Design, synthesis, and evaluation of novel, selective γ-butyrolactones sigma-2 ligands

Nearly 40 years after the first disclosure of sigma receptors, the sigma-2 (σ2) receptor was recently identified as the Transmembrane Protein 97 (TMEM97, also known as MAC30 (Meningioma-associated protein). This macromolecule has been associated with a number of disease states such as schizophrenia, Alzheimer’s disease, neuropathic pain, traumatic brain injury, and cancer. We have recently identified a series of novel, functionalized γ-butyrolactones that are potent σ2 receptor ligands that are drug-like and identified a potential candidate (9z) for future in vivo study.


Introduction
In 1976, W. R. Martin et. al. described their efforts to classify opioids based on their impact on chronic spinal dogs. They observed that exposure to morphine (1), ketocyclazocine, (2), and (rac)-SKF-100047 (3) (Fig. 1) produced different responses in this animal model. They hypothesized that these compounds were engaging three different receptors that they labeled as the µ-opioid receptor (morphine type, MOR), the κ-opioid receptor (ketocyclazocine type, KOR), and the σopioid receptor (SKF-100047 like) [1]. These studies were conducted with racemic material, and follow-up studies with the individual enantiomers of SKF-100047 revealed that (−)-SKF-100047 elicited opioid mediated physiological responses through MOR and KOR, while (+)-SKF-100047's biological activity was produced by interaction with a previously unknown, non-opioid receptor that was designated the sigma receptor (σR) [2,3]. Nearly 17 years later, W. D. Bowen et. al. successfully demonstrated that there were two sub-types of this receptor, sigma-1 (σ 1 ) and sigma-2 (σ 2 ) [4], and in 1996 mammalian σ 1 receptor was cloned and expressed in yeast cells [5]. A crystal structure of the human σ 1 receptor was reported in 2016 [6], but to date, there is no known natural ligand for this receptor.
The true nature of the σ 2 receptor, on the other hand, proved more elusive. In 2017, over 40 years after the original description of the σR, Transmembrane Protein 97 (TMEM97, also known as MAC30 (Meningioma-associated protein) was identified as the σ 2 receptor [7]. The pharmacological role of σ 2 remains unclear, as no natural ligand has been identified. It is known, however, that this receptor protein is present in the endoplasmic reticulum (ER) and lysosomes. There is also some evidence supporting binding of σ 2 to cholesterol in these regions of the cell [8] and that it may play a role the regulation of the Niemann-Pick protein NPC1 [9]. Despite the lack of a clear understanding of the pharmacological role of σ 2 , this macromolecule has been the focus of efforts to develop therapies in a number of disease states such as schizophrenia [10], Alzheimer's disease [11][12][13], neuropathic pain [14], traumatic brain injury [15], and cancer [16,17].
As part of our on-going effort to develop novel, therapeutically useful molecules, we have been exploring the chemical space associated with a series of functionalized γbutyrolactones. In an early publication, we described the identification of muscarinic receptor ligands from this class [18]. Further assessment of this family of compounds by the Psychoactive Drug Screening Program demonstrated that members of this family that have limited binding potency (>10,000 nM) to the family of muscarinic family of receptors, but they are potent σR binders with varying levels of σ 1 /σ 2 selectivity. Given the therapeutic potential of this receptor, a drug discovery program focused on the identification of novel, drug-like functionalized γ-butyrolactones with potent σ 2 binding was established. The synthesis, characterization and preliminary evaluation of these lactones as potential selective σ 2 ligands will be presented.

Results and discussion
Synthesis of substituted γ-butyrolactones was conducted as shown in Scheme 1 utilizing novel methods developed in our laboratory. The synthesis of these compounds begins with disubstituted ester (4). Allylation of (4) under basic conditions (LDA, HMPA) provides (5) which is then converted to (7) using a modified Prins reaction [19], Specifically, reaction of (5) with paraformaldehyde in a hot mixture of acetic and sulfuric acid provided oxepan-2-one (6), which was converted to γ-butyrolactone (7) by sequential treatment with refluxing aqueous NaOH and cold sulfuric acid. Compound 7 was identified as a critical precursor in the preparation of the target series. Conversion of the primary alcohol to the corresponding tosylate (8) followed by displacement with various amines provided the final target molecules (9a, 9b and 9d-w). Alternatively, oxidation of (7) using Jones reagent followed by treatment with thionyl chloride provided the corresponding acid chloride (10), which could be reacted with an appropriate amide provide the final target molecule (9c). Table 1 includes the in vitro binding (K i at σ 1 and σ 2 ), physicochemical properties (MW, TPSA, LogP, solubility), and preliminary data regarding metabolism (human and mouse liver microsomal stability (HLM, MLM), CYP3A4 inhibition). All compounds evaluated herein have acceptable water solubility, and properties that are consistent with Lipinski rule of 5 (MW, cLogP). The compounds prepared and tested have TPSA and cLogP values that indicate they will cross the BBB following oral administration. While many of the compounds were stable in HLMs, only three compounds had T 1/2 values >10 mins in MLMs. Since future in vivo studies will be performed in rodents, this limits the compounds eligible for these studies.

Conclusion
In summary, a series of substituted lactones with drug-like physicochemical properties (MW, TPSA, cLogP) have been investigated as potential selective σ 2 R ligands. We have determined that conversion of either of the piperidine amine units to the corresponding amide causes a significant loss in activity at both σRs (9c, 9d), while replacement of the aliphatic amine of the piperazine is well tolerated (9e). In addition, we have shown that the electronic, steric, and lipophilic character of the ring appended to the piperazine moiety is critical to identifying compounds that are both 1) highly selective for σ 2 over σ 1 , and 2) highly stable in HLM and MLM (9f-9z). Importantly, all of the compounds examined are soluble in aqueous media and none appear to have significant impact on Cyp3A4 activity.
Based on our finding, we have identified (9z) as our preliminary lead compound for future studies and will be advancing this compound into mouse in vivo PK studies. Unlike the other compounds described above, (9z) is a moderate affinity σ 2 ligand (K i = 142 nM), with excellent selectivity for this target over σ 1 (K i = 10,000 nM) and it is highly stable in both MLM and HLM (T 1/2 = 60 min.). We anticipate these studies will help us further evaluate the potential value of this series for the identification of novel therapeutic agents for the treatment of diseases associated with abnormal σ 2 activity [21][22][23] such as schizophrenia, Alzheimer's disease, neuropathic pain, traumatic brain injury, and cancer.

Experimental methods and materials
Reagents were purchased from Fisher Scientific, VWR International, Sigma Aldrich, and Combi-Blocks, Inc. Chromatographic purification of compounds (normal phase and reverse phase) were carried out on a Teledyne Isco Combiflash RF system. H-NMR spectra were obtained on a Bruker 400-MHz NMR. Chemical shift values (δ values) were reported in ppm relative to TMS. For multiplicity, s = singlet, d = doublet, t = triplet, m = multiplet. Purity (%) and mass spectral data were determined with a Waters Agilent 1200 HPLC/MS (Zorbax SB-C18, 2.1 × 30 mm, 3.5 μm, 100% water/0.1% formic acid to 100% acetonitrile/0.1% formic acid over 4.0 min, 1.0 mL/min.) with a diode array detector from 210-400 nm and Agilent 6130 quadrupole MS. All compounds were purified to 95% purity or greater as determined by HPLC/ MS and 1H-NMR. Melting points were recorded on a capillary melting point apparatus.

Sigma-1 and sigma-2 competitive radioligandbinding studies
Competitive binding assays were conducted by the Psychoactive Drug Screening Program (PDSP) at The University of North Carolina, Chapel Hill under the direction of Professor Bryan Roth. Assay conditions can be found in the PDSP assay protocol book at https://pdsp.unc.edu/pdspweb/ content/UNC-CH%20Protocol%20Book.pdf.
A brief description of the assays is provided.
Sigma-2 receptor binding assay K i values for test compounds for the sigma-2 receptor were determined using a filtration assay in a 96 well polypropylene plate using membranes prepared from HEK293T cells stably transfected with the sigma-1 receptor or PC12 cells. The membranes were prepared from cultured cells rinsed with PBS, lysed in cold 50 mM Tris-HCL (pH 7.4), centrifuged at 20000 xg, pellets resuspended in buffer and then stored at −80°C until used. In a final volume of 250 uL of assay buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM EDTA, pH 7.4) the membranes were incubated with 5-7 nM [ 3 H]-1,3-di-(2-tolyl)guanidine ([ 3 H]-DTG, K d = 9.9 nM) and test compound (11 concentrations) at room temperature for 90 min. Nonspecific binding was defined with 10 uM haloperidol. Membranes were then collected by rapid filtration on to filter mats pretreated with 0.3 % polyethyleneimine, washed 4x with cold assay buffer, dried, microscintillant added and then counted in a Microbeta scintillation counter. IC 50 values were determined using a three-parameter non-linear curve fitting program in Prism 4.0 (GraphPad Software). K i values were calculated from the IC 50 values using the Cheng-Prusoff equation [25]. The reference standard haloperidol had a K i = 13.9 nM.
Sigma-1 receptor binding assay K i values for test compounds for the sigma-1 receptor were determined using the sigma-2 method except that membrane from HEK-293 cells stably transfected with the sigma-1 receptor or PC12 cells were used and 2-10 nM [ 3 H]-Pentazocine (K d = 6.5 nM) was the radioligand. Nonspecific binding was defined with 10 uM haloperidol. The reference standard haloperidol had a K i = 3.54 nM.

Aqueous solubility (pH 7.4) assay
Compounds were assessed for their solubility at pH 7.4 using the commercially available Millipore MultiScreenTM Solubility filter system (Millipore, Billerica, MA). Analysis was performed by liquid chromatography tandem mass spectrometry (LC/MS/MS).

Cytochrome P450 3A4 inhibition assay
Compounds were assessed for their ability to inhibit human cytochrome P450 3A4 using testosterone as a substrate and LC/MS/MS analysis. Expressed enzymes was used to minimize non-specific binding and membrane partitioning issues [26].

Microsomal stability assays
Test compounds were assessed for microsomal stability by incubating them at 37°C in the presence of mouse or human liver microsomes and an NADPH regenerating system as described by Yang et al. [27] Microsomal protein content was adjusted to give accurate rates of substrate consumption. Analysis was performed by Liquid Chromatographytandem mass spectrometry (LC/MS/MS) analysis.
Questions regarding this interest may be directed to the Temple University Conflict of Interest Program. No other author has reported conflicts of interest to disclose at the time of publication.
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