D609 was created with the intention of turning it into an antiviral agent. D609 and other Xanthates were first described as broad-spectrum antiviral compounds ^[1]. D609 exists as an enantiomeric pair of four diastereomers, with unknown variable mixtures of these eight isomers in industrial preparations. In antiviral and PC-PLC inhibition assays, isomers have varying biochemical efficacy ^[43]. Lumavita (Basel, Switzerland) is working on an antiviral drug called Letermovir-601 (LMV-601), which is a pure enantiomeric isomer of D609. Over time, LMV-601, a PC-PLC inhibitor, decreased the high-risk Human Papillomavirus (HPV) expression in types 16, 18, and 31, and exacerbated defects of pre-cancer cervical cell ^[6]. Tricyclodecan-9-yl-xanthogenate, an antiviral xanthate compound, can inhibit RNA and DNA viruses in vitro ^[44]. Infectivity assays and Western blot analysis have shown that it can prevent infectious HIV from spreading to chronically infected lymphoma cell (KE37-III) tissue-culture medium ^[45]. HIV-specific proteins, on the other hand, have accumulated intracellularly. At D609 doses that allowed mitotic cell divisions, de novo HIV replication commencement after infection with permissive KE37-1 cells was entirely stopped. Furthermore, the loss of HIV replicative intermediate DNA demonstrated the xanthate compound′s selective anti-viral efficacy. Within these cells, the cellular gene expression of c-myc remains unchanged ^[45].

The human respiratory syncytial virus (RSV) is a cytoplasmic, enveloped virus with negative RNA polarity. In newborns and young children, paramyxovirus is among the common causes of respiratory tract infections ^[46]. In human epithelial cells, the antiviral compound D609 prevents the development of the respiratory syncytial (RS) virus (Hep 2) ^[47]. Viral protein aggregation, the viral phosphorylation of phosphoprotein, infectious particles levels, and extracellular antigens all decreased after treatment with D609. When the polarity was positive or negative, the viral proteins’ relative accumulation was also unbalanced, but there were no variations in viral RNA volume. Furthermore, nucleocapsid formation was not hampered ^[48]. HSV-1 (Herpes simplex virus-1) replication was prevented by D609 without causing cytotoxicity. It inhibited HSV-1 encoded protein kinase and decreased virus-infected cell polypeptide phosphorylation (US3 PK). At concentrations greater than 3.8 µM, virus production decreased by D609, with absolute inhibition at 75.2 µM at or below 1 PFU/cell MOI. D609 can be given up to 7 h after infection and still prevent virus replication. These findings indicate that D609′s antiviral activity is mediated by the protein kinase inhibition and phosphorylation of protein, which influences late HSV replication. As a result of the above research, it is clear that D609 prevents the development of many virus-related diseases ^[49]. The role of D609 in various diseases and its mechanisms are summarized in Figure 3.

D609 has also demonstrated significant potential anti-inflammatory activities ^[50]. Immunization of uveal melanin protein causes experimental melanin–protein induced uveitis (EMIU), which is a form of chronic autoimmune uveitis. The induction of inductive nitric oxide synthase (iNOS) is prevented by D609, a basic inhibitor of phosphatidylcholine-specific phospholipase C ^[51][52]. Thirty-five (Lewis) rats with EMIU were given either PBS or D609 in two separate experiments. D609 had a significant inhibitory effect on EMIU during this study ^[53]. D609-treated eyes had lower peroxide and nitrite levels, higher SOD levels, and lower iNOS mRNA expressions than PBS-treated eyes. The Fas and FasL levels in D609-injected animals′ eyes and lymph nodes increased significantly. D609-treated rats had increased DNA fragmentation in their lymph nodes. D609 inhibited EMIU by quenching NO and initiating programmed cell death, which had formerly been inhibited by NO, as well as displaying anti-inflammatory properties ^[52]. D609, a NO scavenger, was recently shown to be effective at preventing allergic encephalomyelitis in animals ^[54][55]. D609, in addition to inhibiting NO, can also inhibit other inflammatory mediators. By preventing the PC-PLC pathway, D609 inhibits the development of NOS activity. In response to inflammation, the PC-PLC pathway, which plays an important role, is activated ^[56][57]. D609 reduces IL-1α, IL-6, and NO release in endotoxin shock mice, according to Tschaikowsky et al. D609′s inhibition of NO synthesis has a much greater protective effect than D609′s inhibition of other inflammatory mediators. It has also been shown that D609, a PC-PLC and NOS inhibitor, inhibited NO expression in EMIU ^[58]. N9 and BV-2 microglia, RAW 264.7 macrophages, and DITNC1 astrocytes were all significantly inhibited by 100-μM D609 treatment without affecting cell viability ^[32]. D609 may perform an anti-inflammatory function by preventing the proliferation of macrophages/microglia, which are the principal sources of IL-1 and TNF and other pro-inflammatory cytokines. TNF-induced PAR formation is inhibited in D609-treated cultures, according to studies. D609 reduced microglial morphological change, NF-kB transcriptional activation, and PARP-1 activation ^[59].

Ceramide and DAG, linked by the sphingomyelin (SM) synthase pathway, are two-second messengers that control cell proliferation and growth arrest in opposing ways ^[60]. In SM and DAG, SMS transports the phosphocholine group from the PC to the ceramide ^[61]. SMS comes in two varieties: SMS1 is located in the Golgi apparatus, while SMS2 is found in the plasma membrane ^[62][63]. D609 inhibits both forms of SMS. By preventing ceramide injection into SM, inhibiting SMS elevates ceramide levels. Thanks to SMS inhibition, D609 prevented the proliferation of ßFGF-stimulated astrocyte ^[64][65]. According to one study, D609 decreased non-neuronal cell-line proliferation without triggering cell death ^[66]. In BV-2 microglia, D609 therapy elevated the expression of ceramide and p21 levels ^[32][67]. D609 also hypophosphorylated Rb, causing cell-cycle inhibition in the G0/G1 step, and a decrease in proportion of cells in the S-Phase ^[32]. Ceramide′s significance in D609-induced cell-cycle arrests is supported by the exogenous C8-ceramide investigations ^[32].

D609 regulates ceramide formation and cell death caused by death receptors ^[68][69]. Nontoxic concentrations of D609 inhibit sphingomyelin synthase and glucosylceramide synthase in Jurkat cells ^[70]. FasL-induced caspase activation and apoptosis were significantly enhanced by D609 ^[71][72]. Since the authors claim that Bcl-xL overexpression caused D609 outcomes, mitochondrial events were likely involved. The authors conclude that D609 causes cell death in T lymphocyte cells by acting downstream of caspases 8. They believe that in Fasl-induced cell death, a rise occurs due to D609 inhibition of ceramide transfer to complex sphingolipids ^[73]. Anti-HER2 medicines, which block the tumorigenic actions of HER2, do not significantly affect the treatment of HER2-positive EOC (epithelial ovarian cancer) ^[74][75]. Preclinical trial models can be utilized to assess the molecular processes causing HER2 overexpression and oncogenicity, giving rise to new EOC treatments. The enhanced HER2 expression in breast cancer cells is regulated by PC-PLC ^[16]. Researchers found that the inhibition of PC-PLC may be a good target for the tumorigenic effects of increased HER2 expression in EOC41 cells ^[26].

Evidence suggests that oxidative stress has a role in the pathogenesis of Alzheimer′s disease ^[76]. Amyloid-peptide accumulation causes a cascade of oxidative neuron damage and eventually leads to neuronal death, one of the hallmarks of Alzheimer′s disease ^[77][78]. Amyloid-peptide is senile plaque’s major component. It causes damage to nucleic acids, proteins, and membrane lipids in neurons by generating free radicals ^[79][80]. As a result, interest is growing in antioxidant chemicals as they play a protective role in the treatment of AD and other disorders of oxidative stress ^[81][82]. Amid the various antioxidant medications, “thiol-delivering” compounds have received much attention. PC-PLC is inhibited by D609, a compound that mimics the action of glutathione. Recent studies have shown that it can inhibit phosphatidylcholine-specific phospholipase C ^[83][84]. Another study assesses the efficiency of D609 to protect synaptosomes in vivo from amyloid-peptide caused by oxidative stress ^[85][86]. After gerbils were fed D609 or saline solution, synaptosomes were extracted from their brains. The ex vivo treatment of gerbils with synaptosomal preparations derived from D609-injected gerbils with amyloid peptide (1–42) significantly decreased oxidative stress parameters, such as reactive oxygen species, lipid peroxidation (4-hydroxy-2-nonenal), protein oxidation (carbonyl and 3-nitrotyrosine protein), and levels ^[85]. These findings support the idea that amyloid-peptide-induced free-radical control may be a useful therapeutic tool for treating AD and other related oxidative stress diseases ^[87]. Based on the above evidence, D609 is a potent antioxidant that may help cure AD and other related oxidative stress diseases ^{[83][85][88][89]}.

4.6. Cholinergic Neuron Differentiation