Synthesis and antifungal activities of novel 1,3-beta-D-glucan synthase inhibitors. Part 1

Highly potent 1,3-beta-D-glucan synthase inhibitors 10, 11 and 13 have been identified by the chemical modification of the fungicidal macrocyclic lipopeptidolactone, RO-09-3655 (1), isolated from the cultured broth of Deuteromycotinia spp. D-Ornithine derivative (10) showed improved antifungal activity in the systemic candidiasis model in mice and reduced hepatotoxicity in vitro, as compared with 1.     

A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase.

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex.     

Synthesis,spectral characterization,and in vitro antibacterial and antifungal activities of novel 1,3-thiazine-2-amines comprising morpholine nucleus

A collection of 4-(4-morpholinophenyl)-6-aryl-6H-1,3-thiazin-2-amines (20–28) were synthesized and their in vitro antimicrobial activity was investigated. Compound 21 against P. aeruginosa, 23 against B. subtilis, 24 against V. cholerae and P. aeruginosa, 26 against S. aureus and B. subtilis, 27 against B. subtilis and E. coli, and 28 against all tested bacterial strains exerted excellent antibacterial activity. Compound 20 against A. flavus and Rhizopus, 21, 26 against Rhizopus, 22, 27 against Mucor, 23 against A. flavus, 24 against both A. flavus and Mucor, 25 against all tested strains, and 28 against Rhizopus and M. gypseum exerted excellent antifungal activity.     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents

A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC₅₀ of 0.10?mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC₅₀ of 0.18?mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4?μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.     

Synthesis and structure-activity relationships of novel fungal chitin synthase inhibitors

A novel Candida albicans chitin synthase 1 (CaChs1) inhibitor, RO-41-0986 (1) was discovered by random screening. Systematic modification led to the identification of a highly potent CaChs1 inhibitor, RO-09-3024 (2), having strong antifungal activity against Candida spp. in vitro.     

Synthesis and antifungal activities of phenylenedithioureas

A total of 20 new phenylenedithiourea derivatives was synthesized by reaction of phenylenediisothiocyanates with aromatic amines as aminobenzoic, aminosalicylic acid and their derivatives. Their chemical structures were confirmed by elemental analysis, IR spectrometry and 1H NMR. The compounds were screened for in vitro antifungal, antibacterial activities and some of them have strong antifungal activities comparable to the activity observed for ketoconazole.     

Discovery of novel sphingosine kinase-1 inhibitors. Part 2

Building on our initial work, we have identified additional novel inhibitors of sphingosine kinase-1 (SK1). These new analogs address the shortcomings found in our previously reported compounds. Inhibitors 51 and 54 demonstrated oral bioavailability in a rat PK study.     

Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 2: Improvement of in vitro activity

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as IkappaB kinase beta (IKK-beta) inhibitors. Substitution of an aminoalkyl group for the aromatic group at the 4-position on the core pyridine ring resulted in a marked increase in both kinase enzyme and cellular potencies, and provided potent IKK-beta inhibitors with IC(50) values of below 100 nM.     

Synthesis and antiviral activities of novel N-alkoxy-arylsulfonamide-based HIV protease inhibitors

A series of novel N-alkoxy-arylsulfonamide HIV protease inhibitors with low picomolar enzyme activity and single digit nanomolar antiviral activity is disclosed.     

Phosphatases and phosphodiesterases interfere with 1,3-beta-D-glucan synthase activity in pea epicotyl membrane preparations

Soluble and membrane-bound phosphatase and phosphodiesterase activities are present in preparations of 1,3-beta-D-glucan synthase from pea epicotyls. UDP-glucose phosphodiesterase and non-specific alkaline phosphatase could be partially inhibited by N-ethylmaleimide or iodoacetamide and partially removed from membranes by washing. Such treatments helped to prolong 1,3-beta-glucan synthase activity. Nevertheless, the 1,3-beta-D-glucan synthase activity in washed membranes still gradually decreased during incubation in buffer at 30 degrees C. The rate of decay was reduced by adding more specific phosphatase inhibitors, e.g. molybdate, vanadate or fluoride, or by addition of nucleotides, and much of the loss of 1,3-beta-D-glucan synthase activity during preincubation could be restored by addition of phosphatidylethanolamine to the assay mixtures. It is concluded that membrane phospholipid is an essential part of the environment of 1,3-beta-glucan synthase and must be maintained intact in order for the enzyme to remain fully active.     

Novel galbonolide derivatives as IPC synthase inhibitors: design, synthesis and in vitro antifungal activities

A series of novel galbonolide derivatives having a modified methyl enol ether moiety were prepared in total synthetic procedures and evaluated for their in vitro antifungal activities. The antifungal activity was labile to modification of the enol ether functionality and almost all of the modified compounds lacked the activity except for the analogue with an introduction of a methylthio group at the C-6 position, which retained a modest antifungal potency against Cryptococcus neoformans.     

Synthesis and biological evaluation of 3-ethylidene-1,3-dihydro-indol-2-ones as novel checkpoint 1 inhibitors

Chk1 inhibitors have emerged as a novel class of neoplastic agents for abrogating the G2 DNA damage checkpoint arrest. Analogs of the Chk1 inhibitor, 3-ethylidene-1,3-dihydro-indol-2-one, were synthesized and tested in vitro for their inhibitory activities. The most promising compound identified from this series is analog 28, which possesses potent enzymatic and cellular activities.     

Synthesis and antifungal activities of miltefosine analogs

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a?3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5–5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5–3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.     

Synthesis and biological activities of novel beta-carbolines as PDE5 inhibitors

A series of N(2)-furoyl and N(2)pyrimidinyl beta-carbolines was discovered to possess potent inhibitory activity against PDE5. During the synthesis we developed a tandem resin quenching protocol, which allowed us to synthesize large number of target compounds in a rapid fashion. Representative compounds exhibit superior selectivity to sildenafil versus other isozymes of PDEs, and demonstrated in vivo efficacy in increasing introcavernosal pressure in dogs.     

Heteroaryl-O-glucosides as novel sodium glucose co-transporter 2 inhibitors. Part 1

A series of benzo-fused heteroaryl-O-glucosides was synthesized and evaluated in SGLT1 and 2 cell-based functional assays. Indole-O-glucoside 10a and benzimidazole-O-glucoside 18 exhibited potent in vitro SGLT2 inhibitory activity.     

Synthesis and cytotoxic activities of novel copper and silver complexes of 1,3-diaryltriazene-substituted sulfonamides

In this study, a series of 10 novel copper (II) and silver complexes of 1,3-diaryltriazene-substituted sulfonamides was synthesised. All the synthesised ligands and their metal complexes were assessed for in vitro cytotoxicity against human colorectal adenocarcinoma (DLD-1), cervix carcinoma (HeLa), breast adenocarcinoma (MDA-MB-231), colon adenocarcinoma (HT-29), endometrial adenocarcinoma (ECC-1), prostate cancer (DU-145 and PC-3), normal embryonic kidney (HEK-293), normal prostate epithelium (PNT-1A), and normal retinal pigment epithelium (ARPE-19) cells. Most of the metal complexes from the series showed to be more active against all cancerous cells than the uncomplexed 1,3-diaryltriazene-substituted sulfonamides, and lower cytotoxic effects observed on normal cells. Most of the Cu (II) and Ag (I) metal complexes from the presented series showed high cytotoxic activity against HeLa cells with IC₅₀ values ranging from 2.08 to >300?µM. Specifically, compound L₃-Ag showed one of the highest cytotoxicity against all cancer cell lines with IC₅₀ values between 3.30 to 16.18?µM among other tested compounds.     

Synthesis and antibacterial activities of chiral 1,3-oxazinan-2-one derivatives

We report here the design, synthesis, and antibacterial activities of novel classes of compounds containing chiral 1,3-oxazinan-2-ones and oxazolidinones as the basic core structures. These compounds are tertiary amines containing the core structures and two aryl substituents. Several of these molecules exhibit potent antibacterial activities against the tested Gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis. These compounds represent new structure scaffolds and can be further optimized to give new antibacterial agents with structures significantly different from those of existing classes of antibiotics.