AT-527, a Double Prodrug of a Guanosine Nucleotide Analog, Is a Potent Inhibitor of SARS-CoV-2 In Vitro and a Promising Oral Antiviral for Treatment of COVID-19.

The impact of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is global and unprecedented. Although remdesivir has recently been approved by the FDA to treat SARS-CoV-2 infection, no oral antiviral is available for outpatient treatment. AT-527, an orally administered double prodrug of a guanosine nucleotide analog, was previously shown to be highly efficacious and well tolerated in hepatitis C virus (HCV)-infected subjects. Here, we report the potent in vitro activity of AT-511, the free base of AT-527, against several coronaviruses, including SARS-CoV-2. In normal human airway epithelial cells, the concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC90) was 0.47 µM, very similar to its EC90 against human coronavirus (HCoV)-229E, HCoV-OC43, and SARS-CoV in Huh-7 cells. Little to no cytotoxicity was observed for AT-511 at concentrations up to 100 µM. Substantial levels of the active triphosphate metabolite AT-9010 were formed in normal human bronchial and nasal epithelial cells incubated with 10 µM AT-511 (698 ± 15 and 236 ± 14 µM, respectively), with a half-life of at least 38 h. Results from steady-state pharmacokinetic and tissue distribution studies of nonhuman primates administered oral doses of AT-527, as well as pharmacokinetic data from subjects given daily oral doses of AT-527, predict that twice daily oral doses of 550 mg AT-527 will produce AT-9010 trough concentrations in human lung that exceed the EC90 observed for the prodrug against SARS-CoV-2 replication. This suggests that AT-527 may be an effective treatment option for COVID-19.

Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of acylphloroglucinols, and regioselective synthesis of empetrifranzinans from Hypericum roeperianum.

From the ethno-medicinally used leaves of Hypericum roeperianum we isolated a new tricyclic acylphloroglucinol (1), a new tetracyclic acylphloroglucinol (2), and a new prenylated bicyclic acylphloroglucinol (3) together with four known prenylated (4-7) and three known tetracyclic acylphloroglucinol derivatives (8-10). Structure elucidation was based on UV, IR, [α]D(25), 1D- and 2D-NMR experiments. Furthermore, empetrifranzinans A (8) and C (9) were synthesized regioselectively in only two steps. The isolated compounds were evaluated for their cytotoxicity against PC-3 and HT-29 cancer cell lines as well as antibacterial and anthelmintic activities. They were also tested in cell-based assays for cytotoxicity against MT-4 cells and for anti-HIV activity in infected MT-4 cells. Significant anthelmintic activity against Caenorhabditis elegans was exhibited by compound 7 (3-geranyl-1-(2'-methylbutanoyl)-phloroglucinol), which might provide a new lead.

Vaginal expression of efflux transporters and the potential impact on the disposition of microbicides in vitro and in rabbits.

In order to reach sufficiently high tissue concentrations and thus be effective, vaginally applied anti-HIV microbicides that are active at the level of the immune cells must permeate across the cervicovaginal mucosal layer. Cellular efflux transporters, such as Pgp, BCRP, and MRP-2, have been demonstrated to greatly affect drug disposition at different sites in the body including the intestine and the blood-brain barrier; their possible role on drug uptake from the female genital tract, however, has not been elucidated yet. In the present study, the protein expression of Pgp, BCRP, and MRP-2 in endocervical and vaginal tissue of premenopausal women was confirmed by Western blot analysis. To enable the assessment of transporter effects in vitro, the identification of an appropriate cervicovaginal cell line was pursued. The cervical SiHa cell line was observed to express mRNA of the 3 studied transporters, but only MRP-2 was found to be active. Consequently, the established Caco-2 cell line was utilized as an alternative in which the interaction of 10 microbicide candidates with the efflux transporters was studied. Darunavir, saquinavir, and maraviroc were identified as Pgp and MRP-2 substrates. The impact of Pgp on in vivo drug disposition was further examined for the model Pgp substrate talinolol in rabbits. Its vaginal uptake was significantly reduced by Pgp-mediated efflux when formulated in a neutral but not in an acidic gel. Our findings indicate the expression of a functional Pgp transporter in the vaginal mucosa that may severely reduce the vaginal uptake of Pgp substrates, including certain microbicide candidates, especially in women with an increased vaginal pH.

Further SAR studies on bicyclic basic merbarone analogues as potent antiproliferative agents.

Pyrimidopyrimidine derivatives 1 were prepared as rigid thioanalogues of merbarone (a catalytic topoisomerase II inhibitor) and screened as antiproliferative agents against different tumor cell lines. A number of the synthesized compounds emerged as cytotoxic in cell-based assays (MT-4, HeLa and MCF-7 cells) at low micromolar concentrations. In a National Cancer Institute screening, selected member of the series showed a broad spectrum of antiproliferative activity against various tumours (melanoma, renal, CNS, colon and breast cancers). The acid-base and steric properties of the substituent at position 7 of the pyrimidopyrimidine scaffold deeply affected potency. Enzymatic assays evidenced that a subset of tested derivatives efficiently inhibit topoisomerase IIα accordingly to merbarone mechanism of action. However this property does not fully rationalize the cytotoxicity data of the full ligand panel, suggesting that different target(s) should be additionally involved.

Disubstituted thiourea derivatives and their activity on CNS: synthesis and biological evaluation.

A series of new thiourea derivatives of 1,2,4-triazole have been synthesized. The difference in structures of obtained compounds are directly connected with the kind of isothiocyanate (aryl/alkyl). The (1)H NMR, (13)C NMR, MS methods were used to confirm structures of obtained thiourea derivatives. The molecular structure of (1, 17) was determined by an X-ray analysis. Two of the new compounds (8 and 14) were tested for their pharmacological activity on animal central nervous system (CNS) in behavioural animal tests. The results presented in this work indicate the possible involvement of the serotonergic system in the activity of 8 and 14. In the case of 14 is also a possible link between its activity and the endogenous opioid system. All obtained compounds were tested for antibacterial activity against gram-positive cocci, gram-negative rods and antifungal activity. Compounds (1, 2, 5, 7, 9) showed significant inhibition against gram-positive cocci. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Selected compounds (1-13) were examined for cytotoxicity, antitumor, and anti-HIV activity.

3-Aryl-2-[1H-benzotriazol-1-yl]acrylonitriles: a novel class of potent tubulin inhibitors.

During a screening for compounds that could act against Mycobacterium tuberculosis, a series of new cellular antiproliferative agents was identified. The most cytotoxic molecules were evaluated against a panel of human cell lines derived from hematological and solid human tumors. In particular, (E)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-3-(4-methoxyphenyl)acrylonitrile (1) was found to be of a potency comparable to etoposide and greater than 6-mercaptopurine in all cell lines tested. Accordingly, a synthesis of a new series of (E)-2-(5,6-dichloro-1H-benzo[d] [1,2,3]triazol-1-yl)-3-(4-R-phenyl)acrylonitriles was conducted in order to extend the studies of structure-activity relationship (SAR) for this class of molecules. With the aim to evaluate if 3-aryl-2-[1H-benzotriazol-1-yl]acrylonitriles were able to act like tubulin binding agents, the effects on cell cycle distribution of the most active compounds (1, 2a, 3 and 4) were analyzed in K562 cells. A detailed molecular modeling study of the putative binding mode of this series of compounds on tubulin is also reported.

Protective properties of non-nucleoside reverse transcriptase inhibitor (MC1220) incorporated into liposome against intravaginal challenge of Rhesus macaques with RT-SHIV.

In the absence of an effective vaccine against HIV, it is urgent to develop an effective alternative such as a microbicide. Single and repeated applications of MC1220 microbicide were evaluated in macaques. First, animals were given a single application of 0.5% or 1.5% MC1220-containing liposomal gel. A second group were treated with 0.5% MC1220 once a day for 4 days. The control groups were treated by liposomal gel alone. Thirty minutes after the last application, animals were challenged with RT-SHIV. In the first protocol, 2 of 4 animals treated by 0.5% of the MC1220 and 2 of 5 treated by 1.5% were protected. In the second protocol, 3 of 5 treated animals were protected and 5 of 5 controls were infected. The RNA viral load at necropsy was significantly lower (p=0.05) in treated-infected animals than in controls. In both protocols, the number of CD4+ T cells was lower at viremia peak in infected than in protected animals.

Antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives.

Twelve aminoarylazocompounds (A-C) and 46 aryltriazene 7 derivatives (D-G) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. Eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high level, against one or more viruses. A marked activity against BVDV and YFV was prevailing among the former compounds, while the latter type of compounds affected mainly CVB-2 and RSV. None of the active compounds inhibited the multiplication of HIV-1, VSV and VV. Arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are the following; BVDV: 1>7>8>4; YFV: 7>5; CVB-2: 25>56>18; RSV: 14>20>55>38>18>19; HSV-1: 2. For these compounds the EC(50) ranged from 1.6 microM (1) to 12 microM (18), and the S. I. from 19.4 (1) to 4.2 (2). Thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for developing antiviral agents against ss RNA viruses, particularly against RSV and BVDV, which are important human and veterinary pathogens. Finally, molecular modeling investigations indicated that compounds of structure A-C, active against BVDV, could work targeting the viral RNA-dependent RNA-polymerase (RdRp), having been observed a good agreement between the trends of the estimated IC(50) and the experimental EC(50) values.

2-Arylbenzimidazoles as antiviral and antiproliferative agents--Part 2.

In prosecution of an anti-Flaviviridae project a new series of variously substituted 2-diphenyl-benzimidazoles were synthesized and tested in vitro for antiviral and antiproliferative activities. Compounds were tested in cell-based assays against viruses representative of: i) two of the three genera of the Flaviviridae family, i.e. Flaviviruses and Pestiviruses; ii) other RNA virus families, such as Retroviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae; iii) two DNA virus families (Herpesviridae and Poxviridae). The 5-Acetyl-2-(4'-nitrobiphenyl-4-yl)-1H-benzimidazole (24) emerged as potent active lead compound against Yellow Fever Virus (a Flavivirus) (EC(50) = 0.5 microM) and CVB-2 at 1 microM and was not cytotoxic, whereas the other title benzimidazoles showed no antiviral activity at concentrations not cytotoxic for the resting cell monolayers. Among the examined series, the most cytotoxic derivatives (11,12,14,16,18,19,20,21,23,25-30) against mock-infected MT-4 cells (CC50 < 8.0 microM) were evaluated against a panel of human cell lines derived from haematological and solid tumours,using 6-mercaptopurine (6-MP) and etoposide as reference drugs. In particular, compounds 26 and 28 showed a similar potency of 6-MP and etoposide.

Aryl nucleoside H-phosphonates. Part 16: synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters.

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least partially, to intracellular conversion into the corresponding nucleoside 5'-monophosphates, and their efficiency correlated well with the pK(a) values of the aryloxy groups present.

Anti-HIV evaluation of benzo[d]isothiazole hydrazones.

The synthesis and the anti-HIV-1 activity of novel benzo[d]isothiazole hydrazones are reported. Target compounds tested in MT-4 cells cultures for their anti-HIV properties against wild type HIV-1 and HIV strains carrying clinically relevant mutations (EFV(R), Y181C and K103/Y181C) showed good activity against wild type HIV-1 and against the EFV(R) mutant. In terms of SAR the relevant result was that, in the class of benzisothiazole hydrazones, the benzo[d]isothiazol-3(2H)-one moiety (compounds 1 and 4) is an essential structural requirement for the antiretroviral activity.

N-acylated and N,N'-diacylated imidazolidine-2-thione derivatives and N,N'-diacylated tetrahydropyrimidine-2(1H)-thione analogues: synthesis and antiproliferative activity.

Fifty-one acylthioureas (ATUs) incorporating imidazolidine-2-thione or its upper cyclohomologue were prepared by parallel synthesis and evaluated against a high number of human cancer cell lines for antiproliferative activity. ATUs 1o (3,5-dichlorobenzoyl), 1s (2-furoyl), 3s (2-furoyl) and 1t (2-thenoyl) displayed activity against leukemia, melanoma LOX IMVI, non-small cell lung NCI-H522, renal 786-0, CAKI-1, SN12C, UO-31 and breast MCF7, MDA-MB-435, T-47D cancer cell lines in the 0.3-9.7 microM concentration range. Compound 14s exhibited selectivity for melanoma SK-MEL-5 (GI(50)<5 nM); 1s for leukemia MOLT-4 (GI(50): 300 nM); 1q, 3b and 3q for renal cancer UO-31 (GI(50): 70-200 nM); 8s, 9s for non-small cell lung cancer EKVX (GI(50): 300, 10 nM) and 3j for HOP-92 (GI(50): 700 nM) cell line.

In vitro antitumor and antiviral activities of new benzothiazole and 1,3,4-oxadiazole-2-thione derivatives.

A series of new benzothiazole derivatives 6a-h have been synthesized, in five steps, from substituted phenols via the 1,3,4-oxadiazole-2-thiones 5a-h. The in vitro antitumor activity of the compounds obtained was investigated and the benzothiazol derivatives 6d and 6e showed strong effects on leukaemia cell lines CCRF-CEM (CC50=12+/-2 micromol L(-1), 8+/-1 micromol L(-1), respectively). These compounds are leading candidates for further development. The title compounds were tested against representatives of several virus families containing single stranded RNA genomes, either positive-sense (ssRNA+), or negativesense (RNA-), and against double-stranded RNA genomes (dsRNA), as well as some Flaviviridae viruses.

2(3)-Aryl-thio(oxy)-methylquinoxaline derivatives: a new class of P-glycoprotein-mediated drug efflux inhibitors.

A series of quinoxalines variously substituted, namely 3-arylthiomethyl-1,6-dimethylquinoxalin-2-ones (6a-f), 3-arylthiomethyl-1-benzyl-7-trifluoromethylquinoxalin-2-ones (8a-g) and 2-arylthiomethyl-3-benzyloxy-6-trifluoro-methylquinoxalines (10a,b,e-h), were synthesized and compared with previous arylphenoxymethylquinoxalines (1a-f, 2a-f and 3a-b). The purpose was to verify whether the replacement of oxygen with sulphur atom and the insertion of different substituents on the phenyl side chain were able to improve the capability to inhibit the Pgp pump and restore the antiproliferative activity of clinically useful drugs, such as doxorubicin (Doxo), vincristine (VCR) and etoposide (VP16), in drug-resistant human nasopharyngeal carcinoma KB cells (KB(wt), KB(MDR), KB(7D) and KB(V20C)). Furthermore, 2,3-bis(aryloxy-methyl)-6-trifluoromethylquinoxalines (13a-c) were designed with the objective to evaluate the capability of the double side chain to potentiate the antiproliferative activity of the drugs tested. Biological assays showed that title compounds were, in general, endowed with good activity as Pgp inhibitors. In particular compound 3a, bearing 2-CONHPh substituent on phenoxymethyl side chain, resulted the most effective, while the double side chain (compound 13c) gives the ability to inhibit a different MRP pump (a membrane glycoprotein named mrp). Furthermore, we can conclude that replacement of oxygen with sulphur atom did not improve the biological activity.

Design, synthesis and anti flaviviridae activity of N(6)-, 5',3'-O- and 5',2'-O-substituted adenine nucleoside analogs.

During a random screening of representative libraries of nucleoside analogues we discovered that the adenine derivatives FEVB28 and FEG118 were Flaviviridae inhibitors endowed with potency comparable, if not superior, to that of ribavirin. Those studies prompted us to design a new class of protected nucleoside analogs, reported herein, which displays interesting anti-bovine viral diarrhea virus (BVDV) activity and low cytotoxicity in cell-based assays (4, 23, 29 EC(50): 14, 11, 26 microM respectively, CC(50)>100 microM) and appreciable activity in enzyme assays against the RNA dependent RNA polymerase (RdRp) of BVDV (4, 23, 29, RdRp inhibition activity 27, 16, 15 microM respectively). A molecular modeling study was also carried out to highlight the possible interactions between this compounds class and the corresponding hepatitis C virus (HCV) enzyme.

Antiprion activity of cholesterol esterification modulators: a comparative study using ex vivo sheep fibroblasts and lymphocytes and mouse neuroblastoma cell lines.

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

Synthesis and antiproliferative properties of N3/8-disubstituted 3,8-diazabicyclo[3.2.1]octane analogues of 3,8-bis[2-(3,4,5-trimethoxyphenyl)pyridin-4-yl]methyl-piperazine.

A series of novel N(3/8)-disubstituted-3,8-diazabicyclo[3.2.1]octanes in order to improve the in vitro activity of the prototype 3,8-bis[2-(3,4,5-trimethoxyphenyl)pyridyl-4-yl)methylpiperazine (1) were synthesized and evaluated by assays of growth inhibition against several tumor cell lines. Compounds 2a,b,f and m demonstrated not only growth-inhibitory activities against leukemia cancer cells, but also fairly good activities against the growth of certain solid tumors. Among them, 2a is the most potent one with IC(50) values in the low micromolar range. Moreover, compound 2a has been selected for in vitro testing on MCF-7 cell to evaluate the mode of action of this lead compound.

N, N' (4,5-dihydro-1h-imidazol-2-yl)3-aza-1,10-decane-diamine and N, N'(4,5-dihydro-1H-imidazol-2-yl)3-aza-1, 10-dodecane-diamine antagonize cell proliferation as selective ligands towards topoisomerase II.

New alkyl imidazoline derivatives have been synthesized as potential anti-cancer agents. The anti-proliferative activity of these compounds, evaluated against representative human haematological and solid neoplastic cell lines, showed that N, N'-di (4,5-dihydro-1H-imidazol-2-yl)3-aza-1,10-decane-diamine (8) and N, N'-di (4,5-dihydro-1H-imidazol-2-yl)3-aza-1,10-dodecane-diamine (9) were the most active compounds; in fact, they inhibited the cell proliferation at submicromolar concentrations. In enzyme assays, compound 9 turned out to be an inhibitor of topoisomerase II at concentrations comparable with those of the reference topoisomerase II inhibitor, etoposide.

Synthesis of variously substituted 3-phenoxymethyl quinoxalin-2-ones and quinoxalines capable to potentiate in vitro the antiproliferative activity of anticancer drugs in multi-drug resistant cell lines.

Two series of 1,6-dimethyl-3-phenoxymethylquinoxalin-2-ones and 1-benzyl-3-phenoxymethyl-7-trifluoromethylquinoxalin-2-ones, and a series of 2-benzyloxy-3-phenoxymethyl-7-trifluoromethylquinoxaline were synthesized. Their capability to restore/potentiate the antiproliferative activity of clinically useful drugs, such as doxorubicin (Doxo), vincristine (VCR) and etoposide (VP16), in drug-resistant human nasopharyngeal carcinoma KB cells (KB(WT), KB(MDR), KB(7D)and KB(V20C)) was evaluated. In vitro data show that many quinoxalin-2-ones and quinoxalines potentiate the antiproliferative activity of Doxo and VCR in tumor-derived MDR cell lines. In this series, 17a turned out to be the most potent quinoxaline derivative in potentiating the antiproliferative activity of doxorubicin and vincristine against KB(MDR) and KB(V20C) resistant cell lines, respectively.

Amino acid derivatives. Part I. Synthesis, antiviral and antitumor evaluation of new alpha-amino acid esters bearing coumarin side chain.

A series of amino acid esters bearing coumarin (3-15) were synthesized and evaluated, in vitro, against HIV-1, and bovine viral diarrhea virus (BVDV). The in vitro cytotoxicity of 3-10 and 12 was assayed against a panel of tumor cell lines consisting of CD4 human T-cells. Compound 14 showed inhibition of HIV-1 with EC50 > 1.6 microg mL(-1), meanwhile compound 9 exhibited activity against leukaemia (MT4) with CC50 = 24 micromol L(-1).