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February 18, 2010
8:23
Silver nanoparticles against HIV-1
» ‎ Drug Discovery

HIV-1 entry inhibitorMany biological applications of nanoparticles are being explored including biosensors, labels for cells and biomolecules and cancer therapeutics. In 2005, silver nanoparticles were studied for their role against HIV-1 by researcher from the University of Texas, USA.
In this study, three different silver nanoparticles, carbon coated, poly (N-vinyl-2-pyrrolidone) (PVP) coated and bovine serum albumin (BSA) conjugated, were used and the average size distributions for each preparation were 16.19±8.68 nm, 6.53±2.41 nm and 3.12±2.00 nm respectively. High angle annular dark field (HAADF) scanning transmission electron microscopy was used to study the interaction of silver nanoparticles with HIV-1.

It was found that the sizes of nanoparticles that bind with virus were within the range of 1-10 nm as determined by energy-dispersive X-ray spectroscopy (EDS). From this work, authors concluded that silver nanoparticles undergo size dependent interaction with HIV-1 in vitro and suggested that the silver nanoparticles likely interact with the HIV-1 virus via preferential binding to the gp120 glycoprotein knobs.
gp120?
gp120 is a glycoprotein exposed on the surface of the HIV envelope. Entry to the host cell involves the binding of gp120 to the CD4 receptor site on the host cell, process called as “attachment”. Upon binding to CD4 receptor, it promotes further binding to co-receptor like CCR5 or CXCR4. This causes the conformational change in gp120 which facilitate gp41 to unfold and insert its hydrophobic terminus into the host cell membrane. Thereafter gp41 fold back itself which draws virus towards the host cells and promote the fusion of cell membranes. You can check out the video in my previous post explaining the whole process of HIV virus replication.
Thus, an agent like silver nanoparticles, which can interact with the gp120 glycoprotein, would block the entry or fusion of virus to the host cell. It was hypothesized that the exposed sulfur-bearing residues of the glycoprotein knobs would be attractive sites for nanoparticles interaction but the mechanism underlying the HIV-inhibitory activity of silver nanoparticles are fully elucidated recently by Humberto et. al, researcher from the same team. They have done several assays like antiviral activity of silver nanoparticles against various HIV-1 strains, virus adsorption assays, cell-based fusion assays, a gp120/CD4 capture ELISA, time-of-addition experiments, virucidal activity assays with cell-free and cell-associated HIV-1 virus. From these assays, authors come into conclusion that the silver nanoparticles possess anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or viral entry inhibitor. Further pharmacokinetic, pharmacodynamic and toxicological studies in animal models are needed to define the safety parameters before their use.
So, what do you think about the possibility of using silver nanoparticles clinically as an anti-HIV agent?
February 09, 2010
7:36
Photo degradation of rofecoxib, a COX-2 inhibitor
» ‎ Drug Discovery

Characterization of degraded product of RofecoxibResearcher from Monash University, Australia has reported a photocyclization product of rofecoxib. Discovery happened unexpectedly while trying to find new polymorph of rofecoxib. The compound was analyzed and characterized as 6-methylsulphonylphenanthro-[9, 10-C] furan-1 (3H)-one.

“Rofecoxib, a well known COX-2 inhibitor, has been used as anti-inflammatory agent. But due to increased cardiovascular risks, it has been withdrawn from the market.”
Author obtained the degraded product by recrystallization and co-precipitation method. In recrystallization, 10 mg of rofecoxib was dissolved in 2 mL of ethyl acetate and stirred at 65 °C for 15 min. and in case of co-precipitation, 10 mg of rofecoxib was stirred in 2 mL 10% hydroxypropyl-β-cylclodextrin solution at 60 °C for 72 h. Solutions exposed to room light intensity were filtered and left to evaporate at room temperature for one month to get the crystals.

It is likely that most of the compounds on exposure to heat and light for such a long time degrade to give some new products. But the effort put by the author for the identification or characterization of the degraded product is really appreciable.
The compound structure was characterized by using thermoanalytical, elemental analysis, spectroscopic, chromatographic and crystallographic techniques. The underlying mechanism of photo-degradation of rofecoxib has been proposed as below.
This study clearly shows that the rofecoxib is highly light sensitive.
Rofecoxib has potential to increase the cardiovascular risks. Thus, finding of this new rofecoxib derivative may be helpful in the discovery of new biologically active agent.
February 03, 2010
0:18
Promising activity of Sorafenib as a therapeutic option for Osteosarcoma
» ‎ Drug Discovery

Potential therapeutic option for the treatment of metastatic or relapsed Osteosarcoma
Sorafenib, drug approved for the treatment of kidney, liver and thyroid carcinoma, has been studied for its effect on Osteosarcoma (OS) by researcher from University of Torino medical School, A.O, Torino, Italy. It is an orally active biarylureic multi-Kinase inhibitor which can block the ERK 1/2 pathway by targeting Raf-kinases, such as RAF-1 and B-RAF. It is marketed as Nexavar by Bayer pharmaceutical company.
Authors found that Phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) pathways were highly activated in OS cell lines as analyzed by immunohistochemistry. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK 1/2, MCL-1 and P-ERM in a dose-dependent manner.
In chick embryo chorioallantoic membranes (CAM assay), sorafenib blocked the angiogenesis induced by OS supernatants. Matrix metalloproteinases (MMP) are one of the main causes of the invasive phenotype of tumor cells. This study found that sorafenib is able to inhibit MMP2 production by OS cell lines determining the diminished invasiveness potential of OS. VEGF, the principal stimulator of angiogenesis, is also involved in the metastatic behavior of OS. Treatment with sorafenib causes consistent reduction of the concentration of VEGF-A in all cell lines tested. This is most likely due to ERK 1/2 inhibition.In a xenograft OS model, sorafenib causes the reduction in the dimension and number of lung nodules thereby reducing the mouse death rate. Immunohistochemistry analysis revealed that ERK 1/2, MCL-1 and ERM were consistently inhibited in OS xenograft.
The tail vein of SCID mice was injected with human osteosarcoma cell lines (SJSA-1) which give rise to pulmonary colonies within 3 weeks. Treatment with sorafenib for 16 days strongly inhibited tumor colony growth.
Authors concluded that sorafenib has anti-tumoral activity, anti-angiogenic effects in OS and are able to reduce metastatic colony formation of OS cells in lungs. The effect was due to the inhibition of ERK 1/2, MCL-1 and ERM pathways both in vivo and in vitro.This study provides a new insight for the development of therapeutic agents against Osteocarcinoma.
January 28, 2010
9:14
Convenient way of adding reagents-Dropping funnel
» ‎ Drug Discovery

Simple laboratory equipment for adding reagents
Few days back, an undergraduate student had a query on how to add the reagent slowly (drop wise) into the reaction mixture over long period of time in a convenient way. I know most of the synthetic / medicinal chemists are aware of solution for this problem but might be new to freshmen. So I decided to introduce the simple dropping funnel, which can solve above mentioned problem.

Dropping funnel, as shown in the picture, is a glass tube with stopcock at one end. The reagent is kept in the glass tube and its flow is controlled by the stopcock. There is a relief tube (by-pass) which allows the escape of gases evolved during the reaction.

"Simple equipments can make work easy and convenient"
January 22, 2010
6:35
Study of proton pump inhibitors as anti-allergic asthmatic agents
» ‎ Drug Discovery

Study of Omeprazole and Pantoprazole as anti-allergic asthmatic agents
Recently, Choi et. al. reported that proton pump inhibitors have potential as an anti-allergic asthmatic agents. Before going to the study, let me explain in brief about proton pump inhibitors and its mechanism of action.
Proton pump inhibitors (PPIs) like Omeprazole and Pantoprazole are widely used in the treatment of peptic ulcer, gastroesophageal reflux diseases (GERD) and Zollinger-Ellison syndrome. They specifically inhibit the human gastric H+/K+-ATPase (proton pump), which is responsible for the secretion or release of H+ ions in the apical canaliculi of parietal cells. These released H+ ions can combine with Cl- ions to give HCl. So proton pump has been the key target of PPIs. Omeprazole is inactive at neutral pH, but at acidic pH it undergoes protonation to give sulphenic acid and active sulfonamide derivative which actively acts as H+/K+-ATPase inhibitor. As it inactivate the enzyme irreversibly, formation of new H+/K+-ATPase molecules is required for acid secretion.

Mechanism of action of proton pump inhibitors has been well illustrated in the animated clips as shown below.

In this study, authors have found that PPIs like omeprazole and pantoprazole reduced the translationally controlled tumor protein (TCTP) secretion, which are associated with the development of allergic reaction. TCTP has been reported to present in nasal washings, skin blister fluids, and, bronchoalveolar lavage (BAL) fluids during late allergic reaction. They are also responsible for induction of histamine secretion, IL-4, IL-13 from basophils.
Authors believed that modification in intracellular events, such as calcium influx and calcium mobilization due to the blockade of H+/K+-ATPase by PPIs are responsible for modulation of TCTP secretion. TCTP act as a regulator of allergic responses when released from cells. It cannot affect downstream effector cells until it is secreted.
As TCTP has important role in allergic condition, inhibition of TCTP secretion by PPIs has convinced authors to conclude that this can be potentially useful in anti-allergic asthmatic therapies.

Source:
Choi S, Min HJ, Kim M, Hwang ES, Lee K (2009) Proton Pump Inhibitors Exert Anti-Allergic Effects by Reducing TCTP Secretion. PLoS ONE 4(6): e5732. doi:10.1371/journal.pone.0005732
January 16, 2010
6:52
Simple method of recording TLC data
» ‎ Drug Discovery

Simple yet useful method of recording TLC data
Preserving TLC data is important for future record, no doubt. The common way is just by drawing the TLC plate in notebook. Today’s post will explain simple method of transferring the TLC data from glass plate to notebook using sellotape. This method is very simple. What you need is just a sellotape.

Procedure : Apply the sellotape over the TLC plate. Thereafter pull out slowly and carefully. You will find the TLC data attached to sellotape, which you can simply paste in the notebook.

This method is beneficial while recording the stained TLC plate. When you apply the staining agents, you will get different colors for different compounds on heating. If you use common method (sketching TLC plate), either color pen or words are necessary to denote the colors. Second method will easily solve this problem.

You may have confusion why color notation is important? Ok, I will explain with my experience. Once, during the reaction, I checked the TLC but the starting material seems to remain even after long reaction time. I had a question why the starting material hasn’t finished yet? So, I stained and heated the TLC plate. The result was exciting. The color of starting material and the reaction mixture was completely different. I stopped the reaction and did the workup and purification. Finally, from structure analysis it was confirmed as expected compound which has similar Rf value with the starting material.

Simple tricks make your work better.
So, how do you record the TLC data? If you are not using either of these two methods, please share it.
January 11, 2010
23:45
Small molecule inhibitors of HIF-I pathway
» ‎ Drug Discovery

Thiophen-oxadiazole core important for HIF-I signaling pathway inhibitory activity
Recently, researchers from National Institutes of health, Bethesda, US (Xia, M. et. al.,) has identified four promising lead compounds as hypoxia-inducible factor-I (HIF-I) signaling pathway inhibitors. These compounds selectively inhibited both HIF-I activity and vascular endothelia growth factor (VEGF) protein expression. From structure activity relationship study, it is revealed that thiophen-oxadiazole core, rather than the oxadiazole group alone is important for the HIF-I inhibitory activity. For this purpose, they have screened 73,000 compounds by quantitative high-throughput screening (qHTS) method.

Among the four active compounds as shown in above figure, three compounds has same thiophene oxadiazole core (marked in box).
"Hypoxia-inducible factor-I (HIF-I), composed of alpha and beta subunit has significant role for transcriptional regulation of many genes expressed in low oxygen environments (hypoxic). During hypoxic conditions, HIF-I alpha dimerizes with HIF-I beta and translocates to the nucleus where it binds to the hypoxia-response element (HRE) and activates expression of target genes involved in cell growth and survival. Inhibition of the HIF-I signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer agents."
This encourage the authors to investigate the compounds for the potential HIF-I inhibitory activity. As a result, they come up with the discovery of four promising lead compounds. These leads may provide a new insight for the researchers in the development of anticancer agents.
If you know or have some idea about the importance of thiophene-oxadiazole core in other biological activities or HIF-I related informations, please mention in the comment section so that others will get chance to know more about this core.

Source
Xia, M., Bi, K., Huang, R., Cho, M. H., Sakamuru, S., Miller, S. C., Li, H., Sun, Y., Printen, J., Austin, C. P., Inglese, J. Molecular Cancer, 2009, 8, 117.
January 04, 2010
7:33
Reawakening of human theta defensins (retrocyclin) against HIV-1
» ‎ Drug Discovery

Re-awakening human theta defensins (retrocyclin) by aminoglycosides against HIV-1Defensins are the small cysteine-rich cationic antimicrobial peptides that contribute to the host defense against a broad spectrum of pathogens. Position of the cysteine and the disulfide bonding pattern determines the types of defensins as alpha, beta and theta-defensins.Theta defensins such as retrocyclin are the most recently identified defensin, isolated initially from white blood cells and bone marrow of Rhesus macaques. They were produced in Rhesus macaques by the post-translation ligation of two nonapeptides, each derived from a 12 residue precursor called as "demidefensin". Image source: flickr

Retrocyclins are 18 residue cyclic peptides that act as HIV-I entry inhibitors by selectively binding to the C-terminal heptad repeat region on gp41 blocking 6-helix bundle formation.
In human, alpha and beta defensins actively contribute to the innate immune defenses against microbial and viral infections. But human theta-defensin genes have a premature termination codon that blocks translation, thus exist as pseudogenes. As a result, retrocyclins encoded within human genome are not expressed as peptides. Venkataraman et al. from Burnett College of BiomedicalSciences;University of Central florida, U. S. revealed that correction of the premature termination codon in theta-defensin pseudogenes provoke human myeloid cells to produce cyclic, antiviral retrocyclins. They have used aminoglycosides like amikacin, gentamicin and tobramycin to read-through the premature termination codon found in the mRNA transcripts.
Tobramycin was the most effective among them. It produces 26-fold increase in read-through. They also found that cells treated with gentamicin and tobramycin significantly inhibited HIV-1 infection as compared to untreated cells.
"Authors concluded that reawakening of retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-I infection".

Sources:
1. Venkataraman, N., Cole, A. L., Ruchala, P., Waring, A. J., Lehrer, R. I., Stuchlik, O., Pohl, J., Cole, A.M. Plos Biology, 2009, 7, 720.
2. Munk, C., Wei, G., Yang, O. O., Waring, A. J., Wang, W., Hong, T., Lehrer, R. I., Landau, N. R.,Cole, A. M. AIDS Res. Hum. Retroviruses, 2003, 19, 875.
December 27, 2009
7:20
Metal complexes of Salen ligand bearing ferrocenophane
» ‎ Drug Discovery

Combination of chiral metal-salen complexes and ferrocenophane
I hope you had a great Christmas time. Today, in this post, I am going to summarize the synthesis of Metal-Salen complexes bearing a ferrocenophane substituent as reported by Patti et. al. You can check out the previous post on anticancer activity of polyphenolferrocenophane.Chiral N, N'-bis(salicylcidene)ethylendiamine (salen) compounds are a very popular ligands because of their easy formation and rich coordination chemistry with a large variety of metal ions, that has allowed their use as catalysts in different asymmetric reactions. Similarly, ferrocenyl compounds has unique electronic, steric and chemical properties which has been used to synthesize a great variety of derivatives.Interest in the preparation of chiral ferrocenes as well as development of new salen catalysts encouraged authors to synthesize novel compounds bearing both skeleton.Salen3Dstructure source

In this study, first ferrocenophane was synthesized and finally converted to Metal-Salen complexes.
Synthesis of ferrocenophane (4) involves Claisen-schmidt condensation reaction followed by Michael addition reaction. 1, 1’-diacetylferrocene (1) was reacted with aldehyde (2) in the presence of NaOH to give compound 3. Deoxygenation of 3 by treatment with excess borane and removal of benzyl group by reduction over Pd/C and H2 gas yield ferrocenophane (4).Using Riemar-tiemann conditions, a selective conversion of 4 to 5 was achieved. Ligand 6 was prepared by condensation of 5 with (+)-(1R, 2R)-diphenylethylendiamine. Finally, 6 was treated with various metal derivatives to give corresponding metal complexes.

Source:
Molecules, 2009, 14, 4312-4325; doi:10.3390/molecules14114312
December 18, 2009
10:44
Exploration of old drugs for new pharmacological activity
» ‎ Drug Discovery

Study of Ritonavir (HIV protease inhibitor) for anticancer activity

"The most fruitful basis for the discovery of a new drug is to start with an old drug"-by Pharmacologist and Nobel Laureate James Whyte Black.
Starting with the quote by J. W. Black, I would like to emphasize on ritonavir as an example of drugs being explored for new pharmacological activity. Ritonavir, a HIV protease inhibitor, was discovered in 1998 by Kemf et. al., Abott laboratories, Illinois. It is one of the drug (as protease inhibitor) used in highly active antiretroviral therapy (HAART) along with reverse transcriptase inhibitors.

Beside its antiretroviral activity, Ikezoe et. al., reported that ritonavir enhanced the antiproliferative and proapoptic effects of docetaxel in the hormonally independent DU145 prostate cancer cells in vitro and in vivo. Ritonavir blocked the docetaxel-induced expression of CYP3A4, which ultimately enhanced the efficacy of docetaxel, a substrate to CYP3A4. Similarly in 2006, Srirangam et. al discovered that ritonavir inhibits breast cancer growth. They found that effect was through the inhibition of Hsp90 substrates including Akt. In an endeavour to discover the potential of ritonavir as an anticancer agent, Kumar, et. al. recently reported that it possess anti-proliferating activity against ovarian cancer cell line in vitro by the induction of growth arrest and apoptosis. Not only its growth, it inhibited invasion and migration of these cell lines.
From these reports, there is a hope that ritonavir can be a promising candidate for repositioning as an anticancer agent. As it has already been clinically approved for human use, time and cost invested on its toxicological and pharmacokinetic assessments can be avoided. Thus it will be faster and cost effective to bring the drug from bench to bedside.

References
1. Tonse NK Raju. The Lancet, 2000, 355, 1022.
2. Kempf, D. J., Sham, H. L., Marsh, K. C., Flentge, C. A., Betebenner, D., Green, B. E., McDonald, E., Vasavanonda, S., Saldivar, A., Wideburg, N. E., Kati, W. M., Ruiz, L., Zhao, C., Fino, L. M., Patterson, J., Molla, A., Plattner, J. J., Norbeck, D. W. J. Med. Chem., 1998, 41, 602.
3. Ikezoe, T., Hisatake, Y., Takeuchi, T., Ohtsuki, Y., Yang, Y., Said, J. W., Taguchi, H., Koeffler, H. P. Cancer research, 2004, 64, 7426.
4. Srirangam, A., Mitra, R., Wang, M., Gorski, J. C., Badve, S., Baldridge, L. A., Hamilton, J., Kishimoto, H., Hawes, J., Li, L., Orschell, C. M. Clin. Cancer. Res. 2006, 12, 1883.
5. Kumar, S., Bryant, C. S., Chamala, S., Qazi, A., Seward, S., Pal, J., Steffes, C. P., Weaver, D. W., Morris, R., Malone, J. M., Shammas, M. A., Prasad, M., Batchu, R. B. Molecular Cancer, 2009, 8, 26.
"Hope to see your feedback"
December 12, 2009
5:46
Broccoli as a source of anticancer agents
» ‎ Drug Discovery

Anticancer activity of broccoli
Most of the people are aware of healthy benefits of broccoli but the active constituents which makes broccoli to possess anticancer property may not be well known. I hope this piece of writing, in some extent, will make familiar to active anticancer agents found in broccoli. Here, the anticancer effect of Selenium (Se)-enriched broccoli will be highlighted according to the work done by researcher from Gunma University, Japan (Abdulah, et al.). Image: Darwin Bell, Flickr

As a member of Se-accumulator Brassica family, broccoli accumulates Se-methylselenocysteine as the major Se compound when it is germinated in Se-enriched media. Therefore, Se-enriched broccoli accumulates two active anticancer agents: sulforaphane and Se-methylselenocysteine. The anticancer property of Sulforaphane, belonging to isothiocyanates and Se-methylselenocysteine has already been reported (Nishikawa, et. al, and Kim et. al. respectively). Recently, broccoli sprouts have received considerable attention, because they contain ten times more sulforaphane than broccoli florets. Many studies have shown that both cruciferous vegetables and selenium may reduce the incidence of prostate cancer. Thus, in this study, antiproliferative study of a control broccoli sprout extract (CSp) and a Se-enriched broccoli sprout extract (SeSp) was explored. It was found that SeSp extract contain Se in the form of Se-methylselenocysteine. It is a mono-methylated Se and is a precursor of methylselenol, an important metabolite in Se chemoprevention.

Anticancer properties of broccoli sprouts occur through their primary active micronutrient, sulforaphane, by the induction of mitochondrial apoptosis pathway, inhibition of histone deacetylase (HDAC), and the induction of phase II enzymes like glutathione S-transferase (GST) and quinone oxidoreductase 1 (NQO1).
Broccoli sprout extracts inhibit the growth of prostate cancer cell lines in a dose dependent manner. The addition of Se enhances the antiproliferative effects of broccoli sprouts. In all three prostate cancer cell lines tested, SeSp was superior to CSp in inhibiting cell proliferation. The inhibition of carcinogenesis was mediated through the regulation of multiple signaling pathways. It induces a G2/M-phase arrest in prostate cancer cell lines that is accompanied by an increase in sub-G1 cells, which represent a cell death population. Downregulation of Akt/mTOR pathway by SeSp (not CSp), was found to enhance the ability of apoptosis-related proteins to initiate the apoptosis machinery.Authors concluded that Se-enriched broccoli sprout have potential as a chemopreventive agent for prostate cancer management, although further studies will be necessary related to its safety.
References1. Abdulah, R., Faried, A., Kobayashi, K., Yamazaki, C., Suradji, E. W., Ito, K., Suzuki, K., Murakami, M., Kuwano, H., Koyama, H. BMC Cancer, 2009, 9, 414.
2. Kim, T., Jung, U., Cho, D. Y., Chung, A.-S. Carcinogenesis, 2001, 22, 4, 559-565.
3. Nishikawa, T., Tsuno, N. H., Tsuchiya, T., Yoneyama, S., Yamada, J., Shuno, Y., Okaji, Y., Tanaka, J., Kitayama, J., Takahashi, K., Nagawa, H. Ann Surg Oncol. 2009, 16, 534–543.
December 05, 2009
1:57
Thaspine: A promising anticancer agent from Croton Lechleri
» ‎ Drug Discovery

Thaspine as a dual topoisomerase inhibitorThe active alkaloid, thaspine (taspine), from the cortex of the tree Croton lechleri also known as “Sangre de grado” (blood of the dragon in Spanish) has shown to be dual topoisomerase inhibitor. It is also effective in the cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids. Using modified M30-Apoptosense method, Fayad, W., et al, has screened various natural compounds that induce apoptosis of colon carcinoma cells. They found that thaspine induced strong caspase-cleavage of cytokeratin-18 in HCT116 cells. The concentration requirement is similar to that of other cancer therapeutic drugs such as cisplatin, doxorubicin and mechlorethamine for induction of caspase activity of this cell line. Thaspine was also observed to induce tumor apoptosis in vivo. Thaspine induced the apoptosis via mitochondrial apoptosis pathway.
“Thaspine causes reduction of mitochondrial cytochrome C and an increase level in the cytosol (a hallmark of the mitochondrial apoptosis pathway). Thaspine also induced conformational activation of Bak and Bax, Bcl-2 proteins, which are key regulators of the mitochondrial apoptosis pathway.”
Topoisomerase inhibitory activity was done using in vitro enzyme assays. It inhibits both topoisomerase I and II activity at the apoptic concentration (10 µM). Connectivity map analysis showed that thaspine induced a similar gene expression pattern as the topo inhibitors ellipticine (topo II) and camptothecin (topo I) and interestingly, thaspine was also cytotoxic to cell lines overexpresing the PgP and MRP drug efflux transporters.What is Croton lechleri?Croton lechleri grows in the upper Amazon region of Peru, Ecuador, and Colombia. Dark red resin was harvested by cutting or scratching the trunk of the tree (as shown in the video). The resin was used by tribes in the Amazonas basin to stop bleeding, healing wound, treat intestinal disorder, inflammatory diseases and cancer. In the study by Marino, D. S. et. al.,total 15 compounds were isolated from Croton lechleri: 3 megastigmanes, 4 flavan-3-ols, 3 phenylpropanoids, 3 lignans, a clerodane, and the alkaloid thaspine.References1. Fayad, W., Fryknas, M., Brnjic, S., Olofsson, M. H., Larsson, R., Linder, S. Plos one, 2009, 4, 1. 2. Marino, S. D., Gala, F., Zollo, F., Vitalin, S., Fico, G., Visioli, F., Iorizzi, M. Molecules, 2008, 13, 1219.
November 24, 2009
6:18
Active constituents of Saussurea lappa as protein tyrosine phosphatase 1B inhibitors
» ‎ Drug Discovery

Active constituents from Saussurea lappa (costus, kut, kuth) useful in diabetes
Protein-tyrosine phosphatases (PTPases) are enzymes that remove phosphate groups from phosphorylated tyrosine on proteins. Among many PTPases, PTP1B plays an important role in inhibition of insulin signaling. These enzymes bind with insulin receptor (IR) and dephosphorylate the beta subunit leading to the inactivation of IR and termination of insulin signal. They are viewed as potent target for the design of various agents in treating type II diabetes and obesity.
Recently, it has been reported that betulinic acid (1), its methyl ester (2) and guaiane sesquiterpenoids: mokko lactone (3) and dehydrocostuslactone (4) from the roots of Saussurea lappa C.B. Clarke have significant PTP1B inhibitory activity. (Molecules, 2009, 14, 266-272; doi:10.3390/molecules14010266).

In this study total 13 compounds were isolated by bioassay-guided fractionation of a MeOH extract of S. lappa root. PTP1B inhibitory activity was performed for all 13 compounds and among them only above mentioned compounds showed the significant PTP1B inhibition ratio as compared to ursolic acid.

References
1. Dadke, S.; Kusari, J.; Chernoff, J. J. Biol. Chem. 2000, 275, 23642.
2. Johnson, T. O.; Ermolieff, J.; Jirousek, M. R. Nat. Rev. Drug Discov. 2002, 1, 696.
November 17, 2009
23:14
Hydrogenation demonstration
» ‎ Drug Discovery

Demonstration of hydrogenation using balloon
Before going to experimental section, let’s have a brief introduction about hydrogenation. Hydrogenation, addition of hydrogen atoms to a carbon-carbon multiple bonds, is used to reduce or saturate various organic compounds. Hydrogen gas is the common source of hydrogen and palladium on activated charcoal (Pd/C), platinum (Adam’s catalyst) and raney nickel are the most common catalyst. Reduction of alkene to saturated alkane is the simplest hydrogenation. Hydrogenation can also be used to reduce alkyne to alkene and finally to alkane. Use of Pd/C results complete reduction of alkyne to alkane whereas less active catalyst like Lindlar catalyst gives alkene.#Lindlar catalyst: finely divided palladium, precipitated onto calcium carbonate and then deactivated by lead acetate and quinoline.General mechanism of hydrogenation (of alkene) starts with the adsorption of H2 on the catalyst surface. After that alkene forms the complex with catalyst. Insertion of hydrogen into carbon-carbon double bond occurs and finally saturated alkane releases from the catalyst. I think I shall stop on introduction and rather come to the main aim of this post. Actually, I want to show how to setup the hydrogenation reaction using hydrogen balloon and Pd/C catalyst.At first, reagents, solvent and catalyst are kept in the RBF. Before adding catalyst to the solvent be sure to remove the air by flushing with inert gas like nitrogen. Thereafter, one end of the connector is joined to the RBF and another end (opposite to RBF) to the hydrogen balloon. Third outlet is for evacuating the RBF by connecting to the vacuum. Evacuation is repeated for few times and finally the hydrogen gas is passed by opening the upper valve. Evacuation should be done carefully. During my early days, I had bad experience of evacuating the reaction mixture and loosing the compound. The reaction was kept until the completion of starting material (TLC checking). On completion of reaction, Pd/C was removed by filtration through celite, residue washed with appropriate solvent and the filtrate was concentrated to get the desired compound. If necessary, further purification is performed.

I hope it’s helpful for you and easy to understand. Check out the video for more understanding. I was thinking to show the hydrogenation using the hydrogenation apparatus too but unable to do so. I'll try to post it very soon.
November 14, 2009
1:53
Simple but useful laboratory tool: safety disposal can
» ‎ Drug Discovery

Safety disposal can
It’s been a while since I have posted on this blog. These days I am quite busy concentrating on my lab work and not getting much time to prepare for blog post. However, I am sharing the information about the simple but quite useful tool in the laboratory; SOLVENT DISPOSAL CAN.I have been looking for good solvent disposal can for a long time. We were using normal galloon with funnel fitted on top. But because of lack of good airtight cap, unusual smell of organic solvents was surrounding the laboratory. Recently, we have new solvent disposal can with leak tight and airtight cap which eliminates this problem. This is not the only reason we need the well-closed disposal can. Closed disposal can avoids lots of hazard that may occur in laboratory. For e.g. use of closed disposal can prevent escape of various flammable solvents and avoids accidental fire. Expose to organic solvents for long time is harmful for health. Choice of good solvent disposal can saves from these harmful solvent vapors. Besides, the new disposal can has wire mesh within which stops solid materials entering into it (check out the video).

* Frequently magnetic stirrer used to fall into can (at least in my case) while pouring the solvent. Now I don’t need to worry about this, Hooray!!
I hope you will share the information regarding this topic.
November 08, 2009
2:43
Fun with chemistry
» ‎ Drug Discovery

Chemistry can be a fun
I have heard many people complaining chemistry as a boring subject. Hey! just wait, it can be a fun too. Check out the video to find out how it can be so funny? Too hilarious, especially when oxygen was carried away by Hydrogen and water molecule interact with potassium. Even though I got this video long time back, I was not able to share with you guys. So today I am going to share the funny chemistry through this post.

Always going through serious topic? Result: tired.Solution: Let's check out the video and refresh the mind!

PS. It will be great if you leave a comment regarding the funniest part of the video.
November 03, 2009
7:29
Easy way to clean NMR tube
» ‎ Drug Discovery

NMR tube cleaning
Clean NMR tube is one of the factors that determine the fineness of NMR spectra. To clean so many NMR tubes is quite boring. Simple NMR tube solvent jet washer has made the job easy, convenient and fast. Though it looks simple, one can’t ignore it.

Method
The NMR tube solvent jet cleaner is mounted in solvent reservoir. The solvent reservoir is connected to the aspirator (tap water) for generating vacuum. As we can see in the picture, jet cleaner consist of two opening. One is used for putting NMR tube and washing solvent is run through another (image). Generally, tube is washed with distilled water followed by organic solvent (acetone, methylene chloride or chloroform). After washing, NMR tube is taken out, put in flat tray (aluminium foil) and kept in oven for drying (Wilmad-Lab glass recommends 125°C for only 30-45 min). Keeping NMR tube at elevated temperature damage and reshape the tube increasing the camber. It's better to avoid high temperature as much as possible.
Tubes left with samples in them for a long period of time results in more difficult cleaning method. Simple rinsing with water and organic solvent doesn't always remove the degraded or precipitated materials that has been stuck in the inner walls of tube.In such case strong mineral acids such a concentrated nitric acid should be used with precautions.
Check out the video for more understanding.

Process is quite simple but it’s very helpful for those, who are working in the field dealing with NMR. In the same way, I hope this post is helpful for you.
October 27, 2009
9:14
Mitomycin, mode of action and synthetic approach
» ‎ Drug Discovery

Mitomycins
Mitomycins, first discovered in 1958, are isolated from the extracts of genus streptomyces e.g mitomycin C is extracted from the bacterium Streptomyces lavendulae and is far the most known compound of the series. Clinically; they have been used since 1960’s and are class of very potent antibacterial and anti-cancer compounds having a broad activity against a range of tumors. They exert their biological activity through DNA alkylation and cross-linking.Biosynthesis of mitomycins
Mitomycins are biosynthesized by the combination of 3-amino-5-hydroxy benzoic acid (AHBA), D-glucosamine and carbamoyl phosphate. AHBA is also precursor for other antibiotics such as rifamycin and anamycin.Mode of action of mitomycin C

Synthetic approach
Among various synthetic approach, here Danishefsky (mitomycin K) and Fukuyama (mitomycin C) synthetic methods will be explained.
Danishefsky approach for synthesis of mitomycin K (MMK)

Addition of vinyl lithium (1) to aldehyde (2) gave nitro-carbinol (3) Further irradiation at 350 nm induced cycloaddition (hetero Diels- Alder reaction) to give compound 6. [The fused pathway led to the mitomycin C whereas bridged pathway led to FR-series (closely related to mitomycin)]. Compound 6 was oxidized with pyridinium dichromate and reacted with methylthiophenyl azide (frank-azide cycloaddition) to give triazoline 7. Thereafter reduction with L-Selectride of a lactam in the presence of a ketone followed by barton deoxygenation gave compound 8. Thiophenyl component was removed by raney nickel, which facilitate the introduction of N-methyl group of the aziridine to give compound 9. Using Peterson olefination method, exocyclic olefin was introduced and finally p-dimethoxyhydroquinone was oxidized with silver(II)pinacolate to give mitomycin K.

Fukuyama approach for synthesis of mitomycin C (MMC)

Chalcone 1 was coupled with 5-(ethylthio)-2-(trimethylsiloxy) furan in the presence of SnCl4 to give silyl enol ether 2. Compound 2 was subjected to intramolecular azide-olefin cycloaddition to yield tetracyclic aziridine 3. Reduction of 3 with DIBAL in THF followed by acetylation, ozonolysis and oxidation with RuO4 gave aldehyde 4. The aldehyde 4 was reduced with NaBH4 and treatment with trichloroacetyl isocyanate gave N-(trichloroacetyl) carbamate 5. When compound 5 was treated with NH3 in MeOH, it underwent facile ammonolysis and addition of NaBH4 gave compound 6. Methoxy group was introduced under carefully controlled acidic condition using camphor sulfonic acid to give 7. Removal of benzyl group by H2, Pd/C followed by oxidation of the phenol with DDQ gave isomitomycin A (8). Isomytomycin A was treated with saturated NH3 in MeOH to give mitomycin C.

References:
1.Jean-Christophe Andrez, Beilstein Journal of Organic Chemistry, 2009, 5, 33, 1.
2.Tohru Fukuyama ; J.Am.Chem. Soc. 1989, 111, 8303-8304
October 16, 2009
23:51
Flash chromatography
» ‎ Drug Discovery
Flash chromatographyFlash chromatography, simply defined as rapid form of preparative column chromatography, is very useful tool for the separation or purification of compounds. It was first used in 1978 (W.Clark Still et al, 1978, J. Org. Chem, 43, 14, 2923). Previously, air pressure was used but nowadays pre-packed column with automated pumps are developed. The glass columns have been replaced with prepacked plastic cartridges and systems are linked with detectors and fraction collectors.
The distinct advantages of flash chromatography over normal chromatography are:-
- It is faster, less solvent usage and greater flexibility.
- It is more convenient as automatic sample collector is employed. One doesn’t need to stay and change the collecting flask all the time (quite boring job???)
- Even though it seems expensive than normal column, it can be re-used multiple times and saves the time for making column.
- It is also safer for users’ health. Usually while doing normal column chromatography one has to use the silica gel and fill in the glass column which may cause inhalation of silica gels (not safe) but use of prepacked column avoids all these exposure.
Basic things to be considered in flash chromatographyThe solvent system is determined by normal TLC. The solvent system should be taken such that Rf of the desired compound should be in between 0.15 – 0.35, most preferably 0.35. Column volume (CV), number of column volumes require to elute the component from the column can be predicted using the TLC data as CV = 1/Rf. Use of ‘slower’ solvent system can greatly improve the resolution.Sample Loading
Generally, sample can be loaded by two methodsWet loading: In this method, sample is dissolved in the solvent of choice (should be less polar as much as possible and less quantity). The lid of the prepacked column is opened and sample is injected with pipette through the upper opening. Dry loading: It is implied usually for the compound with low solubility. The sample was dissolved in polar solvents (methylene chloride, methanol or chloroform depending upon compounds solubility) and the dry silica gel was added and mixed well. The organic solvent was evaporated and made dry leaving compound adsorbed in the silica gel. The dry silica gel was then poured in samplet cartridge and inserted to prepacked column.*Dry loading avoids the entrance of impurities to the column increasing its durability.CHECK OUT THE VIDEO
Disclaimer: Medchemblog shall have no responsibility, liability or risk for the content or implementation of any of the materials presented.
October 12, 2009
21:58
Neuroprotective Effect of Epigallocatechin-3-gallate
» ‎ Drug Discovery

On previous post (Link), the role of epigallocatechin-3-gallate (EGCG) in cancer was mentioned. Researchers have also studied its neuroprotective effect for Alzheimer disease (Kim, C.Y. et al., Arch. Pharm. Res. 32, 6, 869-881, 2009). Alzheimer's disease (AD), one of the most common types of chronic neurodegenerative disorders, is characterized by progressive loss of neurons and synapses in the brain with the appearance of extraneuronal accumulation of β-amyloid (Aβ) and intraneuronal deposition of hyperphosphorylated tau protein. Aβ peptide, a major component of senile plaques has been regarded to play a crucial role in the development and neuropathogenesis of AD. Various in vitro and in vivo studies indicate that Aβ deteriorates a variety of neuronal and glial functions leading to apoptotic death of these cells. These damages are mediated via oxidative and nitrosative stress. Several studies have demonstrated that Aβ stimulated iNOS induction and subsequent NO production in in vitro cell culture models of microglia and astrocytes. Moreover Aβ has been shown to exert synergistic action with cytokines via an NO-dependent pathway. Therefore, reducing reactive nitrogen species (RNS) level directly or indirectly could be a strategy for prevention and therapeutic intervention in AD.
Recently, cathecins have attracted significant attention because of their potential neuroprotective effects with powerful anti-oxidant and anti-inflammatory properties. Particularly, epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea polyphenol can penetrate blood brain barrier, thereby regarded as a promising candidate for age-related neurodegenerative disorders.
Researchers (Kim, C.Y. et al., Arch. Pharm. Res. 32, 6, 869-881, 2009) investigated whether EGCG could protect the BV2 microglia cells from oxidative and/ or nitrosative cell death caused by Aβ. Aβ treatment led to apoptosis in BV2 cells. EGCG pretreatment effectively ameliorated Aβ-induced cytotoxicity and manifestation of proapoptotic signals. Furthermore, BV2 cells exposed to Aβ underwent nitrosative stress which was effectively suppressed by EGCG pretreatment. Also EGCG treatment fortified cellular GSH pool. All these results suggest that EGCG may have preventive and/ or therapeutic potential in AD patients by augmenting cellular antioxidant defense capacity and attenuating Aβ-mediated oxidative and/ or nitrosative cell death.For further please refer to above mentioned paper.
October 11, 2009
20:51
Role of Gingseng on antiproliferative activity of 5-Fluorouracil
» ‎ Drug Discovery

Panax gingsengAsian ginseng (Panax ginseng C.A meyer) roots are commonly used in oriental countries for various purposes (as adaptogen or tonic, aphrodisiacs, nourishing stimulants, and in the treatment of type II diabetes, hepatitis as well as sexual dysfunction in men). Recently it has also been studied for antiulcer (Choon Sik Jeong et. al., 2003, Arch. Pharm.Res vol 26, No 11, 906-911), antiangiognesis, antiproliferation and apoptosis. This post will cover the enhancement of antiproliferative effect of 5-fluorouracil on human colorectal cancer by Gingseng (Anna B. Fishbein et. al., 2009, Arch.Pharm.Res Vol 32, No 4, 505-513). Before going to the study, let’s talk in brief about the gingseng.
Gingseng are slow growing perennial herbs. Gingseng are classified as Red gingseng (RG) and White ginseng (WG) based on the heat treatment. Former is heat treated or steamed whereas latter one unsteamed. Most of the herbalists prefer RG over WG for the treatment of various conditions. Active constituents of ginseng are various saponins commonly referred as ginsenosides. WG is grown for four to six years, and then peeled and dried to reduce the water content to 12% or less. RG is harvested after six years, is not peeled instead steamed, giving reddish-brown appearance. It is believed that steam treatment change its biochemical composition and prevent breakdown of the active ingredients.
Researchers from Tang center for herbal medicine research, university of Chicago (Anna B. Fishbein et. al., 2009, Arch.Pharm.Res Vol 32, No 4, 505-513) have found that gingseng increases the anti-proliferative effect of 5-fluorouracil (5-FU) on human colorectal cancer (HCT-116). They have found that only RG shows the enhancement of 5-FU activity. They performed the apoptosis of HCT-116 by 5-FU and RG and found that antiproliferative effects of RG and 5-FU observed in the study were not via apoptosis. This suggests that RG has a unique mechanims of action. Analyis of apoptosis was done by flow cytometry after staining with annexin V-FITC/PI. Again they tested effect of 5-FU and RG on HCT-116 cell cycle. 5-FU inhibits cell proliferation through S-phase cell cycle arrest and RG inhibited cell proliferation mostly by arresting cells in G1 phase. When 5-FU was administered with RG, it causes increase in G1 phase arrest as compared to control or 5-FU alone. This implies that synergestic antiproliferative effect of RG and 5-FU may act via multiple mechanisms.It has already been reported that ginsenoside Rg3 in RG contribute to the potent anti-proliferative effect. In this study they tried to find out the main component responsible for the activity but HPLC data didn’t show high content of Rg3. So they concluded that further study is needed for identification of the responsible ginsenoside(s) in RG which may be a promising compound for the adjuvant therapy.
October 08, 2009
13:30
Nobel Prize Chemistry 2009
» ‎ Drug Discovery
Nobel Prize chemistry 2009
The Nobel Prize in chemistry 2009 was shared by three scientist for their pioneering studies "on the structure and function of ribosome". They have revealed the structure of ribosme, key enzyme for protein synthesis, by using X-ray crystallography. Congratulation to nobel laureates for achieving prize with their great contribution

Discovery of ribosome structure is a great achievment and will be a great tool in the field of drug discovery . e.g antibacterial agents, anticancer agents etc."Nobel laureates in chemistry 2009"

Source: [www.mrc-lmb.cam.ac.uk]
- Prof. Venkatraman Ramakrishnan (MRC Laboratory of Molecular Biology Cambridge, United Kingdom)
(Source [www3.weizmann.ac.il]
- Prof. Ada E. Yonath (Weizmann Institute of Science Rehovot, Israel)
Source [www.yale.edu]
- Prof. Thomas A. Steitz (Yale University New Haven, CT ; Howard Hughes Medical Institute, United states of America)
September 29, 2009
21:06
Active compound from Impatiens balsamina
» ‎ Drug Discovery

Active anti-tumor component from leaves of Impatiens balsamina
Impatiens balsamina plants (balsam, LIB) have been widely used as traditional medicine for the treatment of aches, superficial infection, fingernail inflammation, isthmus, fractures etc. Moreover in some areas of China, people take this plant as a vegetable or anti-cancer herb, although there are currently no reports confirming the efficacy of this practice. Researcher from china (Zhi-Shan Ding et. al. , molecules, 2008, 13, 220-229) have reported the in vitro anti-tumor activity of LIB against human hepatocellular carcinoma cell line(HepG2).

As a preliminary study, they have used the Petroleum ether fraction (PEF), chloroform fraction (CHF), chloroform extract (CHE), ethyl acetate fraction (EAF), n-butanol (BUF) and water (WAF) of LIB leaves. MTT assays showed that only PEF, CHF and CHE showed some activity and were choosen for further study. But because of limited sample availability of PEF and CHF, only CHE was further studied. CHE was separate into 6 different fractions CHE1-6. Among them, CHE2 showed the most significant inhibitory activity. CHE2 showed strong cell inhibition effect as compared to curcumin too.
Purification was done for CHE2 to isolate the single active compound and found to be 2-methoxy-1, 4-naphthoquinone (MNQ). To reconfirm the anti-tumor activity of the final compound, it was tested for Hep62 inhibition ration and compared with CHE, CHE2 fractions and curcumin. The results confirmed that the MNQ has the highest activity followed by CHE2 fraction (as it contains MNQ).
Many studies has revealed that MNQ also has notable antimicrobial and antifungal activity.
For experimental sections please refer to the article mentioned above.
September 18, 2009
12:23
Delicious fruit with valuable leaves!
» ‎ Drug Discovery

Delicious fruit to therapeutically valuable leaves...
Mangifera indica L. (Anacardiaceae) is one of the most important tropical plants in the world. It is a large tree whose fruits are liked by many people and commonly called as Mango. Phytochemical research from different parts of M.indica has demonstrated the presence of various polyphenols, phytosterols, flavonoids and this species are widely used for the purpose of analgesic effect, anti-inflammatory, immunostimulant,antioxidant, antidiarrhea etc. Because of its vague biological importance, many researchers are interested in this species.This post, as per the article (Molecules, 2009, 14, 1098; doi: 10.3390/molecules14031098), reveals the antiulcerogenic action of mentioned species’ leaf decoction on different experimental models in rodents. Previously, they have used the flower part for the study but it results reduced availability of the fruits in the market so they changed it to leaf part.
As an initial study acute toxicity was determined. According to the study it was found that the aqueous decoction of M.indica did not show acute toxicity but exhibited gastroprotective effects against several ulcerogenic agents like NSAIDs, HCl/ethanol solution. After receiving positive biological assay results, the active constituents of the decoction were isolated and characterized as ;Mangiferin (C-glucopyranoside of 1, 3, 6, 7-tetrahydroxyxanthone (Mi1)

C-glucosylbenzophenone (Mi2)

Mangiferin is a xanthone with various therapeutic activities including antioxidant. It has been proposed that the catechol moiety with a 6, 7 –dihydroxylated structure, together with its aromatic bonds, is responsible for its antioxidant property (Chem.Pharm.Bull., 1992, 721). Similarly, it has been reported that benzophenones have shown the activity against H.pylori (Mol.Cell.biochem, 2003, 243).

Disclaimer: This experiment has been tested only in animal. So it’s inappropriate and unsafe for human use and is highly discouraged at the moment.
September 14, 2009
8:03
Green tea: role in cancer prevention
» ‎ Drug Discovery

Role of Green tea in cancer preventionA cup of tea in the early morning is quite refreshing. Almost all country in the world has trend of drinking tea. Generally it is divided into three basic categories; non-fermented, semi-fermented and fermented. Among these types, non-fermented tea i. e green tea has many healthy benefits.
Fuzuki et al. has reported that green tea helps in cancer prevention (Cancer lett. 2002, 188, 9). It has been revealed that green tea showed its anti-cancer activity through inhibiton of mitogen-activated protein kinases (MAPK), growth factor-related cell signaling, activation of activator protein 1 ( AP-1) and nuclear factor-B (NF-kappa B), topoisomerase I, matrix metalloproteinases and other potential targets.

Green tea is made with leaves of Camellia sinensis. It originates from China and spread throughout the other asian countries like Japan, Korea and recently to many western countries. Green tea is composed of polyphenols especially catechins, among which the most abundant is epigallocatechin-3-galate (EGCG). EGCG is the most effective chemopreventive agent.

In this paper, the role of EGCG in cancer prevention has been summarized;Role of EGCG in cancer initiation stageOncogene mutation and reactive oxygen species (ROS) plays an important role in cancer initiation. When oncogene mutation occurs, the procacinogen become activated via the activation of phase I enzymes such as the cytochrome P450s. ROS actively participate in the metabolic activation of procarcinogens. EGCC can neutralize these procarcinogens by inhibiting the activity of cytochrome P450 enzymes and modulating ROS. It has been demonstrated that EGCG is a powerful antioxidant.Role of EGCG in cancer promotion stageEGCG possess its anticancer effect by interfering with many signaling pathways and modulating cell cycle.Role of EGCG in cancer progression stageDuring progression of cancer, apoptosis and enzymes like urokinase and metalloproteinase (MMPs) plays an important role. EGCG can inhibit the proliferation of cancer cells by inducing apoptosis and inhibiting the function of these enzymes. Recently Kuzuhara et. al. (J. Biol. Chem. 2006, 282, 17446) proposed that DNA and RNA served as new binding targets of EGCG. Because of its diverse role, EGCG can be developed as multi-targeted anticancer agents and can act as a promising template for the design of various anticancer derivatives.(Source: Molecules 2007, 12, 946-957 )

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