The Berkeleylactones, Antibiotic Macrolides from Fungal Coculture

A carefully timed coculture fermentation of <i>Penicillium fuscum</i> and <i>P. camembertii/clavigerum</i> yielded eight new 16-membered-ring macrolides, berkeleylactones A–H (<b>1</b>, <b>4</b>, <b>6</b>–<b>9</b>, <b>12</b>, <b>13</b>), as well as the known antibiotic macrolide A26771B (<b>5</b>), patulin, and citrinin. There was no evidence of the production of the berkeleylactones or A26771B (<b>5</b>) by either fungus when grown as axenic cultures. The structures were deduced from analyses of spectral data, and the absolute configurations of compounds <b>1</b> and <b>9</b> were determined by single-crystal X-ray crystallography. Berkeleylactone A (<b>1</b>) exhibited the most potent antimicrobial activity of the macrolide series, with low micromolar activity (MIC = 1–2 μg/mL) against four MRSA strains, as well as <i>Bacillus anthracis</i>, <i>Streptococcus pyogenes</i>, <i>Candida albicans</i>, and <i>Candida glabrata</i>. Mode of action studies have shown that, unlike other macrolide antibiotics, berkeleylactone A (<b>1</b>) does not inhibit protein synthesis nor target the ribosome, which suggests a novel mode of action for its antibiotic activity

Propargyl-linked DHFR inhibitors^a inhibit the <i>S. aureus</i> and <i>S. pyogenes</i> DHFR enzymes.

a<p>The generalized scaffold for the propargyl-linked inhibitors is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029434#pone-0029434-g001" target="_blank">Figure 1a</a>.</p>b<p>IC₅₀ values against the DHFR enzymes are reported in nM and represent the average of at least three measurements.</p>c<p>Selectivity is calculated as IC₅₀ (human)/IC₅₀ (pathogen).</p

Propargyl-linked antifolates inhibit the growth of a panel of MRSA strains (MIC values in µg/mL).

a<p>hVISA: heteroresistant vancomycin intermediate <i>S. aureus</i>.</p>b<p>1∶20 reflects the molar ratio of the two components.</p>c<p>The two numbers reflect theindividual MIC values for each component of the mixture.</p

The synthesis of biphenyl analogs delivers compounds 7, 10 and 11.

<p>(a) PhB(OH)₂, Pd(PPh₃)₂Cl₂, Cs₂CO₃, dioxane, 80°C, 91%; (b) Ph₃P = CHOMe, THF; (c) Hg(OAc)₂, KI, THF/H₂O; (d) dimethyl(1-diazo-2 oxopropyl)phosphonate, K₂CO₃, MeOH; 72% over 3 steps (e) 6-ethyl, 5-iodo-2,4-diaminopyrimidine, Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 81%; (f) Br₂, CCl₄, 65%; (g) KOH, MeI, DMSO, 90%; (h) n-butyllithium, acetaldehyde, 53%; (i) MnO₂, 94%; (j) Ph₃P = CHOMe, THF; (k) Hg(OAc)₂, KI, THF/H2O; (l) dimethyl(1-diazo-2 oxopropyl)phosphonate, K₂CO₃, MeOH; 45% over 3 steps; (m) 6-alkyl, 5-iodo-2,4-diaminopyrimidine, Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 72%.</p

The synthesis of derivatives with alternative heterocycles delivers compounds 31, 36 and 37.

<p>(a) Pd(OAc)₂, morpholine, Cs₂CO₃, (2-Biphenyl)di-tert-butyl phosphine, benzene, 80°C,92%; (b)Ph₃P = CHOMe, THF; (c) Hg(OAc)₂, KI, THF/H2O; (d) dimethyl(1-diazo-2-oxopropyl)phosphonate, K₂CO₃, MeOH, 57–69% over 3 steps; (e) 6-alkyl, 5-iodo-2,4-diaminopyrimidine, Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 68–82%, (f) 3-pyridylboronic acid, Pd(PPh₃)₂Cl₂, Cs₂CO₃, dioxane, 80°C, 86%; (g) 4-pyrimidinylboronic acid, Pd(PPh₃)₂Cl₂, Cs₂CO₃, dioxane, 80°C, 76%.</p

Propargyl-linked antifolates potently bind <i>S. aureus</i> DHFR.

<p>a) Depiction of a general scaffold for the propargyl-linked antifolates with the pyrimidine ring (A), phenyl ring (B) and aryl ring (Ar) shown along with possible positions for substitutions (R₆, R_P, R_2′ and R_3′) b) Illustration of compound <b>1</b>, a biphenyl propargyl-linked antifolate, with labeled atom positions b) Active site depiction from the structure of the SaDHFR:NADPH:<b>1</b> ternary complex showing active site residues (orange), NADPH (magenta) and compound <b>1</b> (blue).</p

The synthesis of compounds with a pyridyl ring delivers compounds 24–28.

<p>(a) 4-pyridylboronic acid, Pd(PPh₃)₂Cl₂, Cs₂CO₃, dioxane, 80°C, 82–89%; (b) Ph₃P = CHOMe, THF; (c) Hg(OAc)₂, KI, THF/H2O; (d) dimethyl(1-diazo-2 oxopropyl)phosphonate, K₂CO₃, MeOH; 61–70% over 3 steps (e) 6-alkyl, 5-iodo-2,4-diaminopyrimidine, Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 70–85%.</p

Depictions of <i>S. aureus</i> DHFR bound to ligands reveal interactions with active site residues.

<p><i>S. aureus</i> DHFR is shown bound to NADPH (magenta) and a) compound <b>7</b> (purple), b) compound <b>25</b> (green) and c) a superposition of compounds <b>1</b> (blue) and <b>25</b> (green), as determined from crystal structures.</p

Evaluation of Minimum Bactericidal Concentrations.

<p>Evaluation of Minimum Bactericidal Concentrations.</p

Evaluation of antibacterial and cytotoxicity activity of propargyl-linked antifolates.

a<p>MIC values for MRSA and <i>S. pyogenes</i> are reported in µg/mL (µM).</p>b<p>MIC values for MRSA in the presence of 10% fetal calf serum (FCS) in µg/mL (µM).</p>c<p>MIC values for <i>S. pyogenes</i> in the presence of 10% FCS in µg/mL (µM).</p>d<p>Selectivity values are calculated as IC₅₀ (MCF10)/MIC (pathogen), both values in µM. ND: not determined.</p