Synthesis of Novel N4-Hydrocytidine Analogs as Potential Anti-SARS-CoV-2 Agents

Coronavirus disease 2019 (COVID-19) is an emerging global pandemic with severe morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molnupiravir, an ester prodrug form of N4-hydroxycytidine (NHC), was recently emergency-use approved for the treatment of early SARS-CoV-2 infections. Herein, we report the synthesis and evaluation of a series of novel NHC analogs.


Introduction
The coronavirus pandemic has caused a dual threat to the health and the economy of the U.S. and the world. COVID-19 was first identified in late 2019 in Wuhan, Hubei province, China, resulting in the ongoing 2019-2022 pandemic. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Common symptoms of the disease include fever (88%), dry cough (68%), shortness of breath (19%), and loss of smell (15 to 30%) as well as complications such as pneumonia, bronchitis, viral sepsis, diarrhea, and acute respiratory distress syndrome [1,2]. SARS-CoV-2 is the seventh human coronavirus after 229E, NL63, OC43, HKU1, MERS-CoV, and the original SARS-CoV. Like all coronaviruses, SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus of approximately 30,000 bases in length. Based on the genome organization of SARS-CoV-2, four enzymes are recognized as attractive drug targets, which include the 3CLpro (nsp5), the PLpro (nsp3), RNA helicase (nsp13), and the RNA-dependent RNA polymerase (RdRp, nsp12). The RdRp catalyzes the synthesis of viral RNA and thus plays a central role in the replication and transcription cycle of CoV-2 [3].
Remdesivir was the first nucleoside analog RdRp inhibitor approved by the FDA for the treatment of SARS-CoV-2-infected patients, but its modest to no effect on hospitalization and mortality [4,5] as well as its poor pharmacokinetic properties (it is currently approved for intravenous administration and is therefore limited to hospitalized patients), make remdesivir monotherapy a sub-par option to treat SARS-CoV-2 -infected individuals. Molnupiravir (EIDD-2801), the 5 -isobutyryl ester prodrug form of N 4 -hydroxycytidine (NHC), was emergency-use authorized by the FDA for the treatment of COVID-19 in certain adults, but despite its potent in vitro activity, it shows significant toxicity in in certain cell-based systems, including the induction of mutagenesis in mammalian cells [6][7][8][9]. Consequently, molnupiravir's approval came with a multitude of limitations, including being restricted to patients older than 18 years of age due to its effect on bone and cartilage growth [10] and to non-pregnant women due to potential fetal harm [11]. Finally, it is worth noting that when treatment with molnupiravir was conducted in a well-controlled study, it decreased the risk of hospitalization from COVID-19 by only 30% and its benefit has not been observed in subjects when treatment was initiated after hospitalization due to COVID-19 [12]. The key feature of molnupiravir/NHC is its 4-oxime group, which can either mimic a uridine base pairing with adenosine or a cytidine base pairing with guanosine, depending on its tautomer form (Figure 1). The incorporation of NHC 5 -triphosphate (the active form of molnupiravir) by the viral (and cellular) RdRp during transcription of the viral genome leads to RNA mutations and impairs SARS-CoV-2 replication. Based on this unique attribute, and in order to mitigate molnupiravir's above mentioned liabilities and limitations, we prepared a series of 4-NHOH pyrimidine nucleoside analogs (Compounds 1-13, see Table 1 for structures) and herein report on their evaluation against SARS-CoV-2 in culture. Table 1. Anti-SARS-CoV-2 activity evaluation of compounds 1-13 in Vero cells at 10 µM.

Antiviral Evaluation
The anti-SARS-CoV-2 activity of the 4-NHOH nucleoside analogs 1-13 herein prepared was evaluated at 10 µM following previously reported methods [23]. Briefly, a monolayer of Vero cells in a 96-well cell culture microplate was treated with 10 µM of each compound for 1 h followed by infection with SARS-CoV-2 at 0.1 MOI [24]. After 1 h adsorption at 37 • C, the virus inoculum was removed and the compound or vehicle-containing medium was added to the respected wells. Resultant virus progeny yield was measured 2 days posttreatment from the supernatant of treated infected cells by specific quantitative RT-PCR. While NHC displayed more than 99% inhibition at 10 µM in our assay, compounds 1-13 exhibited no significant inhibition at that same concentration (Table 1). Interestingly, even a small modification of NHC's base (6-aza derivative 10) or a minor modification of its sugar ring (seleno or carbocyclic sugar derivatives 8 and 9, 2 -modified compounds 2-6 or 4 -fluorinated analog 7) completely abrogated NHC's anti-SARS-CoV-2 activity. Similarly, L-NHC (13), 2 ,3 -dideoxy compound 12 and dioxolane analog 11 were inactive at the maximum concentration tested. It is worth noting that none of these compounds displayed toxicity in Vero cells at concentration up to 100 µM while NHC had a CC 50 of 16 µM in these cells.

General Information
Anhydrous solvents were purchased from Millipore Sigma (Milwaukee, WI, USA). All commercially available reagents were used without further purification. Reagents were purchased from commercial sources. All the reactions were carried out under nitrogen in oven-dried glassware unless otherwise noted. Thin layer chromatography was performed on Analtech GHLF silica gel plates. Column chromatography was accomplished on Combiflash Rf200 or via reverse-phase high-performance liquid chromatography. 1 H, 13 C, and 19 F NMR spectra were recorded on a Bruker Ascend 400 spectrometer at rt (400, 101, and 377 MHz) and residual proton solvent signals were used as internal standards. Deuterium exchange and decoupling experiments were utilized to confirm proton assignments. NMR processing was performed with MestReNova (Mestrelab Research, Compostela, Spain) version 14.1.1 24571 or Topspin (Bruker, Berlin, Germany) version 3.5. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), and m (multiplet). Coupling constants (J) are in hertz (Hz). Mass spectra were determined on a Waters Acquity ultraperformance liquid chromatography (UPLC) spectrometer using a SQ detector with electrospray ionization. The purity of final compounds was determined to be >95% using UPLC analyses performed on a Waters Acquity UPLC System with a Kinetex LC column (2.1 mm, 50 mm, 1.7 µm, C18, 100 Å) and further supported by clean NMR spectra. Mobile phase flow was 0.4 mL/min with a 1.20 min gradient from 95% aqueous media (0.05% formic acid) to 95% CH 3 CN (0.05% formic acid) and a 4.5 min total acquisition time. Photodiode array detection was from 190 to 360 nm.   1-((3aR,4R,6R,6aS)

4-(Hydroxyamino)-2 ,3 -dideoxyuridine (12):
To a solution of 23 (164 mg, 0.645 mmol) in pyridine (5 mL), 4-chlorophenyl phosphorodichloridate (0.157 mL, 0.967 mmol) and 1,2,4-triazole (133 mg, 1.93 mmol) were added dropwise at 0 • C. The mixture was stirred at room temperature for 5 days and then concentrated under reduced pressure. The resulting residue was dissolved in DCM (10 mL) and washed with H 2 O (2 × 10 mL) and with a 50% NaHCO 3 solution (5 mL). The organic layer was clarified with Norit, dried over MgSO 4 , and filtered. The filtrate was evaporated to dryness in vacuo to yield 24 as a glassy residue. The crude compound 24 was dissolved in acetonitrile (13 mL) and NH 2 OH in H 2 O (50%, 0.25 mL) was added to the solution. The mixture was stirred for 2 h at room temperature to obtain crude compound 25 which was finally stirred overnight in a saturated solution of methanolic ammonia (10 mL) at room temperature. The volatiles were then evaporated under vacuum and the residue was purified by flash column chromatography (methanol/dichloromethane, 0/100 to 5/95) to obtain compound 12 (

Conclusions
A series of thirteen 4-NHOH pyrimidine nucleoside analogs of NHC/molnupiravir (compounds 1-13) were synthesized and evaluated in vitro for anti-SARS-CoV-2 activity in Vero cells. Unfortunately, none of them displayed significant activity up to 10 µM. These results emphasize, once more, the difficulty in designing antiviral nucleoside analogs for SARS-CoV-2, as a simple modification of a highly active compound can lead to the complete loss of antiviral potency.