Toward New Therapeutics for Skin and Soft Tissue Infections: Propargyl-Linked Antifolates Are Potent Inhibitors of MRSA and Streptococcus pyogenes

Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 µg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin

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

Active antifolates are evaluated against clinical isolates of MRSA.

<p>Active antifolates are evaluated against clinical isolates of MRSA.</p

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

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