2,2'-Cyclocytidine hydrochloride

Scheme 1-Synthesis of 2,2'-Cyclocytidine hydrochloride

10.0 kg of cytidine, 23.6 kg of o-acetylsalicylic chloride and 80.0 kg of acetonitrile were added to the reaction vessel, and the temperature was raised to 75 °C for 1.5 h, and then reduced to room temperature. 20.0 kg of absolute ethanol was added dropwise under the condition of maintaining temperature stability, and after dropwise addition, 50 L of ethanol hydrochloride with a volume concentration of 4 mol/L was added, and the temperature was raised to 60 °C for 7 h. Then it was cooled to 30 °C, 20.0 kg of ethyl acetate was added, the temperature was raised to 35 °C and stirred for 2 h, filtered and dried to obtain 15.7 kg of crude intermediate. The yield was 132.7%, the content was 32.5%, and the purity was (80.19+19.72)%. 48.0 kg of water was added to 12.0 kg of crude intermediate product, the temperature was raised to 74 °C, 7.0 L of ethanol hydrochloride with a volume concentration of 4 mol/L was added, cooled to 50 °C, 18.0 kg of ethanol was added dropwise, cooled to 20 °C and stirred for 2 hours, filtered and dried, and 9.5 kg of refined intermediate was obtained after drying. The yield was 60.5%, the content was 88.7%, and the intermediates in this step were cicetidine hydrochloride and the esters formed by cicitidine hydrochloride and aspirin), and the purity was (96.67+3.32)%.

Scheme 2-Assess whether 2,2'-Cyclocytidine hydrochloride affects the early stages of Viral Invasion

In viral binding experiments, Vero cells were treated with 40 μM 2,2'-Cyclocytidine hydrochloride for 12 h, followed by incubation with VSV at 4 °C (MOI of 10) for 1 h. Wash the cells 3 times with pre-chilled PBS to remove unbound virus. The cells were then divided into two aliquots, and the viral binding efficiency was identified by two methods. A portion of the cells were directly lysed, and the G gene copy was analyzed by qRT-PCR, and the viral RNA content was detected. The other fraction is lysed in fresh Vero cells, and the amount of bound virus is determined with plaque method.

Case Study 1-Inhibition of Hepatitis B virus flaccid circular DNA Synthesis

The synthesis of HBV rcDNA is dependent on the DNA Pol activity of RT and HBV Pol. The activity of RT contributes to the synthesis of the negative strand of rcDNA using pgRNA as a template, while the activity of DNA Pol further expands the positive strand of rcDNA using nsDNA as a template. HepAD38 cells treated with 2,2'-Cyclocytidine hydrochloride showed reduced rcDNA production but did not affect nsDNA synthesis, suggesting that it could inhibit the synthesis of the rDNA positive strand. This may be achieved by competition with normal deoxycytidine, which is hydrolyzed into cytarabine and phosphorylated into a triphosphate form by cellular enzymes incorporated into DNA synthesis, or by direct inhibition of HBV POL activity during positive-strand DNA synthesis, or by both pathways. Interestingly, the non-phosphorylated form of 2,2'-Cyclocytidine hydrochloride also blocks the synthesis of the rcDNA positive strand in cell-free EPR experiments, suggesting that the non-phosphorylated form of 2,2'-Cyclocytidine hydrochloride may also directly inhibit HBV POL activity.

Positive synthesis of rcDNA can be inhibited by 2,2'-Cyclocytidine hydrochloride. (Wang, Xue, et al., 2022)

Case Study 2-Inhibits vesicular stomatitis virus

The antiviral activity of 2,2'-Cyclocytidine hydrochloride against RNA viruses was studied by using vesicular stomatitis virus (VSV), a representative member of rhabdomyovirus, as a model RNA virus. The toxicity of 2,2'-Cyclocytidine hydrochloride to cells was first determined using the Cell Titer-Glo^® Luminescent Cell Viability Assay Kit (Promega, Madison, USA) and its toxicity to 293T cells was found to be minimal even at a high concentration of 1.6 mM. After 2 h treatment of 293T cells at 3 different concentrations (20, 40, and 80 μM), 293T cells were treated with VSV-GFP (VSV expressing the GFP reporter. Persist for an additional 8 hours with an infection multiplicity (MOI) of 0.1. The results showed that viral RNA and protein (VSV-G) were severely reduced after action with 2,2'-Cyclocytidine hydrochloride. In addition, progeny viral titers in the supernatant of infected cells were significantly reduced. The anti-VSV activity of cyclocytidine hydrochloride was further confirmed by immunofluorescence (IFA) and focused reduction method. IFA results showed that with the increase of cyclocytidine hydrochloride concentration, VSV was dose-dependent limited and the foci in the infected cells decreased.

VSV infection is interrupted by cyclocytidine hydrochloride treatment. (Ge, Yunfei, et al., 2020)

2,2'-Cyclocytidine hydrochloride is an anhydride analogue of arabinosecytosine (araC), which can inhibit DNA synthesis by affecting DNA polymerase, and has high antiviral activity against DNA viruses such as herpes simplex virus (HSV) and cytomegalovirus (CMV). Compared with cytarabine, this product has slower metabolism in vivo, longer action time, lower toxicity than cytarabine, and is more stable to heat and other metabolic enzymes.

Inhibits DNA virus synthesis: 2,2'-Cyclocytidine hydrochloride can inhibit the synthesis of HBV rcDNA-positive strand by incorporating DNA strands (triphosphorylated form) or inhibiting HBV POL activity (non-phosphorylated form). High test concentrations (20 mM) may not be suitable for anti-HBV, but the inhibition of HBV POL by 2,2'-Cyclocytidine hydrochloride provides an idea that it can be used in other DNA viruses that rely on the virus or host DNA polymerase. It can inhibit the synthesis of DNA viruses and is used externally for the treatment of herpes simplex virus keratitis.

Applications in neurological research: Cytidine hydrochloride may have potential applications in neuroscience research, such as as a regulator that affects the synthesis or release of neurotransmitters. Its nucleoside structure may enable it to play a role in neuronal metabolic pathways and may affect neuronal activity and signal transduction processes. However, specific to the practical application of cytidine hydrochloride in neuroscience research, further experiments and studies are needed to reveal its exact mechanism and potential.

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