Enhanced Oral Bioavailability of 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA) from its (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL)-Based Prodrugs.

2-(Phosphonomethyl)-pentanedioic acid (2-PMPA) is a potent (IC50 = 300 pM) and selective inhibitor of glutamate carboxypeptidase II (GCPII) with efficacy in multiple neurological and psychiatric disease preclinical models and more recently in models of inflammatory bowel disease (IBD) and cancer. 2-PMPA (1), however, has not been clinically developed due to its poor oral bioavailability (<1%) imparted by its four acidic functionalities (c Log P = -1.14). In an attempt to improve the oral bioavailability of 2-PMPA, we explored a prodrug approach using (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL), an FDA-approved promoiety, and systematically masked two (2), three (3), or all four (4) of its acidic groups. The prodrugs were evaluated for in vitro stability and in vivo pharmacokinetics in mice and dog. Prodrugs 2, 3, and 4 were found to be moderately stable at pH 7.4 in phosphate-buffered saline (57, 63, and 54% remaining at 1 h, respectively), but rapidly hydrolyzed in plasma and liver microsomes, across species. In vivo, in a single time-point screening study in mice, 10 mg/kg 2-PMPA equivalent doses of 2, 3, and 4 delivered significantly higher 2-PMPA plasma concentrations (3.65 ± 0.37, 3.56 ± 0.46, and 17.3 ± 5.03 nmol/mL, respectively) versus 2-PMPA (0.25 ± 0.02 nmol/mL). Given that prodrug 4 delivered the highest 2-PMPA levels, we next evaluated it in an extended time-course pharmacokinetic study in mice. 4 demonstrated an 80-fold enhancement in exposure versus oral 2-PMPA (AUC0-t: 52.1 ± 5.9 versus 0.65 ± 0.13 h*nmol/mL) with a calculated absolute oral bioavailability of 50%. In mouse brain, 4 showed similar exposures to that achieved with the IV route (1.2 ± 0.2 versus 1.6 ± 0.2 h*nmol/g). Further, in dogs, relative to orally administered 2-PMPA, 4 delivered a 44-fold enhanced 2-PMPA plasma exposure (AUC0-t for 4: 62.6 h*nmol/mL versus AUC0-t for 2-PMPA: 1.44 h*nmol/mL). These results suggest that ODOL promoieties can serve as a promising strategy for enhancing the oral bioavailability of multiply charged compounds, such as 2-PMPA, and enable its clinical translation.

Enhanced Brain Delivery of 2-(Phosphonomethyl)pentanedioic Acid Following Intranasal Administration of Its γ-Substituted Ester Prodrugs.

2-(Phosphonomethyl)pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II (GCPII) with efficacy in multiple neurological and psychiatric disease models, but its clinical utility is hampered by low brain penetration due to the inclusion of multiple acidic functionalities. We recently reported an improvement in the brain-to-plasma ratio of 2-PMPA after intranasal (IN) dosing in both rodents and primates. Herein, we describe the synthesis of several 2-PMPA prodrugs with further improved brain delivery of 2-PMPA after IN administration by masking of the γ-carboxylate. When compared to IN 2-PMPA in rats at 1 h post dose, γ-(4-acetoxybenzyl)-2-PMPA (compound 1) resulted in significantly higher 2-PMPA delivery to both plasma (4.1-fold) and brain (11-fold). Subsequent time-dependent evaluation of 1 also showed high brain as well as plasma 2-PMPA exposures with brain-to-plasma ratios of 2.2, 0.48, and 0.26 for olfactory bulb, cortex, and cerebellum, respectively, as well as an improved sciatic nerve to plasma ratio of 0.84. In contrast, IV administration of compound 1 resulted in similar plasma exposure of 2-PMPA versus the IN route (AUCIV: 76 ± 9 h·nmol/mL versus AUCIN: 99 ± 24 h·nmol/mL); but significantly lower nerve and brain tissue exposures with tissue-to-plasma ratios of 0.21, 0.03, 0.04, and 0.04 in nerve, olfactory bulb, cortex, and cerebellum, respectively. In primates, IN administration of 1 more than doubled 2-PMPA concentrations in the cerebrospinal fluid relative to previously reported levels following IN 2-PMPA. The results of these experiments provide a promising strategy for testing GCPII inhibition in neurological and psychiatric disorders.

Application of Pd-Catalyzed Cross-Coupling Reactions in the Synthesis of 5,5-Dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles that Inhibit ALK5 Kinase.

C-H activation of position 3 of a substituted pyrazole ring catalyzed by palladium(II) was straightforward and convenient for arylated or heteroarylated 5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles. Moreover, we introduced simple protection of the nitrogen in the pyridin-2-yl directing group, which otherwise does not allow a cross-coupling reaction, by transformation to the N-oxide. Selected final products were reasonably selective ALK5 kinase inhibitors.

1,3-Dipolar Cycloaddition in the Preparation of New Fused Heterocyclic Compounds via Thermal Initiation.

This paper describes the synthesis of precursors with a benzo[b]furan skeleton for the intramolecular 1,3-dipolar cycloaddition of azomethine ylides prepared from N-substituted 3-allyl-aminobenzo[b]furan-2-aldehydes and secondary amines derived from α-amino acid esters. Reactions were initiated by heating. The products consisted of four fused rings with three stereogenic centers. Their structure and stereochemistry were determined by NMR spectra and X-ray measurements.

A gut-restricted glutamate carboxypeptidase II inhibitor reduces monocytic inflammation and improves preclinical colitis.

There is an urgent need to develop therapeutics for inflammatory bowel disease (IBD) because up to 40% of patients with moderate-to-severe IBD are not adequately controlled with existing drugs. Glutamate carboxypeptidase II (GCPII) has emerged as a promising therapeutic target. This enzyme is minimally expressed in normal ileum and colon, but it is markedly up-regulated in biopsies from patients with IBD and preclinical colitis models. Here, we generated a class of GCPII inhibitors designed to be gut-restricted for oral administration, and we interrogated efficacy and mechanism using in vitro and in vivo models. The lead inhibitor, (S)-IBD3540, was potent (half maximal inhibitory concentration = 4 nanomolar), selective, gut-restricted (AUCcolon/plasma > 50 in mice with colitis), and efficacious in acute and chronic rodent colitis models. In dextran sulfate sodium-induced colitis, oral (S)-IBD3540 inhibited >75% of colon GCPII activity, dose-dependently improved gross and histologic disease, and markedly attenuated monocytic inflammation. In spontaneous colitis in interleukin-10 (IL-10) knockout mice, once-daily oral (S)-IBD3540 initiated after disease onset improved disease, normalized colon histology, and attenuated inflammation as evidenced by reduced fecal lipocalin 2 and colon pro-inflammatory cytokines/chemokines, including tumor necrosis factor-α and IL-17. Using primary human colon epithelial air-liquid interface monolayers to interrogate the mechanism, we further found that (S)-IBD3540 protected against submersion-induced oxidative stress injury by decreasing barrier permeability, normalizing tight junction protein expression, and reducing procaspase-3 activation. Together, this work demonstrated that local inhibition of dysregulated gastrointestinal GCPII using the gut-restricted, orally active, small-molecule (S)-IBD3540 is a promising approach for IBD treatment.

Neutral sphingomyelinase 2 inhibitors based on the pyrazolo[1,5-a]pyrimidin-3-amine scaffold.

Neutral sphingomyelinase 2 (nSMase2) has gained increasing attention as a therapeutic target to regulate ceramide production in various disease conditions. Phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)-pyrrolidin-3-yl)carbamate (PDDC) is a submicromolar nSMase2 inhibitor and has been widely used to study the pharmacological effects of nSMase2 inhibition. Through screening of compounds containing a bicyclic 5-6 fused ring, larotrectinib containing a pyrazolo[1,5-a]pyrimidine ring was identified as a low micromolar inhibitor of nSMase2. This prompted us to investigate the pyrazolo[1,5-a]pyrimidin-3-amine ring as a novel scaffold to replace the imidazo[1,2-b]pyridazine-8-amine ring of PDDC. A series of molecules containing a pyrazolo[1,5-a]pyrimidin-3-amine ring were synthesized and tested for their ability to inhibit human nSMase2. Several compounds exhibited nSMase2 inhibitory potency superior to that of PDDC. Among these, N,N-dimethyl-5-morpholinopyrazolo[1,5-a]pyrimidin-3-amine (11j) was found to be metabolically stable in liver microsomes and orally available with a favorable brain-to-plasma ratio, demonstrating the potential of pyrazolo[1,5-a]pyrimidine ring as an effective scaffold for nSMase2 inhibition.

The combination of immunotherapy and a glutamine metabolism inhibitor represents an effective therapeutic strategy for advanced and metastatic murine pancreatic adenocarcinoma.

Despite constant advances in cancer research, the treatment of pancreatic adenocarcinoma remains extremely challenging. The intratumoral immunotherapy approach that was developed by our research group and was based on a combination of mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA) showed promising therapeutic effects in various murine tumor models, including a pancreatic adenocarcinoma model (Panc02). However, the efficacy of MBTA therapy in the Panc02 model was negatively correlated with tumor size at the time of therapy initiation. Here, we aimed to further improve the outcome of MBTA therapy in the Panc02 model using the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). The combination of intratumoral MBTA therapy and intraperitoneal administration of DON resulted in the complete elimination of advanced Panc02 subcutaneous tumors (140.8 ± 46.8 mm3) in 50% of treated animals and was followed by development of long-term immune memory. In the bilateral Panc02 subcutaneous tumor model, we observed a significant reduction in tumor growth in both tumors as well as prolonged survival of treated animals. The appropriate timing and method of administration of DON were also addressed to maximize its therapeutic effects and minimize its side effects. In summary, our findings demonstrate that the intraperitoneal application of DON significantly improves the efficacy of intratumoral MBTA therapy in both advanced and bilateral Panc02 subcutaneous tumor murine models.

Discovery of tert-Butyl Ester Based 6-Diazo-5-oxo-l-norleucine Prodrugs for Enhanced Metabolic Stability and Tumor Delivery.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) exhibits remarkable anticancer efficacy; however, its therapeutic potential is hindered by its toxicity to gastrointestinal (GI) tissues. We recently reported the discovery of DRP-104, a tumor-targeted DON prodrug with excellent efficacy and tolerability, which is currently in clinical trials. However, DRP-104 exhibits limited aqueous solubility, and the instability of its isopropyl ester promoiety leads to the formation of an inactive M1-metabolite, reducing overall systemic prodrug exposure. Herein, we aimed to synthesize DON prodrugs with various ester and amide promoieties with improved solubility, GI stability, and DON tumor delivery. Twenty-one prodrugs were synthesized and characterized in stability and pharmacokinetics studies. Of these, P11, tert-butyl-(S)-6-diazo-2-((S)-2-(2-(dimethylamino)acetamido)-3-phenylpropanamido)-5-oxo-hexanoate, showed excellent metabolic stability in plasma and intestinal homogenate, high aqueous solubility, and high tumor DON exposures and preserved the ideal tumor-targeting profile of DRP-104. In conclusion, we report a new generation of glutamine antagonist prodrugs with improved physicochemical and pharmacokinetic attributes.

Microglial-Targeted nSMase2 Inhibitor Fails to Reduce Tau Propagation in PS19 Mice.

The progression of Alzheimer's disease (AD) correlates with the propagation of hyperphosphorylated tau (pTau) from the entorhinal cortex to the hippocampus and neocortex. Neutral sphingomyelinase2 (nSMase2) is critical in the biosynthesis of extracellular vesicles (EVs), which play a role in pTau propagation. We recently conjugated DPTIP, a potent nSMase2 inhibitor, to hydroxyl-PAMAM-dendrimer nanoparticles that can improve brain delivery. We showed that dendrimer-conjugated DPTIP (D-DPTIP) robustly inhibited the spread of pTau in an AAV-pTau propagation model. To further evaluate its efficacy, we tested D-DPTIP in the PS19 transgenic mouse model. Unexpectantly, D-DPTIP showed no beneficial effect. To understand this discrepancy, we assessed D-DPTIP's brain localization. Using immunofluorescence and fluorescence-activated cell-sorting, D-DPTIP was found to be primarily internalized by microglia, where it selectively inhibited microglial nSMase2 activity with no effect on other cell types. Furthermore, D-DPTIP inhibited microglia-derived EV release into plasma without affecting other brain-derived EVs. We hypothesize that microglial targeting allowed D-DPTIP to inhibit tau propagation in the AAV-hTau model, where microglial EVs play a central role in propagation. However, in PS19 mice, where tau propagation is independent of microglial EVs, it had a limited effect. Our findings confirm microglial targeting with hydroxyl-PAMAM dendrimers and highlight the importance of understanding cell-specific mechanisms when designing targeted AD therapies.

Discovery of Orally Bioavailable and Brain-Penetrable Prodrugs of the Potent nSMase2 Inhibitor DPTIP.

Extracellular vesicles (EVs) can carry pathological cargo and play an active role in disease progression. Neutral sphingomyelinase-2 (nSMase2) is a critical regulator of EV biogenesis, and its inhibition has shown protective effects in multiple disease states. 2,6-Dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazol-2-yl)phenol (DPTIP) is one of the most potent (IC50 = 30 nM) inhibitors of nSMase2 discovered to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), limiting its clinical development. To overcome DPTIP's PK limitations, we synthesized a series of prodrugs by masking its phenolic hydroxyl group. When administered orally, the best prodrug (P18) with a 2',6'-diethyl-1,4'-bipiperidinyl promoiety exhibited >fourfold higher plasma (AUC0-t = 1047 pmol·h/mL) and brain exposures (AUC0-t = 247 pmol·h/g) versus DPTIP and a significant enhancement of DPTIP half-life (2 h vs ∼0.5 h). In a mouse model of acute brain injury, DPTIP released from P18 significantly inhibited IL-1ß-induced EV release into plasma and attenuated nSMase2 activity. These studies report the discovery of a DPTIP prodrug with potential for clinical translation.

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8+ T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104's effect was CD8+ T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement.

BACKGROUND: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. OBJECTIVE: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. METHODS: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. RESULTS: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. CONCLUSION: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

Identification of Novel Carbonic Anhydrase IX Inhibitors Using High-Throughput Screening of Pooled Compound Libraries by DNA-Linked Inhibitor Antibody Assay (DIANA).

The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA's performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague ("IOCB library"). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA's potential to screen more than 100,000 compounds per day.All DIANA screens showed a high signal-to-noise ratio with a Z' factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.

The Novel Glutamine Antagonist Prodrug JHU395 Has Antitumor Activity in Malignant Peripheral Nerve Sheath Tumor.

The carbon and nitrogen components of glutamine are used for multiple biosynthetic processes by tumors. Glutamine metabolism and the therapeutic potential of glutamine antagonists (GA), however, are incompletely understood in malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma observed in patients with neurofibromatosis type I. We investigated glutamine dependence of MPNST using JHU395, a novel orally bioavailable GA prodrug designed to circulate inert in plasma, but permeate and release active GA within target tissues. Human MPNST cells, compared with Schwann cells derived from healthy peripheral nerve, were selectively susceptible to both glutamine deprivation and GA dose-dependent growth inhibition. In vivo, orally administered JHU395 delivered active GA to tumors with over 2-fold higher tumor-to-plasma exposure, and significantly inhibited tumor growth in a murine flank MPNST model without observed toxicity. Global metabolomics studies and stable isotope-labeled flux analyses in tumors identified multiple glutamine-dependent metabolites affected, including prominent effects on purine synthesis. These data demonstrate that glutamine antagonism is a potential antitumor strategy for MPNST.

Targeting glutamine metabolism enhances tumor-specific immunity by modulating suppressive myeloid cells.

Myeloid cells comprise a major component of the tumor microenvironment (TME) that promotes tumor growth and immune evasion. By employing a small-molecule inhibitor of glutamine metabolism, not only were we able to inhibit tumor growth, but we markedly inhibited the generation and recruitment of myeloid-derived suppressor cells (MDSCs). Targeting tumor glutamine metabolism led to a decrease in CSF3 and hence recruitment of MDSCs as well as immunogenic cell death, leading to an increase in inflammatory tumor-associated macrophages (TAMs). Alternatively, inhibiting glutamine metabolism of the MDSCs themselves led to activation-induced cell death and conversion of MDSCs to inflammatory macrophages. Surprisingly, blocking glutamine metabolism also inhibited IDO expression of both the tumor and myeloid-derived cells, leading to a marked decrease in kynurenine levels. This in turn inhibited the development of metastasis and further enhanced antitumor immunity. Indeed, targeting glutamine metabolism rendered checkpoint blockade-resistant tumors susceptible to immunotherapy. Overall, our studies define an intimate interplay between the unique metabolism of tumors and the metabolism of suppressive immune cells.

Tumor-Targeted Delivery of 6-Diazo-5-oxo-l-norleucine (DON) Using Substituted Acetylated Lysine Prodrugs.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON's toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC0- t = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC0- t = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.

JHU-083 selectively blocks glutaminase activity in brain CD11b+ cells and prevents depression-associated behaviors induced by chronic social defeat stress.

There are a number of clinically effective treatments for stress-associated psychiatric diseases, including major depressive disorder (MDD). Nonetheless, many patients exhibit resistance to first-line interventions calling for novel interventions based on pathological mechanisms. Accumulating evidence implicates altered glutamate signaling in MDD pathophysiology, suggesting that modulation of glutamate signaling cascades may offer novel therapeutic potential. Here we report that JHU-083, our recently developed prodrug of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) ameliorates social avoidance and anhedonia-like behaviors in mice subjected to chronic social defeat stress (CSDS). JHU-083 normalized CSDS-induced increases in glutaminase activity specifically in microglia-enriched CD11b+ cells isolated from the prefrontal cortex and hippocampus. JHU-083 treatment also reverses the CSDS-induced inflammatory activation of CD11b+ cells. These results support the importance of altered glutamate signaling in the behavioral abnormalities observed in the CSDS model, and identify glutaminase in microglia-enriched CD11b+ cells as a pharmacotherapeutic target implicated in the pathophysiology of stress-associated psychiatric conditions such as MDD.

Correction: JHU-083 selectively blocks glutaminase activity in brain CD11b+ cells and prevents depression-associated behaviors induced by chronic social defeat stress.

During this process, we found that we need add the following sentence in the manuscript within the Acknowledgement section.

N-Substituted Prodrugs of Mebendazole Provide Improved Aqueous Solubility and Oral Bioavailability in Mice and Dogs.

Mebendazole (MBZ) was developed as a broad-spectrum anthelmintic but has recently shown efficacy as an anticancer agent. The use of MBZ for cancer, however, is challenging due to its poor solubility leading to poor bioavailability. Herein, we developed a prodrug approach with various N-linked promoieties including acyloxymethyl, aminoacyloxymethyl, and substituted phosphonooxymethyl in attempt to improve these characteristics. Compound 12, containing an (((((isopropoxycarbonyl)oxy)methoxy)phosphoryl)oxy)methyl promoiety, showed a >10 000-fold improvement in aqueous solubility. When evaluated in mice, 12 displayed a 2.2-fold higher plasma AUC0- t and a 1.7-fold improvement in brain AUC0- t with a calculated oral bioavailability of 52%, as compared to 24% for MBZ-polymorph C (MBZ-C), the most bioavailable polymorph. In dogs, 12 showed a 3.8-fold higher plasma AUC0- t with oral bioavailability of 41% compared to 11% for MBZ-C. In summary, we have identified a prodrug of MBZ with better physicochemical properties and enhanced bioavailability in both mice and dog.

ALK5 kinase inhibitory activity and synthesis of 2,3,4-substituted 5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles.

A series of 2,3,4-substituted 5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles (DPPs) was synthesized and evaluated for their ALK5 inhibition activity. The most potent compounds displayed submicromolar IC50 values for ALK5. Preliminary profiling of one of the most active compounds in a panel of 50 protein kinases revealed its selectivity for ALK5. In cells, the compounds caused dose-dependent dephosphorylation of SMAD2, a well-established substrate of ALK5. In addition, the compounds blocked translocation of SMAD2/3 to nuclei of cells stimulated with TGFß and the protein remained predominantly in cytoplasm, further confirming their molecular target. Therefore, novel DPP derivatives proved to be active as ALK5 inhibitors.