Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity.

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

The Novel SSTR3 Agonist ITF2984 Exerts Antimitotic and Proapoptotic Effects in Human Non-Functioning Pituitary Neuroendocrine Tumor (NF-PitNET) Cells.

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984's antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (-40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6.

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.

Characterization of the Cardiac Structure and Function of Conscious D2.B10-Dmdmdx/J (D2-mdx) mice from 16-17 to 24-25 Weeks of Age.

Duchenne muscular dystrophy (DMD) is the most common form of muscle degenerative hereditary disease. Muscular replacement by fibrosis and calcification are the principal causes of progressive and severe musculoskeletal, respiratory, and cardiac dysfunction. To date, the D2.B10-Dmdmdx/J (D2-mdx) model is proposed as the closest to DMD, but the results are controversial. In this study, the cardiac structure and function was characterized in D2-mdx mice from 16-17 up to 24-25 weeks of age. Echocardiographic assessment in conscious mice, gross pathology, and histological and cardiac biomarker analyses were performed. At 16-17 weeks of age, D2-mdx mice presented mild left ventricular function impairment and increased pulmonary vascular resistance. Cardiac fibrosis was more extended in the right ventricle, principally on the epicardium. In 24-25-week-old D2-mdx mice, functional and structural alterations increased but with large individual variation. High-sensitivity cardiac Troponin T, but not N-terminal pro-atrial natriuretic peptide, plasma levels were increased. In conclusion, left ventricle remodeling was mild to moderate in both young and adult mice. We confirmed that right ventricle epicardial fibrosis is the most outstanding finding in D2-mdx mice. Further long-term studies are needed to evaluate whether this mouse model can also be considered a model of DMD cardiomyopathy.

HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling.

BACKGROUND: Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. METHODS: Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. RESULTS: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. CONCLUSIONS: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Improved In Vivo Anti-Tumor and Anti-Metastatic Effect of GnRH-III-Daunorubicin Analogs on Colorectal and Breast Carcinoma Bearing Mice.

Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety for drug delivery systems. The anti-tumor activity of the previously developed GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] conjugate and the novel synthesized GnRH-III-[2ΔHis,3d-Tic,4Lys(Bu),8Lys(Dau=Aoa)] conjugate, containing the anti-cancer drug daunorubicin, were evaluated. Here, we demonstrate that both GnRH-III-Dau conjugates possess an efficient growth inhibitory effect on more than 20 cancer cell lines, whereby the biological activity is strongly connected to the expression of gonadotropin-releasing hormone receptors (GnRH-R). The novel conjugate showed a higher in vitro anti-proliferative activity and a higher uptake capacity. Moreover, the treatment with GnRH-III-Dau conjugates cause a significant in vivo tumor growth and metastases inhibitory effect in three different orthotopic models, including 4T1 mice and MDA-MB-231 human breast carcinoma, as well as HT-29 human colorectal cancer bearing BALB/s and SCID mice, while toxic side-effects were substantially reduced in comparison to the treatment with the free drug. These findings illustrate that our novel lead compound is a highly promising candidate for targeted tumor therapy in both colon cancer and metastatic breast cancer.

HDAC inhibitors as pharmacological treatment for Duchenne muscular dystrophy: a discovery journey from bench to patients.

Earlier evidence that targeting the balance between histone acetyltransferases (HATs) and deacetylases (HDACs), through exposure to HDAC inhibitors (HDACis), could enhance skeletal myogenesis, prompted interest in using HDACis to promote muscle regeneration. Further identification of constitutive HDAC activation in dystrophin-deficient muscles, caused by dysregulated nitric oxide (NO) signaling, provided the rationale for HDACi-based therapeutic interventions for Duchenne muscular dystrophy (DMD). In this review, we describe the molecular, preclinical, and clinical evidence supporting the efficacy of HDACis in countering disease progression by targeting pathogenic networks of gene expression in multiple muscle-resident cell types of patients with DMD. Given that givinostat is paving the way for HDACi-based interventions in DMD, next-generation HDACis with optimized therapeutic profiles and efficacy could be also explored for synergistic combinations with other therapeutic strategies.

Uracil- and Pyridine-Containing HDAC Inhibitors Displayed Cytotoxicity in Colorectal and Glioblastoma Cancer Stem Cells.

Cancer stem cells (CSCs) are a niche of highly tumorigenic cells featuring self-renewal, activation of pluripotency genes, multidrug resistance, and ability to cause cancer relapse. Seven HDACi (1-7), showing either hydroxamate or 2'-aminoanilide function, were tested in colorectal cancer (CRC) and glioblastoma multiforme (GBM) CSCs to determine their effects on cell proliferation, H3 acetylation levels and in-cell HDAC activity. Two uracil-based hydroxamates, 5 and 6, which differ in substitution at C5 and C6 positions of the pyrimidine ring, exhibited the greatest cytotoxicity in GBM (5) and CRC (6) CSCs, followed by the pyridine-hydroxamate 2, with 2- to 6-fold higher potency than the positive control SAHA. Finally, increased H3 acetylation as well as HDAC inhibition directly in cells by selected 2'-aminoanilide 4 and hydroxamate 5 confirmed target engagement. Further investigation will be conducted into the broad-spectrum anticancer properties of the most potent derivatives and their effects in combination with approved, conventional anticancer drugs.

Translation of paclitaxel-induced peripheral neurotoxicity from mice to patients: the importance of model selection.

ABSTRACT: Paclitaxel-induced peripheral neurotoxicity (PIPN) is a potentially dose-limiting side effect in anticancer chemotherapy. Several animal models of PIPN exist, but their results are sometimes difficult to be translated into the clinical setting. We compared 2 widely used PIPN models characterized by marked differences in their methodologies. Female C57BL/6JOlaHsd mice were used, and they received only paclitaxel vehicle (n = 38) or paclitaxel via intravenous injection (n = 19, 70 mg/kg) once a week for 4 weeks (Study 1) or intraperitoneally (n = 19, 10 mg/kg) every 2 days for 7 times (Study 2). At the end of treatment and in the follow-up, mice underwent behavioral and neurophysiological assessments of PIPN. At the same time points, some mice were killed and dorsal root ganglia, skin, and sciatic and caudal nerve samples underwent pathological examination. Serum neurofilament light levels were also measured. The differences in the neurotoxicity parameters were analyzed using a nonparametric Mann-Whitney test, with significance level set at P < 0.05. Study 1 showed significant and consistent behavioral, neurophysiological, pathological, and serological changes induced by paclitaxel administration at the end of treatment, and most of these changes were still evident in the follow-up period. By contrast, study 2 evidenced only a transient small fiber neuropathy, associated with neuropathic pain. Our comparative study clearly distinguished a PIPN model recapitulating all the clinical features of the human condition and a model showing only small fiber neuropathy with neuropathic pain induced by paclitaxel.

Skin equivalents: a tool for the discovery and validation of pharmacodynamic biomarkers.

In the development of targeted oncology drugs, it is important to assess drug effectiveness in individual patients. We evaluated the possibility of reproducing in an ex-vivo system the biological effects observed in vitro and in vivo by the combined administration of two chemotherapeutic drugs, gemcitabine and a small inhibitor of Wee1. We found that modulation of both CDC2 phosphorylation and of a previously-identified gene signature was detectable in human skin equivalents obtained with primary keratinocytes from three individuals. Therefore, we suggest that human skin equivalents could represent a promising tool for the identification and validation of novel pharmacodynamic biomarkers.

The Importance of the "Time Factor" for the Evaluation of Inhibition Mechanisms: The Case of Selected HDAC6 Inhibitors.

Histone deacetylases (HDACs) participate with histone acetyltransferases in the modulation of the biological activity of a broad array of proteins, besides histones. Histone deacetylase 6 is unique among HDAC as it contains two catalytic domains, an N-terminal microtubule binding region and a C-terminal ubiquitin binding domain. Most of its known biological roles are related to its protein lysine deacetylase activity in the cytoplasm. The design of specific inhibitors is the focus of a large number of medicinal chemistry programs in the academy and industry because lowering HDAC6 activity has been demonstrated to be beneficial for the treatment of several diseases, including cancer, and neurological and immunological disorders. Here, we show how re-evaluation of the mechanism of action of selected HDAC6 inhibitors, by monitoring the time-dependence of the onset and relief of the inhibition, revealed instances of slow-binding/slow-release inhibition. The same approach, in conjunction with X-ray crystallography, in silico modeling and mass spectrometry, helped to propose a model of inhibition of HDAC6 by a novel difluoromethyloxadiazole-based compound that was found to be a slow-binding substrate analog of HDAC6, giving rise to a tightly bound, long-lived inhibitory derivative.

LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies.

Identification of a Novel SSTR3 Full Agonist for the Treatment of Nonfunctioning Pituitary Adenomas.

Somatostatin receptor (SSTR) agonists have been extensively used for treating neuroendocrine tumors. Synthetic therapeutic agonists showing selectivity for SSTR2 (Octreotide) or for SSTR2 and SSTR5 (Pasireotide) have been approved for the treatment of patients with acromegaly and Cushing's syndrome, as their pituitary tumors highly express SSTR2 or SSTR2/SSTR5, respectively. Nonfunctioning pituitary adenomas (NFPAs), which express high levels of SSTR3 and show only modest response to currently available SSTR agonists, are often invasive and cannot be completely resected, and therefore easily recur. The aim of the present study was the evaluation of ITF2984, a somatostatin analog and full SSTR3 agonist, as a new potential treatment for NFPAs. ITF2984 shows a 10-fold improved affinity for SSTR3 compared to Octreotide or Pasireotide. Molecular modeling and NMR studies indicated that the higher affinity for SSTR3 correlates with a higher stability of a distorted ß-I turn in the cyclic peptide backbone. ITF2984 induces receptor internalization and phosphorylation, and triggers G-protein signaling at pharmacologically relevant concentrations. Furthermore, ITF2984 displays antitumor activity that is dependent on SSTR3 expression levels in the MENX (homozygous mutant) NFPA rat model, which closely recapitulates human disease. Therefore, ITF2984 may represent a novel therapeutic option for patients affected by NFPA.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

HDAC Inhibition as Potential Therapeutic Strategy to Restore the Deregulated Immune Response in Severe COVID-19.

The COVID-19 pandemic has had a devastating impact worldwide and has been a great challenge for the scientific community. Vaccines against SARS-CoV-2 are now efficiently lessening COVID-19 mortality, although finding a cure for this infection is still a priority. An unbalanced immune response and the uncontrolled release of proinflammatory cytokines are features of COVID-19 pathophysiology and contribute to disease progression and worsening. Histone deacetylases (HDACs) have gained interest in immunology, as they regulate the innate and adaptative immune response at different levels. Inhibitors of these enzymes have already proven therapeutic potential in cancer and are currently being investigated for the treatment of autoimmune diseases. We thus tested the effects of different HDAC inhibitors, with a focus on a selective HDAC6 inhibitor, on immune and epithelial cells in in vitro models that mimic cells activation after viral infection. Our data indicate that HDAC inhibitors reduce cytokines release by airway epithelial cells, monocytes and macrophages. This anti-inflammatory effect occurs together with the reduction of monocytes activation and T cell exhaustion and with an increase of T cell differentiation towards a T central memory phenotype. Moreover, HDAC inhibitors hinder IFN-I expression and downstream effects in both airway epithelial cells and immune cells, thus potentially counteracting the negative effects promoted in critical COVID-19 patients by the late or persistent IFN-I pathway activation. All these data suggest that an epigenetic therapeutic approach based on HDAC inhibitors represents a promising pharmacological treatment for severe COVID-19 patients.

Givinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics.

Glioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time- and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from -25% to -75% of protein levels), and reduction in HDAC activity (-25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclopropyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydroimid azo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 1.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which controls patterning, growth and cell migration in most tissues, but evidence has accumulated showing that many human tumors aberrantly reactivate this pathway. Smoothened antagonists offer opportunities for the treatment of malignancies dependent on the Hh pathway, and the most advanced clinical candidates are demonstrating encourage initial results. A novel series of [6,5]-bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione smoothened antagonists has been identified, and the series has been extensively explored to ascertain the key detriments for activity, demonstrating that the trans-2-phenylcyclopropyl and hydantoin ring systems are critical for potency, while a variety of urea substituents can be tolerated. The combination of these optimal groups gives smoothened antagonists with activity in the low nanomolar range.

N-(2-alkylaminoethyl)-4-(1,2,4-oxadiazol-5-yl)piperazine-1-carboxamides as highly potent smoothened antagonists.

Smoothened (Smo) antagonists are emerging as new therapies for the treatment of neoplasias with aberrantly reactivated hedgehog (Hh) signaling pathway. A novel series of 4-[3-(quinolin-2-yl)-1,2,4-oxadiazol-5-yl]piperazinyl ureas as smoothened antagonists was recently described, herein the series has been further optimized through the incorporation of a basic amine into the urea. This development resulted in identification of some exceptionally potent smoothened antagonists with low serum shifts, however, reductive ring opening on the 1,2,4-oxadiazole in rats limits the applicability of these compounds in in vivo studies.

Identification of a series of 4-[3-(quinolin-2-yl)-1,2,4-oxadiazol-5-yl]piperazinyl ureas as potent smoothened antagonist hedgehog pathway inhibitors.

The Hedgehog (Hh-) signalling pathway is a key developmental pathway and there is a growing body of evidence showing that this pathway is aberrantly reactivated in a number of human tumors. Novel agents capable of inhibiting this pathway are sought, and an entirely novel series of smoothened (Smo) antagonists capable of inhibiting the pathway have been identified through uHTS screening. Extensive exploration of the scaffold identified the key functionalities necessary for potency, enabling potent nanomolar Smo antagonists like 91 and 94 to be developed. Optimization resulted in the most advanced compounds displaying low serum shift, clean off-targets profile, and moderate clearance in both rats and dogs. These compounds are valuable tools with which to probe the biology of the Hh-pathway.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.