Analysis of a polyfluorinated analogue of the nonsteroidal antiandrogen flutamide by nuclear magnetic resonance and liquid chromatography

Introduction. As a result of the search for more active polyfluoro-containing structural analogs of the non-steroidal antiandrogen flutamide against prostate cancer, a number of compounds were obtained, among which 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9-heptadecafluoro-N-(4-nitro-3-(trifluoromethyl)phenyl)nonanamide (code F-4) was of the greatest interest for further study. The necessary data for rigorous proof of the molecular structure of this compound have not previously been obtained. Identification of impurities and quantitative assessment of their content in the F-4 substance have not been conducted. To address these issues, we used nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography with ultraviolet (HPLC-UV) and tandem mass-selective detection (HPLC-MS/MS).

Aim. Comprehensive analysis of the structure of compound F-4 using NMR and HPLC methods, as well as identification and quantitative assessment using them of the content of impurities in samples of the substance.

Materials and methods. The object of the study were laboratory samples of the substance of compound F-4, obtained by acylation of 4-nitro-3-(trifluoromethyl)aniline with perfluoropelargonic acid chloride using a known method. The following were used for the analysis: NMR spectrometer Agilent DD2 NMR System 600; liquid chromatograph Agilent 1200 equipped with diode array detector G1315B; liquid chromatograph Agilent Infinity II equipped with mass spectrometric detector with triple quadrupole G6495C (Agilent Technologies, USA). Both chromatographs used a column Luna C18(2), 250 × 4.6 mm, 5 μm (Phenomenex, USA).

Results and discussion. Structural interpretation of the ¹H, ¹³C, and ¹⁹F NMR spectra of F-4 samples, taking into account the chemical shift values of the signals, their multiplicity, and 2D experimental data, clearly indicates that F-4 has the structure of 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9-heptadecafluoro-N-(4-nitro-3-(trifluoromethyl)phenyl)nonanamide. The ¹H and ¹⁹F NMR spectra of the F-4 substance samples also contained signals of two impurity compounds not related to the starting materials. HPLC-UV method was used to separate them. Using HPLC-MS/MS with electrospray ionization in the negative mode (–ESI), high-intensity m/z signals characteristic of deprotonated molecular ions were recorded for all peaks. The structure of the detected impurities was confirmed using NMR spectroscopy. The results of quantitative determination of the relative impurity content in samples of F-4 substance using ¹H and ¹⁹F NMR methods were virtually identical; the differences between the obtained average values were not statistically significant.

Conclusion. A comprehensive study using NMR and HPLC methods revealed that compound F-4 corresponds to the structure of 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9-heptadecafluoro-N-(4-nitro-3-(trifluoromethyl)phenyl)nonanamide. Two related impurities were identified in samples of substance F-4, the relative content of each impurity did not exceed 1.7 %.

1. Mishugin S. V., Gritskevich A. A., Rusakov I. G. Development and clinical effectiveness of antiandrogens in the treatment of advanced prostate cancer. Medical Council. 2016;(10):34–37. (In Russ.) DOI: 10.21518/2079-701X-2016-10-34-37.

2. Bukharkin B. V., Gridneva Ya. V., Mitin A. A. Nonsteroidal antiandrogens in the treatment of prostate cancer. Russian Medical Journal. 2009;17(22):1497–1499. (In Russ.)

3. Bukharkin B. V., Aksenov A. A. Use of antiandrogens in monotherapy in the treatment of prostate cancer. Canser Urology. 2005;(3):43–47. (In Russ.)

4. Nosov D. A., Volkova M. I., Gladkov O. A., Karabina E. V., Krylov V. V., Matveev V. B., Usychkin S. V., Mitin T. Prostate cancer. Malignant tumors. 2024;14(3s2-2):242–266. (In Russ.) DOI: 10.18027/2224-5057-2024-14-3s2-1.2-10.

5. Ziganshina L. E., Lepakhin V. K., Petrov V. I., Khabrieva R. U., editors. Large reference book of medicine. Moscow: GEOTAR-Media; 2011. 3344 p. (In Russ.)

6. Sternal R., Nugara N. Flutamide. Analytical Profiles of Drug Substances and Excipients. Vol. 27. In: Brittain H. G., editor. Analytical profiles of drug substances. Volume 27. San Diego: Academic Press; 2001. P. 115–157.

7. Ushkalova E. A., Zyryanov S. K., Gopienko I. A. Second-generation androgen receptor inhibitors in therapy of non-metastatic castration resistant prostate cancer: role of safety profile and effect on quality of life. Problems in Oncology. 2021;67(2):217–226. (In Russ.) DOI: 10.37469/0507-3758-2021-67-2-217-226.

8. Kandil S. B., McGuigan C., Westwell A. D. Synthesis and Biological Evaluation of Bicalutamide Analogues for the Potential Treatment of Prostate Cancer. Molecules. 2020;26(1):56. DOI: 10.3390/molecules26010056.

9. Kryukova L. Yu., Moskaleva E. Yu., Astakhov D. V., Posypanova G. A., Rodina A. V., Potashnikov P. F., Vorontsov E. A., Kuznetsov S. L., Kryukov L. N., Severin S. E. Synthesis of polyfluorinated analogues of flutamide and their cytotoxic activity. Problems of Biological, Medical and Pharmaceutical chemistry. 1998;(3):29–32. (In Russ.)

10. Katlinskiy A. V., Severin E. S., Severin S. E., Kryukov L. N., Moskaleva E. Yu., Vorontsov E. A., Smirnova Z. S., Kryukova L. Yu., Belushkina N. N., Kuznetsov S. L., Feldman N. B., Paltsev M. A. Analogues of flutamide: synthesis, biological activity and prospects of medical application. Molecular medicine. 2003;(2):60–64. (In Russ.)

11. Alyautdin R. N., Vorontsov E. A., Ivanov A. A., Kryukov L. N., Kuznetsov S. L., Moskaleva E. Yu., Paltsev M. A., Posypanova G. A., Severin E. S., Severin S. E., Khomyakov Yu. N. 4-Nitro-3-fluoromethylperfluorononanoylanilide (flustat), method if its synthesis and pharmaceutical composition based on thereof. Patent RUS No. 2186057 С1 dated 27.07.2002. Available at: https://patents.google.com/patent/RU2186057C1/en?oq=RU2186057C1. Accessed: 07.02.2024. (In Russ.)

12. Moiseev S. V., Krylov V. I., Masterkova T. V., Yashkir V. A., Bunyatyan N. D. Nuclear magnetic resonance spectroscopy for drug identification, characterization and quantification of impurities in substance of salicylic acid. Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products. Regulatory Research and Medicine Evaluation. 2014;(1):15–19. (In Russ.)

13. Kuz’mina N. E., Moiseev S. V., Yakupov I. Yu., Kuleshova S. I. Quantitative Determination of Major Active Components in Polymyxin B Medicinal Products by NMR Spectroscopy. Regulatory Research and Medicine Evaluation. 2025;15(2):206–212. (In Russ.). DOI: 10.30895/1991-2919-2025-15-2-206-212.

14. Sapon E. S., Lugin V. G., Sovastey O. G., Efremova A. A., Garipov R. M. Application of electrospray ionization HPLC-MS for qualitative analysis of cepholosporin antibiotics. Bulletin of Kazan technological university. 2014;17(17):136–138. (In Russ.)

15. Gottlieb H. E., Kotlyar V., Nudelman A. NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities. The Journal of Organic Chemistry. 1997;62(21):7512–7515. DOI: 10.1021/jo971176v.

16. Zenkevich I. G., Ioffe B. V. Interpretation of mass spectra of organic compounds. Leningrad: Khimiya; 1986. P. 61–65. (In Russ.)