Synthesis and evaluation of bivalent, peptidomimetic antagonists of the αvβ3 integrins

Targeting the integrin α(v)β(3) by directly interfering with its function is considered to be an effective and non-cytotoxic strategy for the treatment of tumor. In this study, a series of bivalent analogs of peptidomimetic integrin antagonists IA 1 and IAC 2 were designed, synthesized, and evaluated for their ability to inhibit the integrin α(v)β(3). All the bivalent ligands exhibited increased potency compared to that of their monomeric counterparts for the integrin α(v)β(3) with low nanomolar range binding affinity. The best bivalent ligand 6 tested in the series has an IC(50)=0.09 nM evaluated by ELISA assay. We conclude that multivalency is providing a useful template for the development novel integrin α(v)β(3) antagonists as potential therapeutics.     

Derivatives of tetrahydroisoquinoline: Synthesis and initial evaluation of novel non-peptide antagonists of the αIIbβ3-integrin

The novel RGDF mimetics were synthesized with the use of 4-(1,2,3,4-tetrahydroisoquinoline-7-yl)amino-4-oxobutyric or 5-(1,2,3,4-tetrahydroisoquinoline-7-yl)amino-5-oxopentanoic acids as a surrogate of Arg-Gly motif. The synthesized compounds have demonstrated a high potency to inhibit platelet aggregation in vitro and to block FITC-Fg binding to α_IIbβ₃ on washed human platelets.     

Synthesis and biological evaluation of a peptide-paclitaxel conjugate which targets the integrin αvβ₆

The integrin α(v)β(6) is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other carcinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds α(v)β(6) and displays minimal affinity for other RGD-binding integrins. Here we report the use of this peptide to actively deliver paclitaxel to α(v)β(6)-positive cells. We synthesized a water soluble paclitaxel-H2009.1 peptide conjugate in which the 2'-position of paclitaxel is attached to the tetrameric peptide via an ester linkage. The conjugate maintains its specificity for α(v)β(6)-expressing NSCLC cells, resulting in selective cytotoxicity. Treatment of α(v)β(6)-positive cells with the conjugate results in cell cycle arrest followed by induction of apoptosis in the same manner as free paclitaxel. However, initiation of apoptosis and the resultant cell death is delayed compared to free drug. The conjugate demonstrates anti-tumor activity in a H2009 xenograft model of NSCLC with efficacy comparable to treatment with free paclitaxel.     

Synthesis and biological evaluation of novel biaryl type α-noscapine congeners

Natural α-noscapine, a known antitussive drug, is also now known to possess weak anticancer efficacy with relatively safe toxicity profile. In this study, we report synthesis and evaluation of novel biaryl type α-noscapine congeners designed by adding aryl unit to the tetrahydroisoquinoline part of natural α-noscapine core. Palladium catalyzed Suzuki cross coupling of 9-bromo α-noscapine with aryl boronic acids was employed using mild and inexpensive reagents to attain desired noscapinoids 5a–g in excellent yields. Screening anti-proliferative activity for new noscapinoids 5b–g, on human cancer cell lines resulted three compounds 5b, 5d and 5f as potent analogues, active against human breast epithelial (MCF-7), human cervix cancer (HeLa) and human lung adenocarcinoma epithelial (A549) cell lines.     

Synthesis and evaluation of cytotoxic effects of novel α-methylenelactone tetracyclic diterpenoids

A series of tetracyclic diterpenoids bearing the α-methylenelactone group have been synthesized and screened for their in vitro anti-tumor activities against six human cancer cell lines. The results showed that compounds 1c, 2a and 2b exhibited significant cytotoxicity superior to the positive control doxorubicin hydrochloride against MDA-MB-231, K562 and HepG2 cell lines. In particular, compound 2b was identified as the most promising anticancer agent against HepG2 cells with IC(50) value of 0.09μM.     

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells

BackgroundIntegrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5β1 and αvβ3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5β1 and αvβ3/β5 purified integrins.MethodsSmall antagonists were either selective for α5β1 integrin, for αvβ3/β5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5β1 integrin in the biological assays.ResultsU87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5β1 or αvβ3/β5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5β1 integrin and was inhibited selectively by α5β1 integrin antagonists but increased by αvβ3/β5 integrin antagonists.ConclusionsWe provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions.General significanceOur data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5β1 integrin-dependent cell migration.     

Synthesis and structure–activity relationship of non-peptidic antagonists of neuropilin-1 receptor

Neuropilins (NRPs) are VEGF-A₁₆₅ co-receptors over-expressed in tumor cells, and considered as targets in angiogenic-related pathologies. We previously identified compound 1, the first non-peptidic antagonist of the VEGF-A₁₆₅/NRP binding, which exhibits in vivo anti-angiogenic and anti-tumor activities. We report here the synthesis and biological evaluations of new antagonists structurally-related to compound 1. Among these molecules, 4a, 4c and 4d show cytotoxic effects on HUVEC and MDA-MB-31 cells, and antagonize VEGF-A₁₆₅/NRP-1 binding. This study confirmed our key structure–activity relationships hypothesis and paved the way to compound 1 ‘hit to lead’ optimization.     

Synthesis and biological evaluation of novel γ-carboline analogues of Dimebon as potent 5-HT6 receptor antagonists

Synthesis, biological evaluation and structure–activity relationships for a series of novel γ-carboline analogues of Dimebon^? are described. Among the studied compounds, γ-carbolines 3{8} and 3{14} have been identified as potent small molecule antagonists of histamine H₁ (IC₅₀ = 0.1 μM) and serotonin 5-HT₆ (IC₅₀ = 0.37 μM) receptors, respectively.     

Distinct Structural Requirements for Interaction of the Integrins α5β1, αvβ5, and αvβ6 with the Central Cell Binding Domain in Fibronectin

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, α5β1, αIIbβ3, and αvβ3, the structural requirements for binding to fibronectin differ. In the present study. we report that two additional integrins, αvβ6. and αvβ5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments. αvβ5, like αvβ3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study. αvβ5 only minimally supported adhesion to intact fibronectin. but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat. alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by αvβ5, as well as adhesion mediated by αvβ6. αvβ5 and αvβ6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.     

Synthesis and pharmacological evaluation of novel benzoylazole-based PPAR α/γ activators

In our search for new PPARα/γ agonists, we designed and synthesized a series of benzoylazole-based carboxylic acids. Compound 9 showed potent PPARγ partial agonistic activity with modest PPARα agonistic activity. The sodium salt of 9 (9Na) demonstrated potent efficacy in lowering both blood glucose and lipids in an animal model without causing significant body weight gain, a well-known side effect associated with PPARγ full agonists.     

Synthesis and evaluation of novel α-amino cyclic boronates as inhibitors of HCV NS3 protease

We have designed and synthesized a novel series of α-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.     

Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT).

Methods

Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed.

Results

Among the 4 probes examined in this study, ¹²⁵I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, ¹²³I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft.

Conclusion

¹²³I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.     

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.     

Synthesis,cytotoxicity and clastogenicity of novel α-aminophosphonic acids

Summary. α-Ethyl-N-(phosphonomethyl) glycine is synthesized and characterized by NMR and FAB spectroscopy. The cytotoxicity, clastogenic and antiproliferative effect of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, sodium salt of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, α-ethyl-α-N-(hydroxyethylamino) methylphosphonic acid, α-ethyl-N-(phosphonomethyl) glycine, α-ethyl-N-(phosphonomethyl) glycine isopropylammonium salt, glyphosate isopropylammonium salt are tested.     

Synthesis and biological evaluation of α,β-unsaturated lactones as potent immunosuppressive agents

Compounds having α,β-unsaturated lactones display a variety of biological activities. Many research groups have tested both natural and unnatural α,β-unsaturated lactones for as-yet undiscovered biological properties. We synthesized α,β-unsaturated lactones with various substituents at the δ-position and studied their immunosuppressive effects, that is, the inhibition of Interleukin-2 (IL-2) production. Among the compounds synthesized, the benzofuran-substituted α,β-unsaturated lactone 4h showed the best inhibitory activity toward IL-2 production in Jurkat e6-1 T lymphocytes (IC(50)=66.9 nM) without cytotoxicity at 10 μM. The results indicated that 4h may be useful as a potent immunosuppressive agent, as well as in IL-2-related studies.     

Design and synthesis of novel dual-cyclic RGD peptides for αvβ3 integrin targeting

The specific binding of RGD cyclic peptide with integrin α_vβ₃ attracts great research interest for tumor-targeting drug delivery. Herein, we designed and synthesized a series of dual-ring RGD-peptide derivatives as a drug carrier for α_vβ₃ targeting. Three novel peptides showed excellent cell adhesion inhibition effect, in which, P3 exhibited 7-fold enhancement in IC₅₀ compared with cyclo(RGDfK). Drug-loaded cytotoxicity experiment and imaging experiment indicated that such dual-cyclic RGD peptides have good tumor targeting effects. This work provides a new strategy for the design of novel RGD peptides.     

Synthesis and biological evaluation of novel tris-chalcones as potent carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase and α-glycosidase inhibitors

A novel class of fluoro-substituted tris-chalcones derivatives (5a-5i) was synthesized from phloroglucinol and corresponding benzaldehydes. A three step synthesis method was followed for the production of these tris-chalcone compounds. The structures of the newly synthesized compounds (5a-5i) were confirmed on the basis of IR, ¹H NMR, ¹³C NMR, and elemental analysis. The compounds’ inhibitory activities were tested against human carbonic anhydrase I and II isoenzymes (hCA I and hCA II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase (α-Gly). These chalcone derivatives had K_i values in the range of 19.58–78.73 nM for hCA I, 12.23–41.70 nM for hCA II, 1.09–6.84 nM for AChE, 8.30–32.30 nM for BChE and 0.93 ± 0.20–18.53 ± 5.06 nM against α-glycosidase. These results strongly support the promising nature of the tris-chalcone scaffold as selective carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase inhibitor. Overall, due to these derivatives’ inhibitory potential on the tested enzymes, they are promising drug candidates for the treatment of diseases like glaucoma, leukemia, epilepsy; Alzheimer’s disease; type-2 diabetes mellitus that are associated with high enzymatic activity of carbonic anhydrase, acetylcholine esterase, butyrylcholinesterase, and α-glycosidase.     

Synthesis,biological evaluation and SAR studies of ursolic acid 3β-ester derivatives as novel CETP inhibitors

Cholesteryl ester transfer protein (CETP) is an attractive therapeutic target for the prevention and treatment of cardiovascular diseases by lowering low-density lipoprotein cholesterol levels as well as raising high-density lipoprotein cholesterol levels in human plasma. Herein, a series of ursolic acid 3β-ester derivatives were designed, synthesized and evaluated for the CETP inhibiting activities. Among these compounds, the most active compound is U12 with an IC₅₀ value of 2.4 μM in enzymatic assay. The docking studies showed that the possible hydrogen bond interactions between the carboxyl groups at both ends of the molecule skeleton and several polar residues (such as Ser191, Cys13 and Ser230) in the active site region of CETP could significantly enhance the inhibition activity. This study provides structural insight of the interactions between these pentacyclic triterpenoid 3β-ester derivatives and CETP protein for the further modification and optimization.     

Synthesis and in vitro evaluation of a novel radioligand for αvβ3 integrin receptor imaging: [18F]FPPA-c(RGDfK)

The development of RGD-based antagonist of αvβ3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the ¹⁸F-labeling of an αvβ3 selective RGD-peptide was successfully prepared. [¹⁸F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N₃)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [¹⁸F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140 min.