Open Access | Peer-reviewed |Research Article
Department Of Pharmaceutical Chemistry, T.V. M College of Pharmacy, Ballari, Karnataka, India – 583103.
Department Of Pharmaceutical Chemistry, Annamacharya College of Pharmacy, Rajampet, Andhra Pradesh, India-516126.
Department Of Pharmaceutical Chemistry, T.V. M College of Pharmacy, Ballari, Karnataka, India – 583103.
Department Of Pharmaceutical Chemistry, T.V. M College of Pharmacy, Ballari, Karnataka, India – 583103.
Published: January 30, 2020 | DOI: 10.5281/zenodo.3647884
2-Azetidinone shows biological activities like anti-bacterial, anti-microbial activity, anti-tubercular activity, and anti-cancer activity. 2-azetdinone derivatives were synthesized by simple procedures. The first step is synthesis of benzohydrazide through nucleophilic substitution reaction between methyl benzoate and hydrazine hydrate. The above formed compound is then treated with substituted aromatic aldehydes in the presence of catalytic amount of concentrated hydrochloric acid with stirring for one hour to give benzohydrazone which results in the formation of Schiff bases.Schiff bases undergone cyclisation in the presence of chloroacetylchloride and diethylenediamine by using ethanol as a solvent upon stirring for 4 hour’s yielded 2-azetidinone derivatives. The in-silico anti-leukemic activity was determined by using the computational tools i.e. “PASS Online”, “AutoDock4.2” and “ADMET” properties by online software’s. Among these six derivatives compounds (AZT-6) was shown more activity when compared with the other five compounds.
Keywords: 2-azetidinone, In-Silico drug design, Anti-leukemic, TPK-BCR-ABL-1, Docking.
|Citation: M. Yaswanth et.al. (2020) In–Silico design, synthesis, characterization and biological evaluation of novel 2-azetidinone derivatives for anti–Leukemic activity. Journal of PeerScientist 2(1): e1000009.|
|Received: November 12, 2019; Accepted January 17, 2020; Published January 30, 2020.|
|Copyright: © 2020 M. Yaswanth et.al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.|
|Data Availability: All relevant data are within the paper and its Supporting Information files.|
|Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.|
|Competing interests: The authors have declared that no competing interests exist.|
|*E-mail: firstname.lastname@example.org | Phone: +91 9059065144|
Tyrosine Protein Kinase BCR-ABL- 1 gene is a chimeric protein which is necessary to play a central role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias, notably chronic myeloid leukemia (CML) . Chronic myeloid leukemia (CML) contains primitive hematopoietic progenitor cells which is a clonal myeloproliferative disorder. The occurrence of chronic myeloid leukemia results by the Philadelphia translocation t (9;22) which fuses the long arm parts of chromosome 9 to chromosome 22 results in the formation of the hybrid gene, BCR-ABL-1. This protein product will be found in more than 95% of chronic myeloid leukemia patients which is a major cause of the disease .
Most significant areas of research in the field of medicinal chemistry were carried out on the heterocyclic compounds. Heterocyclic 2-azetidinones are considered as an important contribution to science and humanity, because they are the constituents of living organisms, natural products, drugs and many more substances which will be more useful to the mankind and society in all walks of life. The attention of the chemists has always drawn over the years for the synthesis of heterocyclic compounds because of their biological properties. Depending upon their physiological and industrial significances they are equally interesting for its theoretical implication for the diversity. Heterocyclic compounds are the large number of drugs introduced in pharmacopoeias every year. Synthesis and evaluation of the heterocyclic compounds has drawn more attention for the chemists and biologists over the years [3-10]. Azetidinones are four membered nitrogen containing heterocyclic’s which are useful substrates in organic chemistry for the design and preparation of biologically active compounds by the adequate fictionalization in the different positions of the ring. The azetidinones are a part of antibiotic structure, figure 1 illustrates the general mechanism of its synthesis. Azetidinones posses various biological activities such as antibacterial [11-13], antifungal , anti-inflammatory [15-16], anti-convulsant , anti-tubercular , anti-cancer and antibiotic activities [19-20].
Figure 1: General mechanism of azetidinones synthesis.
These can also function as enzyme inhibitors and are effective against the central nervous system. These are carbonyl derivatives of azetidines which contain carbonyl group at position-2. So, these are called as 2-azetidinones or more commonly β-lactams. The very first β- lactam was synthesized by H. Straudinger in 1907 via [2+2] cycloaddition reaction of katane and imine and this reaction is termed as the “Staudinger synthesis”. The importance of the β- lactams has been establisted after the discovery of the penicillin by “Alexander Flemming” in 1928 observed bacteriolysis in a nutrient broth at St. Mary’s Hospital in London .
According to the kubinyi most of the drugs in the past were discovered by serendipitously where serendipity plays an important role in finding new drugs [21-23]. At present, the drug discovery is shifted towards drug design, where results depends on understanding the biochemistry of the disease, identifying disease causative proteins, pathways and then designing compounds that are capable of modulating the role of these proteins. In this approach, both experimental and computational methods play an important in the drug discovery and development process via reducing the cost, time and failure chances at late stage of drug discovery pipeline. Some of the benefits of invovling CADD at early stage of drug development are: a. More efficient drug discovery and development process can be achieved by computer based methods. b. Information related to the chemical and biological databases will help in identifying the ligands and targets to optimize novel drugs. c. Drug likeness or pharmacokinetic properties for the chemical compounds should be screened in order to enable early detection of the compounds which are likely to fail in clinical trials and further to enhance detection of promising entities [24-25]. Typical workflow of early stage drug discovery using CADD is shown in figure 2.
Figure 2: Typical workflow of Computer Aided Drug Discovery (CADD):
Results and Discussion
In this present research work, six derivatives of 2- azetidinones were synthesized in three step facile procedure with good yields. All the reactions were monitored by TLC and purification was done by recrystallization process. All the derivatives were characterized using spectral studies like FT-IR spectroscopy,1H-NMR spectroscopy and Mass spectrometry. Six derivatives were screened for In- silico Anti-leukemic activity, based on the in- silico results the title compounds were screened for In- vitro anti-leukemic activity.
In- silico anti-leukemic screening:
In order to prove the alternative hypothesis, In Silico estimation of activity for the synthesized derivatives had been performed using PASS online web resource. The results showed that the studied compounds were having good anti-leukemic activity compared to that of current marketed drugs. This had given the assurance to take the research to further level in future for screening the anti-leukemic activity using other targets.
In- vitro Anti-leukemic activity:
The anti-leukemic activity of the title compounds were done by the MTT assay, the target is Tyrosine protein kinase BCR-ABL-1 Gene which was performed by using Bosutinib as reference standard. Among all the six derivatives the compound AZT-6 exhibited potent anti- leukemic activity when compared with the other derivatives.
In-silico anti-leukemic screening :
Table 1: Estimation of probability of activity by PAAS. (TPKI = Tyrosine protein kinase inhibitor | AL = Anti-leukemic activity)
QSAR, Drug Likeness and Docking results:
Molecular docking studies of TPK-BCR-ABL-1 (PDB ID: 2HZI) with designed potential inhibitors was carried out by using Auto dock 4.2. And the results has been tabulated in table 2. 2D and 3D snapshots depicting the docking poses along with molecular level interactions responsible for the binding have been shown in figure 3 for the standard control drug Bosutinib and six novel 2-Azetidinone derivatives respectively.
Table 2. Docking scores for standard drug and samples:
Figure 3: 2D and 3D docking snapshots of a) Bosutinib, b) AZT-1, c) AZT-2, d) AZT-3, e) AZT-4, f) AZT-5 and g) AZT-6 showing molecular level interactions.
Evaluation of pharmacokinetics, drug likeness and medicinal chemistry friendliness of molecules – Swiss ADME
To be effective as a potent drug, a molecule must reach its target in the body in sufficient concentration, and stay there in a bioactive form long enough for the expected biologic events to occur. Drug development involves assessment of absorption, distribution, metabolism and excretion (ADME) increasingly earlier in the discovery process, at a stage when considered compounds are numerous but access to the physical samples is limited. In that context, computer models constitute valid alternatives to experiments. The Swiss ADME web tool that gives easy efficient input, free access to a pool of fast yet robust predictive models for physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry friendliness, among which in-house proficient methods such as the BOILED Egg, iLOGP and Bioavailability Radar to support drug discovery endeavors. Class: <10 - Insoluble, 10 - Poorly, 6- Moderetly, 4 - soluble, 2 - very, 0 highly. During the time- and resource-consuming processes of drug discovery and development, a large number of molecular structures are evaluated according to very diverse parameters in order to steer the selection of which chemicals to synthesize, test and promote, with the final goal to identify those with the best chance to become an effective medicine for the patients. The molecules must show high biological activity together with low toxicity. Equally important is the access to and concentration at the therapeutic target in the organism. It has been demonstrated that early estimation of ADME in the discovery phase reduces drastically the fraction of pharmacokinetics-related failure in the clinical phases 1. As per the Swiss ADME predictions, results of which were tabulated in tables 2 and 3; all the synthesized 2- Azetidinones are as per Lipinski's rule. Hence all the synthesized compounds have potential drug likeness, leadlikeness, skin permeation and synthetic accessibility.
In-vitro Antileukemic Activity:
Antileukemic activity of the synthesized derivatives was performed by the MTT assay and the results were shown below. As per the results shown in tables 4 and 5 along with the photomicrographs shown in figure 4, among the six derivatives compound six have shown potent activity when compared to other derivatives.
Figure 4: Photomicrograph of MTT assay of the cells treated with a) Bosutinib, b) AZT-1, c) AZT-2, d) AZT-3, e) AZT-4, f) AZT-5 and g) AZT-6.
2-azetidinones derivatives were synthesized, characterized and screened for in-silico antileukemic and in-vitro antileukemic activities using respective standards. In-silico antileukemic screening was performed by PASS online web resource. The results of in-vitro antileukemic screening revealed that compound AZT-6 shown good activity when compared to standard among all the derivatives. This is may be due to the fact that the target TPK-BCR- ABL-1 is hydrophilic in nature and highly polar, but the designed titled compounds are lipophilic in nature and are less polar this may be the one of the reason but not the only reason for rejecting null hypothesis. The results of in - vitro antileukemic screening revealed that the synthesized derivatives have good antileukemic activity when compared that of current marketed drugs. However, further research work need to be carried out to know the relationship between structure and biological activity. The further scope of present research work is to establish the antileukemic activity of the synthesized derivatives on other targets, especially on in-vivo antileukemic activity and QSAR studies
Materials and Methods
Diethylene diamine was purchased from Avra and Hydrazine hydrate from Molychem and other chemicals were purchased from SD FINE Chemicals. All the other chemicals are of AR grade. Purity of the compound was checked by using Pre coated aluminum sheets [n-hexane: Ethylacetate (8:2)] were detected by UV chamber.
General method for the synthesis of studied compounds :
The procedure for the synthesis of compounds consists of three steps.
The methyl benzoate (0.1 mol) in 10 ml of ethanol and hydrazine hydrate (80% 0.1mol) was refluxed for 2.5 hour’s. The completion of the reaction was checked by a thin layer chromatography. Then the mixture was cooled and then filtered. The solid mass was crystallized from ethanol to give hydrazides.
To the above mixture of benzohydrazide (0.01 mol) and various aryl aldehyde (0.01mol) dissolved in 20 ml of water. To the above mixture three drops of concentrated Hydrochloric acid was added thoroughly with continuous stirring for 1hour at room temperature and insoluble solid was generated & washed with water, dried and recrystallized from ethanol.
0.02mol of substituted Schiff base and 0.04mol of chloroacetyl chloride in the presence 0.04 mol of diethylene diamine by taking the 30 ml ethanol as a solvent. The reaction mixture was undergone for continuous stirred for 4 hours. After the completion of reaction the reaction mixture was kept for 48 hours at room temperature. Then concentrated, mixture was cooled, with ice cold water and filtered the mixture. Then the obtained solid was then dried and recrystallised from hot ethanol.
Yellow Solid, Yeild-90%, 1H-NMR: Chemical shift (δ), ppm (500.13 MHz, CDCl3): δ =3, S, 10(H) Ar-H=7.4. IR (Kbr): (C-Cl -Stretch) 805, ( Primary and secondary amine (NH2) Wagging) 850.13, (C-C aromatic Stretch) 1475, (C=O amide and C=O azt ( Stretch) 1563 and 1606, (C-H aromatic (Stretch) 2746, ( Primary and secondary amine (NH2) Stretch) 2936. GC-MS spectrometry results for C16H13ClN2O2: 304.74. Melting point-148-150°c.
Yellow, Yeild-92%, 1H-NMR: Chemical shift (δ), ppm (500.13 MHz, CDCl3): δ =3, S, 1H-CH= 3.73, S, 9(H) Ar-H=7.4. IR (Kbr): (C-Cl -Stretch) 805, ( Primary and secondary amine (NH2) Wagging) 850, (C-C aromatic Stretch) 1430, (C=O amide and C=O azt ( Stretch) 1475.36 and 1913, (C-H aromatic (Stretch) 2748.03, ( Primary and secondary amine (NH2) Stretch) 2937.29. GC-MS spectrometry results for C17H15ClN2O2: 314.77. Melting point-148-149°c.
Light Yellow, Yeild-89%, 1H-NMR: Chemical shift (δ), ppm (500.13 MHz, CDCl3): δ =3, S, S, 10(H) Ar-H-7.4. IR (Kbr): (C-Cl -Stretch) 805, ( Primary and secondary amine (NH2) Wagging) 850, (C-C aromatic Stretch) 1440, (C=O amide and C=O azt ( Stretch) 1475.55 and 1913.17, (C-H aromatic (Stretch) 2748.22, ( Primary and secondary amine (NH2) Stretch) 2937. GC-MS spectrometry results for C16H12Cl2N2O2:: 335.18. Melting point-149-150°c.
Dark Red , Yeild-88%, 1H-NMR: Chemical shift (δ), ppm (500.13 MHz, CDCl3): δ =3, S, S, 9(H) Ar-H=7-8. IR (Kbr): (C-Cl -Stretch) 730, ( Primary and secondary amine (NH2) Wagging) 812, (C=O amide and C=O azt ( Stretch) 1649 and 1705, (C-H aromatic (Stretch) 2935, ( Primary and secondary amine (NH2) Stretch) 3405. GC-MS spectrometry results for C18H18ClN3O2:: 343.81. Melting point-148-150°c.
White, Yeild-85%, 1H-NMR: Chemical shift (δ), ppm (500.13 MHz, CDCl3): δ =3, S, S, 1H,-CH-=4-5, S, 9(H) Ar-H=7-8. IR (Kbr): (C-Cl -Stretch) 849.44, ( Primary and secondary amine (NH2) Wagging) 896.96, (Nitro compound(N-O) assymetric( Stretch) 1293.91, (C=O amide and C=O azt ( Stretch) 1566.65 and 1624.72, (C-H aromatic (Stretch) 2973.43, ( Primary and secondary amine (NH2) Stretch) 3387.06. GC-MS spectrometry results for C16H12ClN3O4:: 345.74. Melting point-148-150°c.
Yellow, Yeild-90%, 1H-NMR: Chemical shift (δ), ppm (500.13 MHz, CDCl3): δ =3, S, S, 1H,-OH-=4, S, 9(H) Ar-H=7-8.4. IR (Kbr): (C-Cl -Stretch) 804.63, ( Primary and secondary amine (NH2) Wagging) 850.77,(C=O amide and C=O azt ( Stretch) 1521.91 and 1612.12, (C-H aromatic (Stretch) 2748.36, ( Primary and secondary amine (NH2) Stretch) 2937. GC-MS spectrometry results for C16H13ClN2O3: 316.74, Melting point-147-150°c.
PASS Online server :
This can be performed through an online web resource called PASS (Prediction of Activity Spectrum of Substances), which is a novel theoretical approach used to screen the novel pharmacological activities of the title compounds. PASS is an online program, which compare the structure of the novel compound with the well known biologically active compounds and predicts the activity of the formulated compounds. By using this thousand’s of compounds can be screened for their novel pharmacological activities. For the prediction of compounds for their novel pharmacological activities the chemical formula was necessary and can be predicted by drawing in chemsketch and submitted into PASS online for the possible mechanism of actions.
Auto Dock is an automated procedure for predicting the interaction of ligands with biomacromolecular targets . The motivation for this work arises from problems in the design of bioactive compounds, and in particular the field of computer-aided drug design.
AutoDock4.2 is parameterized to use a model of the protein and ligand that includes polar hydrogen atoms, but not hydrogen atoms bonded to carbon atoms. An extended PDB format, termed PDBQT, is used for coordinate files, which includes atomic partial charges and atom types. The current Auto Dock force field uses several atom types for the most common atoms, including separate types for aliphatic and aromatic carbon atoms, and separate types for polar atoms that form hydrogen bonds and those that do not. PDBQT files also include information on the tensional degrees of freedom. In cases where specific side chains in the protein are treated as flexible, a separate PDBQT file is also created for the side chain coordinates. Auto Dock Tools, the Graphical User Interface for Auto Dock, may be used for creating PDBQT files from traditional PDB files. Auto Dock Tools includes a number of methods for analyzing the results of docking simulations, including tools for clustering results by conformational similarity, visualizing conformations, visualizing interactions between ligands and proteins, and visualizing the affinity potentials created by Auto Grid details of which has been explained elsewhere in various instances [28-31]. All the docking studies are done using AUTODOCK 4.2 version and the images are rendered using Accelry’s Discovery studio visualizer v4.0 interface.
Antileukemic activity of the synthesized derivatives was performed by the MTT assay and the results were shown below.
This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetra-zolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent (eg. DMSO, Isopropanol) and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.
FBS (Gibco, Invitrogen)Cat No -10270106, Antibiotic – Antimycotic 100X solution (Thermofisher Scientific)-Cat No-15240062, 96- well plates.
|K562 (Leukaemia)||RPMI1640 with low glucose (Cat No-11875-093)|
The cells were seeded at a density of approximately 5×103cells/well in a 96-well flat-bottom micro plate and maintained at 37ºC in 95% humidity and 5% CO2 for overnight. Different concentration (400, 200,100,50,25,12.5 µg/mL) of samples was treated. The cells were incubated for another 48 hours. The cells in well were washed twice with phosphate buffer solution, and 20 µL of the MTT staining solution (5mg/ml in phosphate buffer solution) was added to each well and plate was incubated at 37ºC.After 4h, 100 µL of di- methyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and absorbance was recorded with a 570 nm using micro plate reader [32-33].
Surviving cells (%) = Mean OD of test compound /Mean OD of Negative control ×100.
Using graph Pad Prism Version5.1, we calculate the IC 50 of compounds. Note – DMSO Concentration is less 1.5% in experiments. Concentrations are in duplicates.
MY, PN and MD designed the study. MY executed the work. SB supported MY in executing the designed work. MY, PN, MD and SB analyzed the data. MY wrote the manuscript. MY, PN and MD edited the manuscript. All authors have read and approved the final manuscript.
- Michael W. N. Deininger, Sara Vieira, Rezzeline Mendiola, Beate Schultheis, John M. Goldman and Junia V. Melo, April-2000, BCR-ABLTyrosine Kinase Activity Regulates the Expression of Multiple Genes Implicated in the Pathogenesis of Chronic Myeloid Leukaemia, American Assosciation Of Cancer Research, Volume-60, Issue-7, Page No: 2049-2055.
- M Stanglmaier, M Warmuth, I Kleinlein, S Reis and M Hallek, 2003, The interaction of the Bcr-Abl tyrosine kinase with the Src kinase Hck is mediated by multiple binding domains, Volume-17, Page No: 283-289.
- R. Jaberi, Synthesis of new 2-azetidinone derivatives and related Schiff bases from 3-phenyl-2,3,6-7-tetrahydroimidazo[2,1-b] thiazolo [5,4-d] isoxazole, Organic chemistry research, volume5(1), 2019, page no:42-50.
- Pramod N, Mayuri, Synthesis of novel pyridine containing azetidinone derivatives and their biological evaluation, International Journal of Pharmaceutical Chemistry and Analysis, August-2016, page no: 4-6.
- MN. Kumaraswamy et.al, Synthesis and Pharmacological Investigations of Azetidinones Involving 3-Mercapto-4-amino-5-naphtho[2,1-b]furan-1,2,4-triazole, International journal of pharmaceutical and chemical sciences,volume2(1), 2013, page no:159-168.
- Jagadeeswara Rao et.al, Synthesis, characterization and biological evaluation of the novel benzodioxaphosphole-2-oxide derivatives with aryl substituted 2- azetidinone and 4-thiazolidinone system, Academy of Chemistry of Globe Publications, (2014) 7:1, page no:34-44.
- Pushkal Samadhiya et.al, synthesis of 2-azetidinone derivatives of 6-nitro-1h- indazole and their biological importance, Quim. Nova, Vol. 35, No. 5, 2012, page no:914-919.
- A. Rajasekaran et.al, Synthesis, characterization and biological activity of some novel azetidinones, Journal of Developmental Biology and Tissue Engineering Vol. 2(1), June 2010, page no: 5-13.
- Anil kumar, synthesis, characterization and biological activity of somesubstituted azetidinone derivatives, European journal of biomedical and pharmaceutical sciences, Volume:1, Issue: 3, 2014, page no:592-595.
- Jithesh H T et.al, Synthesis and biological evaluation of 2-azetidinone and thiazolidine-4-one derivatives containing dibenzothiazepine nucleus, Indian Journal of Chemistry, Vol. 57B, page no: 1051-1059,
- Takallum khan, An efficient synthesis and anti- bacterial activity of some novel 2-azetidinone derivatives of 4h-1,2, 4- Triazoles under mild conditions, Journal of heterocyclic chemistry, Volume-55, 2018, page no:1042-1047.
- R. Munavar Sulthana, S. Darlin Quine, Synthesis, characterization, antibacterial evaluation and molecular docking studies of 2-azetidinone derivatives as novel DNA gyrase inhibitors, International Letters of Chemistry, Physics and Astronomy, Vol. 47, Page no: 94-108, 2015.
- Navin B. Patel, Minesh B. Patel, Synthesis and evaluation of antibacterial and antifungal activities of 4-thiazolidinones and 2-azetidinones derivatives from Chalcone, Medicinal chemistry research,2017.
- Apoorva Gupta and Anand K. Halve , Synthesis & Antifungal Screening of Novel Azetidin-2-ones, Open Chemistry Journal, Volume- 2, page no: 1-6, 2015.
- Alex Martin and Abhaiy kumar verma, Synthesis, docking and anti-inflammatory activity of novel series, of azetidinone derivatives, Asian Journal of Research in Biological and Pharmaceutical Sciences, volume:2(3), page no:112-120, 2014.
- Umadevi Racha et.al, Synthesis and Anti-Inflammatory Activity of Some New Schiff’s Bases and Azetidinones, International journal of science and research methodology, volume 4(3), 2016, page no:163-181.
- Archana, Synthesis of Newer Substituted Azetidinone and Thiazolidinone derivatives as potent Anticonvulsant Agents, International Journal of TechnoChem Research, Vol.02, No.02, 2016, page no:121-126.
- Ilango K and Arun Kumar, Synthesis, Antimicrobial and Antitubercular activities of some ovel trihydroxy benzamido azetidin-2-one derivatives, Tropical journal of pharmaceutical research, Volume-10(2), page no: 219-229, 2011.
- Farida Tripodia et.al, Synthesis and Biological Evaluation of New 3-amino-2- azetidinone derivatives as anti-colorectal cancer agents, rsc.li/ med chem comm, 2018, page no:1-10.
- Aakash Deep et.al, Synthesis, antimicrobial and anticancer evaluation of 2- azetidinones clubbed with quinazolinone, Pharmaceutical Chemistry Journal, Vol. 50, No. 1, page no; 24-28, April,
- Le Anh Vu et.al, In-silico Drug Design: Prospective for Drug Lead Discovery, International Journal of Engineering Science Invention, Volume 4 Issue 10, october-2015, Page no: 60-70.
- Ashutosh kumar patidar et.al, Lead Discovery and Lead Optimization: a useful strategy in molecular modification of Lead compound in Analog design, International journal of drug design and discovery, Volume-2, Issue-2, June- 2011, page no:-458-463.
- R. U. Kadam and N. Roy, Recent trends in drug like prediction: A comprehensive review of in- silico methods, Indian journal of pharmaceutical sciences, 69 (5), 2007, page no: 609-615.
- Rupa Guha, Preclinical Pharmacology and toxicology: An important aspect in drug discovery, Advances in clinical toxicology, Medwin publishers, 1(1), 2016, page no: 1-3.
- Le Anh Vu, In-silico Drug Design: Prospective for Drug Lead Discovery,International Journal of Engineering Science Invention, Volume 4 Issue 10, october-2015, Page no: 60-70.1
- A. Filimonov et.al, (2014), Prediction of the biological activity spectra of Organic compounds using the Pass Online Web Resource, Chemistry of Heterocyclic Compounds, Volume – 50, Page No: 444-457
- Goodsell DS, Morris GM, Olson AJ. Automated docking of flexible ligands - Applications of Autodock. Journal of Molecular Recognition. 1996 JanFeb; 9(1): 1–5.
- Swayansiddha Tripathy and Susanta Kumar Sahu (2018) In-silico studies on Molecular Orbital’s, Geometry Optimization and Molecular Docking of Curcumin as an antibacterial drug targets FtsZ protein. Journal of PeerScientist 1(2): e1000006.
- M.M.Prasada Rao et.al. (2018) Novel series of 1, 5 Benzothiazepine skeleton based compounds as anti-cancer agents – In silico and MTT assay based study. Journal of PeerScientist 1(2): e1000008.
- V.S.Viswanadha Murthy.M, V.Girija Sastry, Syed Hussain Basha. (2018) 3,5-dinitrophenyl clubbed azoles against latent tuberculosis- a theoretical mechanistic study. Journal of PeerScientist 1(1): e1000001.
- Reddy, S. V. G., Reddy, K. T., Kumari, V. V., & Basha, S. H. (2015). Molecular docking and dynamic simulation studies evidenced plausible immunotherapeutic anticancer property by Withaferin A targeting indoleamine 2, 3-dioxygenase. Journal of Biomolecular Structure and Dynamics, 33(12), 2695-2709.
- Van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitivity assays: the MTT assay. In Cancer cell culture(pp. 237-245). Humana Press.
- Gerlier, D., & Thomasset, N. (1986). Use of MTT colorimetric assay to measure cell activation. Journal of immunological methods, 94(1-2), 57-63.