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Synthesis, molecular docking and ADMET prediction of novel swertiamarin analogues for the restoration of type-2 diabetes: an enzyme inhibition assay Satyender Kumar , Prakash Niguram , Vedika Bhat , Seema Jinagal , Vinod Jairaj & Neelam Chauhan To cite this article: Satyender Kumar , Prakash Niguram , Vedika Bhat , Seema Jinagal , Vinod Jairaj & Neelam Chauhan (2020): Synthesis, molecular docking and ADMET prediction of novel swertiamarin analogues for the restoration of type-2 diabetes: an enzyme inhibition assay, Natural Product Research, DOI: 10.1080/14786419.2020.1825428 To link to this article: https://doi.org/10.1080/14786419.2020.1825428
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NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1825428
Synthesis, molecular docking and ADMET prediction of novel swertiamarin analogues for the restoration of type-2 diabetes: an enzyme inhibition assay Satyender Kumara , Prakash Niguramb, Vedika Bhatc Vinod Jairajb and Neelam Chauhanc
, Seema Jinagald,
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER) - Ahmedabad, Gandhinagar, Gujarat, India; bDepartment of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER) - Ahmedabad, Gandhinagar, Gujarat, India; cDepartment of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER) - Ahmedabad, Gandhinagar, Gujarat, India; dDepartment of Pharmaceutical Sciences, Baba Mast Nath University - Rohtak, Haryana, India
ABSTRACT
ARTICLE HISTORY
Swertiamarin is a lead, biologically active compound obtained from Enicostemma littorale Blume and known to be identified for the anti-diabetic activity. Present work comprises the synthesis and structural optimization of seven novel swertiamarin analogues and those were not being reported elsewhere till date. Swertiamarin was isolated, followed by modifications that have been accomplished amidst fluorinating, acetylating and oxidizing agents and also performed chromatographic purity and characterization of analogues. Furthermore, the swertiamarin analogues were screened for dipeptidyl peptidase IV (DPP-IV) enzyme inhibition with in silico studies. Besides, the pharmacokinetics and toxicity of analogues were predicted using ADMET software. In a nutshell, the compounds such as SNIPERSV-4 and SNIPERSV-7 have to pose good initial activity (48%) in comparison to standard DPP-IV inhibitor (Sitagliptin). The identified analogues were active against DPP-IV enzyme in preliminary screenings, and these findings would be beneficial for the new age researchers also for the therapy of diabetes.
Received 16 July 2020 Accepted 2 September 2020 KEYWORDS
Swertiamarin; dipeptidyl peptidase-IV (DPP-IV); enzyme inhibition; antidiabetic; fluorination; in silico; ADMET
CONTACT Satyender Kumar [email protected] Present address: Department of Pharmaceutical Sciences, Indira Gandhi University, Meerpur, Rewari, Haryana, India. Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1825428. ß 2020 Informa UK Limited, trading as Taylor & Francis Group
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1. Introduction Type-2 diabetes mellitus (T2DM) is a chronic disease associated with the management of people’s lifestyles. There are many drugs and treatments available in the clinics for the restoration of T2DM (Forbes and Cooper 2013). Amongst all, the DDP-IV inhibitors are accepted worldwide to cure diabetes, perhaps, which acts by reducing glucagon secretion, thereby diminishes blood sugar levels. The likelihood, this DDP-IV enzyme can be identified as a suitable target for drug action and mainly found in the intestine. Meanwhile, which may enhance the number of incretins (GIP and GLP-1) into the intestine helps to maintain the secretion of insulin to lower the elevated levels of blood glucose. (McIntosh et al. 2005; White 2008) The DDP-IV inhibitors such as sitagliptin and saxagliptin were approved orally for the treatment of T2DM to control the hyperglycaemic condition (Unger 2010). Supporting Information, Figure S1 represents different targets showing available therapies and the onset of diabetes. Swertiamarin (Supporting Information Figure S2) is an active constituent obtained from the plant Enicostemma littorale (EL) Blume having biological importance. Several reports mention swertiamarin pharmacological actions and predominantly for the cure of diabetes and hepatic diseases. (Bhattacharya et al. 1976; Yamahara et al. 1991; Kumarasamy et al. 2003; Vishwakarma et al. 2004; Vaidya et al. 2009a, 2009b; Vaijanathappa and Badami 2009; Chiba et al. 2011; Saravanan et al. 2014). The swertiamarin biological actions have effects on cardiac remodeling such as inhibition of NFkB activation; LDL oxidation; inflammation, cell death; as well as lipid peroxidation indicators; and stimulating of antioxidant enzymes by showing the impact on multiple signalling process (Leong et al. 2016). Several synthetic and semi-synthetic derivatives obtained from plants have shown better therapeutic activity after modification, for example, the synthesis of apomorphine from morphine and metformin from galegine is
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being reported (Al-masri et al. 2009; Alexiou and Demopoulos 2010). Initially, galegine had toxic nature and then it was modified with chemical reactions to made metformin for the cure of diabetes (Forbes and Cooper 2013). Also, distinct chemical reactions like esterification, acylation, substitution, cycloadditions, synthesis of oximes and imines, olefinations, fluorination, and palladium (Pd) chemistry have become useful for the preparation of blockbuster drugs including sitagliptin, ciprofloxacin, atorvastatin, fluoxetine. In the market, approximately 20% of the drugs are related to organofluorine compounds; interestingly fluorinated drugs are increasing every year significantly. The incorporation of a fluorine atom at the susceptible location ameliorates metabolic strength and bioavailability of the drug. Some of the fluorinated compounds have been approved by the FDA due to their high potency includes ponatinib, sorafenib, afatinib, regorafenib, vemurafenib, trametinib, clofarabine and sorafenib (Grabley and Thiericke 1999; Reddy 2015). The addition of fluorine to organic compounds improves lipophilicity and membrane permeability (Dinoiu 2007; Plevova et al. 2015). Nowadays, molecular docking studies are playing an imperative role in the initial screenings of synthesized molecules to check their three-dimensional structure and binding capacities of ligands and targets. It is ideally an in silico process and conventionally used to know the binding efficiency of attached protein targets in the course of small chemical drug candidates, which may work on the assumption of molecule actions and strength. Docking performs an integral part throughout the rational design of drugs (Eweas et al. 2014). The thorough literature search evidenced that swertiamarin is having significant medicinal properties, which fulfil the need of the clinical networks by the development of new swertiamarin derivatives. Nevertheless, swertiamarin and its analogues can be a good drug choice for diabetic ailment. However, no reports were available in the public domain on swertiamarin and its analogues for the inhibition of DPP-IV till date. This juicy information prompted us to attempt the development of novel swertiamarin analogues. The present work delineates synthesis and in silico studies of swertiamarin analogues in accordance with the isolation. The chromatographic purity and structural characterization of swertiamarin and its analogues were performed using HPLC, FTIR and NMR studies. Additionally, DDP-IV enzyme inhibition assay (in vitro) was evaluated for the anti-diabetic activity. Finally, pharmacokinetics and toxicity of newly synthesized swertiamarin analogues were predicted by ADMET tool.
2. Results and discussion 2.1. Isolation of swertiamarin Isolation and characterization of swertiamarin were reported in one of our published paper (Kumar and Jairaj 2018). Here, the same procedure was followed for the isolation of swertiamarin and the synthesis of its analogues further.
2.2. Chemistry The published reports mention two analogues and one metabolite of swertiamarin, respectively (Cheema et al. 2014), and therefore, these were showing a reduction in mRNA expression of PPAR-c to show activity toward obesity (Cheema et al. 2014) and
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diabetes (Vaidya et al. 2012). Moreover, the reports conclude that the activity of swertiamarin can be seen by the alteration of chemical groups on its structure. Owing to this, the synthesis process was hypothesized for the development of swertiamarin analogues to amplify the activity of newly synthesized analogues. Then, these properties might be helpful for the therapy of diabetes. Based on the above information, various analogues of swertiamarin were synthesized using different reactions such as fluorination, esterification and epoxidation to potentiate anti-diabetic activity. Scheme 1 (Supporting Information) explains esterification of glycan hydroxyl groups present in swertiamarin (SNIPERSV-1) by the utilization of acetic anhydride (2.1 mM, 3.9 equiv.), pyridine (2.1 mM, 3.9 equiv.) and DMF (2 mL) to protect the alcoholic group as mentioned by Cheema et al. and SNIPERSV-2 was obtained (Cheema et al. 2014). In Scheme 2 (Supporting Information), hydroboration reaction was performed with SNIPERSV-1 and esterified product of SNIPERSV-2 by using BH3-THF (0.36 mM, 1 equiv.), H2O2 (30% aqueous, 0.3 mL), 3M NaOH (to maintain pH 8). Due to the direct use of unprotected swertiamarin, the incorporation of the hydroxyl group on the vinyl position was failed, and this might have transpired owing to the electrophilic environment of the hydroxyl groups which are present on the glycan of the SNIPERSV-1 may lead futile reaction. Hydroboration reaction was accomplished on SNIPERSV-2. Nucleophilic fluorination reaction was performed to attach the fluorine atom at the positions of a hydroxyl group (C-100 , 200 , 300 , 400 , 500 , 600 ) in the glycan part of SNIPERSV-1 by using DAST (diethylaminosulphurtrifluoride) reagent (0.96 mM, 1.8 equiv.; Supporting Information, Scheme 3) and lactone group at (C-8) position in SNIPERSV-2 by using DAST reagent (0.64 mM, 1.8 equiv.; Supporting Information, Scheme 4). Epoxidation of the vinyl group at the positions (C-10 & C-11) of SNIPERSV-1 (Supporting Information, Scheme 5) & SNIPERSV-2 (Supporting Information, Scheme 6) was tried using meta chloroperbenzoic acid (mCPBA) (0.53 mM, 1 equiv), at temperature (37 C) for 8 h and workup was done with DCM and 1M NaOH (for neutralization). Unfortunately, acylation of the SNIPERSV-1 and SNIPERSV-2 was occurred instead of epoxide ring formation. The analysis of 1H-NMR spectrum has unveiled the phenomenon of unexpected anchimeric assistance during the reaction of SNIPERSV-1 and SNIPERSV-2 with mCPBA. The presumption is that this might have taken place due to the reactivity of SNIPERSV-1 and SNIPERSV-2 by benzoic acid formation as a by-product which produced during the reaction. The characterization was performed for all synthesized swertiamarin analogues using spectroscopic techniques. Synthetic routes of different swertiamarin analogues are exemplified in Figure 1.
2.3. Molecular docking studies Molecular docking studies could be performed to swertiamarin and its analogues for the identification of the crystalline structure of DPP-IV in comparison of the standard (sitagliptin). The ligand–receptor association has shown interactions with receptors, namely TYR547 (92.0%) and TYR666 (86.3%) of DPP-IV. The swertiamarin/SNIPERSV-1 was interacted with receptor HIS740 (44.5%); SNIPERSV-2 was interacted with receptor LYS250 (99.3%); SNIPERSV-3 showed interaction with receptor TYR547 (27.9%); SNIPERSV-5 and SNIPERSV-6 have been interacted with receptors like TYR547 (28.7%)
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Figure 1. Schematic representation for the synthesis of swertiamarin analogues.
and TYR547 (28.6%), respectively. The maximum interaction of DDP-IV was observed with an acylated derivative of swertiamarin with the receptor TYR547 (28.7%). There was no interaction of swertiamarin metabolite (gentianin) observed with DDP-IV receptors. The docking (S)-scores were also calculated for sitagliptin and for swertiamarin analogues to check further binding efficiency with DPP-IV. The lower docking scores indicate the greater binding efficiency toward DPP-IV. Hence, some of the swertiamarin analogues had shown more binding interactions with the DPP-IV. The docking scores of different compounds such as sitagliptin (–28.16); SNIPERSV-1 (–14.15); SNIPERSV-2 (–8.79); SNIPERSV-3 (–22.67); SNIPERSV-5 (–23.98); SNIPERSV-6 (–25.16); acylated derivative of swertiamarin (–25.84); and gentianine (–8.99) have been observed and represented in Supporting Information, Figure S3. The results indicate that the interaction with TYR547 receptor of DPP-IV was very much significant for the binding.
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2.4. DPP-IV enzyme inhibition The enzymatic assay (DPP-IV inhibition) was performed in sitagliptin, swertiamarin and its analogues for the evaluation of anti-diabetic potential. The finalized concentrations of DPP-IV used was 2000 mU with l mM substrate for the enzyme inhibition assay and was shown in Supporting Information, Figure S4(a–d). The synthesized swertiamarin analogues were subjected to initial screening to proceed further, and the analogues such as SNIPERSV-1, SNIPERSV-4, and SNIPERSV-7 have shown inhibitory effect at the concentration of 100 mg/mL, as shown in Supporting Information, Figure S5(a). Further screenings were examined at different lower concentrations, i.e. 25, 50 and 75 mg/mL for the active compounds (SNIPERSV-1, SNIPERV-4 and SNIPERSV-7). The % inhibition of the DPP-IV by sitagliptin, swertiamarin and its analogues have been determined, which was depicted in Supporting Information, Figure S5(b). The % inhibition of DPP-IV at different concentrations, i.e. 25, 50, 75 and 100 mg/mL, were obtained for SNIPERSV-1 (0.03, 1.6, 5.6, 15.25); SNIPERSV-4 (0.55. 6.55, 21.03, 45.70); SNIPERSV-6 (0.34, 2.12, 4.88, 16.81); SNIPERSV-7 (0.27, 2.44, 18.28, 47.03); and sitagliptin (positive control) (93, 94, 96, 99), respectively. The enzymatic assay revealed that only two swertiamarin analogues, namely SNIPERSV-4 and SNIPERSV-7, had shown inhibitory activity (48%) concerning to sitagliptin and was shown in Supporting Information, Figure S5(c).
2.5. ADMET prediction of swertiamarin analogues The study of drug-like properties, pharmacokinetics and toxicity of drug substance is of great importance in the early stages of drug discovery and development to screen potential molecules. Conventional methods have complex experimental procedures, expensive and are time-consuming. The evolution of computational approaches for the optimization of pharmacokinetics and toxicity properties may enable the progression of discovery leads to effective and rapid drug candidates. Therefore, in the initial stages, in silico tools are the new option to filter the drug-like molecules. The prediction of pharmacokinetics and toxicity of newly synthesized swertiamarin analogues were assessed by means of Swiss ADME and pkCSM tools, respectively (Ekins et al. 2007; Hughes et al. 2011; Schneider 2013; Sliwoski et al. 2014; Pires et al. 2015). In recent times, the pancreatic beta-cell function was regulated by the gut hormones known to be DPP-IV for the new targets of type 2 diabetes (Hussain et al. 2016). DPP-IV is a proteolytic enzyme which inactivates circulating levels of glucagon-like-peptide1 (GLP1) and is responsible for lowering blood glucose levels (Rotella et al. 2005). It facilitates the proliferation of beta cells, therefore, decreases their apoptosis (Pathak and Bridgeman 2010). The currently available synthetic DPP-IV inhibitor is sitagliptin (gliptins). The drug has good tolerability and usually co-administered with metformin to achieve a balance of fasting as well as postprandial glucose. It was observed that the decrease in HbA1c levels from DPP-IV inhibitor is approximately 0.5–0.7% (Hinnen 2015). Based on this information, the seven new swertiamarin analogues were developed and validated for their activity as DPP-IV inhibitors. As discussed earlier, the prediction of pharmacokinetics and toxicity seems to be a promising strategy in drug discovery and development. When compared with sitagliptin (Merck Canada Inc. 2018), SNIPERSV-3, SNIPERSV-4 and SNIPERSV-7 were found to be orally bioavailable and drug-like
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molecules. These derivatives possess a similar affinity as sitagliptin for TYR547 and TYR666 of DPP-IV enzyme receptor as mentioned in supporting information, Figure S20(a–h). The preliminary data also show approximately 48% DPP-IV enzyme inhibition by SNIPERSV-4 and SNIPERSV-7 proving them as potential anti-diabetic drug candidates. From the in silico toxicity studies, it was observed that none of the derivatives possesses mutagenicity and hepatotoxicity. Only SNIPERSV-2 has shown elongation of QT interval as depicted from hERG I and hERG II inhibition. This property seems to be important in case of combination therapy for diabetes as well as with drugs for comorbidities. Regarding dose values when compared with sitagliptin (Merck Canada Inc. 2018), predicted values of oral rat chronic toxicity and rat LD50 are lower, suggesting the possibility of some toxic effects. However, except SNIPERSV-1 other derivatives have shown higher value predicted human maximum tolerated dose than sitagliptin (100 mg/day). These data might need further validation of in vivo of the derivatives.
2.5.1. In silico pharmacokinetic analysis of swertiamarin analogues Out of seven analyzed analogues, only three were found to be orally bioavailable and drug-like compounds. Bioavailability RADAR graphs for swertiamarin analogues are mentioned in the supporting information, Figure S21(a–h). The same compounds were predicted to have higher GI absorption and BBB permeability. Few of them were predicted as inhibitors of major CYP isoforms CYP2D6 and CYP3A4. None of the analogues was found to be a P-gp substrate. All analyzed analogues were found to have higher negative Log Kp (cm/s) (Supporting information, Table S1). 2.5.2. In silico toxicity analysis of swertiamarin analogues Except SNIPERSV 2 none of the analyzed compounds was found to be toxic when analyzed for five commonly employed toxicity tests. Values for predicted rat LD50, human maximum tolerated dose, oral rat acute, chronic toxicity, T. pyriformis toxicity and Minnow toxicity are mentioned in Supporting information (Table S2). No compound was found to be within a specified range of T. pyriformis toxicity. SNIPERSV 4 and SNIPERSV 7 lie outside the scope of Minnow toxicity. The predicted human maximum tolerated dose for SNIPERSV 1, SNIPERSV 3, SNIPERSV 4 and SNIPERSV 7 were found to be less (Supporting information, Table S2).
3. Experimental 3.1. Synthesis of swertiamarin analogues 3.1.1. Hydroboration of the vinyl group of an esterified product of SNIPERSV-1 Scheme 1 (Supporting information): As per the Cheema et al., the glycan hydroxyl groups (C-10 , 20 , 30 , 40 , 50 , 60 ) of swertiamarin/SNIPERSV-1 were esterified using acetic anhydride (2.1 mM, 3.9 equiv.), pyridine (2.1 mM, 3.9 equiv.) and DMF (2 mL) to protect the alcoholic groups. The reaction was initiated at 0–8 C, and the temperature was gradually raised to 37 C for 24 h following the workup was done with diethyl ether (Supporting information, Figure S6). Scheme 2 (Supporting information): Glycan hydroxyl groups (C-10 , 20 , 30 , 40 , 50 , 60 ) were protected by esterification, and then, hydroboration reaction was performed with
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a vinyl group at 11 positions of SNIPERSV-2. The unprotected swertiamarin was used directly to incorporate the hydroxyl group on the vinyl position, but the effort was failed due to the unprotected molecule. This might occur owing to the electrophilic environment of the hydroxyl groups present on the glycan of the SNIPERSV-1 may lead to a reaction in vain. The hydroboration reaction was executed by using BH3-THF (0.36 mM, 1 equiv.), H2O2 (30% aqueous, 0.3 mL), 3 M NaOH (to maintain pH 8). The reaction was started with a blanket of N2, and then, the temperature was initiated at 0–8 C and raised slowly up to 37 C for 10 h (Supporting information, Figure S6). and diethyl ether used for the workup. During synthesis, the exploration of reaction mechanism was tried for a better understanding of hydroboration. However, it is a wellknown process for the modification of alcohol on any compound and was shown in Supporting Information, Figure S7.
3.1.2. Nucleophilic fluorination of swertiamarin (SNIPERSV-1) hydroxyl groups (C-10 , 20 , 30 , 40 , 50 , 60 ) Scheme 3 (supporting information): Nucleophilic fluorination reaction was carried out for the attachment of fluorine at the hydroxyl group positions (C-10 , 20 , 30 , 40 , 50 , 60 ) in the glycan part of swertiamarin (SNIPERSV-1) by using DAST (diethylaminosulphurtrifluoride) reagent (0.96 mM, 1.8 equiv.). The reaction was started with a blanket of N2 (inert gas) at the temperature of 0–8 C and maintained up to 37 C for 24 h and dichloromethane (DCM) used for the workup. The reaction and mechanism were given in the supporting information, Figure S8 (a,b). 3.1.3. Nucleophilic fluorination of esterified swertiamarin (SNIPERSV-2) lactone group at C-8 Scheme 4 (supporting information): Nucleophilic fluorination reaction was performed to introduce the fluorine at the position (C-8) on the lactone group in SNIPERSV-2 by using DAST (diethylaminosulphurtrifluoride) reagent (0.64 mM, 1.8 equiv.). The reaction was commenced with a blanket of N2 (inert gas) in the temperature range of 0–8 C and maintained up to 37 C for 18 h; subsequently, workup was done with dichloromethane (DCM). The reaction and mechanism are given in supporting information, Figure S9(a,b). 3.1.4. Epoxidation of swertiamarin (SNIPERSV-1) and esterified swertiamarin (SNIPERSV-2) lactone group at C-8 Scheme 5 (supporting information): Epoxidation of the vinyl group at the positions 10 & 11 of SNIPERSV-1 was tried using mCPBA (0.53 mM,1 equiv), at temperature (37 C) for 8 h. The workup was done with DCM and 1 M NaOH (for neutralization), as shown in supporting information, Figure S10. Unfortunately, the acylation of SNIPERSV-1 was observed instead of epoxide ring formation, which was noticed based on the 1H-NMR analysis. The phenomenon of unexpected anchimeric assistance was observed during the reaction of SNIPERSV-1 with meta chloroperbenzoic acid (mCPBA). This may be occurred due to the reactivity of compound SNIPERSV-1 with benzoic acid by-product which formed during the reaction. The schematic representation of the reaction
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mechanism is described in the supporting information, Figure S11, and the obtained product was SNIPERSV-4. Therefore, it confirms the anchimeric assistance phenomenon. Scheme 6 (supporting information): Epoxidation of the vinyl group at positions 10 & 11 SNIPERSV-2 was also tried using mCPBA (0.63 mM, 1.2 equiv), at temperature (37 C) for 8 h and the workup was continued with DCM and 1 M NaOH (for neutralization) as shown in the supporting information, Figure S12. It was observed that the acylation of SNIPERSV-2 was occurred again instead of forming the epoxide ring. The phenomenon of anchimeric assistance was seen once more unexpectedly during the reaction of SNIPERSV-2 with meta chloroperbenzoic acid (mCPBA). Later on, it has been observed the end product named SNIPERSV-7 was identified the same as SNIPERSV-4 upon 1H-NMR and 13C-NMR analysis. The expected and predictive mechanism of reaction mentioned in the Supporting Information, Figure S13. The obtained product from this unexpected mechanism was SNIPERSV-7. So, it seems the products viz SNIPERSV-4 and SNIPERSV-7 were formed due to anchimeric assistance phenomenon. The complete details about the chromatographic conditions, purity profiling (HPLC), yield and characterization (MS, FTIR, 1H-NMR and 13C-NMR) of swertiamarin analogues have been summarized in the supporting information. Representation of HPLC chromatogram, MS, FTIR, 1H-NMR and 13C-NMR for the swertiamarin analogues was done in the supporting information, Figures S14–S19.
3.2. Molecular docking studies Molecular docking study was done by using Human dipeptidyl peptidase-IV (DPP-IV) enzyme (PDB number-1NU8, X-ray diffraction; Resolution (Å) ¼ 2.50) with the help of MOE software. At the end of the docking process, the pose with least score changed into selected from conformations and in every docking procedure the binding orientations turned into a study.
3.3. Pharmacokinetics and toxicity prediction The online tools Swiss ADME (http://www.swissadme.ch/index.php) and pkCSM (http:// biosig.unimelb.edu.au/pkcsm/prediction) were used to predict pharmacokinetics and toxicity. Both require Canonical SMILES of test molecule as input data. The chemical structures were fed in Swiss ADME to generate SMILES.
4. Conclusion Overall, the results possess that swertiamarin analogues have been successfully synthesized and those were subjected to molecular docking studies. The analogues have shown well in silico activity. Soon after, the DPP-IV enzyme inhibitory assay was performed, and the analogues such as SNIPERSV-4 and SNIPERSV-7 have exhibited some inhibitory activity (48%) at the concentration 100 lg/mL in comparison to sitagliptin (positive control) against DPP-IV enzyme. Additionally, ADME properties of analogues
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were assessed for the pharmacokinetics and toxicity. The analysis of pharmacokinetics possesses higher negative Log Kp (cm/s) for all analogues and whereas, SNIPERSV-2 pose toxicity. This is the first study to be reported for the evaluation of anti-diabetic target using swertiamarin and its synthesized analogues.
Disclosure statement No potential conflict of interest was reported by the authors.
Acknowledgements The author Satyender Kumar thankful to Director, NIPER-Ahmedabad, for furnishing such an incredible platform, opportunity and encouragement to carry out research work.
Funding A special note of thanks to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, New Delhi, India for providing research funding.
ORCID Satyender Kumar http://orcid.org/0000-0001-6859-0850 Vedika Bhat http://orcid.org/0000-0003-0371-9945
References Eweas AF, Maghrabi IA, Namarneh AI. 2014. Advances in molecular modeling and docking as a tool for modern drug discovery. Der Pharma Chem. 6:211–228. Al-Masri IM, Mohammad MK, Tahaa MO. 2009. Inhibition of dipeptidyl peptidase IV (DPP IV) is one of the mechanisms explaining the hypoglycemic effect of berberine. J Enzyme Inhib Med Chem. 24(5):1061–1066. Alexiou P, Demopoulos VJ. 2010. Medicinal plants used for the treatment of diabetes and its long-term complications. In: Kokkalou E, editor. Plants Tradit Mod Med Chem Act. Vol. 661. Kerala: Transworld Research Network; p. 69–175. Bhattacharya SK, Reddy PK, Ghosal S, Singh AK, Sharma PV. 1976. Chemical constituents of Gentianaceae XIX: CNS-depressant effects of swertiamarin. J Pharm Sci. 65(10):1547–1549. Cheema S, Goyal R, Vaidya H. 2014. Acetylated and propionated derivatives of swertiamarin have anti-adipogenic effects. J Pharmacol Pharmacother. 5(4):232. Chiba K, Yamazaki M, Kikuchi M, Kakuda R, Kikuchi M. 2011. New physiological function of secoiridoids: neuritogenic activity in PC12h cells. J Nat Med. 65(1):186–190. Dinoiu V. 2007. Chemical fluorination of organic compounds. Rev Roum Chim. 52:219–234. Ekins S, Mestres J, Testa B. 2007. In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. Br J Pharmacol. 152(1):9–20. Forbes JM, Cooper ME. 2013. Mechanisms of diabetic complications. Physiol Rev. 93(1):137–188. Grabley S, Thiericke R. 1999. Bioactive agents from natural sources: trends in discovery and application. Adv Biochem Eng Biotechnol. 64:101–154. Hinnen DA. 2015. Therapeutic options for the management of postprandial glucose in patients with type 2 diabetes on basal insulin. Clin Diabetes. 33(4):175–180.
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Hughes J, Rees S, Kalindjian S, Philpott K. 2011. Principles of early drug discovery. Br J Pharmacol. 162(6):1239–1249. Hussain MA, Akalestou E, Song W-J. 2016. Inter-organ communication and regulation of beta cell function. Diabetologia. 59(4):659–667. Kumar S, Jairaj V. 2018. An effective method for isolation of pure swertiamarin from Enicostemma littorale Blume. Indo Global J Pharm Sci. 08(01):1–8. Kumarasamy Y, Nahar L, Cox PJ, Jaspars M, Sarker SD. 2003. Bioactivity of secoiridoid glycosides from Centaurium erythraea. Phytomedicine. 10(4):344–347. Leong XY, Thanikachalam PV, Pandey M, Ramamurthy S. 2016. A systematic review of the protective role of swertiamarin in cardiac and metabolic diseases. Biomed Pharmacother. 84:1051–1060. McIntosh CHS, Demuth H-U, Pospisilik JA, Pederson R. 2005. Dipeptidyl peptidase IV inhibitors: how do they work as new antidiabetic agents? Regul Pept. 128(2):159–165. R sitagliptin tablets (as sitagliptin phosphate monohydrate). Merck Canada Inc. 2018. JANUVIAV Canada. https://www.merck.ca/static/pdf/JANUVIA-CI_E.pdf Pathak R, Bridgeman MB. 2010. Dipeptidyl peptidase-4 (DPP-4) inhibitors in the management of diabetes. Pharm Ther. 35:509–513. Pires DV, Blundell TL, Ascher DB. 2015. pkCSM: predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. J Med Chem. 58(9):4066–4072. Plevova K, Briestenska K, Colobert F, Mistrıkova J, Milata V, Leroux FR. 2015. Synthesis and biological evaluation of new nucleosides derived from trifluoromethoxy-4-quinolones. Tetrahedron Lett. 56(36):5112–5115. Reddy VP. 2015. Organofluorine Compounds in Biology and Medicine. Newnes. https://books. google.com/books?id=jQ2dBAAAQBAJ&pgis=1 Rotella CM, Pala L, Mannucci E. 2005. Glucagon-like peptide 1 (GLP-1) and metabolic diseases. J Endocrinol Invest. 28(8):746–758. Saravanan S, Hairul Islam VI, Prakash Babu N, Pandikumar P, Thirugnanasambantham K, Chellappandian M, Simon Durai Raj C, Gabriel Paulraj M, Ignacimuthu S. 2014. Swertiamarin attenuates inflammation mediators via modulating NF-jB/I jB and JAK2/STAT3 transcription factors in adjuvant induced arthritis. Eur J Pharm Sci. 56:70–86. Schneider G. 2013. Prediction of Drug-Like Properties. Madame Curie Biosci Database. https:// www.ncbi.nlm.nih.gov/books/NBK6404/ Sliwoski G, Kothiwale S, Meiler J, Lowe EW. 2014. Computational methods in drug discovery. Pharmacol Rev. 66(1):334–395. Unger J. 2010. Incretins: clinical perspectives, relevance, and applications for the primary care physician in the treatment of patients with type 2 diabetes mellitus. Mayo Clin Proc. 85(12 Suppl):S38–S49. Vaidya H, Goyal RK, Cheema SK. 2012. Anti-diabetic activity of swertiamarin is due to an active metabolite, gentianine, that up regulates PPAR-c gene expression in 3T3-L1 cells. Phyther Res. 4:624–627. Vaidya H, Rajani M, Sudarsanam V, Padh H, Goyal R. 2009a. Antihyperlipidaemic activity of swertiamarin, a secoiridoid glycoside in poloxamer-407-induced hyperlipidaemic rats. J Nat Med. 63(4):437–442. Vaidya H, Rajani M, Sudarsanam V, Padh H, Goyal R. 2009b. Swertiamarin: a lead from Enicostemma littorale Blume. for anti-hyperlipidaemic effect. Eur J Pharmacol. 617(1–3): 108–112. Vaijanathappa J, Badami S. 2009. Antiedematogenic and free radical scavenging activity of swertiamarin isolated from Enicostemma axillare. Planta Med. 75(1):12–17. Vishwakarma S, Rajani M, Bagul M, Goyal R. 2004. A rapid method for the isolation of swertiamarin from Enicostemma littorale. Pharm Biol. 42(6):400–403. White JR. 2008. Dipeptidyl peptidase-IV inhibitors: pharmacological profile and clinical use. Clin Diabetes. 26(2):53–57. Yamahara J, Kobayashi M, Matsuda H, Aoki S. 1991. Anticholinergic action of Swertia japonica and an active constituent. J Ethnopharmacol. 33(1–2):31–35.
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Synthesis, molecular docking and ADMET prediction of novel swertiamarin analogues for the restoration of type-2 diabetes: an enzyme inhibition assay Satyender Kumar , Prakash Niguram , Vedika Bhat , Seema Jinagal , Vinod Jairaj & Neelam Chauhan To cite this article: Satyender Kumar , Prakash Niguram , Vedika Bhat , Seema Jinagal , Vinod Jairaj & Neelam Chauhan (2020): Synthesis, molecular docking and ADMET prediction of novel swertiamarin analogues for the restoration of type-2 diabetes: an enzyme inhibition assay, Natural Product Research, DOI: 10.1080/14786419.2020.1825428 To link to this article: https://doi.org/10.1080/14786419.2020.1825428
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NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1825428
Synthesis, molecular docking and ADMET prediction of novel swertiamarin analogues for the restoration of type-2 diabetes: an enzyme inhibition assay Satyender Kumara , Prakash Niguramb, Vedika Bhatc Vinod Jairajb and Neelam Chauhanc
, Seema Jinagald,
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER) - Ahmedabad, Gandhinagar, Gujarat, India; bDepartment of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER) - Ahmedabad, Gandhinagar, Gujarat, India; cDepartment of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER) - Ahmedabad, Gandhinagar, Gujarat, India; dDepartment of Pharmaceutical Sciences, Baba Mast Nath University - Rohtak, Haryana, India
ABSTRACT
ARTICLE HISTORY
Swertiamarin is a lead, biologically active compound obtained from Enicostemma littorale Blume and known to be identified for the anti-diabetic activity. Present work comprises the synthesis and structural optimization of seven novel swertiamarin analogues and those were not being reported elsewhere till date. Swertiamarin was isolated, followed by modifications that have been accomplished amidst fluorinating, acetylating and oxidizing agents and also performed chromatographic purity and characterization of analogues. Furthermore, the swertiamarin analogues were screened for dipeptidyl peptidase IV (DPP-IV) enzyme inhibition with in silico studies. Besides, the pharmacokinetics and toxicity of analogues were predicted using ADMET software. In a nutshell, the compounds such as SNIPERSV-4 and SNIPERSV-7 have to pose good initial activity (48%) in comparison to standard DPP-IV inhibitor (Sitagliptin). The identified analogues were active against DPP-IV enzyme in preliminary screenings, and these findings would be beneficial for the new age researchers also for the therapy of diabetes.
Received 16 July 2020 Accepted 2 September 2020 KEYWORDS
Swertiamarin; dipeptidyl peptidase-IV (DPP-IV); enzyme inhibition; antidiabetic; fluorination; in silico; ADMET
CONTACT Satyender Kumar [email protected] Present address: Department of Pharmaceutical Sciences, Indira Gandhi University, Meerpur, Rewari, Haryana, India. Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1825428. ß 2020 Informa UK Limited, trading as Taylor & Francis Group
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1. Introduction Type-2 diabetes mellitus (T2DM) is a chronic disease associated with the management of people’s lifestyles. There are many drugs and treatments available in the clinics for the restoration of T2DM (Forbes and Cooper 2013). Amongst all, the DDP-IV inhibitors are accepted worldwide to cure diabetes, perhaps, which acts by reducing glucagon secretion, thereby diminishes blood sugar levels. The likelihood, this DDP-IV enzyme can be identified as a suitable target for drug action and mainly found in the intestine. Meanwhile, which may enhance the number of incretins (GIP and GLP-1) into the intestine helps to maintain the secretion of insulin to lower the elevated levels of blood glucose. (McIntosh et al. 2005; White 2008) The DDP-IV inhibitors such as sitagliptin and saxagliptin were approved orally for the treatment of T2DM to control the hyperglycaemic condition (Unger 2010). Supporting Information, Figure S1 represents different targets showing available therapies and the onset of diabetes. Swertiamarin (Supporting Information Figure S2) is an active constituent obtained from the plant Enicostemma littorale (EL) Blume having biological importance. Several reports mention swertiamarin pharmacological actions and predominantly for the cure of diabetes and hepatic diseases. (Bhattacharya et al. 1976; Yamahara et al. 1991; Kumarasamy et al. 2003; Vishwakarma et al. 2004; Vaidya et al. 2009a, 2009b; Vaijanathappa and Badami 2009; Chiba et al. 2011; Saravanan et al. 2014). The swertiamarin biological actions have effects on cardiac remodeling such as inhibition of NFkB activation; LDL oxidation; inflammation, cell death; as well as lipid peroxidation indicators; and stimulating of antioxidant enzymes by showing the impact on multiple signalling process (Leong et al. 2016). Several synthetic and semi-synthetic derivatives obtained from plants have shown better therapeutic activity after modification, for example, the synthesis of apomorphine from morphine and metformin from galegine is
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being reported (Al-masri et al. 2009; Alexiou and Demopoulos 2010). Initially, galegine had toxic nature and then it was modified with chemical reactions to made metformin for the cure of diabetes (Forbes and Cooper 2013). Also, distinct chemical reactions like esterification, acylation, substitution, cycloadditions, synthesis of oximes and imines, olefinations, fluorination, and palladium (Pd) chemistry have become useful for the preparation of blockbuster drugs including sitagliptin, ciprofloxacin, atorvastatin, fluoxetine. In the market, approximately 20% of the drugs are related to organofluorine compounds; interestingly fluorinated drugs are increasing every year significantly. The incorporation of a fluorine atom at the susceptible location ameliorates metabolic strength and bioavailability of the drug. Some of the fluorinated compounds have been approved by the FDA due to their high potency includes ponatinib, sorafenib, afatinib, regorafenib, vemurafenib, trametinib, clofarabine and sorafenib (Grabley and Thiericke 1999; Reddy 2015). The addition of fluorine to organic compounds improves lipophilicity and membrane permeability (Dinoiu 2007; Plevova et al. 2015). Nowadays, molecular docking studies are playing an imperative role in the initial screenings of synthesized molecules to check their three-dimensional structure and binding capacities of ligands and targets. It is ideally an in silico process and conventionally used to know the binding efficiency of attached protein targets in the course of small chemical drug candidates, which may work on the assumption of molecule actions and strength. Docking performs an integral part throughout the rational design of drugs (Eweas et al. 2014). The thorough literature search evidenced that swertiamarin is having significant medicinal properties, which fulfil the need of the clinical networks by the development of new swertiamarin derivatives. Nevertheless, swertiamarin and its analogues can be a good drug choice for diabetic ailment. However, no reports were available in the public domain on swertiamarin and its analogues for the inhibition of DPP-IV till date. This juicy information prompted us to attempt the development of novel swertiamarin analogues. The present work delineates synthesis and in silico studies of swertiamarin analogues in accordance with the isolation. The chromatographic purity and structural characterization of swertiamarin and its analogues were performed using HPLC, FTIR and NMR studies. Additionally, DDP-IV enzyme inhibition assay (in vitro) was evaluated for the anti-diabetic activity. Finally, pharmacokinetics and toxicity of newly synthesized swertiamarin analogues were predicted by ADMET tool.
2. Results and discussion 2.1. Isolation of swertiamarin Isolation and characterization of swertiamarin were reported in one of our published paper (Kumar and Jairaj 2018). Here, the same procedure was followed for the isolation of swertiamarin and the synthesis of its analogues further.
2.2. Chemistry The published reports mention two analogues and one metabolite of swertiamarin, respectively (Cheema et al. 2014), and therefore, these were showing a reduction in mRNA expression of PPAR-c to show activity toward obesity (Cheema et al. 2014) and
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diabetes (Vaidya et al. 2012). Moreover, the reports conclude that the activity of swertiamarin can be seen by the alteration of chemical groups on its structure. Owing to this, the synthesis process was hypothesized for the development of swertiamarin analogues to amplify the activity of newly synthesized analogues. Then, these properties might be helpful for the therapy of diabetes. Based on the above information, various analogues of swertiamarin were synthesized using different reactions such as fluorination, esterification and epoxidation to potentiate anti-diabetic activity. Scheme 1 (Supporting Information) explains esterification of glycan hydroxyl groups present in swertiamarin (SNIPERSV-1) by the utilization of acetic anhydride (2.1 mM, 3.9 equiv.), pyridine (2.1 mM, 3.9 equiv.) and DMF (2 mL) to protect the alcoholic group as mentioned by Cheema et al. and SNIPERSV-2 was obtained (Cheema et al. 2014). In Scheme 2 (Supporting Information), hydroboration reaction was performed with SNIPERSV-1 and esterified product of SNIPERSV-2 by using BH3-THF (0.36 mM, 1 equiv.), H2O2 (30% aqueous, 0.3 mL), 3M NaOH (to maintain pH 8). Due to the direct use of unprotected swertiamarin, the incorporation of the hydroxyl group on the vinyl position was failed, and this might have transpired owing to the electrophilic environment of the hydroxyl groups which are present on the glycan of the SNIPERSV-1 may lead futile reaction. Hydroboration reaction was accomplished on SNIPERSV-2. Nucleophilic fluorination reaction was performed to attach the fluorine atom at the positions of a hydroxyl group (C-100 , 200 , 300 , 400 , 500 , 600 ) in the glycan part of SNIPERSV-1 by using DAST (diethylaminosulphurtrifluoride) reagent (0.96 mM, 1.8 equiv.; Supporting Information, Scheme 3) and lactone group at (C-8) position in SNIPERSV-2 by using DAST reagent (0.64 mM, 1.8 equiv.; Supporting Information, Scheme 4). Epoxidation of the vinyl group at the positions (C-10 & C-11) of SNIPERSV-1 (Supporting Information, Scheme 5) & SNIPERSV-2 (Supporting Information, Scheme 6) was tried using meta chloroperbenzoic acid (mCPBA) (0.53 mM, 1 equiv), at temperature (37 C) for 8 h and workup was done with DCM and 1M NaOH (for neutralization). Unfortunately, acylation of the SNIPERSV-1 and SNIPERSV-2 was occurred instead of epoxide ring formation. The analysis of 1H-NMR spectrum has unveiled the phenomenon of unexpected anchimeric assistance during the reaction of SNIPERSV-1 and SNIPERSV-2 with mCPBA. The presumption is that this might have taken place due to the reactivity of SNIPERSV-1 and SNIPERSV-2 by benzoic acid formation as a by-product which produced during the reaction. The characterization was performed for all synthesized swertiamarin analogues using spectroscopic techniques. Synthetic routes of different swertiamarin analogues are exemplified in Figure 1.
2.3. Molecular docking studies Molecular docking studies could be performed to swertiamarin and its analogues for the identification of the crystalline structure of DPP-IV in comparison of the standard (sitagliptin). The ligand–receptor association has shown interactions with receptors, namely TYR547 (92.0%) and TYR666 (86.3%) of DPP-IV. The swertiamarin/SNIPERSV-1 was interacted with receptor HIS740 (44.5%); SNIPERSV-2 was interacted with receptor LYS250 (99.3%); SNIPERSV-3 showed interaction with receptor TYR547 (27.9%); SNIPERSV-5 and SNIPERSV-6 have been interacted with receptors like TYR547 (28.7%)
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Figure 1. Schematic representation for the synthesis of swertiamarin analogues.
and TYR547 (28.6%), respectively. The maximum interaction of DDP-IV was observed with an acylated derivative of swertiamarin with the receptor TYR547 (28.7%). There was no interaction of swertiamarin metabolite (gentianin) observed with DDP-IV receptors. The docking (S)-scores were also calculated for sitagliptin and for swertiamarin analogues to check further binding efficiency with DPP-IV. The lower docking scores indicate the greater binding efficiency toward DPP-IV. Hence, some of the swertiamarin analogues had shown more binding interactions with the DPP-IV. The docking scores of different compounds such as sitagliptin (–28.16); SNIPERSV-1 (–14.15); SNIPERSV-2 (–8.79); SNIPERSV-3 (–22.67); SNIPERSV-5 (–23.98); SNIPERSV-6 (–25.16); acylated derivative of swertiamarin (–25.84); and gentianine (–8.99) have been observed and represented in Supporting Information, Figure S3. The results indicate that the interaction with TYR547 receptor of DPP-IV was very much significant for the binding.
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2.4. DPP-IV enzyme inhibition The enzymatic assay (DPP-IV inhibition) was performed in sitagliptin, swertiamarin and its analogues for the evaluation of anti-diabetic potential. The finalized concentrations of DPP-IV used was 2000 mU with l mM substrate for the enzyme inhibition assay and was shown in Supporting Information, Figure S4(a–d). The synthesized swertiamarin analogues were subjected to initial screening to proceed further, and the analogues such as SNIPERSV-1, SNIPERSV-4, and SNIPERSV-7 have shown inhibitory effect at the concentration of 100 mg/mL, as shown in Supporting Information, Figure S5(a). Further screenings were examined at different lower concentrations, i.e. 25, 50 and 75 mg/mL for the active compounds (SNIPERSV-1, SNIPERV-4 and SNIPERSV-7). The % inhibition of the DPP-IV by sitagliptin, swertiamarin and its analogues have been determined, which was depicted in Supporting Information, Figure S5(b). The % inhibition of DPP-IV at different concentrations, i.e. 25, 50, 75 and 100 mg/mL, were obtained for SNIPERSV-1 (0.03, 1.6, 5.6, 15.25); SNIPERSV-4 (0.55. 6.55, 21.03, 45.70); SNIPERSV-6 (0.34, 2.12, 4.88, 16.81); SNIPERSV-7 (0.27, 2.44, 18.28, 47.03); and sitagliptin (positive control) (93, 94, 96, 99), respectively. The enzymatic assay revealed that only two swertiamarin analogues, namely SNIPERSV-4 and SNIPERSV-7, had shown inhibitory activity (48%) concerning to sitagliptin and was shown in Supporting Information, Figure S5(c).
2.5. ADMET prediction of swertiamarin analogues The study of drug-like properties, pharmacokinetics and toxicity of drug substance is of great importance in the early stages of drug discovery and development to screen potential molecules. Conventional methods have complex experimental procedures, expensive and are time-consuming. The evolution of computational approaches for the optimization of pharmacokinetics and toxicity properties may enable the progression of discovery leads to effective and rapid drug candidates. Therefore, in the initial stages, in silico tools are the new option to filter the drug-like molecules. The prediction of pharmacokinetics and toxicity of newly synthesized swertiamarin analogues were assessed by means of Swiss ADME and pkCSM tools, respectively (Ekins et al. 2007; Hughes et al. 2011; Schneider 2013; Sliwoski et al. 2014; Pires et al. 2015). In recent times, the pancreatic beta-cell function was regulated by the gut hormones known to be DPP-IV for the new targets of type 2 diabetes (Hussain et al. 2016). DPP-IV is a proteolytic enzyme which inactivates circulating levels of glucagon-like-peptide1 (GLP1) and is responsible for lowering blood glucose levels (Rotella et al. 2005). It facilitates the proliferation of beta cells, therefore, decreases their apoptosis (Pathak and Bridgeman 2010). The currently available synthetic DPP-IV inhibitor is sitagliptin (gliptins). The drug has good tolerability and usually co-administered with metformin to achieve a balance of fasting as well as postprandial glucose. It was observed that the decrease in HbA1c levels from DPP-IV inhibitor is approximately 0.5–0.7% (Hinnen 2015). Based on this information, the seven new swertiamarin analogues were developed and validated for their activity as DPP-IV inhibitors. As discussed earlier, the prediction of pharmacokinetics and toxicity seems to be a promising strategy in drug discovery and development. When compared with sitagliptin (Merck Canada Inc. 2018), SNIPERSV-3, SNIPERSV-4 and SNIPERSV-7 were found to be orally bioavailable and drug-like
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molecules. These derivatives possess a similar affinity as sitagliptin for TYR547 and TYR666 of DPP-IV enzyme receptor as mentioned in supporting information, Figure S20(a–h). The preliminary data also show approximately 48% DPP-IV enzyme inhibition by SNIPERSV-4 and SNIPERSV-7 proving them as potential anti-diabetic drug candidates. From the in silico toxicity studies, it was observed that none of the derivatives possesses mutagenicity and hepatotoxicity. Only SNIPERSV-2 has shown elongation of QT interval as depicted from hERG I and hERG II inhibition. This property seems to be important in case of combination therapy for diabetes as well as with drugs for comorbidities. Regarding dose values when compared with sitagliptin (Merck Canada Inc. 2018), predicted values of oral rat chronic toxicity and rat LD50 are lower, suggesting the possibility of some toxic effects. However, except SNIPERSV-1 other derivatives have shown higher value predicted human maximum tolerated dose than sitagliptin (100 mg/day). These data might need further validation of in vivo of the derivatives.
2.5.1. In silico pharmacokinetic analysis of swertiamarin analogues Out of seven analyzed analogues, only three were found to be orally bioavailable and drug-like compounds. Bioavailability RADAR graphs for swertiamarin analogues are mentioned in the supporting information, Figure S21(a–h). The same compounds were predicted to have higher GI absorption and BBB permeability. Few of them were predicted as inhibitors of major CYP isoforms CYP2D6 and CYP3A4. None of the analogues was found to be a P-gp substrate. All analyzed analogues were found to have higher negative Log Kp (cm/s) (Supporting information, Table S1). 2.5.2. In silico toxicity analysis of swertiamarin analogues Except SNIPERSV 2 none of the analyzed compounds was found to be toxic when analyzed for five commonly employed toxicity tests. Values for predicted rat LD50, human maximum tolerated dose, oral rat acute, chronic toxicity, T. pyriformis toxicity and Minnow toxicity are mentioned in Supporting information (Table S2). No compound was found to be within a specified range of T. pyriformis toxicity. SNIPERSV 4 and SNIPERSV 7 lie outside the scope of Minnow toxicity. The predicted human maximum tolerated dose for SNIPERSV 1, SNIPERSV 3, SNIPERSV 4 and SNIPERSV 7 were found to be less (Supporting information, Table S2).
3. Experimental 3.1. Synthesis of swertiamarin analogues 3.1.1. Hydroboration of the vinyl group of an esterified product of SNIPERSV-1 Scheme 1 (Supporting information): As per the Cheema et al., the glycan hydroxyl groups (C-10 , 20 , 30 , 40 , 50 , 60 ) of swertiamarin/SNIPERSV-1 were esterified using acetic anhydride (2.1 mM, 3.9 equiv.), pyridine (2.1 mM, 3.9 equiv.) and DMF (2 mL) to protect the alcoholic groups. The reaction was initiated at 0–8 C, and the temperature was gradually raised to 37 C for 24 h following the workup was done with diethyl ether (Supporting information, Figure S6). Scheme 2 (Supporting information): Glycan hydroxyl groups (C-10 , 20 , 30 , 40 , 50 , 60 ) were protected by esterification, and then, hydroboration reaction was performed with
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a vinyl group at 11 positions of SNIPERSV-2. The unprotected swertiamarin was used directly to incorporate the hydroxyl group on the vinyl position, but the effort was failed due to the unprotected molecule. This might occur owing to the electrophilic environment of the hydroxyl groups present on the glycan of the SNIPERSV-1 may lead to a reaction in vain. The hydroboration reaction was executed by using BH3-THF (0.36 mM, 1 equiv.), H2O2 (30% aqueous, 0.3 mL), 3 M NaOH (to maintain pH 8). The reaction was started with a blanket of N2, and then, the temperature was initiated at 0–8 C and raised slowly up to 37 C for 10 h (Supporting information, Figure S6). and diethyl ether used for the workup. During synthesis, the exploration of reaction mechanism was tried for a better understanding of hydroboration. However, it is a wellknown process for the modification of alcohol on any compound and was shown in Supporting Information, Figure S7.
3.1.2. Nucleophilic fluorination of swertiamarin (SNIPERSV-1) hydroxyl groups (C-10 , 20 , 30 , 40 , 50 , 60 ) Scheme 3 (supporting information): Nucleophilic fluorination reaction was carried out for the attachment of fluorine at the hydroxyl group positions (C-10 , 20 , 30 , 40 , 50 , 60 ) in the glycan part of swertiamarin (SNIPERSV-1) by using DAST (diethylaminosulphurtrifluoride) reagent (0.96 mM, 1.8 equiv.). The reaction was started with a blanket of N2 (inert gas) at the temperature of 0–8 C and maintained up to 37 C for 24 h and dichloromethane (DCM) used for the workup. The reaction and mechanism were given in the supporting information, Figure S8 (a,b). 3.1.3. Nucleophilic fluorination of esterified swertiamarin (SNIPERSV-2) lactone group at C-8 Scheme 4 (supporting information): Nucleophilic fluorination reaction was performed to introduce the fluorine at the position (C-8) on the lactone group in SNIPERSV-2 by using DAST (diethylaminosulphurtrifluoride) reagent (0.64 mM, 1.8 equiv.). The reaction was commenced with a blanket of N2 (inert gas) in the temperature range of 0–8 C and maintained up to 37 C for 18 h; subsequently, workup was done with dichloromethane (DCM). The reaction and mechanism are given in supporting information, Figure S9(a,b). 3.1.4. Epoxidation of swertiamarin (SNIPERSV-1) and esterified swertiamarin (SNIPERSV-2) lactone group at C-8 Scheme 5 (supporting information): Epoxidation of the vinyl group at the positions 10 & 11 of SNIPERSV-1 was tried using mCPBA (0.53 mM,1 equiv), at temperature (37 C) for 8 h. The workup was done with DCM and 1 M NaOH (for neutralization), as shown in supporting information, Figure S10. Unfortunately, the acylation of SNIPERSV-1 was observed instead of epoxide ring formation, which was noticed based on the 1H-NMR analysis. The phenomenon of unexpected anchimeric assistance was observed during the reaction of SNIPERSV-1 with meta chloroperbenzoic acid (mCPBA). This may be occurred due to the reactivity of compound SNIPERSV-1 with benzoic acid by-product which formed during the reaction. The schematic representation of the reaction
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mechanism is described in the supporting information, Figure S11, and the obtained product was SNIPERSV-4. Therefore, it confirms the anchimeric assistance phenomenon. Scheme 6 (supporting information): Epoxidation of the vinyl group at positions 10 & 11 SNIPERSV-2 was also tried using mCPBA (0.63 mM, 1.2 equiv), at temperature (37 C) for 8 h and the workup was continued with DCM and 1 M NaOH (for neutralization) as shown in the supporting information, Figure S12. It was observed that the acylation of SNIPERSV-2 was occurred again instead of forming the epoxide ring. The phenomenon of anchimeric assistance was seen once more unexpectedly during the reaction of SNIPERSV-2 with meta chloroperbenzoic acid (mCPBA). Later on, it has been observed the end product named SNIPERSV-7 was identified the same as SNIPERSV-4 upon 1H-NMR and 13C-NMR analysis. The expected and predictive mechanism of reaction mentioned in the Supporting Information, Figure S13. The obtained product from this unexpected mechanism was SNIPERSV-7. So, it seems the products viz SNIPERSV-4 and SNIPERSV-7 were formed due to anchimeric assistance phenomenon. The complete details about the chromatographic conditions, purity profiling (HPLC), yield and characterization (MS, FTIR, 1H-NMR and 13C-NMR) of swertiamarin analogues have been summarized in the supporting information. Representation of HPLC chromatogram, MS, FTIR, 1H-NMR and 13C-NMR for the swertiamarin analogues was done in the supporting information, Figures S14–S19.
3.2. Molecular docking studies Molecular docking study was done by using Human dipeptidyl peptidase-IV (DPP-IV) enzyme (PDB number-1NU8, X-ray diffraction; Resolution (Å) ¼ 2.50) with the help of MOE software. At the end of the docking process, the pose with least score changed into selected from conformations and in every docking procedure the binding orientations turned into a study.
3.3. Pharmacokinetics and toxicity prediction The online tools Swiss ADME (http://www.swissadme.ch/index.php) and pkCSM (http:// biosig.unimelb.edu.au/pkcsm/prediction) were used to predict pharmacokinetics and toxicity. Both require Canonical SMILES of test molecule as input data. The chemical structures were fed in Swiss ADME to generate SMILES.
4. Conclusion Overall, the results possess that swertiamarin analogues have been successfully synthesized and those were subjected to molecular docking studies. The analogues have shown well in silico activity. Soon after, the DPP-IV enzyme inhibitory assay was performed, and the analogues such as SNIPERSV-4 and SNIPERSV-7 have exhibited some inhibitory activity (48%) at the concentration 100 lg/mL in comparison to sitagliptin (positive control) against DPP-IV enzyme. Additionally, ADME properties of analogues
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were assessed for the pharmacokinetics and toxicity. The analysis of pharmacokinetics possesses higher negative Log Kp (cm/s) for all analogues and whereas, SNIPERSV-2 pose toxicity. This is the first study to be reported for the evaluation of anti-diabetic target using swertiamarin and its synthesized analogues.
Disclosure statement No potential conflict of interest was reported by the authors.
Acknowledgements The author Satyender Kumar thankful to Director, NIPER-Ahmedabad, for furnishing such an incredible platform, opportunity and encouragement to carry out research work.
Funding A special note of thanks to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, New Delhi, India for providing research funding.
ORCID Satyender Kumar http://orcid.org/0000-0001-6859-0850 Vedika Bhat http://orcid.org/0000-0003-0371-9945
References Eweas AF, Maghrabi IA, Namarneh AI. 2014. Advances in molecular modeling and docking as a tool for modern drug discovery. Der Pharma Chem. 6:211–228. Al-Masri IM, Mohammad MK, Tahaa MO. 2009. Inhibition of dipeptidyl peptidase IV (DPP IV) is one of the mechanisms explaining the hypoglycemic effect of berberine. J Enzyme Inhib Med Chem. 24(5):1061–1066. Alexiou P, Demopoulos VJ. 2010. Medicinal plants used for the treatment of diabetes and its long-term complications. In: Kokkalou E, editor. Plants Tradit Mod Med Chem Act. Vol. 661. Kerala: Transworld Research Network; p. 69–175. Bhattacharya SK, Reddy PK, Ghosal S, Singh AK, Sharma PV. 1976. Chemical constituents of Gentianaceae XIX: CNS-depressant effects of swertiamarin. J Pharm Sci. 65(10):1547–1549. Cheema S, Goyal R, Vaidya H. 2014. Acetylated and propionated derivatives of swertiamarin have anti-adipogenic effects. J Pharmacol Pharmacother. 5(4):232. Chiba K, Yamazaki M, Kikuchi M, Kakuda R, Kikuchi M. 2011. New physiological function of secoiridoids: neuritogenic activity in PC12h cells. J Nat Med. 65(1):186–190. Dinoiu V. 2007. Chemical fluorination of organic compounds. Rev Roum Chim. 52:219–234. Ekins S, Mestres J, Testa B. 2007. In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. Br J Pharmacol. 152(1):9–20. Forbes JM, Cooper ME. 2013. Mechanisms of diabetic complications. Physiol Rev. 93(1):137–188. Grabley S, Thiericke R. 1999. Bioactive agents from natural sources: trends in discovery and application. Adv Biochem Eng Biotechnol. 64:101–154. Hinnen DA. 2015. Therapeutic options for the management of postprandial glucose in patients with type 2 diabetes on basal insulin. Clin Diabetes. 33(4):175–180.
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Hughes J, Rees S, Kalindjian S, Philpott K. 2011. Principles of early drug discovery. Br J Pharmacol. 162(6):1239–1249. Hussain MA, Akalestou E, Song W-J. 2016. Inter-organ communication and regulation of beta cell function. Diabetologia. 59(4):659–667. Kumar S, Jairaj V. 2018. An effective method for isolation of pure swertiamarin from Enicostemma littorale Blume. Indo Global J Pharm Sci. 08(01):1–8. Kumarasamy Y, Nahar L, Cox PJ, Jaspars M, Sarker SD. 2003. Bioactivity of secoiridoid glycosides from Centaurium erythraea. Phytomedicine. 10(4):344–347. Leong XY, Thanikachalam PV, Pandey M, Ramamurthy S. 2016. A systematic review of the protective role of swertiamarin in cardiac and metabolic diseases. Biomed Pharmacother. 84:1051–1060. McIntosh CHS, Demuth H-U, Pospisilik JA, Pederson R. 2005. Dipeptidyl peptidase IV inhibitors: how do they work as new antidiabetic agents? Regul Pept. 128(2):159–165. R sitagliptin tablets (as sitagliptin phosphate monohydrate). Merck Canada Inc. 2018. JANUVIAV Canada. https://www.merck.ca/static/pdf/JANUVIA-CI_E.pdf Pathak R, Bridgeman MB. 2010. Dipeptidyl peptidase-4 (DPP-4) inhibitors in the management of diabetes. Pharm Ther. 35:509–513. Pires DV, Blundell TL, Ascher DB. 2015. pkCSM: predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. J Med Chem. 58(9):4066–4072. Plevova K, Briestenska K, Colobert F, Mistrıkova J, Milata V, Leroux FR. 2015. Synthesis and biological evaluation of new nucleosides derived from trifluoromethoxy-4-quinolones. Tetrahedron Lett. 56(36):5112–5115. Reddy VP. 2015. Organofluorine Compounds in Biology and Medicine. Newnes. https://books. google.com/books?id=jQ2dBAAAQBAJ&pgis=1 Rotella CM, Pala L, Mannucci E. 2005. Glucagon-like peptide 1 (GLP-1) and metabolic diseases. J Endocrinol Invest. 28(8):746–758. Saravanan S, Hairul Islam VI, Prakash Babu N, Pandikumar P, Thirugnanasambantham K, Chellappandian M, Simon Durai Raj C, Gabriel Paulraj M, Ignacimuthu S. 2014. Swertiamarin attenuates inflammation mediators via modulating NF-jB/I jB and JAK2/STAT3 transcription factors in adjuvant induced arthritis. Eur J Pharm Sci. 56:70–86. Schneider G. 2013. Prediction of Drug-Like Properties. Madame Curie Biosci Database. https:// www.ncbi.nlm.nih.gov/books/NBK6404/ Sliwoski G, Kothiwale S, Meiler J, Lowe EW. 2014. Computational methods in drug discovery. Pharmacol Rev. 66(1):334–395. Unger J. 2010. Incretins: clinical perspectives, relevance, and applications for the primary care physician in the treatment of patients with type 2 diabetes mellitus. Mayo Clin Proc. 85(12 Suppl):S38–S49. Vaidya H, Goyal RK, Cheema SK. 2012. Anti-diabetic activity of swertiamarin is due to an active metabolite, gentianine, that up regulates PPAR-c gene expression in 3T3-L1 cells. Phyther Res. 4:624–627. Vaidya H, Rajani M, Sudarsanam V, Padh H, Goyal R. 2009a. Antihyperlipidaemic activity of swertiamarin, a secoiridoid glycoside in poloxamer-407-induced hyperlipidaemic rats. J Nat Med. 63(4):437–442. Vaidya H, Rajani M, Sudarsanam V, Padh H, Goyal R. 2009b. Swertiamarin: a lead from Enicostemma littorale Blume. for anti-hyperlipidaemic effect. Eur J Pharmacol. 617(1–3): 108–112. Vaijanathappa J, Badami S. 2009. Antiedematogenic and free radical scavenging activity of swertiamarin isolated from Enicostemma axillare. Planta Med. 75(1):12–17. Vishwakarma S, Rajani M, Bagul M, Goyal R. 2004. A rapid method for the isolation of swertiamarin from Enicostemma littorale. Pharm Biol. 42(6):400–403. White JR. 2008. Dipeptidyl peptidase-IV inhibitors: pharmacological profile and clinical use. Clin Diabetes. 26(2):53–57. Yamahara J, Kobayashi M, Matsuda H, Aoki S. 1991. Anticholinergic action of Swertia japonica and an active constituent. J Ethnopharmacol. 33(1–2):31–35.
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