From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Desulfinative Alkylation of Heteroarenes via an Electrostatic Electron Donor-Acceptor Complex.

Functionalized pyridine and quinoline rings are important components of numerous bioactive molecules and natural products; however, diversification of these rings often requires de novo heterocycle ring synthesis or demanding reaction conditions. We report a method for desulfinative alkylation of pyridine and quinoline N-methoxide salts that operates under both photocatalytic and electrostatic electron donor-acceptor-mediated pathways. Unlike most EDA-mediated processes, this reaction operates in the absence of light and with the desulfination of the donor compound.

Synthesis of Quinoline Silyloxymethylsulfones as Intermediates to Sulfonyl Derivatives.

Heterocyclic sulfones, sulfonamides, and sulfonyl fluorides constitute an important structural motif in medicinal chemistry. Methods to make six-membered heteroaromatic sulfonyl compounds, however, remain challenging, and most efforts rely on commercial sulfonyl chlorides. We report herein the reaction of sodium tert-butyldimethyl silyloxymethylsulfinate with quinoline N-oxides to selectively furnish C2-substituted sulfones. The silyloxymethylsulfinate can be deprotected to then form sulfonyl fluorides, sulfonamides, and sulfones. This transformation is scalable and has broad applicability to a wide array of quinoline and isoquinoline functionality.

Theoretical and Mechanistic Validation of Global Kinetic Parameters of the Inactivation of GABA Aminotransferase by OV329 and CPP-115.

((S)-3-Amino-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (OV329) is a recently discovered inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), which has 10 times better inactivation efficiency than its predecessor, CPP-115, despite the only structural difference being an endocyclic double bond in OV329. Both compounds are mechanism-based enzyme inactivators (MBEIs), which inactivate GABA-AT by a similar mechanism. Here, a combination of a variety of computational chemistry tools and experimental methods, including quantum mechanical (QM) calculations, molecular dynamic simulations, progress curve analysis, and deuterium kinetic isotope effect (KIE) experiments, are utilized to comprehensively study the mechanism of inactivation of GABA-AT by CPP-115 and OV329 and account for their experimentally obtained global kinetic parameters kinact and KI. Our first key finding is that the rate-limiting step of the inactivation mechanism is the deprotonation step, and according to QM calculations and the KIE experiments, kinact accurately represents the enhancement of the rate-limiting step for the given mechanism. Second, the present study shows that the widely used simple QM models do not accurately represent the geometric criteria that are present in the enzyme for the deprotonation step. In contrast, QM cluster models successfully represent both the ground state destabilization and the transition state stabilization, as revealed by natural bond orbital analysis. Furthermore, the globally derived KI values for both of the inactivators represent the inhibitor constants for the initial binding complexes (Kd) and indicate the inactivator competition with the substrate according to progress curve analysis and the observed binding isotope effect. The configurational entropy loss accounts for the difference in KI values between the inactivators. The approach we describe in this work can be employed to determine the validity of globally derived parameters in the process of MBEI optimization for given inactivation mechanisms.

Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid.

The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1-carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1'-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.

Synthesis of ( S)-3-Amino-4-(difluoromethylenyl)-cyclopent-1-ene-1-carboxylic Acid (OV329), a Potent Inactivator of γ-Aminobutyric Acid Aminotransferase.

( S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (OV329, 1) is being developed for the treatment of epilepsy and addiction. The previous 14-step synthesis of OV329 was low yielding, involved an unselective α-elimination to form the cyclopentene, required the use of tert-butyllithium, and produced toxic selenium byproducts in the penultimate step. A new synthesis, which avoids the aforementioned issues, was carried out on large scale, reducing the step count from 14 to 9 steps and increasing the overall yield from 3.7% to 8.1%.

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. Inhibition of GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, has been established as a possible strategy for the treatment of substance abuse. The raised GABA levels that occur as a consequence of this inhibition have been found to antagonize the rapid release of dopamine in the ventral striatum (nucleus accumbens) that follows an acute challenge by an addictive substance. In addition, increased GABA levels are also known to elicit an anticonvulsant effect in patients with epilepsy. We previously designed the mechanism-based inactivator (1S,3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (2), now called CPP-115, that is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved drug that is an inactivator of GABA-AT. CPP-115 was found to have high therapeutic potential for the treatment of cocaine addiction and for a variety of epilepsies, has successfully completed a Phase I safety clinical trial, and was found to be effective in the treatment of infantile spasms (West syndrome). Herein we report the design, using molecular dynamics simulations, synthesis, and biological evaluation of a new mechanism-based inactivator, (S)-3-amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (5), which was found to be almost 10 times more efficient as an inactivator of GABA-AT than CPP-115. We also present the unexpected crystal structure of 5 bound to GABA-AT, as well as computational analyses used to assist the structure elucidation process. Furthermore, 5 was found to have favorable pharmacokinetic properties and low off-target activities. In vivo studies in freely moving rats showed that 5 was dramatically superior to CPP-115 in suppressing the release of dopamine in the corpus striatum, which occurs subsequent to either an acute cocaine or nicotine challenge. Compound 5 also attenuated increased metabolic demands (neuronal glucose metabolism) in the hippocampus, a brain region that encodes spatial information concerning the environment in which an animal receives a reinforcing or aversive drug. This multidisciplinary computational design to preclinical efficacy approach should be applicable to the design and improvement of mechanism-based inhibitors of other enzymes whose crystal structures and inactivation mechanisms are known.

Regiodivergent addition of phenols to allylic oxides.

The regiodivergent addition of substituted phenols to allylic oxides has been demonstrated using C2-symmetric palladium complexes. Complex phenol donors tyrosine, estradiol, and griseofulvin follow the predictive model.

Syntheses of arnottin I and arnottin II.

Short total syntheses of arnottin I and II were accomplished in 5 and 6 steps, respectively. A sesamol-benzyne cycloaddition with a 3-furyl-benzoate followed by regiospecific lactonization provided rapid, large-scale access to the core of arnottin I. Saponification of arnottin I and hypervalent iodide mediated spirocyclization provided an efficient and direct preparation of racemic arnottin II.

Regiodivergent addition of phenols to allylic oxides: control of 1,2- and 1,4-additions for cyclitol synthesis.

Control of 1,2- and 1,4-addition of substituted phenols to allylic oxides is achieved by intercepting palladium π-allyl complexes. The interconversion of palladium complexes results in the total synthesis of MK 7607, cyathiformine B type, streptol, and a new cyclitol.