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The PD-1 immune checkpoint pathway is a highly validated target for cancer immunotherapy. Despite the potential ad-vantages of small molecule inhibitors over antibodies, the discovery of small molecule checkpoint inhibitors has lagged behind. To discover small molecule inhibitors of the PD-1 pathway, we have utilized a fragment-based approach. Small molecules were identified that bind to PD-L1 and crystal structures of these compounds bound to PD-L1 were obtained.

This letter details the continued chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core by incorporating a 3-amino azetidine amide moiety. The analogs described within this work represent the most potent M4 PAMs reported for this series to date. The SAR to address potency, clearance, subtype selectivity, CNS exposure, and P-gp efflux are described. This work culminated in the discovery of VU6000918, which demonstrated robust efficacy in a rat amphetamine-induced hyperlocomotion reversal model at a minimum efficacious dose of 0.3 mg/kg. 2009 Elsevier Ltd. All rights reserved.

This letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. This article is protected by copyright. All rights reserved.

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins responsible for the regulation of programmed cell death (PCD). Amplification of Mcl-1 is a common genetic aberration in human cancer whose overexpression contributes to the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-apoptotic BH3 containing proteins that achieve high affinity for the target by utilizing four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1 inhibitors using fragment based methods and structure-based design. These novel inhibitors exhibit low nanomolar binding affinities to Mcl-1 and greater than 500-fold selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to Mcl-1 provided detailed information on how these small-molecules bind to the target, and were used extensively to guide compound optimization.

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, has emerged as an attractive target for cancer therapy. Mcl-1 upregulation is often found in many human cancers and is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here, we describe a series of potent and selective tricyclic indole diazepinone Mcl-1 inhibitors that were discovered and further optimized using structure-based design. These compounds exhibit picomolar binding affinity and mechanism-based cellular efficacy, including growth inhibition and caspase induction in Mcl-1-sensitive cells. Thus, they represent useful compounds to study the implication of Mcl-1 inhibition in cancer and serve as potentially useful starting points toward the discovery of anti-Mcl-1 therapeutics.

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.

This letter describes the further exploration of two series of M(1) allosteric agonists, TBPB and VU0357017, previously reported from our lab. Within the TPBP scaffold, either electronic or steric perturbations to the central piperidine ring led to a loss of selective M(1) allosteric agonism and afforded pan-mAChR antagonism, which was demonstrated to be mediated via the orthosteric site. Additional SAR around a related M(1) allosteric agonist family (VU0357017) identified similar, subtle 'molecular switches' that modulated modes of pharmacology from allosteric agonism to pan-mAChR orthosteric antagonism. Therefore, all of these ligands are best classified as bi-topic ligands that possess high affinity binding at an allosteric site to engender selective M(1) activation, but all bind, at higher concentrations, to the orthosteric ACh site, leading to non-selective orthosteric site binding and mAChR antagonism.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp = 0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp > 10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

A new type of polyoxometalate (POM), one with a hydrogen dinitrate group associated with a d-electron-substituted polyoxometalate, FeIII[H(ONO2)2]PW11O395− (1), has been developed that catalyzes highly effective aerobic sulfoxidation (RR′S + 0.5 O2 → RR′SO) under ambient conditions (1 atm of air at room temperature). Comparison of the rates for aerobic sulfoxidation of the mustard simulant 2-chloroethyl ethyl sulfide (CEES) catalyzed by 1 and several other species reported to be catalysts for this class of reactions, including NO+, NO2, (NH4)2CeIV(NO3)6 and AuIIICl2NO3(CH3CN) indicate that 1 is clearly the most effective catalyst. Conversions and selectivities for the desired sulfoxide decontamination product (CEESO in these studies) are both effectively quantitative. The low or nonexistent catalytic activity of FeII(NO)PW11O396−, Fe(NO3)3 and HNO3 argues strongly that nitrosyl and nitrate derivatives of the Fe polyoxometalate and nitric acid are not species important in catalytic turnover. The techniques of single crystal X-ray diffraction, solution NMR, FTIR, TGA, DSC, cyclic voltammetry, elemental and wet chemical analyses were applied to the characterization of 1 and these are collectively consistent with the α-Keggin structure with a strongly associated hydrogen dinitrate group and a more weakly associated nitrate of crystallization.

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Allosteric modulation of AMPA, NR2B, mGlu2, mGlu5 and M1, targeting glutamatergic dysfunction, represents a significant area of research for the treatment of schizophrenia. Of these targets, clinical promise has been demonstrated using muscarinic activators for the treatment of Alzheimer's disease (AD) and schizophrenia. These diseases have inspired researchers to determine the effects of modulating cholinergic transmission in the forebrain, which is primarily regulated by one of five subtypes of muscarinic acetylcholine receptor (mAChR), a subfamily of G-protein-coupled receptors (GPCRs). Of these five subtypes, M1 is highly expressed in brain regions responsible for learning, cognition and memory. Xanomeline, an orthosteric muscarinic agonist with modest selectivity, was one of the first compounds that displayed improvements in behavioral disturbances in AD patients and efficacy in schizophrenics. Since these initial clinical results, many scientists, including those in our laboratories, have strived to elucidate the role of M1 with compounds that display improved selectivity for this receptor by targeting allosteric modes of receptor activation. A survey of selected compounds in this area will be presented.

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins that regulate apoptosis. Amplification of Mcl-1 is found in various cancers, which causes the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through high affinity interactions with pro-apoptotic BH3 containing proteins, Bak and Bax. Thus targeting Mcl-1 with small molecule inhibitors is a promising strategy but a very challenging task. Using fragment-based methods and structure-based design, we discovered a novel class of potent Mcl-1 inhibitors that exhibit selective anti-proliferative activity. New leads containing a tricyclic indole lactam scaffold exhibited dissociation constants of 1000-fold selectivity for Mcl-1 over Bcl-xL and Bcl-2. They also promoted apoptosis only in Mcl-1 sensitive cancer cell lines by activating caspases in a dose-dependent manner. These results provide a strong proof of concept for a selective inhibition of Mcl-1 function as an effective anti-cancer therapy. Finally, our leads also possess desirable pharmaceutical properties including in vivo oral bioavailability and represent an ideal starting point for developing clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers. Citation Format: Taekyu Lee, Zhiguo Bian, Johannes Belmar, Subrata Shaw, James C. Tarr, Bin Zhao, Nick Pelz, DeMarco Camper, Craig M. Goodwin, Allison L. Arnold, John L. Sensintaffar, Carrie F. Browning, Olivia W. Rossanese, Edward T. Olejniczak, Stephen W. Fesik. Discovery of orally bioavailable novel Mcl-1 inhibitors that exhibit selective anti-proliferative activity in Mcl-1 sensitive cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3551.

Mcl-1 (Myeloid cell leukemia 1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and is one of the major acquired resistance mechanisms for many anticancer therapies. Targeting Mcl-1 with small molecules is very challenging because Mcl-1 exerts its effects through protein-protein interactions. Here we describe our continuing efforts in the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. We previously described the discovery of small-molecule Mcl-1 inhibitors containing a 2-indole-carboxylic core that primarily bind to the P2 pocket. In order to enhance potency, we extend our molecules to occupy the entire BH3 binding interface. A number of P4 site hits were identified using a fragment-based screening while the P2 pocket of Mcl-1 was saturated with our previously reported lead. P4 site ligands were then linked to our initial lead guided by the ternary Mcl-1 structure containing both P2 and P4 units using an acylsulfonamide as a synthetic handle that retained the critical charged-charged interaction with R263. X-ray co-crystal structures of new inhibitors occupying both P2 and P4 sites bound Mcl-1 provided detailed information on how these small-molecules bind to the target and guided subsequent potency optimization. From the efforts, we discovered a novel series of 2-indole-acylsulfonamide containing Mcl-1 inhibitors that exhibit low nanomolar binding affinities to the target and greater than 500-fold selectivity over Bcl-xL. These compounds represent useful starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers. Citation Format: Subrata Shaw, James Chris Tarr, Nicholas Pelz, Zhiguo Bian, Johannes Belmar, Bin Zhao, Craig Goodwin, Allison Arnold, John Sensintaffar, Olivia Rossanese, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Discovery of potent 2-Indole-acylsulfonamide Mcl-1 inhibitors using structure guided fragment-based methods. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4331.

This Letter describes the continued optimization of an MLPCN probe molecule (ML137) with a focused effort on the replacement/modification of the isatin moiety present in this highly selective M(1) PAM. A diverse range of structures were validated as viable replacements for the isatin, many of which engendered sizeable improvements in their ability to enhance the potency and efficacy of acetylcholine when compared to ML137. Muscarinic receptor subtype selectivity for the M(1) receptor was also maintained.

The M(1) muscarinic acetylcholine receptor is thought to play an important role in memory and cognition, making it a potential target for the treatment of Alzheimer's disease (AD) and schizophrenia. Moreover, M(1) interacts with BACE1 and regulates its proteosomal degradation, suggesting selective M(1) activation could afford both palliative cognitive benefit as well as disease modification in AD. A key challenge in targeting the muscarinic acetylcholine receptors is achieving mAChR subtype selectivity. Our lab has previously reported the M(1) selective positive allosteric modulator ML169. Herein we describe our efforts to further optimize this lead compound by preparing analogue libraries and probing novel scaffolds. We were able to identify several analogues that possessed submicromolar potency, with our best example displaying an EC(50) of 310 nM. The new compounds maintained complete selectivity for the M(1) receptor over the other subtypes (M(2)-M(5)), displayed improved DMPK profiles, and potentiated the carbachol (CCh)-induced excitation in striatal MSNs. Selected analogues were able to potentiate CCh-mediated nonamyloidogenic APPsα release, further strengthening the concept that M(1) PAMs may afford a disease-modifying role in the treatment of AD.

Herein we report the discovery and SAR of a novel series of M(1) agonists based on the MLPCN probe, ML071. From this, VU0364572 emerged as a potent, orally bioavailable and CNS penetrant M(1) agonist with high selectivity, clean ancillary pharmacology and enantiospecific activity.

In the presence of La(CN)3 a wide range of ketones reacts with acyl silanes to afford α-silyloxyketones.

We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic alpha-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant alpha-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction.

Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones.

Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones.

Metallophosphites derived from tartaric acid catalyze the enantioselective addition of acyl silanes to nitrones. The product α-N-silyloxyamino ketones are obtained in moderate to good yields (36−94%) with excellent enantioselectivity (er ≥ 95:5). The nitrone oxygen is essential in facilitating the silyl transfer that is required for catalyst turnover. This represents, to the best of our knowledge, the first example of C-acylation of nitrones and the most highly enantioselective catalytic addition of an acyl anion equivalent to an azomethine electrophile. The N−O bond of the product may be reductively cleaved under mild conditions to provide N-aryl α-amino ketones with negligible loss of enantiopurity.

Metallophosphites derived from tartaric acid catalyze the enantioselective addition of acyl silanes to nitrones. The product alpha N-silyloxyamino ketones are obtained in moderate to good yields (36-94%) with excellent enantioselectivity (er >= 95:5). The nitrone oxygen is essential in facilitating the silyl transfer that is required for catalyst turnover. This represents, to the best of our knowledge, the first example of C-acylation of nitrones and the most highly enantioselective catalytic addition of an acyl anion equivalent to an azomethine electrophile. The N-O bond of the product may be reductively cleaved under mild conditions to provide N- aryl alpha-amino ketones with negligible loss of enantiopurity.

The speciation of aqueous free chlorine above pH 5 is a well-understood equilibrium of H2O + HOCl ⇌ OCl− + H3O+ with a p Ka of 7.5. However, the identity of another very potent oxidant present at low pH (below 5) has been attributed by some researchers to Cl2 (aq) and by others to H2OCl+. We have conducted a series of experiments designed to ascertain which of these two species is correct. First, using Raman spectroscopy, we found that an equilibrium of H2O + H2OCl+ ⇌ HOCl + H3O+ is unlikely because the “apparent p Ka” increases monotonically from 1.25 to 2.11 as the analytical concentration is increased from 6.6 to 26.2 mM. Second, we found that significantly reducing the chloride ion concentration changed the Raman spectrum and also dramatically reduced the oxidation potency of the low-pH solution (as compared to solutions at the same pH that contained equimolar concentrations of Cl− and HOCl). The chloride ion concentration was not expected to impact an equilibrium of H2O + H2OCl+ ⇌ HOCl + H3O+, if it existed. These observations supported the following equilibrium as pH is decreased: Cl2 (aq) + 2H2O ⇌ HOCl + Cl− + H3O+. The concentration-based equilibrium constant was estimated to be approximately 2.56 × 10−4 M2 in solutions whose ionic strengths were ∼0.01 M. The oxidative potency of the species in low pH solutions was investigated by monitoring the oxidation of secondary alcohols to ketones. These and other results reported here argue strongly that Cl2 (aq) is the correct form of the potent low-pH oxidant in aqueous free-chlorine solutions.