An Improved Synthesis Route of Molnupiravir and its Key Impurities

Abstract

This paper aims to improve the synthetic process of molnupiravir based on previously reported synthetic routes. The route begins with uracil (ML-2), which is protected with an isopropyl group to yield ML-3 (Step 1), followed by an esterification and a triazolation reaction (Steps 2 and 3) to produce ML-5, which, via a hydroxylation reaction and deprotection (Steps 4 and 5), gives the target product ML-1. Nuclear magnetic resonance (¹H NMR) and mass spectra were used for chemical structure identification. There are mainly the following improvements, including: (1) replacing the separate addition of acetone and concentrated H₂SO₄ with 2,2-dimethoxypropane and catalytic p-toluenesulfonic acid monohydrate in Step 1, simplifying the workup operation and reducing the dosage of reaction solvent. (2) Optimize the synthesis conditions of ML-5, reduce the formation of impurities, and improve the purity of the crude product from 43.12 to 85.21%. (3) Three impurities were isolated, two of which are new compounds. This article lays a foundation to obtain molnupiravir with controllable quality and a stable process for the treatment of coronavirus disease 2019.

Keywords

molnupiravir - antiviral drugs - COVID-19 - process improvement

Introduction

Coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus, with common symptoms including fever, sore throat, cough, dyspnea, and fatigue.[1] [2] Molnupiravir is metabolized in the human body into β-D-N ⁴-hydroxycytidine (EIDD-1931), a compound demonstrated to inhibit multiple viruses, including Ebola virus, influenza A and B viruses, norovirus, hepatitis C virus, Venezuelan equine encephalitis virus, respiratory syncytial virus, and chikungunya virus.[3] [4] [5] [6] [7] [8] EIDD-1931 achieves therapeutic effects by inhibiting the continuous synthesis of the virus through increasing the mutation rate of the viral genome.[2] [9]

Molnupiravir was approved by the UK Medicines and Healthcare Products Regulatory Agency (MHRA) on November 4, 2021, becoming the world's first marketed oral anti-COVID-19 virus drug.[10] [11] [12] The drug was recommended by the National Health Commission of China as an anti-COVID-19 drug during the epidemic of COVID-19, and was approved by the National Medical Products Administration of China in December 2022.[13]

There are two routes for the synthesis of molnupiravir. Route 1 was developed by Emory University and begins with uridine ([Scheme 1]).[14] After isopropylidene protection, the intermediate is esterified with isobutyric anhydride to give ML-4, which is converted to ML-5 via a triazolation step, then hydroxylamination followed by deprotection, furnishes ML-1. Industrially, this sequence is hampered by the large solvent volumes required for the initial protection step and by the formation of isomeric impurities during the preparation of ML-5. Route 2 starts from cytidine and proceeds through hydroxyl protection, esterification, hydroxylamination, and deprotection to deliver the target product ([Scheme 1]).[15] In the isolation of ML-6, inorganic salts coprecipitate with the product, making suction filtration laborious. Moreover, the final deprotection with trifluoroacetic acid can cleave the isobutyrate ester, generating additional impurities. Given the above, both routes still require substantial optimization before they can be translated to robust, cost-effective industrial processes. However, in this work, considering the operability of commercial production, we prefer Route 1 for optimization.

Scheme 1 Synthesis of Molnupiravi demonstrated in previous works. Reagents and conditions: (i) 2,2-DMP, acetone, H₂SO₄ (cat.), r.t. (ii) Triethylamine, isobutyric anhydride, DMAP (cat.), acetone, 0°C→r.t. (iii) TEA, 1.2.4-triazole, POCl₃, ACN, 0°C→r.t. (iv) NH₂-OH, IPA, r.t. (v) Formic acid, EtOH, MTBE, IPA, r.t. (vi) 2,2-DMP, H₂SO₄, acetone, 25–30°C. (vii) Isobutyric anhydride, DMAP (cat.), DBU, acetone, 0 to 5°C. (viii) Hydroxylamine sulfate, IPA, 70–80°C. (ix) TFA, H₂O, 50 to 55°C.

Results and Discussion

Compared with the original literature, our optimized Route 1 incorporates the following improvements.

In Step 1, we replaced the separate addition of acetone and concentrated H₂SO₄ with 2,2-dimethoxypropane (DMP) and catalytic p-toluenesulfonic acid monohydrate (TsOH·H₂O). This change cuts the acetone charge to 6 v/m and allows all process-related impurities to be removed by a simple suction filtration, eliminating the need for aqueous washes and solvent swaps. And the yield was improved from 88 to 98.5% ([Table 1]).

Table 1 Condition screening of ML-3

Entry	Solvent	Protecting group	Reagent of acid	Yield of ML-3 (%)[a]
1	Acetone	Acetone	H2SO4	88.0
2	Acetone	2,2-DMP	H2SO4	91.0
3	Acetone	2,2-DMP	TsOH·H2O	98.5
4	IPA	2,2-DMP	TsOH·H2O	82.0
5	ACN	2,2-DMP	TsOH·H2O	87.0
6	THF	2,2-DMP	TsOH·H2O	88.0
7	2-Methyl-THF	2,2-DMP	TsOH·H2O	90.0

^a Determined by an in situ high-performance liquid chromatography assay of the reaction solution.

In Step 3, reaction temperature and POCl₃ stoichiometry were systematically screened. When the batch temperature increased from 10 to 20–25°C, ML-5 content enhanced from 50.5 to 77.6% by an in situ HPLC assay (Entries 1–2, [Table 2]). When the temperature further increased to 30 to 35°C, ML-5 was delivered with a yield of 85.2% (Entry 4). The POCl₃ charge was equally critical: varying the equiv. from 1.3 to 5.2 caused the ML-5 content to fluctuate, with 2.6 equiv., affording the optimal balance (Entries 3–6, [Table 2]).

Table 2 Condition screening of ML-5

Entry	Temperature (℃)	POCl3 (equiv.)	ML-5 content (%)[a]	ML-5-Z1 content (%)[a]	ML-5-Z2 content (%)[a]	ML-5-Z3 content (%)[a]
1	10	2.6	50.52	5.54	6.28	5.33
2	20-25	2.6	77.58	1.22	1.69	1.98
3	30-35	1.3	45.88	0.76	0.64	0.63
4	30-35	2.6	85.21	0.41	0.86	1.01
5	30-35	3.9	70.55	1.35	1.45	15.02
6	30-35	5.2	43.12	1.05	1.34	29.94

^a Determined by an in situ high-performance liquid chromatography assay of the reaction solution.

In addition, three impurities, ML-5-Z1, ML-5-Z2, and ML-5-Z3 ([Fig. 1]), were isolated and identified, among which the impurities of ML-5-Z1 and ML-5-Z3 are new compounds, and no literature reports exist. Although ML-5-Z2 has been reported in the literature,[16] no structural confirmation data were reported. We separated and purified these three impurities, and the structure confirmation data were reported for the first time.

Fig. 1 Chemical structures of three impurities.

In Step 5, a balance had to be made between the removal of the protective group and the remained of the isobutyrate group. Several acid was screened, with formic acid resulting in the highest yield of target product (85% yield, Entry 1, [Table 3]).

Table 3 Condition screening of ML-1

Entry	Solvent	ML-1 yield (%)[a]
1	Formic acid	85
2	HCl	0
3	20% HCl	45
4	TFA	35
5	20% TFA	52
6	Sulfuric acid	0
7	20% Sulfuric acid	38

^a Determined by an in situ high-performance liquid chromatography assay of the reaction solution.

The synthesis route in this work was summarized in [Scheme 2].

Scheme 2 Synthesis of molnupiravir in this work.

Conclusion

This paper optimized the synthesis route of molnupiravir that simplifies downstream processing and prevents the formation of impurities. Additionally, structural confirmation data for three intermediates are reported for the first time. The resulting synthetic route ensures controllable product quality and robust process stability, thereby providing a solid foundation for the quality control of molnupiravir.

Experimental Section

General Experimental Details

The materials and reagents, including uridine, TsOH·H₂O, DMP, acetone, isobutyric anhydride, 1,2,4-triazole, phosphorus oxychloride (POCl₃), triethylamine (TEA), NH₂-OH, formic acid and N,N-dimethylaminopyridine (DMAP), ethyl acetate (EA) (AR), acetonitrile (ACN) (AR), dichloromethane (DCM) (AR), petroleum ether (PE) (AR), isopropyl alcohol (IPA) (AR), and methyl tert-butyl ether (MTBE) (AR) were purchased from Tansoole (Shanghai Titan Scientific Co., Ltd, Shanghai, China).

¹H NMR data were obtained on a Bruker Avance spectrometer (Bruker, Germany) using methanol-d ₄ as a solvent and tetramethylsilane as the internal standard. Chemical shift (δ) is expressed in units of parts per million (ppm). Multiplicities are abbreviated as singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Coupling constants (J) are reported in Hertz (Hz). High-resolution mass spectra data were obtained on an Agilent 6210 MS spectrometer. The X-ray structures of ML-5 and ML-5-Z3 were viewed. The XRD data were collected on a Bruker D8 Venture diffractometer (Bruker, Germany) using Cu Kα (λ = 1.54178 Å) / Mo Kα (λ = 0.71073 Å) radiation at 170 K. The purity of compounds was measured on an Agilent 1260 HPLC system (Agilent, America) and performed according to the reported studies.[17] [18]

Synthesis of ML-4

Acetone (1,800 mL) was added to a jacket vessel with a total capacity of 5 L. The batch temperature was adjusted to 15 to 20°C. ML-2 (1 equiv., 1.23 mol, 300 g), TsOH·H₂O (0.005 equiv., 6.1 mmol, 1.16 g), and DMP (2 equiv., 2.46 mol, 256 g) were added. The reaction mixture was stirred at 15 to 20°C for 12 hours. The resulting slurry was filtered. The filter cake was collected to obtain ML-3, which was used directly in the next step without drying.

Acetone (1,400 mL), ML-3, TEA (6.13 mol, 852 mL), and DMAP (60.9 mmol, 7.44 g) were added to a jacket vessel with a total capacity of 5 L. Acetone (50 mL) was added as a rinse. The batch was cooled to 0 to 5°C. Then, isobutyric anhydride (1.35 mol, 213 g) was added. Acetone (50 mL) was added as a rinse. The mixture was stirred at 0 to 5°C for 1.5 hours, and evaporated in vacuo to give a residue, which was dissolved in EA (600 mL), and washed with saturated aqueous sodium bicarbonate solution (500 mL × 2) and saturated brine (500 mL × 2). The organic layer was dried (Na₂SO₄), filtered, and evaporated in vacuo to give the target product (426.59 g, 97.85% in two-step yield, 98.35% HPLC purity). ML-4 Chemically named “((3aR,4R,6R,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl isobutyrate.” ¹H NMR (400 MHz, MeOD) δ 7.65–7.59 (m, 1H), 5.75 (d, J = 2.0 Hz, 1H), 5.68 (d, J = 8.0 Hz, 1H), 5.06 (dd, J = 6.4, 2.0 Hz, 1H), 4.33–4.24 (m, 3H), 3.31 (dt, J = 3.2, 1.6 Hz, 1H), 2.57 (dt, J = 14.0, 7.0 Hz, 1H), 1.53 (s, 3H), 1.35 (s, 3H), 1.15 (dd, J = 7.0, 1.4 Hz, 6H). MS-ESI (m/z) calcd. for C₁₆H₂₂N₂O₇Na⁺ [M + Na]⁺ 377.1325; found: 377.13.

Synthesis of ML-5

ACN (3,300 mL) was added to a vessel with a total capacity of 10 L, followed by the addition of ML-4 (1 equiv., 0.85 mol, 300 g), 1,2,4-triazole (7.2 equiv., 6.11 mol, 422 g), and TEA (8 equiv., 6.81 mol, 946 mL). ACN (100 mL) was added as a rinse. The batch temperature was adjusted to 0 to 5°C. POCl₃ was added dropwise (2.6 equiv., 2.22 mol, 207 mL). The reaction mixture was stirred at 0 to 10°C for 15 minutes, warmed up to 30 to 35°C, and stirred for another 2 hours. The batch was cooled down to 0 to 10°C. Water (800 mL) was dropped. The reaction mixture evaporated in vacuo to give a residue, which was dissolved in DCM (2,500 mL) and washed with saturated brine (3 L × 3). The organic layer was dried (Na₂SO₄), filtered, and evaporated in vacuo. The residue was purified by column chromatography on a silica gel eluted with PE/EA (1:1–1:10, v/v) to give ML-5 (258.38 g, 75.28% isolated yield, 98.22% HPLC purity). ML-5 Chemically named “((3aR,4R,6R,6aR)-2,2-dimethyl-6-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-1(2H)-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl isobutyrate.” ¹H NMR (600 MHz, MeOD) δ 9.39 (s, 1H), 8.44 (t, J = 6.0 Hz, 1H), 8.26 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 5.90 (d, J = 1.5 Hz, 1H), 5.10 (dd, J = 6.3, 1.7 Hz, 1H), 4.87 (dt, J = 10.7, 5.3 Hz, 1H), 4.56–4.51 (m, 1H), 4.37 (qd, J = 12.0, 4.9 Hz, 2H), 2.50 (dt, J = 14.0, 7.0 Hz, 1H), 1.57 (s, 3H), 1.38 (d, J = 8.3 Hz, 3H), 1.10 (dd, J = 7.9, 7.1 Hz, 6H). MS-ESI (m/z) calcd. for C₁₈H₂₄N₅O₆ ⁺ [M + H]⁺ 406.1721, found: 406.17. MS-ESI (m/z) calcd. for C₁₈H₂₃N₅O₆Na⁺ [M + Na]⁺ 418.1541; found: 428.15.

ML-5-Z1, ML-5-Z2, and ML-5-Z3 were isolated from other batches of products and identified by ¹H NMR.

ML-5-Z1 Chemically named ((3aR,4R,6R,6aR)-2,2-dimethyl-6-(4-oxo-2-(1H-1,2,4-triazol-1-yl)pyrimidin-1(4H)-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl isobutyrate.” ¹H NMR (600 MHz, MeOD) δ 9.45 (s, 1H), 8.81 (d, J = 5.3 Hz, 1H), 8.28 (s, 1H), 7.64 (d, J = 5.3 Hz, 1H), 5.45 (d, J = 2.2 Hz, 1H), 4.66 (ddd, J = 19.7, 6.4, 2.4 Hz, 2H), 4.23 (dd, J = 11.0, 6.7 Hz, 1H), 4.20–4.16 (m, 1H), 2.62–2.57 (m, 1H), 1.50 (d, J = 6.1 Hz, 3H), 1.32 (s, 3H), 1.16 (dd, J = 6.9, 0.6 Hz, 6H). MS-ESI (m/z) calcd. for C₁₈H₂₄N₅O₆ ⁺ [M + H]⁺ 406.1721, found: 406.17.

ML-5-Z2 Chemically named “((3aR,4R,6R,6aR)-2,2-dimethyl-6-(2-oxo-4-(4H-1,2,4-triazol-4-yl)pyrimidin-1(2H)-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl isobutyrate.” ¹H NMR (400 MHz, MeOD) δ 9.22 (s, 2H), 8.36 (d, J = 7.3 Hz, 1H), 6.94 (d, J = 7.2 Hz, 1H), 5.80 (d, J = 1.6 Hz, 1H), 5.00 (dd, J = 6.3, 1.6 Hz, 1H), 4.78 (d, J = 3.6 Hz, 1H), 4.44–4.41 (m, 1H), 4.32–4.23 (m, 2H), 2.42 (dd, J = 14.0, 7.0 Hz, 1H), 1.47 (s, 3H), 1.28 (s, 3H), 1.02 (dd, J = 7.0, 2.5 Hz, 6H). MS-ESI (m/z) calcd. for C₁₈H₂₄N₅O₆ ⁺ [M + H]⁺ 406.1721, found: 406.17.

ML-5-Z3 Chemically named “4-chloro-2-(1H-1,2,4-triazol-1-yl)pyrimidine.” ¹H NMR (400 MHz, MeOD) δ 9.45 (s, 1H), 8.81 (d, J = 5.3 Hz, 1H), 8.27 (d, J = 9.4 Hz, 1H), 7.64 (d, J = 5.3 Hz, 1H). MS-ESI (m/z) calcd. for C₆H₅ClN₅ ⁺ [M + H]⁺ 182.0228, found: 182.02.

Synthesis of ML-6

To a vessel with a total capacity of 10 L was added IPA (4,000 mL), ML-5 (1 equiv., 0.52 mol, 210 g), and another IPA (100 mL) as a rinse. Then, 50% NH₂OH (1 equiv., 6.8 mol, 450 mL) was added. The reaction mixture was agitated at 10 to 20°C for 3 hours and evaporated in vacuo to give a residue, which was dissolved in EA (4 L). The organic phase was washed with saturated brine (4 L × 2), dried over Na₂SO₄, filtered, and evaporated in vacuo to give ML-6 (157.95 g, 82.55% isolated yield, 98.77% HPLC purity).

ML-6 Chemically named “((3aR,4R,6R,6aR)-6-(4-(hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl isobutyrate.” ¹H NMR (600 MHz, MeOD) δ 6.85 (d, J = 8.2 Hz, 1H), 5.69 (t, J = 2.9 Hz, 1H), 5.57 (d, J = 8.2 Hz, 1H), 4.98 (dd, J = 6.5, 2.4 Hz, 1H), 4.79 (dd, J = 6.4, 4.1 Hz, 1H), 4.29–4.24 (m, 2H), 4.23–4.19 (m, 1H), 2.64–2.56 (m, 1H), 1.53 (d, J = 4.8 Hz, 3H), 1.36–1.33 (m, 3H), 1.18–1.14 (m, 6H). MS-ESI (m/z) calcd. for C₁₆H₂₄N₃O₇ ⁺ [M + H]⁺ 370.1609, found: 370.16.

Synthesis of ML-1

To a vessel with a total capacity of 5 L was added formic acid (3,400 mL), ML-6 (1 equiv., 0.41 mol, 150 g), and additional formic acid (100 mL) as a rinse. The mixture was warmed up to 50 to 60°C and stirred at this temperature for 12 hours. The reaction mixture was evaporated in vacuo. Dissolve the residue in MTBE (1 L), evaporate once, and then redissolve the residue in MTBE (0.8 L). The batch was warmed up to 50 to 55°C for 30 minutes. IPA (1 L) was added. The batch was agitated at 50 to 55°C for 2 hours, cooled to room temperature, and filtered. The filter cake was washed with MTBE, dried in a vacuum at 45°C to give ML-1 (113.56 g, 84.92% isolated yield, 98.32% HPLC purity).

ML-1 Chemically named ((2R,3S,4R,5R)-3,4-dihydroxy-5-(4-(hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl isobutyrate.” ¹H NMR (600 MHz, MeOD) δ 6.91 (d, J = 8.2 Hz, 1H), 5.81 (d, J = 4.8 Hz, 1H), 5.61 (d, J = 8.2 Hz, 1H), 4.29 (d, J = 3.5 Hz, 2H), 4.13 (t, J = 4.9 Hz, 1H), 4.08 (q, J = 4.9 Hz, 2H), 2.61 (dt, J = 14.0, 7.0 Hz, 1H), 1.18 (d, J = 1.6 Hz, 3H), 1.17 (d, J = 1.6 Hz, 3H). MS-ESI (m/z) calcd. for C₁₃H₂₀N₃O₇ ⁺ [M + H]⁺ 330.1296, found: 330.13.

Conflict of Interest

None declared.

Supporting Information

¹H NMR and MS spectra of compounds mentioned in the article, as well as X-ray of ML-5 and ML-5-Z3 ([Supplementary Figs. S1]–[S16], available in online version), can be found in the “Supporting Information” section of this article's webpage.

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Publication History

Received: 11 September 2025

Accepted: 30 October 2025

Article published online:
11 December 2025