Sergio Lociuro

Increased hydrophobic interactions of iclaprim with Staphylococcus aureus dihydrofolate reductase are responsible for the increase in affinity and antibacterial activity

OBJECTIVES Iclaprim is a novel 2,4-diaminopyrimidine that exhibits potent, rapid bactericidal activity against major Gram-positive pathogens, including methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus, and is currently in clinical development for the treatment of complicated skin and skin structure infections. An understanding of the known mechanism of resistance to trimethoprim led to the design of this new inhibitor, with improved affinity towards dihydrofolate reductase (DHFR) from S. aureus and clinically useful activity against S. aureus including isolates resistant to trimethoprim. The objective of this study was to characterize the mode of action of iclaprim and its inhibitory properties against DHFR. METHODS The mode of action of iclaprim was assessed by enzymatic analysis, direct binding studies, macromolecular synthesis profiles, synergy and antagonism studies to define its role as an inhibitor of DHFR. The binding properties of iclaprim to DHFR were compared with those of trimethoprim by X-ray crystallography. RESULTS The enzymatic properties, direct binding and X-ray crystallographic studies delineated the mode of interaction with DHFR and the reason for the increased affinity of iclaprim towards the enzyme. The effect of iclaprim on bacterial physiology suggests that iclaprim behaves as a classical antibacterial DHFR inhibitor, as previously documented for trimethoprim. CONCLUSIONS Iclaprim binds and inhibits bacterial DHFR in a similar manner to trimethoprim. However, the increased hydrophobic interactions between iclaprim and DHFR account for increased affinity and, unlike trimethoprim, enable iclaprim to inhibit even the resistant enzyme with nanomolar affinity, thus overcoming the mechanism of trimethoprim resistance. The increased antibacterial activity and lower propensity for resistance make iclaprim a clinically promising and useful inhibitor.

On cardioactive steroids: The synthesis of γ-isobufalin1

Abstract γ-Isobufalin 1 l was synthesized from testosterone. A novel use of α-furans as masking groups of a carboxylic acid is also reported.

In vitro bactericidal activity of iclaprim in human plasma.

ABSTRACT This study evaluated the effect of human plasma on the in vitro bactericidal activity of the novel diaminopyrimidine iclaprim against methicillin (meticillin)-susceptible and -resistant Staphylococcus aureus strains. MICs and minimal bactericidal concentrations (MBCs) of iclaprim, with ∼93% protein binding, were similar in the absence and in the presence of 50% human plasma; MICs and MBCs ranged from 0.06 to 0.125 μg/ml. Furthermore, the activity of iclaprim was not affected by plasma, with ≥99.9% reduction in CFU after 5.0 to 7.6 h.

CHEMICAL MODIFICATION OF THE GE 2270A MACROCYCLE (MDL 62,879)

Abstract Controlled acid hydrolysis of compound 2 resulted in the selective opening of the macrocycle while in basic condition a retro-aldol reaction occurred at the level of phenylserine thiazole. Compound 2 can be easily prepared from the natural antibiotic MDL 62,879. All the compounds prepared resulted less active than MDL 62,879.

Efficacy of Iclaprim against Wild-Type and Thymidine Kinase-Deficient Methicillin-Resistant Staphylococcus aureus Isolates in an In Vitro Fibrin Clot Model

ABSTRACT Iclaprim is a novel diaminopyrimidine antibiotic that is active against methicillin-resistant Staphylococcus aureus (MRSA). However, it is known that the activity of diaminopyrimidines against S. aureus is antagonized by thymidine through uptake and conversion to thymidylate by thymidine kinase. Unlike with humans, for whom thymidine levels are low, thymidine levels in rodents are high, thus precluding the accurate evaluation of iclaprim efficacy in animal models. We have studied the bactericidal activity of iclaprim against an isogenic pair of MRSA isolates, the wild-type parent AW6 and its thymidine kinase-deficient mutant AH1252, in an in vitro fibrin clot model. Clots, which were aimed at mimicking vegetation structure, were made from human or rat plasma containing either the parent AW6 or the mutant AH1252, and they were exposed to homologous serum supplemented with iclaprim (3.5 μg/ml), trimethoprim-sulfamethoxazole (TMP-SMX; 8/40 μg/ml), vancomycin (40 μg/ml), or saline, each of which was added one time for 48 h. In rat clots, iclaprim and TMP-SMX were bacteriostatic against the parent, AW6. In contrast, they were bactericidal (≥3 log10 CFU/clot killing of the original inoculum) against the mutant AH1252. Vancomycin was the most active drug against AW6 (P < 0.05), but it showed an activity similar those of iclaprim and TMP-SMX against AH1252. In human clots, iclaprim was bactericidal against both AW6 and AH1252 strains and was as effective as TMP-SMX and vancomycin (P > 0.05). Future studies of animals using simulated human kinetics of iclaprim and thymidine kinase-deficient MRSA, which eliminate the thymidine-induced confounding effect, are warranted to support the use of iclaprim in the treatment of severe MRSA infections in humans.

Synthesis of 2,5- and 2,4-Cyclohexadiene-1,3-dicarboxylates via Reductive Alkylation of Isophthalates

Abstract Substituted 2,5-(structure 1) and 2,4-(structure 2) cyclohexadiene-1,3-dicarboxylates were prepared via Birch reductive alkylation of substituted isophthalates with alkylating agents such as alkyl and allyl halides, allkyl dihalides, α-halocarboxylates and epoxides. A brief discussion about their reactivity and stability is presented.

Allylic Functionalization of 2,5- and 2,4-Cyclo-Hexadiene-1,3-Dicarboxylates

Abstract A series of 2,4- and 2,5-cyclohexadiene-1,3-dicarboxylates were functionalized at the allylic position via oxidation (SeO2, PDC/t-BuOOH) and halogenation (NBS). The regiochemical outcome for different substrates and reactions was studied and the importance of factors such as reaction mechanism, steric hindrance and reaction intermediates stability was discussed. §Present address: GlaxoWellcomc Medicines Research Centre, Via Fleming 4, Verona, Italy

Dihydrofolate reductase inhibitors as antibacterial agents

Present Address: Hoechst Marion Roussel, Paris, France