Synthesis and evaluation of NHC derivatives and 4'-fluorouridine prodrugs.

ß-D-N4-Hydroxycytidine (NHC) derivatives with structural modifications at the C4', O4' or C6 position and 4'-fluorouridine prodrugs were synthesized and evaluated for their antiviral activities against respiratory syncytial virus (RSV) or influenza virus (IFV) in vitro. The NHC derivatives were found inactive, but 4'-fluorouridine and its prodrugs had potent anti-RSV and anti-IFV activities. 4'-Fluorouridine was proved to be a nucleoside with poor stability, but the tri-ester prodrugs exhibited enhanced stability, especially tri-isobutyrate ester 1a. This prodrug also showed excellent oral pharmacokinetic properties in rats, with potential to be an oral antiviral candidate.

Safety, tolerability, and pharmacokinetics of VV116, an oral nucleoside analog against SARS-CoV-2, in Chinese healthy subjects.

VV116 (JT001) is an oral drug candidate of nucleoside analog against SARS-CoV-2. The purpose of the three phase I studies was to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. Three studies were launched sequentially: Study 1 (single ascending-dose study, SAD), Study 2 (multiple ascending-dose study, MAD), and Study 3 (food-effect study, FE). A total of 86 healthy subjects were enrolled in the studies. VV116 tablets or placebo were administered per protocol requirements. Blood samples were collected at the scheduled time points for pharmacokinetic analysis. 116-N1, the metabolite of VV116, was detected in plasma and calculated for the PK parameters. In SAD, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25-800 mg. T1/2 was within 4.80-6.95 h. In MAD, the accumulation ratio for Cmax and AUC indicated a slight accumulation upon repeated dosing of VV116. In FE, the standard meal had no effect on Cmax and AUC of VV116. No serious adverse event occurred in the studies, and no subject withdrew from the studies due to adverse events. Thus, VV116 exhibited satisfactory safety and tolerability in healthy subjects, which supports the continued investigation of VV116 in patients with COVID-19.

Synthesis and anti-SARS-CoV-2 activity of deuterated GS-441524 analogs.

The COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to spread around the world. GS-441524 is the parent nucleoside of remdesivir which is the first drug approved for the treatment of COVID-19, and demonstrates strong activity against SARS-Cov-2 in vitro and in vivo. Herein, we reported the synthesis of a series of deuterated GS-441524 analogs, which had deuterium atoms up to five at the ribose and the nucleobase moieties. Compared to GS-441524, all the deuterated compounds showed similar inhibitory activities against SARS-CoV-2 in vitro.

Weinreb Amide Approach to the Practical Synthesis of a Key Remdesivir Intermediate.

Currently, remdesivir is the first and only FDA-approved antiviral drug for COVID-19 treatment. Adequate supplies of remdesivir are highly warranted to cope with this global public health crisis. Herein, we report a Weinreb amide approach for preparing the key intermediate of remdesivir in the glycosylation step where overaddition side reactions are eliminated. Starting from 2,3,5-tri-O-benzyl-d-ribonolactone, the preferred route consisting of three sequential steps (Weinreb amidation, O-TMS protection, and Grignard addition) enables a high-yield (65%) synthesis of this intermediate at a kilogram scale. In particular, the undesirable PhMgCl used in previous methods was successfully replaced by MeMgBr. This approach proved to be suitable for the scalable production of the key remdesivir intermediate.

Potency and pharmacokinetics of GS-441524 derivatives against SARS-CoV-2.

The nucleoside metabolite of remdesivir, GS-441524 displays potent anti-SARS-CoV-2 efficacy, and is being evaluated in clinical as an oral antiviral therapeutic for COVID-19. However, this nucleoside has a poor oral bioavailability in non-human primates, which may affect its therapeutic efficacy. Herein, we reported a variety of GS-441524 analogs with modifications on the base or the sugar moiety, as well as some prodrug forms, including five isobutyryl esters, two l-valine esters, and one carbamate. Among the new nucleosides, only the 7-fluoro analog 3c had moderate anti-SARS-CoV-2 activity, and its phosphoramidate prodrug 7 exhibited reduced activity in Vero E6 cells. As for the prodrugs, the 3'-isobutyryl ester 5a, the 5'-isobutyryl ester 5c, and the tri-isobutyryl ester 5g hydrobromide showed excellent oral bioavailabilities (F = 71.6%, 86.6% and 98.7%, respectively) in mice, which provided good insight into the pharmacokinetic optimization of GS-441524.

Pharmacokinetics and tissue distribution of remdesivir and its metabolites nucleotide monophosphate, nucleotide triphosphate, and nucleoside in mice.

Remdesivir (RDV) exerts anti-severe acute respiratory coronavirus 2 activity following metabolic activation in the target tissues. However, the pharmacokinetics and tissue distributions of the parent drug and its active metabolites have been poorly characterized to date. Blood and tissue levels were evaluated in the current study. After intravenous administration of 20 mg/kg RDV in mice, the concentrations of the parent drug, nucleotide monophosphate (RMP) and triphosphate (RTP), as well as nucleoside (RN), in the blood, heart, liver, lung, kidney, testis, and small intestine were quantified. In blood, RDV was rapidly and completely metabolized and was barely detected at 0.5 h, similar to RTP, while its metabolites RMP and RN exhibited higher blood levels with increased residence times. The area under the concentration versus time curve up to the last measured point in time (AUC0-t) values of RMP and RN were 4558 and 136,572 h∙nM, respectively. The maximum plasma concentration (Cmax) values of RMP and RN were 2896 nM and 35,819 nM, respectively. Moreover, RDV presented an extensive distribution, and the lung, liver and kidney showed high levels of the parent drug and metabolites. The metabolic stabilities of RDV and RMP were also evaluated using lung, liver, and kidney microsomes. RDV showed higher clearances in the liver and kidney than in the lung, with intrinsic clearance (CLint) values of 1740, 1253, and 127 mL/(min∙g microsomal protein), respectively.

Synthesis of CBD and Its Derivatives Bearing Various C4'-Side Chains with a Late-Stage Diversification Method.

A novel synthetic route for making (-)-CBD and its derivatives bearing various C4'-side chains is developed by a late-stage diversification method. Starting from commercially available phloroglucinol, the key intermediate (-)-CBD-2OPiv-OTf is efficiently and regioselectively prepared and further undergoes Negishi cross-coupling to furnish (-)-CBD. This approach allowed an efficient synthesis of (-)-CBD in a five-step total 52% yield on a 10 g scale. Furthermore, diversification on the C4'-side chain with this method can be realized in a wide range.

Discovery of pyrimidine nucleoside dual prodrugs and pyrazine nucleosides as novel anti-HCV agents.

To explore the application potential of dual prodrug strategies in the development of anti-HCV agents, a variety of sofosbuvir derivatives with modifications at the C4 or N3 position of the uracil moiety were designed and synthesized. Some compounds exhibited potent anti-HCV activities, such as 4e and 8a-8c with similar EC50 values (0.20-0.22 µM) comparative to that of sofosbuvir (EC50 = 0.18 µM) in a genotype 1b based replicon Huh-7 cell line. Moreover, 8b displayed a good human plasma stability profile, and was easily metabolized in human liver microsomes expectantly. On the other hand, aiming to discover novel anti-HCV nucleosides, pyrazin-2(1H)-one nucleosides and their phosphoramidate prodrugs were investigated. Several active compounds were discovered, such as 25e (EC50 = 7.3 µM) and S-29b (EC50 = 19.5 µM). This kind of nucleosides were interesting and would open a new avenue for the development of antiviral agents.

Continuation of structure-activity relationship study of novel benzamide derivatives as potential antipsychotics.

A series of benzamide derivatives possessing potent dopamine D2 , serotonin 5-HT1A , and 5-HT2A receptor properties were synthesized and evaluated as potential antipsychotics. Among them, 5-(4-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)butoxy)-N-cyclopropyl-2-fluorobenzamide (4k) held the best pharmacological profile. It not only exhibited potent and balanced activities for the D2 , 5-HT1A , and 5-HT2A receptors, but was also endowed with low to moderate activities for the 5-HT2C , H1 , and M3 receptors, suggesting a low propensity for inducing weight gain or diabetes. In animal models, compound 4k reduced phencyclidine-induced hyperactivity with a high threshold for catalepsy or muscle relaxation induction. On the basis of its robust in vitro potency and in vivo efficacy in preclinical models of schizophrenia, 4k was selected as a candidate for further development.

Synthesis and biological investigation of tetrahydropyridopyrimidinone derivatives as potential multireceptor atypical antipsychotics.

In the present study, a series of tetrahydropyridopyrimidinone derivatives, possessing potent dopamine D2, serotonin 5-HT1A and 5-HT2A receptors properties, was synthesized and evaluated as potential antipsychotics. Among them, 3-(2-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (10d) held the best pharmacological profile. It not only exhibited potent and balanced activities for D2, 5-HT1A, and 5-HT2A receptors, but was also endowed with low activities for α1A, 5-HT2C, H1 receptors and hERG channels, suggesting a low propensity for inducing orthostatic hypotension, weight gain and QT prolongation. In animal models, compound 10d reduced phencyclidine-induced hyperactivity with a high threshold for catalepsy induction. On the basis of its robust in vitro potency and in vivo efficacy in preclinical models of schizophrenia, coupled with a good pharmacokinetic profile, 10d was selected as a candidate for further development.

Synthesis, structure-activity relationships, and biological evaluation of a series of benzamides as potential multireceptor antipsychotics.

In the present study, a series of benzamides, endowed with potent dopamine D2, serotonin 5-HT1A and 5-HT2A receptors properties, was synthesized and evaluated as potential antipsychotics. Among them, 3-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)-piperidin-1-yl)butoxy)-N-methylbenzamide (21) and its fluoro-substituted analogue (22) held the best pharmacological binding profiles. They not only presented potent activities for D2, 5-HT1A, and 5-HT2A receptors, but were also endowed with low activities for 5-HT2C, H1 receptors and hERG channels, suggesting a low propensity of inducing weight gain and QT prolongation. In animal models, compounds 21 and 22 reduced phencyclidine-induced hyperactivity with a high threshold for catalepsy induction. It thus provides potential candidates for further preclinical studies.

Combination of miR159 Mimics and Irinotecan Utilizing Lipid Nanoparticles for Enhanced Treatment of Colorectal Cancer.

Colorectal cancer (CRC) ranks as the third most prevalent global malignancy, marked by significant metastasis and post-surgical recurrence, posing formidable challenges to treatment efficacy. The integration of oligonucleotides with chemotherapeutic drugs emerges as a promising strategy for synergistic CRC therapy. The nanoformulation, lipid nanoparticle (LNP), presents the capability to achieve co-delivery of oligonucleotides and chemotherapeutic drugs for cancer therapy. In this study, we constructed lipid nanoparticles, termed as LNP-I-V by microfluidics to co-deliver oligonucleotides miR159 mimics (VDX05001SI) and irinotecan (IRT), demonstrating effective treatment of CRC both in vitro and in vivo. The LNP-I-V exhibited a particle size of 118.67 ± 1.27 nm, ensuring excellent stability and targeting delivery to tumor tissues, where it was internalized and escaped from the endosome with a pH-sensitive profile. Ultimately, LNP-I-V significantly inhibited CRC growth, extended the survival of tumor-bearing mice, and displayed favorable safety profiles. Thus, LNP-I-V held promise as an innovative platform to combine gene therapy and chemotherapy for improving CRC treatment.

A nanoluciferase SFTSV for rapid screening antivirals and real-time visualization of virus infection in mice.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that causes severe hemorrhagic fever in humans, but no FDA-approved specific antivirals or vaccines are available to treat or prevent SFTS. METHODS: The plasmids construction and transfection were performed to generate the recombinant SFTSV harboring the nanoluciferase gene (SFTSV-Nluc). Immunostaining plaque assay was performed to measure viral titers, and DNA electrophoresis and Sanger sequencing were performed to evaluate the genetic stability. Luciferase assay and quantitative RT-PCR were performed to evaluate the efficacy of antivirals in vitro. Bioluminescence imaging, titration of virus from excised organs, hematology, and histopathology and immunohistochemistry were performed to evaluate the efficacy of antivirals in vivo. FINDINGS: SFTSV-Nluc exhibited high genetic stability and replication kinetics similar to those of wild-type virus (SFTSVwt), then a rapid high-throughput screening system for identifying inhibitors to treat SFTS was developed, and a nucleoside analog, 4-FlU, was identified to effectively inhibit SFTSV in vitro. SFTSV-Nluc mimicked the replication characteristics and localization of SFTSVwt in counterpart model mice. Bioluminescence imaging of SFTSV-Nluc allowed real-time visualization and quantification of SFTSV replication in the mice. 4-FlU was demonstrated to inhibit the replication of SFTSV with more efficiency than T-705 and without obvious adverse effect in vivo. INTERPRETATION: The high-throughput screening system based on SFTSV-Nluc for use in vitro and in vivo revealed that a safe and effective antiviral nucleoside analog, 4-FlU, may be a basis for the strategic treatment of SFTSV and other bunyavirus infections, paving the way for the discovery of antivirals. FUNDING: This work was supported by grants from the National Key Research and Development Plan of China (2021YFC2300700 to L. Zhang, 2022YFC2303300 to L. Zhang), Strategic Priority Research Program of Chinese Academy of Sciences (XDB0490000 to L. Zhang), National Natural Science Foundation of China (31970165 to L. Zhang, U22A20379 to G. Xiao), the Science and Technology Commission of Shanghai Municipality (21S11903100 to Y. Xie), Hubei Natural Science Foundation for Distinguished Young Scholars (2022CFA099 to L. Zhang).

Caffeic acid derivatives: a new type of influenza neuraminidase inhibitors.

Recently, many natural products, especially some plant-derived polyphenols have been found to exert antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to build a small compound library with various structures. The enzyme inhibition result indicated that some compounds exhibited moderate activities against NA and compound 15d was the best with IC50=7.2 µM and 8.5 µM against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was important for the activity according to the docking analysis. Besides, compound 15d was found to be a non-competitive inhibitor with Ki=11.5±0.25 µM by the kinetic study and also presented anti-influenza virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhibitors from caffeic acid derivatives to cope with influenza virus.

Further discovery of caffeic acid derivatives as novel influenza neuraminidase inhibitors.

Eight series of compounds, each series containing two to five compounds were prepared by structural modifications of a lead, which was previously discovered as a mild influenza neuraminidase (NA) inhibitor. On the basis of the biological result, a detailed structure-activity relationship (SAR) was derived and discussed. Several caffeic acid derivatives that acted as non-competitive inhibitors were close or superior to the lead and also presented good antiviral activities in cells. Besides, it was interesting to find that modifications of the lead with different strategies could result in selective inhibition against N1 or N2. The preliminary docking analysis indicated that the 150-cavity of the enzymes played an important role in the selective inhibition.

A viral RNA-dependent RNA polymerase inhibitor VV116 broadly inhibits human coronaviruses and has synergistic potency with 3CLpro inhibitor nirmatrelvir.

During the ongoing pandemic, providing treatment consisting of effective, low-cost oral antiviral drugs at an early stage of SARS-CoV-2 infection has been a priority for controlling COVID-19. Although Paxlovid and molnupiravir have received emergency approval from the FDA, some side effect concerns have emerged, and the possible oral agents are still limited, resulting in optimized drug development becoming an urgent requirement. An oral remdesivir derivative, VV116, has been reported to have promising antiviral effects against SARS-CoV-2 and positive therapeutic outcomes in clinical trials. However, whether VV116 has broad-spectrum anti-coronavirus activity and potential synergy with other drugs is not clear. Here, we uncovered the broad-spectrum antiviral potency of VV116 against SARS-CoV-2 variants of concern (VOCs), HCoV-OC43, and HCoV-229E in various cell lines. In vitro drug combination screening targeted RdRp and proteinase, highlighting the synergistic effect of VV116 and nirmatrelvir on HCoV-OC43 and SARS-CoV-2. When co-administrated with ritonavir, the combination of VV116 and nirmatrelvir showed significantly enhanced antiviral potency with noninteracting pharmacokinetic properties in mice. Our findings will facilitate clinical treatment with VV116 or VV116+nirmatrelvir combination to fight coronavirus infection.

[HPLC-MS identification of degradation products of levofloxacin].

The study aims to identify the degradation products of levofloxacin by HPLC-MS. The degradation products of levofloxacin were chromatographed on Agilent Zorbax Extend-C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase was 0.1% ammonium acetate solution (using methanoic acid to adjust to pH 3.5)-acetonitrile at the flow rate of 0.5 mL x min(-1) (gradient elution), the column temperature was 40 degrees C. Descarboxyl levofloxacin, desmethyl levofloxacin and levofloxacin N-oxide were identified through comparing with the standard spectrum and the results of mass spectrometry, i.e. m/z 318.2 was descarboxyl levofloxacin, m/z 348.2 was desmethyl levofloxacin, m/z 378.1 was levofloxacin-N-oxide. This method is simple, fast, accurate and suitable for the identification of degradation products of levofloxacin.

Synthesis and antiviral activity of 2'-deoxy-6'-substituted carbocyclic nucleosides.

A series of 2'-deoxy carbocyclic nucleosides characterized by various 6'-substitutions were synthesized and evaluated for their antiviral activities against three viruses, including hepatitis B virus (HBV), hepatitis C virus, and influenza virus. The in vitro antiviral assays indicated that these nucleosides only showed inhibitory activities against HBV, and the substituted groups at the 6' position significantly affected the activity. Among them, the guanosine analog 2b bearing a 6'-α-hydroxyl methyl group was the most potent compound with an EC50 value of 80 nM. The present study provided useful information for the discovery of antiviral carbocyclic nucleosides.

Practical and Highly Efficient Synthesis of Remdesivir from GS-441524.

A three-step sequence for preparing remdesivir, an important anti-SARS-CoV-2 drug, is described. Employing N,N-dimethylformamide dimethyl acetal (DMF-DMA) as a protecting agent, this synthesis started from (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-furan-2-carbonitrile (GS-441524) and consisted of three reactions, including protection, phosphoramidation, and deprotection. The advantages of this approach are as follows: (1) the protecting group could be removed under a mild deprotection condition, which avoided the generation of the degraded impurity; (2) high stereoselectivity was achieved in the phosphorylated reaction; (3) this synthesis could be performed successively without purification of intermediates. Moreover, the overall yield of this approach on a gram scale could be up to 85% with an excellent purity of 99.4% analyzed by high-performance liquid chromatography (HPLC).

Design, synthesis and biological activity of thiazolidine-4-carboxylic acid derivatives as novel influenza neuraminidase inhibitors.

A series of thiazolidine-4-carboxylic acid derivatives were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus. All the compounds were synthesized in good yields starting from commercially available l-cysteine hydrochloride using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A neuraminidase. The most potent compound of this series is compound 4f (IC(50)=0.14 µM), which is about sevenfold less potent than oseltamivir and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazolidine ring.