd, J = 10.8; 4.5 Hz), 3.32 (1H, m), 2.14 (2H, m), 1.98 (4 H, m), 1.72–131 (12H, m), 1.16 (4H, m), 1.08 (3H, s), 1.01 (3H, s), 1.00 (3H, s), 0.96 (3H, s), 0.91 (3H, s), 0.81 (3H, s), 0.70 (3H, s); 13 C NMR (75 MHz, C5D5N): d 181.8, 170.1, 146.7, 132.4, 132.1, 131.6, 130.8, 130.2, 130.1, 123.9, 84.9, 74.5, 62.0, 56.9, 49.3, 48.2, 48.0, 44.1, 43.7, 43.5, 41.2, 41.0, 39.6, 38.6, 35.8, 34.8, 32.5, 31.5, 31.1, 29.8, 27.7, 25.5, 25.2, 23.0, 19.9, 19.0, 18.9, 16.8; HRMS (ESI+): calcd. for (C38H54ClO4)+ [M+H]+ 609.3632, found 609.3689.

K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

3.1.2.3. (3b)-3-((2S)-2-chloro-2-methylacetoxy)-olean-12-en-28-oic acid (1c). White solid; mp: 266–267 °C; 1H NMR (300 MHz, C5D5N): d 5.50 (1H, s), 4.78 (1H, m), 4.74 (1H, m), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.19 (2H, m), 1.97 (4H, m), 1.74 (3H, q, J = 3.9 Hz), 1.60 (4H, m), 1.48–126 (8H, m), 1.20 (4H, m), 1.04 (3H, s), 1.00 (3H, s), 0.98 (3H, s), 0.94 (3H, s), 0.93 (3H, s), 0.91 (3H, s), 0.85 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.7, 171.5, 146.4, 123.9, 83.9, 56.9, 55.6, 55.3, 49.4, 48.2, 48.0, 43.7, 43.5, 41.2, 39.8, 39.7, 39.6, 38.7, 35.8, 34.8, 32.5, 31.5, 31.1, 29.8, 27.7, 25.3, 25.2, 23.4, 23.0, 19.9, 18.9, 18.4, 16.9; HRMS (ESI+): calcd. for (C33H52ClO4)+ [M+H]+ 547.3476, found 547.3537. 3.1.2.4. (3b)-3-(2,2-Dichloroacetoxy)-olean-12-en-28-oic acid (1d). White solid; mp: 263–265 °C; 1H NMR (300 MHz, C5D5N): d 7.10 (1H, s), 5.49 (1H, s), 4.79 (1H, t, J = 8.7 Hz), 3.32 (1H, dd, J = 9.9; 4.2 Hz), 2.19 (3H, m), 1.97 (3H, m), 1.78–146 (10 H, m), 1.35 (2H, m), 1.25 (4H, m), 1.09 (3H, s), 1.04 (3H, s), 1.00 (3H, s), 0.98 (3H, s), 0.96 (3H, s), 0.92 (3H, s), 0.86 (3H, s); 13C NMR (75 MHz, C5D5N): d 180.8, 165.7, 145.6, 123.0, 85.6, 67.1, 56.0, 55.7, 48.8, 48.5, 47.4, 43.2, 42.9, 42.7, 39.8, 39.0, 38.6, 37.8, 37.7, 34.0, 33.7, 31.7, 29.1, 28.7, 26.9, 24.6, 24.4, 22.2, 19.1, 18.1, 17.5, 16.2; HRMS (ESI+): calcd. for (C32H49Cl2O4)+ [M+H]+ 567.2930, found 567.2997. 3.1.2.5. (3b)-3-(Pivaloyloxy)-olean-12-en-28-oic acid (1e). White solid; mp: 274–276 °C; 1H NMR (300 MHz, C5D5N): d 5.51 (1H, s), 4.67 (1H, dd, J = 5.4; 5.1 Hz), 3.36 (1H, dd, J = 6.7; 4.2 Hz), 2.19 (2H, m), 1.97 (4H, m), 1.85 (4H, m), 1.78–123 (21H, m), 1.11 (3H, s), 1.06 (3H, s), 1.05 (3H, s), 0.98 (3H, s), 0.97 (3H, s), 0.94 (3H, s), 0.86 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.0, 169.5, 146.8, 124.3, 82.2, 57.4, 55.7, 50.0, 48.6, 48.4, 44.4, 44.1, 43.9, 40.0, 40.0, 39.1, 39.0, 36.2, 35.2, 35.1, 35.0, 32.9, 31.5, 31.1, 30.2, 29.3, 28.1, 25.8, 25.7, 25.6, 23.3, 20.4, 19.4, 19.0, 17.3; HRMS (ESI+): calcd. for (C35H57O4)+ [M+H]+ 541.4179, found 541.4237. 3.1.2.6. (3b)-3-((3-Carboxypropanoyl)oxy)-olean-12-en-28-oic acid (1f). White solid; mp: 318–319 °C; 1H NMR (300 MHz, C5D5N): 5.47 (1H, s), 4.78 (1H, dd, J = 11.1; 4.5 Hz), 3.31 (1H, dd, J = 13.2; 4.2 Hz), 2.94 (4H, m), 2.13 (2H, m), 2.05 (3H, m), 1.98 (2H, m), 1.86 (5H, m), 1.61–133 (10H, m), 1.11 (3H, s), 0.98 (3H, s), 0.97 (3H, s), 0.95 (3H, s), 0.94 (3H, s), 0.92 (3H, s), 0.89 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.1, 176.9, 174.5, 146.8, 124.3, 82.8, 57.4, 49.7, 48.6, 48.3, 44.0, 43.9, 41.6, 40.1, 39.9, 39.0, 36.1, 35.2, 35.1, 34.9, 32.9, 32.2, 31.9, 30.2, 30.1, 28.1, 25.8, 25.7, 25.6, 23.8, 20.3, 19.3, 19.0, 17.3; HRMS (ESI+): calcd. for (C34H53O6)+ [M+H]+ 557.3764, found 557.3824.

13

127 (6H, m), 1.11 (3H, s), 0.98 (3H, s), 0.97 (3H, s), 0.95 (3H, s), 0.94 (3H, s), 0.92 (3H, s), 0.89 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.9, 175.2, 173.9, 146.5, 135.4, 134.7, 133.8, 132.9, 132.1, 131.5, 124.0, 83.1, 57.7, 49.2, 48.6, 45.7, 41.1, 40.8, 38.7, 37.4, 37.0, 36.2, 33.2, 32.4, 31.9, 29.9, 29.2, 28.9, 28.2, 27.7, 27.1, 28.1, 25.8, 23.7, 20.3, 19.5, 18.2, 16.9; HRMS (ESI+): calcd. for (C38H53O6)+ [M+H]+ 605.3764, found 605.3833. 3.1.2.9. (3b)-3-(((E)-3-carboxyacryloyl)oxy)-olean-12-en-28-oic acid (1i). White solid; mp: 313–315 °C; 1H NMR (300 MHz, C5D5N): 6.91 (1H, d, J = 6.2 Hz), 6.74 (1H, d, J = 5.8 Hz), 5.49 (1H, s), 4.86 (1H, dd, J = 11.2; 4.5 Hz), 3.32 (1H, dd, J = 13.2; 4.2 Hz), 2.16 (2H, m), 1.91 (2H, m), 1.81 (4H, m), 1.67 (2H, m), 1.59–133 (8H, m), 1.21 (4H, m), 1.11 (3H, s), 0.98 (3H, s), 0.97 (3H, s), 0.95 (3H, s), 0.88 (3H, s), 0.82 (3H, s), 0.81 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.2, 170.8, 166.5, 146.8, 136.5, 131.7, 124.3, 83.1, 57.7, 49.8, 48.7, 48.4, 44.0, 43.9, 41.6, 40.1, 39.9, 39.0, 36.2, 35.2, 35.0, 33.9, 32.9, 31.9, 31.2, 30.1, 28.1, 26.1, 25.7, 25.3, 24.6, 22.3, 19.3, 19.0, 17.2; HRMS (ESI+): calcd. for (C34H51O6)+ [M+H]+ 555.3607, found 555.3693. 3.1.2.10. (3b)-3-(((E)-3-carboxybut-2-enoyl)oxy)-olean-12-en-28-oic acid (1j). White solid; mp: 308–309 °C; 1H NMR (300 MHz, C5D5N): 6.29 (1H, s), 5.49 (1H, s), 4.89 (1H, dd, J = 11.2; 4.5 Hz), 3.32 (1H, dd, J = 13.2; 4.2 Hz), 2.18 (2H, m), 2.03 (3H, s), 1.97 (3H, m), 1.89 (5H, m), 1.59 (4H, m), 1.49–123 (8H, m), 1.11 (3H, s), 0.98 (3H, s), 0.97 (3H, s), 0.95 (3H, s), 0.88 (3H, s), 0.82 (3H, s), 0.81 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.2, 171.8, 167.6, 146.8, 138.1, 136.7, 124.3, 83.5, 57.7, 49.8, 48.6, 48.4, 44.1, 43.9, 42.7, 40.1, 39.9, 39.0, 36.2, 35.2, 35.0, 33.9, 32.9, 31.9, 31.2, 30.1, 28.1, 25.7, 25.4, 24.9, 22.3, 19.3, 19.1, 17.3, 16.2; HRMS (ESI+): calcd. for (C35H53O6)+ [M+H]+ 569.3764, found 569.3831. 3.1.2.11. (2a,3b)-2,3-Bis((thiophene-2-carbonyl)oxy)olean-12-en-28oic acid (2a). Dark red solid; mp: 288–289 °C; 1H NMR (300 MHz, C5D5N): d 8.04 (2H, m), 7.80 (2H, m), 7.19 (2H, m), 5.91 (1H, td, J = 6.7; 4.2 Hz), 5.60 (1H, d, J = 4.2 Hz), 5.42 (1H, t, J = 3.3 Hz), 3.33 (1H, dd, J = 6.7; 4.2 Hz), 2.23 (4H, m), 1.97 (6H, m), 1.64 (4H, m), 1.51–137 (6H, m), 1.35 (3H, s), 1.29 (3H, s), 1.18 (3H, s), 1.06 (3H, s), 1.03 (3H, s), 1.00 (3H, s), 0.97 (3H, s); 13C NMR (75 MHz, C5D5N): d 180.7, 170.8, 164.1, 146.2, 138.1, 136.8, 135.5, 135.1, 134.4, 133.3, 130.4, 129.4, 123.3, 83.9, 73.7, 57.1, 48.6, 48.2, 48.0, 48.6, 44.1, 43.7, 41.6, 40.9, 40.5, 39.1, 35.8, 34.9, 34.7, 32.5, 31.6, 30.4, 29.9, 29.7, 27.7, 25.4, 25.3, 19.3, 17.9, 17.1; HRMS (ESI+): calcd. for (C40H53O6S2)+ [M+H]+ 693.3205, found 693.3293.

3.1.2.7. (3b)-3-Carboxybicyclo[2.2.1]hept-5-ene-2-carbonyloxy-olean12-en-28-oic acid (1g). White solid; mp: 314–316 °C; 1H NMR (300 MHz, C3D6O): d 6.15 (2 H, m), 5.25 (1H, t, J = 3.3 Hz), 4.38 (1H, dd, J = 10.8; 5.1 Hz), 3.35 (2 H, m), 3.11 (2 H, m), 2.90 (1H, dd, J = 14.1; 3.9 Hz), 2.06 (1H, m), 1.97 (1H, m), 1.90 (2H, m), 1.79–163 (8H, m), 1.61–143 (6H, m), 1.38 (2H, m), 1.29 (4H, m), 1.11 (3H, s), 0.98 (3H, s), 0.96 (3H, s), 0.95 (3H, s), 0.92 (3H, s), 0.89 (3H, s), 0.84 (3H, s); 13C NMR (75 MHz, C3D6O): d 177.7, 172.2, 170.9, 143.5, 134.2, 133.7, 121.5, 79.7, 54.7, 47.7, 47.6, 47.0, 46.9, 45.7, 45.5, 45.3, 41.1, 40.8, 38.7, 37.4, 37.0, 36.2, 33.0, 32.1, 31.9, 29.9, 29.2, 28.9, 28.2, 27.7, 27.1, 27.0, 24.8, 22.7, 22.5, 22.3, 17.5, 16.2, 15.9; HRMS (ESI+): calcd. for (C39H57O6)+ [M+H]+ 621.4077, found 621.4133.

3.1.2.12. (2a,3b)-2,3-Bis((2S)-2-chloro-2-phenylacetoxy)olean-12en-28-oic acid (2b). White solid; mp: 286–287 °C; 1H NMR (300 MHz, C5D5N): d 8.14 (4H, m), 7.87 (2H, m), 7.39 (4H, m), 7.07 (1H, s), 6.89 (1H, s), 5.85 (1H, td, J = 6.7; 4.2 Hz), 5.58 (1H, d, J = 4.2 Hz), 5.42 (1H, s), 3.34 (1H, dd, J = 6.7; 4.2 Hz), 2.23–197 (8H, m), 1.87 (4H, m), 1.64 (2H, m), 1.53 (2H, m), 1.44 (4H, m), 1.35 (3H, s), 1.29 (3H, s), 1.18 (3H, s), 1.06 (3H, s), 1.03 (3H, s), 1.00 (3H, s), 0.97 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.0, 167.7, 166.8, 146.9, 137.2, 136.8, 135.2, 135.1, 134.4, 134.1, 133.6, 132.6, 131.2, 130.4, 130.9, 129.4, 124.0, 85.5, 75.7, 67.9, 67.7, 57.1, 48.6, 48.2, 48.0, 48.6, 44.1, 43.7, 41.6, 40.9, 40.5, 39.1, 35.8, 34.9, 34.7, 32.5, 31.6, 30.4, 29.9, 29.7, 27.7, 25.4, 25.3, 19.3, 17.9, 17.1; HRMS (ESI+): calcd. for (C46H59Cl2O6)+ [M+H]+ 777.3610, found 777.3691.

3.1.2.8. (3b)-3-(2-Carboxybenzoyl)oxy-olean-12-en-28-oic acid (1h). White solid; mp: 311–313 °C; 1H NMR (300 MHz, C5D5N): d 8.01 (2 H, m), 7.51 (1H, m), 7.44 (1H, m), 5.40 (1H, s), 4.81 (1H, dd, J = 11.1; 4.5 Hz), 3.19 (1H, dd, J = 13.2; 4.2 Hz), 1.98 (2H, m), 1.92 (2H, m), 1.83 (4H, m), 1.69–153 (6H, m), 1.48 (2H, m), 1.41–

3.1.2.13. (2a,3b)-2,3-Bis((2S)-2-chloro-2-methylacetoxy)olean-12-en28-oic acid (2c). White solid; mp: 276–278 °C; 1H NMR (300 MHz, C5D5N): d 5.79 (2H, m), 5.47 (1H, t, J = 3.3 Hz), 5.11 (2H, m), 3.65 (1H, dd, J = 6.7; 4.2 Hz), 2.44 (6H, m), 2.23 (4H, m), 2.03–192 (8H, m), 1.83 (2H, m), 1.66 (6H, m), 1.45 (6H, s), 1.29 (15H, s); 13C

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K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

NMR (75 MHz, C5D5N): d 180.8, 170.9, 170.4, 145.7, 122.7, 84.3, 72.6, 55.4, 54.5, 54.3, 54.1, 48.6, 48.4, 47.4, 47.2, 44.3, 43.0, 42.7, 40.8, 40.5, 39.1, 35.0, 34.0, 33.9, 33.5, 31.7, 29.0, 26.9, 24.5, 24.4, 22.2, 22.1, 19.0, 18.4, 18.0, 17.0; HRMS (ESI+): calcd. for (C36H55Cl2O6)+ [M+H]+ 653.3297, found 653.3357. 3.1.2.14. (2a,3b)-2,3-Bis(2,2-dichloroacetoxy)olean-12-en-28-oic acid (2d). White solid; mp: 282–284 °C; 1H NMR (300 MHz, C5D5N): d 7.13 (1H, s), 7.07 (1H, s), 5.48 (2H, m), 5.20 (1H, d, J = 10.2 Hz), 3.65 (1H, dd, J = 6.7; 4.2 Hz), 2.33 (4H, m), 2.11–182 (8H, m), 1.73 (2H, m), 1.46 (6H, m), 1.42 (6H, s), 1.18 (3H, s), 1.06 (3H, s), 1.03 (3H, s), 1.00 (3H, s), 0.98 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.0, 166.8, 166.6, 146.9, 123.8, 85.4, 75.7, 67.9, 67.8, 56.4, 49.5, 48.6, 48.4, 45.0, 44.2, 43.8, 42.1, 41.6, 40.4, 36.2, 35.1, 34.7, 32.9, 30.2, 30.1, 29.8, 26.9, 24.5, 23.4, 20.9, 20.2, 19.5, 19.2, 18.2; HRMS (ESI+): calcd. for (C34H49Cl4O6)+ [M+H]+ 693.2205, found 693.2287. 3.1.2.15. (2a,3b)-2,3-Bis(pivaloyloxy)olean-12-en-28-oic acid (2e). White solid; mp: 285–287 °C; 1H NMR (300 MHz, C5D5N): d 5.44 (1H, t, J = 3.3 Hz), 5.31 (1H, m), 5.12 (1H, d, J = 10.3 Hz), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.46 (2H, m), 2.41–2.28 (4H, m), 2.19– 1.88 (8H, m), 1.83 (2H, m), 1.48 (4H, m), 1.41 (3H, s), 1.36 (15H, m), 1.31 (3H, s), 1.29 (3H, s), 1.18 (3H, s), 1.06 (3H, s), 1.03 (3H, s), 1.01 (3H, s), 0.94 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.4, 168.9, 168.2, 145.3, 122.5, 83.8, 71.4, 55.4, 54.5, 54.3, 54.1, 48.6, 48.4, 47.4, 47.2, 44.3, 43.0, 42.7, 40.8, 40.5, 39.1, 35.0, 34.0, 33.9, 33.5, 31.7, 30.2, 29.3, 29.0, 28.1, 26.9, 24.5, 24.4, 22.2, 22.1, 19.0, 18.4, 18.0, 16.8; HRMS (ESI+): calcd. for (C40H65O6)+ [M+H]+ 641.4703, found 641.4789. 3.1.2.16. (2a,3b)-2-((Thiophene-2-carbonyl)oxy)-3-hydroxy-olean12-en-28-oic acid (2f). Dark red solid; mp: 281–283 °C; 1H NMR (300 MHz, C5D5N): d 7.96 (1H, m), 7.69 (1H, m), 7.08 (1H, m), 5.71 (1H, td, J = 6.7; 4.2 Hz), 5.52 (1H, t, J = 3.3 Hz), 3.62 (1H, m), 3.34 (1H, dd, J = 6.7; 4.2 Hz), 2.23 (4H, m), 1.97 (4H, m), 1.79– 1.58 (6H, m), 1.51–1.38 (6H, m), 1.37 (3H, s), 1.29 (3H, s), 1.12 (3H, s), 1.08 (3H, s), 1.02 (6H, s), 0.97 (3H, s); 13C NMR (75 MHz, C5D5N): d 180.5, 162.8, 145.3, 133.9, 133.3, 128.6, 122.6, 79.9, 74.9, 55.8, 48.3, 47.0, 46.9, 44.9, 42.6, 42.4, 40.9, 40.1, 39.0, 34.6, 34.9, 34.7, 33.7, 33.6, 33.5, 31.3, 29.5, 28.7, 26.6, 24.3, 24.1, 19.2, 17.9, 17.7, 16.9; HRMS (ESI+): calcd. for (C35H51O5S)+ [M+H]+ 583.3379, found 583.3451.

45.9, 44.2, 42.5, 42.4, 41.7, 40.6, 36.2, 35.2, 35.0, 32.9, 31.0, 30.2, 28.1, 25.8, 25.7, 25.6, 23.8, 23.5, 20.7, 19.4, 19.3, 18.4; HRMS (ESI+): calcd. for (C33H52ClO5)+ [M+H]+ 563.3425, found 563.3497. 3.1.2.19. (2a,3b)-2-(2,2-Dichloroacetoxy)-3-hydroxy-olean-12-en-28oic acid (2i). White solid; mp: 288–290 °C; 1H NMR (300 MHz, C5D5N): d 6.63 (1H, s), 5.48 (1H, t, J = 3.3 Hz), 5.63 (1H, td, J = 6.7; 4.2 Hz), 3.51 (1H,d, J = 9.9 Hz), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.23 (4H, m), 2.11–1.91 (8H, m), 1.88 (2H, m), 1.84–1.61 (6H, m), 1.42 (6H, s), 1.18 (3H, s), 1.06 (3H, s), 1.03 (3H, s), 1.00 (3H, s), 0.98 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.3, 168.6, 146.9, 123.8, 81.4, 74.7, 67.4, 56.7, 49.5, 48.2, 48.4, 46.1, 44.2, 44.0, 42.1, 41.6, 40.6, 36.4, 35.5, 34.7, 32.9, 30.2, 30.1, 29.8, 26.9, 24.5, 23.4, 20.9, 20.2, 19.5, 19.4, 18.7; HRMS (ESI+): calcd. for (C32H49Cl2O5)+ [M+H]+ 583.2879, found 583.2947. 3.1.2.20. (2a,3b)-2-(Pivaloyloxy)-3-hydroxy-olean-12-en-28-oic acid (2j). White solid; mp: 283–285 °C; 1H NMR (300 MHz, C5D5N): d 5.62 (1H, td, J = 6.7; 4.2 Hz), 5.49 (1H, t, J = 3.3 Hz), 3.52 (1H, d, J = 10.2 Hz), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.36 (2H, m), 2.33 (3H, m), 2.11 (6H, m), 1.83 (2H, m), 1.48 (4H, m), 1.41 (3H, s), 1.36 (9H, m), 1.31 (3H, s), 1.29 (3H, s), 1.18 (3H, s), 1.06 (3H, s), 1.03 (3H, s), 1.01 (3H, s), 0.94 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.4, 168.9, 168.2, 145.3, 122.5, 83.8, 71.4, 55.4, 54.5, 54.3, 54.1, 48.6, 48.4, 47.4, 47.2, 44.3, 43.0, 42.7, 40.8, 40.5, 39.1, 35.0, 34.0, 33.9, 33.5, 31.7, 30.2, 29.3, 29.0, 28.1 26.9, 24.5, 24.4, 22.2, 22.1, 19.0, 18.4, 18.0, 16.8; HRMS (ESI+): calcd. for (C35H57O5)+ [M+H]+ 555.4128, found 555.4198. 3.1.2.21. (2a,3b)-2,3-Bis((3-carboxypropanoyl)oxy)olean-12-en-28oic acid (2k). White solid; mp: 252–253 °C; 1H NMR (300 MHz, C5D5N): d 5.72 (1H, td, J = 6.7; 4.2 Hz), 5.61 (1H, d, J = 4.2 Hz), 5.43 (1H, s), 3.33 (1H, dd, J = 6.7; 4.2 Hz), 2.23 (4H, m), 2.09 (4H, m), 2.04–1.91 (6H, m), 1.86 (2H, m), 1.72 (4H, m), 1.66 (2H, m), 1.53 (2H, m), 1.49 (2H, m), 1.39 (2H, m), 1.26 (6H, s), 1.10 (3H, s), 1.06 (3H, s), 1.04 (3H, s), 1.01 (3H, s), 0.99 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.3, 176.4, 173.6, 172.1, 170.8, 145.8, 122.9, 83.1, 73.7, 61.5, 56.6, 54.2, 49.3, 48.2, 44.6, 44.1, 43.5, 43.0, 42.5, 41.5, 40.4, 36.1, 35.4, 35.1, 34.8, 32.9, 31.4, 30.7, 30.4, 30.1, 28.1, 25.8, 25.6, 23.3, 20.0, 19.1, 18.2, 17.1; HRMS (ESI+): calcd. for (C38H57O10)+ [M+H]+ 673.3873, found 673.3926.

3.1.2.17. (2a,3b)-2-((2S)-2-chloro-2-phenylacetoxy)-3-hydroxyolean-12-en-28-oic acid (2g). Dark red solid; mp: 279–280 °C; 1H NMR (300 MHz, C5D5N): d 7.94 (2H, m), 7.66 (1H, m), 7.19 (2H, m), 6.87 (1H, s), 5.62 (1H, td, J = 6.7; 4.2 Hz), 5.53 (1H, t, J = 3.3 Hz), 3.67 (1H, m), 3.34 (1H, dd, J = 6.7; 4.2 Hz), 2.23 (6H, m), 1.97 (4H, m), 1.81 (4H, m), 1.57 (2H, m), 1.41 (4H, m), 1.37 (3H, s), 1.29 (3H, s), 1.12 (3H, s), 1.08 (3H, s), 1.04 (3H, s), 1.01 (3H, s), 0.98 (3H, s); 13C NMR (75 MHz, C5D5N): d 180.8, 163.9, 145.4, 136.3, 135.7, 134.1, 132.6, 130.4, 130.8, 123.1, 81.5, 74.7, 65.4, 57.1, 48.6, 48.2, 48.0, 48.6, 44.1, 43.7, 41.6, 40.9, 40.5, 39.1, 35.8, 34.9, 34.7, 32.5, 31.6, 30.4, 29.9, 29.7, 27.7, 25.4, 25.3, 19.3, 18.2, 17.3; HRMS (ESI+): calcd. for (C38H54ClO5)+ [M+H]+ 625.3582, found 625.3651.

3.1.2.22. (2a,3b)-2,3-Bis(3-carboxybicyclo[2.2.1]hept-5-ene-2-carbonyloxy)olean-12-en-28-oic acid (2l). White solid; mp: 256– 257 °C; 1H NMR (300 MHz, CDCl3): d 6.41 (2H, m), 6.33 (2H, m), 6.24 (1H, td, J = 6.7; 4.2 Hz), 6.17 (1H, d, J = 4.2 Hz), 5.28 (1H, s), 4.88 (4H, m), 4.11 (2H, m), 3.34 (1H, dd, J = 6.7; 4.2 Hz), 3.23 (2 H, m), 2.12 (4H, m), 2.07 (4H, m), 2.01 (2H, m), 1.86 (4H, m), 1.69 (2H, m), 1.61 (2H, m), 1.53 (2H, m), 1.47 (4H, m), 1.37 (3H, s), 1.29 (3H, s), 1.18 (3H, s), 1.09 (3H, s), 1.03 (3H, s), 1.00 (3H, s), 0.89 (3H, s); 13C NMR (75 MHz, CDCl3): d 184.0, 183.5, 178.0, 173.0, 172.6, 143.6, 136.2, 135.1, 134.8, 133.1, 122.4, 80.9, 73.4, 60.4, 55.0, 54.4, 49.1, 48.7, 48.4, 47.8, 47.5, 46.9, 46.6, 46.4, 46.0, 45.4, 43.4, 41.7, 41.6, 40.7, 39.7, 39.5, 39.2, 38.4, 38.2, 33.8, 33.0, 29.7, 28.5, 27.6, 26.0, 23.6, 22.9, 21.1, 17.0, 16.9, 16.4, 16.3; HRMS (ESI+): calcd. for (C48H65O10)+ [M+H]+ 801.4499, found 801.4573.

3.1.2.18. (2a,3b)-2-((2S)-2-chloro-2-methylacetoxy)-3-hydroxyolean-12-en-28-oic acid (2h). Dark red solid; mp: 280–282 °C; 1H NMR (300 MHz, C5D5N): d 5.46 (2H, m), 4.68 (1H,m), 3.49 (1H,d, J = 9.9 Hz), 3.28 (1H, dd, J = 6.7; 4.2 Hz), 2.12 (4H, m), 1.96 (2H, m), 1.87–1.71 (6H, m), 1.69 (3H, d, J = 6.9 Hz), 1.58 (4H, m), 1.39 (4H, m), 1.31 (3H, s), 1.20 (3H, s), 1.12 (3H, s), 1.03 (3H, s), 1.00 (3H, s), 0.97 (3H, s), 0.92 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.0, 172.2, 146.8, 124.1, 81.3, 77.3, 56.0, 55.6, 49.9, 48.6, 48.4,

3.1.2.23. (2a,3b)-2,3-Bis((2-carboxybenzoyl)oxy)olean-12-en-28-oic acid (2m). White solid; mp: 255–257 °C; 1H NMR (300 MHz, C5D5N): d 8.05 (4H, m), 7.49 (2H, m), 7.42 (2H, m), 5.85 (1H, td, J = 6.7; 4.2 Hz), 5.56 (1H, d, J = 4.2 Hz), 5.40 (1H, s), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.12 (2H, m), 2.09 (4H, m), 2.03 (2 H, m), 1.92 (2H, m), 1.72 (4H, m), 1.66 (2H, m), 1.53 (2H, m), 1.39 (2H, m), 1.26 (6H, s), 1.10 (3H, s), 1.06 (3H, s), 1.04 (3H, s), 1.01 (3H, s), 0.99 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.0, 175.3, 172.6,

K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

170.5, 170.0, 146.9, 136.0, 134.5, 133.3, 132.9, 132.7, 132.5, 131.6, 131.5, 131.2, 130.9, 124.0, 84.0, 73.9, 62.2, 57.2, 54.4, 49.7, 48.5, 45.6, 44.1, 43.8, 42.1, 41.6, 41.1, 40.3, 36.1, 35.2, 35.1, 34.8, 32.9, 30.7, 30.1, 28.1, 25.8, 25.6, 23.3, 20.0, 19.1, 18.2, 16.2; HRMS (ESI+): calcd. for (C46H57O10)+ [M+H]+ 769.3873, found 769.3953. 3.1.2.24. (2a,3b)-2,3-Bis-(((E)-3-carboxyacryloyl)oxy)olean-12-en28-oic acid (2n). White solid; mp: 256–257 °C; 1H NMR (300 MHz, C5D5N): d 6.32 (2H, m), 6.21 (2H, m), 5.76 (1H, td, J = 6.7; 4.2 Hz), 5.55 (1H, d, J = 4.2 Hz), 5.48 (1H, s), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.23 (4H, m), 2.03 (4 H, m), 1.92 (2 H, m), 1.72 (4H, m), 1.66 (2 H, m), 1.53 (2H, m), 1.49 (2H, m), 1.35 (3H, s), 1.24 (3H, s), 1.11 (3H, s), 1.06 (3H, s), 1.04 (3H, s), 1.01 (3H, s), 0.92 (3H, s); 13C NMR (75 MHz, C5D5N): d 182.0, 177.3, 175.6, 173.3, 171.8, 145.8, 135.1, 135.0, 133.6, 133.1, 122.9, 83.1, 73.7, 61.5, 56.6, 54.2, 49.3, 48.2, 44.6, 44.1, 43.5, 42.5, 41.5, 40.6, 40.4, 36.1, 35.4, 35.1, 34.8, 32.9, 31.4, 28.1, 25.8, 25.6, 23.3, 20.0, 19.1, 17.2; HRMS (ESI+): calcd. for (C38H53O10)+ [M+H]+ 669.3560, found 669.3633. 3.1.2.25. (2a,3b)-2,3-Bis-(((E)-3-carboxybut-2-enoyl)oxy)olean-12en-28-oic acid (2o). White solid; mp: 250–252 °C; 1H NMR (300 MHz, C5D5N): d 6.42 (2 H, m), 5.76 (1H, td, J = 6.7; 4.2 Hz), 5.55 (1H, d, J = 4.2 Hz), 5.48 (1H, s), 3.35 (1H, dd, J = 6.7; 4.2 Hz), 2.35 (3H, s), 2.31 (3H, s), 2.23 (4H, m), 2.13 (4H, m), 1.87 (2H, m), 1.71 (4H, m), 1.70 (2H, m), 1.53 (2H, m), 1.49 (2H, m), 1.35 (3H, s), 1.24 (3H, s), 1.11 (3H, s), 1.06 (3H, s), 1.04 (3H, s), 1.01 (3H, s), 0.92 (3H, s); 13C NMR (75 MHz, C5D5N): d 181.8, 176.1, 175.2, 173.9, 172.4, 146.3, 136.2, 136.0, 134.6, 134.2, 123.2, 81.6, 72.9, 61.4, 55.6, 54.7, 49.5, 46.2, 44.6, 44.5, 43.5, 42.6, 42.0, 41.7, 40.4, 36.1, 35.4, 35.1, 34.8, 32.9, 31.4, 28.1, 25.8, 25.6, 23.3, 20.0, 19.1, 17.8, 16.4, 15.9; HRMS (ESI+): calcd. for (C40H57O10)+ [M+H]+ 697.3873, found 697.3933. 3.2. Biological activity 3.2.1. Antibacterial activity All the tested compounds were found to be active towards the used bacteria. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentration (MBC) of the compounds were determined referring to the results of the microdilution method (Table 3). The results showed that compound 2 (MIC = 15–30 mg/mL; CMB = 25–50 mg/mL) was significantly more active than compound 1 (MIC = 25–90 mg/mL; CMB = 50– 300 mg/mL). This effect may be explained by the importance of the additional hydroxyl group in position 2 and probably the R stereochemistry of the corresponding carbon. In most cases, the compounds having sulfur atom (1a, 2a and 2f) and chlorine atom (1b–d, 2b–d, 2g–i) showed an interesting inhibitory activity against most Gram-positive and Gram-negative bacteria at very low MICs within a range of 5–25 lg/ml. To our delight, the latter compounds exhibited a comparable antibacterial potential with that of the positive control (Imipenem) against practically all the used bacteria (Table 3). The compounds 1a, 1c, 2b–d, 2h and 2i could inhibit E. faecalis at lower concentrations (MIC = 5 lg/mL) whereas higher amounts (MIC = 5–15 lg/mL) of these compounds were required for the inhibition of S. aureus. Compounds 1a–d, 2c, 2d, 2h and 2i might be the major active compounds against E. coli and exhibited a similar activity (MIC = 5 lg/mL and MBC = 15–30 lg/mL). The six latter compounds 1c, 1d, 2c, 2d, 2h and 2i had a 1-chloroethyl and a dichloromethyl acyl substitute. The comparison of CMB values of the analogs 2a and 2f with the same sulphurated acyl group at both C-2 and C-3 and at C-2, respectively revealed that their antibacterial potential varied depending on the bacterial species tested. Indeed, S. aureus was

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found to be more sensitive to the diacylated derivative 2a (CMB = 15 lg/mL) than to its monoacylated analog 2f (CMB = 50 lg/mL) whereas these same analogs induced an opposite effect against E. faecalis. It should be also noticed that the chlorinated diacylated 2c (CMB = 25 lg/mL) derivative was more active than its chlorinated monoacyl analogous 2h towards S. aureus (CMB = 100 lg/mL), whereas both compounds exhibited nearly the same effect against the rest of the bacteria used. On the other hand, the present results also indicated that the derivatives 1f–j and 2k–o prepared using cyclic anhydrides had a quite similar antibacterial spectrum but relatively lower if compared to the rest of the acylated analogs without additional carboxylic acid function. Considering our experimental conditions, we did not record any antibacterial activity for 1e, 1j and 2j compounds probably due to their insolubility in DMSO. As a result, the oleanolic acid esters exhibited comparable antibacterial potential with that of maslinic acid esters. A good number of natural triterpenoids were reported as has been proven before in literature to possess a wide range of antimicrobial activities; however, in most cases and to get effective, a comparatively higher amount of compounds were usually required. Hichri et al. (2003) described the antibacterial activity of the acetylated oleanolic acid towards S. aureus, E. coli and P. aeruginosa which showed a weaker antibacterial activity than that observed with our esters (1a–i). This finding shows that the nature of the acyl moiety introduced influences the activity. Scalon Cunha et al. (2007) evaluated some natural triterpenoids including oleanolic and ursolic acids and some of their derivatives as well with MIC values in the range of 30–80 lg/mL against different Streptococcus species. Prachayasittikul, Saraban, Cherdtrakulkiat, Ruchirawat, and Prachayasittikul (2010) also assessed the antimicrobial activity of certain bioactive triterpenoids against Grampositive pathogens (MICs 64–256 lg/mL). Recently, Kiplimo, Koorbanally, and Chenia (2011) isolated several triterpenoids from Veronica auriculifera, biologically active against both Gram-positive and Gram-negative pathogenic bacteria with MIC values around 250 lg/mL. In the current study, it is noteworthy that the synthesized compounds having sulfur and chlorine atoms exhibited interesting antibacterial activities. The nature of the acyl group and its position on the ring A of the triterpene acid significantly influences the antibacterial activity.

3.2.2. Antifungal activity The antifungal results (Table 4) revealed that the synthesized compounds showed variable degrees of inhibition against the tested fungi. The triterpenoid derivatives: (3b)-3-((thiophene-2-carbonyl)oxy)-olean-12-en-28-oic acid (1a) and (2a,3b)-2,3-bis((thiophene-2-carbonyl)oxy)olean-12-en-28-oic acid (2a) were the most active compounds, they exhibited a good antifungal activity against all the tested fungal species especially P. italicum (IZ = 22.5 and 24 mm, respectively). However, (2a,3b)-2-((thiophene-2-carbonyl)oxy)-3-hydroxy-olean-12-en-28-oic acid (2f) was found to be also active against P. italicum (IZ = 14 mm), P. digitatum (IZ = 15.5 mm), T. harzianum (IZ = 16 mm) and A. niger (IZ = 19 mm). Furthermore, the screening of the antifungal potential of the tested compounds revealed that most of them have displayed a good inhibitory effect against T. harzianum (IZ = 9.5–22 mm). The compound 2k was found to be active only against T. harzianum with an IZ value of 16 mm. Only compounds 2m and 2n exhibited an antifungal effect towards A. flavus (IZ = 15 and 19 mm, respectively). The results obtained showed that the esterification of either compounds 1 or 2 by acid chlorides or cyclic anhydrides greatly improves their antifungal activities. It was also found that the

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K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

Table 3 Antibacterial activity of compounds 1, 2 and their acylated derivatives 1a–j and 2a–o expressed in MIC and MBC (lg/mL). Compounds

Gram-positive bacteria S. aureus

1 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 2 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n 2o Imipenem

Gram-negative bacteria E. faecalis

E. coli

MIC

MBC

MIC

MBC

95 ± 2 15 ± 0.8 25 ± 1 15 ± 1 15 ± 0.9 – 50 ± 2 90 ± 2 30 ± 1.5 30 ± 1 – 15 ± 1 5 ± 0.4 15 ± 0.7 5 ± 0.5 5 ± 0.6 150 ± 3 15 ± 2 100 ± 2.5 10 ± 1 5 ± 0.2 – 30 ± 2 300 ± 8 300 ± 5 50 ± 4 50 ± 2 15 ± 1

300 ± 8 30 ± 1 100 ± 5 25 ± 2 30 ± 2 – 150 ± 6 500 ± 8 90 ± 2 250 ± 5 – 25 ± 2 15 ± 1 30 ± 1 25 ± 1.5 50 ± 2 – 50 ± 2 – 100 ± 5 50 ± 2 – 90 ± 1 – – 90 ± 3 60 ± 2 25 ± 2

30 ± 1 5 ± 0.4 15 ± 1 5 ± 0.6 15 ± 2 – 50 ± 4 50 ± 2 30 ± 1 30 ± 3 – 30 ± 2 10 ± 0.6 5 ± 0.4 5 ± 0.4 5 ± 0.5 100 ± 3 10 ± 0.2 25 ± 1 5 ± 0.1 5 ± 0.4 – 30 ± 4 50 ± 3 30 ± 3 50 ± 5 100 ± 3 15 ± 2

150 ± 3 25 ± 2 25 ± 1 25 ± 2 30 ± 2 – 150 ± 5 150 ± 4 250 ± 3 150 ± 2 – 50 ± 2 50 ± 4 30 ± 2 25 ± 2 25 ± 2 – 25 ± 1 50 ± 2 25 ± 2 50 ± 5 – 50 ± 2 150 ± 5 60 ± 3 150 ± 2 – 50 ± 3

P. aeruginosa

MIC 90 ± 1 5 ± 0.5 5 ± 0.4 5 ± 0.5 5 ± 0.9 – 50 ± 1 150 ± 2 150 ± 5 100 ± 2 – 15 ± 0.4 10 ± 0.4 15 ± 0.2 5 ± 0.9 5 ± 0. 2 300 ± 7 10 ± 0.2 15 ± 1 5 ± 0.5 5 ± 0.5 – 50 ± 5 50 ± 5 50 ± 3 25 ± 1 100 ± 1 25 ± 2

MBC

MIC

MBC

150 ± 1 15 ± 2 25 ± 2 25 ± 2 15 ± 0.8 – 150 ± 5 – – – – 50 ± 5 50 ± 5 30 ± 2 30 ± 1 25 ± 2 – 50 ± 2 30 ± 4 25 ± 2 30 ± 2 – 100 ± 4 150 ± 2 300 ± 5 150 ± 2 – 50 ± 2

25 ± 2 5 ± 0.4 15 ± 0.5 15 ± 0.5 5 ± 0.8 – 50 ± 2 50 ± 4 300 ± 2 300 ± 5 – 15 ± 0.9 25 ± 2 5 ± 0.6 5 ± 0.4 5 ± 0.4 150 ± 5 15 ± 2 10 ± 0.03 15 ± 0.2 5 ± 0.02 – 50 ± 2 90 ± 3 25 ± 3 50 ± 5 50 ± 2 5 ± 0.1

50 ± 1 15 ± 2 30 ± 2 25 ± 2 50 ± 2 – 100 ± 5 150 ± 5 – – – 30 ± 2 50 ± 2 30 ± 2 25 ± 4 15 ± 0.8 – 50 ± 2 25 ± 3 25 ± 2 25 ± 2 – 100 ± 1 300 ± 2 150 ± 4 100 ± 1 90 ± 2 25 ± 1

MIC (lg/mL), minimum inhibitory concentration, i.e., the lowest concentration of the compound to inhibit the growth of bacteria completely. MBC (lg/mL), minimum bacterial concentration, i.e., the lowest concentration of the compound for killing the bacterial completely.

Table 4 Antifungal activity of compounds 1, 2 and their acylated derivatives 1a–j and 2a–o expressed in inhibition zone (mm). Compounds

1 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 2 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n 2o FONG DMSO _:

Zone of inhibition (mm) Aspergillus flavus

Aspergillus niger

Penicillium digitatum

Penicillium italicum

Trichoderma harzianum

_ 9.5 ± 1 _ _ _ _ _ _ _ _ _ _ 9 ± 0.5 _ _ _ _ _ _ 12 ± 0.5 _ _ _ _ 15 ± 0.8 19 ± 1 _ 40 ± 2 _

_ 10 ± 1 _ 9 ± 0.5 _ _ _ _ _ _ _ 9 ± 0.4 13.5 ± 0.5 _ _ _ _ 19 ± 0.3 _ _ _ _ _ _ _ _ _ 28 ± 1 _

13.5 ± 0.5 9.5 ± 0.5 _ 9±1 _ 11 ± 0.2 _ 19 ± 0.5 _ _ _ _ 11.5 ± 1 _ 15 ± 2 _ _ 15.5 ± 0.4 _ _ _ _ _ 9 ± 0.05 13 ± 0.1 _ _ 45 ± 3 _

15 ± 1 22.5 ± 0.2 _ _ _ 15 ± 0.2 _ _ _ _ _ 13 ± 0.5 24 ± 0.4 _ _ _ _ 14 ± 0.2 _ _ _ 13 ± 0.2 _ _ _ _ _ 43 ± 2 _

15 ± 2 13 ± 1 _ 22 ± 2 11 ± 1 11 ± 1 _ 15.5 ± 2 10 ± 1 10 ± 2 _ 9.5 ± 1 16 ± 2 9.5 ± 2 9.5 ± 1 _ _ 16 ± 0.5 13 ± 0.5 _ 10 ± 0.5 _ 16 ± 0.5 9.5 ± 0.5 _ 17 ± 0.5 _ 34 ± 1 _

No inhibition zone observed. FONG: Carbendazim 0.5 mg/mL.

K. Chouaïb et al. / Food Chemistry 183 (2015) 8–17

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