Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Application of Covalent Binding Body Burden in the HµREL Human Hepatocyte Coculture Model for Reactivity Risk Assessment of Metabolically Low Turnover Drugs.

The human hepatocyte suspension model has been a valuable tool to study covalent binding (CVB) for compounds that form reactive metabolites. However, accurately measuring CVB values with the suspension model becomes challenging for metabolically low turnover compounds. In this study, we evaluated the HµREL human hepatocyte coculture model relative to existing literature using human hepatocyte suspension for drugs of known drug-induced liver injury category. Our results indicate that this coculture model provides ample metabolic turnover to reproducibly measure CVB. It is sufficiently robust to apply a predefined 1 mg/day CVB body burden threshold for risk assessment to guide our discovery programs, allowing for expanded coverage to include metabolically low turnover compounds.

Characterization of JNJ-2482272 [4-(4-Methyl-2-(4-(Trifluoromethyl)Phenyl)Thiazole-5-yl) Pyrimidine-2-Amine] As a Strong Aryl Hydrocarbon Receptor Activator in Rat and Human.

[4-(4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-yl)pyrimidine-2-amine] (JNJ-2482272), under investigation as an anti-inflammatory agent, was orally administered to rats once daily at 60 mg/kg for 6 consecutive days. Despite high plasma exposure after single administration (Cmax of 7.1 µM), JNJ-2482272 had plasma concentrations beneath the lower limit of quantification (3 ng/ml) after 6 consecutive days of dosing. To determine if JNJ-2482272 is an autoinducer in rats, plated rat hepatocytes were treated with JNJ-2482272 for 2 days. The major hydroxylated metabolites of JNJ-2482272 were isolated and characterized by mass spectrometry and NMR analyses. Compared with the vehicle-treated cells, a concentration-dependent increase was observed in the formation of phase I- and II-mediated metabolites coinciding with greater expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in rat hepatocytes. CYP1A1, CYP1A2, CYP1B1, and UGT1A6 transcripts were predominantly induced, suggesting that JNJ-2482272 is an activator of the aryl hydrocarbon receptor (AhR). In a human AhR reporter assay, JNJ-2482272 demonstrated potent AhR activation with an EC50 value of 0.768 nM, a potency more comparable to the strong AhR activator and toxin 2,3,7,8-tetrachloro-dibenzodioxin than to weaker AhR activators 3-methylcholanthrene, ß-naphthoflavone, and omeprazole. In plated human hepatocytes, JNJ-2482272 induced CYP1A1 gene expression with an EC50 of 20.4 nM and increased CYP1A activity >50-fold from basal levels. In human recombinant P450s, JNJ-2482272 was exclusively metabolized by the CYP1 family of enzymes and most rapidly by CYP1A1. The summation of these in vitro findings bridges the in vivo conclusion that JNJ-2482272 is a strong autoinducer in rats and potentially in humans through potent AhR activation. SIGNIFICANCE STATEMENT: Drugs that induce their own metabolism (autoinducers) can lack sustained exposures for pharmacology and safety assessment hindering their development. JNJ-2482272 is demonstrated herein as a strong aryl hydrocarbon receptor (AhR) activator and CYP1A autoinducer, explaining its near complete loss of exposure after repeat administration in rat, which is likely translatable to human (if progressed further) considering its nanomolar potency comparable to "classical" AhR ligands like 2,3,7,8-tetrachloro-dibenzo-dioxin despite bearing a "nonclassical" drug structure.

The influence of the Or and Carotene Hydroxylase genes on carotenoid accumulation in orange carrots [Daucus carota (L.)].

KEY MESSAGE: The Or and CH genes are necessary for the accumulation of high amounts of ß-carotene and other carotenoid pigments in carrot roots, in addition to the Y and Y2 genes. Carrot taproot color results from the accumulation of various carotenoid and anthocyanin pigments. Recently, the Or gene was identified as a candidate gene associated with the accumulation of ß-carotene and other provitamin A carotenoids in roots. The specific molecular mechanisms involved with this process, as well as the interactions between Or and the other genes involved in this process are not well understood. In order to better characterize the effect that Or alleles have on conditioning the accumulation of carotenoids in roots, we analyzed an F3 family fixed homozygous recessive for y and y2, derived from a cross between an orange carrot and a white wild carrot, segregating for the two known Or alleles, which we name Orc and Orw. QTL mapping across three different environments revealed that the accumulation of several carotenoids was associated with the Orc allele, with consistent patterns across environments. A second QTL on chromosome 7, harboring a carotene hydroxylase gene homologous to Lut5 in Arabidopsis, was also associated with the accumulation of several carotenoids. Two alleles for this gene, which we name CHc and CHw, were discovered to be segregating in this population. Our study provides further evidence that Or and CH are likely involved with controlling the accumulation of ß-carotene and may be involved with modulating carotenoid flux in carrot, demonstrating that both were important domestication genes in carrot.

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.

Population genomics identifies genetic signatures of carrot domestication and improvement and uncovers the origin of high-carotenoid orange carrots.

Here an improved carrot reference genome and resequencing of 630 carrot accessions were used to investigate carrot domestication and improvement. The study demonstrated that carrot was domesticated during the Early Middle Ages in the region spanning western Asia to central Asia, and orange carrot was selected during the Renaissance period, probably in western Europe. A progressive reduction of genetic diversity accompanied this process. Genes controlling circadian clock/flowering and carotenoid accumulation were under selection during domestication and improvement. Three recessive genes, at the REC, Or and Y2 quantitative trait loci, were essential to select for the high α- and ß-carotene orange phenotype. All three genes control high α- and ß-carotene accumulation through molecular mechanisms that regulate the interactions between the carotenoid biosynthetic pathway, the photosynthetic system and chloroplast biogenesis. Overall, this study elucidated carrot domestication and breeding history and carotenoid genetics at a molecular level.

Discovery of a Series of Substituted 1H-((1,2,3-Triazol-4-yl)methoxy)pyrimidines as Brain Penetrants and Potent GluN2B-Selective Negative Allosteric Modulators.

Herein, we describe a series of substituted 1H-((1,2,3-triazol-4-yl)methoxy)pyrimidines as potent GluN2B negative allosteric modulators. Exploration of several five- and six-membered heterocycles led to the identification of O-linked pyrimidine analogues that possessed a balance of potency and desirable ADME profiles. Due to initial observations of metabolic saturation, early metabolite identification studies were conducted on compound 18, and the results drove further iterative optimization efforts to avoid the formation of undesired saturating metabolites. The comprehensive investigation of substitution on the pyrimidine moiety of the 1H-1,2,3-triazol-4-yl)methoxy)pyrimidines allowed for the identification of compound 31, which demonstrated high GluN2B receptor affinity, improved solubility, and a clean cardiovascular profile. Compound 31 was profiled in an ex vivo target engagement study in rats at a 10 mg/kg oral dose and achieved an ED50 of 1.7 mg/kg.

A Phase 1 First-in-Human Pharmacokinetic and Pharmacodynamic Study of JNJ-64264681, a Covalent Inhibitor of Bruton's Tyrosine Kinase.

JNJ-64264681 is an irreversible covalent inhibitor of Bruton's tyrosine kinase. This phase 1, first-in-human, 2-part (single-ascending dose [SAD]; multiple-ascending dose [MAD]) study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD; Bruton's tyrosine kinase occupancy [BTKO]) of JNJ-64264681 oral solution in healthy participants. For SAD (N = 78), 6 increasing doses of JNJ-64264681 (4-400 mg) or placebo were evaluated in fasted males. The effects of sex, food, and a capsule formulation were evaluated in separate cohorts. For MAD (N = 27), sequential cohorts of male and female participants received 36/100/200 mg JNJ-64264681 once daily for 10 days. JNJ-64264681 exposure (peak concentration; area under the concentration-time curve) was less than dose proportional from 4 mg to 36 mg. Dose-normalized area under the concentration-time curves following the 36 mg and 100 mg doses were generally similar. The mean terminal half-life was 1.6-13.2 hours. With multiple doses, steady state was achieved by day 2. A semimechanistic PK/PD model was developed using the first 5 SAD cohorts' data to predict %BTKO in MAD cohorts. PK/PD model guided dose-escalation, and all participants in the 200/400 mg single-dose cohorts achieved ≥90% BTKO at 4 hours after dosing (peak) with prolonged occupancy. As BTKO data became available from MAD cohorts, it was found that observed BTKO data were consistent with model predictions. JNJ-64264681 showed no safety signals of concern. Overall, safety, tolerability, PK, BTKO, and PK/PD modeling guided the rationale for dose selection for the subsequent first-in-patient lymphoma studies.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

Discovery of a Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase with Oral Anti-Inflammatory Activity.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

Is News Surveillance related to Cancer Knowledge in Underserved Adults? Testing Three Versions of the Cognitive Mediation Model.

The cognitive mediation model (CMM) proposes indirect paths to news learning such that news surveillance increases news learning through attention to the news and elaboration about the news. But there is a need for additional research that tests key postulates of the CMM especially for media targeting underserved populations. The present study tested three versions of the CMM to model ethnic newspaper learning within a low-income, Spanish-speaking population (N = 150). The original CMM was not supported by the data as elaboration was not related to knowledge; however, a simplified version of the CMM (surveillance → attention → knowledge) was supported. Moreover, a serial mediation model that included a measure of health maven was supported such that news surveillance was positively related to knowledge through maven-ness and attention.

The Genetic Diversity of Cranberry Crop Wild Relatives, Vaccinium macrocarpon Aiton and V. oxycoccos L., in the US, with Special Emphasis on National Forests.

Knowledge of the genetic diversity in populations of crop wild relatives (CWR) can inform effective strategies for their conservation and facilitate utilization to solve agricultural challenges. Two crop wild relatives of the cultivated cranberry are widely distributed in the US. We studied 21 populations of Vaccinium macrocarpon Aiton and 24 populations of Vaccinium oxycoccos L. across much of their native ranges in the US using 32 simple sequence repeat (SSR) markers. We observed high levels of heterozygosity for both species across populations with private alleles ranging from 0 to 26. For V. macrocarpon, we found a total of 613 alleles and high levels of heterozygosity (HO = 0.99, HT = 0.75). We also observed high numbers of alleles (881) and levels of heterozygosity (HO = 0.71, HT = 0.80) in V. oxycoccos (4x). Our genetic analyses confirmed the field identification of a native population of V. macrocarpon on the Okanogan-Wenatchee National Forest in the state of Washington, far outside the previously reported range for the species. Our results will help to inform efforts of the United States Department of Agriculture Agricultural Research Service (USDA-ARS) and the United States Forest Service (USFS) to conserve the most diverse and unique wild cranberry populations through ex situ preservation of germplasm and in situ conservation in designated sites on National Forests.

Substituted Azabicyclo[2.2.1]heptanes as Selective Orexin-1 Antagonists: Discovery of JNJ-54717793.

The orexin system consists of two neuropeptides (orexin-A and orexin-B) that exert their mode of action on two receptors (orexin-1 and orexin-2). While the role of the orexin-2 receptor is established as an important modulator of sleep wake states, the role of the orexin-1 receptor is believed to play a role in addiction, panic, or anxiety. In this manuscript, we describe the optimization of a nonselective substituted azabicyclo[2.2.1]heptane dual orexin receptor antagonist (DORA) into orally bioavailable, brain penetrating, selective orexin-1 receptor (OX1R) antagonists. This resulted in the discovery of our first candidate for clinical development, JNJ-54717793.

Design, Synthesis, and Preclinical Evaluation of 3-Methyl-6-(5-thiophenyl)-1,3-dihydro-imidazo[4,5-b]pyridin-2-ones as Selective GluN2B Negative Allosteric Modulators for the Treatment of Mood Disorders.

Selective inhibitors of the GluN2B subunit of N-methyl-d-aspartate receptors in the ionotropic glutamate receptor superfamily have been targeted for the treatment of mood disorders. We sought to identify structurally novel, brain penetrant, GluN2B-selective inhibitors suitable for evaluation in a clinical setting in patients with major depressive disorder. We identified a new class of negative allosteric modulators of GluN2B that contain a 1,3-dihydro-imidazo[4,5-b]pyridin-2-one core. This series of compounds had poor solubility properties and poor permeability, which was addressed utilizing two approaches. First, a series of structural modifications was conducted which included replacing hydrogen bond donor groups. Second, enabling formulation development was undertaken in which a stable nanosuspension was identified for lead compound 12. Compound 12 was found to have robust target engagement in rat with an ED70 of 1.4 mg/kg. The nanosuspension enabled sufficient margins in preclinical toleration studies to nominate 12 for progression into advanced good laboratory practice studies.

1H-Pyrrolo[3,2-b]pyridine GluN2B-Selective Negative Allosteric Modulators.

Herein, we disclose a series of selective GluN2B negative allosteric modulators containing a 1H-pyrrolo[3,2-b]pyridine core. Lead optimization efforts included increasing brain penetration as well as decreasing cytochrome P450 inhibition and hERG channel binding. The series was also optimized to reduce metabolic turnover in human and rat. Compounds 9, 25, 30, and 34 have good in vitro GluN2B potency and good predicted absorption, but moderate to high projected clearance. They were assessed in vivo to determine their target engagement. All four compounds achieved >75% receptor occupancy after an oral dose of 10 mg/kg in rat. Compound 9 receptor occupancy was measured in a dose-response experiment, and its ED50 was found to be 2.0 mg/kg.

Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators.

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

Carotenoid Presence Is Associated with the Or Gene in Domesticated Carrot.

Carrots are among the richest sources of provitamin A carotenes in the human diet, but genetic variation in the carotenoid pathway does not fully explain the high levels of carotenoids in carrot roots. Using a diverse collection of modern and historic domesticated varieties, and wild carrot accessions, an association analysis for orange pigmentation revealed a significant genomic region that contains the Or gene, advancing it as a candidate for carotenoid presence in carrot. Analysis of sequence variation at the Or locus revealed a nonsynonymous mutation cosegregating with carotenoid content. This mutation was absent in all wild carrot samples and nearly fixed in all orange domesticated samples. Or has been found to control carotenoid presence in other crops but has not previously been described in carrot. Our analysis also allowed us to more completely characterize the genetic structure of carrot, showing that the Western domesticated carrot largely forms one genetic group, despite dramatic phenotypic differences among market classes. Eastern domesticated and wild accessions form a second group, which reflects the recent cultivation history of carrots in Central Asia. Other wild accessions form distinct geographic groups, particularly on the Iberian peninsula and in Northern Africa. Using genome-wide Fst , nucleotide diversity, and the cross-population composite likelihood ratio, we analyzed the genome for regions putatively under selection during domestication and identified 12 regions that were significant for all three methods of detection, one of which includes the Or gene. The Or domestication allele appears to have been selected after the initial domestication of yellow carrots in the East, near the proposed center of domestication in Central Asia. The rapid fixation of the Or domestication allele in almost all orange and nonorange carrots in the West may explain why it has not been found with less genetically diverse mapping populations.

Lead Optimization of 5-Aryl Benzimidazolone- and Oxindole-Based AMPA Receptor Modulators Selective for TARP γ-8.

Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead 4 was optimized for potency and brain penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC50 = 9.7). Following oral dosing in rats, 18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED50 = 0.6 mg/kg, plasma EC50 = 9 ng/mL).

A Dipolar Cycloaddition Reaction To Access 6-Methyl-4,5,6,7-tetrahydro-1H-[1,2,3]triazolo[4,5-c]pyridines Enables the Discovery Synthesis and Preclinical Profiling of a P2X7 Antagonist Clinical Candidate.

A single pot dipolar cycloaddition reaction/Cope elimination sequence was developed to access novel 1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridine P2X7 antagonists that contain a synthetically challenging chiral center. The structure-activity relationships of the new compounds are described. Two of these compounds, (S)-(2-fluoro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 29) and (S)-(3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 35), were found to have robust P2X7 receptor occupancy at low doses in rat with ED50 values of 0.06 and 0.07 mg/kg, respectively. Compound 35 had notable solubility compared to 29 and showed good tolerability in preclinical species. Compound 35 was chosen as a clinical candidate for advancement into phase I clinical trials to assess safety and tolerability in healthy human subjects prior to the initiation of proof of concept studies for the treatment of mood disorders.