Synthesis and structural elucidation of 2,3-dimethylnaphthazarin ester derivatives

A simple and efficient synthesis of bis-ester substituted 2,3-dimethylnaphthazarins is described. The preparation of 5-O ,8-O -bis-ester derivatives of 2,3-dimethylnaphthazarin was achieved via double esterification reaction of 5,8-dihydroxy-2,3-dimethylnaphthazarin with a range of acyl chlorides under mild reaction conditions. Additionally, 5-O - mono ester and unsymmetrical 5-O ,8-O -bis-ester derivatives of 2,3-dimethylnaphthazarins were successfully synthesized. The identities of new compounds were confirmed by 1 H NMR, 13 C NMR, FTIR, HRMS and X-ray single-crystal diffraction techniques


Introduction
][6] Well known naphthazarin-based compounds, such as Shikonin and its enantiomer Alkannin (Figure 1) have been isolated from in the roots of Lithospermum erythrorhizon and Arnebia euchroma. 5,7Among the naphthazarin derivatives, 2,3-dimethylnaphthazarin (DMN) is of significant synthetic interest due to its biological activity. 8In particular, bis-benzoyl ester dimethylnapthazarin derivatives have been reported to possess borderline inhibitory activity against leukemia P388 cells. 8Apart from the pharmaceutical importance, DMN-based compounds have been employed as charge-transfer complexes. 9Consequently, naphthazarin-containing analogues have been recognized as being of value and their synthesis has thus been targeted by organic chemists.Limited synthetic approaches have been reported for the synthesis of DMN and its derivatives.One of the oldest methods was reported by Rodriguez in 1984 which involved a multi-step procedure including an initial cycloaddition between 2,3-dimethyl-1,3-butadiene and p-benzoquinone.This step was followed by acetylation and chromium trioxide oxidation of the resulting adducts and subsequent hydrolysis of the resultant naphthazarin diacetate. 9In a similar manner, DMN has also been prepared from 1,3-butadiene and 2,3dimethylquinone. 9The most efficient, and more recent synthesis of DMN has been achieved in a one-pot process by the direct reaction of 2,3-dimethylmaleic anhydride and 1,4-dimethoxybenzene, in the presence of aluminum chloride and sodium chloride. 10The main advantage of this latter method is that the DMN could be synthesized in relatively high yield from inexpensive commercial chemicals.
In conjunction with the synthetic efforts focused on naphthazarin, the presence of the hydroxyl groups, located at the C5 and C8 positions on the naphthazarin aromatic ring, should allow the synthesis of a range of targeted mono-and bis-ester compounds.2] However, (selective) derivatization of the parent DMN has rarely been studied in the literature, 10 presumably due to the harsh reaction conditions and multi-step processes involved in the previously reported synthetic routes.The current study presents the development of a general method for the synthesis of bis-ester 2,3-dimethylnaphthazarin derivatives from reaction of DMN with acyl chlorides under mild reaction conditions.The reaction is facilitated by the phenoxy groups located at the C5 and C8 positions and yields both mono-ester and unsymmetrical bis-ester derivatives of the parent 2,3dimethylnaphthazarin.

Results and Discussion
In earlier studies, the synthesis of bis-acetyl and bis-benzoyl ester derivatives of 2,3-dimethylnapthazarin 5 and 12 were prepared via multi-step reactions, requiring harsh reaction conditions and long reaction times.For example, Cheng and coworkers reported the synthesis of bis-acetyl-2,3-dimethylnaphthazarin 5 in three steps starting from building the naphthoquinone platform via a Diels-Alder reaction of 2,3-dimethyl-1,3-butadiene and p-benzoquinone, followed by acetylation and oxidation with chromium trioxide. 8The yield of the reaction was mentioned as extremely low and no characterization data was reported for the bis-acetate product 5.Using the same synthetic route, the group also reported the synthesis of bis-benzoyl derivative 12 in three steps using benzoyl chloride as an acylation reagent.The overall yield for this reaction sequence was likewise very low, as the yield from the final oxidation was only 14%.In addition, the total reaction time was reported to be 32 hours.The detailed characterization data for derivative 12 was also not reported.In 1986, Rodriguez and coworkers reported the synthesis of the bis-acetyl ester of 2,3-dimethylnaphthazarin 5 following the same reaction sequence. 13After building the naphthoquinone structure using a Diels-Alder reaction, treatment of the resulting adduct with acetic anhydride in acetic acid under reflux for 3 hours and subsequent oxidation with chromium trioxide generated product 5.The final oxidation step provided bis-acetyl-2,3-dimethylnaphthazarin 5 in 35% yield and 1 H NMR characterization data of product 5 was provided.To best of our knowledge, there is no other study in the literature for the synthesis of symmetrical bis-ester derivatives of the parent 2,3dimethylnapthazarin.In addition to this, the preparation of mono-ester or unsymmetrical bis-ester derivatives of DMN have not been reported thus far.
In order to bridge this gap, DMN 3 was synthesized by following a literature procedure 10 involving a double Friedel-Craft acylation reaction between 1,4-dimethoxybenzene 1 and 2,3-dimethylmaleic anhydride 2 in the presence of molten form of aluminum chloride-sodium chloride, providing DMN 3 in an isolated yield of 37% (Scheme 1).

Scheme 1. Synthesis of 2,3-dimethylnaphthazarin.
With DMN 3 in hand, reaction conditions for its double esterification were investigated using acetyl chloride 4 as an acylation reagent.Initially, one equivalent of naphthazarin 3 was treated with three equivalents of base at room temperature, followed by four equivalents of acetyl chloride and the reaction progress was monitored by TLC analysis.When potassium carbonate or pyridine was used as the base, only a trace amount of the target product 5 was isolated.When the base was changed to triethylamine, the reaction gave the targeted bis-ester 5 in 28% yield along with its mono-ester, 5-O-acetyl-2,3-dimethylnaphthazarin 6, in 4% yield.Following optimization, it was found that the treatment of naphthazarin 3 with three equivalents of triethylamine at room temperature for 30 minutes, followed by the addition of acetyl chloride 4 at 0 o C and subsequent warming to room temperature provided the highest yield of the target product 5 in 45% yield, along with the 5-O-acetyl-mono-ester 6 in 3% yield (Scheme 2).The structures of products 5 and 6 were elucidated by a detailed one-and two-dimensional NMR spectroscopy investigation, as well as being fully characterized using FTIR, HRMS and finally confirmed by single crystal X-ray analysis (Figure 2).Scheme 2. Optimized reaction conditions for the double esterification of 2,3-dimethylnaphthazarin 3.
The 1 H NMR spectrum of the bis-ester 5 at room temperature in CDCl3 is consistent with that reported in the literature. 13The most important evidence for successful double esterification of 3 is the disappearance of both of the phenolic OH signals which initially appear as a singlet at  12.63 ppm due to intramolecular hydrogen-bonding interaction between the carbonyl oxygen of quinonoid ring and the proton of phenolic hydroxyl group proton in the aromatic ring of 3. The appearance of a new singlet signal at  2.44 ppm in the proton NMR spectrum of 5 was associated with the protons of the two acetyl ester CH3 group.In the 13 C NMR spectrum of the bis-product 5, the appearance of new signals in the high field region was associated with acetyl methyl CH3 carbons ( 21.2 ppm), while additional signals in the low field region were associated with the acetyl ester carbonyl C=O carbons ( 169.5 ppm), proving that the double esterification of 3 has taken place.Highresolution mass spectrometry indicated that the molecular ion of product 5 has a mass to charge ratio of 325.0676, consistent with the molecular formula C16H14O6 [M+Na] + .The structure of bis-ester 5 was further confirmed by single crystal X-ray crystallography analysis (Figure 2).
The new minor mono-ester 6 was also fully characterized.HRMS showed that the molecular ion of product 6 has a mass to charge ratio of 260.0686, consistent with the molecular formula C14H12O5 [M] + .Finally, an analytical sample of mono-ester 6 was successfully recrystallized and its structure was also confirmed by single crystal X-ray (Figure 2).The structure of mono-ester 6 was elucidated with further NMR experiments, namely APT NMR, 2dimensional 1 H -1 H (COSY), and 1 H-13 C (HMQC, HMBC) correlation experiments in CDCl3 at room temperature.
In the 1 H NMR spectrum of 6, only one phenolic OH signal was observed at  12.62 ppm as a singlet.This signal proved that the single esterification took place.As mono-ester 6 is not symmetrical, unlike the starting material DMN 3 and bis-ester 5, both of the methyl group CH3 protons on the quinonoid ring give different proton resonances as singlets at  2.15 and 2.12 ppm.The 13 C NMR spectrum of mono-ester 6 showed the methyl CH3 carbons on quinonoid ring at different chemical shifts of  13.2 and 12.3 ppm.The signals appearing at  21.2 ppm and 169.8 ppm associated with the acetyl ester methyl carbon CH3 and carbonyl carbon C=O, respectively.It is worth to note that while the carbonyl carbon signal (C1) on the quinonoid ring shifted to downfield region and appears at  189.9 ppm, the other carbonyl carbon signal (C4) resonates at  182.9 ppm, which is further upfield when compared to the starting material 3 ( 186.5 ppm) (Table 1).It shows that the carbonyl oxygen C=O at C1 is intramolecularly hydrogen bonded with the phenolic OH proton of the aromatic ring, whereas the C4 carbonyl oxygen is not.Additionally, carbon atoms attached to the phenolic hydroxyl group showed significant chemical shift differences.For example, the phenoxy group attached to carbon C8 of mono-ester 6 resonated at  159.9 ppm, similar to DMN 3 (Table 1).However, once the phenolic hydroxyl group was converted into the corresponding ester derivative 5, the carbon atom (C5) signal at the aromatic ring appeared in the far upfield region at  142.6 ppm, as compared to DMN 3 ( 158.6) (Table 1).Another important chemical shift difference was observed at the fused carbon atoms such as C10, which is next to the acetyl substituted carbon (C5) on the aromatic ring, which resonated downfield at  122.1 ppm, whereas carbon C9, neighboring to phenolic hydroxyl substituted carbon (C8), resonated at  114.8 ppm.On the contrary, the fused carbon signals of bis-ester 5 appeared in the far downfield region at  124.7 ppm (Table 1).
In order to extend this study to generate a variety of new 2,3-dimethylnaphthazarin bis-ester derivatives, various commercially available aromatic acyl , such as benzoyl, p-methyl-, p-bromo-, p-methoxy-, and pnitrobenzoyl chlorides (7-11), were also used (Table 2).The esterification reaction was performed using the optimum reaction conditions as described earlier.The reaction progress was monitored by TLC analysis and the reaction was worked up once no further change was observed on the TLC.It was found that the reaction with p-bromobenzoyl chloride 9 was completed in 1.5 hours, whereas the reaction with benzoyl chloride 7 took 22 hours; both of them gave bis-ester 14 and 12 in 30% and 23% yield, respectively, as sole products (Table 2).The reaction with p-methylbenzoyl chloride 8 resulted in bis-adduct 13 as major product in 30% yield, along with mono-ester 17 in 5% yield, while the reaction with p-nitrobenzoyl chloride 11 gave bis-ester 16 and mono-ester 19 products, 21% and 27%, respectively.In contrast, the reaction between 2,3-dimethylnapthazarin 3 and pmethoxybenzoyl chloride 10 resulted in mono-ester 18 in 73% yield as a major product along with bis-ester 15 in 13% yield as the minor product.Following the successful synthesis of symmetrical bis-ester derivatives of DMN (5,12-16), the synthesis of an unsymmetrical bis-ester of DMN was examined.By using similar reaction conditions, that is treating one equivalent of DMN 3 with one equivalent of acetyl chloride 4 in THF, 5-O-acetyl-mono-ester 6 was generated in 36% yield, bis-ester 5 in 29% yield, while 3% of unreacted DMN 3 was recovered (Scheme 3).One equivalent of mono-ester 6 was then treated with one and a half equivalents of benzoyl chloride 7 at room temperature, in the presence of one and a half equivalents of triethylamine and THF, to result in the unsymmetrical bis-ester 20 in 17% yield (Scheme 3).This product 20 was fully characterized using spectroscopic methods and its structure was also confirmed by single crystal x-ray crystallography.

Conclusions
In summary, a variety of new ester derivatives of 2,3-dimethylnaphthazarin was achieved under mild reaction conditions in one step.These new dimethylnaphthazarin structures have considerable potential for development in areas including medicinal chemistry.Additionally, mono-ester derivatives create an opportunity to generate a library of unsymmetrical bis-substituted naphthazarins, and this work is currently in progress.

Experimental Section
General Information.Reactions were performed under a positive pressure of nitrogen or argon in oven or flame dried glassware, unless otherwise specified.All reagents and solvents were obtained from commercial sources and appropriately purified, if necessary.Anhydrous THF was dried over sodium wire and distilled from sodium benzophenone ketyl. 1 H and 13 C NMR spectra were recorded at 298 K on a Varian 500 MHz NMR spectrometer.The deuterated solvent (deutero chloroform (CDCl3) for NMR spectroscopy was obtained from Merck.The central line of the residual chloroform (CHCl3 in CDCl3) (δ = 7.26 ppm) and triplet (δ = 77.10ppm) was used for 1 H NMR and 13 C NMR spectra as an internal reference, respectively.The following abbreviations and combinations thereof are used to describe 1 H NMR spectra multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br.= broad.Assignment of proton signals was assisted by COSY experiment where necessary.Assignment of carbon signals was assisted by APT, HMBC, and HSQC experiment where necessary.IR spectra were recorded on between 4000 and 650 cm -1 using a Perkin Elmer Spectrum 100 FT-IR spectrometer with an attenuated total reflection (ATR) accessory.Mass spectra were recorded on a Thermo ORBITRAP Q-EXACTIVE mass spectrometer (Bremen, Germany) equipped with a Troyasil (Istanbul Turkiye) C18 column (150 x 3 mm i.e., 3 μm particle size) for measurements of HRMS.Melting points were measured using a Stuart SMP10 melting point apparatus.Analytical TLC was performed with Merck silica gel plates, precoated with silica gel 60 F254 (0.2 mm) on aluminium sheets and visualized using UV fluorescence (λmax = 254 nm).Flash column chromatography employed Merck Kieselgel 60 (230-400 mesh) silica gel.