(M1030-04-24) Synthesis of Rhenium Tricarbonyl Phenformin Complex as an Anticancer Theragnostic Agent and Development of LC-MS/MS Assay for Pharmacokinetic Study

Purpose: Phenformin is a biguanide drug that has been used as an antidiabetic agent for many years. Recent studies have suggested that phenformin is a potential anticancer agent due to its ability to inhibit mitochondrial complex I and induce cell death in cancer cells. In this project, we pursue two aims: 1) we synthesized Re tricarbonyl phenformin complex: Re-(CO)₃-Phenformin (Re-Phen) as an anticancer theragnostic agent and 2) developed a sensitive LC-MS/MS method for the evaluation of pharmacokinetics and biodistribution of the synthesized Re-Phen in rodents.

Methods: The [NEt₄]₂[Re(CO)₃Br₃] was synthesized by combining [Re(CO)₅Br] with NEt₄Br in diglyme at elevated temperatures (115 °C), followed by filtration and washing of the precipitate with diethyl ether and diglyme. Excess NEt₄Br was removed by suspending the resulting solid in ethanol. The obtained product was then reacted with phenformin hydrochloride in methanol to form Re-Phen. The product was purified by preparative HPLC, and characterized using mass spectrometry (MS), analytical HPLC, and NMR. (Figure 1). Fourier transform infrared spectroscopy (FTIR) (Nicolet iS10, US) was performed for the characterization of specific chemical functional groups in the samples. The measurements were run in 0.4 cm⁻¹ resolution, at 400–4000 cm⁻¹ scanning range, and gathered 32 scans per spectrum. For the chromatographic separation, a Kinetex C18 column (1.7 μm, 100 × 2.10 mm) was utilized with isocratic elution at a flow rate of 0.3 mL/min and a total runtime of 10 minutes. The samples used for calibration standards (CS) were prepared by adding respective working solutions to blood. Re-Phen was calibrated at concentrations ranging from 1.95 ng/ml to 500 ng/ml. For each sample, 10 µL of a 2 µg/ml internal standard (D₅-Phenformin) was added to 10 µL of a blood sample, and the mixture was vortexed for 30 seconds. The samples were then added with 200 µL of acetonitrile with 0.1% (v/v) formic acid (FA), and the resulting mixture was centrifuged at 18,000 xg for 15 minutes. The clear supernatant was collected and transferred into 1.5 ml vials and evaporated to dryness before reconstituting in 200 µL of mobile phase (40:60, Methanol with 0.1% FA: Water with 0.1% FA). The sample was placed in an autosampler rack to be injected into the chromatographic system for isocratic elution. Analytes were ionized through positive electrospray ionization using the API 5500-Qtrap triple quadrupole mass spectrometer (Applied Biosystem/AB SCIEX).

Results: After conjugation with phenformin, the resulting powder was purified using preparative HPLC, and its properties were analyzed by MS (Figure 1). The reaction yield was approximately 40%. To confirm the identity of Re-Phen, we compared the FT-IR spectra of pure phenformin with Re(CO)₃-phenformin (Figure 2). FTIR spectrum of the pure phenformin showed the characteristic broad bands absorption at around 3299 and 3154 cm⁻¹ due to the N-H stretching of primary amine and N-H stretching of the secondary amine, respectively. After the conjugation of Re with phenformin, the intensity of both peaks of primary and secondary amines decreased significantly. The absorption of C-N Stretching vibration at a peak around 1143 cm^-1 was observed after the coordination of the ligand to the metal where the electron density of the C=N shifted towards the metal, and the nature of the double bond changed to a single bond and appeared as another peak in the 1185 cm^-1 region. Other peaks included 3026 (aromatic, –CH), 2932 and 2922 (aliphatic, –CH), 2018 and 1861 (C< ![if !msEquation] >< ![if !vml] >< ![endif] >< ![endif] >O), 1640 (C=N Stretching), 1552 (N-H Bending primary amin), 1513 (C=C Aromatic), 1422 (C-H Bending Methyl), 848 and 791 (Re-N), 726 (C-Re). Re-Phen was tested in a panel of pancreatic cancer cell lines (Kras* murine PDAC and MiaPaca-2 Human PDAC). Re-Phen showed a higher cytotoxic effect in Miapaca-2 cells (IC₅₀= 755 µM) when compared to Phen treatment (IC₅₀= 4.02 mM). Re-Phen also showed a higher cytotoxic effect in kras* cells (IC₅₀= 410 µM) when compared to Phen (IC₅₀= 2.36 mM) (Figure 3). Whole blood samples were spiked as surrogates in LC-MS/MS method validation. The assay linearity was established at the range of 1.95 (LLOQ)-500 ng/ml, and the accuracy and precision were validated. The assay will be used in the quantification of Re-Phen and the possible degradation product of phenformin in blood and other biological matrices in preclinical cancer models after dosing of Re-Phen.

Conclusion: A stable and aqueous soluble compound was synthesized and that can be used in future efficacy studies in preclinical cancer models. Re-Phen has potential anti-cancer activity as demonstrated in pancreatic cancer cell lines. An LC-MS/MS assay for the quantification of phenformin and Re-Phen in blood was developed and demonstrated linearity with LLOQ of 1.95 ng/mL, accuracy, and precision over a wide range of concentrations up to 500 ng/mL. Further, in vivo, proof-of-concept efficacy studies are in progress to establish the efficacy and safety of Re-Phen in cancer animal models.

Figure 1. Synthesis of (NEt4)2[Re(CO)3Br3] (a).Conjugation of tricarbonyl rhenium with phenformin (Re-Phen) (b), and high-resolution mass spectroscopy characterization of Re-Phen after purification by preparative HPLC (c)

Figure 2. Infrared Spectroscopy of Re-Phen and Phenformin.

Figure 3. Cytotoxicity of Re-Phen in Kras* murine PDAC and MiaPaca-2 Human PDAC for 24 hours, measured by the MTS assay.