Isothiocyanates as Tubulin Polymerization Inhibitors-Synthesis and Structure-Activity Relationship Studies.

Among the various substances that interfere with the microtubule formation process, isothiocyanates (ITCs) are the group of compounds for which the binding mode and mechanism of action have not yet been explained. To better understand the structure-activity relationship of tubulin-isothiocyanate interactions, we designed and synthesized a series of sixteen known and novel, structurally diverse ITCs, including amino acid ester-derived isothiocyanates, bis-isothiocyanates, analogs of benzyl isothiocyanate, and phosphorus analogs of sulforaphane. All synthesized compounds and selected natural isothiocyanates (BITC, PEITC, AITC, and SFN) were tested in vitro to evaluate their antiproliferative activity, tubulin polymerization inhibition potential, and influence on cell cycle progression. The antiproliferative activity of most of the newly tested compounds exceeded the action of natural isothiocyanates, with four structures being more potent as tubulin polymerization inhibitors than BITC. As a confirmation of anti-tubulin activity, the correlation between polymerization inhibition and cell cycle arrest in the G2/M phase was observed for the most active compounds. In light of the biological results indicating significant differences in the impact of structurally diverse isothiocyanate on tubulin polymerization, in silico analysis was conducted to analyze the possible mode of isothiocyanate-tubulin binding and to show how it can influence the polymerization reaction.

Boronic acid-based arginase inhibitors in cancer immunotherapy.

Arginase is an enzyme that converts l-arginine to l-ornithine and urea in the urea cycle. There are two isoforms of arginase in mammals: ARG-1 and ARG-2. l-Arginine level changes occur in patients with various types of affliction. An overexpression of arginase leads to the depletion of arginine and then to inhibition of the growth of T and NK cells, and in effect to the tumor escape of the immune response. Based on those observations, an inhibition of arginase is proposed as a method to improve anti-tumor immune responses (via an activation and proliferation of T and NK cells). Boronic acid derivatives as arginase inhibitors are leading, potential therapeutic agents for the treatment of several diseases. All these compounds are derived from the original 2-(S)-amino-6-boronohexanoic acid (ABH), the first boronic acid arginase inhibitor proposed by Christianson et al. This article focuses on the review of such sub-class of arginase inhibitors and highlights their SAR and PK properties. It covers molecules published until early 2020, including patent applications.

New diaryl ω-(isothiocyanato)alkylphosphonates and their mercapturic acids as potential antibacterial agents.

Thirty-four novel, diaryl ω-(isothiocyanato)alkylphosphonates with chlorine atom and methoxy, dimethoxy, methylsulfanyl, or methoxycarbonyl groups at ortho, meta, or para positions of the phenyl ring, and with an unbranched alkyl chain (n = 2-6) were designed and synthesized in a one-pot reaction in 11-76% yields. All isothiocyanates thus generated were evaluated for the first time for antibacterial activity on Pseudomonas aeruginosa and Staphylococcus aureus bacterial strains, and had satisfactory antibacterial activity in most cases. The highest activity, similar to that of reference gentamicin activity against S. aureus, was seen in compounds 9 and 13 (1.5 ±â€¯0.1 and 2.5 ±â€¯0.2 µM, respectively), whereas for P. aeruginosa more than half of tested compounds proved to be more effective than gentamicin. Additionally, selected isothiocyanates (9, 13, 18, and 23) were transformed in 52-73% yields into mercapturic acids 42-45, which also exhibited satisfactory antibacterial effect against S. aureus strain.

Phosphorus-containing isothiocyanate-derived mercapturic acids as a useful alternative for parental isothiocyanates in experimental oncology.

A series of phosphonates, phosphinates and phosphine oxides isothiocyanate-derived mercapturic acids were synthesized. A temperature dependence dynamic proton decoupled 31P NMR studies indicated that in most cases the compounds were obtained as a mixture of rotamers. Moreover, biologically relevant reversibility of mercapturic acids synthesis from the parental isothiocyanates was confirmed. All compounds were evaluated ashighly active antiproliferative agents in vitro in human colon cancer cell lines (LoVo and its doxorubicin-resistant subline LoVo/DX). The cell cycle progression and caspase-3 activity analyses revealed compounds moderate activity as apoptosis inducers and their poor influence on cell cycle progression in the LoVo cells. Our results confirm that isothiocyanate-derived mercapturic acids present a reasonable alternative for the parental compounds, and can replace them in the future studies on isothiocyanates potential as anticancer agents.

Design, Synthesis, and Evaluation of ω-(Isothiocyanato)alkylphosphinates and Phosphine Oxides as Antiproliferative Agents.

A series of 21 novel, structurally diverse ω-(isothiocyanato)alkylphosphinates and phosphine oxides (ITCs) were designed and synthesized in moderate to good yields. The synthesized compounds were evaluated for in vitro antiproliferative activity using LoVo and LoVo/DX cancer cell lines. The biological activity of the synthesized compounds was higher than that of natural isothiocyanates such as benzyl isothiocyanate or sulforaphane. The antiproliferative activity of selected ITCs was also tested on selected cancer cell lines: A549, MESSA and MESSA/DX-5, HL60 and HL60MX2, BALB/3T3, and 4T1. These compounds were assessed for their mechanism of action as inducers of cell-cycle arrest and apoptosis. Ethyl (6-isothiocyanatohexyl)(phenyl)phosphinate (71) was tested in vivo on the 4T1 cell line and demonstrated moderate antitumor activity, similar to that benzyl isothiocyanate and cyclophosphamide.

Novel phosphonate analogs of sulforaphane: Synthesis, in vitro and in vivo anticancer activity.

A library of over forty, novel, structurally diverse phosphonate analogs of sulforaphane (P-ITCs) were designed, synthesized and fully characterized. All compounds were evaluated for antiproliferative activity in vitro on Lovo and LoVo/DX colon cancer cell lines. All compounds exhibited high antiproliferative activity, comparable or higher to the activity of naturally occurring benzyl isothiocyanate and sulforaphane. Assessment of the mechanisms of action of selected compounds revealed their potential as inducers of G2/M cell cycle arrest and apoptosis. Further antiproliferative studies for selected compounds with the use of a set of selected cell lines derived from colon, lung, mammary gland and uterus as well as normal murine fibroblasts were performed. In vivo studies of the analyzed phosphonate analogs of sulforaphane showed lower activity in comparison with those of benzyl isothiocyanate. Our studies demonstrated that newly synthesized P-ITCs can be used for as a starting point for the synthesis of novel isothiocyanates with higher anticancer activity in the future.

Synthesis and biological activity of diisothiocyanate-derived mercapturic acids.

This Letter deals with new non-natural diisothiocyanates, their mercapturic acid derivatives-conjugated with N-acetylcysteine as well as their antiproliferative activity towards human colon cancer cell lines and their inhibitory potency towards histone deacetylase activity. The activity of analysed isothiocyanates is not significantly different than their N-acetylcysteine conjugates. In comparison to simple mono-isothiocyanate analogues, aliphatic diisothiocyanates and their conjugates are much more active than the simple presence of two isothiocyanate functionalities could indicate.

Friedel-Crafts-type reaction of pyrene with diethyl 1-(isothiocyanato)alkylphosphonates. Efficient synthesis of highly fluorescent diethyl 1-(pyrene-1-carboxamido)alkylphosphonates and 1-(pyrene-1-carboxamido)methylphosphonic acid.

Friedel-Crafts-type reaction of pyrene with diethyl 1-(isothiocyanato)alkylphosphonates promoted by trifluoromethanosulfonic acid afforded diethyl 1-(pyrene-1-carbothioamido)alkylphosphonates in 83-94% yield. These compounds were transformed, in 87-94% yield, into the corresponding diethyl 1-(pyrene-1-carboxamido)alkylphosphonates by treatment with Oxone(®). 1-(Pyrene-1-carboxamido)methylphosphonic acid was obtained in a 87% yield by treating the corresponding diethyl phosphonate with Me3Si-Br in methanol. All of the synthesized amidophosphonates were emissive in solution and in the solid state. The presence of a phosphonato group brought about an approximately two-fold increase in solution fluorescence quantum yield in comparison with that of a model N-alkyl pyrene-1-carboxamide. This effect was tentatively explained by stiffening of the amidophosphonate lateral chain which was caused by the interaction (intramolecular hydrogen bond) of phosphonate and amide groups. The synthesized phosphonic acid was soluble in a biological aqueous buffer (PBS, 0.01 M, pH 7.35) and was strongly emissive under these conditions (λem = 383, 400 nm, τ = 18.7 ns, ΦF > 0.98). Solid-state emission of diethyl 1-(pyrene-1-carboxamido)methylphosphonate (λmax = 485 nm; ΦF = 0.25) was assigned to π-π aggregates, the presence of which was revealed by single-crystal X-ray diffraction analysis.

Differentiation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids by EI mass spectrometry and density functional theory.

Electron ionization mass spectrometry and density functional theory (DFT) calculations have been used to study the fragmentation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids. The loss of a diethoxyphosphoryl group and the elimination of diethyl phosphonate were found to be competitive fragmentation processes, which can be used to differentiate both stereoisomers. Selective deuterated analogs and product- and precursor-ion mass spectra allowed the elucidation of the fragmentation mechanisms. The structures of the transition states and product ions were optimized using the density functional theory (DFT), and free energy calculations confirmed the observed differences in the formation and relative intensities of specific fragment ions.

Synthesis and antiproliferative activity of novel α- and ß-dialkoxyphosphoryl isothiocyanates.

A series of 15 mostly new dialkoxyphosphoryl alkyl and aralkyl isothiocyanates were synthesized using two alternative strategies, and their in vitro antiproliferative activity against several cancer cell lines (including drug resistant) is here demonstrated. The IC(50) values measured for the new compounds are within the range of 6.3-21.5 µM, and they are quite similar to the activity of two best and most extensively investigated natural benzyl isothiocyanate (A) and phenethyl isothiocyanate (B). Preliminary studies utilizing the cell cycle and reduced glutathione level analysis performed on A549 lung cancer cell line using representative compounds revealed important differences in the mechanism of action possibly correlated with their chemical properties. Hydrophobic compounds react mainly with the cytosolic glutathione reduced leading to its depletion, causing an oxidative stress and cell cycle arrest in G0/G1 phase. On the other hand, hydrophilic compounds cause moderate cell cycle arrest and massive cell death associated with moderate reduced glutathione depletion. These suggest that significant changes in the chemical structure of isothiocyanates, which do not lead to the significant changes in antiproliferative activity, but simultaneously cause a differences in the mechanism of action are possible.

Stereochemical effects in fragmentation of diastereoisomers of protected diethyl 1,2-diamino-alkylphosphonates.

Diastereoisomers of diethyl 5-substituted (2-thioxo-imidazolidin-4-yl)phosphonates, which can be regarded as protected diethyl 1,2-diaminoalkylphosphonates, have been analyzed by electron ionization mass spectrometry. Significant differences in the fragmentation of cis- and trans-diastereoisomers were found. The stereospecificity of the elimination of diethyl phosphonate and the loss of the diethoxyphosphoryl group were studied using specific labeled compounds and collision-induced dissociation. The relative abundances of ions formed via these fragmentation processes can be used for differentiation of both diastereoisomers.

N-carbamate protected alpha-amidoalkyl-p-tolylsulfones: convenient substrates in the aza-Morita-Baylis-Hillman reaction.

An efficient and practical one-pot approach to aza-Morita-Baylis-Hillman adducts has been developed. The reaction occurs between N-Boc or N-Cbz imines, generated in situ from stable and easy to handle N-Boc or N-Cbz protected alpha-amidoalkyl-p-tolylsulfones, and electron-deficient alkenes in the presence of DABCO. The presented procedure eliminates the use of the relatively unstable N-carbamate imines prior to the coupling reaction. The reaction is limited to alpha-amidosulfones derived from aromatic and heteroaromatic aldehydes.

The assignment of the absolute configuration of diethyl hydroxy- and aminophosphonates by 1H and 31P NMR using naproxen as a reliable chiral derivatizing agent.

The assignment of the absolute configuration of hydroxy- and aminophosphonates by their double derivatization with commercially available naproxen is presented. The correlation between the spatial arrangement around the stereogenic carbon center and the signs of the DeltadeltaRS allows determination of the absolute configuration of hydroxy- and aminophosphonates by simple comparison of the 1H and 31P NMR spectra of the (R)- and (S)-naproxen ester or amide derivatives. Extensive conformational analysis (theoretical calculations, low-temperature experiments) supported by the NMR studies of structurally diverse naproxen esters and amides of hydroxy- and aminophosphonates proved that a simplified model can be successfully used.