Discovery of novel benzothiophene derivatives as potent and narrow spectrum inhibitors of DYRK1A and DYRK1B.

The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.

Sulfonyl(thio)urea derivative induction of insulin secretion is mediated by potassium, calcium, and sodium channel signal transduction.

AIM: To investigate the mechanism of action of sulfonyl(thio)urea derivative (SD) on glycemia and on insulin secretion in pancreatic islets. METHODS: Wistar rats were divided into hyperglycemic control group, rats received 4 g/kg body weight glucose plus sitagliptin 10 mg/kg (p.o.); hyperglycemic plus SD 10 mg/kg (p.o.); hyperglycemic plus SD plus sitagliptin. Blood was collected before glucose overloading (zero time), and at 15, 30, 60, and 180 min after glucose, from the afore mentioned groups for glycemia and glucagon-like peptide 1 (GLP-1) measurements and intestinal disaccharidases activity. Pancreatic islets were isolated for the calcium influx and insulin secretion in in vitro studies. RESULTS: SD reduced glycemia and increased GLP-1 secretion, while inhibited sucrase and lactase activity. This SD (1.0 and 10.0 µM) stimulated calcium influx in a similar percentile to that of glibenclamide, and in a nonsynergic manner. In addition, the trigger effect of SD on calcium influx was through the K+ -ATP-dependent channels, and partially by activating voltage-dependent K + channels and voltage-dependent calcium channels. Furthermore, SD-stimulated Na + and Ca 2+ entry, induced by the transient receptor potential ankyrin 1 and by modulation of Na + /Ca 2+ exchange. The activation of these pathways by SD culminated in in vitro insulin secretion, reinforcing the critical role of K + -ATP channels in the secretagogue effect of SD. CONCLUSIONS: SD diminish glycemia by inducing GLP-1 secretion and inhibiting disaccharidases. To our knowledge, this is the first report of an insulin secretagogue effect of SD that is mediated by potassium and calcium, as well as sodium, signal transduction.

Mechanism of action of camphoryl-benzene sulfonamide derivative on glucose uptake in adipose tissue.

The aim of the present study was to investigate the mechanism of action of a sulfonamide derivative on glucose uptake in adipose tissue, as well as to characterize the effects of this compound on intestinal disaccharidases and advanced glycation end-products (AGEs) formation. Camphoryl-benzene sulfonamide (CS) was able to stimulate glucose uptake in isolated adipocytes, adipose tissue, and in soleus muscle. The stimulatory effect of the compound (10 µM) on glucose uptake on adipose tissue was blocked by diazoxide, wortmannin, U73122, colchicine, and N-ethylmaleimide. On the other hand, the effects of CS were not blocked by glibenclamide, an inhibitor of the K+ -ATP channel, or even by the inhibitor of protein p38 MAPK, SB 203580. In vivo, this compound reduced intestinal disaccharidase activity, while, in vitro, CS reduced the formation of AGEs at 7, 14, and 28 days of incubation. The stimulatory effect of CS on glucose uptake requires the activation of the K+ -ATP channel, translocation, and fusion of GLUT4 vesicles to the plasma membrane on adipocytes for glucose homeostasis. In addition, the inhibition of disaccharidase activity contributes to the glucose homeostasis in a short-term as well as the remarkable reduction in AGE formation indicates that the CS may prevent of complications of late diabetes.

Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis protein tyrosine phosphatase A inhibitors.

Mycobacterium tuberculosis secretes two protein tyrosine phosphatases as virulence factors, PtpA and PtpB. Inhibition studies of these enzymes have shown significant attenuation of the M. tuberculosis growth in vivo. As PtpA mediates many effects on the regulation of host signaling ensuring the intracellular survival of the bacterium we report, for the first time, thiosemicarbazones as potential novel class of PtpA inhibitors. Several compounds were synthesized and biologically evaluated, revealing interesting results. Enzyme kinetic assays showed that compounds 5, 9 and 18 are non-competitive inhibitors of PtpA, with Ki values ranging from 1.2 to 5.6 µM. Modeling studies clarified the structure-activity relationships observed in vitro and indicated a possible allosteric binding site in PtpA structure. To the best of our knowledge, this is the first disclosure of potent non-competitive inhibitors of PtpA with great potential for future studies and development of analogues.

Solid lipid nanoparticles improve octyl gallate antimetastatic activity and ameliorate its renal and hepatic toxic effects.

Metastasis is the main cause of cancer-related death and requires the development of effective treatments with reduced toxicity and effective anticancer activity. Gallic acid derivatives have shown significant biological properties including antitumoral activity as shown in a previous study with octyl gallate (G8) in vitro. Thus, the aim of this work was to evaluate the antimetastatic effect of free and solid lipid nanoparticle-loaded G8 in mice in a lung metastasis model. Animals inoculated with melanoma cells presented metastasis in lungs, which was significantly inhibited by treatment with G8 and solid lipid nanoparticle-loaded G8, named G8-NVM. However, G8-treated mice showed an increase in several toxicological parameters, which were almost completely circumvented by G8-NVM treatment. This study supports the need for pharmacological studies on new potential medicinal plants to treat cancer and can provide new perspectives on using nanotechnology to improve biological activities while decreasing the chemotherapy toxicological effects of anticancer drugs.

Incretinomimetic and Insulinomimetic Effect of (2E)-N'-(1'-Naphthyl)-3,4,5-Trimethoxybenzohydrazide for Glycemic Homeostasis.

To characterize the role and the mechanism of action of (2E)-N'-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide (BZD) on incretin secretion, glucose uptake in skeletal muscle and α-glucosidase activity on intestine, targets for glucose homeostasis. It was assayed on glucose tolerance test (GTT) to analyze GLP-1 secretion and the activity of DPP-4 enzyme in vitro. In skeletal muscle, mechanism of action on glucose uptake was carried out by in vitro experiments. The activity of intestinal disaccharidases was performed after in vivo and in vitro experiments. The compound improved the glucose tolerance around 30%, 25%, and 20% at 15, 30, and 60 min, respectively and potentiated the sitagliptin effect, an inhibitor of the enzyme that removes GLP-1, about 50, 45, and 54% at 15, 30, and 60 min, respectively. Additionally, BZD did not modify the activity of DPP-4 enzyme. The acute effect of BZD on glucose uptake is mediated by increasing GLUT4 expression (around 140%) and its translocation to the plasma membrane in soleus muscle. The genomic effect as well as GLUT4 translocation involve the activation of PI-3K and MAPK pathways and require the microtubules integrity to the complete stimulatory effect of this compound on glucose uptake. Beyond, BZD acts in an alternative target to ameliorate glycaemia, intestinal disaccharidases. In a whole, these data point an incretino- and insulinomimetic effect of the compound for glycemic control.

Investigation of cellular mechanisms involved in apoptosis induced by a synthetic naphthylchalcone in acute leukemia cell lines.

We have previously reported the cytotoxic effects of chalcone A1, derived from 1-naphthaldehyde, in leukemia cell lines. On the basis of these findings, the main aim of this study was to elucidate some of the molecular mechanisms involved in apoptosis induced by chalcone A1 toward K562 and Jurkat cells. In both cell lines, chalcone A1 decreased the mitochondrial membrane potential, increased the expression of Bax proapoptotic protein, and decreased the expression of Bcl-2 antiapoptotic protein (resulting in the inversion of the Bcl-2/Bax ratio), which indicates the involvement of the intrinsic pathway. In addition, chalcone A1 increased the expression of FasR in Jurkat cells, which also indicates the involvement of the extrinsic pathway in this cell line. The results also showed an increased expression of effector caspase-3 and cleaved PARP-1 and a decreased expression of IAP protein survivin, which are consistent with apoptotic cell death. The decreased expression of Ki67 suggests that the mechanism involved in cell death induced by chalcone A1 also involves a decrease in cell proliferation. In ex-vivo experiments, chalcone A1 reduced the cell viability of blast cells collected from eight patients with different types of acute leukemia, confirming the cytotoxicity results found in vitro. The results obtained so far are very promising and further studies need to be carried out so that chalcone A1 can be used as a prototype for the development of new antileukemia agents.

Synthetic compounds from an in house library as inhibitors of falcipain-2 from Plasmodium falciparum.

Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkyl-esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5 ± 0.8 µM, 9.5 ± 0.2 µM and 4.9 ± 1.3 µM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.

Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles.

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.

Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita.

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4'-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC50) of 171 µg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC50 of 260 µg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ~50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita.

Antimetastatic activity and low systemic toxicity of tetradecyl gallate in a preclinical melanoma mouse model.

Gallates with eight or more carbon atoms in the lateral chain show potent anticancer activity against various cell lines. However, studies regarding the in vivo antimelanoma activity of tetradecyl gallate (C(14)) have not yet been reported. In this study an evaluation of the ability of C(14) to inhibit metastasis, using lung metastases as a model, was carried out. The experimental mouse melanoma model was established by intravenous injection of metastatic B16F10 melanoma cells. The systemic toxicity of C(14) was evaluated in vivo by monitoring the weight, survival, biochemical and hematological parameters, and through histological analysis. It was observed that C(14) decreased lung metastasis in vivo by 80% and increased the survival rate of the animals without toxic effects. Additionally, C(14) induced cytotoxic effects on B16F10 cells, inhibited the inter-cellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression, and significantly decreased cell adhesion. These results reveal that C(14) has potent antimetastatic ability and is a good candidate for further study as a potential therapeutic agent for tumor metastases.

Antibacterial activity of chalcones, hydrazones and oxadiazoles against methicillin-resistant Staphylococcus aureus.

The increase in antibiotic resistance due to multiple factors has encouraged the search for new compounds which are active against multidrug-resistant pathogens. In this context, chalcones, dihydrochalcones, hydrazones and oxadiazoles were tested against Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus (MRSA) isolates, which were obtained from clinical laboratories and were characterized as MRSA using traditional and molecular methods. Among 65 tested compounds, two chalcones, one dihydrochalcone and two hydrazones were active against MRSA. Based on the minimal inhibitory concentration and cytotoxicity, hydrazones provided a better selectivity index than chalcones. Active hydrazones are promising antibiotic-like substances and they should be the subject of further microbiological studies.

Trimethoxy-chalcone derivatives inhibit growth of Leishmania braziliensis: synthesis, biological evaluation, molecular modeling and structure-activity relationship (SAR).

In this work we described the synthesis, the antileishmanial activity and the molecular modeling and structure-activity relationship (SAR) evaluations of a series of chalcone derivatives. Among these compounds, the methoxychalcones 2h, 2i, 2j, 2k and 2l showed significant antileishmanial activity (IC(50)<10 µM). Interestingly 2i (IC(50)=2.7 µM), 2j (IC(50)=3.9 µM) and 2k (IC(50)=4.6 µM) derivatives presented better antileishmanial activity than the control drug pentamidine (IC(50)=6.0 µM). Our SAR study showed the importance of methoxy di-ortho substitution at phenyl ring A and the relationship between the frontier orbital HOMO coefficients distribution of these molecules and their activity. The most active compounds 2h, 2i, 2j, 2k, and 2l fulfilled the Lipinski rule-of-five which theoretically is important for good drug absorption and permeation through biological membranes. The potential profile of 2j (IC(50)=3.9 µM and CC(50)=216 µM) pointed this chalcone derivative as a hit compound to be further explored in antileishmanial drug design.

Design, synthesis and antihypertensive evaluation of novel codrugs with combined angiotensin type 1 receptor antagonism and neprilysin inhibition.

The multifactorial etiology of hypertension has promoted the research of blood pressure-lowering agents with multitarget actions to achieve better clinical outcomes. We describe here the discovery of novel dual-acting antihypertensive codrugs combining pharmacophores with angiotensin type 1 (AT1) receptor antagonism and neprilysin (NEP) inhibition. Specifically, the codrugs combine the AT1 antagonists losartan or its carboxylic acid active metabolite (E-3174) with selected monocarboxylic acid NEP inhibitors through a cleavable linker. The resulting codrugs exhibited high rates of in vitro conversion into the active molecules upon incubation with human/rat liver S9 fractions and in vivo conversion after oral administration in rodents. Moreover, the acute effects of one of the designed codrugs (3b) was confirmed at the doses of 10, 30 and 60 mg/kg p.o. in the spontaneous hypertensive rat (SHR) model, showing better antihypertensive response over 24 hours than the administration of an equivalent fixed-dose combination of 15 mg/kg of losartan and 14 mg/kg of the same NEP inhibitor used in 3b. The results demonstrate that the codrug approach is a plausible strategy to develop a single molecular entity with combined AT1 and NEP activities, aiming at achieving improved pharmacokinetics, efficacy and dosage convenience, as well as reduced drug-drug interaction for hypertension patients. In addition, the developability of the codrug should be comparable to the one of marketed AT1 antagonists, most of them prodrugs, but bearing only the AT1 pharmacophore.

Discovery of ACH-000143: A Novel Potent and Peripherally Preferred Melatonin Receptor Agonist that Reduces Liver Triglycerides and Steatosis in Diet-Induced Obese Rats.

The modulation of melatonin signaling in peripheral tissues holds promise for treating metabolic diseases like obesity, diabetes, and nonalcoholic steatohepatitis. Here, several benzimidazole derivatives have been identified as novel agonists of the melatonin receptors MT1 and MT2. The lead compounds 10b, 15a, and 19a demonstrated subnanomolar potency at MT1/MT2 receptors, high oral bioavailability in rodents, peripherally preferred exposure, and excellent selectivity in a broad panel of targets. Two-month oral administration of 10b in high-fat diet rats led to a reduction in body weight gain similar to dapagliflozin with superior results on hepatic steatosis and triglyceride levels. An early toxicological assessment indicated that 10b (also codified as ACH-000143) was devoid of hERG binding, genotoxicity, and behavioral alterations at doses up to 100 mg/kg p.o., supporting further investigation of this compound as a drug candidate.

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: kinetics, molecular modeling, toxicity and effect on growth.

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes pi stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents.

Brain-wide mapping of c-fos expression in the single prolonged stress model and the effects of pretreatment with ACH-000029 or prazosin.

Post-traumatic stress disorder (PTSD) is a mental health condition that is triggered by a stressful event, with symptoms including exaggerated startle response, intrusive traumatic memories and nightmares. The single prolonged stress (SPS) is a multimodal stress protocol that comprises a sequential exposure to physical restraint, forced swimming, predator scent and ether anesthesia. This procedure generates behavioral and neurobiological alterations that resemble clinical findings of PTSD, and thus it is commonly used to model the disease in rodents. Here, we applied c-fos mapping to produce a comprehensive view of stress-activated brain regions in mice exposed to SPS alone or to SPS after oral pretreatment with the serotonin-noradrenaline receptor dual modulator ACH-000029 or the α1-adrenergic blocker prazosin. The SPS protocol evoked c-fos expression in several brain regions that control the stress-anxiety response, including the central and medial amygdala, the bed nucleus of the stria terminalis, the pallidum, the paraventricular hypothalamus, the intermediodorsal, paraventricular and central medial thalamic nuclei, the periaqueductal gray, the lateral habenula and the cuneiform nucleus. These effects were partially blocked by pretreatment with prazosin but completely prevented by ACH-000029. Collectively, these findings contribute to the brain-wide characterization of neural circuits involved in PTSD-related stress responses. Furthermore, the identification of brain areas regulated by ACH-000029 and prazosin revealed regions in which SPS-induced activation may depend on the combined or isolated action of the noradrenergic and serotonergic systems. Finally, the dual regulation of serotonin and α1 receptors by ACH-000029 might represent a potential pharmacotherapy that can be applied in the peri-trauma or early post-trauma period to mitigate the development of symptoms in PTSD patients.

The serotonergic and alpha-1 adrenergic receptor modulator ACH-000029 ameliorates anxiety-like behavior in a post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a severe chronic mental illness that develops in individuals exposed to life-threatening trauma and is characterized by hyperarousal, flashbacks and nightmares. The serotonergic (5-HT) and noradrenergic (NE) systems are deeply involved in the pathogenesis of PTSD. We have previously reported a novel anxiolytic compound, ACH-000029, that modulates 5-HT and α1-adrenergic receptors and induces acute anxiolytic-like effects in rodents. Here, we investigated the potential of ACH-000029 to prevent anxiety-like behavior in the single prolonged stress (SPS) PTSD model. Mice were subjected to the SPS procedure, followed by a 7-day treatment with ACH-000029 and, for comparison, with the α1-adrenergic antagonist prazosin. Animals were behaviorally assessed using social interaction, elevated plus maze and open field tests. Interestingly, treatment with ACH-000029 but not with prazosin ameliorated the SPS-induced sociability impairment and anxiety-like behavior. The brain-wide c-fos mapping, used as a surrogate for brain activity, indicated the brain structures that were altered by SPS and putatively involved in the anxiolytic-like effect of ACH-000029. The SPS protocol produced long-lasting impairment of regions involved in stress-anxiety response, such as the amygdala, prefrontal cortex, globus pallidus and superior colliculus. ACH-000029 treatment reversed the SPS-induced c-fos changes in the globus pallidus, lateral septum and entorhinal cortex and exclusively modulated c-fos levels in subregions from the retrosplenial cortex, cerebellum, superior colliculus and ventromedial hypothalamus. These results support the hypothesis that the dual regulation of 5-HT and α1-adrenergic receptors is required to alleviate PTSD symptoms and suggest a possible role of ACH-000029 as a PTSD treatment.

Preclinical characterization of ACH-000029, a novel anxiolytic compound acting on serotonergic and alpha-adrenergic receptors.

Anxiety disorders are serious and common mental diseases, yet there is still a need for the development of more effective anxiolytics with better safety profiles than benzodiazepines and serotonin reuptake inhibitors. The serotonergic and noradrenergic systems have reciprocal interactions and are intricately related to the pathogenesis of anxiety. In this study, the anxiolytic-like effect of the novel compound ACH-000029, 3-(2-(4-(2-methoxyphenyl) piperazine-1-yl) ethyl) quinazoline-4(3H)-one, is reported. This compound acts at selected serotonergic (5-HT1A and 5-HT1D partial agonism and 5-HT2A antagonism) and α-adrenergic (α-1A, 1B and 1D antagonism) receptors, with good selectivity over other G-protein-coupled receptors. ACH-000029 exhibited high blood-brain barrier permeation and acute anxiolytic effects in the marble burying (MB) and light-dark box (LDB) models of anxiety over the dose ranges of 8-32 mg/kg i.p. and 16-30 mg/kg p.o. The anxiolytic activity was comparable to that observed for serotonin reuptake inhibitors (paroxetine and fluoxetine) and benzodiazepines (alprazolam, diazepam and clobazam). The analysis of the whole-brain c-fos expression following oral dosing showed that ACH-000029 regulated regions highly associated with the processing of environmental stimuli and anxiety behavior, such as the amygdala, paraventricular nucleus of the thalamus, retrosplenial dorsal, pallidum, bed nuclei of the stria terminalis, and locus ceruleus. No safety concerns were identified for ACH-000029 in the functional observational battery up to 50 mg/kg i.p. and in the nonprecipitated withdrawal test up to 30 mg/kg p.o. twice daily for 20 days. This work supports the further development of ACH-000029 as a drug candidate for the treatment of anxiety disorders. The analysis of the in vitro pharmacology and brain regions regulated by this compound may also lead to the exploration of other indications within the psychiatry field.