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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10593-021-02927-1. © Springer Science+Business Media, LLC, part of Springer Nature 2021.

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).[1-6] 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.[8-10]

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 JC–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 (4JC–N) which is due to the interaction between 13С-2 and 15N-5 atoms. In addition, a set of cross peaks with the corresponding JC–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

13C NMR spectrum (175 MHz, DMSO-d6)		15N NMR spectrum (71 MHz, DMSO-d6)
Atom	δ, ppm (J, Hz)*	Atom	δ, ppm, J, Hz
C-2	166.4 dddd 3JС2–D < 1*** 2JC2–C2' = 1.2** 1JС2–N1 = 4.7 (×) 4JС2–N5 ~ 0.4 (×)	N-1	259.1 dd 3JN1–C2' = 2.6 1JN1–C2 = 4.7
C-2'	13.3 sept dd 1JС2'–D = 21.6 2JC2'–C2 = 1.2** 3JС2'–N1 = 2.6 (×)	N-3*4 N-4*4	233.0 s 294.0 s
C-3a	160.7 dd 2JС3a–N5 = 2.1 (×) 2JС3a–N1 = 0.4	N-5	397.0 d 2JN5–N6' = 6.3
C-6	145.1 ddd 1JС6–N5 = 1.8 (×) 1JС6–N6' 23.4 (×) 3JС6–N1 1.6 (×)	N-6'	367.9 d 2JN5-N6' = 6.3
C-7	143.5 dddd 3JC7–C2 = 7.0 2JС7–N5 = 1.3 2JС7–N6' = 5.3 (×) 2JС7– = 3.6 (×)	N-8*4	226.0 s

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 (2JC–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 (1JC–D) heteronuclear interaction. The presence of the 1JC–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 1JC2–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 method[17] 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)-3[13] (15N enrichment 98%) and aminoguanidine (15N)-4[18] (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- C,2- N)triazole (( C, 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, 1JH–N = 107.6, 2JH–C = 10.0, NH). 13C NMR spectrum, δ, ppm (J, Hz): 152.5 (CNH2, 2JC–C = 5.5); 162.8 (CS, 1JC–N = 12.5). 15N NMR spectrum, δ, ppm (J, Hz): 192.2 (1JС–N = 12.5). Found, m/z: 119.0233 [M+H]+. C13СH5N315NS. Calculated, m/z: 119.0233.

5-Amino-3-( H )methylsulfanyl-1,2,4-(3- C,2- N)-triazole (( H , C, 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, 1JC–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-( N)nitro(2- C,1,5- N )[1,2,4]-triazolo[5,1- ][1,2,4]triazin-7(4 )-one sodium salt dihydrate (( H , С , N )-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)).

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