Facile synthesis of rapamycin-peptide conjugates as mTOR and Akt inhibitors.

A simple and straightforward process for the synthesis of rapamycin peptide conjugates in a regio and chemoselective manner was developed. The methodology comprises the tagging of chemoselective functionalities to rapamycin and peptides which enables the conjugation of free peptides, without protecting the functionality of the side chain amino acids, in high yield and purity. From this methodology, we successfully conjugate free peptides containing up to 15 amino acids. Rapamycin is also conjugated to the peptides known for inhibiting the kinase activity of Akt protein. These conjugates act as dual target inhibitors and inhibit the kinase activity of both mTOR and Akt.

Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling.

BACKGROUND: The mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking. METHODS: Protein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion. RESULTS: Here, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling. CONCLUSIONS: Our study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.

Regioselective ß-Csp3-Arylation of ß-Alanine: An Approach for the Exclusive Synthesis of Diverse ß-Aryl-ß-amino Acids.

An approach for the synthesis of a variety of new ß-aryl-ß-amino acids has been developed via a palladium-catalyzed auxiliary-directed regioselective Csp3-H arylation of the unactivated ß-methylene bond of ß-alanine. The use of 8-aminoquinoline amide as an auxiliary efficiently directs the desired regioselective ß-Csp3-H functionalization. The developed protocol enables the easy and straightforward access to several high-value ß-aryl-ß-amino acids useful for peptide engineering, starting from inexpensive and readily available ß-alanine precursors in moderate to excellent yields.

Pyrrolidine ring puckering and prolyl amide bond configurations of 2-methyl-allo-hydroxyproline-based dipeptides.

An expeditious method for the synthesis of homo and heterochiral dipeptides containing l-alanine and d/l 2-methyl allo-hydroxyl prolines was developed using direct aminolysis of bicyclic lactones derived from d/l alanine. The impact of C-2 methylation and its spatial orientation on the pyrrolidine ring puckering and prolyl amide bond configuration was ascertained by solution NMR studies. The present studies reveal that C-2 methylation causes the prolyl amide bond to exist exclusively in the trans geometry in both homo- and heterochiral dipeptides. However, the spatial orientation of the C-2 methyl group and its i + 2 position in appropriately capped model dipeptides may nucleate into a turn like structure.

AzaGly-Appended Peptidomimetics Structurally Related to PTR6154 as Potential PKB/Akt Inhibitors.

We report the synthesis and biological and physiochemical properties of a series of azaGly-appended peptidomimetics. We have developed a simple and facile synthesis for azapeptides on solid support without any side reaction. The azaGly is inserted by in situ reaction of disuccinimidyl carbonate with free amine followed by treatment of hydrazine hydrate at room temperature. The new series of peptidomimetics was prepared by azaGly scanning of heptapeptide Arg-Pro-Arg-Nle-Tyr-Dap-Nle (Akt-01), a GSK-3ß-derived Akt inhibitor. The azaGly-appended peptides showed significant improvement in biological activity and serum stability, with retention of conformation as evidenced by NMR and CD studies. The results clearly demonstrate that azaGly-appended peptides are new peptidomimetics. Their synthesis makes this approach highly useful for the development of novel peptidomimetics of therapeutic potential.

Stereoselective Synthesis of (R)-3-Methylthalidomide by Piperidin-2-one Ring Assembly Approach.

A simple and stereoselective synthesis of 3-methylthalidomide, a configurationally stable thalidomide analog, is presented. Herein we describe the synthesis of (R)-3-methylthalidomide starting from (S)-alanine by piperidin-2-one ring assembly approach in high yield and enantiomeric purity without using a chiral auxiliary or reagent. Starting from (R)-alanine, the corresponding (S)-3-methylthalidomide can be prepared using the same methodology.

Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant strain.

Emergence and spread of multidrug resistant strains of Plasmodium falciparum has severely limited the antimalarial chemotherapeutic options. In order to overcome the obstacle, a set of new side-chain modified 4-aminoquinolines were synthesized and screened against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum. The key feature of the designed molecules is the use of methylpiperazine linked α, ß(3)- and γ-amino acids to generate novel side chain modified 4-aminoquinoline analogues. Among the evaluated compounds, 20c and 30 were found more potent than CQ against K1 and displayed a four-fold and a three-fold higher activity respectively, with a good selectivity index (SI=5846 and 11,350). All synthesized compounds had resistance index between 1.06 and >14.13 as against 47.2 for chloroquine. Biophysical studies suggested that this series of compounds act on heme polymerization target.

Synthesis of chiral chloroquine and its analogues as antimalarial agents.

In this investigation, we describe a new approach to chiral synthesis of chloroquine and its analogues. All tested compounds displayed potent activity against chloroquine sensitive as well as chloroquine resistant strains of Plasmodium falciparum in vitro and Plasmodium yoelii in vivo. Compounds S-13 b, S-13c, S-13 d and S-13 i displayed excellent in vitro antimalarial activity with an IC50 value of 56.82, 60.41, 21.82 and 7.94 nM, respectively, in the case of resistant strain. Furthermore, compounds S-13a, S-13c and S-13 d showed in vivo suppression of 100% parasitaemia on day 4 in the mouse model against Plasmodium yoelii when administered orally. These results underscore the application of synthetic methodology and need for further lead optimization.

Augmentation of antileishmanial efficacy of miltefosine in combination with tuftsin against experimental visceral leishmaniasis.

Current drugs for the treatment of visceral leishmaniasis are inadequate, and their efficacies are also compromised due to suppression of immune function during the course of infection. Miltefosine is the only promising orally active antileishmanial drug, but due to its long half-life, there is risk of development of resistance. To overcome these problems, efforts are needed to develop combination therapy of miltefosine with effective immunostimulating agents where a decrease of parasitic burden and simultaneous enhancement of adaptive immunity can be achieved. In the present study, we have explored the antileishmanial efficacy of a subcurative dose of miltefosine in combination with free as well as liposomal palmitoyl tuftsin (p-tuftsin) using a Leishmania donovani/BALB/c mouse model. When miltefosine (2.5 mg/kg for 5 days) was given with free p-tuftsin, the inhibitory effect was significantly increased from 49.6% to 66% (P < 0.01), which was further enhanced up to 81% (P < 0.001) when given after liposomal encapsulation of p-tuftsin. Significant enhancement in parasitic inhibition (93%, P < 0.01) was witnessed when animals were co-administered with liposomal p-tuftsin + 5 mg/kg × 5 days dose of miltefosine (72.1%). Enhancement in the production of Th1 cytokines (IL-12, TNF-α, and IFN-γ), reactive oxygen, and nitrogen metabolites was witnessed in the combination group. A remarkable increase in phagocytosis index was also observed indicating overall immunological enhancement to antileishmanial activity of miltefosine by p-tuftsin.

A facile and chemoselectivity in synthesis of 4-chloro-N-(4-((1-hydroxy-2-methylpropan-2-yl)oxy)phenethyl)benzamide, the alcohol derivative of Bezafibrate.

A facile method for the reduction of carboxylic acid group of Bezafibrate, an approved drug, is described. The selective reduction of carboxylic acid group to corresponding alcohol was carried out by activation of the carboxylic acid moiety via mixed anhydride followed by the addition of stoichiometric amount of NaBH4 and methanol to obtain the first alcohol variant of Bezafibrate. The reaction was completed in 5-10 min in excellent yield and purity. The new alcohol derivative was characterized by spectroscopic methods. This is the first report on this new molecule.

Chemoselective Reduction of Fenofibric Acid to Alcohol in the Presence of Ketone by Mixed Anhydride and Sodium Borohydride.

A highly efficient and facile protocol for the selective reduction of carboxylic acid of Fenofibric acid to corresponding alcohol was developed. The selective reduction was carried out by activation of carboxylic acid by mixed anhydride followed by the reaction of sodium borohydride in presence of methanol. This is the first example of chemoselective reduction of carboxylic acid to alcohol in presence of a ketone without any external catalyst or ligand in a single step. The reaction offers wide applicability for the selective carboxylic group reduction methodology. The chemoselective reduction was demonstrated by the reduction of Fenofibric acid, an active metabolite of the drug Fenofibrate, to corresponding alcohol in excellent selectivity, yield, and purity.

Synthesis and Antimalarial Activity of 4-Methylaminoquinoline Compounds against Drug-Resistant Parasite.

A series of novel 4-aminoquinoline analogues bearing a methyl group at 4-aminoquinoline moiety were synthesized via a new and robust synthetic route comprising in situ tert-butoxycarbonyl (Boc) deprotection-methylation cascade resulting in the corresponding N-methylated secondary amine using Red-Al and an efficient microwave-assisted strategy for the fusion of N-methylated secondary amine with 4-chloroquinoline nucleus to access the series of novel 4-N-methylaminoquinoline analogues. The new series of compounds were evaluated for their antimalarial activity in in vitro and in vivo models. Among 21 tested compounds, 9a-i have shown a half-maximal inhibitory concentration (IC50) value less than 0.5 µM (i.e., <500 nM) against both chloroquine-sensitive strain 3D7 and chloroquine-resistant strain K1 of Plasmodium falciparum with acceptable cytotoxicity. Based on the in vitro antimalarial activity, selected compounds were screened for their in vivo antimalarial activity against Plasmodium yoelii nigeriensis (a multidrug-resistant) parasite in Swiss mice. Most of the compounds have shown significant inhibition on day 4 post infection at the oral dose of 100 mg/kg. Compound 9a has shown 100% parasite inhibition on day 4, and out of five treated mice, two were cured till the end of the experiment. The present study suggests that 4-methylamino substitution is well tolerated for the antiplasmodial activity with reduced toxicity and therefore will be highly useful for the discovery of a new antimalarial agent against drug-resistant malaria.

Tuftsin-Bearing Liposomes Co-Encapsulated with Doxorubicin and Curcumin Efficiently Inhibit EAC Tumor Growth in Mice.

BACKGROUND: Targeted multidrug-loaded delivery systems have emerged as an advanced strategy for cancer treatment. In this context, antibodies, hormones, and small peptides have been coupled to the surface of drug carriers, such as liposomes, polymeric and metallic nanoparticles loaded with drugs, as tumor-specific ligands. In the present study, we have grafted a natural macrophage stimulating peptide, tuftsin, on the surface of the liposomes (LPs) that were loaded with doxorubicin (DOX) and/or curcumin (CUR), by attaching to its C-terminus a palmitoyl residue (Thr-Lys-Pro-Arg-CO-NH-(CH2)2-NH-COC15H31, P.Tuft) to enable its grafting within the liposome's bilayer. METHODS: The prepared drug-loaded liposomes (DOX LPs, CUR LPs, DOX-CUR LPs, P.Tuft-LPs, P.Tuft-DOX LPs, P.Tuft-CUR LPs, P.Tuft-DOX-CUR LPs) were thoroughly characterised in terms of particle size, drug content, encapsulation efficiency and structural properties using UV-visible spectroscopy, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). The anti-cancer activity and drug toxicity of the liposomal formulations were examined on Ehrlich ascites carcinoma (EAC) tumor-induced mice model. RESULTS: A significant reduction in the tumor weight and volume was observed upon treating the tumor-bearing mice with palmitoyl tuftsin-grafted dual drug-loaded liposomes (P.Tuft-DOX-CUR LPs), as compared to the single drug/peptide-loaded formulation (DOX LPs, CUR LPs, DOX-CUR LPs, P.Tuft- LPs, P.Tuft-DOX LPs, P.Tuft-CUR LPs). Western blot analysis revealed that the tumor inhibition was associated with p53-mediated apoptotic pathway. Further, the biochemical and histological analysis revealed that the various liposomal preparation used in this study were non-toxic to the animals at the specified dose (10mg/kg). CONCLUSION: In conclusion, we have developed a targeted liposomal formulation of P.Tuftsin-bearing liposomes co-encapsulated with effective anti-cancer drugs such as doxorubicin and curcumin. In experimental animals, tumor inhibition by P.Tuft-DOX-CUR LPs indicates the synergistic therapeutic effect of the peptide and the dual drug.

Synthesis and biological activity of Ub2 derived peptides as potential host-directed antitubercular therapy.

The correlation of mycobactericidal property of macrophages with its potential to deliver bacteria to hydrolytic lysosomes, augmented with ubiquitin-derived peptides (Ub2), activates the process of autophagy. This leads to the formation of phagolysosomes supported by factor involving increased cationic charges which regulate the acidic pH causing elimination of Mycobacterium. To better understand this interaction of cationic-rich ubiquitin-derived peptides with mycobacteria and to identify putative mycobacterial intrinsic resistance mechanisms for phagolysosome formation, we have synthesized a new series of Ub2 peptides, wherein the Gly residues are replaced with azaGly with the aim to improve metabolic stability. In addition to that a new methodology is reported for the synthesis of heteroaryl tethered peptides using azaGly as a linker. We have demonstrated that positive puncta (directly proportional to the acidification of lysosome) in cytosol was significantly increased after 6 hours on the treatment of macrophage with Ub2 peptide derivatives (1, 6, 10, and 11) causing the higher intensity of lysosome observed through LysoTracker Red Dye. The circular dichroism spectral studies are carried out in water and water:TFE mixture and demonstrated that the Ub2 peptides have helix-forming tendency in the presence of TFE. The recognizable intracellular killing of Mycobacterium tuberculosis by Ub2 peptides provides a new approach for host-directed therapy.

Immunogenicity and Protective Efficacy of T-Cell Epitopes Derived From Potential Th1 Stimulatory Proteins of Leishmania (Leishmania) donovani.

Development of a suitable vaccine against visceral leishmaniasis (VL), a fatal parasitic disease, is considered to be vital for maintaining the success of kala-azar control programs. The fact that Leishmania-infected individuals generate life-long immunity offers a viable proposition in this direction. Our prior studies demonstrated that T-helper1 (Th1) type of cellular response was generated by six potential recombinant proteins viz. elongation factor-2 (elF-2), enolase, aldolase, triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and p45, derived from a soluble antigenic fraction (89.9-97.1 kDa) of Leishmania (Leishmania) donovani promastigote, in treated Leishmania patients and golden hamsters and showed significant prophylactic potential against experimental VL. Moreover, since, it is well-known that our immune system, in general, triggers production of specific protective immunity in response to a small number of amino acids (peptide), this led to the identification of antigenic epitopes of the above-stated proteins utilizing immunoinformatics. Out of thirty-six, three peptides-P-10 (enolase), P-14, and P-15 (TPI) elicited common significant lymphoproliferative as well as Th1-biased cytokine responses both in golden hamsters and human subjects. Further, immunization with these peptides plus BCG offered 75% prophylactic efficacy with boosted cellular immune response in golden hamsters against Leishmania challenge which is indicative of their candidature as potential vaccine candidates.

Inhibiting wild-type and C299S mutant AKR1B10; a homologue of aldose reductase upregulated in cancers.

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

New acylides: synthesis of 3-O-[gamma-(4-oxo-2-aryl-thiazolidin-3-yl)butyryl]erythromycin A derivatives.

In search of new erythromycin derivatives 3-O-[gamma-(4-oxo-2-aryl-thiazolidin-3-yl)butyryl]erythromycin A derivatives have been synthesized. The 3-hydroxy group was derivatised to a primary amine and subsequently the thiazolidinone nucleus was generated at the amino functionality through DCC mediated one-pot three-component reaction in good yields.

IAP Proteins Antagonist: An Introduction and Chemistry of Smac Mimetics under Clinical Development.

BACKGROUND: Smac mimetics (also known as IAP antagonist) are a new class of targeted drugs having a goal to suppress the IAPs, reestablishing the apoptotic pathways and inducing cancer cell death. Therefore, development of Smac mimetics was considered an attractive strategy for the development of new anticancer drugs. Lots of reviews have come in yesteryears which mainly discussed the biology of IAPs and their role in cancer development. None of these reviews focused on the chemical synthesis of Smac mimetics. METHODS: Literature study was done by using standard bibliographic search engines like scifinder, pubmed etc. The characteristic features of screened articles were described in the review. RESULTS: The review gives an introduction of IAP proteins and Smac mimetics. Readers will gain an overview of the development of Smac mimetics with representative examples of both monovalent and bivalent Smac mimetics as anticancer agents and an understanding of their structure-activity relationships. Chemical synthesis of biologically important Smac mimetics was discussed briefly in this review. CONCLUSION: Small molecules that mimic Smac are continuously progressing towards clinical development. Smac mimetics are generally well tolerated and have demonstrated rapid suppression of their target (the IAPs), activation of apoptosis and anti-tumor activity. Continuous research has been done to generate even more insight into the function of IAP proteins to significantly enhance the therapeutical potential of Smac mimetics.

Synthesis and Chiral Separation of Fibratol, Isopropyl 2-(4-((4-chlorophenyl)(hydroxyl) methyl)-phenoxy)-2-methylpropanoate.

Practical synthetic route for the formation of enantiomeric mixture of Isopropyl 2-(4-((4-chlorophenyl)(hydroxyl)methyl)phenoxy)-2-methylpropanoate (Fibratol 2a/b) from isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoate (Fenofibrate 1) has been developed. Method has also been established for the chiral separation of enantiomers of Fibratol 2a/b that is synthesized using the route mentioned above. The optical activity determined for enantiomerically separated Fibratol (2a) and Fibratol (2b) are -5.2° and 8.0° which reflect their ability to rotate plane polarized light counterclockwise (levo) and clockwise (dextro), respectively.

Diastereoselective Synthesis of 5-Heteroaryl-Substituted Prolines Useful for Controlling Peptide-Bond Geometry.

A versatile diastereoselective Friedel-Crafts alkylation reaction of heteroaryl systems with a cyclic enecarbamate for the preparation of 5-heteroaryl-substituted proline analogues in good yields has been developed. These heterocyclic tethered cyclic amino acid building blocks constitute important structural motifs in many biologically active molecules. The impact of the substitution on proline cis/trans isomerization was explored by carrying out solution conformational studies by NMR on 5-furanyl-substituted proline-containing peptides. Conformational analysis revealed that the peptide bond is constrained in an exclusively trans conformation.