Bis(4-((E)-3,5–Diacetoxystyryl)phenyl)nonanedioate

Abstract

Resveratrol is a natural stilbene known for its wide range of biological activities, including antioxidant, anti-inflammatory, and anti-aging effects. However, its application in cosmetics and dermatology is limited by poor stability and bioavailability. Azelaic acid is a natural carboxylic acid employed in cosmetics for its tyrosinase inhibition activity and for cutaneous hyperpigmentation disorders. In this work, we report a concise chemoenzymatic procedure for the synthesis of a novel hybrid molecule combining acetylated resveratrol and azelaic acid. This methodology offers a valuable route for the development of new bioactive compounds for potential cosmetic and dermatological applications.

1. Introduction

Resveratrol (3,5,4′-trihydroxystilbene, 1) is a naturally occurring phenolic compound renowned for its wide range of biological activities [1,2,3,4]. Resveratrol is biosynthesized almost entirely in grape skins and over 70 plant species [1,5] and found in red wine. Its multifunctional properties include antioxidant [6], anti-inflammatory [7], cardioprotective [1], antimicrobial [8], neuroprotective [9], and anticancer effects [10,11,12]. It is also increasingly used in cosmetics and dermatology for its anti-aging benefits [13].

In recent years, various natural compounds have been extensively studied for their inhibitory effects on key metabolic enzymes (pancreatic lipase, α-glucosidase, and α-amylase). Structural modifications of these molecules have often led to an enhancement in inhibitory activity towards metabolic enzymes [14,15,16,17,18,19,20]. Whitin this framework, the inhibition of tyrosinase—an enzyme crucial in melanin biosynthesis—has also gained significant attention, particularly in skin depigmentation and hyperpigmentation treatments. Although resveratrol’s direct effect on melanin synthesis is limited [21], it shows potential as a supportive agent in hyperpigmentation treatments due to its ability to inhibit tyrosinase [22]. Resveratrol inhibits tyrosinase through multiple mechanisms, including transcriptional and post-transcriptional regulation [23]. Even though resveratrol is a strong inhibitor of tyrosinase, its use is limited due to its chemical instability, low solubility and, in particular, low lipo-permeability [5]. For this reason, resveratrol can be conjugated with other molecules to improve liposolubility, stability, and skin absorption [24]. Triacetyl resveratrol (2), a more lipophilic and less cytotoxic derivative of 1, showed a lower in vitro tyrosinase inhibition compared with 1 (Figure 1), but it proved more effective than 1 in the inhibition of cellular melanin biosynthesis [25]. Furthermore, it has been demonstrated that triacetyl resveratrol acts as a ‘prodrug’, releasing the active resveratrol upon its hydrolysis by the cellular esterases. Thus, more lipophilic compounds containing ester bonds can be attractive candidates for effective skin-whitening agents in terms of efficacy, safety, and stability [22,25].

Azelaic acid (3)—a dicarboxylic acid naturally found in grains—has long been recognized for its dermatological uses [26]. It acts as a competitive inhibitor of tyrosinase for l-tyrosine, selectively interfering with melanin production [27]. Nowadays, azelaic acid is widely used in topical formulations not only for acne and rosacea but also in the treatment of lentigo maligna melanoma, due to its ability to inhibit melanogenesis [28].

Herein, we report a concise synthesis of a novel conjugate between resveratrol and azelaic acid via ester bond. The new compound may represent a prodrug of two molecules with potential tyrosinase inhibitory activity.

2. Results

In the effort to develop a novel biofunctionalized azelaic derivative with resveratrol, a chemoenzymatic synthesis was planned according to the conditions reported in Scheme 1. Namely, the synthetic route begins with the acetylation of 1, followed by a chemoenzymatic butanolysis under mild conditions [29]. The 3,5-diacetylated resveratrol was involved in the Steglich reaction, thus affording a di-functionalized product, as suggested by MS spectrum and elemental analysis. ¹H and ¹³C NMR spectra indicated the formation of a symmetrical compound centered at position 5′′, with a resveratrol-to-azelaic-acid ratio of 2:1. Key ¹H NMR signals in the aromatic region arise from the resveratrol scaffold, with characteristic peaks at δ 7.43 (H-2′/6′), 7.11 (H-2/6), 7.08 (H-3′/5′), and 6.82 (H-4), consistent with the substitution pattern of resveratrol derivatives. The methyl protons of the acetyl groups appear as a singlet at δ 2.29, integrating for 12 protons, confirming the presence of four acetyl groups. Additionally, the aliphatic region shows multiplets and triplets corresponding to the azelaic acid chain, with representative signals at δ 2.57 (H-2′′), 1.78 (H-3′′), and 1.45 (H-4′′/5′′), consistent with the expected pattern of a saturated dicarboxylic acid backbone. Analogously, the ¹³C NMR spectrum with the presence of only one signal at δ 172.2, diagnostic for a sp2 C of an ester (C-1′′), and the deshielded signal related to C-4′ (δ 150.6) suggested azelaic acid was esterified with two resveratrol molecules via OH at the C-4′ position. The analysis of the HMBC spectrum allowed the discrimination of some resonances. For instance, the carbons and protons involved in the olefin (C-7 and C-8) were unambiguously assigned as the signal at δ 127.1 (C-7) correlated with the doublet at δ 7.11 assigned to protons H-2/6, whereas the carbon signal at δ 129.7 was assigned as C-8 as it correlated with the doublet at δ 7.43 (H-2′′/6′). The signal at 134.4 of a quaternary carbon showed a correlation with proton signals at δ 7.08 (H-3′/5′) and δ 6.96 (H-7); thus, it was unambiguously assigned as C-1′. The signal at 150.6 showed a correlation with proton signals at δ 7.08 (H-3′/5′) and 7.43 (H-2′/6′), and it was assigned as C-4′, whereas the carbon signal at 151.4 was assigned as C-3/5 for the HMBC correlation with H-2/6 and H-4 signals. All the HMBC correlations are reported in Table S1. The combination of these data confirms the formation of the symmetric, difunctionalized resveratrol-azelaic acid diester 5, validating the success of the synthetic strategy.

3. Materials and Methods

All chemicals used were of reagent grade and employed without any additional purifications. Solvents and reagents were purchased from Sigma-Aldrich (Milan, Italy), Merck (Darmstadt, Germany), and Tokyo Chemical Industry TCI (Eschborn, Germany). The principal solvents utilized included ethanol, butanol, dichloromethane, methyl tert-butyl ether (MTBE), 4-dimethylaminopyridine (DMAP), N,N′-dicyclohexylcarbodiimide (DCC), hexane, and ethyl acetate. Enzymatic catalysis was carried out using Pseudomonas cepacia lipase (PsL). Preparative liquid chromatography was conducted on silica gel (particle size 63–200 μm; Merck, Darmstadt, Germany) employing dichloromethane and hexane as eluent systems. NMR spectra were recorded on a Varian Unity Inova spectrometer (Milan, Italy), operating at 500 MHz for ¹H and 125 MHz for ¹³C, including also g-COSY, g-HSQC, and g-HMBC analysis conducted at a constant temperature of 300 K in CDCl₃. High-resolution mass spectra were collected using a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) fitted with an electrospray ionization (ESI) source operating in positive mode. A full scan was acquired over an m/z range of 150 to 1000.

3.1. Synthesis of (E)-5-(4-Acetoxy Styryl)-1,3-Phenylene Diacetate (2)

Resveratrol (600.1 mg, 2.63 mmol), sodium bicarbonate (1.33 g, 15.78 mmol), and acetic anhydride (3.98 g, 38.98 mmol) were stirred at room temperature for six hours under constant magnetic agitation. The reaction was monitored by TLC. To stop the reaction, NaHCO₃ was removed by filtration on cotton, and the residue diluted in dichloromethane was partitioned with water. The organic residue taken to dryness was purified by flash chromatography on Silica-DIOL eluted in CH₂ Cl₂, to yield the expected compound 2 with 77.4% yield. R_f = 0.59. Silica-DIOL is a functionalized silica with diol groups that shows less polarity compared to regular silica gel and, being neutral, can be used to purify acid-labile compounds as an ester. Spectroscopic data were in agreement with those previously reported [29].

3.2. Synthesis of (E)-5-(4-Hydroxystyryk)-1,3-Phenylene Diacetate (4)

Compound 2 (720.0 mg; 2.03 mmol) was first dissolved in 35.0 mL of TBME, then 2.7 mL of n-BuOH was added. Finally, 651.2 mg of PsL was added to the reaction mixture and incubated at 37 °C for 3 h. The enzyme was removed from the solution by filtration and washed with ethyl acetate. The filtrate was taken to dry, and the desired product 4 was obtained as a grainy slightly yellow powder without any chromatographic separation (624.0 mg, 98,4% yield). R_f = 0.43. Spectroscopic data were in agreement with those previously reported [29].

3.3. Synthesis of Bis (4-((E)-3,5–Diacetoxystyryl)phenyl)nonanedioate (5)

Compound 4 (300.1 mg; 0.96 mmol) was solubilized in dry CH₂Cl₂ (5.0 mL), and then DMAP (20.0 mg; 0.16 mmol), azelaic acid (151.1 mg; 0.80 mmol), and DCC (248.0 mg; 1.20 mmol) were added. The reaction mixture was stirred at room temperature for four hours and subsequently diluted in dichloromethane and filtered. The residue was partitioned with water. The organic residue, taken to dryness, was purified by flash chromatography on silica eluted with CH₂Cl₂: n-hexane (90:10%) to yield the expected compound 5 with 36.6% yield. R_f = 0.84. ¹HNMR (500 MHz; CDCl₃): δ 7.43 (d, J = 8.5 Hz, 4 H, H2′/H6′), 7.11 (d, J = 2 Hz, 4 H, H2/H6), 7.08 (d, J = 8 Hz, 4 H, H3′/5′), 7.05 (d, J = 16.5 Hz, 2 H, H8), 6.95 (d, J = 16 Hz, 2 H, H7), 6.82 (bt, J = 2 Hz, 2 H, H4), 2.57 (t, J = 7.5 Hz, 4 H, H2′′), 2.29 (s, 12 H, H1′′′/H4′′′), 1.78 (t, J = 7.5 Hz, 4 H, H3′′), 1.45 (t, J = 4 Hz, 6 H, H4′′/H5′′). ¹³CNMR (125 MHz; CDCl₃): δ 172.2 (C, C1′′), 169.0 (C, C2′′′/C3′′′), 151.4 (C, C3/C5), 150.6 (C, C4′), 139.6 (C, C1), 134.4 (C, C1′), 129.7 (CH, C8), 127.7 (CH, C2′/C6′), 127.1 (CH, C7), 121.9 (CH, C3′/C5′), 117.0 (CH, C2/C6), 114.4 (CH, C4), 34.4 (CH2, C2′′), 28.9 (CH2, C4′′),28.9 (CH2, C5′′), 24.8 (CH2, C3′′), 21.2 (CH3, C1′′′/C4′′′). HRMS (-): calcd for C₄₅H₄₅O₁₂ [M + H]⁺: 777.2911, found: 777.2932. Elemental analysis: found: C 68.66; H 6.01; O, 25.33, required: C 69.58; H 5.71; O 24.71.

4. Conclusions

A new molecule formed by the conjugation of acetylated resveratrol and azelaic acid was successfully synthesized under mild conditions, leading to the desired product with an overall 19.9% yield. This method highlights the potential of enzymatic catalysis for the efficient and sustainable synthesis of novel bioactive compounds.