Antiviral drug Triazavirin, selectively labeled with ²H, ¹³C, and ¹⁵N stable isotopes. Synthesis and properties

Isotope-labeled antiviral drug Triazavirin containing ²H, ¹³C, and ¹⁵N atoms in its structure has been synthesized. ¹³C²H₃I and KS¹³CN served as donors of ¹³C isotopes. The use of ¹³С-MeI containing ²H atoms made it possible to additionally incorporate deuterium labels into the structure of the compound. The ¹⁵N atoms were incorporated using ¹⁵N-enriched sodium nitrite, aminoguanidine carbonate, and ethyl nitroacetate. The resulting ²H₃,¹³C₂,¹⁵N₃-Triazavirin was characterized by NMR spectroscopy.

Triazavirin (TZV) (1) is used to treat diseases caused by various types of influenza A and B viruses, including the pandemic H5N1 (avian influenza virus).^1,2,3,4,5,6 Research to expand the spectrum of action of this drug is ongoing. The data obtained make it possible to classify TZV as a firstline drug in the treatment of tick-borne viral encephalitis.⁷ In addition, TZV (1) is undergoing clinical trials as an antiviral agent against the pandemic strain of coronavirus COVID-19.^8,9,10

To evaluate the new data obtained on the activity of TZV, more complete information on the mechanism of its action, bioavailability, and biological transformation would be required. To solve these problems, it is advisable to use compounds enriched in stable isotopes (²H, ¹³C, and ¹⁵N) which can be used as internal standards for chromato-mass spectrometry.^11,12 This strategy makes it possible to investigate blood, serum, and other biological fluids for the presence of the studied biologically active compounds and their metabolites. The ability to analyze the concentrations of a compound and its metabolites is necessary for pharmacokinetic studies and in the selection of effective doses of drugs.

Previously, we proposed the synthesis of labeled TZV (²H₃,¹⁵N₃)-1 containing ²H and ¹⁵N atoms in its structure (Scheme 1, labeled atoms are indicated in bold). The incorporation of stable isotopes was based on the reaction of the diazonium salt (²H,¹⁵N₂)-2 with ethyl nitroacetate (¹⁵N)-3.¹³ It is important to note that this compound was used as an internal standard in studying the effect of TZV (1) on the aggregation of natural peptides prone to selfassociation, as exemplified by the β-amyloid peptide (βAP) fragment accumulating in the brains of patients with Alzheimer's disease, and the cytolytic peptide of bee venom melittin.¹⁴

Scheme 1

This work presents the preparation of labeled TZV (²H₃,¹³C₂,¹⁵N₃)-1 containing two additional ¹³C atoms (Scheme 2). In this case, the inclusion of ¹³C isotopes in the structures of biologically active compounds makes it possible to additionally involve the method of ¹³C NMR spectroscopy for the study of metabolic and pharmacokinetic pathways.^15,16

Scheme 2

Enriched potassium thiocyanate and (²H₃,¹³C)-MeI were used as donors of ¹³C atoms. Treatment of aminoguanidine carbonate (¹⁵N)-4 (¹⁵N, 98%) with labeled KSCN (¹³C, 95– 98%) gave 5-amino-3-mercaptotriazole (¹³С,¹⁵N)-5 in 60% yield (Scheme 2). The presence of 13C and 15N atoms in the structure of (¹³С,¹⁵N)-5 was confirmed by the registration of the molecular ion [M+H]⁺ peak with a monoisotopic mass of 119.0233 Da in the high-resolution mass spectrum. The reaction of compound (¹³С,¹⁵N)-5 with labeled MeI (99.5% 2H, 99% ¹³C) gave aminotriazole (²H₃,¹³C₂,¹⁵N)-6 in 61% yield. The incorporation of additional ²H and ¹³C labels in this case was proved by mass spectrometry ([M+H]⁺ m/z 137.0612). The reaction of aminotriazole (²H₃,¹³C₂,¹⁵N)-6 with 15N-nitrous acid generated from enriched NaNO₂ (98%, ¹⁵N) resulted in the formation of diazonium salt (²H₃,¹³C₂,¹⁵N₂)-2. Subsequent treatment with ethyl nitroacetate (15N)-3 (15N, 98%) afforded isotopically enriched triazolotriazine sodium salt dihydrate (²H₃,¹³C₂,¹⁵N₃)-1 in 43% yield.

Studying compound (²H₃,¹³C₂,¹⁵N₃)-1 by NMR spectroscopy it was found that in the ¹³C NMR spectrum the signals of all carbon atoms have additional splitting characterized by the constants of the spin-spin interaction ¹³С–¹⁵N (JC–N) with labeled ¹⁵N atoms. The quantitative measurement of J_C–N was carried out on the basis of a set of one-dimensional ¹³C NMR experiments recorded with selective decoupling from ²H and ¹⁵N atoms (Figs. S3–S5, Supplementary information file). The values of the measured ¹³С–¹⁵N coupling constants are presented in Table 1. The presence of spin-spin interactions between the ¹³С-2 and ¹³С-2' atoms with the ¹⁵N-1 atom was additionally confirmed by the data of the ¹³С–¹⁵N HMBC two-dimensional spectrum in which the corresponding cross peaks are present (Fig. 1). Analysis of the ¹³С–¹⁵N HMBC two-dimensional spectrum also showed the presence of a long-range constant (⁴J_C–N) which is due to the interaction between ¹³С-2 and ¹⁵N-5 atoms. In addition, a set of cross peaks with the corresponding J_C–N constants between the C-3a, C-6, and C-7 atoms with a natural content of the ¹³C isotope and ¹⁵N-enriched N-1, N-5, and N-6' atoms was observed in the ¹³С–¹⁵N HMBC spectrum.

Table 1. Chemical shifts and SSCC of signals in the ¹³С and ¹⁵N NMR spectra of compound (²H₃,¹³C₂,¹⁵N₃)-1

Figure 1.

Fragments of the ¹³C–¹⁵N HMBC spectrum of compound (²H₃,¹³C₂,¹⁵N₃)-1 and the corresponding segments of the one-dimensional ¹³C and ¹⁵N NMR spectra. The intensity of the fragments of the one-dimensional ¹³C NMR spectrum shown in the panels on the right is increased relative to the fragment on the left panel. The ¹³C–¹⁵N HMBC spectrum showing correlations of ¹⁵N nuclei with ¹³C-2 nucleus was obtained with selective decoupling from ¹⁵N-1 nucleus (the signal area is denoted by a dotted circle).

Analysis of the ¹³С–¹⁵N HMBC spectrum also indicated the possible signal frequencies of the N-3, N-4, and N-8 atoms with the natural abundance of the ¹⁵N isotope (Fig. 1, Table 1). Using the one-dimensional ¹³C NMR spectrum acquired with decoupling from ²H and ¹⁵N atoms, the geminal coupling constant ¹³С–¹³С (²J_C–С) between the labeled ¹³С-2 and ¹³С-2' atoms was recorded (Table 1). At the same time, the recording of the one-dimensional ¹³C NMR spectrum with decoupling only from ¹⁵N atoms made it possible to estimate the ²H–¹³C (¹J_C–D) heteronuclear interaction. The presence of the ¹J_C–D constant in the signal of the C-2' atom (Table 1) unambiguously proves the presence of deuterium atoms in the structure of (²H₃,¹³C₂,¹⁵N₃)-1. The signal of the 2'-¹³C₂H₃ group was observed as a doublet in the one-dimensional ²H NMR spectrum at ~2.65 ppm (Fig. S1, Supplementary information file). The upfield component of the doublet was partially overlapped by the signal from the solvent DMSO-d₆.

The one-dimensional ¹⁵N NMR spectrum of compound (²H₃,¹³C₂,¹⁵N₃)-1 showed three signals of labeled ¹⁵N nitrogen atoms (Table 1, Fig. 1) presented as a doublet of doublets at 259.1 ppm (N-1) and two doublets at 397.0 and 367.9 ppm (N-5 and N-6', respectively). The assignment of signals in the one-dimensional ¹⁵N NMR spectrum was carried out on the basis of measuring the ¹³С–15N coupling constant and analysis of chemical shifts.¹³ The ¹⁵N–¹⁵N spin-spin interaction is reflected in the structure of the signals of the N-5 and N-6' atoms. The multiplicity of the resonance signal of the ¹⁵N-1 atom is explained by the presence of two ¹³C–¹⁵N coupling constants with labeled carbon atoms. This conclusion was confirmed by acquiring one-dimensional ¹⁵N NMR spectra recorded with selective decoupling from the ¹³C-2 and ¹³C-2' atoms (Fig. S2, Supplementary information file). These experiments made it possible to quantitatively measure the direct constant ¹J_C2–N1 (Table 1).

To conclude, we have developed methods that allow, in addition to the ²H and ¹⁵N atoms, to selectively incorporate ¹³C isotopes into the structure of the antiviral drug Triazavirin. As a result, a sample was obtained containing three types of stable isotopes at once which was characterized by NMR spectroscopy. The practical use of labeled Triazavirin can significantly expand the possibilities in a comprehensive study of the bioavailability, pharmacokinetics, and metabolic pathways of this antiviral drug using mass spectrometry and NMR spectroscopy. This, in turn, can contribute to a more rational choice of treatment strategies.

Experimental

¹Н, ¹³C, and ¹⁵N NMR (700, 175, and 71 MHz, respectively) and ¹³C–¹⁵N HMBC spectra of compound (²H₃,¹³C₂,¹⁵N₃)-1 were acquired on a Bruker Avance 700 spectrometer equipped with a triple resonance sensor (¹H, ¹³C, ¹⁵N) in DMSO-d₆, using residual solvent signals (2.50 ppm for ¹Н nuclei) or solvent signals (40.11 ppm for ¹³С nuclei) as internal standard; liquid NН₃ (for ¹⁵N nuclei) was used as external standard. For measuring ¹³С–¹³С and ¹³С–¹⁵N SSCCs, a previously developed method¹⁷ of nonlinear approximation of line shapes in one-dimensional ¹³C NMR spectra recorded with and without decoupling from ²H and ¹⁵N nuclei was used. For selective decoupling of ¹⁵N nuclei, adiabatic pulses (WURST-20) with a length of 10–20 ms and an inversion range of ~ 1 kHz (14 ppm) for ¹⁵N nuclei were used. The decoupling of the ²H nuclei was carried out by the WALTZ-16 wideband sequence. The ¹³C–¹⁵N HMBC two-dimensional spectra were recorded using delays for the transfer of magnetization between the ¹³C and ¹⁵N nuclei in the range of 50–100 ms. In some cases, selective decoupling of the ¹⁵N-1 nucleus was achieved using saturation of the corresponding ¹⁵N frequency during magnetization transfer and ¹³C detection. ¹Н, ¹³C, and ¹⁵N NMR spectra for compounds (¹³C,¹⁵N)-5 and (²H₃,¹³C,¹⁵N)-6 were registered on a Bruker Avance II spectrometer (400, 100, and 41 MHz, respectively), DMSO-d₆ solvent. TMS was used as internal standard (for ¹³С, ¹Н nuclei), liquid ammonia was used as the external standard (for ¹⁵N nuclei). High-resolution mass spectra were recorded on a Finnigan LTQ FT mass spectrometer equipped with a 7 Tesla superconducting magnet and an Ion Max electrospray ionizer. Melting points were determined in open capillaries on a Stuart SMP3 apparatus.

Ethyl nitroacetate (¹⁵N)-3¹³ (¹⁵N enrichment 98%) and aminoguanidine (¹⁵N)-4¹⁸ (¹⁵N enrichment 98%) were synthesized according to the previously described methods. Labeled MeI (²H, 99.5% and ¹³С, 99%) was supplied by Aldrich. Enriched with stable isotopes sodium nitrate (¹⁵N, 98%) and potassium thiocyanate (¹³С, 95–98%) were supplied by Cambridge Isotope Laboratories.

5-Amino-3-mercapto-1,2,4-(3- ¹³ C,2- ¹⁵ N)triazole (( ¹³ C, ¹⁵ N)-5). (¹⁵N)-Aminoguanidine carbonate (¹⁵N)-4 (0.56 g, 4.00 mmol) KS¹³CN (¹³C, 95–98%; 0.40 g, 4.00 mmol), NH₄Cl (0.05 g, 0.80 mmol), and water (0.18 ml) were mixed together. The mixture was heated and stirred at 90°С for 2 h, then concentrated HCl (2.70 mmol, 0.46 ml) was added dropwise over 2 h and heating was continued at 100°С for 1 h. Then, a solution of KOH (0.25 g, 4.40 mmol) in H₂O (0.25 ml) was added to the reaction mixture. The mixture was heated at 100°С for 2 h, cooled, filtered, and the filtrate was acidified with concentrated HCl to pH 2. The formed precipitate was filtered off and washed with H₂O. The resulting labeled product (¹³C,¹⁵N)-5 (0.283 g, 60%, ¹³C 95–98%, ¹⁵N 98%) was used in further syntheses without purification. 1H NMR spectrum, δ, ppm (J, Hz): 5.74 (2H, br. s, NH₂); 12.06 (1H, br. s, NH); 12.25 (1H, dd, 1J_H–N = 107.6, ²J_H–C = 10.0, NH). ¹³C NMR spectrum, δ, ppm (J, Hz): 152.5 (CNH₂, ²J_C–C = 5.5); 162.8 (CS, ¹J_C–N = 12.5). ¹⁵N NMR spectrum, δ, ppm (J, Hz): 192.2 (¹J_С–N = 12.5). Found, m/z: 119.0233 [M+H]⁺. C¹³СH₅N₃¹⁵NS. Calculated, m/z: 119.0233.

5-Amino-3-( ² H ₃ )methylsulfanyl-1,2,4-(3- ¹³ C,2- ¹⁵ N)-triazole (( ² H ₃ , ¹³ C, ¹⁵ N)-6). Labeled 5-amino-3-mercapto-1,2,4-triazole (¹³C,¹⁵N)-5 (0.118 g, 1.00 mmol) was added to a solution of KOH (0.062 g, 1.10 mmol) in H₂O (2.00 ml). The solution was then cooled to 0°С, ¹³C²H₃I (²H 99.5%, ¹³C 99%; 0.160 g, 1.10 mmol) was added, and the mixture was stirred for 6 h. After evaporation of H₂O under reduced pressure at a temperature not exceeding 60°C to about half of the initial volume, the formed precipitate was filtered off, washed with ice-cold H₂O, and dried. The obtained 5-amino-3-methylmercapto-1,2,4-triazole (²H₃,¹³C,¹⁵N)-6 (0.082 g, 61%, 2H 99.5%, 13C 99%, 15N 98%) was used in further syntheses without purification. ¹H NMR spectrum, δ, ppm: 5.94 (2H, br. s, NH₂); 11.88 (1H, br. s, NH). ¹³C NMR spectrum, δ, ppm (J, Hz): 13.0 (sept, ¹J_C–D = 21.4, C₂H₃); 156.3 (CS); 157.9 (CNH₂). The SSCC for ¹³С–¹⁵N was not observed due to signal broadening. ¹⁵N NMR spectrum, δ, ppm: 261.4. Found, m/z: 137.0612 [M+H]⁺. C¹³С₂H₄²H₃N₃¹⁵NS. Calculated, m/z: 137.0612.

2-(2H3)Methylsulfanyl-6-( ¹⁵ N)nitro(2- ¹³ C,1,5- ¹⁵ N ₂ )[1,2,4]-triazolo[5,1- с ][1,2,4]triazin-7(4 H )-one sodium salt dihydrate (( ² H ₃ , ¹³ С ₂ , ¹⁵ N ₃ )-1). Labeled 5-amino-3-methylmercapto-1,2,4-triazole (²H₃,¹³С₂,¹⁵N)-6 (0.2 g, 1.47 mmol) was added to a mixture of H₂O (2.00 ml) and concentrated HCl (0.25 ml). The resulting mixture was cooled to –5°С, and a solution of Na¹⁵NO₂ (0.111 g, 1.50 mmol) in H₂O (1.00 ml) was added dropwise. The reaction mixture was stirred for 10 min and added to a cooled (0°С) solution of ethyl ¹⁵N-nitroacetate ((¹⁵N)-3) (0.30 ml) in 17% aqueous Na₂CO₃ (4.00 ml). The reaction mixture was stirred for 2 h at room temperature, the formed precipitate was filtered and recrystallized from 50% AcOH. Yield 0.185 g (43%, 2H 99%, ¹³C 99%, ¹⁵N 98%), yellow crystals, mp >300°C. Found, m/z: 259.0090 [M+H]. C₃¹³C₂H²H₃N₃¹⁵N₃O₃SNa. Calculated, m/z: 259.0124. Found, %: C 20.99; H 3.41; N 29.31. C₃¹³C₂²H₃N₃¹⁵N₃O₃SNa·2H₂O. Calculated, %: C 21.09; H 3.42; N 29.58.

Supplementary information file containing ²H, ¹³C, and ¹⁵N NMR spectra of compound (²H₃,¹³С₂,¹⁵N₃)-1 is available at the journal website at http://link.springer.com/journal/10593.

This work was supported by the Russian Foundation for Basic Research (grant 20-03-00842) and the Ministry of Science and Higher Education of the Russian Federation (project No. FEUZ-2020-0058 (N687.42B.223/20)).