JLK1486, a N,N-[(8-hydroxyquinoline)methyl]-substituted benzylamine analogue, inhibits melanoma proliferation and induces autophagy.

OBJECTIVES: To investigate anti-proliferatory activity of a selected N,N-[(8-hydroxyquinoline)methyl]-substituted benzylamine (JLK1486) on melanoma cells and to characterize its mechanism of cell population growth inhibition. MATERIALS AND METHODS: In vitro cultures of B16F10 (mouse melanoma) cells were used as a model to characterize anti-proliferatory activity of JLK1486 using MTT growth assay, trypan blue viability assessment, cell cycle analysis, melanin production, ß-galactosidase and acridine orange staining. RESULTS: Proliferating B16F10 and also MeWo (human melanoma) cells were strongly growth inhibited by JLK1486, displaying IC50 values of 196 nm and 110 nm respectively. Anti-proliferatory effects were independent of cell death and were characterized by a distinct accumulation of cells in G0 /G1 phase. Tyrosinase activity and relative melanin content remained unchanged indicating that the anti-proliferatory activity was not due to phenotype differentiation. Although treated B16F10 cells stained strongly positive for senescence marker ß-galactosidase, cells regained near normal proliferatory activity after removal of JLK1486. Increased acridine orange staining and presence of perinuclear vacuoles suggested induction of autophagy in B16F10 cells. Furthermore, JLK1486 pre-treatment completely abolished melphalan and antimycin A-induced apoptosis. CONCLUSION: JLK1486 provides a promising chemical scaffold to develop new anti-melanoma drugs or combination therapies, due to its potent inhibition of cell proliferation and induction of autophagy, at pharmacologically relevant concentrations.

New N(4)-substituted piperazine naphthamide derivatives as BACE-1 inhibitors.

Synthesis and enzymatic evaluation of new series of N(4)-substituted piperazine naphthamide derivatives as BACE-1 inhibitors for the treatment of Alzheimer's disease are reported.

New 3'-azido-3'-deoxythymidin-5'-yl O-(4-hydroxyalkyl or -alkenyl or -alkylepoxide) carbonate prodrugs: synthesis and anti-HIV evaluation.

New 5'-O-carbonate prodrugs of zidovudine (AZT) have been synthesized in order to enhance its uptake by HIV-1 infected cells, to improve its anti-HIV potency, and to optimize the intramolecular cyclic rearrangement process related to the 5'-O-(4-hydroxybutyl) carbonate moiety. Evidence of this prodrug rearrangement was confirmed by comparison of the serum half-lives of the 3'-azido-3'-deoxythymidin-5'-yl O-(4-hydroxyalkyl or -alkenyl or -alkylepoxide) carbonate prodrugs with our thermodynamic predictions. Interestingly, these 5'-O-carbonate prodrug series show increased anti-HIV potencies in conjunction with, or without, reduced cytotoxicity as compared to AZT that lead to a gain in selectivity indexes. The cytotoxicity of AZT could be reduced with these 5'-O-carbonate prodrug series by delaying the 5'-O-glucuronidation of AZT, which is one of the major limitations of AZT.

New protease inhibitors prevent gamma-secretase-mediated production of Abeta40/42 without affecting Notch cleavage.

We have designed new non-peptidic potential inhibitors of gamma-secretase and examined their ability to prevent production of amyloid-beta 40 (Abeta40) and Abeta42 by human cells expressing wild-type and Swedish-mutant beta-amyloid precursor protein (betaAPP). Here we identify three such agents that markedly reduce recovery of both Abeta40 and Abeta42 produced by both cell lines, and increase that of C99 and C83, the carboxy-terminal fragments of betaAPP that are derived from beta-and alpha-secretase, respectively. Furthermore, we show that these inhibitors do not affect endoproteolysis of endogenous or overexpressed presenilins. These inhibitors are totally unable to affect the mDeltaEnotch-1 cleavage that leads to generation of the Notch intracellular domain (NICD). These represent the first non-peptidic inhibitors that are able to prevent gamma-secretase cleavage of betaAPP without affecting processing of mDeltaEnotch-1 or endoproteolysis of presenilins. The distinction between these two proteolytic events, which are both prevented by disruption of presenilin genes, indicates that although they are intimately linked with betaAPP and Notch maturation, presenilins are probably involved in the control of maturation processes upstream of enzymes that cleave gamma-secretase and Notch.

New 3'-azido-3'-deoxythymidin-5'-yl O-(omega-hydroxyalkyl) carbonate prodrugs: synthesis and anti-HIV evaluation.

Prodrugs of zidovudine (AZT) have been synthesized in an effort to enhance its uptake by HIV-1 infected cells and its anti-HIV activity. The 5'-OH function of AZT was functionalized with various enzymatically labile alkyl groups using specific procedures. The prodrug moieties included 5'-O-carbonate, 5'-O-carbamate, and 5'-O-ester. Analogues of the 3'-azido-3'-deoxythymidin-5'-yl O-(omega-hydroxyalkyl) carbonate series were particularly interesting since they were rearranged through an intramolecular cyclic process during their enzymatic hydrolysis. Evidence of this prodrug rearrangement was confirmed by comparison of the serum half-lives of 5'-O-carbonate prodrugs with their corresponding 5'-O-ester- and 5'-O-carbamate-AZT prodrugs. Interestingly, the anti-HIV-1 activities (EC(50)) of 3'-azido-3'-deoxythymidin-5'-yl O-(4-hydroxybutyl) carbonate 10 in acutely infected MT-4 cells and in peripheral blood mononuclear cells (PBMCs) were 0.5 nM and 0.78 nM, respectively. Compound 10 was 30 to 50 times more potent than its parent drug AZT. Our results suggest that the specific intramolecular rearrangement associated with the 3'-azido-3'-deoxythymidin-5'-yl O-(omega-hydroxyalkyl) carbonate prodrugs could explain the remarkable anti-HIV-1 activity of this series of AZT prodrugs. Prodrug 10 may therefore have better clinical potential than AZT for the treatment of AIDS.

Use of proline bioisosteres in potential HIV protease inhibitors: phenylalanine-2-thiophenoxy-3-pyrrolidinone: synthesis and anti-HIV evaluation.

The synthesis of new phenylalanine-2-thiophenoxy-3-pyrrolidinones is described. Anti-HIV recombinant protease assays and HIV infected cell culture assays (observation of syncytia) demonstrated the potent anti-HIV activity of this new class of pseudopeptides.

Synthesis and anti-HIV evaluation of new 2',3'-dideoxy-3'-thiacytidine prodrugs.

A series of anti-HIV prodrugs possessing various polyaminated side arms have been developed. The incorporation of a N-Boc protected monoamine or diamine side arm into the backbone of the 2',3'-dideoxy-3'-thiacytidine 1 (BCH-189) provided an increase in antiviral potency, which could be several orders magnitude greater than the parent drug (1) depending on the cell culture systems used (MT-4 or MDMs). Twenty six 2',3'-dideoxy-3'-thiacytidine prodrugs which differ from each other by the length, the nature of the 5'-O function and the 5'-O or/and N-4 position on the nucleoside moiety were synthesized. Among this new series of prodrugs, several congeners (12c and 12a) were found to inhibit HIV-1 replication in cell culture with 50% effective concentrations EC50 of 10 and 50 nM respectively, in MT-4 cells. Compound 12c was found more active on infected MDMs cells with 50% effective concentration of 0.01 nM. The synthesis and the antiviral properties of these compounds are discussed.

New anti-HIV derivatives: synthesis and antiviral evaluation.

A small focused library of 18 compounds incorporating the motif 1,3-(N,N'-dibenzyl)diamino-2-propanol has been synthesized, using adapted synthetic methodologies. These series of compounds were evaluated for their in vitro anti-HIV activity on infected MT4 cells (syncytium formation observation). Some of the new synthesized compounds show potent anti-HIV activities. EC50 values for compounds (31, 40, 34, 37 and 46) range from 0.1 to 1 microM. In order to determine at which level these new derivatives interfere with the HIV replicative cycle, inhibition assays on recombinant HIV protease and HIV integrase have been performed. None of the compounds were found active on these two enzymatic targets. Experiments are in progress in order to identify their biological target within the HIV replicative cycle.

Design and evaluation of hepatitis B virus inhibitors.

Antiviral therapy of chronic hepatitis B remains a major clinical problem worldwide. The design of new nucleoside analogs that inhibit hepatitis B virus (HBV) replication allowed their evaluation in in vitro and in vivo experimental models of HBV infection. This research has led to the discovery of the anti-HBV activity of lamivudine and its approval for the therapy of chronic hepatitis B. However, due to the development of viral resistance, strategies based on the combination of new inhibitors of HBV replication with immune modulatory approaches are urgently required.

Synthesis and biological evaluation of polyaminated 2',3'-dideoxy-3'-thiacytidine prodrugs.

The syntheses and biological evaluation of polyaminated 2',3'-dideoxy-3'-thiacytidine have been performed. A new lead was found to increase the in vitro antiviral potency (syncitia formation on MT-4 cell line) of two order magnitude greater than the parent nucleoside drug. Moreover, the in vitro activity on HIV macrophages was found to be more than 3 log greater than the activity of the parent drug 1.

New polyazamacrocycle-nucleoside conjugates: synthesis, anti-HIV evaluation and interaction with CXCR-4 coreceptor.

We report the synthesis of new conjugates that incorporate in their structure bis-tetraazamacrocycle coupled with AZT via enzymolabile bond. Two series of bis-polyazamacrocycles-AZT conjugates were designed, synthesized and evaluated for their antiviral effect in vitro as well as their capability to bind to CXCR-4 coreceptor.

New bicyclam-AZT conjugates: design, synthesis, anti-HIV evaluation, and their interaction with CXCR-4 coreceptor.

We report the synthesis of mono- and bis-tetraazamacrocycle-AZT conjugates. All new compounds were screened for their ability to inhibit HIV-1 replication in MT4 cell line and were compared to AZT alone. It appears that N-protected covalent prodrugs are equipotent to AZT as inhibitor of HIV replication, while N-deprotected analogues exhibit both higher activity and selectivity against HIV-infected cells. The most active antiviral compounds 27, 28, 34, and 35 were then tested for their binding capability to CXCR-4 receptor. N-Boc analogues 27 and 34 were only weakly effective; in contrast, N-deprotected conjugates 28 and 35 were antagonists to 12G5 mAb binding until 0.05 and 5 microg/mL, respectively. The stability of compound 28 in human plasma was evaluated, and half-life was found to be approximately 8 h in the described conditions. All these results seem to demonstrate the confidence of our prodrug approach, with analogue 28 emerging as the best candidate as lead compound in HIV-1 polytherapy perspective.

Synthesis and anti-HIV activity of alpha-thiophenoxy-hydroxyethylamide derivatives.

A series of new anti-HIV derivatives containing a novel alpha-thiophenoxyhydroxyethylamide core have been synthesized, using S-phenylbenzenethiosulfonate as the thiosulfenylating reagent. Some of the new synthesized compounds (1a, 1c, 1g, 1i, 1j and 1l) inhibited HIV replication in cell culture assays (syncytia formation) with effective concentrations (EC(50)) ranging from 0.1-1 microM. Incorporation of thiophenoxy substitution within various pseudomimetic peptide backbones provided a series of highly potent HIV inhibitors.

Tri-N-Boc-tetraazamacrocycle-nucleoside conjugates: synthesis and anti-HIV activities.

As far as linear N-Boc-polyamines conjugates elicited remarkable anti-HIV activity, the synthesis and anti-HIV properties of cyclic N-Boc-polyamines conjugates such as tetraazamacrocycle-nucleoside were studied. These new conjugates include an ester linkage between the two moieties. They were synthesized using Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate coupling reagent, in the case of N-alkyl polyazamacrocycle derivatives, or through direct condensation of the acyl chloride derivative with nucleoside in the case of N-acyl polyazamacrocycle compounds. None of the new conjugates presented anti-HIV activity greater than that of the corresponding parent nucleosides.

Synthesis and anti-HIV activities of symmetrical N1,N3-dibenzyl-2-hydroxy-propane derivatives.

We report the synthesis and the anti-HIV activities of new C2-symmetrical and achiral N1,N3-dibenzyl-2-hydroxy-propane isosteres. Some of them showed significant inhibitory activity with respect to HIV-infected MT4 cells (compound 6a and 7a, IC50 = 0.1 microM). These new structurally simple compounds represent new synthons which can be suitable for combinatorial chemistry purposes.

Design, synthesis and structure relationships of new N,N',N",N"'-tetrakis (omega-amino alkyl) tetraazamacrocycles.

A number of N,N',N",N"'-tetrakis (omega-aminoalkyl) tetraazamacrocycles and related compounds were synthesized and evaluated for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) and duck hepatitis B virus (DHBV) replication. The activity of these compounds was found to be highly dependent upon structural features: (i) the length of the alkyl linker connecting the nitrogen atoms of the macrocyclic ring to the exocyclic nitrogen atoms of the terminal amino groups (five methylenes favoured antiviral activity); (ii) substitution of the terminal amino groups of the linker reduced antiviral activity; and (iii) the size of the tetraazamacrocyclic ring (14 or 15 atoms) did not markedly affect the antiviral activity. Some analogues were potent inhibitors of HIV-1 replication, with anti-HIV activity similar to that of biscyclam (JM 2763). In contrast, other analogues were found to be highly toxic in duck hepatocyte primary culture, the 2.2.15 cell line and to a lesser extent in MT-4 cells. Structural parameters, macrocyclic ring size and metal-chelating ability have been used to develop a structure-activity relationship model in order to aid the design of antiviral molecules derived from N,N',N",N"'-tetrakis (omega-aminoalkyl) tetraazamacrocycles.

Syntheses of new modified Phe-Pro peptides. Use of proline replacements in potential HIV inhibitors.

The syntheses consisting of replacement of proline amino acid by a 3-pyrrolidinone ring in Phe-Pro analogues are described. Preliminary anti-HIV studies demonstrated the potential activity of this new class of compounds.

Selective inhibition of the duck hepatitis B virus by a new class of tetraazamacrocycles.

The antiviral activity of a new class of N,N,N',N",NA'''-pentakis (omega-aminoalkyl) tetraazamacrocycles was evaluated in primary duck hepatocyte cultures infected with the duck hepatitis B virus (DHBV). Three of the four tested compounds were able to selectively inhibit DHBV replication by acting at an early step of the hepadnavirus infection but were associated with significant toxicity.

Inhibition of human immunodeficiency virus type 1 replication by phosphonoformate- and phosphonoacetate-2',3'-dideoxy-3'-thiacytidine conjugates.

The synthesis of potential "combined prodrugs" where phosphonoformic acid (PFA) or phosphonoacetic acid (PAA) was attached to the 5'-O or N4 position of 2',3'-dideoxy-3'-thiacytidine (BCH-189) is described. The anti-HIV-1 activity of 11 analogues which included carboxylic ester or phosphoric ester linkages of PFA or PAA to BCH-189 was determined in MT-4 cells. Of these compounds, the IC50 of analogues 3, 4, 6, and 7 ranged from 0.2 to 100 microM, while IC50 for BCH-189 in this system was 0.1 microM. In vitro hydrolysis of the various esters or amides in human plasma indicated that these agents were relatively stable in the presence of plasma esterases with t1/2 values of up to 120 min. Moreover, lipophilicity of these compounds (partition coefficient) was determined in order to establish correlation between lipophilicity and diffusion of BCH-189 analogues into the cells. The active compounds may exert their effects by extracellular or intracellular hydrolysis to the corresponding antiviral agent BCH-189, but intrinsic anti-HIV-1 activity of some of PAA and PFA adducts, themselves, may also be involved.