Discovery and Optimization of Potent, Selective, and Brain-Penetrant 1-Heteroaryl-1H-Indazole LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease.

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.

Prolylcarboxypeptidase (PrCP) inhibitors and the therapeutic uses thereof: a patent review.

INTRODUCTION: Prolylcarboxypeptidase (PrCP) is a serine protease that produces or degrades signaling proteins in several important pathways including the renin-angiotensin system (RAS), kallikrein-kinin system (KKS) and pro-opiomelanocortin (POMC) system. PrCP has the potential to be a therapeutic target for cardiovascular, inflammatory and metabolic diseases. Numerous classes of PrCP inhibitors have been developed by rational drug design and from high-throughput screening hits. These inhibitors have been tested in mouse models to assess their potential as new therapeutics. Areas Covered: This review covers the relevant studies that support PrCP as a target for drug discovery. All the significant patent applications and primary literature concerning the development of PrCP inhibitors are discussed. Expert Opinion: The pathways where PrCP is known to operate are complex and many aspects remain to be characterized. Many potent inhibitors of PrCP have been tested in vivo. The variable results obtained from in vivo studies with PrCP inhibitors suggest that additional understanding of the biochemistry and the required therapeutic inhibitor levels is necessary. Additional fundamental research into the signaling pathways is likely required before the true therapeutic potential of PrCP inhibition will be realized.

Pyrazoles as non-classical bioisosteres in prolylcarboxypeptidase (PrCP) inhibitors.

Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.

Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors.

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.

Gold-catalyzed formation of heterocycles - an enabling new technology for medicinal chemistry.

Gold-catalyzed transformations allow efficient access to a wide scope of heterocyclic structures that serve as building blocks and pharmacophores in medicinal chemistry. Compared with other transition metal and Lewis acid catalysis, gold catalysis presents mechanistic divergence, excellent functional group tolerance and/or operational advantages. Emergent applications of gold catalysis have played a key role in the synthesis of biologically active molecules including a drug candidate.

A new class of prolylcarboxypeptidase inhibitors, part 1: discovery and evaluation.

A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.

A new class of prolylcarboxypeptidase inhibitors, part 2: the aminocyclopentanes.

A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert-butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC(50) values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.

Discovery of aminoheterocycles as potent and brain penetrant prolylcarboxypeptidase inhibitors.

Efforts were dedicated to develop potent and brain penetrant prolylcarboxypeptidase (PrCP) inhibitors by replacing the amide group of original leads 1 and 2 with heterocycles. Aminopyrimidines including compound 32a were identified to display good PrCP inhibitory activity (32a, IC(50)=43 nM) and impressive ability to penetrate brain in mice (brain/plasma ratio: 1.4).

The discovery of non-benzimidazole and brain-penetrant prolylcarboxypeptidase inhibitors.

Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC(50) values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.

A method for the reductive scission of heterocyclic thioethers.

A mild, chemoselective, and generally high-yielding method for the reductive scission of heterocyclic thioethers is described. Suitable heterocycles have a thioether substituent at the 2-position relative to a ring heteroatom. The convenient and straightforward method is demonstrated with reactants which are not compatible with the standard Raney nickel conditions such as sulfides, sulfones, and thiophenes. In addition, benzyl esters, benzyl amides, and benzyl carbamates are tolerated by the reductive reaction conditions.

A direct synthesis of oxazoles from aldehydes.

An expedient method for the direct conversion of aldehydes to 2,4-disubstituted oxazoles is presented. The method relies on the oxidation of an oxazolidine formed from the condensation of serine with an aldehyde and proceeds through a 2,5-dihydrooxazole intermediate. In contrast to standard methods that start from carboxylic acids, the use of aldehydes as starting materials does not require intermediate purification and affords the oxazoles under relatively mild conditions.

Identifying a resistance determinant for the antimitotic natural products disorazole C1 and A1.

Disorazoles are macrocyclic polyketides first isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. Both the major fermentation product disorazole A(1) and its much rarer companion disorazole C(1) exhibit potent cytotoxic activity against many human tumor cells. Furthermore, the disorazoles appear to bind tubulin uniquely among known antimitotic agents, promoting apoptosis or premature senescence. It is uncertain what conveys tumor cell sensitivity to these complex natural products. Therefore, we generated and characterized human tumor cells resistant to disorazole C(1). Resistant cells proved exceedingly difficult to generate and required single step mutagenesis with chronic stepwise exposure to increasing concentrations of disorazole C(1). Compared with wild-type HeLa cells, disorazole C(1)-resistant HeLa/DZR cells were 34- and 8-fold resistant to disorazole C(1) and disorazole A(1) growth inhibition, respectively. HeLa/DZR cells were also remarkably cross-resistant to vinblastine (280-fold), paclitaxel (2400-fold), and doxorubicin (47-fold) but not cisplatin, suggesting a multidrug-resistant phenotype. Supporting this hypothesis, MCF7/MDR cells were 10-fold cross-resistant to disorazole C(1). HeLa/DZR disorazole resistance was not durable in the absence of chronic compound exposure. Verapamil reversed HeLa/DZR resistance to disorazole C(1) and disorazole A(1). Moreover, HeLa/DZR cells expressed elevated levels of the drug resistance ATP-binding cassette ABCB1 transporter. Loss of ABCB1 by incubation with short interfering RNA restored sensitivity to the disorazoles. Thus, the multidrug resistance transporter ABCB1 can affect the cytotoxicity of both disorazole C(1) and A(1). Disorazole C(1), however, retained activity against cells resistant against the clinically used microtubule-stabilizing agent epothilone B.

Microtubule binding and disruption and induction of premature senescence by disorazole C(1).

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A(1), was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C(1) and found it retained potent antiproliferative activity against tumor cells, causing prominent G(2)/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C(1) produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C(1) inhibited purified bovine tubulin polymerization, with an IC(50) of 11.8 +/- 0.4 microM, and inhibited [3H]vinblastine binding noncompetitively, with a K(i) of 4.5 +/- 0.6 microM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C(1), with a K(i) of 10.6 +/- 1.5 microM, indicating that disorazole C(1) bound tubulin uniquely among known antimitotic agents. Disorazole C(1) could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Cellular analysis of disorazole C and structure-activity relationship of analogs of the natural product.

Structure-activity analyses of synthetic disorazole C(1) and eight of its analogs indicate that the presence of a vinyl oxirane moiety or a tetraene sequence is not necessary for potent cytotoxic and antimitotic properties. Using an automated multiparameter fluorescence-based cellular assay to simultaneously probe the effects of disorazole analogs on cellular microtubules, mitotic arrest, and cytotoxicity, we found that disorazole C(1) enhanced the mitotic index and chromatin condensation and arrested cells in the G2/M phase of the cell cycle. All structural analogs and synthesis precursors of disorazole C(1) were at least two orders of magnitude less potent than the parent compound, thus indicating that both the functional group array and the three-dimensional conformation of the parent compound are critical for interaction with the biological target. We conclude that disorazole C(1) is a potent inducer of mitotic arrest and hypothesize that this biological activity may be mediated by microtubule perturbation.

Synthesis and hetero-Michael addition reactions of 2-alkynyl oxazoles and oxazolines.

Stereoselective conjugate additions of alcohols, amines, thiols, and halides to C(2)-alkynyl oxazoles and oxazolines provide a versatile entry to heterocyclic building blocks.

Total synthesis of (-)-disorazole C1.

The antimitotic natural product disorazole C1 was isolated in 1994 from the fermentation broth of the myxobacterium Sorangium cellulosum. We have developed a highly convergent and stereoselective total synthesis of this compound which establishes its relative and absolute configuration. Key features of our synthesis include a highly convergent strategy and selective functional group manipulations that minimize decomposition of the sensitive polyene macrodiolide.