Regiospecific synthesis by copper- and ruthenium-catalyzed azide–alkyne 1,3-dipolar cycloaddition, anticancer and anti-inflammatory activities of oleanolic acid triazole derivatives

1 Introduction

A review of the literature showed that some triterpenes and their derivatives have been associated with various pharmacological activities. They have shown good antitumoral (Schwarz and Csuk, 2010), antioxidant (Chiung et al., 2010), anti-inflammatory (Bednarczyk-Cwynar et al., 2012), antiviral (Grishko et al., 2014), antibacterial and antifungal (Chouaïb et al., 2015; Hichri et al., 2003) activities.

Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, 1) is a natural pentacyclic triterpenoid compound (Fig. 1), widely distributed throughout the vegetable kingdom (Guo et al., 2015; Liu, 2005). This compound and some of its derivatives have been reported to possess various biological activities such as anticancer (Srivastava et al., 2010), gastroprotective (Sánchez et al., 2006), anti-Alzheimer (Abdalla et al., 2012), antidiabetic (Chen et al., 2006), hepatoprotective (Yim et al., 2001) and many other important activities. Thus all the pharmacological properties of oleanolic acid were reviewed by Liu (1995), who mentioned also its hepatoprotective, anti-neoplastic and anti-hyperlipidemic properties and emphasized its low toxicity. Similarly, oleanolic acid has been marketed in China as an oral drug for human liver disorders (Liu, 2005).

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Figure 1 Chemical structure of oleanolic acid 1.

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On the other hand, triazoles, an important class of N-heterocycles, are employed in many pharmaceutical products (Farajzadeh and Khoshmaram, 2013). In particular, 1,2,3-triazoles are of great importance in medicinal chemistry because they can act as pharmacophores and linkers between two or more substances of interest in molecular hybridization approaches (Agalave et al., 2011). 1,2,3-Triazoles are among heterocyclic compounds that attract the attention of organic chemists (Singh et al., 2010) due to their broad spectrum of applications in biochemical, medicinal chemistry and material sciences. Several of these triazoles are drugs and have a wide range of biological activities such as anti-HIV (Kirschberg et al., 2008), antiviral (Aufort et al., 2008), cytotoxic (Kamal et al., 2008) and antibacterial (Wang et al., 2010) effects. Rashid et al. (2013) suggest that ursolic acid–triazolyl derivatives show an anticancer activity against a panel of human cancer cell lines including A-549 (lung), MCF-7 (breast), HCT-116 (colon), THP-1 (leukemia) and a normal epithelial cell line (FR-2) using sulforhodamine-B assay. Most of these compounds exhibited better anticancer activity against all the tested cancer cell lines compared to positive controls, 4,5-fluorouracil and mitomycin-C. According to previous reports, it has been observed that keeping polar substituents at the C-28 position of the triterpene skeleton enhanced its anti-tumor potential against BGC-823 cell lines (Bai et al., 2011). Recently, Wei et al. (2015) suggest that the incorporation of a 1,2,3-triazole moiety at C-28 position of oleanolic acid 1 was useful to improve its cell proliferation inhibition against human cancer cell lines.

The 1,3-dipolar cycloaddition is considered the most important and versatile synthetic route for a wide range of 1,2,3-triazoles and triazolines. The conventional synthesis of 1,2,3-triazoles relies on the Huisgen [3 + 2] cycloaddition between alkynes and organic azides usually provides a mixture of 1,4- and 1,5-disubstituted regioisomers (Norris et al., 1996; Talekar and Wightman, 1997). In general, the attempts to separate these regioisomers by chromatography, with some exceptions, were not successful. The growing use of catalyzed azide–alkyne cycloaddition named the “click” reaction, allowing the easy access to regiospecific 1,2,3-triazoles, motivated their use in the drug discovery field (Giffin et al., 2008). In view of the preceding literature data, we were encouraged to synthesize oleanolic acid-(C-28)triazolyl derivatives as a contribution to develop more effective therapeutic agents by employing click chemistry protocol. Click chemistry enables a modular approach to generate these novel pharmacophores utilizing a collection of reliable chemical reactions. The Huisgen 1,3-dipolar cycloaddition of alkynes and azides (AAC) has appeared as a powerful linking reaction to give substituted-1,2,3-triazoles. This reaction when catalyzed with Copper (I) (CuAAC) leads exclusively to the 1,4-regioisomer (Kumar et al., 2009). On the other hand, the ruthenium-catalyzed version of the reaction (RuAAC), affords mainly the 1,5-regioisomer (Takasu et al., 2010).

We report herein the synthesis of 1,4- and new 1,5-disubstituted-1,2,3-triazole derivatives from oleanolic acid 1 (Fig. 1) isolated from pomace olive (Olea europaea L.) cultivar: Chemlali, under ultra-sonication conditions (6.8 g (3.4 mg/g DW)) (Chouaïb et al., 2015). They are prepared via well established Cu(I)-catalyzed azide–alkyne (CuAAC) and Ru(II)-catalyzed azide–alkyne (RuAAC) cycloadditions which were conducted for the first time under microwave and solvent free conditions. Oleanolic acid 1 and most obtained derivatives were evaluated for their anticancer and anti-inflammatory activities in vitro.

2 Results and discussion

The precursors 2a–g were synthesized from the appropriate anilines using a two-step procedure by diazotization with sodium nitrite in acidic conditions followed by displacement with sodium azide. The desired aromatic azides 2a–g were obtained in yields ranging from 85% to 98%. On the other hand, compound 1 was subjected to oxidation using Jones reagent at 0 °C that resulted in the formation of C-3 oxidized derivative 3 in quantitative yield (94%). The propargylation of the carboxyl group at C-28 in dry DMF using NaH as catalyst provided the propargylated ester 4 in excellent yield (98%). The 3-oxo group of Compound 4 was selectively reduced to C-3 hydroxyl group with sodium borohydride under microwave irradiation (250 W, 3 min), to obtain the dipolarophile 5 in 99% yield (Scheme 1).

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Scheme 1 Synthesis of the 1,4- and 1,5-triazolyl (6a–g, 7a–g) derivatives. Reagents and conditions: (a) Jone’s oxidation (CrO₃, H₂SO₄/acetone), 0 °C, 94%, (b) propargyl bromide, NaH, dry DMF, rt, 98%, (c) NaBH₄, CH₃OH/THF, Microwave (250 W, 3 min), 99%, (d) Cuprous iodide (CuI), Et₃N, solvent free, Microwave (200 W, 2–4 min), 87–98%, (e) [Cp^∗RuCl(PPh₃)₂], DMF, Microwave (250 W, 3–6 min), 84–96%.

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Although the Cu(I)-catalyzed Huisgen 1,3-dipolar cyclization usually proceeds in high yields, it has been reported to be highly dependent on the substrate and reaction conditions. Using phenylazide (2a, R = H) as a model azide, the most suitable conditions to carry out the cyclization with alkyne 5 were explored. The use of 2 equiv of phenylazide is here necessary because of its possible dimerization into secondary amine (Lange et al., 1998; An et al., 2004). The most relevant results of this study are shown in Table 1.

As shown in Table 1  0.5 equiv of CuI under microwave and solvent free conditions in the presence of triethylamine as a base (1 equiv. referred to the starting alkyne) was essential to obtain the higher yield of the desired 1,2,3-triazole in minimum reaction time (entry 5). Interestingly, a further increase in the amount of CuI did not improve the yield (entry 6). However, the excess of Cu(I) probably caused a diminution in the performance due to the deposition of copper species on the dipole and the low solubility of cuprous iodide in triethylamine. The optimal condition (Table 1, entry 5) found was applied in a regiospecific approach using Huisgen 1,3-dipolar cycloaddition reaction (CuAAC) of the alkyne 5 with various aromatic azides 2a–g in the presence of cuprous iodide (CuI) and Et₃N resulting into the formation of regiospecific 1,4-substituted-triazolyl derivatives 6a–g in excellent yields (Scheme 1).

Table 1 Optimization of Cu(I)-catalyzed 1,3-dipolar cyclization for the synthesis of 1,2,3-triazoles.^a

Entry	CuI (equiv)b	Solvent	Et3N (equiv)b	Condition	Time (h)	Yield (%)d
1	0.3	DMF	0.1	rt	8	40
2	0.1	DMF	0	rt	8	Traces
3	0.5	DMF	1	110 °C	8	85
4	0.5	DMF/H2O (1:1)	1	Microwavec	0.1	90
5	0.5	Solvent free	1	Microwavec	0.033	92
6	2	Solvent free	1	Microwavec	0.1	50

Bold in entry highlights the optimal reaction conditions.

a Alkyne 5 (2 mmol) and two equivalents of phenylazide 2a.

b Referred to the starting alkyne 5.

c The reaction was performed at 200 W.

d Isolated yield after column chromatography (EP-EtOAc), based on the starting dipolarophile 5.

All the reactions were carried under microwave irradiation (200 W) completed within 2–4 min. The desired compounds 6a–g were obtained in yields ranging from 87% to 98% under microwave and solvent free conditions. This method compared to that cited in the literature (Wei et al., 2015) for the preparation of the same compounds has the advantage of being simple and the products can be obtained from the reaction mixture by simple extraction.

The structure of the 1,4-substituted triazole regioisomers were evidenced by their spectral data. The ¹H NMR spectra of compounds 6a–g showed a singlet at δ_H 7.96–8.02 attributable to the proton H-5 of the triazole moiety and signals at the aromatic region (δ_H 7.00–7.70) relative to the aromatic protons introduced by the azides used. These structures were further supported by ¹³C and DEPT NMR spectra, which showed all the expected carbon signals corresponding to oleanolic acid–triazolyl derivatives, essentially the aromatic carbons resonating at δ_C 114.2–159.3 introduced by the azides used.

The resulting 1,4-regioisomers were evidenced from the NOE between H-5_triazole/H_methylene and H-5_triazole/H_arom, and the absence of NOE between H_methylene/H_arom, and such regiospecificity agrees with that cited in the literature (Kumar et al., 2009; Li et al., 2014). High Resolution Mass Spectrometry (HRMS) data of all the formed derivatives were also in agreement with the proposed structures.

On the other hand, we turned our attention to the ruthenium–catalyzed version of the reaction (RuAAC), which is less extensively described and led mainly to 1,5-regioisomers (Boren et al., 2008). Generally, the common approaches to 1,5-regioisomers were based on Grignard reagents (Krasinski et al., 2004), or 1-trimethylsilylalkynes (Coats et al., 2005). Microwave heating allows a significant acceleration of the reaction from 8 h to 2 min (Table 1). Working under microwave irradiation in DMF, we first investigated the optimal conditions for the cycloaddition of alkyne 5 and phenylazide 2a using different concentrations of the catalyst Cp^∗RuCl(PPh₃)₂ (Table 2).

Table 2 Optimization of RuAAC under microwave conditions (250 W).^a

Entry	Catalyst (mol%)b	Solvent	Irradiation time (min)	Conversion (%)
1	1	DMF	10	50
2	3	DMF	10	80
3	5	DMF	4	100
4	5	Solvent free	10	No reaction

Bold in entry highlights the optimal reaction conditions.

a Alkyne 5 (2 mmol) and two equivalents of phenylazide 2a.

b Referred to the starting alkyne.

The total conversion of oleanolic acid–alkyne derivative 5 was obtained after only 4 min of reaction with 5% catalyst (entry 3). For lower catalyst loading, conversion was not complete, even after 10 min reaction (Table 2, entries 1 and 2). Moreover, when we worked under solvent free conditions, there was no reaction (entry 4).

With these optimized conditions in hand, the dipolarophile 5 was reacted with various aromatic azides and 5 mol% Cp^∗RuCl(PPh₃)₂ catalyst. All the reactions were performed under microwave conditions in DMF and completed within 3–6 min. The desired new compounds 7a–g were obtained in yields ranging from 84% to 96% (Scheme 1).

The reaction was conducted with complete regiospecificity and most of the yields remained good for the whole synthetic sequence. The structure of the prepared compounds (7a–g) was established on the basis of their analytical data.

In addition of the signals corresponding to the protons introduced by oleanolic acid–alkyne 5, new signals related to the azides used were observed in ¹H NMR spectra. Examination at 300 MHz showed in particular a singlet at δ_H 7.82–8.17 (H-4 of the triazole ring) and also other signals at δ_H 7.05–7.93 (aromatic protons). The ¹³C NMR spectra confirmed the above spectral data by the observation of new signals at δ_C 114.2–160.1 relative to the aromatic carbons and the triazoles formed.

The 1,5-regiochemistry thus adopted was confirmed by NOESY spectra showing the NOE between H-4_triazole/H_methylene and H_methylene/H_arom, and the absence of NOE between H-4_triazole/H_arom, and such observation was in agreement with the proposed structures and with that indicated in the literature (Takasu et al., 2010). The HRMS data of the formed compounds 7a–g were in concordance with the established structures.

3 Anticancer activity

The cytotoxic activity of oleanolic acid 1 and most of its triazole derivatives were studied using cultured murine EMT-6 (Breast) and human SW480 (colon) cancer cell lines. Doxorubicin, etoposide, 5-fluorouracil and methotrexate (10 μM) were used as anticancer references in this study. Compounds 6b (4-OCH₃) and 6c (4-Cl) (1,4-regioisomers) and 7b (4-OCH₃) (1,5-regioisomer) were found to be the most promising. From the percentage viability and IC₅₀ data (Tables 3 and 4) and compared to most of its derivatives, oleanolic acid 1 displayed the highest anticancer activity, exhibiting potent anti-proliferative activity toward breast (EMT-6) (Viability (%/Control) = 7% (100 μM); IC₅₀ = 66.6 ± 0.7 μM) and colon (SW480) (Viability (%/Control) = 13% (100 μM); IC₅₀ = 76.0 ± 3.0 μM) cancer cells. The anticancer activity of oleanolic acid 1 disappeared at lower concentrations (30 and 10 μM). The results given in Tables 3 and 4, show that the junction of oleanolic acid 1 to triazole according to the two regiochemistry approaches (1,4- and 1,5-) through the linker methylene did not improve its anticancer activity.

For the 1,4-regioisomers, compounds 6b and 6c exhibited the highest anticancer activity against breast cancer cells (IC₅₀ = 91.8 ± 3.3 and 76.0 ± 3.7 μM, respectively). Toward colon cell lines, only 6c displayed a remarkable effect (IC₅₀ = 29.6 ± 1.7 μM), and it was found more than twice as active as oleanolic acid 1 (IC₅₀ = 76.0 ± 3.0 μM). The positive mesomeric effect (+M) of the methoxy group in 6b and chlorine atom in 6c may explain their activity compared to that of the rest of analogues from the same series. In the contrary, in the case of compounds 6e, 6f and 6g, the presence of 4-NO₂–C₆H₄, 3-CH₃–C₆H₄ and naphthyl, respectively attached to their triazole moieties appears to reduce considerably the anticancer activity against the two cancer cell lines used.

For the 1,5-regioisomers, only 7f, bearing a 3-CH₃–C₆H₄ in its triazole moiety, was found to be most active against Breast (EMT-6) cells (Viability (%/Control) = 60 ± 1% (100 μM); IC₅₀ = 60 ± 1 μM) followed by 7e (4-NO₂) (Viability (%/Control) = 74 ± 3% (100 μM); IC₅₀ = 74 ± 3 μM). Compound 7b (4-OCH₃–C₆H₄) and 7c (4-Cl–C₆H₄) displayed less activity than 7f and 7e against the same cell line ((IC₅₀ = 82.8 ± 1.7 and 94 ± 3 μM, respectively).

These results showed clearly the contribution of the nature of the aromatic system attached to the triazole ring in this activity.

All compounds 7 tested did not exhibit any activity toward the used cancer cell lines (IC₅₀ > 100 μM) toward (SW480) colon cancer cell lines.

The results obtained show that the difference in activity of the regioisomers 6 (1,4-) and 7 (1,5-) is undoubtedly due to both the regiochemistry and the nature of the substituent borne by the aromatic ring attached to the triazole.

4 Anti-inflammatory activity

The anti-inflammatory activity of the tested compounds was studied using LPS-stimulated human peripheral blood mononuclear cells (PBMCs). Three anti-inflammatory references were tested in the same conditions: ZVAD (5 μM), dexamethasone (DEXA, 1 μM) and prednisolone (PRED, 70 μM). Secretion IL-1β was measured only in the culture supernatants of PBMCs treated with compounds that showed no or low toxicity, using a sandwich enzyme-linked immunosorbent assay (ELISA) method (eBioscience, San Diego, USA), according to manufacturer instructions.

The anti-inflammatory results (Table 4) revealed that the synthesized compounds showed variable degrees of % IL-1β production. This finding showed that the introduction of the 1,4- and 1,5-triazole moieties in compound 1 did not considerably improve its anti-inflammatory activity. In the series of 1,4-regioisomers, compound 6b was found to be relatively the most active (% IL-1β production = 71 ± 15; 100 μM). The significant positive mesomeric effect (+M) of the methoxy group in para position of the aromatic ring attached to the triazole moiety in compound 6b compared to that exerted by the chlorine atom in compound 6c and by the methyl group in compound 6f at the same position may explain the difference in activity between these three derivatives. On the other hand, it has been found that compounds 6e and 6g both bearing a group with a negative mesomeric effect (−M) (4(NO₂–C₆H₄) and naphtyl, respectively) did not exhibit any activity.

On the other hand, the results showed that only 7f (3-CH₃; 1,5-regioisomer) (% IL-1β production = 79 ± 2; 100 μM) exhibited a slightly higher anti-inflammatory activity than oleanolic acid 1 (% IL-1β production = 88 ± 3; 100 μM). Compounds 7b, 7c and 7e were found to be not anti-inflammatory and the triazole system in its 1,5-regiochemistry has not improved remarkably the activity of oleanolic acid 1 (see Table 5).

5 Conclusion

In summary, oleanolic acid 1 was quantitatively isolated from pomace olive (O. europaea L.) cultivar: Chemlali (6.8 g (3.4 mg/g DW)) under ultra-sonication conditions. On the other hand, we used the Copper(I) iodide catalytic system for CuAAC and the Cp^∗RuCl(PPh₃)₂ for RuAAC under microwave conditions to reach hitherto new natural oleanolic acid derivatives bearing 1,4 and 1,5-disubstituted triazole, respectively. The cooperative effect of the catalyst and the microwave activation afforded the desired compounds in a few minutes in high yields. This effective approach allows an easy access to a series of 1,4-regioisomers under CuAAC and 1,5-disubstituted-triazolo derivatives under RuAAC. Oleanolic acid 1 and most of the prepared derivatives were evaluated for the anticancer and anti-inflammatory activities. Oleanolic acid 1 and compounds 6c (4-Cl; 1,4-regioisomer) and 7e (4-NO₂; 1,5-regioisomer) showed the highest anticancer activity against breast (EMT-6) and 6c was found more than twice as active as oleanolic acid 1 toward colon (SW480) cancer cells. In this context, the regiochemistry and the mesomeric effect appear involved in this activity. On the other hand, 6b (4-OCH₃; 1,4-regioisomer) and 7f (3-CH₃; 1,5-regioisomer) showed a slightly higher anti-inflammatory activity than oleanolic acid 1.

6 Experimental section

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 μm, sds). HRMS spectra were acquired with an ESI-TOF (LCT Premier XE, Waters) using the reflectron mode. Leucine-enkephaline peptide was employed as a lockmass for the LockSpray. ¹H (300 MHz) and ¹³C (75 MHz) NMR spectra were recorded on a Bruker AM-300 spectrometer, using CDCl₃ and CD₃OD as solvents. The microwave was a Biotage AB Initiator EXP EU with a maximum power of 800 W (2450 MHz). The chemical shifts (δ) are reported in ppm relative to the residual non-deuterated solvent as internal standard and coupling constants were measured in Hz.