Polyhydroxylated azetidine iminosugars: Synthesis,glycosidase inhibitory activity and molecular docking studies

An efficient and practical strategy for the synthesis of unknown azetidine iminosugars (2S,3R,4S)-2-((R)-1,2-dihydroxyethyl)-3-hydroxy-4-(hydroxymethyl)azetidine 2, (2S,3r,4R)-3-hydroxy-2,4-bis(hydroxymethyl)azetidine 3 and (2S,3R,4S)-3-hydroxy-4-(hydroxymethyl)-N-methylazetidine-2-carboxylic acid 4, starting from the d-glucose has been reported. The methodology involves preparation of the 3-amino-N-benzyloxycarbonyl-3-deoxy-6-O-tert-butyldimethylsillyl-1,2-O-isopropylidene-α-d-glucofuranose 9, which was converted to the C-5-OMs derivative 11. Intramolecular nucleophilic displacement of the C-5-OMs group with in situ generated 3-amino functionality provided the required key azetidine ring skeletons 10 with additional hydroxymethyl group. Removal of 1,2-acetonide protection, followed by reduction and hydrogenolysis afforded azetidine iminosugar 2. Alternatively, removal of 1,2-acetonide group and chopping of C1-anomeric carbon gave C2-aldehyde that on reduction or oxidation followed by hydrogenolysis gave 2,4-bis(hydroxymethyl) azetidine iminosugars 3 and N-methylazetidine-2-carboxylic acid 4 respectively. The glycosidase inhibitory activity of 2–4 iminosugars was screened against various glycosidase enzymes and compared with a standard miglitol. Amongst synthesized targets, the compound 2 was found to be more potent amyloglucosidase inhibitor than miglitol. These results were supported by molecular docking studies.     

Nucleic acid-specific photoactivation of oligodeoxyribonucleotides labeled with deuterated dihydro-N,N,N′,N′-tetramethylrhodamine using green light

We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N′,N′-tetramethylrhodamine (F*) with oligodeoxyribonucleotides and a 2′-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD₄ and conjugates of oligonucleotides with N,N,N′,N′-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the F* is oxidized forming fluorescent F. This reaction is >30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells.     

Identification of diacylglycerol-O-(N,N,N-trimethyl)-homoserine in the halotolerant alga, Dunaliella parva

An unusual zwitterionic polar lipid component isolated from the halotolerant alga, Dunaliella parva Lerche, has been identified as 1(3),2-diacylglyceryl-3(1)-O-4′-(N,N,N-trimethyl) homoserine by means of infrared spectrometry, ¹H- and ¹³C-NMR spectrometry and field desorption mass spectrometry of the intact lipid, as well as by its TLC mobilities and staining behaviour. Mass spectrometry of this lipid indicated the presence of the following major molecular species: 16:0–18:0 (24%); 18:3–18:3 (19%); 16:0–18:2 (16%); 16:0–18:1 (8%).     

Design,synthesis and biological evaluation of N,N-3-phenyl-3-benzylaminopropanamide derivatives as novel cholesteryl ester transfer protein inhibitor

A series of N,N-3-phenyl-3-benzylaminopropanamide derivatives were identified as novel CETP (cholesteryl ester transfer protein) inhibitors. In our previous study, lead compound L10 was discovered by pharmacophore-based virtual screening (Dong-Mei Zhao et al., 2014). Based on L10 (IC₅₀ 8.06 μM), compound HL6 (IC₅₀ 10.7 μM) was discovered following systematic structure variation and biological tests. Further optimization of the structure–activity relationship (SAR) resulted in N,N-3-phenyl-3-benzylaminopro panamides derivatives as novel CETP inhibitors. They were synthesized and evaluated against CETP by BODIPY-CE fluorescence assay. Among them, HL16 (IC₅₀ 0.69 μM) was a highly potent CETP inhibitor in vitro. In addition, HL16 exhibited favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. The molecular docking of HL16 into the CETP was performed. The binding mode demonstrated that HL16 occupied the CETP binding site and formed interactions with the key amino acid residues.     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

Mercaptoethanesulfonic acid (CoM imitator) interaction studies with nickel(II) complexes of pyridyl groups containing tetradentate ligands: Synthesis, structure, spectra and redox properties

Nickel(II) complexes of N,N′-dimethyl-N,N′-bis(pyridyl-2yl-methyl)ethylene-diamine (L¹), N,N′-dimethyl-N,N′-bis(pyridyl-2-ylmethyl)-1,2-diaminopropane (L²) and N,N′-dimethyl-N,N′-bis(pyridyl-2-ylmethyl)-1,3-diaminopropane (L³) were prepared and their spectroscopic and redox properties studied. The distorted octahedral structure was determined for [NiL³ClCH₃OH](ClO₄) by using X-ray crystallography. The electronic spectral behavior of the complexes at different pHs was analyzed; it is shown that a new band grew at the expense of the other band intensity in acid media. The redox properties of ligands and their complexes show the peaks of Ni(II) → Ni(III) and Ni(II) → Ni(0) as these were detected at low concentration while Ni(II) → Ni(I) process was detectable clearly at high concentration. Furthermore, the interaction studies of 2-mercaptoethanesulfonic acid as a simulator of coenzyme M reductase (CoM) with NiN₄ chromophores are discussed.     

Design,synthesis and biological evaluation of N2,N4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives as HIV-1 NNRTIs

A series of N₂,N₄-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs) was designed and synthesized by a facile route. The biological assay results showed that five most potent compounds displayed inhibitory activity against HIV-1 at low micromolar concentrations (EC₅₀ = 5.1–8.9 μM). Structure–activity relationship analysis indicated that N₂-(3-halogenated-benzyl) analogues were more potent than N₂-(unsubstituted-benzyl) analogues. The N₄-substitutions contributed to the antiviral activity in the following order: 2-/3-cyano substituted benzyl > 2-/3-halogenated benzyl > non-substituted benzyl > 4-halogenated benzyl. Docking studies of the representative compound revealed the binding conformation of these compounds and provided critical insights for the further development of PTTD analogues.     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

Inhibition of P-glycoprotein-mediated Multidrug Resistance (MDR) by N,N-bis(cyclohexanol)amine aryl esters: further restriction of molecular flexibility maintains high potency and efficacy

Conformational modulation of the aryl portion of a set of N,N-bis(cyclohexanol)amine aryl esters (1a-d) that are potent Pgp-dependent MDR inhibitors has been performed. Toward this end the trans-3-(3,4,5-trimethoxyphenyl)acrylic acid present in set 1 was substituted with 3-(3,4,5-trimethoxyphenyl)propanoic and 3-(3,4,5-trimethoxyphenyl)propiolic moieties to give sets 2 and 3, respectively. While the introduction of 3-(3,4,5-trimethoxyphenyl)propanoic moiety resulted in a definite drop in potency and efficacy, esterification with 3-(3,4,5-trimethoxyphenyl)propiolic acid gave four isomers (3a-d) that maintain high potency and possess optimal efficacy. These results are discussed in terms of conformational flexibility of the different sets of compounds.     

Phosphono-sphingomyelins. I: Synthesis of ceramide (trimethyl) aminoethyl phosphonate

The synthesis of the phosphono-analogue of sphingomyelin is described. The N-acyl-D-erythro-sphingosyl-1-(N,N,N-trimethyl-2-aminoethyl) phosphonate was obtained by phosphonylation of N-acyl-3-O-benzoyl-D-erythro-sphingosine with (2-bromoethyl)phosphonic acid chloride and triethylamine, subsequent quaternisation with anhydrous trimethylamine and benzene at 55–60°C for four days, and finally, consecutive removal of the protective group by mild alkaline hydrolysis. Comparison of the CD spectra of both, natural sphingomyelin and its phosphono-analogue, confirmed that their structures and configurations were identical.     

The Novel Bacterial N-Demethylase PdmAB Is Responsible for the Initial Step of N,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

The environmental fate of phenylurea herbicides has received considerable attention in recent decades. The microbial metabolism of N,N-dimethyl-substituted phenylurea herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. The pdmAB genes in Sphingobium sp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted phenylurea herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genes pdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity of pdmAB. Without its cognate electron transport components, expression of PdmAB in Escherichia coli, Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp. strain RW1 greatly enhanced the catalytic activity of PdmAB in E. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved in O- or N-demethylation reactions, indicating that PdmAB is a distinct bacterial RO N-demethylase.     

N,O-Dialkyl deoxynojirimycin derivatives as CERT START domain ligands

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.     

Discovery of novel N,N-3-phenyl-3-benzylaminopropionanilides as potent inhibitors of cholesteryl ester transfer protein in vivo

Epidemiological studies have identified that the risk of cardiovascular events increases due to the decreased levels of high density lipoprotein-cholesterol and the elevated levels of low density lipoprotein-cholesterol. Herein, we report a novel series of N,N-3-phenyl-3-benzylaminopropionanilide derivatives, which were identified as potent cholesteryl ester transfer protein (CETP) inhibitor. The initial lead compound L10 (IC₅₀ 8.06 μM) was found by pharmacophore-based virtual screening (Dong-Mei Zhao et al., Chin. Chem. Lett. 2014, 25, 299). After systematic structure variation and biological testing against CETP, two different series were identified as scaffolds for potent CETP inhibitors. One is N,N-3-phenyl-3-benzylaminopropanamide derivatives, which were investigated in our previous paper (Bioorg. Med. Chem. 2015, doi: http://dx.doi.org/10.1016/j.bmc.2015.12.010). The most potent compound HL16 in that series has the IC₅₀ of 0.69 μM. The other series is N,N-3-phenyl-3-benzylaminopropionanilide derivatives, which was investigated in current study. Further optimization of the structure–activity relationship (SAR) resulted in H16 (IC₅₀ 0.15 μM), which was discovered as a potent CETP inhibitor in vitro by BODIPY-CE fluorescence assay. In addition, the results of pharmacodynamics studies showed that H16 exhibited both favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. It also has an excellent stability in rat liver microsomal.     

The crystal and molecular structures of dichloro [N,O-(D,L)-diaminopropionic acid] platinum(II) and Dichloro [N,O-(L)-lysine] platinum(II) monohydrate

K₂PtCl₄ reacts with L-lysine and with D,L-diaminiopropionic acid (Dap) forming the neutral complexes [PtCl₂(N,O-Lys)]·H₂0 (1) and [PtCl₂(N,O-Dap)], (2) respectively.Compound 1 is monoclinic, space group P2₁ with a = 11.262(3), b = 11.041(2), c = 9.690(2) Å, β = 102.07(5)°, V = 1178(1) ų and Z = 4. Compound 2 is monoclinic, space group P2₁/n with a = 8.777(1), b = 10.615(2), c = 7.947(1) Å, β = 94.98(3)°, V = 738(1) ų and Z = 4. In both compounds, the zwitterionic ligands form an N,O-five membered chelate with the platinum atom. Structures 1 and 2 were refined to R values of 3.3% and 6.3% respectively.     

N4-Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine analogue, (S)-HPMP-5-azaC: Chemistry and antiviral activity

Even number fatty acid residues—docosanoyl (behenoyl) and stearoyl were selected for introduction to the N⁴-position of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMPC, cidofovir), and its 5-azacytosine counterpart, (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMP-5-azaC) with the aim to prepare a new type of lipophilic prodrugs. The study on the influence of these modifications to the stability and biological activity of both antivirals was performed. Different reactivity of both systems towards acylation reactions was also found: the 4-NH₂ group of cidofovir was more reactive compared to that of HPMP-5-azaC. In 5-azacytosine derivatives, we found mostly a destabilizing effect of the N⁴-acylation but this could be compensated by a positive influence of the esterification of the phosphonate group. Chemical stability of the 5-azacytosine moiety in the HPMP series is increasing in the following order: HPMP-5-azaC < cyclic HPMP-5-azaC < HPMP-5-azaC esters. From the view of prodrug development, the best chemical stability was observed in case of the double prodrug 7: the N⁴-behenoyl derivative of the hexadecyloxyethyl ester of cyclic HPMP-5-azaC. The free phosphonic acid (N⁴-behenoyl-HPMPC) appeared to be a more potent and selective inhibitor of herpesvirus replication than the parent HPMPC derivative.     

A kinetic analysis of peroxidase-catalyzed N,N-dimethylaniline dimerization

The horseradish peroxidase/hydrogen peroxide-supported oxidation of N,N-dimethylaniline is shown to proceed along three separate primary pathways; N-demethylation to N-metnylaniline, dimerization to N,N,N′,N′-tetramethyl-p,p′-benzidine and its oxidation products, and another dimerization to an unidentified water-soluble product, possibly an N-oxide. The first pathway predominates at low concentrations of peroxidase, while the dimerizations predominate at higher enzyme concentrations. These studies were made possible by the development of a very sensitive fluorimetric assay for N,N,N′,N′-tetramethyl-p,p′-benzidine. Kinetic studies of the dimerizations show them to be second-order with peroxidase concentration at low enzyme concentrations (less than 0.1 μg/3 ml), but near first order at higher enzyme concentrations. The dimerizations appear to involve interactions between pairs of enzyme-substrate or enzyme-intermediate complexes.     

N,N'-Dialkylaminoalkylcarbonyl (DAAC) prodrugs and aminoalkylcarbonyl (AAC) prodrugs of 4-hydroxyacetanilide and naltrexone with improved skin permeation properties

N,N′-Dialkylaminoalkylcarbonyl (DAAC) and aminoalkylcarbonyl (AAC) prodrugs of phenolic drugs acetaminophen (APAP) and naltrexone (NTX) are reported. The effects of incorporation of a basic amine group into the promoiety of an acyl prodrug of a phenolic drug on its skin permeation properties are also presented. DAAC-APAP prodrugs were synthesized via a three-step procedure starting with haloalkylcarbonyl esters which were reacted with five different amines: dimethylamine, diethylamine, dipropylamine, morpholine, and piperidine. The spacing between the amino group and the carbonyl group of the acyl group was 1-3 CH₂. After the hydrolysis of the ester, the carboxylic acid product was subsequently coupled with the parent drug via a dicyclohexyl carbodiimide (DCC) mediated coupling to yield the DAAC-APAP-HCl prodrugs in excellent yields. The AAC prodrugs were synthesized using commercially available Boc-protected amino acids using DCC or EDCI as coupling agents. The yields of the prodrugs synthesized using these two different methods have been compared. Half-lives (t_1/2) of a few members of the DAAC and AAC series were measured in buffer (pH 6.0, 20 mM). The members evaluated in hydrolysis experiments exhibit a t_1/2 range of 15-113 min. Among AAC-APAP prodrugs, the isopropyl group in valinate-APAP-HCl exerted a steric effect that increased the t_1/2 value for this prodrug compared to alaninate-APAP-HCl or prolinate-APAP-HCl. The 2-morpholinylacetate-APAP prodrug was able to achieve twice the flux of APAP in in vitro diffusion cell experiments through hairless mouse skin.     

Cytotoxic palladium complexes of bioreductive quinoxaline N1,N4-dioxide prodrugs

Four new palladium(II) complexes with the formula Pd(L)₂, where L are quinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and ¹H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd(L1)₂ and Pd(L2)₂, where L1 is 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N¹,N⁴-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd(L3)₂, where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N¹,N⁴-dioxide, resulted in vitro more potent cytotoxin in hypoxia (P = 5.0 μM) than the corresponding free ligand (P = 9.0 μM) and tirapazamine (P = 30.0 μM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd(L2)₂ introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd(L1)₂ and Pd(L3)₂ produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd(L3)₂ showed a more marked effect than Pd(L1)₂. Indeed, for the highest doses assayed, Pd(L3)₂ was even able to introduce positive supercoils on the plasmid DNA.     

N6-dipeptide derivatives of N12-ribosyl-indolo[2,3-a]carbazole

N6-derivatives of N12-indolo[2,3-a]pirrolo[3,4-c]carbazole-5,7-dione are synthesized as potential antitumor agents. The pyrrol N6 atom of these compounds is included into the dipeptide residue of the general formula >N6-(CH₂) _n -CO-Ala/βAla-OMe (n = 2 or 3). These compounds are synthesized by the reaction in DMF at 130°C between 13-methyl-12-(2,3,4-tri-O-acetyl-β-D-ribopyranosyl)indolo[2,3-a]furano[3,4-c]carbazole-5,7-dione and dipeptides containing the free N-terminal amino group. The nitrogen atom of the peptide amino group replaces O6 in the furan ring and is embedded as the N6 imide nitrogen atom of pyrrol. The ability of the compounds obtained to inhibit the growth of the SKOV3 human ovarian carcinoma cells was studied. The only derivative containing the >N6-(CH₂)₃-CO-L-Ala-OMe radical showed the cytotoxic activity with an inhibitory concentration of IC₅₀ = 8 μM.