Category: Preclinical Development
Purpose: Sigma receptors comprise of two subtypes, sigma-1 and sigma-2. Sigma-2 receptors are highly upregulated in cancer cells compared to normal tissues and, as such, Sigma-2 receptor ligands have great potentials as both cancer therapeutics and imaging tools.1,2 CM572 is a known benzoxazolone derivative bearing a reactive isothiocyanate moiety designed to selectively and irreversibly bind sigma-2 receptor pocket.3 However, the stock solution of CM572 showed a time dependent variation in binding results, while maintaining a similar cytotoxicity profile. Thus, we decided to investigate any possible degradation and confirm any identified major degradation product through chemical synthesis.
Methods: Cell culture: Human SK-N-SH neuroblastoma cells were grown in MEM with 10% FBS and 10 mg/L insulin at 37°C and 5% CO2, in a humidified environment. Binding assay: [3H](+)-pentazocine was used to label sigma-1 receptors, and [3H]DTG (in the presence of unlabeled (+)-pentazocine) was used to label sigma-2 receptors in rat liver membranes. Sigma-2 ligand concentrations of 0.5 nM to 10 μM were used in competition assays. Ki values were determined using GraphPad Prism.
MTT assay: Cells were plated at 15,000 cells/well in a 96-well plate and were allowed to adhere overnight. Cells were then treated with compounds for the indicated time period. MTT reagent was allowed to metabolize for 3 hours, prior to the addition of detergent for 2 hours. Absorbance of formazan was read at 570 nm.
LC/MS: High resolution mass spectrometric characterizations were carried out on the Agilent 6530 Accurate Mass Q-TOF LC/MS system. The compounds were diluted to a concentration of 10 mM in DMSO. LC/MS was run by Tun-Li Shen at the Brown University Chemistry Core.
NMR: NMR spectra were recorded using a Varian Mercury-Plus 400 spectrometer with a broad-band probe and a z-axis gradient. Deuterated solvents were purchased from Fisher Scientific.
Synthesis: CM572 was synthesized as previously described.4 CM572 was then reacted with its precursor CM571 to afford the final compound MAM03055A
Results: In vitro cytotoxicity and binding assays were performed using a 10 mM stock solution of CM572 in DMSO and the stock was stored at -20 °C. CM572 showed time dependent binding differences which were initially overlooked because no significant difference was evident during the routine cytotoxicity assays. The stock solution was then analyzed by LC/MS at different time points and showed the conversion of CM572 into a new peak (CM572/HN) with m/z 406. A 1H NMR degradation study was performed on a solution of CM572 in DMSO-d6 showing a significant shift in the signals corresponding to the aromatic portion, hence indicating that the chemical modification should occur near this region of the molecule. HRMS analysis of the mixture allowed the identification of CM572/HN as a dimeric analog of CM572. It was then hypothesized that the isocyonate group of CM572 degraded in presence of water to the corresponding amine to form the other sigma receptor ligand CM571 which by condensation with another molecule of CM572 led to the formation of CM572/HN. To prove our hypothesis CM571 and CM572 were synthesized as previously reported in the literature and reacted together to obtain MAM03055A (former unknown CM572/HN). CM572/HN and MAM03055A showed identical HRMS isotopic fingerprint and overlapping 1H NMR signals. The pure MAM03055A showed similar cytotoxicity to CM572 and maintained a sigma 1/2 binding profile more similar to the selective CM572 than the non-selective CM571.
Conclusion: After the observation of time dependent changes in binding values for a stock solution of CM572, the formation of the degradation compound CM572/HN was hypothesized. The investigation of the degradation was carried out by LC/MS, HRMS and 1H NMR, and led to the identification and generation of the dimeric compound MAM03055A. MAM03055A showed a similar cytotoxicity profile to CM572, despite lacking the possibility for irreversible binding. The chemical instability of the isothiocyanate group indicates the poor practicality of use of such reactive alkylating species and suggests the need to investigate alternative electrophilic moieties. At the same time, the difference between CM572 and MAM03055A opens questions about a more detail understanding of the pharmacophoric requirements for this larger molecule and open the interest for further modifications (e.g. heterodimers or different linkers). (*M. Mottinelli and C. Liu contributed equally to this work)
REF: 1) Cancer Res. 55: 408–413 (1995); 2) J. Med. Chem. 56: 7137-7160 (2013); 3) J. Pharmacol. Exp. Ther. 354: 203-212 (2015); 4) U.S. Patent US 8686008 B2. 2014 Apr 1.
Cheri Liu– Providence, Rhode Island
Hilary Nicholson– Providence, Rhode Island
Eric Zhong– Providence, Rhode Island
Wayne Bowen– Providence, Rhode Island
Christopher McCurdy– Professor of Medicinal Chemistry Director, UF Translational Drug Development Core, Department of Medicinal Chemistry, College of Pharmacy, University of Florida; Department of Biomolecular Sciences, University of Mississippi, University; UF Translational Drug Development Core, University of Florida, Gainesville, Florida