Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models.

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

New Oligonucleotide 2'-O-Alkyl N3'→P5' (Thio)-Phosphoramidates as Potent Antisense Agents: Physicochemical Properties and Biological Activity.

We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2'-O-alkyl ribonucleotide N3'→P5' phosphoramidate (2'-O-alkyl-NPO) and (thio)-phosphoramidite (2'-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2'-O-alkyl modifications such as 2'-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3'-amino sugar-phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3'-endo sugar pucker conformation. Moreover, 2'-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2'-deoxy NPOs. In addition, the relative lipophilicity of the 2'-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3'-O- counterparts. The 2'-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2'-O-MOE-NPS gapmers demonstrated higher activity than the 3'-O-containing 2'-O-MOE counterpart. The properties of 2'-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.

Synthesis and Anti-HCV Activity of Sugar-Modified Guanosine Analogues: Discovery of AL-611 as an HCV NS5B Polymerase Inhibitor for the Treatment of Chronic Hepatitis C.

Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 µM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5'-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.

Structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs.

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

Distinct Effects of T-705 (Favipiravir) and Ribavirin on Influenza Virus Replication and Viral RNA Synthesis.

T-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virus-infected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. In addition, the cellular nucleotide pools as well as direct inhibition of the viral polymerase enzyme were quantified. We demonstrate that the anti-influenza virus effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at ≥50 µM, whereas exposure to lower drug concentrations induces formation of noninfectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is 2-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2'-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases.

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2'-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2'-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

The ambiguous base-pairing and high substrate efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate towards influenza A virus polymerase.

T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3'-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus.

Synthesis and antiviral evaluation of 6-(alkyl-heteroaryl)furo[2,3-d]pyrimidin-2(3H)-one nucleosides and analogues with ethynyl, ethenyl, and ethyl spacers at C6 of the furopyrimidine core.

Sonogashira coupling strategies were employed to synthesize new furo[2,3-d]pyrimidin-2(3H)-one (FuPyrm) 2'-deoxynucleoside analogues. Partial or complete reduction of ethyne-linked compounds afforded ethenyl- and ethyl-linked derivatives. Levels of inhibition of varicella-zoster virus (VZV), human cytomegalovirus (HCMV), a broad range of other DNA and RNA viruses, and several cancer cell lines were evaluated in cell cultures. The anti-VZV potency decreased with increasing rigidity of the side chain at C6 of the FuPyrm ring in the order dec-1-yn-1-yl < dec-1-en-1-yl < decan-1-yl. In contrast, compounds with a rigid ethynyl spacer between C6 of the FuPyrm ring and a 4-alkylphenyl moiety were more potent inhibitors of VZV than the corresponding derivatives with an ethyl spacer. Replacement of the phenyl moiety in 6-(4-alkylphenyl) derivatives with a pyridine ring (in either regioisomeric orientation) gave analogues with increased solubility in methanol but reduced anti-VZV potency, and replacement with a pyrimidine ring reduced the anti-VZV activity even further. The pyridine-ring-containing analogues were approximately 20-fold more potent inhibitors of VZV than acyclovir but were approximately 6-fold less potent than BVDU and approximately 60-fold weaker than the most active 6-(4-pentylphenyl)-substituted prototype.

Synthesis and biological evaluation of 6-(alkyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-one base and nucleoside derivatives.

Derivatives of the 2'-deoxynucleoside of furo[2,3-d]pyrimidin-2(3H)-one with long-chain alkyl (or 4-alkylphenyl) substituents at C6 exhibit remarkable anti-VZV (varicella-zoster virus) potency and selectivity, and analogous 2',3'-dideoxynucleoside derivatives show anti-HCMV (human cytomegalovirus) activity. We now report a synthetic approach that enables the preparation of long-chain 6-(alkyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-ones in which the rodlike acetylene spacer replaces the 4-substituted-phenyl ring at C6. Analogues with methyl, beta-d-ribofuranosyl, beta-d-arabinofuranosyl, and 2-deoxy-beta-d-erythro-pentofuranosyl substituents at N3 have been prepared. Long-chain derivatives at C6 in the 2'-deoxynucleoside series showed virus-encoded nucleoside kinase-sensitive anti-VZV activity. Surprisingly, 3-methyl-6-(octyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-one (prepared as a negative anti-VZV test control) exhibited anti-HCMV activity, which supports the possibility of development of non-nucleoside anti-HCMV agents originating from uncomplicated derivatives of such bicyclic ring systems.

Synthesis of 5'-triphosphate mimics (P3Ms) of 3'-azido-3',5'-dideoxythymidine and 3',5'-dideoxy-5'-difluoromethylenethymidine as HIV-1 reverse transcriptase inhibitors.

3'-Azido-3',5-dideoxythymidine 5'-phosphonate and 3',5'-dideoxy-5'-difluoromethylenethymidine 5'-phosphonate were prepared by multistep syntheses. The nucleoside 5'-phosphonates were converted to their triphosphates and triphosphate mimics (P3Ms) containing beta,gamma-difluoromethylene, beta,gamma-dichloromethylene, or beta,gamma-imodo by condensation with pyrophosphate or pyrophosphate mimics, respectively. Inhibition of HIV-1 reverse transcriptase by the nucleoside P3Ms is briefly discussed.

Synthesis of 2',3'-dideoxynucleoside 5'-alpha-P-borano-beta,gamma-(difluoromethylene)triphosphates and their inhibition of HIV-1 reverse transcriptase.

The triphosphates of antiviral 2',3'-dideoxynucleosides (ddNs) are the active chemical species that inhibit viral DNA synthesis. The inhibition involves incorporation of ddNMP into DNA and subsequent chain termination. A conceivable strategy for antiviral drugs is to employ nucleoside 5'-triphosphate mimics that can entirely bypass cellular phosphorylation. AZT 5'-alpha-R(P)-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaB-betagammaCF(2)TP) has been identified as a potent inhibitor of HIV-1 reverse transcriptase (HIV-1 RT). This work was aimed at confirming that 5'-alphaB-betagammaCF(2)TP is a useful generic triphosphate moiety and can render antiviral ddNs with potent inhibitory effects on HIV-1 RT. Thus, 10 ddNs were converted to their 5'-alphaB-betagammaCF(2)TPs via a sequence (one-pot) of reactions: formation of an activated phosphite, formation of a cyclic triphosphate, boronation, and hydrolysis. Other synthetic routes were also explored. All ddN 5'-alphaB-betagammaCF(2)TPs tested exhibited essentially the same level of inhibition of HIV-1 RT as the corresponding ddNTPs. A conclusion can be made that 5'-alphaB-betagammaCF(2)TP is a generic and promising triphosphate mimic (P3M) concerning HIV-1 RT inhibition and serum stability. It is anticipated that use of 5'-alphaB-betagammaCF(2)TP as P3M moiety will lead to the discovery of a new class of anti-HIV agents.

Synthesis of AZT 5'-triphosphate mimics and their inhibitory effects on HIV-1 reverse transcriptase.

In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.