Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.     

Bioreductively-activated prodrugs for targeting hypoxic tissues: elimination of aspirin from 2-nitroimidazole derivatives.

2-Nitroimidazoles were synthesised substituted with aspirin or salicylic acid, as leaving groups linked through the (imidazol-5-yl)methyl position. Activation of aqueous solutions by CO2*- (a model one-electron reductant) resulted in release of aspirin or salicylate, probably via the 2-hydroxyaminoimidazole. The analogous 2-nitroimidazole with bromide as leaving group eliminated bromide in < 1 ms via the radical-anion.     

Nitrobenzyl radical metabolites from microsomal reduction of nitrobenzyl chlorides

The o-, m-, and p-nitrobenzyl chlorides are reduced aerobically and anaerobically by NADPH and rat hepatic microsomes. Under aerobic conditions, these nitro anion radicals reduce oxygen to superoxide as demonstrated by oxygen consumption and spin trapping of superoxide with 5,5-dimethyl-1-pyrroline N-oxide. At low oxygen concentration, the p- and o-nitro anion radicals undergo intramolecular electron transfer and decompose to carbon-centered nitrobenzyl radicals, which can be spin-trapped with t-nitrosobutane. The p-nitrobenzyl (o-nitrobenzyl) radical adduct was characterized by a nitrogen hyperfine splitting of 16.5 G (17.1 G) and two equivalent beta-hydrogen hyperfine splittings of 10.6 G (14.4 G). The spin trap 5,5-dimethyl-1-pyrroline N-oxide also yields adducts characteristic of carbon-centered free radicals. This unimolecular decomposition is much faster than the disproportionation decay, which is characteristic of most nitro anion radicals, and the primary o- and p-nitrobenzyl chloride anion radicals never achieve detectable concentrations. The nitrobenzyl radical trapping is not inhibited by metyrapone or CO. In contrast, the m-nitrobenzyl anion radical does achieve a detectable steady-state concentration, which is increased 20% by either metyrapone or a CO atmosphere.     

Mass spectral analysis of peptides containing nitrobenzyl moieties

Summary By means of MALDI-TOF analysis of peptides containing a nitrobenzyl moiety, we have observed that the predominant signal often corresponds to ions that have eliminated multiple oxygen atoms, [M–O_n+H]⁺, and not to the protonated molecular ion, [M+H]⁺. Matrix selection for handling these types of molecules appears to be important. The MALDI-TOF matrices, such as sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) and -cyano-4-hydroxycinnamic acid, resulted in the appearance of significant levels of the photodeoxygenated species, whereas 4-hydroxyazobenzene-2-carboxylic acid appeared to moderately and in some cases completely suppress the photodeoxygenation reaction. This phenomenon was independent of laser intensity. Photodeoxygenation was not observed with electrospray or fast atom bombardment ionization techniques. Therefore, use of MALDI should be avoided with peptides containing a nitrobenzyl moiety and similar moieties that are prone to reactive photochemistry.     

Design and synthesis of factor Xa inhibitors and their prodrugs

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency (14, IC(50)=0.028 microM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.     

Plasma-mediated release of morphine from synthesized prodrugs

Two morphine prodrugs (‘PDA’ and ‘PDB’) were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5–10 min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse > rat > guinea pig > human). Morphine’s release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.     

Antiprotozoal activity of dicationic 3,5-diphenylisoxazoles,their prodrugs and aza-analogues

Fifty novel prodrugs and aza-analogues of 3,5-bis(4-amidinophenyl)isoxazole and its derivatives were prepared. Eighteen of the 24 aza-analogues exhibited IC₅₀ values below 25 nM against Trypanosoma brucei rhodesiense or Plasmodium falciparum. Six compounds had antitrypanosomal IC₅₀ values below 10 nM. Twelve analogues showed similar antiplasmodial activities, including three with sub-nanomolar potencies. Forty-four diamidines (including 16 aza-analogues) and the 26 prodrugs were evaluated for efficacy in mice infected with T. b. rhodesiense STIB900. Six diamidines cured 4/4 mice at daily 5 mg/kg intraperitoneal doses for 4 days, giving results far superior to pentamidine and furamidine. One prodrug attained 3/4 cures at daily 25 mg/kg oral doses for 4 days.     

Antibiotic resistance plasmids from Serratia marcescens and their elimination by DNA-gyrase inhibitors

We studied the effects of ciprofloxacin on 73 strains of Serratia marcescens. In the first place, we have tested their plasmidic content: 76% of Serratia marcescens strains contained plasmids by electrophoresis, and 29% of these plasmids were self-transmissible by conjugation. Secondly, we studied the plasmid stability with regard to ciprofloxacin. We obtained a spontaneous cure with 19% of plasmids, and ciprofloxacin, at very low concentrations (0.4 mg/l), increased the rate of cure and more efficiently than novobiocin, a compound used as a known curing agent.     

Abacavir prodrugs: microwave-assisted synthesis and their evaluation of anti-HIV activities

The synthesis of a new series of abacavir prodrugs involving N2-substitution with various substituted benzaldehyde and ketone derivatives is described. The in vitro anti-HIV activities indicated that compound (3-(2-(4-methylaminobenzylideneamino)-6-(cyclopropylamino)-9H-purin-9-yl)cyclopentyl)methanol (3) was found to be most potent compound with EC50 of 0.05 microM and CC50 of >100 microM with selectivity index of >2000. Compound 3 was found to be 32 times more potent than the parent drug (EC50 of 1.6 microM). At pH 7.4, 37 degrees C, the hydrolytic t1/2 ranged between 120 and 240 min.     

Synthesis of 7-trifluoromethyl-7-deazapurine ribonucleoside analogs and their monophosphate prodrugs

Abstract

Novel 7-trifluoromethyl-7-deazapurine ribonucleoside analogs (13a-c) and their Protides (15a-c) were successfully synthesized from ribolactol or 1-α-bromo-ribose derivatives using Silyl-Hilbert-Johnson or nucleobase-anion substitution reactions followed by key aromatic trifluoromethyl substitution. Newly prepared compounds were evaluated against a panel of RNA viruses, including HCV, Ebola or Zika viruses.     

Development of enzymatically cleavable prodrugs derived from dendritic polyglycerol

In this Letter we report the synthesis and in vitro studies of cleavable polymer–drug conjugates derived from dendritic polyglycerol and maleimide-bearing prodrugs of doxorubicin and methotrexate that are cleaved by cathepsin B. Cleavage properties and cytotoxicity of the new conjugates are presented.     

The chemical modification of immunoglobulins for accelerating their elimination from the blood flow during radioimmunodiagnosis

Immunoglobulins were modified by diethylenetriaminepentaacetic acid anhydride and sulfosuccinimidy 16-(biotinamido) hexanoate and were conjugated with modified polylysine. Biodistribution of the samples was observed before and after avidin injection. Samples containing no polylysine accumulate in reticuloendothelial system, protein conjugate with polylysine concentrate in kidneys. The results demonstrate that drug distribution depends on the type of modification.     

Bacteriophage and their lysins for elimination of infectious bacteria

When phages were originally identified, the possibility of using them as antibacterial agents against pathogens was immediately recognized and put into practise based on the knowledge available at the time. However, with the advent of antibiotics a decline in the use of phage as therapeutics followed. Phages did, however, become more useful in the study of fundamental aspects of molecular biology and in the diagnostic laboratory for the identification of pathogenic bacteria. More recently, the original application of phage as therapeutics to treat human and animal infections has been rekindled, particularly in an era where antibiotic resistance has become so problematic/commonplace. Phage lysins have also been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections. Indeed the past decade has seen a considerable amount of research worldwide focused on the engineering of phages as antibacterial agents in a wide range of applications. Furthermore, the US Food and Drug Administration and/or the US Department of Agriculture have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods. Such developments have prompted this review into the status of phage research as it pertains to the control of infectious bacteria.     

Tumor escape from immune elimination

A simple kinetic model is proposed for the interaction between a tumor and the immune system. Special attention is given to the phenomenon of “sneaking through” and the associated phenomena of tumor regression and recurrence. Sneaking through refers to the situations in which small antigenic tumors grow progressively, medium-sized tumors are rejected and large ones break through again. The combination of two factors is proposed as being essential for explaining this behavior: (a) The dependence of the immune response on antigen dose. (b) The negative intervention of immunosuppressors, or inhibitory factors.The immune response is described here as a repeated antigen-dependent stimulation of lymphoid cells to proliferate, with a parallel process of antigen-dependent differentiation to a terminal phase. For too small or too large antigen doses the growth in the number of cells is counter-balanced by loss from the proliferative pool through natural decay or by enhanced differentiation, respectively. When this is combined with the blocking of resting and proliferating precursor cells by factors originating from tumor cells, computer simulations demonstrate that the model is able to account for the observed patterns of tumor behavior. The model allows for a discussion of the significance of various biological parameters and is amenable for testing.     

Circular dichroism measurements of benzyl L-aspartate–nitrobenzyl L-aspartate copolymers and their use in detecting and characterizing preferred polymer conformations

We have examined the nature of the circular dichroism band at 330 mμ for a series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate. The circular dichroism band arises from an electronic transition in the nitroaromatic group. In order to interpret the effect quantitatively, we employed a simplified statistical treatment and curve fitting for six copolymers. Both approaches gave consistent results, which indicates that the dichroism comes from pairwise interactions between two nitrobenzyl groups. We constructed a molecular model that meets the constraints and requirements of the analyses developed in this paper. In this model, it is proposed that the main chain forms a right-handed α-helix and that nitrobenzyl groups separated by four residues interact with each other.     

Induction of SENP1 in Endothelial Cells Contributes to Hypoxia-driven VEGF Expression and Angiogenesis

SENP1 (SUMO-specific protease 1) has been shown to be essential for the stability and activity of hypoxia-inducible factor 1 (HIF-1α) under hypoxia conditions. However, it is unknown how SENP1 activation and hypoxia signaling are coordinated in the cellular response to hypoxia. Here, we report the essential role of SENP1 in endothelial cells as a positive regulator of hypoxia-driven VEGF production and angiogenesis. SENP1 expression is increased in endothelial cells following exposure to hypoxia. Silencing of HIF-1α blocks SENP1 expression in cell response to hypoxia. Mutation of the hypoxia response element (HRE) on the Senp1 promoter abolishes its transactivation in response to hypoxia. Moreover, silencing of SENP1 expression decreases VEGF production and abrogates the angiogenic functions of endothelial cell. We also find that the elongated endothelial cells in embryonic brain section and vascular endothelial cells in embryonic renal glomeruli in Senp1^−/− mice are markedly reduced than those in wild-type. Thus, these results show that hypoxia implies a positive feedback loop mediated by SENP1. This feedback loop is important in VEGF production, which is essential for angiogenesis in endothelial cells.     

Hypoxia-driven proliferation of embryonic neural stem/progenitor cells--role of hypoxia-inducible transcription factor-1alpha

We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1alpha, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O(2), 10% O(2), and 3% O(2)) for 3 days. The results showed that hypoxia, especially 10% O(2), promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1alpha mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O(2). To further elucidate the potential role of HIF-1alpha in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1alpha, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1alpha mRNA was used to knock down HIF-1alpha. We found that overexpression of HIF-1alpha caused the same proliferative effect on NPCs under 20% O(2) as under 10% O(2). In contrast, knockdown of HIF-1alpha inhibited NPC proliferation induced by 10% O(2). These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1alpha was critical in this process.