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A series of new lipoic acid derivatives were designed and synthesized as multitarget ligands against Alzheimer's disease. In particular, analogues combining both lipoic acid and cysteine core structures were synthesized. The antioxidant properties of these compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS(•+) ) radical cation scavenging assays and ferrous ion chelation. The antioxidant potential of the synthesized compounds was also evaluated in a cellular context and compared to α-lipoic acid and its reduced form, dihydrolipoic acid. The antioxidant effects observed for these compounds in vitro confirmed the importance of free thiol functions for effective antioxidant capacities. However, these promising in vitro results were not mirrored by the antioxidant activity in T67 cell line. This suggests that multiple factors are at stake and warrant further investigations., (© 2014 John Wiley & Sons A/S.)

In the present work, the capacity of new pro-GSH molecules to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the immune response to Th1 in ovalbumin (Ova)-sensitized mice were examined. The molecules were the N-butanoyl GSH derivative, GSH-C4, and a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA), I-152. In vitro, 2h-incubation with both molecules was found to increase intra-macrophage thiol content; in vivo, Ova-sensitized mice pre-treated by intraperitoneal administration of the pro-GSH molecules showed an increase in plasma anti-Ova IgG2a and IgG2b, characterizing Th1 immune response, and a decrease in IgG1, typical of the Th2 response. Such findings were connected to a shift to a Th1 response also involving splenocyte IFN-γ production as revealed by ELISPOT assay and higher levels of IL-12 in circulation. Although immune responses are in vivo mediated both by dendritic cells and macrophages, the data reported in this paper corroborate the suggestion that the pro-GSH molecules, increasing the intra-cellular thiol pool, modulate the Th1/Th2 balance favouring Th1-type responses and may be employed as Th1-directing adjuvants in new vaccination protocols and as immunomodulators in those diseases where Th1 response patterns are compromised in favour of Th2., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Four novel N-isobutyryl-L-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test, and deoxyribose assay) and their metal binding capacity was evaluated by the ethidium bromide fluorescence binding assay. These results were compared with those obtained with their pro-GSH acetyl analogues recently developed in our laboratory. We observed that most of these compounds exhibit free radical-scavenging activities similar to those of Trolox, but always superior than NAC. While none of these new derivatives had pro-GSH activities, they displayed anti-HIV properties in human monocyte-derived macrophages infected in vitro. The present study demonstrates that these new N-isobutyryl derivatives, which are expected to have a greater bioavailability than their acetyl analogues, may have useful applications in HIV infection in respect to their antioxidant and anti-HIV activities.

We synthesized a series of N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine (10) analogues bearing various acyl groups on thiol cysteine or cysteamine residues, to investigate the structure-activity relationship for pro-GSH and anti-HIV properties in human macrophages. The S-substituents were ranked in the following order of efficacy: H > or = acetyl > isobutyryl > pivaloyl > benzoyl. We found that none of these derivatives had pro-GSH or antiviral activities in vitro higher than that of 10, but several displayed similar levels of anti-HIV activity, making them possible candidates for use as adjuvant therapies in conjunction with HAART, for treating neurological aspects of HIV infection.

Oxidative stress and glutathione deficiency seem to play a major role in the pathogenesis of HIV infection, as suggested by the increased survival of HIV-infected patients treated with N-acetylcysteine, a prodrug of glutathione. However, beneficial effects of GSH-replenishing drugs are restricted in vivo by the high concentrations needed to obtain biological effects and their low bioavailability. In this study, we evaluated the antiretroviral and antioxidant activities of new more lipophilic GSH-replenishing molecules, in macrophages infected in vitro with HIV-1. In these experimental conditions, a prodrug of N-acetylcystéine and beta-mercaptoethylamine, I-152 demonstrated a potent anti-HIV activity, increased intracellular GSH level, and decreased TNF-alpha production. Altogether, these results suggest that I-152 could be beneficial as adjuvant therapy of antiretrovirals in HIV-infected patients, especially in those with damages to the central nervous system or with mitochondrial damages associated with highly active antiretroviral therapy.

RFCNU [(chloro-2-ethyl)-1-(ribofuranosylisopropylidene-2',3'-paranitrobenzoate-5')-3- nitrosourea], RPCNU [(chloro-2-ethyl)-1-(ribopyranosyltriacetate-2',3',4')-3-nitrosourea] and CCNU [(chloro-2-ethyl)-1-cyclohexyl-3-nitrosourea) were found cytotoxic in vitro for human blood lymphocytes from healthy donors at doses higher than 50 micrograms/ml. Blood lymphocytes from different individuals displayed different behaviors both in their sensitivity to nitrosoureas and in their proliferative response to mitogenic lectins. With pokeweed-mitogen-stimulated lymphocytes the main effect of all three drugs (10 micrograms/ml) was an increase of [3H]-thymidine uptake. This facilitation effect was found only in half of the experiments with phytohemagglutinin-stimulated lymphocytes, while the stimulation by concanavalin A was inhibited under the same conditions. The hypothesis that nitrosoureas might be selectively cytotoxic for a suppressor cell subpopulation might explain the observed increase of thymidine uptake.

1. The major tissue metabolites of the radioprotector S-acetyl-N-glycl cysteamine (I) labelled with 14C on the cysteamine group, were quantified and identified in normal tissues and EMT6 tumours implanted in mice, by chromatographic comparison with authentic reference compounds. 2. In all tissues the radioprotector undergoes rapid deacetylation and hydrolysis leading to the formation of cysteamine, which is the main metabolite involved in radioprotection. A major part of this metabolite is reversibly inactivated by binding to endogenous SH. 3. The differential radioprotection of healthy tissues versus EMT6 tumour is explained both by a lower uptake of radioprotector, and a weaker deacetylation and hydrolysis rate, in the tumour cells.

1. The disposition of S-acetyl-N-glycyl cysteamine (I) labelled with 14C on the cysteamine group (label 1), the glycyl group (label 2) and the acetyl group (label 3) has been studied in mice bearing EMT6 tumours. 2. Label 1 was mainly excreted in urine (63.1% dose in 24 h). Label 2 elimination was both in urine (36.0% dose in 24 h) and in expired air as 14CO2 (12.1% dose in 24 h). Label 3 was essentially eliminated in expired air as 14CO2 (55.4% dose in 24 h). 3. Tissue distribution studies of label 1 and label 2 showed that concentrations in tissues were higher than blood concentration as early as 10 min after administration. Whichever label was used, only little radio-activity was found in EMT6 tumour and brain. 4. Analysis of the urinary elimination products showed the presence of unchanged I and of cystamine, N-acetylcystamine, N-acetyl-S-methyl cysteamine sulphoxide and taurine. I is a prodrug of cysteamine which is released after deacetylation and hydrolysis of the amide bond. A metabolic pathway is proposed for this new radioprotective agent.

Some N-(dipeptidyl)-S-acetylcysteamine and N,N'-(dipeptidyl)cystamine salt derivatives were synthesized and evaluated as candidate radioprotector agents. Toxicity and radioprotective activity as the dose reduction factor (DRF) were determined in vivo on mice and compared to N-glycyl-S-acetylcysteamine trifluoroacetate. One of the most interesting compounds of this series was N-glycylglycyl-S-acetylcysteamine trifluoroacetate (8).

Examined by flow cytofluorometric analysis, the DNA distribution of EMT 6 tumor cells was highly perturbed after one hour of in vitro incubation with: RPCNU, RFCNU, chlorozotocin (CZT) or 185 (CNCC), four new nitrosourea derivatives. After the treatment with chlorozotocin (20 micrograms/ml) and CNCC (50 micrograms/ml), most of cells were in G2 + M phase and this accumulation lasted more than 48 hours without any restoration before 72 hours. RPCNU (20 micrograms/ml) and RFCNU (50 and 65 micrograms/ml) induced and accumulation of cells in G2 + M phase during 24 hours. The normal state was regained after 48 hours. These reduced rate of progression of the cells through S phase and the G2 block observed after exposure to the new compounds, should, in part, explain their antitumoral activity.

We have examined the pharmacological characteristics of three dinitrosated isomers of N,N'-bis[N(2-chloroethyl)-N-carbamoyl]cystamine [CNCC-(D), 1C1G1325] differing in the relative positions of the nitroso substituents [CNCC-(C), (1,1' dinitroso); CNCC-(S), 3,3' dinitroso); and CNCC-(M), (1,3'-dinitroso)] and which were designed to be subject to preferential bioreductive activation in hypoxic tumors. The decomposition products of the isomers formed under physiological conditions [both in the absence and in the presence of dithiothreitol (DDT)] were identified and quantified. For example, CNCC-(S) in phosphate buffer, pH 7.0, and 37 degrees gave rise to 2-chloroethylisocyanate, bis(2-chloroethyl)urea and bis(2-hydroxyethyl)disulfide, whereas in the presence of DTT it afforded 2-chloroethylisocyanate, bis(2-chloroethyl)urea, bis(2-hydroxyethyl) disulfide, thiirane and 2-mercaptoethanol. Control aqueous decomposition profiles were performed with two known metabolites of CNCC, namely 3-(2-chloroethyl)-1-(2-thioethyl)-1-nitrosourea and 3-(2-chloroethyl)-1-(2-methylthioethyl)-1-nitrosourea. CNCC-(C) caused 20% interstrand cross-linking of lambda-DNA in 2 hr, whereas in the presence of DTT the extent of cross-linking increased to 38% in the same time period. In contrast, isomer (S) showed no detectable cross-linking in 7 hr. This thiol potentiation of cross-linking which is observed with other 2-chloroethylnitrosoureas is explained by nucleophilic attack at the carbonyl group and subsequent stereoelectronically controlled decomposition of the tetrahedral intermediate. The relative extents of carbamoylating activity of the CNCC isomers were obtained using a [14C]-lysine assay which showed (S) approximately equal to (M) greater than (C). Inhibition of glutathione reductase for both Walker 256 resistant (WR) and Walker 256 sensitive (WS) strains showed that isomer (S) inactivated the enzyme more effectively than isomer (C) in accord with the carbamoylating activity results. The higher carbamoylators (S) and (M) also showed greater effects on the intracellular thiol pools in both WR and WS cells indicative of sulfhydryl conjugation and efflux and/or inhibition of the GSH metabolic enzymes. In vitro cytotoxicity studies with human DU 145 prostatic carcinoma cells showed the isomer cytotoxicity was (M) greater than (C) greater than (S) over a 24-hr incubation period. The reduced cytotoxic potential of CNCC-(S) in both the Walker 256 cells and in the human prostatic carcinoma cells may be a function of an interaction between GSH and the drug thereby protecting other more critical nucleophilic targets within the nucleus.

A practical and convenient method for synthesizing antitumor compounds, N-alkyl-N-nitrosoureas, regioselectively nitrosated on the nitrogen atom bearing the alkyl group is proposed. N-Alkyl-N-nitrosocarbamates are interesting intermediates in these syntheses and yield, by reaction with amino compounds, the regioselectively nitrosated N-alkyl-N-nitrosoureas. As an interesting example, N,N'-bis[(2-chloroethyl)nitrosocarbamoyl]cystamine, a new attractive oncostatic derivative, has been prepared. The cytotoxic activity of these various compounds were tested on L1210 leukemia.