O-Aryl α,ß-d-ribofuranosides: synthesis & highly efficient biocatalytic separation of anomers and evaluation of their Src kinase inhibitory activity.

A series of peracetylated O-aryl α,ß-d-ribofuranosides have been synthesized and an efficient biocatalytic methodology has been developed for the separation of their anomers which was otherwise almost impossible by column chromatographic or other techniques. The incubation of 2,3,5-tri-O-acetyl-1-O-aryl-α,ß-d-ribofuranoside with Lipozyme® TL IM immobilized on silica led to the selective deacetylation of only one acetoxy group, viz the C-5'-O-acetoxy group of the α-anomer over the other acetoxy groups derived from the two secondary hydroxyl groups present in the molecule and also over three acetoxy groups (derived from one primary and two secondary hydroxyls of the ß-anomer). This methodology led to the easy synthesis of both, α- and ß-anomers of O-aryl d-ribofuranosides. All the arylribofuranosides were screened for inhibition of Src kinase. 1-O-(3-Methoxyphenyl)-ß-d-ribofuranoside exhibited the highest activity for inhibition of Src kinase (IC(50)=95.0µM).

Synthesis, anticancer activities, and cellular uptake studies of lipophilic derivatives of doxorubicin succinate.

A number of lipophilic 14-substituted derivatives of doxorubicin were synthesized through conjugation of doxorubicin-14-hemisuccinate with different fatty amines or tetradecanol to enhance the lipophilicity, cellular uptake, and cellular retention for sustained anticancer activity. The conjugates inhibited the cell proliferation of human leukemia (CCRF-CEM, 69-76%), colon adenocarcinoma (HT-29, 60-77%), and breast adenocarcinoma (MDA-MB-361, 66-71%) cells at a concentration of 1 µM after 96-120 h of incubation. The N-tetradecylamido derivative of doxorubicin 14-succinate (10) exhibited consistently comparable antiproliferative activity to doxorubicin in a time-dependent manner (IC(50) = 77 nM in CCRF-CEM cells). Flow cytometry analysis showed a 3-fold more cellular uptake of 10 than doxorubicin in SK-OV-3 cells. Confocal microscopy revealed that the conjugate was distributed in cytoplasmic and perinuclear areas during the first 1 h of incubation and slowly relocalized in the nucleus after 24 h. The cellular hydrolysis study showed that 98% of compound 10 was hydrolyzed intracellularly within 48 h and released doxorubicin.

4-Aryl-4H-naphthopyrans derivatives: one-pot synthesis, evaluation of Src kinase inhibitory and anti-proliferative activities.

BACKGROUND: A series of 2-amino-4-aryl-4H-benzo[h or f]chromene-3-carbonitrile derivatives were synthesized and evaluated for inhibition of Src kinase and cell proliferation in breast carcinoma (BT-20) cell lines. METHODS: The one-pot, three-component reaction of α or ß-naphthol, malonitrile and an aromatic aldehyde in the presence of diammonium hydrogen phosphate was afforded the corresponding 2-amino-4-aryl-4H-benzo[h or f]chromene-3-carbonitrile derivatives, All target compounds were evaluated for inhibition of Src kinase and cell proliferation in breast carcinoma (BT-20) cell lines. RESULTS: Among all tested compounds, unsubstituted 4-phenyl analog 4a showed Src kinas inhibitory effect with IC50 value of 28.1 µM and was the most potent compound in this series. In general, the compounds were moderately active against BT-20. 3-Nitro-phenyl 4e and 3-pyridinyl 4h derivatives inhibited the cell proliferation of BT-20 cells by 33% and 31.5%, respectively, and found to be more potent compared to doxorubicin (25% inhibition of cell growth). CONCLUSION: The data indicate that 4-aryl-4H-naphthopyrans scaffold has the potential to be optimized further for designing more potent Src kinase inhibitors and/or anticancer lead compounds.

4-Aryl-4H-chromene-3-carbonitrile derivatives: evaluation of Src kinase inhibitory and anticancer activities.

Src kinase mutations and/or overexpression have been implicated in the development of a number of human cancer including colon, breast, and lung cancers. Thus, designing potent and selective Src kinase inhibitors as anticancer agents is a subject of major interest. A series of 4-aryl substituted derivatives of 2-amino-7-dimethylamino-4H-chromene-3-carbonitrile were synthesized using one-pot reaction of appropriate substituted aromatic aldehydes, malononitrile, and 3-(dimethylamino)phenol in the presence of piperidine. All 23 compounds were evaluated for inhibition of Src kinase and cell proliferation in human colon adenocarcinoma (HT-29) and leukemia (CCRF-CEM) cell lines. Among the tested compounds, 2-chlorophenyl- (4c), 3-nitrophenyl- (4h), 4-trifluoromethyphenyl- (4i), and 2,3-dichlorophenyl- (4k) substituted chromenes showed Src kinase inhibitory effect with IC(50) values of 11.1-18.3 µM. Compound 4c was relatively selective against Src (IC(50) = 11.1 µM), when compared with selected kinases, epidermal growth factor receptor (EGFR, IC(50) > 300 µM), C-terminal Src kinase (Csk, IC(50) = 101.7 µM), and lymphocyte-specific protein tyrosine kinase (Lck, IC(50) = 46.8 µM). 3-Chlorophenyl substituted thiazole (4v) and 2-chlorophenylsubstituted thiazole (4u) chromene derivatives inhibited the cell proliferation of HT-29 and CCRF-CEM by 80% and 50% respectively, at a concentration of 50 µM. The data indicate that 4H-chromene-3-carbonitrile scaffold has the potential to be optimized further for designing more potent Src kinase inhibitors and/or anticancer lead compounds.

Hydrophobicity drives the cellular uptake of short cationic peptide ligands.

Short cationic linear peptide analogs (LPAs, prepared as Arg-C( n )-Arg-C( n )-Lys, where C( n ) represents an alkyl linkage with n = 4, 7 or 11) were synthesized and tested in human breast carcinoma BT-20 and CCRF-CEM leukemia cells for their application as targeting ligands. With constant LPA charge (+4), increasing the alkyl linkage increases the hydrophobic/hydrophilic balance and provides a systematic means of examining combined electrostatic and hydrophobic peptide-membrane interactions. Fluorescently conjugated LPA-C(11) (F-LPA-C(11)) demonstrated significant uptake, whereas there was negligible uptake of the shorter LPAs. By varying temperature (4°C and 37°C) and cell type, the results suggest that LPA-C(11) internalization is nonendocytic and nonspecific. The effect of LPA binding on the phase behavior, structure, and permeability of model membranes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine (DPPC/DPPS, 85/15) was studied using differential scanning calorimetry (DSC), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence leakage studies to gain insight into the LPA uptake mechanism. While all LPAs led to phase separation, LPA-C(11), possessing the longest alkyl linkage, was able to penetrate into the bilayer and caused holes to form, which led to membrane disintegration. This was confirmed by rapid and complete dye release by LPA-C(11). We propose that LPA-C(11) achieves uptake by anchoring to the membrane via hydrophobicity and forming transient membrane voids. LPAs may be advantageous as drug transporter ligands because they are small, water soluble, and easy to prepare.

Synthesis and biological evaluation of fatty acyl ester derivatives of 2',3'-didehydro-2',3'-dideoxythymidine.

A number of 5'-O-fatty acyl derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) were synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cytotoxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these conjugates, 5'-O-12-azidododecanoyl derivative of d4T (2), displaying EC(50) = 3.1-22.4 µM, showed 4- to 9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through ß-alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cellular uptake and anti-HIV activity of stavudine.

Fatty acyl amide derivatives of doxorubicin: synthesis and in vitro anticancer activities.

Doxorubicin is extensively used in anticancer therapy. Doxorubicin is highly hydrophilic, has short half-life, and its use is associated with severe side effects at high doses. Fatty acyl amide derivatives of doxorubicin were synthesized with the expectation to improve the lipophilicity and anticancer activity of the drug. The lipophilicity was enhanced with the increase in chain length of fatty acyl moiety. Conjugation of 4'-amino group with fatty acids through an amide bond reduced the anticancer activity in leukemia, breast, ovarian, and colon cancer cell lines, suggesting that the presence of free amino group is required for anticancer activity of doxorubicin. Dodecanoyl-doxorubicin derivative was consistently the most effective among the synthesized derivatives and inhibited the proliferation of colon (HT-29) and ovarian (SK-OV-3) cancer cells by 64% and 58%, respectively, at a concentration of 1 µM after 96 h incubation.

Synthesis of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates and evaluation of their Src kinase inhibitory and anticancer activities.

A series of two classes of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates were synthesized using click chemistry approach. All compounds were evaluated for inhibition of Src kinase and human ovarian adenocarcinoma (SK-Ov-3), breast carcinoma (MDA-MB-361), and colon adenocarcinoma (HT-29). Hexyl triazolyl-substituted 3-phenylpyrazolopyrimidine exhibited inhibition of Src kinase with an IC(50) value of 5.6 µM. 4-Methoxyphenyl triazolyl-substituted 3-phenylpyrazolopyrimidine inhibited the cell proliferation of HT-29 and SK-Ov-3 by 73% and 58%, respectively, at a concentration of 50 µM.

Click chemistry inspired one-pot synthesis of 1,4-disubstituted 1,2,3-triazoles and their Src kinase inhibitory activity.

Two classes of 1,4-disubstituted 1,2,3-triazoles were synthesized using one-pot reaction of α-tosyloxy ketones/α-halo ketones, sodium azide, and terminal alkynes in the presence of aq PEG (1:1, v/v) using the click chemistry approach and evaluated for Src kinase inhibitory activity. Structure-activity relationship analysis demonstrated that insertion of C(6)H(5)- and 4-CH(3)C(6)H(4)- at position 4 for both classes and less bulkier aromatic group at position 1 in class 1 contribute critically to the modest Src inhibition activity (IC(50) = 32-43 µM) of 1,4-disubstituted 1,2,3-triazoles.

Synthesis and evaluation of fatty acyl ester derivatives of cytarabine as anti-leukemia agents.

Cytarabine is a chemotherapeutic agent predominately used for the treatment of acute myeloid leukemia and lymphoblastic leukemia. Cytarabine is a polar nucleoside, has a short plasma half-life, and its use is associated with severe side effects. Fatty acyl derivatives of cytarabine were synthesized with the expectation to improve cellular uptake and generate derivatives with a longer duration of action. Multi-step protection and deprotection reactions of hydroxyl and amino groups and conjugation with a fatty acid (i.e., myristic acid and 12-thioethyldodecanoic acid) afforded 5'-O-substituted, 2'-O-substituted, and 2',5'-disubstituted fatty acyl derivatives of cytarabine. 2',5'-Dimyristoyl derivative of cytarabine was found to inhibit the growth of CCRF-CEM cells by approximately 76% at concentration of 1 microM after 96 h incubation.

Platelet-derived growth factor receptor beta inhibition increases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity: imatinib and TRAIL dual therapy.

BACKGROUND: There is a crucial need for better therapeutic approaches for the treatment of Ewing sarcoma (EWS). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in EWS cells in vitro. However, in vivo, acquired resistance to TRAIL is a major limiting factor. Platelet-derived growth factor receptor-beta (PDGFR-beta) is highly expressed on EWS cells. Thus, the authors evaluated whether PDGFR-beta blockade could sensitize EWS cells to TRAIL-induced apoptosis in vitro and in vivo. METHODS: The effect of combined imatinib mesylate (Gleevec) and TRAIL on EWS cell growth and apoptosis was tested in vitro. Stable PDGFR-beta knockdown in EWS cells was achieved by short-hairpin RNA transduction, and TRAIL sensitivity of these cells was measured. Expression of death receptors was measured by fluorescence-activated cell-sorting (FACS) analysis, and caspase 8 activity was evaluated by Western blot analysis. An orthotopic human xenograft model of EWS growth and spontaneous metastasis in nude mice was used to assess the in vivo affect of combined imatinib mesylate and TRAIL. RESULTS: Imatinib mesylate induced a significant TRAIL proapoptotic effect in EWS cells in vitro. Specific PDGFR-beta silencing in EWS cells enhanced the effects of TRAIL, possibly through an increase in the expression of death receptors 4 and 5. The combination of imatinib mesylate and TRAIL significantly inhibited the growth of primary tumors and decreased the incidence of spontaneous EWS pulmonary metastasis compared with either drug alone. CONCLUSIONS: PDGFR-beta blockade combined with TRAIL resulted in antihuman EWS activity in vitro and in vivo, suggesting the possibility that combining these treatments will improve anti-EWS therapy.

Synthesis, Src kinase inhibitory and anticancer activities of 1-substituted 3-(N-alkyl-N-phenylamino)propane-2-ols.

A series of 3-(N-alkyl-N-phenylamino)propan-2-ol derivatives were synthesized from epichlorohydrine in a multi-step strategy and were evaluated as Src kinase inhibitors. First, epoxy ring opening of epichlorohydrine was carried out in the presence of N-alkylanilines to yield 3-(N-alkyl-N-phenylamino)-1-chloro-propan-2-ol derivatives using Ca(OTf)2 as catalyst based on our previous studies [1]. Second, ring closure was performed under basic conditions to afford N-epoxymethyl N-alkylaniline derivatives. Finally, the epoxide ring opening with four different secondary amines and three nucleobases afforded the final products, i.e., a series of beta-amino alcohols. All compounds were screened for their inhibitory activity against Src kinase and anticancer activity on human breast carcinoma cells, BT-20 cell line. Among all compounds, 3-N-methyl-N-phenylamino-1-(pyrrolidin-1-yl)propan-2-ol (13b) exhibited the highest inhibitory potency (IC50=66.1 microM) against Src kinase. Structure-activity relationship studies suggested that the incorporation of bulky groups at position 1 and N-substitution with groups larger than methyl moiety, reduced the inhibitory potency of the compound significantly. Compounds 3-(N-ethyl-N-phenylamino-)-1-(4-methylpiperazin-1-yl)propan-2-ol (14c) and 3-(N-ethyl-N-phenylamino)-1-(thymine-1-yl)propan-2-ol (17) were found to inhibit the growth of breast carcinoma cells by approximately 45-49% at concentration of 50 microM.

Inhibiting platelet-derived growth factor beta reduces Ewing's sarcoma growth and metastasis in a novel orthotopic human xenograft model.

BACKGROUND: Despite aggressive therapy, Ewing's sarcoma (ES) patients have a poor five-year overall survival of only 20-40%. Pulmonary metastasis is the most common form of demise in these patients. The pathogenesis of pulmonary metastasis is poorly understood and few orthotopic models exist that allow study of spontaneous pulmonary metastasis in ES. MATERIALS AND METHODS: We have developed a novel orthotopic xenograft model in which spontaneous pulmonary metastases develop. While the underlying biology of ES is incompletely understood, in addition to the EWS-FLI-1 mutation, it is known that platelet-derived growth factor receptor beta (PDGFR-beta) is highly expressed in ES. Hypothesizing that PDGFR-beta expression is indicative of a specific role for this receptor protein in ES progression, the effect of PDGFR-beta inhibition on ES growth and metastasis was assessed in this novel orthotopic ES model. RESULTS: Silencing PDGFR-beta reduced spontaneous growth and metastasis in ES. CONCLUSION: Preclinical therapeutically relevant findings such as these may ultimately lead to new treatment initiatives in ES.

Cellular uptake of gold nanoparticles directly cross-linked with carrier peptides by osteosarcoma cells.

Nanoparticles have been extensively used for a variety of biomedical applications and there is a growing need for highly specific and efficient delivery of the nanoparticles into target cells and subcellular location. We attempted to accomplish this goal by modifying gold particles with peptide motif's that are known to deliver a 'cargo' into chosen cellular location specifically, we intended to deliver nanogold particles into cells through chemical cross-linking with different peptides known to carry protein into cells. Our results suggest that specific sequence of such 'carrier peptides' can efficiently deliver gold nanoparticles into cells when chemically cross-linked with the metal particles.

Engineering a noncarrier to a highly efficient carrier peptide for noncovalently delivering biologically active proteins into human cells.

Noncovalent protein delivery into cells via peptide carriers is an emerging concept. Only a handful of such peptides are known. To address various limitations associated with protein delivery for therapeutic purposes, a greater number of different delivery peptides would be required. No general method exists for creating such peptides. By combining a sequence of 16 lysine residues (K16) with the signal peptide (SP) sequence of Kaposi's fibroblast growth factor (K-FGF), we have synthesized a peptide (K16SP) that efficiently and noncovalently delivers functionally intact proteins (immunoglobulin G molecules, beta-galactosidase, and green fluorescent protein) into mammalian cells. The peptides K16 and SP each alone did not show any noncovalent protein-carrying capacity. K16SP appears to be nontoxic to cells and three to four times more efficient than a commercially available peptide reagent. Our approach offers proof-of-concept of a general strategy for creating a diverse array of peptide carriers for eventual therapeutic applications.

Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma.

Deciphering the molecular basis of cancer is critical for developing novel diagnostic and therapeutic strategies. To better understand the early molecular events involving osteogenic sarcoma (OGS), we have initiated a program to identify potential tumor suppressor genes. Expression profiling of total RNA from ten normal bone cell lines and eleven OGS-derived cell lines by microarray showed 135-fold lower expression of FRZB/sFRP3 mRNA in OGS cells compared to bone cells; this down-regulation of Frzb/sFRP3 mRNA expression was found to be serum-independent. Subsequently, fourteen OGS biopsy specimens showed nine-fold down-regulation of Frzb/sFRP3 mRNA expression compared to expression in eight normal bone specimens as determined by microarray. FRZB /sFRP3 protein level was also found to be at a very low level in 4/4 OGS cell lines examined. Quantitation by RT-PCR indicated approximately 70% and approximately 90% loss of Frzb/sFRP3 mRNA expression in OGS biopsy specimens and OGS-derived cell lines respectively, compared to expression in bone (p<0.0001). Hybridization experiments of a cDNA microarray containing paired normal and tumor specimens from nineteen different organs did not show any significant difference in the level of Frzb/sFRP3 mRNA expression between the normal and the corresponding tumor tissues. Exogenous expression of FRZB/sFRP3 mRNA in two OGS-derived cell lines lacking endogenous expression of the mRNA produced abundant mRNA from the exogenous gene, eliminating degradation as a possibility for very low level of FRZB/sFRP3 mRNA in OGS specimens. Results from PCR-based experiments suggest that the FRZB/sFRP3 gene is not deleted in OGS cell lines, however, karyotyping shows gross abnormalities involving chromosome 2 (location of the FRZB gene) in five of twelve OGS-derived cell lines. Together, these data suggest a tumor-suppressive potential for FRZB/sFRP3 in OGS.

The use of microorganisms for the formation of metal nanoparticles and their application.

Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. The development of reliable experimental protocols for the synthesis of nanomaterials over a range of chemical compositions, sizes, and high monodispersity is one of the challenging issues in current nanotechnology. In the context of the current drive to develop green technologies in material synthesis, this aspect of nanotechnology is of considerable importance. Biological systems, masters of ambient condition chemistry, synthesize inorganic materials that are hierarchically organized from the nano- to the macroscale. Recent studies on the use of microorganisms in the synthesis of nanoparticles are a relatively new and exciting area of research with considerable potential for development. This review describes a brief overview of the current research worldwide on the use of microorganisms in the biosynthesis of metal nanoparticles and their applications.

Superparamagnetic nanoparticle-supported enzymatic resolution of racemic carboxylates.

Candida rugosa lipase immobilized on maghemite nanoparticles demonstrated high stereoselectivity in kinetic resolution of racemic carboxylates and improved long-term stability over its parent free enzyme, allowing the supported enzyme to be repeatedly used for a series of chiral resolution reactions.

Enzyme mediated extracellular synthesis of CdS nanoparticles by the fungus, Fusarium oxysporum.

The biosynthesis of Q-state CdS nanoparticles by reaction of aqueous CdSO4 solution with the fungus, Fusarium oxysporum, is demonstrated. Nanoparticle formation proceeds by release of sulfate reductase enzymes by the fungus, conversion of sulfate ions to sulfide ions that subsequently react with aqueous Cd2+ ions to yield highly stable CdS nanoparticles. Elucidation of an enzymatic pathway using fungi opens up the exciting possibility of developing a rational, biosynthesis strategy for nanomaterials over a range of chemical compositions which is currently not possible.