The synthesis and SAR of 4-methoxy-3-(piperidin-4-yl) benzamides identified after a high-throughput screen of the MLPCN library is reported. SAR was explored around the 3-piperidine subtituent as well as the amide functionality of the reported compounds. Starting from the initial lead compounds, 1-7, iterative medicinal chemistry efforts led to the identification of ML352 (10m). ML352 represents a potent and selective inhibitor of CHT based on a drug-like scaffold.
MLPCN; ML352; Choline transporter; CHT; Structure-activity relationship
Hyperarousal and sleep disturbances are common, debilitating symptoms of post-traumatic stress disorder (PTSD). PTSD patients also exhibit abnormalities in quantitative electroencephalography (qEEG) power spectra during wake as well as rapid eye movement (REM) and non-REM (NREM) sleep. Selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacological treatment for PTSD, provide modest remediation of the hyperarousal symptoms in PTSD patients, but have little to no effect on the sleep–wake architecture deficits. Development of novel therapeutics for these sleep–wake architecture deficits is limited by a lack of relevant animal models. Thus, the present study investigated whether single prolonged stress (SPS), a rodent model of traumatic stress, induces PTSD-like sleep–wake and qEEG spectral power abnormalities that correlate with changes in central serotonin (5-HT) and neuropeptide Y (NPY) signaling in rats. Rats were implanted with telemetric recording devices to continuously measure EEG before and after SPS treatment. A second cohort of rats was used to measure SPS-induced changes in plasma corticosterone, 5-HT utilization, and NPY expression in brain regions that comprise the neural fear circuitry. SPS caused sustained dysregulation of NREM and REM sleep, accompanied by state-dependent alterations in qEEG power spectra indicative of cortical hyperarousal. These changes corresponded with acute induction of the corticosterone receptor co-chaperone FK506-binding protein 51 and delayed reductions in 5-HT utilization and NPY expression in the amygdala. SPS represents a preclinical model of PTSD-related sleep–wake and qEEG disturbances with underlying alterations in neurotransmitter systems known to modulate both sleep–wake architecture and the neural fear circuitry.
Single prolonged stress (SPS); post-traumatic stress disorder (PTSD); electroencephalography (EEG); FKBP5; serotonin (5-HT); neuropeptide Y (NPY)
This Letter describes the natural product guided synthesis of unnatural analogs of the marine bromopyrrole alkaloid dispyrin, and the resulting SAR of H3 antagonism. Multiple rounds of iterative parallel synthesis improved human H3 IC50 ~33-fold, and afforded a new class of H3 antagonists based on the novel bromotyramine core of dispyrin.
H3 antagonist; Dispyrin; Marine natural product; Alkaloid
We report the discovery and SAR of two novel series of imidazopyrimidinones and dihydroimidazopyrimidinones as metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs). Exploration of several structural features in the western and eastern part of the imidazopyrimidinone core and combinations thereof, revealed compound 4a as a mGlu5 PAM with good in vitro potency and efficacy, acceptable drug metabolism and pharmacokinetic (DMPK) properties and in vivo efficacy in an amphetamine-based model of psychosis. However, the presence of CNS-mediated adverse effects in preclinical species precluded any further in vivo evaluation.
Positive allosteric modulator (PAM); Metabotropic glutamate receptor 5 (mGlu5); Imidazopyrimidinone; Dihydroimidazopyrimidinone; Schizophrenia
A short, high-yielding protocol involving the enantioselective α-chlorination of aldehydes has been developed for the enantioselective synthesis of C2-functionalized aziridines and N-alkyl terminal azetidines from a common intermediate. This methodology allows for the rapid preparation of functionalized aziridines in 50–73% overall yields and 88–94% ee, and azetidines in 22–32% overall yields and 84–92% ee. Moreover, we developed a scalable and cost-effective route to the key organocatalyst (54% overall yield, >95% dr).
organocatalysis; azetdine; aziridine; enantioselective
This article describes the discovery and development of the first highly selective, small molecule antagonist of the muscarinic acetylcholine receptor subtype I (mAChR1 or M1). An M1 functional, cell-based calcium-mobilization assay identified three distinct chemical series with initial selectivity for M1 versus M4. An iterative parallel synthesis approach was employed to optimize all three series in parallel, which led to the development of novel microwave-assisted chemistry and provided important take home lessons for probe development projects. Ultimately, this effort produced VU0255035, a potent (IC50 = 130 nM) and selective (>75-fold vs. M2-M5 and > 10 μM vs. a panel of 75 GPCRs, ion channels and transporters) small molecule M1 antagonist. Further profiling demonstrated that VU0255035 was centrally penetrant (BrainAUC/PlasmaAUC of 0.48) and active in vivo, rendering it acceptable as both an in vitro and in vivo MLSCN/ MLPCN probe molecule for studying and dissecting M1 function.
muscarinic; acetylcholine; mAChR; M1; antagonist
Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5) have exciting potential as therapeutic agents for multiple brain disorders. Translational studies with mGlu5 modulators have relied on mGlu5 allosteric site positron emission tomography (PET) radioligands to assess receptor occupancy in the brain. However, recent structural and modeling studies suggest that closely related mGlu5 allosteric modulators can bind to overlapping but not identical sites, which could complicate interpretation of in vivo occupancy data, even when PET ligands and drug leads are developed from the same chemical scaffold. We now report that systemic administration of the novel mGlu5 positive allosteric modulator VU0092273 displaced the structurally related mGlu5 PET ligand, [18F]FPEB, with measures of in vivo occupancy that closely aligned with its in vivo efficacy. In contrast, a close analog of VU0092273 and [18F]FPEB, VU0360172, provided robust efficacy in rodent models in the absence of detectable occupancy. Furthermore, a structurally unrelated mGlu5 negative allosteric modulator, VU0409106, displayed measures of in vivo occupancy that correlated well with behavioral effects, despite the fact that VU0409106 is structurally unrelated to [18F]FPEB. Interestingly, all three compounds inhibit radioligand binding to the prototypical MPEP/FPEB allosteric site in vitro. However, VU0092273 and VU0409106 bind to this site in a fully competitive manner, whereas the interaction of VU0360172 is noncompetitive. Thus, while close structural similarity between PET ligands and drug leads does not circumvent issues associated with differential binding to a given target, detailed molecular pharmacology analysis accurately predicts utility of ligand pairs for in vivo occupancy studies.
This letter describes the continued optimization of the MLPCN probe ML375, a highly selective M5 negative allosteric modulator (NAM), through a combination of matrix libraries and iterative parallel synthesis. True to certain allosteric ligands, SAR was shallow, and the matrix library approach highlighted the challenges with M5 NAM SAR within in this chemotype. Once again, enantiospecific activity was noted, and potency at rat and human M5 were improved over ML375, along with slight enhancement in physiochemical properties, certain in vitro DMPK parameters and CNS distribution. Attempts to further enhance pharmacokinetics with deuterium incorporation afforded mixed results, but pretreatment with a pan-P450 inhibitor (1-aminobenzotriazole; ABT) provided increased plasma exposure.
M5; Muscarinic receptor; Negative allosteric modulator; Matrix library; Pharmacokinetics
Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure.
Muscarinic receptor 1; mAChR; M1; positive allosteric modulator (PAM)
Previous studies have shown that activation of mouse MrgC11, a G-protein coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large scale screen has been carried out to isolate small molecule allosteric agonists of MrgX1, the human homologue of MrgC11. The goal of this study is to improve the efficacy and potency of the positive allosteric modulators with therapeutic implications of anti-chronic pain. Here, we report an iterative parallel synthesis effort and a structure-activity relationship of a series of arylsulfonamides, which led to the discovery of the first positive allosteric modulator (PAM) of MrgX1, ML382.
MrgX1; ML382; chronic pain; positive allosteric modulator; MLPCN; molecular probes
This letter describes the on-going SAR efforts based on two scaffolds, a PLD1-biased piperidinyl benzimidazolone and a PLD2-biased piperidinyl triazaspirone, with the goal of enhancing PLD inhibitory potency and isoform selectivity. Here, we found that addition of an α-methyl moiety within the PLD2-biased piperidinyl triazaspirone scaffold abolished PLD2 preference, while the incorporation of substituents onto the piperdine moiety of the PLD1-biased piperidinyl benzimidazolone, or replacement with a bioisosteric [3.3.0] core, generally retained PLD1 preference, but at diminished significance. The SAR uncovered within these two allosteric PLD inhibitor series further highlights the inherent challenges of developing isoform selective PLD inhibitors.
PLD; Phospholipase D; isoform; inhibitors; phosphodiesterase
Further chemical optimization of the halopemide-derived family of dual PLD1/2 inhibitors afforded ML395 (VU0468809), a potent, >80-fold PLD2 selective allosteric inhibitor (cellular PLD1, IC50 >30,000 nM, cellular PLD2, IC50 = 360 nM). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).
phospholipase D; PLD2; inhibitor; antiviral; lipid
functional multiaddition high throughput screen and
subsequent iterative parallel synthesis effort identified the first
highly selective and CNS penetrant glucagon-like peptide-1R (GLP-1R)
positive allosteric modulator (PAM). PAM (S)-9b potentiated low-dose exenatide to augment insulin secretion
in primary mouse pancreatic islets, and (S)-9b alone was effective in potentiating endogenous GLP-1R to
reverse haloperidol-induced catalepsy.
Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer's disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M4 (M4), we developed a homogenous, multiparametric, 1,536 well assay to measure M4 receptor agonism, positive allosteric modulation (PAM) and antagonism in a single well. This assay yielded a Z’ of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M1 and M5 subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M4 selective primary agonists, 1,450 PAMs and 2,389 antagonist hits. Concentration response analysis identified 25 selective agonists, 4 PAMs and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.
Of the eight metabotropic glutamate (mGlu) receptor subtypes, only mGlu7 is expressed presynaptically at the Schaffer collateral (SC)-CA1 synapse in the hippocampus in adult animals. Coupled with the inhibitory effects of Group III mGlu receptor agonists on transmission at this synapse, mGlu7 is thought to be the predominant autoreceptor responsible for regulating glutamate release at SC terminals. However, the lack of mGlu7-selective pharmacological tools has hampered direct testing of this hypothesis. We used a novel, selective mGlu7-negative allosteric modulator (NAM), ADX71743, and a newly described Group III mGlu receptor agonist, LSP4-2022, to elucidate the role of mGlu7 in modulating transmission in hippocampal area CA1 in adult C57BL/6J male mice. Interestingly, although mGlu7 agonists inhibit SC-CA1 EPSPs, we found no evidence for activation of mGlu7 by stimulation of SC-CA1 afferents. However, LSP4-2022 also reduced evoked monosynaptic IPSCs in CA1 pyramidal cells and, in contrast to its effect on SC-CA1 EPSPs, ADX71743 reversed the ability of high-frequency stimulation of SC afferents to reduce IPSC amplitudes. Furthermore, blockade of mGlu7 prevented induction of LTP at the SC-CA1 synapse and activation of mGlu7 potentiated submaximal LTP. Together, these data suggest that mGlu7 serves as a heteroreceptor at inhibitory synapses in area CA1 and that the predominant effect of activation of mGlu7 by stimulation of glutamatergic afferents is disinhibition, rather than reduced excitatory transmission. Furthermore, this mGlu7-mediated disinhibition is required for induction of LTP at the SC-CA1 synapse, suggesting that mGlu7 could serve as a novel therapeutic target for treatment of cognitive disorders.
glutamate; hippocampus; LTP; mGlu; mGlu7; mGlur7
D (PLD) hydrolyses cellular lipids to produce the
important lipid second messenger phosphatidic acid. A PLD enzyme expressed
by Pseudomonas aeruginosa (PldA) has
been shown to be important in bacterial infection, and NAPE-PLD has
emerged as being key in the synthesis of endocannabinoids. In order
to better understand the biology and therapeutic potential of these
less explored PLD enzymes, small molecule tools are required. Selective
estrogen receptor modulators (SERMs) have been previously shown to
inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a
library of SERM analogues, additional parallel synthesis, and evaluation
in multiple PLD assays, we discovered a novel desketoraloxifene-based
scaffold that inhibited not only the two mammalian PLDs but also structurally
divergent PldA and NAPE-PLD. This finding represents an important
first step toward the development of small molecules possessing universal
inhibition of divergent PLD enzymes to advance the field.
is a rational, scientific approach to compare
the value (in terms of both cost and patient outcome) of one medication
or drug therapy regimen to another. The impact of this new approach
on both the practicing medicinal chemist and broader drug discovery
efforts is considered.
Pharmacoeconomics; cost benefit; outcomes
Metabotropic glutamate receptor 7
(mGlu7) is a member
of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both
excitatory and inhibitory synapses, and activation of the receptor
regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological
and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia.
Here we report two new pan-group III mGlu positive allosteric modulators,
VU0155094 and VU0422288, which show differential activity at the various
group III mGlus. Additionally, both compounds show probe dependence
when assessed in the presence of distinct orthosteric agonists. By
pairing studies of these nonselective compounds with a synapse in
the hippocampus that expresses only mGlu7, we have validated
activity of these compounds in a native tissue setting. These studies
provide proof-of-concept evidence that mGlu7 activity can
be modulated by positive allosteric modulation, paving the way for
future therapeutics development.
Allosteric modulator; metabotropic glutamate
receptor; electrophysiology; hippocampus
we report the structure–activity relationship of
a chiral morpholine-based scaffold, which led to the identification
of a potent and selective dopamine 4 (D4) receptor antagonist.
The 4-chlorobenzyl moiety was identified, and the compound was designated
an MLPCN probe molecule, ML398. ML398 is potent against the D4 receptor with IC50 = 130 nM and Ki = 36 nM and shows no activity against the other dopamine
receptors tested (>20 μM against D1, D2S, D2L, D3, and D5). Further in vivo studies showed that ML398 reversed cocaine-induced
hyperlocomotion at 10 mg/kg.
Dopamine 4 receptor antagonist; ML398; addiction; MLPCN
Transcriptional repression of E-cadherin is a hallmark of Epithelial-to-Mesenchymal Transition (EMT) and is associated with cancer cell invasion and metastasis. Understanding the mechanisms underlying E-cadherin repression during EMT may provide insights into the development of novel targeted therapeutics for cancer. Here, we report on the chemical probe, ML327, which de-represses E-cadherin transcription, partially reverses EMT, and inhibits cancer cell invasiveness and tumor cell migration in vitro and in vivo. Induction of E-cadherin mRNA expression by ML327 treatment does not require de novo protein synthesis. RNA sequencing analysis revealed that ML327 treatment significantly alters expression of over 2,500 genes within three hours in the presence of the translational inhibitor, cycloheximide. Network analysis reveals Hepatocyte Nuclear Factor 4-alpha (HNF4α) as the most significant upstream transcriptional regulator of multiple genes whose expressions were altered by ML327 treatment. Further, small interfering RNA-mediated depletion of HNF4α markedly attenuates the E-cadherin expression response to ML327. In summary, ML327 represents a valuable tool to understand mechanisms of EMT and may provide the basis for a novel targeted therapeutic strategy for carcinomas.
small molecule; E-cadherin; EMT
identification of sites on receptors topographically distinct
from the orthosteric sites, so-called allosteric sites, has heralded
novel approaches and modes of pharmacology for target modulation.
Over the past 20 years, our understanding of allosteric modulation
has grown significantly, and numerous advantages, as well as caveats
(e.g., flat structure–activity relationships, species differences,
“molecular switches”), have been identified. For multiple
receptors and proteins, numerous examples have been described where
unprecedented levels of selectivity are achieved along with improved
physiochemical properties. While not a panacea, these novel approaches
represent exciting opportunities for tool compound development to
probe the pharmacology and therapeutic potential of discrete molecular
targets, as well as new medicines. In this Perspective, in commemoration
of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship (LindsleyC. W.Adventures in allosteric drug discovery. Presented at the 246th National Meeting of the American Chemical
Society, Indianapolis, IN, September 10, 2013; The 2013
Portoghese Lectureship), several vignettes of drug discovery
campaigns targeting novel allosteric
mechanisms will be recounted, along with lessons learned and guidelines
that have emerged for successful lead optimization.
high throughput screen identified a novel chemotype
for the positive allosteric modulation (PAM) of the muscarinic acetylcholine
receptor (mAChR) subtype 5 (M5). Application of rapid analog,
iterative parallel synthesis efficiently optimized M5 potency
to arrive at the most potent M5 PAMs prepared to date and
provided tool compound 8n (ML380) demonstrating modest
CNS penetration (human M5 EC50 = 190 nM, rat
M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).
Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.
High-throughput screening; glucagon-like peptide-1 receptor; calcium; positive allosteric modulator; agonist
Development of SAR in an aryl ether series of mGlu5 NAMs leading to the identification of pyrazine analog VU0431316 is described in this Letter. VU0431316 is a potent and selective non-competitive antagonist of mGlu5 that binds at a known allosteric binding site. VU0431316 demonstrates an attractive DMPK profile, including moderate clearance and good bioavailability in rats. Intraperitoneal (IP) dosing of VU0431316 in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu5 antagonists and other anxiolytics, produced dose proportional effects.