Semi-synthesis of a novel hybrid isoxazolidino withaferin via chemoselective and diastereoselective 1,3-dipolar nitrone cycloaddition reaction

Abstract A facile, atom-economic synthesis of isoxazilidino withaferin, a novel hybrid of withaferin A, has been accomplished via two-step reaction of nitrone synthesis followed by nitrone 1,3-dipolar cycloaddition. The reaction is highly chemoselective (preferential reaction only on one of the two double bonds present on withaferin A) and diastereoselective affording exclusively the cis-fused products. The structure was determined by detailed analysis of 1D, 2D NMR and mass spectral data. Graphical Abstract

Our objective of this work is to introduce a new molecular entity as Isoxazolidino Withaferin, a novel hybrid of withaferin A and isoxazolidine. The principle of this concept is to combine partial or whole structures of molecules to create new, possibly more active, molecular entities (Mehta and Singh 2002;Tietze et al. 2003).  Figure 1. Some of the drug containing isoxazolidine ring system.

Results and discussion
Semi-synthesis of the Isoxazolidino Withaferin hybrid is performed in a two-step reaction. The first step is the synthesis of nitrone and the second step is nitrone 1,3- dipolar cycloaddition reaction on ring A unsaturation of withaferin A (1). Due to unavailability of the n-phenyl hydroxylamine in commercial market, prior to the semisynthesis n-phenyl hydroxylamine was synthesized from nitrobenzene as per the literature procedure of reduction using Zn, NH 4 Cl (Kamm 1925). Synthesis of nitrones (2A-2C) was performed as per the literature report with N-phenyl hydroxylamine and various 4-substituted benzaldehyde in ethanol under room temperature in dark condition for overnight (Br€ uning et al. 1966). The cycloaddition reactions were accomplished using nitrone analogs (2A-2C) and withaferin-A (1) in 1:1 mixture of refluxing butanol and xylene at 120 C (Scheme 1). Around 38-40 hours were required for the reaction to produce products 3A-3C in moderate (60-65%) yields. With increasing the temperature and time of reflux, the yield is not increased. The identity of all the products of the cycloaddition reaction was confirmed by detailed mass, NMR spectrometric data analysis. Assignment of the site of 1,3-dipolar cycloaddition on withaferin A was confirmed on the basis of disappearance of characteristic double bond signals of ring A of withaferin A in the NMR spectrum of 3A. The careful analysis of the NMR spectral data of the products revealed that the signals relating to B, C and D ring system and d lactone ring remained unaltered. However, the chemical shifts for the nuclei belonging to the a,b-unsaturated ketone containing A ring were distinctly shifted. The shift was observed with C-2 and C-3, suffering a profound alteration in the resonance position from d 131.3 and 141.9 to d 58.5 and 78.1 respectively. It must be pointed out that withaferin A numbering has been maintained for the basic skeleton for ease in correlation.
Following the success of the semi-synthesis of isoxazolidino withaferin A hybrids (3A-3C) with the above mentioned 4-substituted benzaldehydes, a dinitrone (4) species was prepared using glyoxal followed by the synthesis of a dinitrone adduct (5) via double nitrone cycloaddition in one pot condition at around 60% yield (Scheme 2). A critical observation of the HMBC spectrum of withaferin-dinitrone adduct (5) revealed the crucial evidence in support of the cycloaddition as the correlation between signals of C-1 (d 208.4) and H-2 (d 4.03), H-3 (d 5.03), H-1 00 (d 4.61); between C-2 00 (i.e., the isooxazolidine carbon attached to nitrogen, d 68.4) and H-2 (d 4.03). Further the COSY relationship between H-1 00 of isoxazolidine (d 4.61) and H-2 (d 4.03), and between H-2 (d 4.03) and H-3 (d 5.03) strongly support the mode of addition. Strong NOESY relationship of H-2 (d 4.03) with both H-3 (d 5.03) and b-oriented C-19 methyl proton (d 1.31) suggests that both H-2 and H-3 are b oriented (Figure 6). From a mechanistic point of view, it is expected that the phenyl group of C-substituted N-phenyl nitrone does prefer to be a-oriented to avoid dipole-dipole repulsion with C-1 carbonyl group of withaferin A. The reaction proceeds in a highly stereoselective manner as it furnished b-orienting cis fused ring junction hydrogens due to the presence of the C-4 b-orienting hydroxyl group, which guides the addition mode from a-face. Moreover, the reaction also proceeds through a regioselective as well as a chemoselective pathway; only unsaturation of ring A in withaferin A took part in the reaction, but unsaturation present in lactone ring of WA remain unreactive due to steric hindrance of 24 th position methyl group and 25 th position hydroxymethyl group on withaferin A.

General experimental procedure
Withaferin A used in reaction was isolated from the methanol extract of leaves of the medicinal plant Withania somnifera by column chromatography followed by crystallization. All chemicals employed for reactions were purchased from Alfa-Aesar/Sigma-Aldrich Company and used as received without further purification. All other solvents and chromatographic adsorbents were purchased from E. Merck (Germany) and SRL (India) Ltd. unless otherwise indicated. TLC analysis was performed on Merck 60 F254  silica gel TLC plates using varying percentages of methanol in chloroform as solvent system and spots were identified using UV indicator (254 nm) followed by staining in iodine vapour. Flash chromatography was performed using YAMAZEN AKROS smart flash chromatography system. NMR spectra were recorded using a Bruker spectrophotometer operating at 600 MHz for 1 H and 150 MHz for 13 C respectively in CDCl 3 . HPLC analysis was done in Shimadzu HPLC with SPD-M20A PDA detector using a C18 column. HRMS data of the compounds were obtained from Agilent 6545 Q-TOF LC/ MS instrument.

Synthesis of a-(4substituted phenyl) N-phenyl nitrone
Solution was prepared of freshly prepared 1g. of pure N-phenyl hydroxylamine in 15 ml. of ethanol in a 100 ml. erlenmeyer flask and react with equimolar amount of 4substituted (R ¼ NO 2 , Br, Cl) benzaldehyde (exothermic reaction) in 40-60 C. The flask was stoppered and kept overnight at room temperature in the dark. The colorless needles of a-(4-substituted phenyl) N-phenyl nitrone are collected on a Buchner funnel and washed once with 20 ml. of ethanol. The yield of the product obtained is 1.90g (85-87%).

Synthesis of withaferin -nitrone hybrids
In a 100 ml. round bottom flask provided with a reflux condenser 500 mg. previously prepared pure nitrone and an equimolar amount of withaferin A were taken in 25 ml of butanol and xylene 1:1 mixture. The flask was heated at 120 C for 40 hours. After compilation of reaction as evident from TLC, the solvent was removed in rotary evaporator and the crude product was subjected to flash chromatography using increasing concentration of methanol in chloroform as eluant. The product was crystallized from chloroform-methanol mixture.

Synthesis of withaferin-dinitrone adduct
In a 50 ml. round bottom flask provided with a reflux condenser 500 mg. previously prepared pure dinitrone and 2 equivalent amount of withaferin A were taken in 20 ml of butanol and xylene 1:1 mixture. The flask was heated at 120 C for 40 hours. After compilation of reaction as evident from TLC, the solvent was removed in rotary evaporator and the crude product was subjected to flash chromatography using increasing concentration of methanol in chloroform as eluant. The product was crystallized from chloroform-methanol mixture.

Conclusion
In conclusion, a facile, atom-economic synthesis of isoxazilidino withaferin, a novel hybrid of withaferin A has been achieved via two-step reaction, generation of nitrone and nitrone 1,3-dipolar cycloaddition on withaferin A. The reaction is highly chemoselective (preferential reaction only on one of the two double bonds present on withaferin A), regioselective and diastereoselective affording exclusively the cis-fused products. The structure was determined by thorough analysis of 1D and 2D NMR and mass spectral data.