key: cord-0798228-xa38kzpt
authors: Wei, Daibao; Hu, Tianwen; Zhang, Yumin; Zheng, Wei; Xue, Haitao; Shen, Jingshan; Xie, Yuanchao; Aisa, Haji A.
title: Potency and Pharmacokinetics of GS-441524 Derivatives against SARS-CoV-2
date: 2021-08-11
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2021.116364
sha: 5b5ad2b17c189df90724de263551c5e12a4f5884
doc_id: 798228
cord_uid: xa38kzpt

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.

The coronavirus disease 2019 (COVID- 19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that was firstly identified in December of 2019. 1 The disease has developed into a global pandemic, leading to more than 182 million confirmed cases and 3.9 million deaths till July 2, 2021. SARS-CoV-2 is a novel positive-sense single-stranded RNA virus, sharing 79.5% genetic sequence identity with SARS-CoV. 2 Evidences have indicated that this new virus is more contagious than SARS-CoV, and has a long incubation period, which poses a great challenge to control the pandemic. 3 Structurally, remdesivir is a phosphoramidate prodrug that is designed to improve the intracellular nucleoside triphosphate conversion efficiency. 7 This kind of nucleoside prodrugs shows liver-targeting properties and has been widely applied in the development of antiviral drug against hepatitis virus infections. 8 For the treatment of COVID-19, RDV is administered by intravenous (IV) injection mainly due to its specific pharmacokinetic (PK) properties. After IV administration in human, RDV is found to be rapidly metabolized to its predominant metabolite GS-441524 ( Figure 1) that persists in the circulation with t 1/2 of about 27 h. 9 GS-441524 is a 1′-cyano substituted pyrrolotriazine C-glycoside with broad-spectrum antiviral activities. 10 This nucleoside also displays potent anti-SARS-CoV-2 activity in different cells, especially in the primary human epithelial cells. 11 Of note, it was recently reported that GS-441524 could effectively inhibit SARS-CoV-2 infection in mouse models, 12 and has a synergic antiviral efficacy with the 3CL protease inhibitor, GC376. 13 Currently, GS-441524 is deemed as a promising anti-SARS-CoV-2 therapeutic, and is being investigated under preclinical study.

In addition to GS-441524, some other pyrrolotriazine C-nucleosides have been previously reported, and several of them proved to be potent antiviral agents. It was also indicated that the substituents on the sugar or the base moiety could significantly affect the antiviral activities. 7, 10, 14 Given the high demand of effective anti-SARS-CoV-2 agents, in this work, we synthesized a variety of GS-441524 analogs with modifications at the sugar 1′, 2′ position or the base 6, 7 position, and investigated their anti-SARS-CoV-2 activities in Vero E6 cells. In another aspect, in order to improve the 

Compounds 1a and 1b were prepared from GS-441524 by halogenation using Nchlorosuccinimide and N-iodosuccinimide in DMF, respectively. Synthesis of compounds 1c and 1d is shown in Scheme 1. Treatment of 8 with Selectfluor in acetonitrile at room temperature afforded 9, which was then debenzylated with BCl 3 to afford the desired 7-fluoro derivative 1c. The 7-CN derivative 1d was synthesized in three steps from 8, bromination, cyano-substitution and deprotection. The two intermediates were treated with methanesulfonic acid to give the cyclized products (19 and 21) followed by debenzylation to give 2d (two stereoisomers in ratio of 5:1) and 2e (two stereoisomers in ratio of 1:1). 2f derived from the debenzylation of 8 as a byproduct, and 3a was prepared according to the reported method. 15 Synthesis of 4c, 5a, 5b and 6 was shown in scheme 5. Selective 3', 5'-hydroxy group protection of 29 with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl) followed by exocyclic amino protection with N, N-dimethylformamide dimethyl acetal (DMF-DMA) afforded 31. The intermediate 31 was used for synthesis of compounds 4c, 5a and 5b. Methylation of 31 with methyl iodide in DMF afforded 32, and the subsequent removal of the amino and the hydroxyl protecting groups afforded 4c.

Condensation of 31 with isobutyryl chloride and Boc-L-Val under general reaction conditions gave 33 and 34, respectively. After removal of the TIPDS group with TBAF, the acyl groups were completely migrated from the 2'-position to the 3'-position ， which was unambiguously confirmed by single crystal X-ray crystallography (Fig. S43 X-ray single-crystal structure of 35). The similar migration had been reported in previous literature. 16 

All of the synthesized GS-441524 derivatives were screened for antiviral activity (1a and 1d) . Moreover, we designed the phosphoramidate prodrug of 1c, but it exhibited decreased antiviral activity (7, 82% inhibition at 10 µM). RDV also had a much weaker antiviral activity (EC 50 = 2.0 µM) than GS-441524, which was mainly due to the limited formation of the active nucleoside triphosphate form in Vero E6 cells. 18 With respect to the 1'-modifications, all the six derivatives (2a-2f) did not show any inhibition of SARS-CoV-2 replication at the concentration of 5.0 µM. The C1'cyano group was very important for the anti-SARS-CoV-2 activities, as evidenced by a previous research. 18 Recently, it was revealed that the C1'-cyano group of RDV or GS-441524 nucleoside monophosphate could cause a translocation barrier, which played a crucial role in the termination of viral RNA elongation. 19 3a derived by the removal of the C1'-cyano group of GS-441524 was highly toxic to Vero E6 cells even at low concentrations, and its antiviral activity was hard to be determined. 3b bearing a hydroxyamino group was designed according to the structure of β-D-N 4hydroxycytidine, a highly potent anti-SARS-CoV-2 nucleoside, but this nucleoside did not show any antiviral activity. 20 For the other three compounds (4a-4c), to our disappointment, they were all inactive against SARS-CoV-2. 

Adsorption of nucleosides in the gastrointestinal tract largely depended on the transporters, and a number of approved antiviral nucleoside drugs are given by oral route in the parent nucleoside form. 21 As for GS-441524, its oral bioavailability varies greatly in different species with F values of 33% in rats, 85% in dogs and 8.3% in cynomolgus monkeys. 22 Recently, a PK study of GS-441524 in a volunteer showed that a high oral dose (750 mg) for three times a day could achieve the exposure that was required to completely suppress the replication of SARS-CoV-2 in vivo. 23 GS-441524 contained multiple hydrophilic groups, but it was very slightly soluble in water (pH = 6.0) with a solubility of 0.105 mg/ml at 37 ℃ determined in our lab. Based on these evidences, we concluded that the oral bioavailability of GS-441524 in human would be low, and a suitable prodrug of GS-441524 may serve as a better candidate for clinical development. In order to improve the oral bioavailability of GS-441524, at first, we designed and synthesized five prodrugs, including two mono-isobutyric acid esters (5a and 5c), two L-valine esters (5b and 5d), and one carbamate 6. Compounds 5a-5d were found to exhibit stronger anti-SARS-CoV-2 activities than GS-441524, which likely resulted from their improved cellular permeability. The carbamate 6 showed a significant decrease in the anti-SARS-CoV-2 activity with only 65% inhibition rate at the concentration of 10 µM. This kind of prodrug was previously applied for the modification of R1479, an anti-HCV nucleoside, and proved to be unable to efficiently release the parent nucleoside. 24 The low antiviral activity of 6 also indicated that this prodrug could not be efficiently converted to the nucleoside in Vero E6 cells.

Then, we investigated the stability of four ester prodrugs (5a-5d) in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). All of them were found to be stable in SGF with pepsin, but only the 3'-isobutyryl ester 5a showed good stability in SIF with pancreatin ( Figure 3 ). At 4 h post incubation in SIF, there remained 100%, 34%, 2% and 9% of the parent esters for 5a, 5b, 5c and 5d, respectively. It seemed that the two 3'-esters exhibited a higher resistance to in vitro hydrolysis in SIF than the corresponding 5'-esters. Next, we evaluated the PK properties of three esters (5a, 5b and 5c) in mice. Due to the hepatic-first pass metabolism, and the potential ester hydrolysis in mouse plasma that contained high level of esterases, all the parent esters were hardly detected in mouse plasma. As shown in Table 3 , 5a and 5c had excellent oral bioavailabilities in mice with F values of 71.6% and 86.6%, respectively, which were much higher that of GS-441524 (F = 15.7% reported by us recently), 25 and that of 5b (F = 27.5%). There was little difference between the bioavailabilities of 5a and 5c, even though 5a had a better stability in SIF. This was likely due to the rapid oral adsorption of 5a and 5c in view of the short T max of the nucleoside metabolite (0.33 h and 0.42 h, respectively).

Considering the low oral bioavailability of 5b, the valine ester prodrug may be not a good choice for GS-441524 modification. Besides 5a and 5c, we also synthesized two di-isobutyrates (5e and 5f) and one tri-isobutyrate (5g). 5g was obtained as a white foam.

To obtain a good solid form, salt formation as a widely used approach to improve solid state properties was applied. Among the salts formed by hydrochloric acid, sulphuric acid or hydrobromic acid, only hydrobromide salt (5g·HBr) was obtained as a wellcrystallized salt. In addition, 5g·HBr had low hygrogscopicity and good chemical stability. Afterwards, the PK study of 5g·HBr was conducted in mice. To our delight, 5g·HBr also had a good oral bioavailability with the F value of 98.7%. Indeed, oral administration of 5g·HBr afforded a relatively lower (nearly 1.3-fold, calculated by the molar dose) plasma exposure of GS-441524 compared with that of 5a and 5c. For the three GS-441524 ester prodrugs, further PK studies especially in monkeys would be necessary to determine the optimal candidate. Table 3 . Single-dose PK parameters for GS-441524 in mice. Calculation of PK parameters for GS441524 following oral (50 mg/Kg) and intravenous (25 mg/Kg) administration of the ester prodrugs (5a, 5b, 5c and 5g·HBr) in CD-1 mice (N = 3 per group). 

An acute toxicity study of compound 5c was conducted with ICR mice. 

In summary, a series of pyrrolotriazine C-nucleosides and several GS-441524 prodrugs were synthesized and evaluated for their anti-SARS-CoV-2 activity in Vero E6 cell line. Among these novel nucleosides, only the fluoro-substituted GS-441524 analog showed moderate anti-SARS-CoV-2 activity. GS-441524 has been considered a promising oral anti-SARS-CoV-2 candidate currently evaluated in early-stage clinical trials, but its therapeutic potential may be limited due to the poor oral adsorption. Our study showed that the oral bioavailability of GS-441524 could be significantly improved by the means of the ester prodrug strategy. With respect to the three ester prodrugs (5a, 5c and 5g·HBr) that had high oral bioavailability in mice, further PK studies especially in monkeys would be necessary to determine the optimal prodrug candidate.

All commercially available chemicals and solvents were directly used without further purification. All reactions were monitored by thin layer chromatography (TLC) on silica gel plates (GF-254). Low-resolution mass spectra (LRMS) were measured on a Thermo Fisher FINNIGAN LTQ spectrometer. 1 (1.5 mL). The mixture was allowed to warm to rt, and 20% aqueous potassium sodium tartrate solution (50 mL) was added. The resulting solution was extracted with dichloromethane, dried over Na 2 SO 4 and concentrated. The residue was purified on silica gel to give 12 (2.1 g, 52%) as a white solid. To a solution of 12 (600 mg, 1.06 mmol, 1.0 eq.) in ethanol (10 mL) was added sodium borohydride (104 mg, 2.76 mmol, 2.6 eq.) at 0 ℃. After stirring for 2 h at rt, the reaction was quenched with acetic acid.

The resulting solution was concentrated, and the residue was partitioned between water and ethyl acetate. The organic layer was dried, concentrated, and purified on silica gel to afford 13 (480 mg, 80%) as a white solid. 1 A mixture of 13 (480 mg, 0.85 mmol, 1.0 eq.), 10% Pd/C (60 mg) and formic acid (2 mL) in methanol (6 mL) was stirred under hydrogen atmosphere overnight. The mixture was filtered, and the filtrate was concentrated to give a crude product, which was purified on reverse silica gel to give 2a (50 mg, 20%) as a white solid. 1 A solution of hydroxylamine hydrochloride (540 mg, 7.8 mmol, 30 eq.) in water (3 mL) was prepared, and adjusted to pH= 6 with 10% aqueous NaOH solution. Then 3a (69 mg, 0.26 mmol, 1.0 eq.) was added and the mixture was stirred at 40 ℃ overnight.

After evaporation, the residue was purified on reverse silica gel to afford 3b (52 mg, 71%) as an off-white solid. 1 Compound 4b was prepared following the same procedure as described for 4a. 13 23 pyridine (10 mL) and DMF-DMA (1.63 g, 13.72 mmol, 4.0 eq.) were added. The mixture was stirred at rt overnight, and concentrated to give crude 37 as an oil. A mixture of the crude, isobutyric anhydride (1.08 g, 6.86 mmol, 2.0 eq.), triethylamine (1.04 g, 10.29 mmol, 3.0 eq.) and DMAP (420 mg, 3.43 mmol, 1.0 eq.) in dichloromethane (10 mL) was stirred at rt overnight. After workup, 38 was obtained as an oil which was treated with a mixture of ethanol (20 mL) and acetic acid (6.17 g, 102.90 mmol, 30 eq.) at 50 ℃ overnight. The resulting solution was concentrated and the residue was partitioned between water and ethyl acetate. The organic layer was concentrated, and the residue was purified on silica gel to give 5c (818 mg, 66% over three steps) as a white solid. 1 Following the procedure for the synthesis of 5b, 5d (80 mg, 20% over four steps) was obtained as a white solid from 29 (290 mg, 1 mmol). 1 To a solution of 5c (360 mg, 1 mmol) and trimethyl orthoisobutyrate (370 mg, 2.5 mmol, 2.5 eq.) in acetonitrile (5 mL) was added p-toluenesulfonic acid monohydrate (230 mg, 1.2 mmol, 1.2 eq.). The mixture was stirred at rt for 4 h, and then neutralized with saturated NaHCO 3 solution. The resulting solution was partitioned between water and ethyl acetate. The organic layer was evaporated, and the residue was dissolved in m/z = 621. 22 [M+1] + . crystallographic data of

The colorless crystal was grown by slow evaporation in ethyl acetate solution.

Diffraction intensity data were acquired with a CCD area detector with graphite- 

The artificial gastric and intestinal juice were prepared according to Pharmacopeia of USA. The artificial gastric juice was prepared by dissolving 2.0 g of sodium chloride and 3.2 g of pepsin (3.2 g) into 600 mL pure water, then adding 7.0 mL of concentrated hydrochloric acid and diluted with water to 1 L, which was finally adjusted to pH 1.2.

The artificial intestinal juice was prepared by firstly dissolving 6.8 g potassium dihydrogen phosphate in 250 mL water, then mixing with 77 mL sodium hydroxide solution (0.2 M) and 500 mL water with 10 g pancreatin, which was finally adjusted to pH 6.8 and diluted with water to 1 L. The compounds (5a-5d) were dissolved with DMF to make stock solutions (2 μM). Then 2 μL the above solution was added to a 1.7 mL microcentrifuge tube, followed by the addition of 398 μL artificial gastric or intestinal juice. The mixture was incubated for 24 h at 37 ℃. At the predetermined time point (0, 1, 2, 3, 4, 6 and 24 h) incubations were terminated by adding 450 μL ice-cold acetonitrile. The resulting mixtures were centrifuged and the supernatants were subjected to HPLC analysis.

The pre-seeded Vero E6 cells (5×10 4 cells/well) were treated with compound at indicated doses for 1 h, then infected with SARS-CoV-2(nCoV-2019BetaCoV/Wuhan/WIV04/2019) at multiplicity of infection (MOI) of 0.05 for 2h.

After removal of the supernatant of the mixture, the infected cells were washed with phosphate buffered saline (PBS), and cultured in fresh drug containing medium for 24 h. Then the supernatant of the mixture was collected and subjected to real-time fluorescence quantitative PCR (qRT-PCR) analysis for quantification of viral copy number which was used for the calculation of inhibition rate of tested compounds. The half-maximal effective concentration (EC 50 ) was calculated with Graphpad Prism software 8.0. The cytotoxicities of compounds to the Vero E6 cells were determined using the cell counting kit-8 (CCK-8) colorimetric assay.

The PK studies were conducted at SIMM-Servier Joint Laboratory. The animals (N = 3 for CD-1 mice) were fasted for 12 h before the administration of tested 

For the acute toxicity study, 15 male ICR mice (28 -36 g) were randomized into five treatment groups, with three animals per group. The animals were dosed once with 5c at 50 mg/kg, 100 mg/kg, 200 mg/kg, 500 mg/kg, 1000 mg/kg by oral gavage, respectively (formulated with 5% DMSO, 5% Solution Hs15 and 90% saline). After administration, the mice were observed for 7 days (general clinical observation, body weight, and food consumption) and sacrificed at day 8.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

mL) was added, and the resulting mixture was stirred at rt for 1 h. After workup, the residue was purified on silica gel to give 5e (328 mg, 76%) as a white solid. 1 H NMR (600 MHz

MS m/z = 432

mL) was stirred at 60 ℃ for 1 h. The resulting solution was co-evaporated with ethanol and toluene, successively. Then, the obtained oil was treated with isopropanol to give 41 (2.0 g, 84%) as a white solid

500 MHz, DMSO-d 6 ) δ 8.15-7.89 (m, 3H), 6.94(d, J =4.73,1H), 6.79 (d, J =4.65,1H), 6.01 (d, J = 5.7 Hz, 1H), 5.44 (dd, J = 5.7, 3.1 Hz, 1H)

25 mmol, 0.5 eq.) were suspended in dichloromethane (5 mL), and then isobutyryl chloride (240 mg, 2.25 mmol, 4.5 eq.) was added dropwise in an ice bath. The mixture was stirred at rt overnight. The resulting solution was washed aqueous 1

M HCl solution, saturated aqueous NaHCO 3 solution, then dried, and concentrated to afford 44 (223 mg, 80 %) as a white solid

DMSO-d 6 ) δ 8.04 (s, 1H), 7.98 (s, 2H), 7.94 (s, 1H)

To a solution of 30 (150 mg, 0.28 mmol) in anhydrous dichloromethane (2 mL) was added pyridine (265 mg, 3.36 mmol, 12 eq.) and trimethylchlorosilane (91 mg, 0.84 mmol, 3.0 eq.) at 0 ℃The mixture was stirred for 1 h, and then pentyl chloroformate (128 mg, 0.84 mmol, 3.0 eq.) was added. After stirring for 2 h at rt, the resulting solution was poured into water, and extracted with dichloromethane. The organic layer was washed with saturated aqueous NaHCO 3 solution and brine, dried and concentrated to afford 36 as an oil. To a solution of 36 in tetrahydrofuran (2 mL) was added TBAF (1 M in tetrahydrofuran, 0.56 mL, 0.56 mmol, 2.0 eq.) at rt, and the mixture was stirred at rt for 2 h. After workup, the residue was purified on silica gel to give 6 (55 mg, 48% over two steps) as a white solid. 1 H NMR (600 MHz

yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl

24-7.14 (m, 3H), 6.73 (s, 1H), 6.45 (d, J = 6.0 Hz, 1H)

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This work was supported by the grant from the Shanghai Science and Technology Committee in China (Number: 21S11903100).