Synthesis and evaluation of novel flexible nucleoside analogues against H1N1 influenza virus

Xiyao Hu; Yufei Yan; Shuli Liang; Fengxia Ren; Zixing Yu; Yabin Song; Jingchao Cheng; Xiaoping Liu; Baogang Wang; Weiguo Shi

doi:10.25259/AJC_352_2025

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Original Article

ARTICLE IN PRESS

doi:

10.25259/AJC_352_2025

Synthesis and evaluation of novel flexible nucleoside analogues against H1N1 influenza virus

Xiyao Hu^a,b, Yufei Yan^b,d, Shuli Liang^c, Fengxia Ren^b, Zixing Yu^b, Yabin Song^c, Jingchao Cheng^b, Xiaoping Liu^d, Baogang Wang^c,*, Weiguo Shi^b,*

aDepartment of Medicinal Chemistry, Qingdao University, No.308 Ningxia Road, Qingdao, Shandong Province, China

bState Key Laboratory of National Security Specially Needed Medicines, 27 Tai-Ping Road, Beijing, China

cState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medicine Sciences, Academy of Military Sciences, 20 Dong-Da Street, Beijing, China

dKey Laboratory of Structure-Based Drug Design & Discovery of the Ministry of Education, Shenyang Pharmaceutical University, Shenyang, China

* Corresponding authors: E-mail address: wbg024@126.com (B. Wang), shiwg1988@126.com (W. Shi)

Received: 2025-04-11, Accepted: 2025-12-09, Epub ahead of print: 2026-03-27,

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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

Flexible nucleoside analogues (fleximers) are viewed as a new class of inhibitors against numerous highly lethal RNA virus infections and pandemics. In this work, we chemically synthesized a series of novel fleximers featuring a flexible base moiety connected to a pentose sugar ring and tested them for activity against H1N1 influenza virus. The novel flexible nucleoside analogues exhibit low micromolar levels of antiviral activity. The most potent 7c is able to potently inhibit the multiplication of the H1N1 influenza strains with an in vitro anti-H1N1 EC₅₀ of 1.371 μM. Most importantly, 7c demonstrated an excellent safety profile with cytotoxicity CC₅₀ >200μM and an in vivo mouse LD₅₀ > 2000 mg/kg. As such, 7c could serve as a novel lead compound for the further development of antiviral drug candidate.

Keywords

Flexible nucleoside analogues

H1N1 influenza virus

Lead compound

Nucleoside

RNA virus

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1. Introduction

In recent years, the outbreaks of highly pathogenic RNA viruses, such as influenza viruses and SARS-CoV-2, have posed significant global public health challenges [1,2]. The emerging and re-emerging of RNA viruses with approximately 2–3 novel variant strain annually, highlights the urgent need for new antiviral therapeutics [3]. There are challenges for the finding of novel antiviral agents such as rapid drug resistance, the difficulty for the development of broad-spectrum antiviral compounds and the limited therapeutic window [4-6]. Therefore, new chemical entities with diverse molecular scaffolds need to be identified and confirmed as novel candidates against various RNA virus [7-9].

Nucleoside analogues remain the most important and effective class of anti-RNA viral agents. Chemical modification of nucleoside molecules represents an efficient method for obtaining the novel antiviral drugs [10-12]. To date, over 40 nucleoside analogues have been approved by the FDA for the treatment of various viral infections [13].

Here, we chemically synthesized a series of novel flexible nucleoside analogues feature of a flexible base moiety connected to a pentose sugar ring and tested them for activity against H1N1 influenza virus. By splitting the rigid purine base of nucleoside analogues into a five-membered imidazole ring and a six-membered pyrimidine ring, connected via a flexible C–C bond, the free rotation of the C–C bond increases flexibility, enabling the flexible nucleoside to interact with more target sites [14-18]. Compared to the acyclic fleximers, the novel flexible five-membered ring nucleoside analogues we described here expand the molecular scaffold diversity of nucleotide monomers, which is crucial for design and synthesis of both antiviral nucleoside agents and other nucleic acid therapeutics.

2. Materials and Methods

2.1. Chemistry

TLC analysis was performed on pre-coated silica plates (GF254) using UV light (λ=254 nm) for reaction monitoring. ¹H NMR spectra and ¹³C NMR spectra were recorded on a 600 MHz spectrometer and a 151 MHz spectrometer, respectively, in which DMSO-d₆ and Methanol-d₄ were used as the solvent. Quantitative analysis relied on TMS as the calibration standard. Chemical shifts (δ) and coupling constants (J values) are expressed in ppm and Hz, respectively. Peak multiplicity is recorded as follows: m (multiplet), s (singlet), d (doublet), t (triplet), and q (quartet). The Agilent 1260/G6230A mass spectrometer generated high-resolution mass spectra (HRMS) profiles for all synthesized compounds. Accurate masses are reported for the molecularion [M + H]⁺. The sample purity was analyzed on an Agilent HPLC system using a reversed-phase C-18 column (250 mm × 4.6 mm × 5 μm). All analyses were conducted at a 25°C and used a 1 mL/min flow rate. The HPLC eluent conditions were as follows: initially, a mixture of 10% MeOH/ 90% water was used; over a period of 30 min, the gradient of MeOH increased to 90%. The UV detector was performed at 215nm, the injection volume was 1 μL. All reactions were carried out with commercially available reagents/solvents in their native purity.

2.2. Synthesis of compounds

2.2.1. (2R, 3R, 4R, 5R)-2-((Benzoyloxy)methyl)-5-(4,5-diiodo-1H-imidazol-1-yl) tetra hydrofuran-3,4-diyl dibenzoate (2)

Under nitrogen, compound 1 and 4, 5-diiodoimidazole (3.20 g, 9.918 mmol) were mixed in anhydrous CH₃CN (90 mL), followed by N, O-bis(trimethylsilyl)acetamide (5 mL, 19.8 mmol), stirred at 25°C for 4 h and cooled it to 0°C, trimethylsilyl triflate (2 mL, 10.910 mmol,) added dropwise. The solvent was removed after stirred at 60°C overnight, the unpurified product redissolved in CH₂Cl₂ washing with saturated NaHCO₃ solution, saturated salt solution, dried with anhydrous Na₂SO₄. Purification by flash chromatography (PE/EA 7:2) afforded compound 2 as off-white solid (7.00 g, 92.4%).

2.2.2. 2-((2R, 3R, 4S, 5R)-2-(4, 5-Diiodo-1H-imidazol-1-yl)-5-(hydroxymethyl)tetra hydrofuran-3,4-diol) (3)

2 (5.00 g, 6.545 mmol) in methanol (130 mL) was mixed with MeONa (0.71 g, 13.090 mmol) under N₂ condition. Following 1 h of continuous stirring, the reaction was complete. Chromatographic purification (20:1 DCM / MeOH) afforded coumpound 3 as brown oil (2.58 g, 87.2%).

2.2.3. 1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl) -4,5-diiodo-1H-imidazole (4)

60% NaH (0.45 g, 11.065 mmol) and 3 (2.00 g, 4.426 mmol) in anhydrous DMF (80 mL) were mixed at 0°C with N₂ condition. Stirring 2 h, BnBr (2 mL, 17.704 mmol) and tetrabutylammonium iodide (0.16 g, 0.443 mmol) were added. After mixed 18 h, the solvent was removed. Flash chromatography (PE : EA = 5 : 1) to get 4 as white solid (2.53 g, 79.0%).

2.2.4. 1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)-4-iodo-1H-imidazole (5)

1 M EtMgBr (1.66 mL, 1.662 mmol) was instilled into an anhydrous THF solution of 4 (1.00 g, 1.385 mmol) at 0°C under argon. Stirring for 30 min, the reaction was terminated by saturated NH₄Cl (aq). Flash chromatography (PE : EA = 3 : 1) to afford 5 as yellow oil (0.46 g, 55.2%).

2.2.5. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-2,4-dimethoxypyrimidine (6a)

Compound 5 (1.00 g, 1.680 mmol) dissolved in anhydrous THF (80 mL), 2, 4-dimethoxypyrimidin-5-ylboronic acid pinacol ester (0.49 g, 1.850 mmol), PdCl₂(PPh₃)₂ (0.26 g, 0.370 mmol) and K₂CO₃ (0.79 g, 5.710 mmol) were mixed sequentially with N₂, deionized water 10 mL was introduced, refluxed for 4 h. After solvent removing, residuum extracted with EA, washed with brine. Flash chromatography (PE : EA = 1 : 2) afforded 6a as a brown oil (0.82 mg, 80.4%).

2.2.6. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-4-methylpyrimidin-2-amine (6b)

Similar to the synthesis of 6a, compound 5 (1.00 g, 1.680 mmol) was reacted with 2-amino-4-methylpyrimidin-5-ylboronic acid pinacol ester (0.44 mg, 1.850 mmol). Compound 6b was isolated as brown oil (0.83 g, 85.5%).

2.3. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-4-(trifluoromethyl)pyrimidin-2-amine (6c)

Compound 5 (1.00 g, 1.680 mmol) and 2-amino-4-(trifluoromethyl)pyrimidin-5-ylboronic acid pinacol ester (0.54 g, 1.850 mmol) underwent Suzuki coupling under identical conditions as 6a. Chromatographic purification (PE/EA = 1:2) yielded 6c as brown oil (0.80 g, 75.4%).

2.3.1. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)pyrimidin-2-amine (6d)

Compound 5 (1.00 g, 1.680 mmol) was subjected to the same reaction sequence using 2-aminopyrimidin-5-ylboronic acid pinacol ester (0.41 g, 1.850 mmol). Column chromatography (PE/EA = 1:2) provided 6d as brown oil (0.82 g, 85.7%).

2.3.2. (2R, 3R, 4S, 5R)-2-(4-(2, 4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5- (hydroxylmethyl)tetrahydrofuran-3,4-diol (7a)

6a (0.70 g, 1.150 mmol) in anhydrous CH₂Cl₂ solution (50 mL), boron trichloride (4 mL, 4.610 mmol) incorporated into the mixture at -30°C. A methanol quench (2 mL) was applied to the system. 7a was purified by column chromatography (10:1 DCM/MeOH) as white solid (0.21 g, 55.0% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₁₄H₁₈N₄O₆ [M + H]⁺ 339.1304, found 339.1305. ¹H NMR (600 MHz, DMSO-d₆) δ 8.86 (s,1H), 7.97 (d, J = 1.3 Hz, 1H), 7.74 (d, J = 1.3 Hz, 1H), 5.61 (d, J = 5.7 Hz, 1H), 5.44 (br s, 1H), 5.18 (br s, 1H), 5.09–5.07 (m, 1H), 4.20 (t, J = 5.5 Hz, 1H), 4.09–4.08 (m, 1H), 4.05 (s, 3H), 3.97–3.94 (m, 1H), 3.93 (s, 3H), 3.63 (dt, J = 11.7, 3.5 Hz, 1H), 3.57 (dt, J = 11.7, 4.0 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.7, 163.5, 155.0, 137.2, 133.1, 116.9, 110.0, 90.1, 85.9, 75.9, 70.9, 61.7, 54.9, 54.4.

2.3.3. (2R, 3R, 4S, 5R)-2-(4-(2-Amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5- (hydroxymethyl)tetrahydrofuran-3,4-diol (7b)

Following the protocol for 7a, Compound 6b (0.50 g, 0.866 mmol) was subjected to identical conditions. Purification via column chromatography (DCM/MeOH = 10:1) yielded 7b as yellow solid (0.13 g, 47.0% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₁₃H₁₇N₅O₄ [M + H]⁺ 308.1359, found 308.1413. ¹H NMR (600 MHz, DMSO-d₆) δ 8.45 (s, 1H), 7.94 (d, J = 0.9 Hz, 1H), 7.52 (d, J = 0.9 Hz, 1H), 6.48 (s, 2H), 5.59 (d, J = 5.8 Hz, 1H), 5.43 (br s, 1H), 5.17 (br s, 1H), 5.02 (t, J = 5.4 Hz, 1H), 4.24–4.22 (m, 1H), 4.08 (t, J = 4.2 Hz, 1H), 3.91 (q, J = 3.8 Hz, 1H), 3.64–3.60 (m, 1H), 3.57–3.53 (m, 1H), 2.43 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 163.4, 161.8, 156.5, 137.0, 136.5, 116.3, 114.4, 89.5, 85.3, 75.2, 70.4, 61.4, 23.4.

2.3.4. (2R, 3R, 4S, 5R)-2-(4-(2-Amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol- 1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(7c)

Compound 6c (0.80 g, 1.267 mmol) was processed analogously to 7a. Chromatographic purification (DCM/MeOH = 10:1) obtained 7c as yellow solid (0.26 g, 57.5% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₁₃H₁₄F₃N₅O₄ [M + H]⁺ 362.1076, found 362.1074. ¹H NMR (600 MHz, DMSO-d₆) δ 8.67 (s, 1H), 7.96 (d, J = 1.1 Hz, 1H), 7.27 (s, 2H), 7.52 (s, 1H), 5.59 (d, J = 5.7 Hz, 1H), 5.40 (d, J = 6.2 Hz, 1H), 5.16 (d, J = 4.3 Hz, 1H), 4.99 (t, J = 5.2 Hz, 1H), 4.18 (q, J = 5.5 Hz, 1H), 4.05 (q, J = 3.7 Hz, 1H), 3.90 (q, J = 3.7 Hz, 1H), 3.60 (ddd, J = 11.8, 5.3, 3.8 Hz, 1H), 3.53 (ddd, J = 11.8, 5.1, 3.8 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.1, 161.9, 149.9 (q, J = 33.6 Hz), 136.8, 133.8, 121.2 (q, J = 276.5 Hz), 115.8 (q, J = 3.2 Hz), 114.3, 89.6, 85.5, 75.5, 70.5, 61.3.

2.3.5. (2R, 3R, 4S, 5R)-2-(4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxyl methyl)tetrahydrofuran-3,4-diol (7d)

Using the same procedure as for 7a, Compound 6d (0.50 g, 0.888 mmol) was converted to 7d. After column chromatography (DCM/MeOH = 10:1), yellow solid 7d was obtained (0.13 g, 46.1% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₁₂H₁₅N₅O₄ [M + H]⁺ 294.1202, found 294.1196. ¹H NMR (600 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.20 (s, 1H), 7.90 (d, J = 1.3 Hz, 1H), 7.73 (d, J = 1.3 Hz, 1H), 6.80 (s, 1H), 6.59 (s, 2H), 5.55 (d, J = 5.9 Hz, 1H), 5.20 (s, 1H), 5.05 (s, 1H), 4.22 (t, J = 6.6 Hz, 1H), 3.92 (t, J = 3.9 Hz, 1H), 3.89 (q, J = 4.0 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.12, 152.83, 140.69, 139.50, 126.37, 115.37, 90.57, 85.65, 76.28, 72.15, 61.71.

2.3.6. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2,4-dimethoxypyrimidin-5-yl)-1H- imidazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (8a)

7a (0.30 g, 0.887 mmol), 4-dimethylaminopyridine (0.11 g, 0.090 mmol) and acetic anhydride (0.36 g, 3.548 mmol) in anhydrous DMF (40 mL) were mixed and stirred for 2 h at rt. Then, water and EA (1 : 1) were added to the mixture. The EA phase was washed twice with saturated NaHCO₃ solution, saturated salt solution, dried with anhydrous Na₂SO₄.The crude sample purified with 200–300 mesh silica gel column chromatography (20 : 1, DCM : MeOH). Compound 8a was provided as white solid (0.24 g, 59.5% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₀H₂₄N₄O₉ [M + H]⁺ 465.1621, found 465.1620. ¹H NMR (600 MHz, CD₃OD) δ 8.84 (s, 1H), 7.98 (d, J = 1.3 Hz, 1H), 7.69 (d, J = 1.3 Hz, 1H), 6.03 (d, J = 5.8 Hz, 1H), 5.52 (t, J = 5.7 Hz, 1H), 5.44 (dd, J = 5.5, 4.0 Hz, 1H), 4.48 (q, J = 3.6 Hz, 1H), 4.39 (dd, J = 12.5, 3.2 Hz, 1H), 4.36 (dd, J = 12.5, 3.9 Hz, 1H), 4.16 (s, 3H), 4.03 (s, 3H), 2.14 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 172.0, 171.4, 171.1, 168.6, 165.2, 156.3, 138.0, 135.1, 117.1, 110.0, 89.2, 81.9, 75.7, 72.0, 64.3, 55.5, 54.9, 20.7, 20.4, 20.2.

2.3.7. (2R, 3R, 4R, 5R)-2-(4-(2, 4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((propionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate (8b)

8a (0.30 g, 0.887 mmol) was solubilized in DMF anhydrous (10 mL), propionic anhydride (0.46 g, 3.548 mmol) was added under conditions analogous to those for 7a. Chromatographic purification (20:1 DCM/MeOH) provided 8b as white solid (0.29 g, 64.6% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₃H₃₀N₄O₉ [M + H]⁺ 507.2091, found 507.2090. ¹H NMR (600 MHz, CD₃OD) δ 8.85 (s, 1H), 7.66 (d, J = 1.3 Hz, 1H), 7.57 (d, J = 1.3 Hz, 1H), 6.04 (d, J = 8.1 Hz, 1H), 5.56 (t, J = 5.7 Hz, 1H), 5.47 (dd, J = 5.5, 3.9 Hz, 1H), 4.60 (q, J = 3.6 Hz, 1H), 4.50 (dd, J = 12.5, 3.5 Hz, 1H), 4.43 (dd, J = 12.5, 3.9 Hz, 1H), 4.18 (s, 3H), 4.04 (s, 3H), 2.48–2.42 (m, 6H), 1.17 (t, J = 3.5 Hz, 3H), 1.14 (t, J = 3.5 Hz, 3H), 1.12 (t, J = 3.5 Hz, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 177.72, 177.72,177.72, 156.28,156.28, 137.95, 133.81, 133.12, 129.95, 117.10, 89.14, 81.98, 75.63, 71.93, 64.21, 55.47, 54.96, 31.70,31.70, 29.80, 18.55,18.55 18.55.

2.3.8. (2R, 3R, 4R, 5R)-2-(4-(2, 4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8c)

Replacing acetic anhydride with isobutyric anhydride (0.56 g, 3.548 mmol), 7a (0.30 g, 0.887 mmol) was acylated in anhydrous DMF. Prepared according to the procedure for 8a, 8c was obtained as yellow solid (0.30 g, 61.7% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₆H₃₆N₄O₉ [M + H]⁺ 549.2540, found 549.2558. ¹H NMR (600 MHz, CD₃OD) δ 8.87 (s, 1H), 8.02 (d, J = 1.3 Hz, 1H), 7.69 (d, J = 1.3 Hz, 1H), 6.12(d, J = 6.1 Hz, 1H), 5.54 (t, J = 5.6 Hz, 1H), 5.47 (dd, J = 5.8,4.0 Hz, 1H), 4.58–4.51 (m, 1H),4.50 (dd, J = 11.8, 4.5 Hz, 1H), 4.45 (dd, J = 11.9, 4.4 Hz, 1H), 4.17 (s, 3H), 4.08 (s, 3H),2.39–2.34 (m, 3H) 1.26 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 177.21, 176.62, 176.24, 165.55, 164.46, 155.30, 141.36, 140.56, 123.80, 111.05, 88.13, 80.50, 78.72, 74.37, 64.27, 54.68, 52.93, 34.30, 34.17, 34.16, 19.02, 18.64, 18.63.

2.3.9. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2-amino-4-methylpyrimidin-5-yl)-1H- imidazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (8d)

7b (0.40 g, 1.302 mmol) and DMAP (0.02 mg, 0.130 mmol) in DMF anhydrous (40 mL) was treated with acetic anhydride (0.53 g, 5.208 mmol) and stirred for 2 h at rt, then added H₂O/EA (1:1, 50 mL) and EA (3 × 30 mL). The organic solution phase was separated and washed. After purification (20:1 DCM/MeOH) yielded white solid 8d (0.39 g, 69.2% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₁₉H₂₃N₅O₇ [M + H]⁺ 434.1675, found 434.1692. ¹H NMR (600 MHz, CD₃OD) δ 8.36 (s, 1H), 8.00 (d, J = 1.3 Hz, 1H), 7.41 (d, J = 1.3 Hz, 1H), 6.03 (d, J = 5.7 Hz, 1H), 5.53 (t, J = 5.7 Hz, 1H), 5.42 (dd, J = 5.6, 4.2 Hz, 1H), 4.46 (q, J = 3.9 Hz, 1H), 4.41 (dd, J = 12.3, 4.1 Hz, 1H), 4.37 (dd, J = 12.3, 3.4 Hz, 1H), 2.45 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 172.1, 171.4, 171.1, 167.3, 163.4, 158.5, 138.8, 138.0, 117.9, 116.3, 89.3, 81.8, 75.7, 71.9, 64.3, 23.3, 20.7, 20.4, 20.2.

2.3.10. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((propionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate (8e)

Using propionic anhydride (0.68 g, 5.208 mmol) instead of acetic anhydride, 7b (0.40 g, 1.302 mmol) was processed as above described for 8d. White solid 8e was obtained (0.38 g, 61.7% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₂H₂₉N₅O₇ [M + H]⁺ 476.2145, found 476.2143. ¹H NMR (600 MHz, DMSO-d₆) δ 8.45 (s, 1H), 8.00 (d, J = 1.2 Hz, 1H), 7.55 (d, J = 1.2 Hz, 1H), 6.55 (s, 2H), 6.04 (d, J = 6.1 Hz, 1H), 5.55 (t, J = 6.0 Hz, 1H), 5.40 (dd, J = 5.9, 3.7 Hz, 1H), 4.35 (dq, J = 7.1, 3.5 Hz, 2H), 4.32–4.29 (m, 1H), 2.41 (s, 3H), 2.40–2.32 (m, 6H), 1.06 (t, J = 7.5 Hz, 3H), 1.03 (t, J = 7.5 Hz, 3H), 1.00 (t, J = 7.5 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.93, 173.26, 173.00, 165.86, 161.89, 157.03, 136.46, 116.43, 114.78, 114.73, 87.85, 80.46, 74.23, 70.38, 62.77, 29.31, 26.74, 26.57, 26.39, 7.90, 7.84, 7.73.

2.3.11. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8f)

7b (0.40 g, 1.302 mmol) underwent acylation with isobutyric anhydride (0.82 g, 5.208 mmol) in anhydrous DMF. Analogous workup to 8a provided a white solid 8f (0.50 g, 74.2% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₅H₃₅N₅O₇ [M + H]⁺ 518.2614, found 518.2610. ¹H NMR (600 MHz, DMSO-d₆) δ 8.44 (s, 1H), 7.99 (d, J = 1.3 Hz, 1H), 7.52 (d, J = 1.3 Hz, 1H), 6.54 (s, 2H), 6.05 (d, J = 6.1 Hz, 1H), 5.55–5.40 (m, 2H), 4.36 (t, J = 4.1 Hz, 1H), 4.33 (d, J = 5.0 Hz, 2H), 2.63 –2.54 (m, 3H), 2.41 (s, 3H), 1.14 (d, J = 3.5 Hz, 3H), 1.13 (d, J = 3.5 Hz, 3H), 1.11 (d, J = 6.9 Hz, 3H), 1.09 (d, J = 7.0 Hz, 3H), 1.07 (d, J = 7.0 Hz, 3H), 1.05 (d, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 177.21, 176.62, 176.24, 161.27, 160.71, 152.68, 141.11, 140.36, 125.25, 119.14, 88.13, 80.50, 78.72, 74.37, 64.27, 34.30, 34.17, 34.16, 26.77, 19.02, 18.64, 18.63.

2.3.12. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2-amino-4-(trifluoromethyl)pyrimidin- 5-yl)-1H-imidazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (8g)

7c (0.40 g, 1.110 mmol) dissolved in anhydrous DMF (40 mL), DMAP (0.01 g, 0.110 mmol), and acetic anhydride (0.45 g, 4.430 mmol) were mixed and stirred for 2h, 50 mL of a 1:1 H₂O/EA mixture was introduced to dilute the reaction system. The organic solution phase was separated and washed. Column chromatography (20:1 DCM/MeOH) gave a white solid 8g (0.36 g, 67.0% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₁₉H₂₀F₃N₅O₇ [M + H]⁺ 488.1393, found 488.1388. ¹H NMR (600 MHz, CD₃OD) δ 8.58 (s, 1H), 8.00 (d, J = 1.3 Hz, 1H), 7.42, (s, 1H), 6.04 (d, J = 5.8 Hz, 1H), 5.48 (t, J = 5.7 Hz, 1H), 5.41 (dd, J = 5.5, 4.0 Hz, 1H), 4.48 (q, J = 3.6 Hz, 1H), 4.41 (dd, J = 12.4, 3.7 Hz, 1H), 4.36 (dd, J = 12.4, 3.7 Hz, 1H), 2.13 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 172.0, 171.4, 171.1, 163.9, 163.5, 153.7 (q, J = 33.8 Hz), 138.0, 136.0, 122.4 (q, J = 275.9 Hz), 117.2 (q, J = 3.5 Hz), 115.2, 89.2, 81.9, 75.9, 71.9, 64.3, 20.6, 20.4, 20.2.

2.3.13. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol- 1-yl)-5-((propionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate (8h).

Substituting acetic anhydride with propionic anhydride (0.58 g, 4.430 mmol), 7c (0.40 g, 1.110 mmol) was reacted under identical conditions to 8g. Isolation by chromatography afforded a white solid 8h (0.40 g, 68.1% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₂H₂₆F₃N₅O₇ [M + H]⁺ 530.1862, found 530.1858. ¹H NMR (600 MHz, CD₃OD) δ 8.57 (s, 1H), 8.00 (d, J = 1.4 Hz, 1H), 7.41 (s, 1H), 6.03 (d, J = 5.9 Hz, 1H), 5.50 (t, J = 5.7 Hz, 1H), 5.43 (dd, J = 5.5, 3.9 Hz, 1H), 4.48 (q, J = 3.5 Hz, 1H), 4.43 (dd, J = 12.4, 3.6 Hz, 1H), 4.37 (dd, J = 12.4, 3.6 Hz, 1H), 2.46–2.37 (m, 6H), 1.16 (t, J = 7.6 Hz, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.11 (t, J = 7.6 Hz, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 175.3, 174.7, 174, 164.0, 163.5, 153.8 (q, J = 33.7 Hz), 138.0, 136.0, 122.4 (q, J = 275.9 Hz), 117.2 (q, J = 3.2 Hz), 115.2, 89.2, 82.1, 75.8, 71.9, 64.3, 28.1, 28.0, 27.8, 9.3, 9.3, 9.2.

2.3.14. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol- 1-yl)-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8i)

7c (0.40 g, 1.110 mmol) was treated with isobutyric anhydride (0.70 g, 4.430 mmol) in anhydrous DMF. Analogous workup to 8g, white solid 8i was obtained (0.40 g, 63.7% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₅H₃₂F₃N₅O₇ [M + H]⁺ 571.2254, found 572.2332. ¹H NMR (600 MHz, CD₃OD) δ 8.56 (s, 2H), 8.01 (d, J = 1.3 Hz, 1H), 7.41 (d, J = 1.3 Hz, 1H), 6.05 (d, J = 6.1 Hz, 1H), 5.48 (t, J = 5.8 Hz, 1H), 5.42 (dd, J = 5.4, 3.6 Hz, 1H), 4.49 (q, J = 3.4 Hz, 1H), 4.44 (dd, J = 12.4, 3.6 Hz, 1H), 4.35 (dd, J = 12.4, 3.0 Hz, 1H), 2.68–2.57 (m, 3H), 1.22 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 178.0, 177.1, 176.8, 164.0, 163.5, 153.8 (q, J = 33.8 Hz), 137.9, 136.0, 122.4 (q, J = 276.0 Hz), 117.1 (q, J = 3.2 Hz), 115.2, 89.1, 82.3, 75.8, 71.8, 64.4, 35.2, 35.0, 34.9, 19.4, 19.3, 19.2, 19.1, 19.1, 19.1.

2.3.15. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2-aminopyrimidin-5-yl)-1H-imidazol- 1-yl)tetrahydrofuran-3,4-diyl diacetate (8j)

7d (0.30 g, 1.020 mmol) solution of anhydrous DMF (40 mL) was sequentially treated with DMAP (0.01 mg, 0.10 mmol), acetic anhydride (0.42 g, 4.090 mmol). The reaction was stirred 2h and quenched with H₂O/EA (1:1, 40 mL). Purification yielded a white solid 8j (0.27 g, 62.2% yield, >98.0% HPLC purity). HR-ESIMS (ESI) m/z: calcd. for C₁₈H₂₁N₅O₇ [M + H]⁺ 420.1519, found 420.1518. ¹H NMR (600 MHz, CD₃OD) δ 8.63 (s, 2H), 7.97 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 1.3 Hz, 1H), 6.028 (d, J = 5.5 Hz, 1H), 5.50 (t, J = 5.5 Hz, 1H), 5.43 (dd, J = 5.6, 4.4 Hz, 1H), 4.50 (s, 1H), 4.47 (q, J = 4.0 Hz, 1H), 4.40–4.39 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 170.6, 169.9, 169.7, 162.2, 155.9, 154.8, 154.8, 137.0, 117.3, 111.7, 87.9, 80.2, 74.3, 70.4, 62.8, 19.2, 18.9, 18.8.

2.3.16. (2R, 3R, 4R, 5R)-2-(4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-((pro pionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate(8k)

Using propionic anhydride (0.53 g, 4.080 mmol), 7d (0.30 g, 1.020 mmol) was acylated as per the 8j procedure. White solid 8k was isolated (0.28 g, 59.5% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₁H₂₇N₅O₇ [M + H]⁺ 462.1988, found 462.1985. ¹H NMR (600 MHz, CD₃OD) δ 8.62 (s, 2H), 7.98 (d, J = 1.4 Hz, 1H), 7.65 (d, J = 1.4 Hz, 1H), 6.02 (d, J = 5.5 Hz, 1H), 5.52 (t, J = 5.5 Hz, 1H), 5.45 (dd, J = 5.6, 4.3 Hz, 1H), 4.46 (q, J = 3.8 Hz, 1H), 4.41 (t, J = 3.5 Hz, 2H), 2.46–2.36 (m, 6H), 1.16 (t, J = 7.6 Hz, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.11 (t, J = 7.5 Hz, 3H). ¹³C NMR (151 MHz,Methanol-d₄) δ 175.4, 174.7, 174.5, 163.7, 156.3, 156.3, 138.5, 129.4, 118.72, 113.2, 89.4, 81.7, 75.8, 71.8, 64.2, 28.2, 28.0, 27.8, 9.4, 9.3, 9.2.

2.3.17. (2R, 3R, 4R, 5R)-2-(4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-((isobutyryl oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8l)

Reaction of 7d (0.30 g, 1.020 mmol) with isobutyric anhydride (0.65 g, 4.080 mmol,) in anhydrous DMF, followed by the 8j protocol, provided white solid 8l (0.31 g, 61.0% yield, >98.0% HPLC purity). HR-ESIMS m/z: calcd. C₂₄H₃₃N₅O₇ [M + H]⁺ 504.2458, found 504.2460. ¹H NMR (600 MHz, CD₃OD) δ 8.68 (s, 2H), 7.97 (d, J = 1.3 Hz, 1H), 7.65 (d, J = 1.4 Hz, 1H), 6.03 (s, 1H), 5.51 – 5.50 (m, 1H), 5.44 – 5.43 (m, 1H), 4.57 (s, 1H), 4.42 (dd, J = 4.0, 2.3 Hz, 1H), 4.39 (dd, J = 3.3, 1.3 Hz, 1H), 2.64 (m, 3H), 1.21 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 177.21, 176.62, 176.24, 162.12, 152.83, 140.64, 139.52, 126.47, 115.37, 88.04, 80.50, 78.72, 74.37, 64.27, 34.30, 34.17, 34.16, 19.02, 18.64, 18.63.

2.4. Statistical analysis

Prism 8 software (GraphPad, San Diego, CA) was used as statistical analyses. Data are expressed as mean ± SEM. Concentration-response curves were analyzed by nonlinear regression (three-parameter model) to determine EC₅₀ values. To compare treatment effects versus baseline activity, paired two-tailed Student’s t-tests were employed. A probability value of less than 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Synthesis of novel flexible nucleosides

HP083, an acyclic flexible nucleoside molecule reported by the Seley-Radtke laboratory, has demonstrated broad-spectrum antiviral activity at low micromolar levels against various RNA viruses, including EBOV, MERS-CoV, ZIKV, and DENV [19-21]. We report a novel class of flexible nucleoside analogues containing a five-membered sugar ring (Figure 1). Structural modifications were strategically implemented at the 2- and 4-positions of the pyrimidine moiety, incorporating amino, trifluoromethyl, methyl, and methoxygroups to optimize interactions with target active sites. The substitutions should enhance the mutual effects through such as hydrogen bond, ionic interactions mediated by charged groups and electronic effects. Ester prodrug derivatization was also applied to the sugar hydroxyl groups to enhance the metabolic stability and oral bioavailability of the target compounds.

Chemical structures of HP083 and novel flexible nucleosides.

The synthesis of the novel fleximers commence with 1-acetoxy-2, 3, 5- tribenzoyloxy-1-β-D-ribofuranose 1 as the starting material. Under the catalysis of bis(trimethylsilyl)acetamide (BSA) and trimethylsilyl trifluoromethanesulfonate (TMSOTf), 1 was coupled with 4,5-diiodoimidazole to yield compound 2. Subsequently, 2 underwent a deprotection reaction in a sodium methoxide methanol solution to afford compound 3. The three hydroxyl groups of 3 were then protected as benzyl ethers, resulting in compound 4.

Next, under Grignard reaction conditions, using tetrabutylammonium iodide (TBAI) as a catalyst, the iodine at the C-5 position of 4 was removed to generate the key intermediate 5. Compound 5 was then subjected to Suzuki coupling reactions with various pyrimidine boronic acid pinacol ester ligands to produce compounds 6a–6d. Finally, the benzyl protecting groups in 6a–6d were removed using boron trichloride (BCl₃), yielding the target compounds 7a–7d.The hydroxyl groups of 7a–7d were further esterified with different anhydrides to generate the ester prodrug target compounds 8a–8l (Scheme 1), (Table 1).

Synthesis of novel fleximers. Reagents and conditions: a) TMSOTf, r.t. 4 h, BSA, 60°C, N2, 18 h; b) MeONa, MeOH, 0°C, 1 h; c) NaH, 0°C, 2 h; TBAI, BnBr, r.t., 12 h; d) EtMgBr, 0°C, Ar, 1 h; e) Pd(pph3)4, K2CO3, H2O, N2, 85°C, 4 h; f) BCl3, –78°C, 2h; g) DMAP, DMF, r.t., 1 h.

Table 1. Chemical substituent group of flexible nucleosides.

Compound	R₁	R₂	R₃	R₄	R₅
7a	OCH₃	OCH₃	H	H	H
7b	NH₂	CH₃	H	H	H
7c	NH₂	CF₃	H	H	H
7d	NH₂	H	H	H	H
8a	OCH₃	OCH₃	acetyl	acetyl	acetyl
8b	OCH₃	OCH₃	propionyl	propionyl	propionyl
8c	OCH₃	OCH₃	isobutyryl	isobutyryl	isobutyryl
8d	NH₂	CH₃	acetyl	acetyl	acetyl
8e	NH₂	CH₃	propionyl	propionyl	propionyl
8f	NH₂	CH₃	isobutyryl	isobutyryl	isobutyryl
8g	NH₂	CF₃	acetyl	acetyl	acetyl
8h	NH₂	CF₃	propionyl	propionyl	propionyl
8i	NH₂	CF₃	isobutyryl	isobutyryl	isobutyryl
8j	NH₂	H	acetyl	acetyl	acetyl
8k	NH₂	H	propionyl	propionyl	propionyl
8l	NH₂	H	isobutyryl	isobutyryl	isobutyryl

3.2. In vitro antiviral assays

The antiviral activity of the novel flexible nucleoside compounds was evaluated using an H1N1 influenza virus model constructed with MDCK (Madin-Darby canine kidney) cells. All experiments were conducted under biosafety level 2 (BSL-2) laboratory conditions. Compound cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8) cell viability assay. The antiviral activity against H1N1 virus of the compounds was quantitatively assessed using the neutral red uptake assay [22].

3.2.1. Cytotoxicity assay

To evaluate cell viability, the CCK-8 assay was employed to analyze the cytotoxicity of the target compounds in MDCK cells Briefly, MDCK cells (1×10⁴ cells/well) were allowed to adhere in 96-well microplates and cultured for 12 h under standard conditions until a confluent monolayer formed. The test compounds were diluted in virus isolation serum-free medium, starting at a concentration of 400.0 µM, followed by 2-fold serial dilutions to generate 8 concentration gradients, with the highest concentration being 200 µM. Each concentration of the test samples (100 µL) was added to the cell plate along with 100 µL of virus isolation serum-free medium, which was subjected to 37°C/5% CO₂ conditions for a 48 h incubation period. Upon completion of incubation, aspiration of the supernatant and then used two PBS washing cycles (5 min each) to cleanse cellular residues. Addition of 10 µL CCK-8 reagent per well was performed, after which the plates underwent 4 h of incubation. Finally, using a 96-well plate-compatible detector, light absorption at 450 nm was assessed. The cell viability was calculated. The CC₅₀ values (half-maximal cytotoxic concentration) were determined using GraphPad Prism 8 software.

3.2.2. In vitro anti-H1N1 virus assay

The in vitro anti-H1N1 influenza virus activity was evaluated using the MDCK cell model. Remdesivir served as the positive control. Both virus control (medium only), and cell control (virus without compound) were included in each assay plate. MDCK cells were plated in 96-well culture dishes (5×10⁴ cells/well), and cultured at 37°C with 5% CO₂ for 24 h. Prior to the experiment, the stored H1N1 virus was thawed and diluted to a working concentration of 10 to 100 TCID₅₀. in virus growth medium at 2× the highest test concentration and then 3-fold serially diluted to generate test concentrations. Each concentration was tested in triplicate. Equal volumes of virus suspension and compound dilutions were mixed, and 200 µL of the mixture was added to each well of pre-plated MDCK cells. The cells were incubated at 37°C with 5% CO₂ for 4 days, and cytopathic effect (CPE) was monitored daily. When more than 85% of the virus control wells exhibited CPE, the supernatants were discarded, and the cells were washed twice with PBS (pH 7.4). Subsequently, 100 µL of neutral red staining solution was added to each well and incubated for 3–5 h. After staining, wells washed 2–3 times with PBS, acidic ethanol 100 µL was added to elute dye, and absorbance at 540 nm was measured using a microplate spectrophotometer. The experimental data were analyzed using GraphPad Prism 8 software to calculate the EC₅₀ values (half-maximal effective concentration) and evaluate the antiviral activity of the target compounds.

The cytotoxicity and in vitro anti-H1N1 influenza virus activity tests of the novel flexible nucleoside analogues revealed that most of the new compounds exhibited low cytotoxicity and potent antiviral activity (Table 2). Except for compounds 8i (CC₅₀ = 148.2) and 8l (CC₅₀ = 156.6), which exhibited moderate cytotoxicity, the remaining compounds showed no significant cytotoxic effects, with CC₅₀ values exceeding 200 μM. Among the tested compounds, nine exhibited EC₅₀ values below 10 μM, with three flexible nucleoside analogues 7c, 8g, and 8h displaying low micromolar-level antiviral activity (7c EC₅₀ = 1.371 μM; 8g EC₅₀ = 2.637 μM; 8h EC₅₀ = 2.213 μM). Their anti-H1N1 influenza virus activity was significantly potent than that of the positive control drug remdesivir (EC₅₀ = 11.510 μM). Notably, 7c demonstrated a therapeutic index (CC₅₀/EC₅₀) greater than 145, indicating excellent safety.

Table 2. Anti-H1N1 virus activity of flexible nucleosides in infected MDCK cells.

Compound	CC₅₀ (μM) ^a	EC₅₀ (μM) ^b	Therapeutic index (TI)^c
7a	>200	17.140	>11.6
7b	>200	5.262	>38.0
7c	>200	1.371	>145.8
7d	>200	15.830	>12.6
8a	>200	3.165	>63.1
8b	>200	14.190	>14.0
8c	>200	4.747	>42.1
8d	>200	4.001	>49.9
8e	>200	6.626	>38.1
8f	>200	6.997	>28.5
8g	>200	2.637	>74.8
8h	>200	2.213	>90.3
8i	148.2	>100	<1.4
8j	>200	>100	<2.0
8k	>200	>100	,2.0
8l	156.6	>100	<1.5
Remdesivir^d	>200	11.510	>17.3

^a CC₅₀: half-maximal cytotoxic concentration. Samples were assessed in triplicate (n = 3 wells) within one independent experiment. Values indicated as “>200 µM” indicate that ≥ 50% cell viability remained at the highest concentration tested.

^b EC₅₀: half-maximal effective concentration. Samples were assessed in triplicate (n = 3 wells) within one independent experiment. Values reported as “> 100 µM” indicate that ≤ 50% antiviral effect was achieved at the highest concentration tested.

^c Ttherapeutic index (TI = CC₅₀/EC₅₀).

^d Positive control.

Structure-activity analysis revealed that the five-membered sugar ring is essential for antiviral efficacy, likely due to enhanced target binding flexibility compared to acyclic analogs. Modifications at the pyrimidine C2 position revealed that amino-substituted analogues tended to display enhanced antiviral potency relative to their methoxy counterparts. At the C4 position, activity trends appeared to correlate with substituent electronic and steric properties, where trifluoromethyl analogues generally showed superior activity over methyl and unsubstituted derivatives. This observation may be attributed to enhanced hydrophobic interactions with conserved polymerase residues, though further mechanistic studies are required to confirm this hypothesis. Ester prodrug derivatization of the sugar hydroxyl groups preserved antiviral activity in most cases, with ethyl esters showing the most favorable balance of potency and metabolic stability. The improved activity profile of ethyl esters could potentially stem from its improved physicochemical properties, such as cell membrane permeability, metabolic stability, and bioavailability.

3.2.3. In vivo metabolic evaluation of 7c

The pharmacokinetic profile of 7c was evaluated in Sprague-Dawley (SD) rats. Plasma samples were collected to determine the drug concentration of 7c, and its pharmacokinetic parameters were calculated based on the obtained data.

Table 3 and Figure 2 indicate that 7c exhibits favorable pharmacokinetic characteristics in SD rats. After administration, the plasma concentration of 7c rapidly increased, peaked, and then gradually declined, consistent with the typical pharmacokinetic profile of intravenous administration. The t_1/2 was calculated to be 3.6477 h, and the total exposure (AUC_0∼t) over 0 to 24 h averaged 1230.1193 h·ng/mL. The observed clearance (Cl_obs) averaged 26.9593 mL/min/kg. Although some individual variability in pharmacokinetic parameters was observed among the rats, the overall differences were minimal. The results suggest that 7c has a stable and predictable pharmacokinetic profile in vivo.

Table 3. Pharmacokinetic parameters of 7c in plasma following a single intravenous administration to SD rats (2 mg·kg⁻¹, N = 3).

Animal	t_1/2 (h)	C₀ (ng/mL)	AUC_0∼t (h*ng/mL)	AUC_{0∼t_D_obs} (hkgng/mL/mg)	Vz_obs (L/kg)	Cl_obs (mL/min/kg)
MR1	3.701	2566.797	1113.075	556.538	9.454	29.506
MR2	3.819	2831.813	1372.652	686.326	7.906	23.912
MR3	3.423	3391.725	1204.631	602.316	8.137	27.460
Mean	3.6477	2930.1117	1230.1193	615.0600	8.4990	26.9593
SD	0.2033	421.1574	131.6522	65.8258	0.8351	2.8304
CV%	5.57	14.37	10.70	10.70	9.83	10.50
N	3	3	3	3	3	3

MR: Male rat. SD: Standard deviation. CV%: Coefficient of variation%. N: Number. t₁/₂: Drug half-life. C₀: Initial concentration. AUC₀₋t: Area under the concentration - time curve from time zero to time t. AUC₀₋t D obs: Area under the concentration - time curve from time zero to time t, Dose, observed. Vz obs: Observed apparent volume of distribution. Cl obs: Observed clearance

Plasma concentration-time curve of 7c (Mean ± SD, N = 3).

3.3. Molecular docking studies

The antiviral target of remdesivir is the RNA-dependent RNA polymerase (RdRp), while the mechanism of action of our newly designed flexible nucleoside analogues remains unclear. Due to their structural similarity to remdesivir and the fact that the PB1-PB2 subunit of the H1N1 virus is a critical component of RdRp, we selected the PB1-PB2 subunit protein (PDB: 2ZTT) as the target for molecular docking to preliminarily explore its potential mechanism of action [23].

The experimental protocol follows: RdRp protein of H1N1 influenza virus (PDB: 2ZTT) was selected as the target for molecular docking using AutoDock Vina, with results visualized in PyMOL and Discovery Studio. The protein structure was preprocessed by removing water molecules, adding hydrogen atoms, and optimizing the structure. Target compounds were constructed in Chem3D, energy-minimized, and assigned charges. The docking search space was set to the protein active site, with exhaustiveness = 9 for accuracy. This approach evaluated compound binding to PB1-PB2, providing insights into their antiviral mechanism.

Molecular docking analysis indicated that compound 7c bound strongly to H1N1 influenza virus RdRp (PDB: 2ZTT), with a calculated binding energy of -7.7 kcal/mol, suggesting potential inhibition of viral replication (Table 4). Further analysis of the binding mode demonstrated that multiple hydrogen bonds and hydrophobic interactions were formed between compound 7c and the protein’s active site, which collectively stabilized the complex structure (Figure 3). Specifically, The trifluoromethyl (CF₃) group at the 4-position of the pyrimidine ring formed a hydrogen bond interaction with Glu A: 697 in the RdRp protein, while the 1-position of the pyrimidine ring established conventional hydrogen bonds with residues Tyr A: 689, Cys A: 693, and Mse B: 28. Additionally, Hydrogen bond relations between the 5’-OH group of the sugar and Phe A: 700 were observed, consistent with canonical base pairing. The hydrogen bonding and hydrophobic interactions between 7c and the RdRp active site residues suggest a competitive inhibition mechanism against viral RNA synthesis.

Table 4. Binding energy of 7c with H1N1 influenza virus proteins.

Compound	7a	7b	7c	7d	8a	8b	8c	8d	8e	8f	8g	8h	8i	8j	8k	8l	Remdesivir
Binding energy/(kcal/mol)	-6.9	-7.4	-7.7	-7.3	-6.7	-6.2	-6.5	-7.1	-6.6	-6.2	-6.8	-6.5	-7.2	-6.9	-6.6	-6.6	-7.0

Binding of 7c with RdRp Protein of H1N1 influenza virus.

4. Conclusions

Nucleoside analogs remain the cornerstone of antiviral therapies for emerging and re-emerging highly lethal RNA viral diseases and pandemics. Flexible nucleoside analogues represent a new class of candidate against various RNA virus. Herein, the novel flexible five-membered ring nucleoside analogues exhibit low micromolar levels of anti-H1N1 influenza virus activity. The most potent analogue 7c is able to potently inhibit the multiplication of the H1N1 influenza strains with a very minute in vitro anti-H1N1 EC₅₀ of 1.371 μM. Most importantly, 7c demonstrated an excellent safety profile with little to no cytotoxicity CC₅₀ >200 μM and an in vivo mouse LD₅₀ > 2000 mg/kg (see supplement data). Furthermore, 7c showed favorable metabolic stability, with a half-life (t_1/2) of 3.6477h intravenous administration in rats. As such, 7c could serve as a novel lead compound for the further development of antiviral drug candidate. However, more work needs to be done to fully elucidate these novel compounds’ mechanism of action, detailed SAR studies and activity against other RNA viruses.

CRediT authorship contribution statement

Xiyao Hu, Yufei Yan: Writing–original draft, Investigation, Chemical synthesis, Biological evaluation. Shuli Liang: Writing–original draft, Supervision, Biological evaluation. Fengxia Ren: Writing–original draft, Supervision, Biological evaluation. Zixing Yu: Writing–original draft, Chemical synthesis. Yabin Song: original draft, Investigation, Molecular docking. Jingchao Cheng: Writing–original draft, Chemical synthesis. Xiaoping Liu: Writing–review & editing, Supervision, Conceptualization. Baogang Wang: Writing–review & editing, Supervision, Conceptualization. Weiguo Shi: Writing–review & editing, Supervision, Conceptualization.

Declaration of competing interest

No potential conflicts of interest, whether financial or personal, were identified that might affect the objectivity of this scholarly work.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

References

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1. Introduction

2. Materials and Methods

2.1. Chemistry
2.2. Synthesis of compounds
2.3. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-4-(trifluoromethyl)pyrimidin-2-amine (6c)
2.4. Statistical analysis

3. Results and Discussion

3.1. Synthesis of novel flexible nucleosides
3.2. In vitro antiviral assays
3.3. Molecular docking studies

4. Conclusions

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[1] Koonin E.V., Kuhn J.H., Dolja V.V., Krupovic M.. Megataxonomy and global ecology of the virosphere. The ISME Journal. 2024;18:wrad042. https://doi.org/10.1093/ismejo/wrad042
[Google Scholar]

[2] Koonin E.V., Krupovic M., Agol V.I.. The Baltimore classification of viruses 50 years later: How does it stand in the light of virus evolution? Microbiology and Molecular Biology Reviews : MMBR. 2021;85:e0005321. https://doi.org/10.1128/MMBR.00053-21
[Google Scholar]

[3] van Leur S.W., Heunis T., Munnur D., Sanyal S.. Pathogenesis and virulence of flavivirus infections. Virulence. 2021;12:2814-2838. https://doi.org/10.1080/21505594.2021.1996059
[Google Scholar]

[4] Kausar S., Said Khan F., Ishaq Mujeeb Ur Rehman M., Akram M., Riaz M., Rasool G., Hamid Khan A., Saleem I., Shamim S., Malik A.. A review: Mechanism of action of antiviral drugs. International Journal of Immunopathology and Pharmacology. 2021;35:20587384211002621. https://doi.org/10.1177/20587384211002621
[Google Scholar]

[5] Kumari R., Sharma S.D., Kumar A., Ende Z., Mishina M., Wang Y., Falls Z., Samudrala R., Pohl J., Knight P.R., Sambhara S.. Antiviral approaches against influenza virus. Clinical Microbiology Reviews. 2023;36:e0004022. https://doi.org/10.1128/cmr.00040-22
[Google Scholar]

[6] Domingo E., García-Crespo C., Lobo-Vega R., Perales C.. Mutation rates, mutation frequencies, and proofreading-repair activities in RNA virus genetics. Viruses. 2021;13:1882. https://doi.org/10.3390/v13091882
[Google Scholar]

[7] Du S., Hu X., Menéndez-Arias L., Zhan P., Liu X.. Target-based drug design strategies to overcome resistance to antiviral agents: Opportunities and challenges. Drug Resistance Updates : Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy. 2024;73:101053. https://doi.org/10.1016/j.drup.2024.101053
[Google Scholar]

[8] Ishikawa T., Konishi E.. Potential chemotherapeutic targets for Japanese encephalitis: Current status of antiviral drug development and future challenges. Expert Opinion on Therapeutic Targets. 2015;19:1379-1395. https://doi.org/10.1517/14728222.2015.1065817
[Google Scholar]

[9] Kim S., Han K.H., Ahn S.H.. Hepatitis C virus and antiviral drug resistance. Gut and Liver. 2016;10:890-895. https://doi.org/10.5009/gnl15573
[Google Scholar]

[10] Shaw M.L., Klumpp K.. Successes and challenges in the antiviral field. Current Opinion in Virology. 2013;3:483-486. https://doi.org/10.1016/j.coviro.2013.08.015
[Google Scholar]

[11] Al-Majed A.A., Bakheit A.H.H., Al-Qahtani B.M., Al-Kahtani H.M., Abdelhameed A.S.. Emtricitabine. Profiles of Drug Substances, Excipients, and Related Methodology. 2020;45:55-91. https://doi.org/10.1016/bs.podrm.2019.10.003
[Google Scholar]

[12] Chang J.Y., Balch C., Puccio J., Oh H.S.. A narrative review of alternative symptomatic treatments for herpes simplex virus. Viruses. 2023;15:1314. https://doi.org/10.3390/v15061314
[Google Scholar]

[13] Tompa D.R., Immanuel A., Srikanth S., Kadhirvel S.. Trends and strategies to combat viral infections: A review on FDA approved antiviral drugs. International Journal of Biological Macromolecules. 2021;172:524-541. https://doi.org/10.1016/j.ijbiomac.2021.01.076
[Google Scholar]

[14] Seley-Radtke K.. Flexibility-Not just for yoga anymore! Antiviral Chemistry & Chemotherapy. 2018;26:2040206618756788. https://doi.org/10.1177/2040206618756788
[Google Scholar]

[15] Yates M.K., Raje M.R., Chatterjee P., Spiropoulou C.F., Bavari S., Flint M., Soloveva V., Seley-Radtke K.L.. Flex-nucleoside analogues - Novel therapeutics against filoviruses. Bioorganic & Medicinal Chemistry Letters. 2017;27:2800-2802. https://doi.org/10.1016/j.bmcl.2017.04.069
[Google Scholar]

[16] Chudinov M.V.. Nucleoside analogs with fleximer nucleobase. Chemistry of Heterocyclic Compounds. 2020;56:636-643. https://doi.org/10.1007/s10593-020-02713-5
[Google Scholar]

[17] Seley K.L., Salim S., Zhang L.. “Molecular chameleons”. Design and synthesis of c-4-substituted imidazole fleximers. Organic Letters. 2005;7:63-66. https://doi.org/10.1021/ol047895v
[Google Scholar]

[18] Seley K.L., Zhang L., Hagos A., Quirk S.. “Fleximers”. Design and synthesis of a new class of novel shape-modified nucleosides 1. The Journal of Organic Chemistry. 2002;67:3365-3373. https://doi.org/10.1021/jo0255476
[Google Scholar]

[19] Seley-Radtke K., Deval J.. Advances in antiviral nucleoside analogues and their prodrugs. Antiviral Chemistry & Chemotherapy. 2018;26:2040206618781410. https://doi.org/10.1177/2040206618781410
[Google Scholar]

[20] Thames J.E., Waters C.D., Valle C., Bassetto M., Aouadi W., Martin B., Selisko B., Falat A., Coutard B., Brancale A., Canard B., Decroly E., Seley-Radtke K.L.. Synthesis and biological evaluation of novel flexible nucleoside analogues that inhibit flavivirus replication in vitro. Bioorganic & Medicinal Chemistry. 2020;28:115713. https://doi.org/10.1016/j.bmc.2020.115713
[Google Scholar]

[21] Peters H.L., Jochmans D., de Wilde A.H., Posthuma C.C., Snijder E.J., Neyts J., Seley-Radtke K.L.. Design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity. Bioorganic & Medicinal chemistry Letters. 2015;25:2923-2926. https://doi.org/10.1016/j.bmcl.2015.05.039
[Google Scholar]

[22] Genzel Y., Reichl U.. Continuous cell lines as a production system for influenza vaccines. Expert Review of Vaccines. 2009;8:1681-1692. https://doi.org/10.1586/erv.09.128
[Google Scholar]

[23] Sugiyama K., Obayashi E., Kawaguchi A., Suzuki Y., Tame J.R., Nagata K., Park S.Y.. Structural insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase. The EMBO Journal. 2009;28:1803-1811. https://doi.org/10.1038/emboj.2009.138
[Google Scholar]

Synthesis and evaluation of novel flexible nucleoside analogues against H1N1 influenza virus

Abstract

Keywords

Flexible nucleoside analogues

H1N1 influenza virus

Lead compound

Nucleoside

RNA virus

1. Introduction

2. Materials and Methods

2.1. Chemistry

2.2. Synthesis of compounds

2.2.1. (2R, 3R, 4R, 5R)-2-((Benzoyloxy)methyl)-5-(4,5-diiodo-1H-imidazol-1-yl) tetra hydrofuran-3,4-diyl dibenzoate (2)

2.2.2. 2-((2R, 3R, 4S, 5R)-2-(4, 5-Diiodo-1H-imidazol-1-yl)-5-(hydroxymethyl)tetra hydrofuran-3,4-diol) (3)

2.2.3. 1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl) -4,5-diiodo-1H-imidazole (4)

2.2.4. 1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)-4-iodo-1H-imidazole (5)

2.2.5. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-2,4-dimethoxypyrimidine (6a)

2.2.6. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-4-methylpyrimidin-2-amine (6b)

2.3. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)-4-(trifluoromethyl)pyrimidin-2-amine (6c)

2.3.1. 5-(1-((2R, 3R, 4R, 5R)-3, 4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran- 2-yl)-1H-imidazol-4-yl)pyrimidin-2-amine (6d)

2.3.2. (2R, 3R, 4S, 5R)-2-(4-(2, 4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5- (hydroxylmethyl)tetrahydrofuran-3,4-diol (7a)

2.3.3. (2R, 3R, 4S, 5R)-2-(4-(2-Amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5- (hydroxymethyl)tetrahydrofuran-3,4-diol (7b)

2.3.4. (2R, 3R, 4S, 5R)-2-(4-(2-Amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol- 1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(7c)

2.3.5. (2R, 3R, 4S, 5R)-2-(4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxyl methyl)tetrahydrofuran-3,4-diol (7d)

2.3.6. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2,4-dimethoxypyrimidin-5-yl)-1H- imidazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (8a)

2.3.7. (2R, 3R, 4R, 5R)-2-(4-(2, 4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((propionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate (8b)

2.3.8. (2R, 3R, 4R, 5R)-2-(4-(2, 4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8c)

2.3.9. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2-amino-4-methylpyrimidin-5-yl)-1H- imidazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (8d)

2.3.10. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((propionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate (8e)

2.3.11. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5- ((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8f)

2.3.12. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2-amino-4-(trifluoromethyl)pyrimidin- 5-yl)-1H-imidazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (8g)

2.3.13. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol- 1-yl)-5-((propionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate (8h).

2.3.14. (2R, 3R, 4R, 5R)-2-(4-(2-Amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol- 1-yl)-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8i)

2.3.15. (2R, 3R, 4R, 5R)-2-(Acetoxymethyl)-5-(4-(2-aminopyrimidin-5-yl)-1H-imidazol- 1-yl)tetrahydrofuran-3,4-diyl diacetate (8j)

2.3.16. (2R, 3R, 4R, 5R)-2-(4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-((pro pionyloxy)methyl)tetrahydrofuran-3,4-diyl dipropionate(8k)

2.3.17. (2R, 3R, 4R, 5R)-2-(4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-((isobutyryl oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8l)

2.4. Statistical analysis

3. Results and Discussion

3.1. Synthesis of novel flexible nucleosides

3.2. In vitro antiviral assays

3.2.1. Cytotoxicity assay

3.2.2. In vitro anti-H1N1 virus assay

3.2.3. In vivo metabolic evaluation of 7c

3.3. Molecular docking studies

4. Conclusions

CRediT authorship contribution statement

Declaration of competing interest

Declaration of generative AI and AI-assisted technologies in the writing process

References

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