Tuning the Biological Activity of PI3Kδ Inhibitor by the Introduction of a Fluorine Atom Using the Computational Workflow.

As a member of the class I PI3K family, phosphoinositide 3-kinase δ (PI3Kδ) is an important signaling biomolecule that controls immune cell differentiation, proliferation, migration, and survival. It also represents a potential and promising therapeutic approach for the management of numerous inflammatory and autoimmune diseases. We designed and assessed the biological activity of new fluorinated analogues of CPL302415, taking into account the therapeutic potential of our selective PI3K inhibitor and fluorine introduction as one of the most frequently used modifications of a lead compound to further improve its biological activity. In this paper, we compare and evaluate the accuracy of our previously described and validated in silico workflow with that of the standard (rigid) molecular docking approach. The findings demonstrated that a properly fitted catalytic (binding) pocket for our chemical cores at the induced-fit docking (IFD) and molecular dynamics (MD) stages, along with QM-derived atomic charges, can be used for activity prediction to better distinguish between active and inactive molecules. Moreover, the standard approach seems to be insufficient to score the halogenated derivatives due to the fixed atomic charges, which do not consider the response and indictive effects caused by fluorine. The proposed computational workflow provides a computational tool for the rational design of novel halogenated drugs.

The design of experiments (DoE) in optimization of an aerobic flow Pd-catalyzed oxidation of alcohol towards an important aldehyde precursor in the synthesis of phosphatidylinositide 3-kinase inhibitor (CPL302415).

Herein, we describe the development of a green, scalable flow Pd-catalyzed aerobic oxidation for the key step in the synthesis of CPL302415, which is a new PI3Kδ inhibitor. Applying this environmental-friendly, sustainable catalytic oxidation we significantly increased product yield (up to 84%) and by eliminating of workup step, we improved the waste index and E factor (up to 0.13) in comparison with the stoichiometric synthesis. The process was optimized by using the DoE approach.

Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II-Benzimidazole Derivatives.

Phosphoinositide 3-kinase (PI3K) is the family of lipid kinases participating in vital cellular processes such as cell proliferation, growth, migration, or cytokines production. Due to the high expression of these proteins in many human cells and their involvement in metabolism regulation, normal embryogenesis, or maintaining glucose homeostasis, the inhibition of PI3K (especially the first class which contains four subunits: α, ß, γ, δ) is considered to be a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE) or multiple sclerosis. In this work, we synthesized a library of benzimidazole derivatives of pyrazolo[1,5-a]pyrimidine representing a collection of new, potent, active, and selective inhibitors of PI3Kδ, displaying IC50 values ranging from 1.892 to 0.018 µM. Among all compounds obtained, CPL302415 (6) showed the highest activity (IC50 value of 18 nM for PI3Kδ), good selectivity (for PI3Kδ relative to other PI3K isoforms: PI3Kα/δ = 79; PI3Kß/δ = 1415; PI3Kγ/δ = 939), and promising physicochemical properties. As a lead compound synthesized on a relatively large scale, this structure is considered a potential future candidate for clinical trials in SLE treatment.

Design, Synthesis, and Development of pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part I-Indole Derivatives.

Phosphoinositide 3-kinase δ (PI3Kδ), a member of the class I PI3K family, is an essential signaling biomolecule that regulates the differentiation, proliferation, migration, and survival of immune cells. The overactivity of this protein causes cellular dysfunctions in many human disorders, for example, inflammatory and autoimmune diseases, including asthma or chronic obstructive pulmonary disease (COPD). In this work, we designed and synthesized a new library of small-molecule inhibitors based on indol-4-yl-pyrazolo[1,5-a]pyrimidine with IC50 values in the low nanomolar range and high selectivity against the PI3Kδ isoform. CPL302253 (54), the most potent compound of all the structures obtained, with IC50 = 2.8 nM, is a potential future candidate for clinical development as an inhaled drug to prevent asthma.

Hydrogenation of ß-Keto Sulfones to ß-Hydroxy Sulfones with Alkyl Aluminum Compounds: Structure of Intermediate Hydroalumination Products.

ß-Hydroxy sulfones are important in organic synthesis. The simplest method of ß-hydroxy sulfones synthesis is the hydrogenation of ß-keto sulfones. Herein, we report the reducing properties of alkyl aluminum compounds R3Al (R = Et, i-Bu, n-Bu, t-Bu and n-Hex); i-Bu2AlH; Et2AlCl and EtAlCl2 in the hydrogenation of ß-keto sulfones. The compounds i-Bu2AlH, i-Bu3Al and Et3Al are the at best reducing agents of ß-keto sulfones to ß-hydroxy sulfones. In reactions of ß-keto sulfones with aluminum trialkyls, hydroalumination products with ß-hydroxy sulfone ligands [R2AlOC(C6H5)CH2S(O)2(p-R1C6H4]n [where n = 1,2; 2aa: R = i-Bu, R1 = CH3; 2ab: R = i-Bu, R1 = Cl; 2ba: R = Et, R1 = CH3; 2bb: R = Et, R1 = Cl] and {[Et2AlOC(C6H5)CH2S(O)2(p-ClC6H4]∙Et3Al}n3bb were obtained. These complexes in the solid state have a dimeric structure, while in solutions, they appear as equilibrium monomer-dimer mixtures. The hydrolysis of both the isolated 2aa, 2ab, 2ba, 2bb and 3bb and the postreaction mixtures quantitatively leads to pure racemic ß-hydroxy sulfones. Hydroalumination reaction of ß-keto sulfones with alkyl aluminum compounds and subsequent hydrolysis of the complexes is a simple and very efficient method of ß-hydroxy sulfones synthesis.

Towards deeper understanding of multifaceted chemistry of magnesium alkylperoxides.

Despite considerable progress in the multifaceted chemistry of non-redox-metal alkylperoxides, the knowledge about magnesium alkylperoxides is in its infancy and only started to gain momentum. Harnessing the well-defined dimeric magnesium tert-butylperoxide [(f5BDI)Mg(µ-η2:η1-OOtBu)]2 incorporating a fluorinated ß-diketiminate ligand, herein, we demonstrate its transformation at ambient temperature to a spiro-type, tetranuclear magnesium alkylperoxide [(f5BDI)2Mg4(µ-OOtBu)6]. The latter compound was characterized by single-crystal X-ray diffraction and its molecular structure can formally be considered as a homoleptic magnesium tert-butylperoxide [Mg(µ-OOtBu)2]2 terminated by two monomeric magnesium tert-butylperoxides. The formation of the tetranuclear magnesium alkylperoxide not only contradicts the notion of the high instability of magnesium alkylperoxides, but also highlights that there is much to be clarified with respect to the solution behaviour of these species. Finally, we probed the reactivity of the dimeric alkylperoxide in model oxygen transfer reactions like the commonly invoked metathesis reaction with the parent alkylmagnesium and the catalytic epoxidation of trans-chalcone with tert-butylhydroperoxide as an oxidant. The results showed that the investigated system is among the most active known catalysts for the epoxidation of enones.

Formylation of a metathesis-derived ansa[4]-ferrocene: a simple route to anticancer organometallics.

Formylation of ansa[4]-ferrocene, obtained through the ruthenium-catalysed olefin metathesis, yields two separable, planar chiral 1,3- and 1,2-ansa-ferrocene aldehydes. Single-crystal X-ray structure analysis reveals that both regioisomers crystallize with spontaneous resolution of the racemate in the chiral P212121 space group with one molecule in the asymmetric unit. The major 1,3-isomer was further transformed into a conjugate with 1,2,3-triazole and uracil using "click" chemistry as the key synthetic step. This inorganic-organic hybrid displays anticancer activity (MCF-7, A549, MDA-MB-231 cell lines) with EC50 values comparable to those for cisplatin.

Sulfone derivatives enter the cytoplasm of Candida albicans sessile cells.

Since our study showed that sulfone derivatives' action mode creates a lesser risk of inducing widespread resistance among Candida spp., we continued verifying sulfones' antifungal activity using the following newly synthesized derivatives: bromodichloromethy-4-hydrazinyl-3-nitrophenyl sulfone (S1), difluoroiodomethyl-4-hydrazinyl-3-nitrophenyl sulfone (S2), and chlorodifluoromethyl-4-hydrazinyl-3-nitrophenyl sulfone (S3). As the mechanism by which sulfones gain access to the cytoplasm has not been elucidated yet, in order to track S1-3, we coupled their hydrazine group with BODIPY (final S1-3 BODIPY-labelled were named SB1-3). This approach allowed us to follow the vital internalization and endocytic routing of SB1-3, while BODIPY interacts primarily with fungal surfaces, thus confirming that S1-3 and their counterparts SB1-2 behaved as non-typical agents by damaging the cell membrane and wall after being endocytosed (SB1-3 fluorescence visible inside the unlysed sessile cells). Thus greatly decreasing the likelihood of the appearance of strains resistance. Core sulfones S1-3 are a promising alternative not only to treat planktonic C. albicans but also biofilm-embedded cells. In the flow cytometric analysis, the planktonic cell surface was digested by S1-3, which made the externalized PS accessible to AnnexinV binding and PI input (accidental cell death ACD). The occurrence of ACD as well as apoptosis (crescent-shaped nuclei) and anoikis of sessile cells (regulated cell death by 100%-reduction in attachment to epithelium) was assessed through monitoring the AO/PI/HO342 markers. CLSM revealed the invasion of S1-3 and SB1-3 in C. albicans without inducing cell lysis. This was a novel approach in which QCM-D was used for real-time in situ detection of viscoelastic changes in the C. albicans biofilm, and its interaction with S1 as a representative of the sulfones tested. S1 (not toxic in vivo) is a potent fungicidal agent against C. albicans and could be administered to treat invasive candidiasis as a monotherapy or in combination with antifungal agents of reference to treat C. albicans infections.

New antifungal 4-chloro-3-nitrophenyldifluoroiodomethyl sulfone reduces the Candida albicans pathogenicity in the Galleria mellonella model organism.

Candida albicans represents an interesting microorganism to study complex host-pathogen interactions and for the development of effective antifungals. Our goal was to assess the efficacy of 4-chloro-3-nitrophenyldifluoroiodomethyl sulfone (named Sulfone) against the C. albicans infections in the Galleria mellonella host model. We assessed invasiveness of CAI4 parental strain and mutants: kex2Δ/KEX2 and kex2Δ/kex2Δ in G. mellonella treated with Sulfone. We determined that KEX2 expression was altered following Sulfone treatment in G. mellonella-C. albicans infection model. Infection with kex2Δ/kex2Δ induced decreased inflammation and minimal fault in fitness of larvae vs CAI4. Fifty percent of larvae died within 4-5 days (P value < 0.0001) when infected with CAI4 and kex2Δ/KEX2 at 109 CFU/mL; survival reached 100% in those injected with kex2Δ/kex2Δ. Larvae treated with Sulfone at 0.01 mg/kg 30 min before infection with all C. albicans tested survived infection at 90-100% vs C. albicans infected-PBS-treated larvae. Hypersensitive to Sulfone, kex2Δ/kex2Δ reduced virulence in survival. KEX2 was down-regulated when larvae were treated with Sulfone: 30 min before and 2 h post-SC5314-wild-type infection respectively. kex2Δ/kex2Δ was able to infect larvae, but failed to kill host when treated with Sulfone. Sulfone can be used to prevent or treat candidiasis. G. mellonella facilitates studding of host-pathogen interactions, i.e., testing host vs panel of C. albicans mutants when antifungal is dosed.

Novel Sulfones with Antifungal Properties: Antifungal Activities and Interactions with Candida spp. Virulence Factors.

Since candidiasis is so difficult to eradicate with an antifungal treatment and the existing antimycotics display many limitations, hopefully new sulfone derivatives may overcome these deficiencies. It is pertinent to study new strategies such as sulfone derivatives targeting the virulence attributes of C. albicans that differentiate them from the host. During infections, the pathogenic potential of C. albicans relies on the virulence factors as follows: hydrolytic enzymes, transcriptional factors, adhesion, and development of biofilms. In the article we explored how the above-presented C. albicans fitness and virulence attributes provided a robust response to the environmental stress exerted by sulfones upon C. albicans; C. albicans fitness and virulence attributes are fungal properties whose inactivation attenuates virulence. Our understanding of how these mechanisms and factors are inhibited by sulfones has increased over the last years. As lack of toxicity is a prerequisite for medical approaches, sulfones (non-toxic as assessed in vitro and in vivo) may prove to be useful for reducing C. albicans pathogenesis in humans. The antifungal activity of sulfones dealing with these multiple virulence factors and fitness attributes is discussed.

Unprecedented Variety of Outcomes in the Oxygenation of Dinuclear Alkylzinc Derivatives of an N,N-Coupled Bis(ß-diketimine).

Reactions between O2 and organometallics with non-redox-active metal centers have received continuous interest for over 150 years, although significant uncertainties concerning the character and details of the actual mechanism of these reactions persist. Harnessing dinuclear three-coordinate alkylzinc derivatives of an N,N-coupled bis(ß-diketimine) proligand (LH2 ) as a model system, we demonstrate for the first time that a slight modification of the reaction conditions might have a dramatic influence on the oxygenation reaction outcomes, leading to an unprecedented variety of products originating from a single reaction system, that is, partially and fully oxygenated zinc alkoxides, zinc alkylperoxides, and zinc hydroxide compounds. Our studies indicate that accessibility of the three-coordinate zinc center by the O2 molecule, coupled with the lower reactivity of Zn-Me vs. Zn-Et units towards dioxygen, are key factors in the oxygenation process, providing a novel tetranuclear methyl(methoxy)zinc {[L][ZnMe][Zn(µ-OMe]}2 and zinc ethoxide {[L][Zn(µ-OEt)]2 }2 . Remarkably, oxygenation of three-coordinate alkylzinc [L][ZnR]2 complexes at ambient temperature afforded a unique hydroxide {[L][Zn(µ-OH)]2 }2 . Oxygenation of the [L][ZnEt]2 complex in the presence of 4-methylpyridine (py-Me) at low temperature led to the isolation of a dinuclear zinc ethylperoxide [L][Zn(OOEt)(py-Me)]2 , which nicely substantiates the intermediacy of an unstable zinc alkylperoxide in the formation of the subsequent zinc alkoxide and hydroxide compounds. Finally, our investigations provide compelling evidence that a non-redox-active metal center plays a crucial role in the oxygenation process through assisting in single-electron transfer from an M-C bond to an O2 molecule. Although the oxygenation of zinc alkyls occurs by radical pathways, the reported results stand in clear contradiction to the widely accepted free-radical chain mechanism.

Effect of serine protease KEX2 on Candida albicans virulence under halogenated methyl sulfones.

AIM: The effect of KEX2 mutations on C. albicans virulence and resistance to halogenated methyl sulfones was assessed. MATERIALS & METHODS: The mechanism of action of sulfones was studied using flow cytometry and microscopy. Expression of KEX2 and SAP5 was assessed using quantitative Real-Time-PCR. 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide and lactate dehydrogenase assays were elaborated to study, respectively, metabolism of Candida treated with sulfones and their cytotoxicity against tissues. Inflammatory response was detected by ELISA. RESULTS: Lysosome permeabilization and dose-dependent programmed cell death under sulfones were noted. KEX2 induction depended on halogenomethylsulfonyl groups, which affected cell wall biosynthesis and adhesion. CONCLUSION: Sulfones treatment reduced Candida pathogenicity in Galleria mellonella. Sulfones are an alternative for antifungal therapies due to their safety profile and antibiofilm activity.

Role of Virulence Determinants in Candida albicans' Resistance to Novel 2-bromo-2-chloro-2-(4-chlorophenylsulfonyl)-1-phenylethanone.

We investigated the role of KEX2, SAP4-6, EFG1, and CPH1 in the virulence of Candida under a novel compound 2-bromo-2-chloro-2-(4-chlorophenylsulfonyl)-1-phenylethanone (Compound 4). We examined whether the exposure of C. albicans cells to Compound 4, non-cytotoxic to mammalian cells, reduces their adhesion to the human epithelium. We next assessed whether the exposure of C. albicans cells to Compound 4 modulates the anti-inflammatory response (IL-10) and induces human macrophages to respond to the Candida cells. There was a marked reduction in the growth of the sap4Δsap5Δsap6Δ mutant cells when incubated with Compound 4. Under Compound 4 (minimal fungicidal concentration MFC = 0.5-16 µg/mL): (1) wild type strain SC5314 showed a resistant phenotype with down-regulation of the KEX2 expression; (2) the following mutants of C.albicans: sap4Δ, sap5Δ, sap6Δ, and cph1Δ displayed decreased susceptibility with the paradoxical effect and up-regulation of the KEX2 expression compared to SC5314; (3) the immune recognition of C. albicans by macrophages and (4) the stimulation of IL-10 were not blocked ex vivo. The effect of deleting KEX2 in C. albicans had a minor impact on the direct activation of Compound 4's antifungal activity. The adhesion of kex2Δ is lower than that of the wild parental strain SC5314, and tends to decrease if grown in the presence of a sub-endpoint concentration of Compound 4. Our results provide evidence that SAP4-6 play a role as regulators of the anti-Candida resistance to Compound 4. Compound 4 constitutes a suitable core to be further exploited for lead optimization to develop potent antimycotics.

Oxygenation Chemistry of Magnesium Alkyls Incorporating ß-Diketiminate Ligands Revisited.

Despite the fact that extensive research has been carried out, the oxygenation of alkyl magnesium species still remains a highly unexplored research area and significant uncertainties concerning the mechanism of these reactions and the composition of the resulting products persist. This case study compares the viability of the controlled oxygenation of alkylmagnesium complexes supported by ß-diketiminates. The structural tracking of the reactivity of (N,N)MgR-type complexes towards O2 at low temperature showed that their oxygenation led exclusively to the formation of magnesium alkylperoxides (N,N)MgOOR. The results also highlight significant differences in the stability of the resulting alkylperoxides in solution and demonstrate that [(BDI)Mg(µ-η2 :η1 -OOBn)]2 (in which BDI=[(ArNCMe)2 CH]- and Ar=C6 H3 iPr2 -2,6) can be easily transformed to the corresponding magnesium alkoxide [(BDI)MgOBn]2 at ambient temperature, whilst [(F3 BDI)Mg(µ-OOtBu)]2 (in which F3 BDI=[(ArNCMe)2 CH]- and Ar=C6 H2 F3 -2,4,6) is stable under similar conditions. The observed selective oxygenation of (N,N)MgR-type complexes to the corresponding (N,N)MgOOR alkylperoxides strongly contradicts the widely accepted radical-chain mechanism for the oxygenation of the main-group-metal alkyls. Furthermore, either the observed transformation of the alkylperoxide [(BDI)MgOOBn]2 to the alkoxide [(BDI)MgOBn]2 as well as the formation of an intractable mixture of products in the control reaction between the alkylperoxide [(F3 BDI)MgOOtBu]2 and the parent alkylmagnesium [(F3 BDI)MgtBu] complex are not in line with the common wisdom that magnesium alkoxide complexes' formation results from the metathesis reaction between MgOOR and Mg-R species. In addition, a high catalytic activity of well-defined magnesium alkylperoxides, in combination with tert-butyl hydroperoxide (TBHP) as an oxygen source, in the epoxidation of trans-chalcone is presented.

Antifungal Effect of Novel 2-Bromo-2-Chloro-2-(4-Chlorophenylsulfonyl)-1-Phenylethanone against Candida Strains.

We investigated the antifungal activity of novel a 2-bromo-2-chloro-2-(4-chlorophenylsulfonyl)-1-phenylethanone (compound 4). The synthesis of compound 4 was commenced from sodium 4-chlorobenzene sulfinate and the final product was obtained by treatment of α-chloro-ß-keto-sulfone with sodium hypobromite. The sensitivity of 63 clinical isolates belonging to the most relevant Candida species toward compound 4 using the method M27-A3 was evaluated. We observed among most of the clinical strains of C. albicans MIC ranging from 0.00195 to 0.0078 µg/mL. Compound 4 at 32 µg/mL exhibited fungicidal activity against nine Candida strains tested using the MFC assay. Compound 4 displayed anti-Candida activity (with clear endpoint) against 22% of clinical strains of Candida. Under compound 4, Candida susceptibility and tolerance, namely paradoxical effect (PG), was found for only two clinical isolates (C. glabrata and C. parapsilosis) and reference strain 14053 using both M27-A3 and MFC method. We found that compound 4 does not induce toxicity in vivo against larvae of Galleria mellonella (≥97% survival) and it displays reduced toxicity on mammalian cells in vitro (< CC20 at 64 µg/mL). Furthermore, XTT assay denoted clear metabolic activity of sessile cells in the presence of compound 4. Thus, the effect of compound 4 on formed C. albicans biofilms was minimal. Moreover, strain 90028 exhibited no defects in hyphal growth on Caco-2 monolayer under compound 4 influence at MIC = 16 µg/mL. The MIC values of compound 4 against C. albicans 90028, in medium with sorbitol did not suggest that compound 4 acts by inhibiting fungal cell wall synthesis. Our findings with compound 4 suggest a general strategy for antifungal agent development that might be useful in limiting the emergence of resistance in Candida strains.

Susceptibility of Candida albicans to new synthetic sulfone derivatives.

The influence of halogenated methyl sulfones, i.e. bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named halogenated methyl sulfone 1), dichloromethyl-4-chloro-3-nitrophenyl sulfone (halogenated methyl sulfone 2), and chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (halogenated methyl sulfone 3), on cell growth inhibition, aspartic protease gene (SAP4-6) expression, adhesion to epithelium, and filamentation was investigated. Antifungal susceptibility of the halogenated methyl sulfones was determined with the M27-A3 protocol in the range of 16-0.0313 µg/mL. Adherence to Caco-2 cells was performed in 24-well plates; relative quantification was normalized against ACT1 in cells after 18 h of growth in YEPD and on Caco-2 cells. SAP4-6 expression was analyzed using RT-PCR. Structure-activity relationship studies suggested that halogenated methyl sulfone 1 containing bromodichloromethyl or dichloromethyl function at C-4 (halogenated methyl sulfone 2) of the phenyl ring showed the best activity (100% cell inhibition at 0.5 µg/mL), while hydrazine at C-1 (halogenated methyl sulfone 3) reduced the sulfone potential (100% = 4 µg/mL). SAP4-6 were up- or down-regulated depending on the strains' genetic background and the substitutions on the phenyl ring. Halogenated methyl sulfone 2 repressed germination and affected adherence to epithelium (P ≤ 0.05). The tested halogenated methyl sulfones interfered with the adhesion of Candida albicans cells to the epithelial tissues, without affecting their viability after 90 min of incubation. The mode of action of the halogenated methyl sulfones was attributed to the reduced virulence of C. albicans. SAP5 and SAP6 contribute to halogenated methyl sulfones resistance. Thus, halogenated methyl sulfones can inhibit biofilm formation due to their interference with adherence and with the yeast-to-hyphae transition.

Development of zinc alkyl/air systems as radical initiators for organic reactions.

This paper reports a series of comparative experiments on the activity of carbon- and oxygen-centred radical species in a model reaction of the radical addition of THF to imines mediated by a series of zinc alkyl/air reaction systems. The study strongly contradicts the notion that generally R˙ radicals are the initiating species in organic reactions mediated by R n M/air systems, and simultaneously demonstrates that oxygen-centred radical species are the key intermediates responsible for the initiation process. In addition, a new efficient RZn(L)/air initiating system for radical organic reactions exampled by a model reaction of radical addition of THF to imines is developed. Moreover, the isolation and structural characterization of the first zinc alkylperoxide supported by a carboxylate ligand, [Zn4(µ3-OOtBu)3(µ4-O)(O2CEt)3]2, as well as the novel octanuclear zinc oxo(alkoxide) aggregate with entrapped O-THF species, [Zn4(µ4-O)(µ3-2-O-THF)(O2CEt)5]2, provide clear mechanistic signatures for the mode of function of the RZn(O2CR')/air system.

New synthetic sulfone derivatives inhibit growth, adhesion and the leucine arylamidase APE2 gene expression of Candida albicans in vitro.

The successful preventing and effective treatment of invasive Candida albicans infections required research focused on synthesis of new classes of agents and antifungal activity studies. Bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named compound 6); dichloromethyl-4-chloro-3-nitrophenyl sulfone (named 7); and chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (named 11) on inhibition of planktonic cells' growth, leucine arylamidase APE2 gene expression, and adhesion to epithelial cells were investigated. In vitro anti-Candida activities were determined against wild-types, and the morphogenesis mutants: Δefg1 and Δcph1. MICs of compounds 6, 7 and 11 (concentrated at 0.25-16µg/ml) were determined using the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document). APE2 expression was analyzed using RT-PCR; relative quantification was normalized against ACT1 in cells growth in YEPD and on Caco-2 cell line. Adherence assay of C. albicans to Caco-2 was performed in 24-well-plate. The structure activity relationship suggested that sulfone containing hydrazine function at C-1 (compound 11) showed higher antifungal activity (cell inhibition%=100 at 1-16µg/ml) than the remaining sulfones with chlorine at C-1. Δcph1/Δefg1 was highly sensitive to compound 11, while the sensitivity was reduced in Δcph1/Δefg1::EFG1 (%=100 at 16-fold higher concentration). Compound 11 significantly affected adherence to epithelium (P ⩽0.05) and hyphae formation. The APE2 up-regulation plays role in sulfones' resistance on MAP kinase pathway. Either CPH1 or EFG1 play a role in the resistance mechanism in sulfones. The strain-dependent phenomenon is a factor in the sulfone resistance mechanism. Sulfones' mode of action was attributed to reduced virulence arsenal in terms of adhesiveness and pathogenic potential related to the APE2 expression and morphogenesis.

Sulfone derivatives reduce growth, adhesion and aspartic protease SAP2 gene expression.

Fungal virulence factors represent a strategy for the design of new compounds with effective activities against Candida spp. Dichloromethyl-4-chloro-3-nitrophenylsulfone (named Compound 1) and chlorodibromomethyl-4-hydrazino-3-nitrophenylsulfone (Compound 2) versus Candida albicans virulence factors (SAP2 expression and adhesion to Caco-2 cell line) were investigated. Candida albicans SC5314 and its mutants: Δsap9, Δsap10, Δsap9/10 were used. MICs of the Compounds (concentrated at 0.0313-16 µg/ml) were determined using M27-A3. Percentage of cell inhibition was assessed spectrophotometrically (OD405) after 48 h at 35 °C. The SAP2 expression was analyzed with the use of RT-PCR; relative quantification was normalized against ACT1 in cells grown in YEPD and on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. Compound 1 showed higher activity (% = 100 at 4 µg/ml) than Compound 2 (MIC90 = 16 µg/ml). Dichloromethyl at the para position of the phenyl ring exerted anti-Candidal potential. Under Compound 1, SAP2 was down-regulated in all the strains (P ≤ 0.05). Conversely, SAP2 was over-expressed in Δsap9-10 (untreated cells) compared with the wild-type. The Compounds significantly affected adherence to epithelium (P ≤ 0.05). The tested sulfones interfered with the adhesion of C. albicans cells to the epithelial tissues without affecting their viability after 90-min of incubation. The Compounds' mode of action was attributed to the reduced adhesiveness and the lower SAP2 expression. Saps9-10 play a role in C. albicans adhesion and they can be involved in the sulfone resistance mechanisms.

Synthesis and in vitro antibacterial activity of 5-halogenomethylsulfonyl- benzimidazole and benzotriazole derivatives.

A series of 5-halogenomethylsulfonylbenzimidazole and benzotriazole derivatives was synthesized as potential antibacterial agents. A new method of synthesis of benzimidazoles was developed. The antimicrobial activities of these compounds were tested against a series of reference and clinical strains. The reference strains include Gram-positive bacteria (S. aureus, S. epidermidis, B. subtilis, B. cereus, E. hirae, M. luteus) and Gram-negative rods (E. coli, P. vulgaris, P. aeruginosa, B. bronchiseptica). The clinical strains include six methicillin-resistant (MRSA) and two methicillin-sensitive (MSSA) S. aureus strains, one methicillin-resistant S. epidermidis, three E. faecalis and two E. faecium strains. Compound with trifluoromethyl- substituent at C-2 position (4) displayed significant antibacterial activities comparable with nitrofurantoin against four strains, and higher against Micrococcus luteus. MIC values for clinical Staphylococci strains (MRSA) were 12.5-25 µg/mL; for Enterococcus strains these values were 50-100 µg/mL. MIC values for reference and clinical strains of Staphylococcus and Enterococcus group were similar, respectively.