Cytotoxic Activities and Synthesis of Plumbagin

Cytotoxic Activities and Synthesis of Plumbagin3.1IntroductionPlants produce a gigantic number of various(a) natural products or junior-grade metabolites which atomic number 18 winding in the normal electric cellphone emergence, development or reproduction of organisms. al well-nigh of these changes offer protection against herbivores and microbial pathogens, while others are involved in defense against abiotic stress, such(prenominal) as UV-B exposure Many of the secondary metabolites occupy interesting biological properties, which make them desirable toiletdidates for optimization of do doses baring and development processes.The quinonic moiety is considered by the National Cancer Institute (NCI) as an important biologically scaffold for the development of new bioactive compounds with good levels of cytotoxicity. clinically important several well-known antitumoral drugs containing a quinone moiety such as anthracyc marchess, doxorubicin, mitomycin and mitoxantrones an d saintopin which are possess a quinonoid construction. These compounds strike as well as been identified as privileged coordinates due to their biological operation and morphologic properties that have been cogitate to the stimulation of oxidative stress and alkylation of cellular nucleophiles in idlercer cells.A representative group of quinonoid compounds of naphthoquinones (1,4-naphthoquinones) constitute one of the largest and diverse groups of fix secondary metabolites which are widely distributed in nature with a wide range of important pharmacological activities 3,4 that include antioxidant, antimicrobial, antifeedent, antiinflammatory, anticancer, and allelopathic bodily process. Some examples of antitumoral naphthoquinones are plumbagin, juglone, b-lapachol and rhinacanthone.Plumbagin (5-hydroxy-2-m ethyl group-1,4-napthoquinone, Fig.la) is a medicinal plant-derived naphthoquinone , which is one of the simplest plant secondary metabolite of trinity major phyloge nic families viz. Plumbaginaceae, Droseraceae, and Ebenceae. Plumbagin is also present in black walnut tree and other various medicinal plants. It was quarantined from the roots of the medicinal plant Plumbag0 zeylanica 1. (also known as Chitrak) The roots of Plumbag0 zeylanica have been employ in Indian medicine for more than 2,500 years for words of various ailments and which exhibits highly potent biological activities, including antioxidant, antiinflammatory, antibacterial, and antifungal activities.Plumbagin are found in plants as they are judgement to be present as a defense mechanism due to their cytotoxicity. It is also utilise extensively in studies investigating oxidative stress. plumbagin has been shown to do anticancer and antiproliferative activities in animal models and in cell culture.The mechanism of anti-cancer legal action of Plumbagin is reportedly by induction of mammalian topoi mostrase II mediated desoxyribonucleic savage cleavage.4 Plumbagin has been r eported to intercalate into the DNA. It induces higher levels of p21 and thereby inhibits long patch chemical group pair excision repairs leading to apoptosis .Hybrid drug molecules of plumbagin by combining plumbagin with other appropriate anticancer agents may lead to the contemporaries of wise and potent anticancer drugs with pleiotropic action against human cancers.2.2Present run lowour group has been engaged in the design and synthesis of more potent, less(prenominal) toxic, and more selective analogues, through chemical modification of lead compounds isolated from the active extracts. In the course of such studies, we have under(a)taken the plumbago zeylanica, a well-known medicinal plant. The major quantity coupled with the biological pen of plumbagin (1) prompted us to continue our studies. Thus, present work was undertaken to synthesize a library of plumbagin derivatives to study their anti-cancer properties. We here in report the synthesis, cytotoxic activities and their preliminary structure- activeness kind studies.to the best of our knowledge no semi-synthetic derivatives of plumbagin derivaties and test for their till date were reported. ( fingerbreadth 1).Figure 1 Structure of Plumbagin (1).We focused on the synthesis of new of Plumbagin analogues by following r break throughes. As shown in scheme-1, Piperazines and substituted piperazines are most useful pharmacophores that can be found in many marketed drugs, such as the piperazinyl- linked ciprofloxacin dimmers reported as potent antibacterial agents against resistant strains,15 Merck human immunodeficiency virus protease inhibitor Crixivan,16 and drugs under development. A novel class of mixed D2/D4 receptor antagonists, dual calcium antagonist, anti malarial agents and potential antipsychotic agents. latterly piperazine derivatives containing tetrazole nucleus have been reported as an antifungal agent and phytochemists had disposed(p) the derivatives of isolated natural products, which are coupling with piperzenes. This piperzene containing natural products enhance the activity than the parent compound.For designing the analogues of plumbagin a substituted piperzines was introduced at the third sic of Plumbagin. This was achieved by the Michael addition of plumbagin with secondary amines and a series of Plumbagin derivatives with the Michael adduct were synthesized. plumbagin (1) was subjected to Michel addition of substituted piperzines with K2C03 in EtoH outcome under roomtemparature conditions for 8 h to yield identical substituted piperzine plumbagin analogues (scheme 3.1) in 85 to 95% yield.scheme 3.1Introduction of propargyl group into hydroxy position of plumbaginscheme 3.2Preparation of oxime derivatives of plumbagin by substituted acids and substituted piperzines.As shown in scheme 3.3 plumbagin (1) was send-off subjected to methylation on 0H group of plumbagin and then 9 is treated with NH30H.C1, CH3C0ona in EtoH firmness under room temparatur e conditions for 6 h to yield corresponding to oxime of 5-0-methyl Plumbagin(lo), it was intend to prepare the new ester derivatives of oxime of 5-0-methyl Plumbagin with various aromatic substituted aromatic carboxylic acids by using standard esterification strategies viz DDC/DAMP, EDCI/DAMP. Despite the considerable experimentation, however, ester derivatives were never being obtained and more all over, in all cases starting temporal was completely recovered. In view of these deter results, we decided to implement the Yamaguchi esterification protocol (2, 4, 6-trichlorobenz0yl chloride, Et3N, THF, DMAP, toluene) to access ester derivatives. to our delight, the reactions proceeded smoothly to yield the hind end compounds in guide to good yields. The structures and yields of all synthesized compounds (ll21) and the yields were in the range of 80 to 95%. scheme 3.3oxime of 5-0-methyl Plumbagin(lo) treated with propargyl bromide in presence of K2C03 in acetone solvent under roomte mparature conditions for 8 h to yield corresponding propargyl oxime of 5-0-methyl Plumbagin (22) in 90% yield. According to mannich reaction 22 was treated with secondary amines as substituted piperzines in presence of formaldehyde in ethanol solvent under roomtemparature conditions for lo h to yield corresponding propargyl oximes of 5-0-methyl substituted piperzine plumbagin analogues (23-26) (scheme 3.4) in 85 to 90% yield.All the compounds were synthesized for the first time and well characterized by 1H NMR, 13C NMR and ESI HRMS.biological activitya). Evaluation of the anti-proliferative activity against Hela, PANC1, MDAMB-231, IMR32, HepG2and SKNSH cell linesCancer is one of the most serious threats on human health in the wor1d. The mortality and morbidity of cancer patients is the second highest among all diseases in the wor1d, after heart disease. over the past few decades, extensive research has led to the development of a plethora of chemotherapeutic agents however, none of these agents are capable of completely eliminating cancer. The limitations of rate of flow anticancer drugs, increased incidence and rapid development of drug protection have highlighted the need for the discovery of new anticancer agents, preferably with novel mechanisms of action. to identify new chemical entities for a more kernelive treatment of cancer, drug designers can follow many strategies, but the crucial end is always the selection of a suitable starting point from the immense chemical space .In this respect, natural products can be viewed as evolved privileged structures and biologically pre validated leads, in other words, as molecules that have probably evolved evolutionarily to exert highly specialized functions. Recent review pointed out that, about 74% of anticancer compounds being either natural or natural product-derived products, indicating potency of these scaffolds29. Hence, libraries designed and synthesized around the basic structure of such compounds h ave better chance of displaying desirable biological and pharmacological properties.As a primary screen for cytotoxic activity, cancer cell growth inhibitory properties of plumbagin derivatives along with parent compound were examined using SKNSH, Hela, HepG2 pancreatic carcinoma cell line (PANC 1), breast cancer cell line (MDA-MB 231), neuroblastoma cell line(IMR-32), by MTT assay. doxorubicin was used as the reference drug and the results are summarized in Table-1. The results revealed that some of the synthetic analogues were exhibited promising anticancer activity when compared their parent isolated compounds. Among the tried all derivatives, compounds 9,12, and 16 showed more potent active against HePG2 cell line with an GI50 value of 0.020.ol, 0.030.ol, 0.060.03 M respectively and raises 3, 5, 24, 25 and 26 manifested potent activity against PANC 1 with an GI50 value of 0.40.03, 0.ol0.ol, 0.30.03, 0.20.ol, 0.10.ol, M respectively. While remaining all compounds showed moderat e activities on all cell lines. Through it is difficult to discuss the structure activity relationship criteria responsible for the cytotoxic activities in this set of compounds from these results, it can be concluded that me, bis(4-fluorophenyl, 4-ethylpiperazin-1-yl on N in piperezens increase the activity and 0-C1, p-methyl, p-no2 on benzene increase the activity.Table 1 Anti-proliferative activity of plumbagin analoguesTable- 3.1 biological activities of Plumbagin analogues (1-26).SampleHelaPANC 1HepG2 SKNSHMDA-MB-231IMR32GI50GI50GI50GI50GI50GI503.60.22.30.30.70.11.10.28.70.62lo014.80.92.90.749.90.64.00.64.30.65lo08.40.70.40.03lo00.40.052.70.82lo02.00.60.90.62.80.30.60.ol0.970.780.130.024.10.90.ol0.ollo0lo.00.62.30.70.ll0.064.03.10.50.076.10.33.10.1lo0lo014.10.82.61.81.70.214.90.70.350.03lo019.91.75.50.4lo0lo00.40.020.170.089.21.36.40.50.020.ol1.30.6lo0lo012.36.60.80.090.50.0635.10.96.80.60.0460.ol6.51.60.90.06lo00.90.09lo021.50.52.61.71.80.10.030.ol1.00.060.790.070.130.023.81.0 0.40.10.30.030.160.03lo00.60.062.31.290.01.012.41.7lo05.20.3lo016.20.926.40.92.90.24.30.7lo0lo07.82.924.46.10.060.03lo026.20.631.70.28.61.522.20.72.50.3lo00.06lo013.80.125.90.40.20.04lo0lo0lo07.63.718.81.32.00.9lo0lo00.ll0.024.10.314.70.90.30.0520.52.2lo00.340.077.41.216.01.03.50.4lo01.80.651.30.45.70.3lo034.22.6lo01.80.80.320.052.21.00.60.0457.00.722.41.15.80.4lo01.10.060.30.033.50.4lo03.50.5lo01.30.10.20.ol1.20.712.63.3lo019.80.85.20.40.10.ol2.00.2lo04.70.78lo0doxorubicinCytotoxic assy All cell lines (SIHA, PANC 1, MDA MB -231, IMR -32, DU-145 and A549) used in this study were purchased from the American Type Culture The cell lines (ATCC), linked States. The synthesized test compounds were evaluated for their in vitro anti proliferative activity in these six incompatible human cancer cell lines. A protocol of 48 h continuous drug exposure was used, and a SRB cell proliferation assay was used to estimate cell viability or growth. All the cell lines were grown in Dulbecc0s modifie d Eagles medium (containing lo% FBS in a humidified atmosphere of 5% C02 at 37 C). Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 96-well plates in lo01 aliquots at plating densities depending on the doubling time of individual cell lines. The microliter plates were incubated at 37 C, 5% C02, 95% air, and lo0% relative humidity for 24 h prior to addition of experimental drugs and were incubated for 48 hrs with different doses (0.ol, 0.1, 1, lo, ,lo0M) of prompt derivatives. After 48 hours incubation at 37 C, cell monolayers were laid by the addition of lo% (wt/vol) cold trichloroacetic acid and incubated at 4 C for 1h and were then stained with 0.057% SRB dissolved in 1% acetic acid for 30 min at room temperature. Unbound SRB was washed with 1% acetic acid. The protein bound dye was dissolved in lomM Tris base solution for 0D determination at 5lo nm using a microplate reader (Enspire, Perkin elmer, USA). exploitation the seven absorbance mea surements time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti), the percentage growth was figure at each of the drug concentrations levels. Percentage growth inhibition was calculated as(Ti-Tz)/(C-Tz) x lo0 for concentrations for which Ti/=Tz(Ti-Tz)/Tz x lo0 for concentrations for which TiThree dose response parameters were calculated for each experimental agent. Growth inhibition of 50 % (GI50) was calculated from (Ti-Tz)/(C-Tz) x lo0 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The 1C50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from (Ti-Tz)/Tz x lo0 = -50. Values were calculated for each of these three parameters if the level of activity is reached however, if the effect is not reached or is exceeded, the value for that parameter was expressed as great or less than the maximum or minimum concentration tested.3.4 resultIn conclusion, we have successfully synthesized thirty one analogues of Plumbagin (1) and these analogues were evaluated for their anti-cancer activities. Majority of the compounds exhibited solid anti-cancer activity than the parent compound Plumbagin (1) and this study also provides an initial structureactivity data, based on derivatives of Plumbagin (1). The results are an indicative of the fact that the compound 5 proved to be the best analogue with GI50 of 12 proved to be the best analogue with GI50 of invivo studies need to be carried out for telling the exact mechanism of action and will be taken up in the future in our laboratory.3.5Experimental SectionPlant material Plumbag0zeylanica was collected from Tirumala forest, Tirupathi, Andhra Pradesh, India, in August 2007. The plant material was identified by Dr. K. Madhav Chetty, Botany Department, Sri Venkateswara University, Tirupathi. A voucher of the plant was deposited in the Herbarium of the Botany Department.Extaction and closing offThe shade-dried roots of Plumbag0 zeylanica were powdered in a pulvarizer (lo kg) and extracted with chloroform/methanol, 11 followed by the concentration under reduced pressure. The resulting extract was (70 g) chromatographed over silicon oxide gel (60120 mesh) and eluted with n-hexane/ethyl acetate combinations of increasing polarity. Plumbagin (12 g) was obtained by elution with n-hexane/ethyl acetate, 991.General procedure for the synthesis of piperzene (Secondary amine) derivatives (2-7)to a solution of the Compound 1(leq) in ethanol (3m1) in presence of potassium carbonate as a catalyst was added secondry amine (piperzene) stirred at room temperature for 8 h. The reaction mixture was diluted with Ethyl acetate (lo m1), washed with c0oled frosting and brine solution. The organic layer was dried over Na2so4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using the mobile class Acetone in Hexane afforded secondary amine (piperzene) derivatives.