Synthesis and photoprotective properties of new salicylic and vanillic acid derivatives

Article history: Received January 2, 2017 Received in revised form March 1, 2017 Accepted March 21, 2017 Available online March 21, 2017 A simple one-step procedure for synthesis of new derivatives of phenolic acids was developed. As the starting materials salicylic acid, vanillic acid and alkyl haloalkanoates were applied. The reactions were carried out in N,N-dimethylformamide (DMF) in the presence of anhydrous potassium carbonate. Conditions for regioselective synthesis of target compounds were established. The esters 3a-h were obtained in great yields and were characterized by MS, 1H and 13C NMR spectra. Their photoprotective activity was evaluated in vitro by spectrophotometric method. The study revealed that the tested compounds are moderate UVB absorbers, but could be used to augment the effect of other photoprotective agents. Their SPF values were in the range from 3.63 to 4.26 for salicylates 3a-d and from 3.03 to 3.51 for vanillates. © 2017 Growing Science Ltd. All rights reserved.


Introduction
People outdoors are exposed to solar radiation which consists of 56% of infrared light photons, 39% of visible light, and 5% of UV light. 1 Despite the fact that UV radiation is a minority stream of light that reaches the Earth's surface, it can cause the harmful effects on the human body.Excessive exposure to ultraviolet radiation is a major factor causing the skin cancer development. 2,3e solar UV radiation can be subdivided into three ranges: UVA (320-400 nm), UVB (280-320 nm), and UVC (190-280 nm). 2 UVC radiation does not reach the earth's surface because its rays are blocked in the stratosphere by the ozone layer, so it is not of biological relevance. 4Unlike both UVA and UVB rays pass through the atmosphere and are able to cause biological effects. 5e ultraviolet light can induce various changes in the skin, both acute and chronic.Acute effects include vitamin D synthesis and pigmentation, which are positive and desirable by people during relaxation in the sun.6][7] Moreover, acute UV irradiation, even during a single exposure induces damage of DNA. 7Cutaneous malignant melanoma -the most lethal of the skin cancers, is frequently related with sporadic acute burning exposure to sunlight. 2,8Non-melanoma skin cancers are initiated in the majority of cases by long-term chronic sunlight exposure. 2Ultraviolet B causes direct photochemical damage of DNA leading to the creation of mutations.In contrast, ultraviolet A acts indirectly generating of reactive oxygen species. 2,9,10Long-term sunlight exposure is also associated with the chronic effects such as the premature cutaneous aging. 6,9,11e risk of the potential problems connected with the exposure to ultraviolet light can by limited by wearing protective clothes or avoidance of sun exposure, especially during the time when diseaseinducing solar radiation is relatively intense (from 11 am to 4 pm). 2,6Unfortunately, this solution seems to be unacceptable in the global society, so the use of topical sunscreens is the only way to prevent cellular damage in the skin or reduce it to a minimum. 2,6,9otoprotective agents used in sunscreens can be classified into organic (chemical) and inorganic (physical) on the basis of their mechanism of action. 12Organic agents absorb UV radiation with excitation to a higher energy state and returning to the stable state release insignificant amount of heat or emit fluorescent radiation. 2,12,133][14] Inorganic agents, primarily titanium dioxide and zinc oxide, reflect and scatter UV radiation increasing the optical pathway of the photons in the sunscreen formulation leading to higher efficiency of the organic photoprotective agents. 2,12,13In order to provide a high level of sun protection against a wide range of the UV spectrum multiple compounds are usually incorporated into sunscreening cosmetics -no single photoprotective agent can provide a high sun protection factor 6,13 at the level required by consumers and regulatory agencies, e.g.FDA in the USA or the Commission of the European Communities in the EU.For this reason the list of new photoprotective substances is developed and different combinations of sunscreens have become the subject of producers' interests. 6,13aring the above in mind, the aim of this work was the synthesis of phenolic acids derivatives as the new photoprotective compounds.According to literature, sunscreen chemicals are generally aromatic compounds containing an electron-acceptor group conjugated with an electron-releasing group in ortho or para position. 15,16Our research work describes the synthesis of salicylates with free hydroxyl group in position ortho and vanillates with hydroxyl group in position para.

Synthesis of salicylic and vanillic acid esters (3a-h)
The new salicylic and vanillic acid derivatives (3a-h) were obtained by reaction of particular acids with alkyl haloalkanoates in alkaline conditions (Scheme 1).Phenolic acids have two functional groups -carboxyl and hydroxyl, so different products could be formed.The initial aim was to establish parameters favourable to obtain esters with free hydroxyl group.For this purpose vanillic acid (1b) was treated with methyl 2-bromobutanoate (2b) at equimolar ratio (Scheme 2).The reactions were carried out in N,N-dimethylformamide (DMF) -an aprotic polar solvent in the presence of anhydrous potassium carbonate at 93-96 °C.The advantageous conditions of synthesis were determined by a single-factor experimental method.The reaction time and 1b:K2CO3 molar ratio were changed.The selectivity of reaction and yields of target compound 3f are presented in Table 1.Influence of potassium carbonate on both selectivity and yield was observed.The molar ratio of the vanillic acid to potassium carbonate of 1.0:1.0 in the entries 1-3 led to formation of 1-methoxy-1oxobut-2-yl vanillate (3f) in yield of 67-69%.Under these conditions compound 4f was also formed as a by-product.Reduction of the amount of potassium carbonate in the entries 4-6 resulted in an increase selectivity of the process.At the molar ratio of reactants of 1:0.5 (1b:K2CO3) the pure product 3f was obtained.It was observed that the reaction time is an important factor that affects the final product yield.Conducting process for 30 min the yield of 74% was achieved.The rise of process efficiency to 97% was observed when longer reaction time was applied.Comparing the results from trials 5 and 6 it can be stated that reaction time 1 hour should be preferred from an economic point of view.
The established conditions were applied to synthesis of esters 3a-h.The target products were obtained in excellent yields (89-97%).Salicylates 3b-d and vanillates 3e-h are new compounds.Their structures were confirmed by 1 H NMR, 13 C NMR and MS spectra.Compound 3a was already described in the literature and was obtained by the insertion reaction of ethyl diazoacetate to salicylic acid in fluorinated alcohols. 17ccording to the literature, [18][19][20][21][22] synthesis of phenolic acid esters usually includes three steps: protection of hydroxyl group, esterification and removal of the protecting group.As the blocking agents benzyl halides or acetic anhydride are often applied to protect hydroxyl group. 22They are removed after the esterification process by hydrogenolysis or hydrolysis, respectively.Unfortunately, application of protecting groups is inconvenient in industrial production.Multiple-step syntheses raise the costs of the production and are unfriendly for environment due to the need for additional materials and energy.In contrast, the proposed method is one-stage and occurs in good yields.

Photoprotective properties
The obtained derivatives of salicylic and vanillic acids were screened for photoprotective activity.This property is specified by the index called as the sun protection factor (SPF).The sun protection factor is expressed as the ratio of the minimum dose of UV radiation causes erythema on skin protected by a sunscreen product to the minimum dose of UV radiation causes erythema on the same unprotected skin.This parameter could be also tested in vitro.In our work spectrophotometric method was applied.For this purpose the obtained compounds were dissolved in ethyl alcohol and UV spectra in the range from 210 to 350 nm were recorded.Exemplary spectra of compounds 3a and 3e are presented in Fig. 1.
The tested substances absorb energy in the range of UVB and UVC.The salicylic acid derivatives (3a-d) spectra showed the maxima of absorption at 238 and 308 nm wavelength.The band at 308 nm is characteristic for aromatic carboxylic acids substituted with hydroxyl group in ortho position.The hydroxyl group in position para in compounds 3e-h resulted in the presence of absorption band at 263 nm.Moreover, vanillic acid contains methoxy group, so in the spectra of 3e-h the band at 293 nm was observed.
The photoprotective properties of the obtained compounds against UVB radiation were determined by the method developed by Mansur et al. 23 using homomenthyl salicylate (homosalate) as a standard.This method assumes that a sunscreen formulation containing 8% homosalate presents a SPF value of 4. The obtained values for compounds 3a-h are presented in Fig. 2. The photoprotective properties of salicylates 3a-d were similar to homosalate.The SPF values for these compounds were in the range from 3.63 to 4.26.It was observed that the length of alkyl chain (R4) have influence on the photoprotective properties of salicylates 3a-d -SPF values decreased with the increase of alkyl chain length.In contrast, for vanillates (3e-h) this relationship was reversed.Furthermore, this group of esters were less effective against UVB radiation -their SPF values were in the range from 3.03 to 3.51.However these compounds can be also used as the ingredients of photoprotective agents mixture in the sunscreen formulations.The advantage of the obtained vanillic acid esters is their flavor -mild and pleasant with spicy, floral and honey characteristics.

Conclusions
We have reported an efficient method for the preparation of salicylic and vanillic acid derivatives.Conditions for selective synthesis of 1-alkoxy-1-oxoalkan-2-yl salicylates (3a-d) and 1-alkoxy-1- oxoalkan-2-yl vanillates (3e-h) in the one-step procedure were established.As the most favourable the time of reaction of 1 hour and the molar ratio of phenolic acid to potassium carbonate 1.0:0.5 were recognized.The target products were obtained in excellent yields (89-97%).
The photoprotective properties of the obtained compounds 3a-h were tested in vitro using spectrophotometric method.Compound 3a was described in the literature 17 but it was never considered as a photoprotective agent.The SPF values of salicylates 3a-d were in the range from 3.63 to 4.26.Vanillates 3e-h were less effective against UVB radiation -their SPF values were in the range from 3.03 to 3.51.Both salicylates and vanillates are moderate UVB absorbers, but they could be used to augment the effect of other UVB absorbers.
For determination of sun protection factor (SPF) homomenthyl salicylate (homosalate) -certified reference material purchased from Fluka and ethanol for spectroscopy were used.
The GC-MS analysis of the obtained products diluted with acetone was carried out using an Agilent 6890 gas chromatograph equipped with an Agilent 5973 Network Mass Selective Detector (MSD).The separation was effected using a HP-5MSI capillary column with bonded (5% phenyl)methylpolysiloxane stationary phase (30 m  0.25 mm I.D., 0.25 μm film thickness).The GC oven temperature was programmed: initial temperature 60 °C (hold for 3 min); ramp rate 10 °C/min; final temperature 300 °C (hold for 10 min).Helium was used as a carrier gas at a constant flow rate of 1.2 mL/min.The mass selective detector was working in electron impact mode (70 eV).The mass spectra were scanned in the range from 50 to 500 m/z. 1 H NMR and 13 C NMR spectra were recorded in CDCl3 with TMS as an internal standard on a TM Bruker DPX 400 spectrometer (400 MHz).
Melting points were determined using a Boetius apparatus and are uncorrected.
UV spectra of the obtained compounds were recorded on a GBC UV/VIS 916 double beam spectrophotometer using 1 cm quartz cuvettes, and ethanol as a blank.

Determination of sun protection factor (SPF)
The photoprotective properties of the prepared compounds was tested in vitro based on spectrophotometric analysis of dilute solutions. 23For this purpose the solutions of compounds 3a-h and homomenthyl salicylate (homosalate) in ethanol were prepared.The concentration of these solutions was 16 µg/mL.The absorbance of samples in solution form was measured in wavelength range of 290 to 320nm, every 5 nm wavelength interval.The following equation was applied to calculate the SPF: where: CF -correction factor, EEλ -erythemogenic effect of radiation at wavelength λ, Iλ -intensity of solar light at wavelength λ, and Aλ -absorbance of the solution at wavelength λ.
The values of EEλ × Iλ are constants determined by Sayre et.al and known from the literature. 26,27hey are presented in Table 2. Correction factor was determined experimentally taking into account the value of SPF for homomenthyl salicylate is equal to 4. 23

Fig. 1 .
Fig. 1.The exemplary UV spectra of the obtained esters 3a and 3e

Fig. 2 .
Fig. 2. The SPF values determined for compounds 3a-h (the mean values of three determinations)