Spermine Derivatives of Indole-3-carboxylic Acid, Indole-3-acetic Acid and Indole-3-acrylic Acid as Gram-Negative Antibiotic Adjuvants.

The discovery of new antibiotic adjuvants is an attractive option for overcoming antimicrobial resistance. We have previously reported the discovery of a bis-6-bromoindolglyoxylamide derivative of spermine as being able to enhance the action of antibiotics against Gram-negative bacteria but suffers from being cytotoxic and red-blood cell haemolytic. A series of analogues was prepared exploring variation of the indolglyoxylamide unit, to include indole-3-acrylic, indole-3-acetic and indole-3-carboxylate units, and evaluated for antibiotic enhancing properties against a range of Gram-negative bacteria, and for intrinsic antimicrobial, cytotoxic and haemolytic properties. Two spermine derivatives, bearing 5-bromo-indole-3-acetic acid (17) and 5-methoxy-indole-3-acrylic acid (14) end groups were found to exhibit good to moderate antibiotic adjuvant activities for doxycycline towards the Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae, but with more modest intrinsic antimicrobial activity and greatly reduced cytotoxic and haemolytic properties. The mechanism of action of the latter derivative identified its ability to disrupt the outer membranes of bacteria and to inhibit the AcrAB-TolC efflux pump directly or by inhibiting the proton gradient.

Electrophoresis and Biosensor-Based DNA Interaction Analysis of the First Paraben Derivatives of Spermine-Bridged Cyclotriphosphazenes.

Cancer is the uncontrolled growth of abnormal cells via malignant cell division and rapid DNA replication. While DNA damaging molecules can cause cancer, their role as anticancer drugs are very significant. For this purpose, the novel series of paraben substituted spermine bridged(dispirobino) cyclotriphosphazene compounds 2-6 were synthesized for the first time, and their structures were characterized by various spectroscopic techniques. The solid-state structures and geometries of compounds 2-6 were determined using single-crystal X-ray structural analysis. In addition, it was confirmed by TGA that all compounds 1-6 showed high thermal stability. Two methods were used in order to investigate DNA interaction properties of the targeted molecules. While biosensor-based screening test that measures DNA hybridization efficiency on a biochip surface, the agarose gel electrophoresis method examines the effect of compounds on plasmid DNA structure. The results collected from the automated biosensor device and agarose gel electrophoresis have indicated that compounds 1, 5, and 6 showed higher DNA damage than the compounds 2-4. According to the biosensor results, compounds 1, 5, and 6 showed 85%, 69%, and 77% activity, respectively.

Spermine-NBD as fluorescent probe for studies of the polyamine transport system in Leishmania donovani.

This study describes the synthesis of fluorescent probes as potential substrates for the polyamine transport system (PTS) of Leishmania donovani. A competitive radioassay was used to determine the most efficient probe. We observed that the conjugate spermine-nitrobenzofurazan (Spm-NBD) was able to compete with [3H]-spermidine in L. donovani at a potent IC50 of 60 µM.

An oxaliplatin(iv) prodrug-based supramolecular self-delivery nanocarrier for targeted colorectal cancer treatment.

A redox-responsive supramolecular nanocarrier was constructed from the self-assembly of spermine modified cyclodextrin and oxaliplatin prodrug. The nanocarrier could preferentially accumulate in polyamine transporter over-expressing HCT116 cells, releasing drugs under a reducing intracellular environment to maximize anticancer treatment.

Biochemical evaluation of the anticancer potential of the polyamine-based nanocarrier Nano11047.

Synthesizing polycationic polymers directly from existing drugs overcomes the drug-loading limitations often associated with pharmacologically inert nanocarriers. We recently described nanocarriers formed from a first-generation polyamine analogue, bis(ethyl)norspermine (BENSpm), that could simultaneously target polyamine metabolism while delivering therapeutic nucleic acids. In the current study, we describe the synthesis and evaluation of self-immolative nanocarriers derived from the second-generation polyamine analogue PG-11047. Polyamines are absolutely essential for proliferation and their metabolism is frequently dysregulated in cancer. Through its effects on polyamine metabolism, PG-11047 effectively inhibits tumor growth in cancer cell lines of multiple origins as well as in human tumor mouse xenografts. Promising clinical trials have been completed verifying the safety and tolerance of this rotationally restricted polyamine analogue. We therefore used PG-11047 as the basis for Nano11047, a biodegradable, prodrug nanocarrier capable of targeting polyamine metabolism. Following exposure of lung cancer cell lines to Nano11047, uptake and intracellular degradation into the parent compound PG-11047 was observed. The release of PG-11047 highly induced the polyamine catabolic enzyme activities of spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX). By contrast, the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis and a putative oncogene, was decreased. Consequently, intracellular levels of the natural polyamines were depleted concurrent with tumor cell growth inhibition. This availability of Nano11047 as a novel drug form and potential nucleic acid delivery vector will potentially benefit and encourage future clinical studies.

Practical Synthesis of Spermine, Thermospermine and Norspermine.

Polyamines, such as spermine (1), thermospermine (2) and norspermine (3), are widely distributed in nature, and have multiple biological activities. In addition, many of their conjugates have potential for pharmacological use. Here, we present a solid-phase synthesis using our nitrobenzenesulfonyl (Ns) strategy, which can provide 1, 2 and 3 on a gram scale. This approach should be suitable for facile construction of a diverse library of polyamines.

Synthesis and anticancer evaluation of spermatinamine analogues.

Spermatinamine was isolated from an Australian marine sponge, Pseudoceratina sp. as an inhibitor of isoprenylcysteine carboxyl methyltransferase (Icmt), an attractive and novel anticancer target. Herein, we report the synthesis of spermatinamine analogues and their cytotoxic evaluation against three human cancer cell lines, that is, cervix adenocarcinoma (HeLa), breast adenocarcinoma (MCF-7), and prostate carcinoma (DU145). Analogues 12, 14 and 15 were found to be the most potent against one or more cell lines with the IC50 values in the range of 5-10 µM. The obtained results suggested that longer polyamine linker along with aromatic oxime substitution provided the most potent analogue compounds against cancer cell lines.

Synthesis of novel cationic spermine-conjugated phosphotriester oligonucleotide for improvement of cell membrane permeability.

A spermine-conjugated ethyl phosphotriester oligonucleotide was obtained by solid-phase synthesis based on phosphoramidite chemistry. The ethyl phosphotriester linkage was robust to exonuclease digestion and stable in fetal bovine serum. Cell membrane permeability of the spermine-conjugated ethyl phosphotriester oligonucleotide was studied by fluorescence experiments. The effective cell penetrating potency of the spermine-conjugated ethyl phosphotriester oligonucleotide was determined by confocal laser scanning microscopy and measurement of intracellular fluorescence intensity.

Tritiation and characterization of several polyamine natural products.

Methods are presented to tritiate the polyamines putrescine and spermine.

Functional properties of Claramine: a novel PTP1B inhibitor and insulin-mimetic compound.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-ß (IRß), Akt and GSK3ß. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

Star-shaped tetraspermine enhances cellular uptake and cytotoxicity of T-oligo in prostate cancer cells.

PURPOSE: An oligonucleotide termed 'T-oligo' having sequence homology with telomere overhang has shown cytotoxicity in multiple cancers. We have demonstrated that T-oligo can induce apoptosis in androgen independent prostate cancer cell line DU-145. In this report, we evaluate the use of star-shaped tetraspermine (SSTS) for delivery of T-oligo. METHODS: SSTS was synthesized from spermine and its intrinsic cytotoxicity towards DU-145 cells was compared with spermine and branched polyethyleneimine (bPEI). Atomistic molecular dynamic (MD) simulations were conducted to understand binding and complexation of spermine and SSTS with T-oligo. Complexation was also determined using gel electrophoresis and SYBR gold assay. Complexes were characterized for size, cellular uptake and antiproliferative effect. RESULTS: SSTS exhibited significantly lower toxicity than spermine and bPEI. Its affinity towards T-oligo was significantly higher than spermine as determined by experimental studies and confirmed by MD simulations and it formed stable complexes (TONPs) with T-oligo. TONPs facilitated cellular uptake and nuclear accumulation of T-oligo and their cytotoxic potential was observed at concentration several folds lower than that required for T-oligo alone. CONCLUSION: SSTS significantly enhanced therapeutic benefits associated with the use of T-oligo and can be developed as a delivery vehicle for its in-vivo therapeutic applications.

Design, synthesis and in vitro antikinetoplastid evaluation of N-acylated putrescine, spermidine and spermine derivatives.

A structure-activity relationship study on polyamine derivatives led to the synthesis and the determination of antikinetoplastid activity of 17 compounds. Among them, a spermidine derivative (compound 13) was specifically active in vitro against Leishmania donovani axenic amastigotes (IC50 at 5.4µM; Selectivity Index >18.5) and a spermine derivative (compound 28) specifically active against Trypanosoma brucei gambiense (IC50 at 1.9µM; Selectivity Index >52).

Influence of oligospermines architecture on their suitability for siRNA delivery.

Spermines are naturally abundant polyamines that partially condense nucleic acids and exhibit the proton-sponge effect in an acidic environment. However, spermines show a limited efficiency for transfecting nucleic acids because of their low molecular weight. Therefore, spermines need to be modified to be used as nonviral vectors for nucleic acids. Here, we synthesized linear bisspermine as well as a linear and dendritic tetraspermine with different molecular architectures. These oligospermines were self-assembled into polyplexes with siRNA. The structure-activity relationship of the oligospermines was evaluated in terms of their efficiency for delivering siRNA into a nonsmall cell lung carcinoma cell line. Oligospermines displayed minimal cytotoxicity but efficient siRNA condensation and showed better stability against polyanions than polyethylenimine. The morphology of the polyplexes was strongly affected by the oligospermine architecture. Linear tetraspermine/siRNA polyplexes showed the best gene-silencing efficiency among the oligospermines tested at both the mRNA and protein expression levels, indicating the most favorable structure for siRNA delivery.

Macrophage mannose receptor-specific gene delivery vehicle for macrophage engineering.

Macrophages are the most plastic cells in the hematopoietic system and they exhibit great functional diversity. They have been extensively applied in anti-inflammatory, anti-fibrotic and anti-cancer therapies. However, the application of macrophages is limited by the efficiency of their engineering. The macrophage mannose receptor (MMR, CD206), a C-type lectin receptor, is ubiquitously expressed on macrophages and has a high affinity for mannose oligosaccharides. In the present study, we developed a novel non-viral vehicle with specific affinity for MMR. Mannan was cationized with spermine at a grafted ratio of ∼12% to deliver DNA and was characterized as a stable system for delivery. This spermine-mannan (SM)-based delivery system was evaluated as a biocompatible vehicle with superior transfection efficiency on murine macrophages, up to 28.5-fold higher than spermine-pullulan, 11.5-fold higher than polyethylenimine and 3.0-fold higher than Lipofectamine™ 2000. We confirmed that the SM-based delivery system for macrophages transfection was MMR-specific and we described the intracellular transport of the delivery system. To our knowledge, this is the first study using SM to demonstrate a mannose receptor-specific gene delivery system, thereby highlighting the potential of a novel specific non-viral delivery vehicle for macrophage engineering.

[Stable analogues of coenzyme-substrate complex of spermidine/spermine-N1-acetyltransferase reaction. synthesis, interaction with the enzyme].

Convenient two-step synthesis of conjugates of HS-CoA and D-pantetheine with aminooxy analogues of Spm, Spd and Put was suggested. The use of acetone linker provided target conjugates with quantitative yields. The activity of CoA-derived "bisubstrate" inhibitors being active at microM concentrations was at least 100 times better than that of corresponding derivatives of D-pantetheine.

Antitumor effects and preliminary systemic toxicity of ANISpm in vivo and in vitro.

Polyamines as a vector to ferry toxic agents have attracted attention, and naphthalimide-polyamine conjugates show potent activity and tumor cell selectivity. The present study was carried out to evaluate the antitumor effects and preliminary systemic toxicity of ANISpm, a novel 3-amino-naphthalimide-spermine conjugate. The polyamine transport system recognition of ANISpm, supported by α-difluoromethylornithine (DFMO)/spermidine (Spd) experiments, is in accordance with its potent cell selectivity between human hepatoma HepG2 cells and normal QSG7701 hepatocyte. The antiproliferative effect is because of ANISpm-induced cell apoptosis, a common characteristic of both naphthalimide and polyamine analogs. Various apoptotic assessment assays have shown that ANISpm can induce apoptosis through the PI3K/Akt signal pathway. The apoptotic signaling cascade involves Akt inactivation, which results in a series of cellular events. The downstream pathway includes Bad dephosphorylation, dissociation of 14-3-3 and Bad, and binding to Bcl-xL, which triggers the disruption of the mitochondrial membrane, release of cytochrome c, and caspases' cascade activation. Furthermore, the Akt/mTOR signal pathway is also involved in ANISpm-mediated cell-cycle arrest. Additive DFMO or Spd, which only enhances or attenuates ANISpm-mediated cell apoptosis, respectively, does not alter the signal pathway. In addition, preliminary toxicology evaluation showed that ANISpm had no obvious system toxicity at a dose of 2.5 mg/kg, which exerted potent antitumor activity in vivo, especially hematotoxicity. Thus, ANISpm merits further investigation as a potential chemotherapeutic agent against hepatocellular carcinoma.

Discovery and preliminary structure-activity relationship analysis of 1,14-sperminediphenylacetamides as potent and selective antimalarial lead compounds.

Screening of synthesized and isolated marine natural products for in vitro activity against four parasitic protozoa has identified the ascidian metabolite 1,14-sperminedihomovanillamide (orthidine F, 1) as being a non-toxic, moderate growth inhibitor of Plasmodium falciparum (IC(50) 0.89 µM). Preliminary structure-activity relationship investigation identified essentiality of the spermine polyamine core and the requirement for 1,14-disubstitution for potent activity. One analogue, 1,14-spermine-di-(2-hydroxyphenylacetamide) (3), exhibited two orders of magnitude increased anti-P. f activity (IC(50) 8.6 nM) with no detectable in vitro toxicity. The ease of synthesis of phenylacetamido-polyamines, coupled with potent nM levels of activity towards dual drug resistant strains of P. falciparum makes this compound class of interest in the development of new antimalarial therapeutics.

Synthesis of bisethylnorspermine lipid prodrug as gene delivery vector targeting polyamine metabolism in breast cancer.

Progress in the development of nonviral gene delivery vectors continues to be hampered by low transfection activity and toxicity. Here we proposed to develop a lipid prodrug based on a polyamine analogue bisethylnorspermine (BSP) that can function dually as gene delivery vector and, after intracellular degradation, as active anticancer agent targeting dysregulated polyamine metabolism. We synthesized a prodrug of BSP (LS-BSP) capable of intracellular release of BSP using thiolytically sensitive dithiobenzyl carbamate linker. Biodegradability of LS-BSP contributed to decreased toxicity compared with nondegradable control L-BSP. BSP showed a strong synergistic enhancement of cytotoxic activity of TNF-related apoptosis-inducing ligand (TRAIL) in human breast cancer cells. Decreased enhancement of TRAIL activity was observed for LS-BSP when compared with BSP. LS-BSP formed complexes with plasmid DNA and mediated transfection activity comparable to DOTAP and L-BSP. Our results show that BSP-based vectors are promising candidates for combination drug/gene delivery.

Syntheses of pseudoceramines A-D and a new synthesis of spermatinamine, bromotyrosine natural products from marine sponges.

Herein we report the total syntheses of pseudoceramine A-D (2-5) and spermatinamine (1) isolated from the marine sponge Pseudoceratina sp. Direct acyl substitution of α-hydroxyiminoesters with amine nucleophiles was developed as a key transformation. The synthetic compounds confirm the reported structures and importantly gives access to non-symmetrical spermine based natural products carrying two different bromotyrosine building blocks. Our new synthesis of spermatinamine is two steps shorter and more efficient than the previously reported sequence.