A novel stilbene-like compound that inhibits melanoma growth by regulating melanocyte differentiation and proliferation.

Melanoma is the most aggressive form of skin cancer. Current challenges to melanoma therapy include the adverse effects from immunobiologics, resistance to drugs targeting the MAPK pathway, intricate interaction of many signal pathways, and cancer heterogeneity. Thus combinational therapy with drugs targeting multiple signaling pathways becomes a new promising therapy. Here, we report a family of stilbene-like compounds called A11 that can inhibit melanoma growth in both melanoma-forming zebrafish embryos and mouse melanoma cells. The growth inhibition by A11 is a result of mitosis reduction but not apoptosis enhancement. Meanwhile, A11 activates both MAPK and Akt signaling pathways. Many A11-treated mouse melanoma cells exhibit morphological changes and resemble normal melanocytes. Furthermore, we found that A11 causes down-regulation of melanocyte differentiation genes, including Pax3 and MITF. Together, our results suggest that A11 could be a new melanoma therapeutic agent by inhibiting melanocyte differentiation and proliferation.

Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray.

The mechanism of action for a new lead stilbene compound coded SK-03-92 with bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) is unknown. To gain insight into the killing process, transcriptional profiling was performed on SK-03-92 treated vs. untreated S. aureus. Fourteen genes were upregulated and 38 genes downregulated by SK-03-92 treatment. Genes involved in sortase A production, protein metabolism, and transcriptional regulation were upregulated, whereas genes encoding transporters, purine synthesis proteins, and a putative two-component system (SACOL2360 (MW2284) and SACOL2361 (MW2285)) were downregulated by SK-03-92 treatment. Quantitative real-time polymerase chain reaction analyses validated upregulation of srtA and tdk as well as downregulation of the MW2284/MW2285 and purine biosynthesis genes in the drug-treated population. A quantitative real-time polymerase chain reaction analysis of MW2284 and MW2285 mutants compared to wild-type cells demonstrated that the srtA gene was upregulated by both putative two-component regulatory gene mutants compared to the wild-type strain. Using a transcription profiling technique, we have identified several cellular pathways regulated by SK-03-92 treatment, including a putative two-component system that may regulate srtA and other genes that could be tied to the SK-03-92 mechanism of action, biofilm formation, and drug persisters.

Pharmacokinetic/Toxicity Properties of the New Anti-Staphylococcal Lead Compound SK-03-92.

Because of the potential of a new anti-staphylococcal lead compound SK-03-92 as a topical antibiotic, a patch, or an orally active drug, we sought to determine its safety profile and oral bioavailability. SK-03-92 had a high IC50 (125 µg/ml) in vitro against several mammalian cell lines, and mice injected intraperiteonally at the highest dose did not exhibit gross toxicity (e.g. altered gait, ungroomed, significant weight loss). Single dose (100 µg/g) pharmacokinetic (PK) analysis with formulated SK-03-92 showed that peak plasma concentration (1.64 µg/ml) was achieved at 20-30 min. Oral relative bioavailability was 8%, and the drug half-life was 20-30 min, demonstrating that SK-03-92 is likely not a candidate for oral delivery. Five-day and two-week PK analyses demonstrated that SK-03-92 plasma levels were low. Multi-dose analysis showed no gross adverse effects to the mice and a SK-03-92 peak plasma concentration of 2.12 µg/ml with the presence of significant concentrations of breakdown products 15 min after dosing. SK-03-92 appeared to be very safe based on tissue culture and mouse gross toxicity determinations, but the peak plasma concentration suggests that a pro-drug of SK-03-92 or preparation of analogs of SK-03-92 with greater bioavailability and longer half-lives are warranted.

Zebrafish heart failure models for the evaluation of chemical probes and drugs.

Heart failure is a complex disease that involves genetic, environmental, and physiological factors. As a result, current medication and treatment for heart failure produces limited efficacy, and better medication is in demand. Although mammalian models exist, simple and low-cost models will be more beneficial for drug discovery and mechanistic studies of heart failure. We previously reported that aristolochic acid (AA) caused cardiac defects in zebrafish embryos that resemble heart failure. Here, we showed that cardiac troponin T and atrial natriuretic peptide were expressed at significantly higher levels in AA-treated embryos, presumably due to cardiac hypertrophy. In addition, several human heart failure drugs could moderately attenuate the AA-induced heart failure by 10%-40%, further verifying the model for drug discovery. We then developed a drug screening assay using the AA-treated zebrafish embryos and identified three compounds. Mitogen-activated protein kinase kinase inhibitor (MEK-I), an inhibitor for the MEK-1/2 known to be involved in cardiac hypertrophy and heart failure, showed nearly 60% heart failure attenuation. C25, a chalcone derivative, and A11, a phenolic compound, showed around 80% and 90% attenuation, respectively. Time course experiments revealed that, to obtain 50% efficacy, these compounds were required within different hours of AA treatment. Furthermore, quantitative polymerase chain reaction showed that C25, not MEK-I or A11, strongly suppressed inflammation. Finally, C25 and MEK-I, but not A11, could also rescue the doxorubicin-induced heart failure in zebrafish embryos. In summary, we have established two tractable heart failure models for drug discovery and three potential drugs have been identified that seem to attenuate heart failure by different mechanisms.

Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species, including M. tuberculosis.

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 µg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.

Antibacterial spirobisnaphthalenes from the North American cup fungus Urnula craterium.

Urnucratins A-C (1-3), which possess an unusual bisnaphthospiroether skeleton with one oxygen bridge and one C-C bridge and represent a new subclass of bisnaphthalenes, were isolated from the North American cup fungus Urnula craterium. Their structures, including absolute configurations, were determined by means of HRMS, NMR, and quantum chemical CD calculations. Urnucratin A (1) was found to be active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and Streptococcus pyogenes with MIC values of 2, 1, and 0.5 µg/mL, respectively.

Synthesis and minimum inhibitory concentrations of SK-03-92 against Staphylococcus aureus and other gram-positive bacteria.

Staphylococcus aureus causes hundreds of thousands of infections and thousands of deaths per year in the United States. The emergence of methicillin-resistant S. aureus (MRSA), including community-associated methicillin-resistant S. aureus (CA-MRSA), has added to the problem. As MRSA continue to evolve, they are becoming resistant to more classes of antibiotics. In the past 20 years, only three new antibiotics have been approved for human use (linezolid, daptomycin, and tigecycline), and resistance to these three drugs has already emerged. New antibiotics are needed, and we have developed a promising drug candidate that may be applicable to treating MRSA, among other gram-positive bacterial infections. We have identified a novel synthetic drug, coded SK-03-92, that shows broad in vitro efficacy against a variety of gram-positive bacterial strains that include a number of strains of S. aureus. Besides the activity against gram-positive bacteria, this new drug also exhibits activity against Mycobacterium strains.

Base-mediated stereospecific synthesis of aryloxy and amino substituted ethyl acrylates.

The stereospecific synthesis of aryloxy and amino substituted E- and Z-ethyl-3-acrylates is of interest because of their potential in the polymer industry and in medicinal chemistry. During work on a copper-catalyzed cross-coupling reaction of ethyl (E)- and (Z)-3-iodoacrylates with phenols and N-heterocycles, we discovered a very simple (nonmetallic) method for the stereospecific synthesis of aryloxy and amino substituted acrylates. To study this long-standing problem on the stereoselectivity of aryloxy and amino substituted acrylates, a series of O- and N-substituted nucleophiles was allowed to react with ethyl (E)- and (Z)-3-iodoacrylates. Screening of different bases indicated that DABCO (1,4-diazabicyclo[2.2.2]octane) afforded successful conversion of ethyl (E)- and (Z)-3-iodoacrylates into aryloxy and amino substituted ethyl acrylates in a stereospecific manner. Herein are the details of this DABCO-mediated stereospecific synthesis of aryloxy and amino substituted E- or Z-acrylates.

A new class of potential anti-tuberculosis agents: Synthesis and preliminary evaluation of novel acrylic acid ethyl ester derivatives.

Novel acrylic acid ethyl ester derivatives were synthesized and evaluated as potential agents against Mycobacterium species. A versatile and efficient copper-catalyzed coupling process was developed and used to prepare a library of substituted acrylic acid ethyl ester analogs. Minimum inhibitory concentration assays indicated that two of these compounds 3 and 4 have greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Moreover, members of this new class of compounds appear to exhibit a specific anti-mycobacterial effect and do not inhibit the growth of the other Gram-positive or Gram-negative species tested.

Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity.

BACKGROUND: Acinetobacter baumannii and species within the Burkholderia cepacia complex (BCC) are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat A. baumannii and BCC infections. METHODS: The aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against A. baumannii and Burkholderia cepacia in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the in vitro activity of the purified compounds. RESULTS: Only three crude extracts (0.9%) had activity against A. baumannii and B. cepacia. Compounds from two of these extracts had MICs greater than 128 microg/ml, and further analyses were not performed. From the third extract, prepared from Leucopaxillus albissimus, 2-aminoquinoline (2-AQ) was isolated. This compound exhibited a modest MIC in vitro against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 microg/ml), and a weak MIC (128 microg/ml) against A baumannii. The IC50 against a murine monocyte line was 1.5 mg/ml. CONCLUSION: The small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 microg/ml) for both A. baumannii and BCC strains suggests that 2-AQ is not suitable for further drug development in its current form.

Antibacterial compounds from mushrooms II: lanostane triterpenoids and an ergostane steroid with activity against Bacillus cereus isolated from Fomitopsis pinicola.

Anti- Bacillus cereus bioassay-guided fractionation of a crude extract of the American mushroom, Fomitopsis pinicola, was performed using thin-layer chromatography, Sephadex LH-20 column chromatography, and preparative-scale HPLC. Five lanostane triterpenoids (1-5) and one ergostane steroid (6) were isolated and identified. Compound 1 is a new lanostane triterpenoid, and its structure was determined using 1D and 2D NMR experiments, HR-MS, and physical data. Each of the purified compounds (1-6) was tested for antibacterial activity against B. cereus using standard MIC assays. Compounds 1-6 had MIC values of 32, 16, 32, 32, 128, and 64 microg/mL, respectively.

Antibacterial compounds from mushrooms I: a lanostane-type triterpene and prenylphenol derivatives from Jahnoporus hirtus and Albatrellus flettii and their activities against Bacillus cereus and Enterococcus faecalis.

Antibacterial bioassay-guided fractionation of two American mushroom species, Jahnoporus hirtus and Albatrellus flettii, led to the isolation and identification of their major antibacterial constituents: 3,11-dioxolanosta-8,24( Z)-diene-26-oic acid (1) from J. hirtus and confluentin (2), grifolin (3), and neogrifolin (4) from A. flettii. Compound 1 is a new lanostane-type triterpene. All purified compounds were evaluated for their ability to inhibit the growth of Bacillus cereus and Enterococcus faecalis using standard MIC assays. Compounds 1- 4 demonstrated MIC values of 40, 20, 10, and 20 microg/mL, respectively, against B. cereus and MIC values of 32, 1.0, 0.5, and 0.5 microg/mL, respectively, against E. faecalis. Thus, one novel compound and three others were shown to possess antimicrobial activities against these gram-positive bacteria employed as surrogates for more virulent and dangerous pathogens.

New classes of Gram-positive selective antibacterials: inhibitors of MRSA and surrogates of the causative agents of anthrax and tuberculosis.

An antimicrobial phenolic stilbene, (E)-3-hydroxy-5-methoxystilbene, 1 was recently isolated from the leaves of Comptonia peregrina (L.) Coulter and shown to possess inhibitory activity against several Gram-positive bacteria, including isolates of methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium bovis BCG, and avirulent Bacillusanthracis (Sterne strain), among others. These results prompted the design and synthesis of two new classes of compounds, phenoxystyrenes and phenothiostyrenes, as analogs of the natural antimicrobial stilbene. These and additional stilbenoid analogs were synthesized using new, efficient, copper-mediated coupling strategies. Minimum inhibitory concentration (MIC) antimicrobial assays were performed on all compounds prepared. These preliminary structure-activity relationship studies indicated that both new classes of synthetic analogs, as well as the stilbenes, show promising activity against Gram-positive bacteria when at least one phenolic moiety is present, but not when absent. The potencies of the phenolic phenoxystyrenes and phenothiostyrenes were found to be comparable to those of the phenolic stilbenes tested.

Stereo- and regiospecific cu-catalyzed cross-coupling reaction of vinyl iodides and thiols: a very mild and general route for the synthesis of vinyl sulfides.

A mild and efficient method for the copper-catalyzed formation of vinylic carbon-sulfur bonds has been developed. The desired vinyl sulfides are obtained in good to excellent yields, with full retention of stereochemistry. This method is particularly noteworthy given its mild reaction conditions, simplicity, and generality, as well as low cost of the catalyst system.

'Hybrid' benzofuran-benzopyran congeners as rigid analogs of hallucinogenic phenethylamines.

Phenylalkylamines that possess conformationally rigidified furanyl moieties in place of alkoxy arene ring substituents have been shown previously to possess the highest affinities and agonist functional potencies at the serotonin 5-HT(2A) receptor among this chemical class. Further, affinity declines when both furanyl rings are expanded to the larger dipyranyl ring system. The present paper reports the synthesis and pharmacological evaluation of a series of 'hybrid' benzofuranyl-benzopyranyl phenylalkylamines to probe further the sizes of the binding pockets within the serotonin 5-HT(2A) agonist binding site. Thus, 4(a-b), 5(a-b), and 6 were prepared as homologs of the parent compound, 8-bromo-1-(2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-aminopropane 2, and their affinity, functional potency, and intrinsic activity were assessed using cells stably expressing the rat 5-HT(2A) receptor. The behavioral pharmacology of these new analogs was also evaluated in the two-lever drug discrimination paradigm. Although all of the hybrid isomers had similar, nanomolar range receptor affinities, those with the smaller furanyl ring at the arene 2-position (4a-b) displayed a 4- to 15-fold greater functional potency than those with the larger pyranyl ring at that position (5a-b). When the furan ring of the more potent agonist 4b was aromatized to give 6, a receptor affinity similar to the parent difuranyl compound 2 was attained, along with a functional potency equivalent to 2, 4a, and 4b. In drug discrimination experiments using rats trained to discriminate LSD from saline, 4b was more than two times more potent than 5b, with the latter having a potency similar to the classic hallucinogenic amphetamine 1 (DOB).

Substituted hexahydrobenzodipyrans as 5-HT2A/2C receptor probes.

A pair of substituted hexahydrobenzodipyrans was designed as molecular probes for determining the steric restrictions of the agonist binding site of serotonin 5-HT2A and 5-HT2C receptors. The rationale for the design of these receptor ligands, their chemical synthesis, rat behavioral pharmacology in the two-lever drug discrimination assay using LSD-trained rats, affinity for cloned rat 5-HT2A and 5-HT2C receptors and agonist functional activities are reported.