Evaluation of New Dihydrophthalazine-Appended 2,4-Diaminopyrimidines against Bacillus anthracis: Improved Syntheses Using a New Pincer Complex.

The synthesis and evaluation of ten new dihydrophthalazine-appended 2,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl)-piperidine]palladium(II), allows the final Heck coupling to be performed at 90 °C using triethylamine as the base. These milder conditions have been used to achieve improved yields for new and previously reported substrates with functional groups that degrade or react at the normal 140 °C reaction temperature.

Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis.

The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the central aromatic ring and (2) modified at the central ring ether groups. Structures 4a-b incorporating an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH2CH3 vs CH3) on the central ring oxygen atoms (R(2) and R(3)) had a minimal impact on the inhibition. Comparison of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives (19 b and 20a-b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition (MICs 0.

Identification of novel potential antibiotics against Staphylococcus using structure-based drug screening targeting dihydrofolate reductase.

The emergence of multidrug-resistant Staphylococcus aureus (S. aureus) makes the treatment of infectious diseases in hospitals more difficult and increases the mortality of the patients. In this study, we attempted to identify novel potent antibiotic candidate compounds against S.

Synthesis and biological evaluation of 2,4-diaminopyrimidine-based antifolate drugs against Bacillus anthracis.

Due to the innate ability of bacteria to develop resistance to available antibiotics, there is a critical need to develop new agents to treat more resilient strains. As a continuation of our research in this area, we have synthesized a series of racemic 2,4-diaminopyrimidine-based drug candidates, and evaluated them against Bacillus anthracis. The structures are comprised of a 2,4-diaminopyrimidine ring, a 3,4-dimethoxybenzyl ring, and an N-acryloyl-substituted 1,2-dihydrophthalazine ring.

The structure and competitive substrate inhibition of dihydrofolate reductase from Enterococcus faecalis reveal restrictions to cofactor docking.

We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim.

Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase.

Background: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme.

Inhibition of bacterial dihydrofolate reductase by 6-alkyl-2,4-diaminopyrimidines.

(±)-6-Alkyl-2,4-diaminopyrimidine-based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies revealed attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125-8 μg mL(-1) range for both organisms.

Synthesis and biological activity of substituted 2,4-diaminopyrimidines that inhibit Bacillus anthracis.

A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against Bacillus anthracis using previously reported (±)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory concentration (MIC) value of 1-3 μg/mL, as the standard. In the current work, the corresponding isobutenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest MICs with values of 0.5 μg/mL and 0.

High-throughput screening of a diversity collection using biodefense category A and B priority pathogens.

One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute-based high-throughput screening (HTS) 96-well-based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 µg/mL.

Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets.

Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major setback. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA).

Crystal structure of Bacillus anthracis dihydrofolate reductase with the dihydrophthalazine-based trimethoprim derivative RAB1 provides a structural explanation of potency and selectivity.

Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target.

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase.

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides.

Establishment of a method for evaluating intracellular antibiotic efficacy in Brucella abortus-infected Mono Mac 6 monocytes.

Background: Brucellae produce chronic and often lifelong infections in natural hosts. The persistent nature of these infections is predominantly due to the capacity of these bacteria to maintain intracellular residence in host macrophages. Successful antimicrobial therapy requires eradication of brucellae from this intracellular niche.

In vitro efficacy of new antifolates against trimethoprim-resistant Bacillus anthracis.

Bacillus anthracis is innately resistant to trimethoprim (TMP), a synthetic antifolate that selectively inhibits several bacterial dihydrofolate reductases (DHFRs) but not human DHFR. Previously, we were able to confirm that TMP resistance in B. anthracis (MIC > 2,048 microg/ml) is due to the lack of selectivity of TMP for the B.

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase.

Dihydrofolate reductase (EC 1.5.1.

Genetic basis for sulfonamide resistance in Bacillus anthracis.

Natural resistance of field strains of Bacillus anthracis to drugs from the sulfonamide class of antimicrobials that act by inhibiting dihydropteroate synthase (DHPS) has been reported. Though the structure of B. anthracis DHPS has been determined, its connection to the apparent intrinsic sulfonamide resistance of the bacterium has not been established.

Efficacy of rifabutin-loaded microspheres for treatment of Mycobacterium avium-infected macrophages and mice.

Two poly(DL-lactide-co-glycolide) microsphere formulations (A, 10% wt/wt, and B, 23% wt/wt, 1-10 microns) were evaluated for intracellular delivery of rifabutin using the J774 murine and Mono Mac 6 (MM6) human monocytic cell lines. Within 7 days, formulation A released 100% in both cell lines and B released 53 and 67% in the J774 and MM6, respectively. Intracellular release of rifabutin with both formulations caused significant reduction of intracellularly replicating Mycobacterium avium (MAC).

Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim.

Bacillus anthracis is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for B. anthracis Sterne is >2,048 but < or =4,096 microg/ml.

Microsphere technology for chemotherapy of mycobacterial infections.

The purpose of this review article is to examine the various studies that have evaluated microspheres for delivery of antimycobacterial drugs. Some of the studies strictly involve the development and evaluation of microspheres for use in antimycobacterial drug delivery, whereas others actually use drug-loaded microspheres to treat mycobacterial infections in cell lines and small animals. Although there is a potential to use microspheres to treat a variety of mycobacterial infections, it appears that most of the studies so far have focused on the etiological agent of tuberculosis, Mycobacterium tuberculosis.

Sustained release characteristics of rifampin-loaded microsphere formulations in nonhuman primates.

Controlled release rifampin-loaded microspheres were evaluated for the first time in nonhuman primates. Animals received either 2.0 g of a large formulation (10-150 microm, 23 wt% rifampin) injected subcutaneously at Day 0 (118-139 mg rifampin/kg), 4.

Metabolism of 2-methyladenosine in Mycobacterium tuberculosis.

2-Methyladenosine (methyl-Ado) has selective activity against Mycobacterium tuberculosis (M. tuberculosis). In an effort to better understand its mechanism of action, we have characterized its metabolism in M.