Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.


Introduction
The extracellular calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) [1]. Its main and best-described physiological role is monitoring and regulating free ionized blood calcium (Ca 2+ ) concentration [2]. However, the CaSR has several other functions independent of calcium homeostasis. It regulates physiological and pathophysiological processes, such as inflammation, airway constriction, renal and intestinal water transport, cardiovascular effects, neuronal development and function, and Briefly, substituted phenol 1 was deprotonated in refluxing acetone, using an excess of potassium carbonate. Commercially available (S)-nosyl epoxide 2 was then added at r.t., and the mixture was refluxed in acetone overnight, to afford the pure intermediate 3 in high yield after flash column chromatography purification. The epoxide ring in 3 was then regioselectively opened with [1,1-dimethyl-2-(2-naphthalenyl)ethyl]amine at the less hindered position, by heating the two reagents in anhydrous EtOH at 80 °C in a sealed tube for 72 h, to afford the enantiomerically pure product NPS S-2143 in good yield after flash column chromatography purification. The pure hydrochloride salt of NPS S-2143 was finally obtained by treating NPS S-2143 with an excess of a concentrated hydrochloric acid solution, while stirring in MeOH at room temperature for 1 h.

Crystal Structure of NSP S-2143.HCl
The crystal structure of NPS S-2143.HCl was determined by small molecule crystallography, which confirmed the desired (S) absolute configuration of the chiral center. In addition to two independent chloride anions, the asymmetric unit of the crystal structure comprises two independent cations, C1-C24, N1, N2, O1, O2, Cl1 ( Figure 2a) and C25-C48, N3, N4, O3, O4, Cl2 (Figure 2b). Both are the cations of NPS S-2143 (with chiral centres located on atoms C9 and C33), which differ in their assumption of different conformations, as illustrated by Figure 2 and the torsion angles in Table S1. When packed into the crystal, aromatic rings of the chlorobenzonitrile moieties of neighbouring cations are stacked in the a-axis direction with centroid-to-centroid separation of ca. 3.6 Å . The molecules involved are also bridged by N-H…Cl and O-H…Cl hydrogen bonds with each chloride ion interacting with two N-H and one O-H groups ( Figure S1 and Table S2). Briefly, substituted phenol 1 was deprotonated in refluxing acetone, using an excess of potassium carbonate. Commercially available (S)-nosyl epoxide 2 was then added at r.t., and the mixture was refluxed in acetone overnight, to afford the pure intermediate 3 in high yield after flash column chromatography purification. The epoxide ring in 3 was then regioselectively opened with [1,1-dimethyl-2-(2-naphthalenyl)ethyl]amine at the less hindered position, by heating the two reagents in anhydrous EtOH at 80 • C in a sealed tube for 72 h, to afford the enantiomerically pure product NPS S-2143 in good yield after flash column chromatography purification. The pure hydrochloride salt of NPS S-2143 was finally obtained by treating NPS S-2143 with an excess of a concentrated hydrochloric acid solution, while stirring in MeOH at room temperature for 1 h.

Crystal Structure of NSP S-2143.HCl
The crystal structure of NPS S-2143.HCl was determined by small molecule crystallography, which confirmed the desired (S) absolute configuration of the chiral center. In addition to two independent chloride anions, the asymmetric unit of the crystal structure comprises two independent cations, C1-C24, N1, N2, O1, O2, Cl1 ( Figure 2a) and C25-C48, N3, N4, O3, O4, Cl2 (Figure 2b). Both are the cations of NPS S-2143 (with chiral centres located on atoms C9 and C33), which differ in their assumption of different conformations, as illustrated by Figure 2 and the torsion angles in Table S1. When packed into the crystal, aromatic rings of the chlorobenzonitrile moieties of neighbouring cations are stacked in the a-axis direction with centroid-to-centroid separation of ca. 3.6 Å. The molecules involved are also bridged by N-H . . . Cl and O-H . . . Cl hydrogen bonds with each chloride ion interacting with two N-H and one O-H groups ( Figure S1 and

Enantiospecific Inhition of the CaSR via NPS 2143
With NPS S-2143 now available, we proceeded to test the synthetized compound in vitro by individual-cell Ca 2+ -imaging performed on HEK-293 cells stably transfected with the human CaSR, which is the most common cell model for evaluating the receptor and agents targeting it. Pre-incubation of the cells with a concentration of 100 nmol/L of NPS R-2143 (the selective enantiomer of the compound) significantly suppressed the intracellular Ca 2+ -response elicited by 5 mmol/L extracellular Ca 2+ , while the newly synthetized (unselective) NPS S-2143 had no such effect (Figure 3a-d). Together, these results showed that the clean S enantiomer of NPS 2143 is indeed not active on the CaSR. It could thus be used for the following specificity experiments in colorectal cancer cell.

Enantiospecific Inhition of the CaSR via NPS 2143
With NPS S-2143 now available, we proceeded to test the synthetized compound in vitro by individual-cell Ca 2+ -imaging performed on HEK-293 cells stably transfected with the human CaSR, which is the most common cell model for evaluating the receptor and agents targeting it. Pre-incubation of the cells with a concentration of 100 nmol/L of NPS R-2143 (the selective enantiomer of the compound) significantly suppressed the intracellular Ca 2+ -response elicited by 5 mmol/L extracellular Ca 2+ , while the newly synthetized (unselective) NPS S-2143 had no such effect (Figure 3a-d). Together, these results showed that the clean S enantiomer of NPS 2143 is indeed not active on the CaSR. It could thus be used for the following specificity experiments in colorectal cancer cell.

CaSR Gene Induction and Pro-Inflammatory Responses in Colon Cancer Cells Are Mediated through the CaSR
Having established the different activities of the two enantiomers of NPS 2143, we evaluated the specificity of the enantiomers of calcimimetics and calcilytic and their combination in a cell model of colorectal cancer. Here, we used HT29 colon cancer cells, which were modified to stably express the CaSR via lentiviral infection (HT29 CaSR-GFP ) using empty vector infected cells as the control (HT29 GFP ).
To test directly whether the calcimimetic-induced effects on CaSR expression were indeed mediated via specific activity of the drug on the receptor or whether unspecific effects of the drug were (partly) responsible as well, we performed a series of tightly controlled single and combination treatment experiments with the selective and nonselective enantiomers of the calcimimetic NPS 568 and the calcilytic NPS 2143. To ensure proper interaction of the calcilytic with the receptor, cells were always pre-treated with NPS 2143 (or vehicle) for 30 min before their treatment with NPS 568 (if applicable). The total treatment time after the addition of NPS 568 was 4 h. Here, we used 1 µmol/L of each compound, as the initial results using NPS R-568 and NPS R-2143 in these transfected colon cancer cells were obtained using drug concentrations that were previously deemed to be CaSR-selective [5,9,[14][15][16].

CaSR Gene Induction and Pro-Inflammatory Responses in Colon Cancer Cells are Mediated through the CaSR
Having established the different activities of the two enantiomers of NPS 2143, we evaluated the specificity of the enantiomers of calcimimetics and calcilytic and their combination in a cell model of colorectal cancer. Here, we used HT29 colon cancer cells, which were modified to stably express the CaSR via lentiviral infection (HT29 CaSR-GFP ) using empty vector infected cells as the control (HT29 GFP ).
To test directly whether the calcimimetic-induced effects on CaSR expression were indeed mediated via specific activity of the drug on the receptor or whether unspecific effects of the drug were (partly) responsible as well, we performed a series of tightly controlled single and combination treatment experiments with the selective and non-selective enantiomers of the calcimimetic NPS 568 and the calcilytic NPS 2143. To ensure proper interaction of the calcilytic with the receptor, cells were always pre-treated with NPS 2143 (or vehicle) for 30 min before their treatment with NPS 568 (if applicable). The total treatment time after the addition of NPS 568 was 4 h. Here, we used 1 µ mol/L of each compound, as the initial results using NPS R-568 and NPS R-2143 in these transfected colon cancer cells were obtained using drug concentrations that were previously deemed to be CaSR-selective [5,14-16] [9]. The CaSR has a rather unique property in that its expression increases in response to stimulation (rather than decreases as most receptors do due to desensitization and internalization). This process can be observed directly at the cell membrane, where it is called agonist-driven insertional signaling (ADIS) [17], but also at the mRNA level.
Treatment of HT29 CaSR-GFP cells with R-568 evoked a large (~5-fold) increase in CaSR gene expression after 4 h of treatment. In contrast, the non-selective enantiomer of the calcimimetic, S-568, did not have any effect on CaSR gene expression. Neither the selective nor the non-selective enantiomers of NPS 2143 had any effect on CaSR gene expression, either by themselves or in combination. Importantly, pre-treatment with the selective NPS R-2143 completely abolished the CaSR gene induction by NPS R-568. On the other hand, pre-treatment with the non-CaSR-selective enantiomer NPS S-2143 did not have any effect on NPS R-568-induced CaSR gene expression. The most prominent orthosteric agonist of the CaSR, Ca 2+ , also upregulated CaSR gene expression, and was used as a positive control ( Figure 4a). None of the compounds or their combinations had any effect on CaSR expression levels in HT29 GFP cells, which was expected as the expression levels of the CaSR in the non-CaSR transfected HT29 GFP cells are negligible (Figure 4b). Taken together, these results showed that both NPS 568 and NPS 2143 affected CaSR gene expression in a highly enantiospecific manner. This strongly indicates that the induction of CaSR gene expression is indeed induced directly via the CaSR itself.
pre-treatment with the non-CaSR-selective enantiomer NPS S-2143 did not have any effect on NPS R-568-induced CaSR gene expression. The most prominent orthosteric agonist of the CaSR, Ca 2+ , also upregulated CaSR gene expression, and was used as a positive control (Figure 4a). None of the compounds or their combinations had any effect on CaSR expression levels in HT29 GFP cells, which was expected as the expression levels of the CaSR in the non-CaSR transfected HT29 GFP cells are negligible (Figure 4b). Taken together, these results showed that both NPS 568 and NPS 2143 affected CaSR gene expression in a highly enantiospecific manner. This strongly indicates that the induction of CaSR gene expression is indeed induced directly via the CaSR itself. Next, we wanted to assess whether the observed enantiospecific effects of CaSRmodulation could be observed on a CaSR-influenced effector gene. As mentioned before, we have recently shown that the modulation of the CaSR with NPS R-568 led to dramatic changes in their gene expression patterns. One of the most strikingly upregulated families of genes were involved in inflammation. Thus, we investigated the effect of the abovementioned single enantiomers and their combinations on IL-8 gene expression, as one of the most prominent members of the previously observed pro-inflammatory genes. Next, we wanted to assess whether the observed enantiospecific effects of CaSRmodulation could be observed on a CaSR-influenced effector gene. As mentioned before, we have recently shown that the modulation of the CaSR with NPS R-568 led to dramatic changes in their gene expression patterns. One of the most strikingly upregulated families of genes were involved in inflammation. Thus, we investigated the effect of the abovementioned single enantiomers and their combinations on IL-8 gene expression, as one of the most prominent members of the previously observed pro-inflammatory genes.
We observed the same pattern for IL-8 gene expression as for CaSR in HT29 CaSR-GFP cells. NPS R-568 induced a strong upregulation of IL-8, while the unspecific NPS S-586 showed no effect whatsoever. Both enantiomers of the calcilytic NPS 2143 did not affect IL-8 gene expression, and neither did the combination of the two unselective enantiomers NPS S-568 and NPS S-2143. Again, pre-treatment of the cells with NPS R-2143 inhibited NPS R-568-induced IL-8 gene expression, while NPS S-2143 was not able to suppress this upregulation. The orthosteric CaSR agonist Ca 2+ also induced a robust upregulation of IL-8 gene expression (Figure 5a). In HT29 GFP cells, none of the substances led to any change in IL-8 gene expression compared with the vehicle control ( Figure 5b).
Finally, we investigated whether changes in IL-8 gene expression were mirrored by changes at the secreted protein level. We, therefore, performed an ELISA experiment on cell culture supernatants from treated HT29 CaSR-GFP and HT29 GFP cells and observed the same effect on the protein level as we did on the mRNA level. NPS R-568 and 5 mmol/L Ca 2+ induced upregulation of IL-8 secretion by the HT29 CaSR-GFP cells. The pre-incubation with NPS R-2143 prevented this upregulated secretion, while the unspecific NPS S-2143 did not (Figure 6a). No effects on IL-8 secretion were observed in HT29 GFP cells lacking the CaSR (Figure 6b). Interestingly, baseline IL-8 levels were already higher in vehicle-treated HT29 CaSR-GFP cells than in HT29 GFP cells.
IL-8 gene expression, and neither did the combination of the two unselective enantiomers NPS S-568 and NPS S-2143. Again, pre-treatment of the cells with NPS R-2143 inhibited NPS R-568-induced IL-8 gene expression, while NPS S-2143 was not able to suppress this upregulation. The orthosteric CaSR agonist Ca 2+ also induced a robust upregulation of IL-8 gene expression (Figure 5a). In HT29 GFP cells, none of the substances led to any change in IL-8 gene expression compared with the vehicle control (Figure 5b). Finally, we investigated whether changes in IL-8 gene expression were mirrored by changes at the secreted protein level. We, therefore, performed an ELISA experiment on cell culture supernatants from treated HT29 CaSR-GFP and HT29 GFP cells and observed the same effect on the protein level as we did on the mRNA level. NPS R-568 and 5 mmol/L Ca 2+ induced upregulation of IL-8 secretion by the HT29 CaSR-GFP cells. The pre-incubation with NPS R-2143 prevented this upregulated secretion, while the unspecific NPS S-2143 did not (Figure 6a). No effects on IL-8 secretion were observed in HT29 GFP cells lacking the CaSR (Figure 6b). Interestingly, baseline IL-8 levels were already higher in vehicle-treated HT29 CaSR-GFP cells than in HT29 GFP cells.

Discussion
We were able to prove unequivocally that the CaSR mediates the Ca 2+ -and NPS R-568-induced increase in the expression of the inflammation marker IL-8 in HT29 CaSR-GFP cells. To verify this, we used both the R and S enantiomers of the calcimimetic NPS 568 and of the calcilytic NPS 2143 and compared their effects in the isogenic cell lines HT29 CaSR-GFP and HT29 GFP , differing only in the expression of CaSR. As, to our knowledge, no S enantiomer of NPS 2143 was available, we synthesized it and thus obtained a very useful tool for further pharmacological studies on the CaSR.
After ensuring that the synthesized NPS S-2143 is indeed significantly less active than 2+ 2+

Discussion
We were able to prove unequivocally that the CaSR mediates the Ca 2+ -and NPS R-568induced increase in the expression of the inflammation marker IL-8 in HT29 CaSR-GFP cells.
To verify this, we used both the R and S enantiomers of the calcimimetic NPS 568 and of the calcilytic NPS 2143 and compared their effects in the isogenic cell lines HT29 CaSR-GFP and HT29 GFP , differing only in the expression of CaSR. As, to our knowledge, no S enantiomer of NPS 2143 was available, we synthesized it and thus obtained a very useful tool for further pharmacological studies on the CaSR.
After ensuring that the synthesized NPS S-2143 is indeed significantly less active than R-2143 in preventing extracellular Ca 2+ -induced intracellular Ca 2+ release, we assessed the effectiveness of the modulators in regulating CaSR expression, a well-known effect of these compounds. Indeed, extracellular Ca 2+ and the well-characterized calcimimetic NPS R-568 significantly induced CaSR expression, which was inhibited by R-2143. None of the S enantiomers affected CaSR expression, proving that the cells were responsive only to the R modulators. In the same cells, we measured the IL-8 expression at both the mRNA and protein levels. Numerous studies have shown that IL-8 is a pro-inflammatory cytokine [18] in the intestine [19,20], and thus a valid marker for a pro-inflammatory response in our cell model. Therefore, we needed to prove that our contradictory observation [9] that the activation of the CaSR induces IL-8 expression is real, and indeed mediated by the CaSR. The results of the Iamartino study [9] were already very convincing, as the observed upregulation of several inflammatory markers was seen only in the cells expressing the CaSR but not in the cells with undetectable CaSR levels. While the observation that R-568 induced the expression of these markers suggested that the CaSR mediates this effect, we needed unequivocal proof. Indeed, we could show that only the actively binding R-568, at the same concentration of 1 µmol/L as used in the aforementioned study, induced the expression of IL-8, and NPS S-568 was unable to do so. The effect of NPS R-568 was inhibited by NPS 2143 in a similar stereospecific manner: only R-2143 prevented the R-568-dependent induction, whereas NPS S-2143 did not.
Several studies suggested that calcilytics could be used in the treatment of inflammatory diseases, such as asthma [14,21]. Thus, the newly synthesized S-2143 could become a highly useful tool in experiments of proof of principle in other tissues and organs as well.
The first generation of calcimimetics, such as NPS R-568, has one chiral carbon and acts stereoselectively on the CaSR. Regardless of the parameter assessed, the R enantiomer was always more potent (10-to 100-fold, depending on the measured variable) compared to the S enantiomer. At concentrations that maximally activate the CaSR, NPS 568 inhibits ion channels, but this effect is not stereospecific, meaning that both the R and S enantiomers have similar effects [22]. The calcilytic NPS 2143 also acts on the CaSR in a stereoselective manner and again, the R enantiomer is more potent [12,22]. Thus, testing that the compounds used in our experiment produce the expected effect in a stereospecific manner ascertained that the effect was CaSR-mediated. Moreover, none of the compounds had any effect on IL-8 (or CaSR) expression in the HT29 GFP cells lacking detectable levels of endogenous CaSR, suggesting that at the concentrations used, the observed effects were not due to the activation of any other possible targets (e.g., voltage-gated Ca 2+ channels).
To understand the role of the CaSR in a cell is demanding, because Ca 2+ , its main physiological ligand, signals not only through the CaSR, but also by binding to other molecules (e.g., ion channels). Therefore, it is important to combine the right pharmacological approach with the most appropriate experimental design. However, this is challenging in cells where the primary role of the receptor is not clear and Ca 2+ affects numerous biological processes. The biological process we had to test was given (regulation of the expression of IL-8) and the specificity of the drugs used in this study has already been tested either on parathyroid cells and/or on HEK-CaSR cells (a model broadly used in the field).
Our study is a good example for testing the involvement of the CaSR in molecular processes. It is the first study, to our knowledge, that uses enantiomer pairs and combinations of positive and negative modulators to prove the involvement of a target in the signaling process in intestinal cells. Many natural (endogenous) ligands of the CaSR signal through numerous alternative pathways. Most of the pharmacological modulators have structures very similar to drugs acting on other molecules (e.g., Ca 2+ -channel or β-blockers).
Therefore, it is difficult to exclude the possibility that the effects seen are mediated through other pathways, even more so when the concentrations used are higher than those needed to modulate CaSR activity. The S enantiomers could prove very useful in such cases, as they could be used to test if the effect seen is indeed caused by the binding of the drug to the CaSR.
The role of the CaSR in the gastrointestinal tract is complex. The clinical use of calcimimetics, both oral and intravenously applied, is associated with substantial gastrointestinal side effects, such as vomiting and nausea [11], and the U.S. Food and Drug Administration (FDA) has updated the label of cinacalcet to include the risk for upper gastrointestinal bleeding (FDA ID: 4097661). The actual, direct role of the gastrointestinal CaSR in (patho-)physiology and in these adverse effects is, however, still not fully understood. Several studies have shown that the CaSR is directly involved in intestinal fluid secretion [23,24]. However, the involvement of the CaSR in intestinal inflammation is still unclear [6]. The most frequent approaches to verify the role of CaSR in physiology and pathophysiology are either genetic, by comparing the phenotype of the mice lacking the CaSR to their wild type controls [25,26], or pharmacological approaches, using specific CaSR activators (e.g., R-568) or inhibitors (e.g., NPS 2143). One of the limitations of the genetic approach is that the organism might develop mechanisms to compensate for the loss of the CaSR. The limitations of the pharmacological approaches are that often the doses of the pharmacological compounds are too high [27], raising the question as to how this affects the specificity of the effect. The potent and selective pharmacological modulators of the CaSR (NPS R-568 and NPS R-2143) have been proven useful in uncovering new functions of the CaSR at different sites throughout the body [28]. However, it is important to use these compounds in a concentration that is selective to the CaSR, as at high concentrations, they will affect cellular responses independent of the CaSR [22]. In our study, we combined a genetic approach (but instead of knocking out, we introduced the CaSR in the cells) with the pharmacological approach, by using compounds at concentrations that are still specific for the CaSR and taking advantage of the stereospecificity of the used calcimimetic and calcilytic.
Our study demonstrates that in the HT29 CaSR-GFP cells, the major activators of the CaSR, Ca 2+ and R-568, induced and did not inhibit the expression of the inflammatory cytokine IL-8. Our results unequivocally confirm the CaSR as a mediator of the enhanced inflammatory gene expression in these cells and are quadruple controlled: (1) a positive CaSR modulator induced inflammatory gene expression, (2) a negative CaSR modulator prevented this upregulation, (3) the unspecific enantiomers of these compounds did not exhibit these effects, and (4) none of these effects were observed in cells not expressing the CaSR.
These data also suggest that in native cells, where no endogenous CaSR is present, the drugs will not affect the expression of these inflammatory markers. This would be in line with our observation in an in vivo study, where neither the calcimimetic cinacalcet nor NPS R-2143 affected the expression of the inflammatory marker IL-6 in the colon of mice treated per gavage for 2 weeks with these modulators (unpublished results/manuscript in preparation). We now have a better understanding of the role of the CaSR in mediating inflammatory responses and with NPS S-2143, a valuable tool for controlling pharmacological CaSR modulation experiments

Preparation of NPS S-2143-Synthetic Chemistry
All solvents and reagents were used as obtained from commercial sources, unless otherwise indicated. All solvents used for chromatography were HPLC grade (Fisher Scientific, Loughborough, UK). All reactions were performed under a nitrogen atmosphere. 1 H and 13 C-NMR spectra were recorded with a Bruker Avance III HD spectrometer (Bruker, Coventry, UK) operating at 500 MHz for 1 H and 125 MHz for 13 C, with Me 4 Si as the internal standard. Deuterated chloroform was used as the solvent for NMR experiments. 1 H chemical shifts values (δ) are referenced to the residual non-deuterated components of the NMR solvents (δ = 7.26 ppm for CHCl 3 , etc.). The 13 C chemical shifts (δ) are referenced to CDCl 3 (central peak, δ = 77.0 ppm). TLC was performed on silica gel 60 F254 plastic sheets. Normal-phase automated flash column chromatography was performed using a Biotage Isolera system (Biotage, Hengoed, UK). UPLC-MS analysis was conducted on a Waters UPLC system (Waters, Wilmslow, UK) with both Diode Array detection and Electrospray (+ ve and − ve ion) MS detection. The stationary phase was a Waters Acquity UPLC BEH C18 1. Potassium carbonate (17.6 mmol, 3 eq.) was added to a stirring solution of 2-chloro-6-hydroxybenzonitrile 1 (5.9 mmol, 1 eq.) in dry acetone (60 mL), and the mixture was heated to reflux for 30 min. After cooling to room temperature, commercially available (S)-nosyl epoxide 2 (5.9 mmol, 1 eq.) was added to the reaction, and stirring was continued while heating to reflux overnight. After cooling to room temperature, the reaction mixture was filtered, and the filtrate was dried under vacuum. The crude residue was purified by flash column chromatography on silica gel (n-hexane/ethyl acetate 100:0 to n-hexane/ethyl acetate 0:100 v/v) to afford the pure title compound as a white solid in 98% yield. 1  A solution of (S)-epoxide 3 (3.3 mmol, 1 eq.) and [1,1-dimethyl-2-(2-naphthalenyl)ethyl]amine (3.3 mmol, 1 eq.) in dry EtOH (17 mL) was heated to 80 • C in a sealed tube for 72 h. The mixture was then dried under vacuum, and the crude residue was purified by flash column chromatography on silica gel (n-hexane/ethyl acetate 100:0 to n-hexane/ethyl acetate 0:100 v/v) to afford the pure title compound as a colourless oil in 73% yield. 1 13

Crystal Structure Determination
Single-crystal XRD data were collected at room temperature on an Agilent SuperNova Dual Atlas diffractometer (Agilent Technologies XRD Products, Yarnton, UK) with a mirror monochromator using Cu (λ = 1.5418 Å) radiation. The crystal structure was solved using SHELXS [29] and refined using SHELXL2018 [30]. Non-hydrogen atoms were refined with anisotropic displacement parameters, and hydrogen atoms were inserted in idealized positions. A riding model was used with Uiso(H) set at 1.2 or 1.5 times the Ueq(C,N,O) values of the atoms to which the H atoms are bonded. The asymmetric unit contains two cations and two chloride anions. The naphthalene moieties of both independent cations were modelled as disordered, and the components refined to roughly equal occupancy. The crystallographic and refinement parameters are as follows: C 24

Other Compounds and Reagents
NPS R-568 and NPS R-2143 were obtained commercially (Tocris Bioscience/Bio-Techne Ltd., Abingdon, UK), NPS S-568 was a kind gift from Amgen, UK. All basic compounds were obtained from Merck (Darmstadt, DE), unless otherwise stated.

Calcium Imaging Experiments
HEK293 cells stably transfected with the human CaSR (HEK-CaSR, a kind gift of Dr. Donald Ward [31]) were cultured on poly-D-lysine coated 13 mm coverslips, and calcium imaging experiments were performed as described previously [21]. In brief, the medium of the cells was removed, and cells were loaded with 3 µmol/L Fura 2-AM (Thermo Fisher Scientific, Waltham, MA, USA) in extracellular buffer containing 1.0 mmol/L Ca 2+ for 45 min at 37 • C. Cells were then washed with ECS at room temperature and preincubated with buffer containing 0.2 mmol/L Ca 2+ and 100 nmol/L NPS R/S-2143 or 0.1% DMSO for 15 min. Cells were imaged on an inverted Olympus IX71 fluorescence microscope (Olympus, Southend-on-Sea, UK). A rapid perfusion system was then used to alter extracellular Ca 2+ from 0.2 (2 min, baseline) to 5 (3 min, stimulation) to 0.2 mmol/L Ca 2+ (2 min), with each buffer containing either 100 nmol/L NPS R/S-2143 or 0.1% DMSO. Fluorescence of individual cells (~30-70 per experiment) at 340 and 380 nm was acquired every 2 s. For analysis, the background fluorescence was subtracted for both wavelengths, and the average F340/F380 ratio of the baseline of each cell was then subtracted from the maximum response of each cell during the stimulation phase. The average of these differences in one experiment counted as one biological repetition.

Colon Cancer Cells
We used lentiviral stably transduced colon cancer cell lines HT29 CaSR-GFP and HT29 GFP [9]. The parent HT-29 cell line was obtained commercially (HTB-38™) from the American Type Culture Collection (ATCC, Manassas, VA, USA). Transduced cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, 100 U/mL Pen-Strep, 2 mmol/L L-glutamine and 10 mmol/L Hepes. Of note, the medium by itself contains 1.8 mmol/L Ca 2+ , the complete (FCS supplemented) medium contains~2 mmol/L Ca 2+ . To select for transduced cells, 0.5 µL/mL puromycin (all Thermo Fisher Scientific) was added to the cell medium. For the treatments, cells were seeded into 6-well plates and grown to