Design and Synthesis of Novel Isoxazole Tethered Quinone-Amino Acid Hybrids

A new series of isoxazole tethered quinone-amino acid hybrids has been designed and synthesized involving 1,3-dipolar cycloaddition reaction followed by an oxidation reaction using cerium ammonium nitrate (CAN). Using this method, for the first time various isoxazole tethered quinone-phenyl alanine and quinone-alanine hybrids were synthesized from simple commercially available 4-bromobenzyl bromide, propargyl bromide, and 2,5-dimethoxybenzaldehyde in good yield.


Introduction
Compounds containing the quinone group present an important class of biologically active molecules that are widespread in nature [1][2][3]. The discoveries of antibiotic [4,5] and antitumor [6] properties assigned to several natural quinones have raised interest among scientists for use as pharmaceuticals. While antibiotics display an enormous diversity in chemical structures, quinone antibiotics such as Adriamycin, Mitomycin C, and Streptonigrin deserve special attention [7][8][9][10]. In this context, search of new molecules containing quinone moiety has always fascinated the organic as well as medicinal chemist.
Isoxazole derivatives are an important class of heterocyclic pharmaceuticals and bioactive natural products because of their significant and wide spectrum of biological activities, including potent and selective antagonism of the NMDA receptor and anti-HIV activity . [11,12]. It shows antihyperglycemic [13], analgesic [14], anti-inflammatory [15], antifungal [16], and antibacterial activity [17]. 3,5-Disubstituted isoxazole derivatives which are biological active include muscimol, dihydromuscimol, micafungin, and cycloserine [18,19]. Unnatural amino acids, the nonproteinogenicamino acids that occur either naturally or chemically synthesized, have been used widely as chiral building block. They have been also used as molecular scaffolds in constructing combinatorial libraries [20]. They represent a powerful tool in drug discovery when incorporated into therapeutic peptidomimetics and peptide analogs [21]. The seminal work on the synthesis of unnatural amino acids has been done by O'Donnell and Maruoka independently, which accelerated the application of this class of amino acid for practical applications [22,23].
Synthesis of hybrid natural products has gained momentum in recent years [24][25][26]. It is expected that combining features of more than one biologically active natural segment in a single molecule may result in pronounced pharmacological activity while retaining high diversity and biological relevance. There are a few reports describing the preparation of quinone-hybrid with other natural products. For example, quinone-amino acids [27], sugar-oxasteroidquinone [28], quinone-annonaceous acetogenins [29], and conduritol-carba-sugar [30] hybrids have been described using different synthetic protocol. In our continuation endeavour to prepare novel hybrid molecules containing variety of natural products [31], we developed interest in the synthesis of novel isoxazole tethered quinone-amino acid hybrid natural products, and herein we report our initial results. Depending on the hybrid pattern, hybrid molecules containing amino acids and either quinone or isoxazole were prepared by various groups using different methods (Figure 1). For example, AMPA ( -amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) is a type of glutamatergic ion channels in the central nervous system which can be considered as isoxazole-amino acid hybrid. A series of novel AMPA analogues were prepared in order to evaluate it as drug candidates for neurological disorder [32]. Abenquine D is an amino acid quinone hybrid which is composed of an amino acid linked to an N-acetyl-amino benzoquinone. Abenquines A-D are new bioactive secondary metabolites found in the fermentation broth of Streptomyces sp. stain DB634 which was isolated from the soils of Chilean highland of Atacama desert. The abenquines show inhibitory activity against bacteria, dermatophytic fungi, and phosphodiesterase type 4b [33]. It is noteworthy to mention here that amino acid attached to the quinone is relevant to the enzyme inhibitory activity. Similarly, IRL 3461 is a potent and bifunctional ET A /ET B endothelin antagonist. IRL 346a is an isoxazoleamino acid hybrid prepared from 4-methyl-acetophenone in nine steps synthetic protocol [34]. Katritzky et al. have prepared naphthoquinone-amino acid conjugates starting from naphthoquinone and L-amino acids by a Michael type mechanism in aqueous ethanol solution at RT in the presence of triethylamine [35]. Kotha group has used a "building block approach" to synthesize the quinone-amino acid hybrids through ethylene cross-enyne metathesis and Diels-Alder reaction as the key step [27]. But there are no reports of isoxazole tethered quinone amino acid hybrids as per the literature search. To the best of our knowledge, this is the first report on the synthesis of new series of isoxazole tethered quinone-amino acid hybrid natural products.
In view of the importance of these three classes of natural products, we have designed a new class of hybrid structures 1 or 2 ( Figure 2) in an effort to combine the activity of amino acid moiety and the quinone unit using isoxazole ring as linker. These hybrids may have significant biological activity and so an efficient strategy to these hybrid molecules would allow us to construct diverse hybrid analogues.

Materials and Methods
All reactions were carried out in oven-dried glassware with magnetic stirrers under an argon atmosphere. THF was dried over Na/benzophenone and DCM was dried over CaH 2 . Commercially available chemicals were purchased from Sigma-Aldrich and Alfa Aesar. EtOAc and pet ether were distilled before use. All melting points were taken in open capillaries and are uncorrected. Analytical thin-layer chromatography (TLC) was performed on commercially available Merck TLC Silica gel 60 F 254 . Silica gel column chromatography was performed on silica gel 60 (spherical 100-200 m). FTIR spectra were recorded on Perkin-Elmer FT/IR-4000 spectrophotometer and only the characteristic peaks are reported. Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer. 1 H NMR spectra were recorded on Varian-400 (400 MHz) spectrometer. Chemical shifts of 1 H NMR spectra were reported relative to tetramethylsilane.

Experimental Procedure for the Preparation of Methyl 2-
To a solution of compound 3b (3.5 g, 10.26 mmol) in triethylamine (20 mL), PdCl 2 (PPh 3 ) 2 (0.359 g, 0.51 mmol), CuI (0.048 g, 0.25 mmol), and trimethylsilylacetylene (1.20 g, 12.31 mmol) were added under argon atmosphere and heated at 90 ∘ C in a sealed tube for 12 h. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). After completion of the reaction, the reaction mixture was filtered. The filtrate was evaporated to give the crude reaction mixture which was charged on silica gel column. The column was eluted with 20% ethyl acetate/pet ether to give the compound 4b (

Experimental Procedure for the Preparation of Methyl 2-((tert-Butoxycarbonyl)amino)-3-(4-ethynylphenyl)propanoate (5a).
To a solution of compound 4a (0.800 g, 2.13 mmol) in THF (10 mL), 1 M TBAF in THF (4.26 mL, 4.26 mmol) was added at −70 ∘ C and stirred for 2 h. The progress of the reaction was monitored by TLC analysis (20% ethyl acetate/pet ether). After the reaction was complete, the reaction mixture was quenched with water (10 mL) and extracted with ethyl acetate thrice. The organic layers were combined and washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent in high vacuum gave the compound 5a (0.650 g, 95% yield) as light yellow solid.

Experimental Procedure for the Preparation of Methyl 3-(4-Ethynylphenyl)-2-pivalamidopropanoate (5b).
To a solution of compound 4b (1.0 g, 3.84 mmol) in THF (20 mL), 1 M TBAF in THF (3.8 mL, 7.66 mmol) was added at −70 ∘ C and stirred for 2 h. The progress of the reaction was monitored by TLC analysis (20% ethyl acetate/pet ether). After the reaction was complete, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate thrice. The organic layers were combined, washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent in high vacuum gave the compound 5b (0.650 g, 82% yield) as offwhite solid.

Experimental
Procedure for the Preparation of 2,5-Dimethoxybenzaldehyde Oxime (7a). To a solution of compound 6a (1 g, 6.02 mmol) in MeOH (10 mL), NaOAc (0.98 g, 12.04 mmol) and NH 2 OH⋅HCl (0.62 g, 9.03 mmol) were added under nitrogen atmosphere. Then the reaction mixture was stirred at RT for 2 h. The progress of the reaction was monitored by TLC analysis (20% ethyl acetate/pet ether).
After completion of the reaction, the solvent was evaporated, quenched with water (20 mL), and extracted with ethyl acetate thrice. The organic layers were combined, washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent in high vacuum gave the compound 7a

Experimental Procedure for the Preparation of Methyl 3-(4-(3-(2,5-Dimethoxy-4-methylphenyl)isoxazol-5-yl)phenyl)-2-pivalamidopropanoate (8d).
To a solution of compound 7b (0.15 g, 0.76 mmol) in dichloromethane (10 mL), compound 5b (0.24 g, 0.84 mmol), triethylamine (0.116 g, 1.15 mmol), and NaOCl (9-12% in water, 10 mL) were added at 0 ∘ C under nitrogen atmosphere. Then the reaction mixture was stirred Journal of Amino Acids 5 at RT for 12 h. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). After completion of the reaction, water (10 mL) was added and the reaction mixture was extracted with dichloromethane thrice. The organic layers were combined, washed with water, brined, and dried over anhydrous Na 2 SO 4 . The solvent was evaporated to give the crude reaction which was charged on silica gel column. Elution of the column with 25% ethyl acetate/pet ether gave the compound 8d (0.31 g, 83% yield) as light yellow liquid. IR

Experimental Procedure for the Preparation of Methyl 2-
To a solution of compound 8a (0.15 g, 0.31 mmol) in acetonitrile (6 mL) and H 2 O (1 mL), CAN (0.511 g, 0.93 mmol) was added and the reaction mixture was stirred at RT for 1 h. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). After completion of the reaction, water (10 mL) was added and extracted with ethyl acetate thrice. The organic layers were combined, washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent in high vacuum gave the compound 9a (0.12 g, 85% yield) as yellow solid.

Experimental Procedure for the Preparation of Methyl
To a solution of compound 8b (0.2 g, 0.42 mmol) in acetonitrile (8 mL) and H 2 O (2 mL), CAN (0.705 g, 1.28 mmol) was added and the reaction mixture was stirred at RT for 1 h. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). After completion of the reaction, water (20 mL) was added and extracted with ethyl acetate thrice. The organic layers were combined, washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent in high vacuum gave the compound 9b (0.136 g, To a solution of compound 8c (0.23 g, 0.46 mmol) in acetonitrile (10 mL) and H 2 O (2 mL), CAN (0.76 g, 1.39 mmol) was added and the reaction mixture was stirred at RT for 1 h. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). After completion of the reaction, water (20 mL) was added and extracted with ethyl acetate thrice. The organic layers were combined, washed with water, brined, and dried over anhydrous

Experimental Procedure for the Preparation of 5-(Bromomethyl)-3-(2,5-dimethoxyphenyl)isoxazole (16a).
To a solution of compound 15a (1.5 g, 6.38 mmol) in dichloromethane (15 mL), phosphorous tribromide (2.59 g, 9.57 mmol) was added at 0 ∘ C under nitrogen atmosphere. Then the reaction mixture was stirred at RT for 16 hr. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). Then, water (10 mL) was added and the reaction mixture was extracted with dichloromethane. The combined organic layer was washed with water, brined, and dried over anhydrous Na 2 SO 4 . The solvent was evaporated and the crude reaction mixture was purified by column chromatography to give the compound 16a (1.

Experimental Procedure for the Preparation of 5-(Bromomethyl)-3-(2,5-dimethoxy-4-methylphenyl)isoxazole (16b).
To a solution of compound 15b (1.0 g, 4.0 mmol) in dichloromethane (20 mL), phosphorous tribromide (1.62 g, 6.0 mmol) was added at 0 ∘ C under nitrogen atmosphere. Then the reaction mixture was stirred at RT for 16 hr. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). Then, water (20 mL) was added and the reaction mixture was extracted with dichloromethane. The combined organic layer was washed with water, brined, and dried over anhydrous Na 2 SO 4 . The solvent was evaporated and the crude reaction mixture was purified by column chromatography to give the compound 16b

Experimental Procedure for the Preparation of Methyl 2-Amino-3-(3-(2,5-dimethoxy-4-methylphenyl)isoxazol-5-yl) propanoate (18b).
To a solution of compound 17b (0.5 g, 1.03 mmol) in diethyl ether (10 mL), 1 M HCl (10 mL) was added at 0 ∘ C. Then the reaction mixture was stirred at RT for 16 hr. The progress of the reaction was monitored by TLC analysis (10% methanol/chloroform). The layers were separated and the aqueous layer was basified with aqueous ammonia until PH 10 and extracted with ethyl acetate. The combined organic layer was washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent gave the compound 18b

Experimental Procedure for the Preparation of Methyl 2-((tert-Butoxycarbonyl)amino)-3-(3-(2,5-dimethoxyphenyl) isoxazol-5-yl)propanoate (19a).
To a solution of compound 18a (0.300 g, 0.98 mmol) in dichloromethane (15 mL), triethylamine (0.19 g, 1.98 mmol) was added. Then (Boc) 2 O (0.23 g, 1.07 mmol) was added and the reaction mixture was stirred at RT for 16 hr. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). Then, water (10 mL) was added and the reaction mixture was extracted with dichloromethane. The combined organic layer was washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent gave the crude compound which was purified by column chromatography to give the compound 19a (0.380 g, 95% yield) as light brown solid.

Experimental Procedure for the Preparation of Methyl 3-(3-(2,5-Dimethoxyphenyl)isoxazol-5-yl)-2-pivalamidopropanoate (19b).
To a solution of compound 18a (0.300 g, 0.98 mmol) in DCM (15 mL), dimethylaminopyridine (0.012 g, 0.098 mmol) was added. Then pivaloyl chloride (0.356 g, 2.94 mmol) was added and the reaction mixture was stirred at RT for 16 hr. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). Then, water (10 mL) was added and the reaction mixture was extracted with dichloromethane. The combined organic layer was washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent gave the crude compound which was purified by column chromatography to give the compound 19b (0.300 g, 78% yield) as light yellow liquid. IR

Experimental Procedure for the Preparation of Methyl 3-(3-(2,5-Dimethoxy-4-methylphenyl)isoxazol-5-yl)-2-pivalamidopropanoate (19d).
To a solution of compound 18b (0.3 g, 0.93 mmol) in dichloromethane (10 mL), dimethylaminopyridine (0.011 g, 0.093 mmol) was added. Then pivaloyl chloride (0.34 g, 2.81 mmol) was added and the reaction mixture was stirred at RT for 16 hr. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). Then, water (10 mL) was added and the reaction mixture was extracted with dichloromethane. The combined organic layer was washed with water, brined, and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent gave the crude compound which was purified by column

Experimental Procedure for the Preparation of Methyl 2-((tert-Butoxycarbonyl)amino)-3-(3-(4-methyl-3,6-dioxocy
To a solution of compound 19c (0.2 g, 0.47 mmol) in acetonitrile (10 mL) and H 2 O (2 mL), CAN (0.52 g, 0.95 mmol) was added and the reaction mixture was stirred at RT for 1 h. The progress of the reaction was monitored by TLC analysis (30% ethyl acetate/pet ether). Then, water (10 mL) was added and extracted with ethyl acetate. The combined organic layer was washed with water, brined, and dried over anhydrous

Results and Discussion
Our proposed strategy was based on a simple and lucid cycloaddition reaction [42][43][44][45] of alkyne 5 with oxime 7 to prepare isoxazole-amino acid hybrids 8 (Scheme 1). The alkyne 5 was prepared in five steps using the protocol developed by O'Donnell et al. starting from N-(diphenylmethylene) glycine methyl ester [46,47] using appropriate benzyl or propargyl halides. Subsequent oxidation of the suitable placed methoxy group in the aromatic ring of compound 8 will provide isoxazole tethered quinoneamino acid hybrids 9.
To start with, the compound 3a or 3b was prepared according to literature procedure [48] starting from 4bromobenzyl bromide. Then the compound 3a or 3b reacted with TMS-acetylene in the presence of CuI/Et 3 N/ PdCl 2 (PPh 3 ) 2 in reflux condition to give compound 4a or 4b (Scheme 1). Then TMS group was deprotected using TBAF to give the key acetylenic amino acid ready for cycloaddition reaction. 2,5-Dimethoxy benzaldehyde 6a or 6b reacted with hydroxylamine hydrochloride (NH 2 OH⋅HCl) to produce the oxime derivative 7a or 7b. The compound 7a or 7b was subjected to the key 1,3-dipolar cycloaddition reaction with actetylenic amino acid 5a or 5b in the presence of NaOCl/Et 3 N in DCM as solvent. The isoxazoles 8a-d were smoothly formed using this Huisgen's one pot protocol. We did not observe the formation of any other isomer and nitrile oxide dimerization product in this reaction. Any undesired by-products resulting from aromatic halogenations reaction were not observed in our case. Then the compound 8a was oxidized with CAN to give the desired isoxazole tethered quinone-amino acid 9a in very good yield. The compound 9a was characterized by 1 H-NMR, 13 C-NMR, and HRMS. For example, the characteristic isoxazole proton at 7.1 and quinone proton at 6.8 in 1 H-NMR confirms the oxidation of compound 8a to generate compound 9a. The two carbonyl peaks at 186.8 and 185.2 in 13 C-NMR spectrum validate the benzoquinone moiety. Using similar sequence (Scheme 1) the target compounds 9bd (Table 1) were prepared and characterized by spectral data. The isoxazole tethered quinone-amino acids show equilibrium between hydroquinone (9ah) and benzoquinone (9a) in liquid chromatography mass spectrometry (LC-MS) analysis condition as shown in Figure 3. It may be possible that benzoquinone forms a reduced species during the ionization process in LC-MS condition [49].
Encouraged by this result, we turn our attention to the preparation of propargyl amino acid as starting material. Various acetylene building blocks containing an amino acid moiety were prepared from Schiff-base N-(diphenylmethylene)glycine ester 10 using the literature procedure [50]. Thus, alkylation of 10 with propargyl bromide 11 in the presence of K 2 CO 3 /CH 3 CN in reflux condition gave the propargylated derivative 12. Then the compound 12 was subjected to 1,3-dipolar cycloaddition reaction with the oxime 7a in the presence of NCS/NaHCO 3 in ethyl acetate as solvent to give very low yield of the compound 17a (Scheme 2). We tried couple of conditions to improve the yield of compound 17a but without any success. Maybe either steric factor or the instability of compound 12 is the main reason behind the low yield of the cycloaddition product. Then we turn our attention to the compound 13 which was prepared from compound 12 by hydrolysis reaction followed by protection of the amino group. We tried several conditions (Table 2) to improve the yield of the compound 19b without any success.
Alternatively we plan to introduce the amino acid at the end of reaction sequence to prepare the isoxazole tethered quinone-amino acid hybrid (Scheme 3). Thus, the oxime derivative 7a or 7b was subjected to cycloaddition reaction with propargyl alcohol 14 using NCS/NaHCO 3 condition in ethyl acetate as solvent. Under this reaction condition, the 3-aryl-isoxazole derivative 15a or 15b was prepared in good 4a R = Boc (76%) 4b R = Piv (48%) 5a R = Boc (95%) 5b R = Piv (82%) 8a R 1 = H, R = Boc (71%) 8b R 1 = H, R = Piv (58) 8c R 1 = Me, R = Boc (80%) 8d R 1 = Me, R = Piv (83) 9a R 1 = H, R = Boc (85%) 9b R 1 = H, R = Piv (72) 9c R 1 = Me, R = Boc (95%) 9d R 1 = Me, R = Piv (89) yield. Then the hydroxyl methyl group in the compound 15a or 15b was converted to bromo methyl group using PBr 3 /DCM condition to give the compound 16a or 16b. Gratifyingly, the key step alkylation reaction of 10 with compound 16a in the presence of K 2 CO 3 /CH 3 CN in reflux condition gave the 3,5-substituted isoxazole derivative 17a or 17b. The compound 17a was hydrolyzed in the presence of 1 N HCl/diethyl ether and the resulting amino ester 18a was protected with either Boc 2 O or pivaloyl chloride to obtain Boc derivative 19a or pivaloyl derivative 19b, respectively. Then the compound 19a was oxidized with CAN to give the desired target isoxazole tethered quinone amino acid 20a in very good yield. The compound 20a was characterized by 1 H-NMR, 13 C-NMR, and HRMS spectral data. In a similar sequence the target compounds 20b-d were prepared and characterized by spectral data. The purity of the final isoxazole tethered quinone amino acids (Table 3) was obtained by LC-MS analysis which showed equilibrium between quinone and hydroquinone as observed earlier (Figure 3). It is noteworthy to mention here that previously inaccessible quinone amino acids (19a-d and 20a-d) containing isoxazole moiety were synthesized in very good yield. As indicated in Table 3, the proting group (Boc or pivaloyl) has no effect on the yield of cycloaddition as well as oxidation reaction to give the isoxazole tethered quinone amino acids.
In conclusion, we have developed an efficient and simple method to synthesize isoxazole tethered quinone-amino acid hybrids using 1,3-dipolar cycloaddition and oxidation reactions as key steps in good yields. We believe that this methodology will find a widespread application for the synthesis of 2-aryl-benzoquinone and its derivatives. Further application of this methodology for the synthesis of isoxazole tethered quinone-peptide hybrid as well as preparation of tetrazole tethered quinone-amino acid hybrid is undergoing in our group.