Improving properties of the nucleobase analogs T-705/T-1105 as potential antiviral

Abstract

In this minireview we describe our work on the improvement of the nucleobase analogs Favipiravir (T-705) und its non-fluorinated derivative T-1105 as influenza and SARS-CoV-2 active compounds. Both nucleobases were converted into nucleotides and then included in our nucleotide prodrugs technologies cycloSal-monophosphates, DiPPro-nucleoside diphosphates and TriPPPro-nucleoside triphosphates. Particularly the DiPPro-derivatives of T-1105-RDP proved to be very active against influenza viruses. T-1105-derivatives in general were found to be more antivirally active as compared to their T-705 counterpart. This may be due to the low chemical stability of all ribosylated derivatives of T-705. The ribosyltriphosphate derivative of T-1105 was studied for the potential to act as a inhibitor of the SARS-CoV-2 RdRp and was found to be an extremely potent compound causing lethal mutagenesis. The pronucleotide technologies, the chemical synthesis, the biophysical properties and the biological effects of the compounds will be addressed as well.

Keywords: Favipiravir; Influenza; Nucleoside analog; Nucleoside triphosphates; Prodrug; Pronucleotides; RNA polymerases; SARS-CoV-2; TriPPPro.

Fig. 1

Mode of action of Favipiravir (T-705) and T-1105 and the corresponding nucleoside prodrug approach.

Fig. 2

Elongation reaction with the PT substrate in with each 50 μM of GTP, UTP and CTP in the absence of ATP (left) shows rapid addition of the first uracil followed by slow misincorporation of the GTP:U mismatch. If 1 μM T-1105-RTP it gets incorporated on a similar timescale as the native GTP:U mismatch (right).

Scheme 1

Synthesis of T-705-ribonucleoside-5′-monophosphate 3: (A) HMDS, (NH₄)₂SO₄, 60 min, 140°C; (B) tetra-O-acetyl-β-D-ribofuranose, SnCl₄, CH₃CN, 7 h, rt.; (C) Bu₂SnO, MeOH, 24 h, 80°C, overall yield: 40%. (D) phosphorous oxychloride, pyridine, H₂O, 30 min, 0°C. (E) addition of 2, 20 min, 0°C. (F) ice H₂O, 30 min, aqueous saturated NH₄HCO₃ (pH 7.0), overall yield: 50%.

Scheme 2

Synthesis of T-1106 5: (A) tetra-O-acetyl-β-d-ribofuranose, N,O-bis(trimethylsilyl)acetamid, CH₃CN, 30 min, rt.; (B) trimethylsilyl trifluoromethansulfonate, CH₃CN, 44 h, rt.; (C) MeOH, H₂O, Et₃N, 6 h, rt., overall yield: 55%.

Scheme 3

Synthesis of T-1106-DP 12 and -TP 13: (A) DMTrCl, C₅H₅N, CH₃CN, 2 h, rt.; (B) TEA, Ac₂O, C₅H₅N, CH₃CN, 16 h, rt., 75% over two steps; (C) CH₃OH, p-toluenesulfonic acid, 10 min, rt., 84%; (D) 1. bis(9H-fluoren-9-ylmethyl)-diisopropylaminophosphite (in CH₂Cl₂), DCI, CH₃CN, 15 min, rt.; 2. TBHP, CH₃CN, 10 min, rt.; (E) TEA, CH₃CN, 72 h, rt.; then CH₃OH/H₂O/TEA 7:3:1, 24 h, rt., 72% over steps (D–E); (F) bis(9H-fluoren-9-ylmethyl)-diisopropylamino-phosphite (in CH₂Cl₂), DCI, DMF, 10–20 min, rt.; (G) TBHP, DMF, 5 min, rt.; (H) TEA, CH₃CN, 10–20 min, rt.; (I) TEA, H₂O, CH₃CN, 24–48 h, rt.

Scheme 4

Synthesis of cycloSal-d4TMP triesters 18 and T-1106-cycloSal-prodrugs 20,21. Method A: PCl₃, pyridine, Et₂O, − 10°C, 2 h, 50–85%; Method B: (i) d4T, DIPEA, CH₃CN, − 20°C to rt., 1 h; (ii) TBHP, CH₃CN, − 20°C to rt., 1 h, 50–73%; Method C: diisopropylamine, Et₂O, 0°C, 30 min, 50–85%; Method D: (i) d4T, pyridinium chloride, tetrazole or imidazolium triflate, CH₃CN, 0°C, 30 min; (ii) TBHP, CH₃CN, rt., 1 h, 70–95%; Method E: O₂, toluene, rt., 16 h, quant; Method F: POCl₃, TEA, CH₂Cl₂, − 60°C to rt., 61%; Method G: d4T, pyridine, − 50°C, 4 h, 83–95%; Method H: (i) cycloSal-phosphochloridite, TEA, CH₃CN, DMF, 15–20 min, rt.; (ii) TBHP, CH₃CN, DMF, 1–2 h, rt.; (iii) TFA, CH₂Cl₂, TFA, 1 h, rt. CycloSal-T-1105-RMP prodrug 21 was obtained in 87% purity.

Scheme 5

Hydrolysis pathways of the cycloSal-d4TMP triesters 18 (left) and T-1106-cycloSal-prodrugs 20,21 (right).

Fig. 3

Chemical hydrolysis of cycloSal-pronucleotides 20,21 in pH 7.3 phosphate buffer at 37°C.

Scheme 6

Synthesis and hydrolysis of cycloSal-AZTDPs 24.

Scheme 7

Synthesis of the DiPPro-d4TDPs 28.

Scheme 8

Hydrolysis mechanism of DiPPro-d4TDPs 28.

Scheme 9

Synthesis of non-symmetric DiPPro-nucleoside diphosphates 34.

Scheme 10

Hydrolysis pathways of non-symmetric DiPPro-d4TDPs 34.

Fig. 4

Metabolic profiles of T-1106-DiPPro-prodrug 36a (A) and T-1106-TriPPPro-compound 52a (B). T-1106-prodrugs 36a (A) and 52a (B) were incubated at 450 μM in the absence or presence of PLE (3 U per mL).

Fig. 5

Metabolic conversion of T-1106-DiPPro-prodrugs 36 and T-1106-TriPPPro-prodrugs 52 in MDCK and MDCK-TG^res cell extracts. Prodrugs 36,52 (500 μM) were incubated in cell extract and after 2 h, proteins were precipitated.

Scheme 11

Synthesis and hydrolysis mechanism of T-1106-DiPPro-prodrugs 36.

Scheme 12

Synthesis of TriPPPro-prodrugs 38 using via P(III) and P(V) chemistry. (i) Triethylamine, THF, 0 C-rt, 20 h; (ii) 1. 5-chlorosaligenylchlorophosphite, N,N-diisopropylethylamine, CH₃CN, − 20 C-rt, 3 h, 2. t-BuOOH in n-decane, 0 C-rt, 30 min; (iii) (H₂PO₄)Bu₄N, DMF, rt., 20 h; (iv) 1. DCI, CH₃CN, rt., 1 min, 2. t-BuOOH in n-decane, 0 C-rt,15 min; (v) pyridine, 38°C, 2 h; (vi) 1. NCS, CH₃CN, rt., 2 h, 2. (H₂PO₄)Bu₄N, CH₃CN, rt., 1 h (vii) 1. TFAA, Et₃N, CH₃CN, 0 C, 10 min, 2. 1-methylimidazole, Et₃N, CH₃CN, 0 C-rt, 10 min, 3. NMP, rt., 1–3 h.

Scheme 13

Synthesis of mono-masked NTPs 43.

Scheme 14

Hydrolysis mechanism of TriPPPro-prodrugs 38 in PBS (pH 7.3) (shown for d4T as an example).

Scheme 15

Synthesis of TriPPPro-prodrugs 47 (d4T as an example) and intermediates 51 via H-phosphonate route.

Scheme 16

Hydrolysis and delivery mechanism of TriPPPro-d4TTPs 47.

Scheme 17

Synthesis and hydrolysis mechanism of T-1106-TriPPPro-prodrugs 52.