Isolation of phytochemicals from Dolichandrone atrovirens followed by semisynthetic modification of ixoside via azomethine ylide cycloaddition; computational approach towards chemo-selection

Abstract Aims of the study were the phytochemical investigation and chemical transformation of isolated compounds of medicinal plant listed in ‘Ayurveda’ like Dolichandrone atrovirens, endemic to Indian subcontinents. From chloroform extract of D. atrovirens four compounds; Ursolic acid (1), Maslinic acid (2), Lupeol (3), β-sitosterol (4) and from methanol extract five compounds; β-sitosterol-3-O-β-D-glucopyranoside (5), 10-O-trans-p-Methoxycinnamoylcatalpol (6), Kaempferol-3-O-β-D-glucopyranoside (7), 6-O-[6"(S)-hydroxy-2",6"dimethyl-2"(E)-7"-octadienoyl] catalpol (8) and Ixoside (9) were isolated. Ixoside was used for the semi-synthetic modification via azomethine ylide cycloaddition leading to novel spiro-oxindolo-pyrrolizidine adduct. The structures of novel adducts were elucidated by analysis of IR, MS and 1 D/2D NMR data. Furthermore, to confirm the chemo selection of only one double bond, we performed density functional theory (DFT) calculation, which confirms the chemo selectivity. In addition, in-silico ADME studies and atom-additive approach based on SASA was also examined for the molecules which suggest that they may be potential future candidates for drug discovery. Graphical Abstract


Introduction
Natural products hold an upper hand in terms of chemical as well as pharmacological diversity which is beyond the limits of current synthetic chemistry. Till now, total 1881 small molecules have been approved, of which 60% drug substances and most of the FDA approved drugs (Newman and Cragg 2020) are either coming from nature or inspired from natural product scaffold. Gratifying nature always will continue to be a vital source of molecular complexity-diversity, for development of new chemical entity.
In our current work we have explored the chemical diversity of one of the medicinal plant listed in 'Ayurveda' like Dolichandrone atrovirens, also known as Wavy Trumpet Flower. Found particularly in parts of Tamil Nadu, over the sandy soils of river beds in south India and is endemic to Indian subcontinents. However, literature search reveals that although traditionally D. atrovirens is an important medicinal plant but in chemical study it remains neglected. Leave and bark extracts of the plants are widely used by the local tribes and native medical practitioners to treat various chronic disorders, like diabetes in Indian folk medicine (Kayarohanam and Kavimani 2015). Genus of this flowering plant belongs to the family Bignoniaceae and a variety of pharmacological activities of this ethno medicinally important family has been reported like trypanocidal, mosquito larvicidal (Ghosh et al. 2012), anti-oxidant (Hussain et al. 2016), antidiabetic (Govindappa et al. 2013), anti-microbial (Choudhary et al. 2013), anti-cancer (Mostafa et al. 2016), antinociceptive (Kameshwaran et al. 2012), and neurotrophic activities (Rana et al. 2011). Members of Bignoniaceae are a huge reservoir of variety of secondary metabolites like saponins (Ge et al. 2009), flavonoids (Blatt et al. 1998;Nassar et al. 2013), quinines (Zofou et al. 2012), napthoquninoes (Epifano et al. 2014), alkaloids (Binutu and Lajubutu 1994), sitosterols (Zhang et al. 2017), iridoids (Castillo and Rossini 2010), terpenes (Hashem et al. 2014) and steroids (Wang et al. 2011).
atrovirens to synthesize series of spirooxindolo-pyrrolizidine derivatives. To the best of our knowledge, for chemical transformations there were no reports found on the a, b unsaturated carboxylic acid containing double bond of ixoside. Therefore, the introduction of spirooxindolo-pyrrolizidine skeleton into the iridoid core would be an interesting strategy to construct a library with structural diversity and novelty in a single molecular framework and may increase their biological activities or create new medicinal properties. Isolation followed by semi-synthesis of spirooxindole-3,2 0 -pyrrolidine compounds containing iridoid glycoside (ixoside) moieties using one pot azomethine ylide 1,3-dipolar cycloaddition reaction for lead generation is the major goal of this communication.
Owing to a wide range of therapeutic outcomes, ixoside have attracted the attention of natural product chemists. The high abundance of ixoside from the crude extract and the presence of functionalities (9) motivate us to focus on semi-synthetic modification to generate highly diversified novel compounds. In this research, six analogs were prepared through azomethine ylide cycloaddition reaction to form novel pyrrolizidine fused spiro-oxindolo analogues of ixoside. The cycloaddition reactions were accomplished using ixoside, a-amino acid and substituted isatins in equivalent amounts in 1:1 mixture of refluxing butanol and xylene at 120 C. Products 12 A-12F, were formed in 35-40% even after 24-30 hours of reaction (Scheme 1).
Products were successfully separated by column chromatography on silica gel (60-100 mesh) and further purification has been done by means of preparative RP-HPLC (Reversed Phase-High Pressure Liquid Chromatography) using (Pursuit 5 C18 (250 mm Â 21.2 mm, 5 mm) (Agilent Technologies, USA) column, MeOH: H 2 O (70:30) as mobile phase, in flow rate of 15 mL/min, (254 nm, Rt 10.8 min). All the synthesized analogs of the cycloaddition reactions were confirmed by detailed IR, Mass, 1 D and 2 D NMR spectrometric data analysis. Scheme 1. Synthesis of spiro-oxindolo pyrrolizidine adducts of ixoside (Reaction Condition: ixoside, L-Proline and substituted isatin in 1:1:1 mixture, solubilised in 10 ml butanol: xylene (1:1 ratio) reflux at 120 C, 24 hr) Assignment of the site of 1,3-dipolar cycloaddition on ixoside was confirmed on the basis of disappearance of characteristic double bond signals of five membered ring of ixoside in the NMR spectrum of isolated compounds. The careful analysis of the NMR spectral data of the products revealed that the chemical shifts for the nuclei belonging to the a, b-unsaturated carboxylic acid containing 5 membered rings were distinctly shifted. The shift was observed with C7 and C8, suffering profound alteration from downfield to up field resonance position from d 147.5 and 136.4 to d 53.9 and 74.5, respectively as evident from 2 D NMR analysis. It must be pointed out that ixoside numbering has been maintained for the basic skeleton for ease in correlation. The crucial evidence in support of this addition came from the observed HMBC correlation ( Figure S1 Figure S1).
From a mechanistic point of view, it was expected that reaction does prefer to be on the a, b-unsaturated carboxylic acid containing unit of 6 membered ring. But the reaction proceeds through a highly chemo-selective pathway; via involving only a, b-unsaturated carboxylic acid containing unit of 5 membered ring in ixoside while a, b-unsaturated carboxylic acid containing unit of 6 membered ring remain unreactive, might be because of electron rich environment due to electron donation from ring oxygen functionality. Moreover, the reaction also proceeded in a highly stereo-selective manner as it furnished cis fused ring system via syn addition (Scheme S1). To confirm the selectivity on five membered ring double bond investigations was done via theoretical calculation. Quantum chemical calculations based on Density Functional Theory (DFT) methods were carried out using the Jaguar module implemented in Schrodinger 2020-1. The geometries of all the reactants, transition states (TS), and two expected products were fully optimized using B3LYP method and 6-31 G Ã þ basis sets in the gas phase (Bochevarov et al. 2013). For all the geometries, frequencies were calculated analytically in order to confirm the nature of stationary points. Computed transition state was confirmed by the presence of only one imaginary frequency ( Figure S2). To reduce the computational time, the glucose ring was omitted from the structures and converted to methoxy group. The use of this truncated model is justified as the reaction is happening away from this moiety. The pharmacokinetic study (Table S1) concluded that all the synthesised novel spiro-oxindole hybrids are within the acceptable range for various properties of surface area and ADME (Table S2) and show better value than the parent molecule (Ixoside). So, these derivatives can be considered as novel leads for the further study.

General experimental procedure
Column chromatography was performed on silica gel (60-120 mesh, 100-200 mesh and Diaion HP-20). Flash chromatography was performed using YAMAZEN AKROS smart flash chromatography system. HPLC analysis was done in Shimadzu HPLC with SPD-M20A PDA detector using a C18 column. Preparative HPLC was performed on Agilent 1260 Infinity II series with a Pursuit 5 C18, 5 mm, 250 Â 21.2 mm column with guard column Agilent 5 prep-C18, 50 Â 21.2 mm and PDA detector. HPLC-grade methanol and water were obtained from RANKEM Mumbai, India. IR spectra were recorded as KBr pellets using a FTIR PERKIN ELMER spectrometer version 10.5.1. HRMS data of the compounds were obtained from Agilent 6545 Q-TOF LC/MS instrument (positive mode) in the electro spray ionisation mode. The NMR spectra were recorded using a JEOL 400 MHz spectrometer (Tokyo, Japan) operating at 400 MHz for 1 H and 100 MHz for 13 C using CD 3 OD and CDCl 3 as solvent and the chemical shifts are reported in d value in parts per million (ppm) and the coupling constants (J) are given in hertz (Hz). All other chemicals employed for reactions were purchased from Alfa-Aesar. All other solvents and chromatographic absorbents were procured from E. Merck (Darmstadt, Germany) and SRL Ltd. (Mumbai, India) unless otherwise indicated. Thin-layer chromatography was performed on pre-coated silica gel 60 F254 aluminium sheets (E. Merck, Darmstadt, Germany) using various solvent systems (1%, 5% and 10% MeOH in CHCl 3 and 5:3:1:1 (ethyl methyl ketone: ethyl acetate: formic acid: water) and spots were developed using UV irradiation, iodine and Liebermann-Burchard reagent.

Extraction and isolation
Leaves of D. atrovirens were collected from Tamilnadu, India. Dried leaves of D. atrovirens (1.5 kg) were powdered and extracted successively with CHCl 3 and CH 3 OH by using Soxhlet extraction process. The chloroform and methanol extract were concentrated under reduced pressure by using rotary evaporator to give 90 g and 210 g respectively. Chloroform extract (90 g) was chromatographed over silica gel (100-200 mesh) and graded elution was carried out using hexane: chloroform and chloroform: methanol. Fractions having similar spots on TLC were combined to obtain 6 major fractions. Fractions were subjected to re-chromatography on silica gel (100-200 mesh) to obtain four compounds from chloroform extract as compound 1 (100 mg), compound 2 (55 mg), compound 3 (60 mg) and compound 4 (150 mg).
The dried methanol extract (210 g) was partitioned between water-saturated n-BuOH and water. The organic layer was evaporated to dryness under reduced pressure using a rotary evaporator to yield a dark-green residue (140 g). The 140 g extract was chromatographed on silica gel (100-200 mesh) with graded elution of CHCl 3 -MeOH (100:0 to 50:50) and fractions giving similar spots on TLC combined together to obtain 5 major fractions. Fractions were subjected to re-chromatography over silica gel (100-200 mesh) and to get five compounds as compound 5 (200 mg), compound 6 (358 mg), compound 7 (125 mg), compound 8 (38 mg) and compound 9 (180 mg).

DFT study
In order to understand energetic behaviour of this reaction, the activation energy barrier for 1,3 dipolar cycloaddition was computed using density functional theory and details of energetic are given in ( Figure S2). The activation barrier associated with the formation of S3 product is found to be 15 kcal/mol. In addition, in the formation of cycloaddition product S3, 4.46 kcal/mol energy is released. For alternative product S4, we have tried several times to get suitable transition state, unfortunately, we were not able trace it. The formation of product S4 is endothermic by 7.50 kcal/mol. Hence, overall, the formation of product S3 is thermodynamically is favoured by 11.97 kcal/ mol compared to product S4, on the potential energy surface.

Pharmacokinetic prediction of semi-synthesised molecules and ixoside
The semi-synthesised and parent molecules are subjected to predict the ADME properties using QikProp module of Schrodinger suite (2020). All calculation was carried out on a HP Z 40-Linux workstation with a 3.5 GHz Intel Xeon 32 core processor. A set of ADMET-related properties were calculated by using the QikProp program (Schr€ odinger 2020) running in normal mode (Schrodinger release. 2020).

Conclusion
In summary, we have isolated nine compounds from the leaves of D. atrovirens and characterised using detailed spectral analysis (NMR, Mass) and compared with the literature to finalize the structure of compounds. In addition, a facile semi-synthesis of novel spiro-oxindolo pyrrolizidine adducts of ixoside has been achieved via an intermolecular 1,3-dipolar azomethine ylide cycloaddition reaction. Reactions were done with different isatin derivatives, proline and ixoside to afford 6 new compounds which were fall in acceptable range of ADMET properties. The reaction is highly chemo and stereo selective confirmed via DFT study, performed in an efficient and practical way in which only one of the two a, b-unsaturated carboxylic acid containing double bonds took part in reaction to form novel spiro-oxindolo pyrrolizidino water soluble adducts. The structures of the products were determined by thorough analysis of IR, mass data and 1 D/2D NMR. The synthesized water-soluble molecules can be a promising potential candidate for drug discovery.