Structural Modification and Biological Evaluation of 2,8-Disubstituted Adenine and Its Nucleosides as A2A Adenosine Receptor Antagonists: Exploring the Roles of Ribose at Adenosine Receptors.

Building on the preceding structural analysis and a structure-activity relationship (SAR) of 8-aryl-2-hexynyl nucleoside hA2AAR antagonist 2a, we strategically inverted C2/C8 substituents and eliminated the ribose moiety. These modifications aimed to mitigate potential steric interactions between ribose and adenosine receptors. The SAR findings indicated that such inversions significantly modulated hA3AR binding affinities depending on the type of ribose, whereas removal of ribose altered the functional efficacy via hA2AAR. Among the synthesized derivatives, 2-aryl-8-hexynyl adenine 4a demonstrated the highest selectivity for hA2AAR (Ki,hA2A = 5.0 ± 0.5 nM, Ki,hA3/Ki,hA2A = 86) and effectively blocked cAMP production and restored IL-2 secretion in PBMCs. Favorable pharmacokinetic properties and a notable enhancement of anticancer effects in combination with an mAb immune checkpoint blockade were observed upon oral administration of 4a. These findings establish 4a as a viable immune-oncology therapeutic candidate.

Structure-Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A2A/A3 Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity.

Based on hA2AAR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA2AAR agonists into antagonists while maintaining affinity toward hA3AR. The final compounds of 2,8-disubstituted-N6-substituted 4'-thionucleosides, or 4'-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA2AAR, including 5d with the highest affinity (Ki,A2A = 7.7 ± 0.5 nM). The hA2AAR-5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA3AR. Structural SAR features and docking studies supported different binding modes at A2AAR and A3AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo. Overall, this study suggests that the novel dual A2AAR/A3AR nucleoside antagonists would be promising drug candidates for immune-oncology.

GPCR Agonist-to-Antagonist Conversion: Enabling the Design of Nucleoside Functional Switches for the A2A Adenosine Receptor.

Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson's disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds.

Antiproliferative and Antimigration Activities of Fluoro-Neplanocin A via Inhibition of Histone H3 Methylation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is among the most aggressive and potentially metastatic malignancies. Most affected patients have poor clinical outcomes due to the lack of specific molecular targets on tumor cells. The upregulated expression of disruptor of telomeric silencing 1-like (DOT1L), a histone methyltransferase specific for the histone H3 lysine 79 residue (H3K79), is strongly correlated with TNBC cell aggressiveness. Therefore, DOT1L is considered a potential molecular target in TNBC. Fluoro-neplanocin A (F-NepA), an inhibitor of S-adenosylhomocysteine hydrolase, exhibited potent antiproliferative activity against various types of cancer cells, including breast cancers. However, the molecular mechanism underlying the anticancer activity of F-NepA in TNBC cells remains to be elucidated. We determined that F-NepA exhibited a higher growth-inhibitory activity against TNBC cells relative to non-TNBC breast cancer and normal breast epithelial cells. Moreover, F-NepA effectively downregulated the level of H3K79me2 in MDA-MB-231 TNBC cells by inhibiting DOT1L activity. F-NepA also significantly inhibited TNBC cell migration and invasion. These activities of F-NepA might be associated with the upregulation of E-cadherin and downregulation of N-cadherin and Vimentin in TNBC cells. Taken together, these data highlight F-NepA as a strong potential candidate for the targeted treatment of high-DOT1L-expressing TNBC.

6'-ß-Fluoro-Homoaristeromycin and 6'-Fluoro-Homoneplanocin A Are Potent Inhibitors of Chikungunya Virus Replication through Their Direct Effect on Viral Nonstructural Protein 1.

Alphaviruses are arthropod-borne, positive-stranded RNA viruses capable of causing severe disease with high morbidity. Chikungunya virus (CHIKV) is an alphavirus that causes a febrile illness which can progress into chronic arthralgia. The current lack of vaccines and specific treatment for CHIKV infection underscores the need to develop new therapeutic interventions. To discover new antiviral agents, we performed a compound screen in cell culture-based infection models and identified two carbocyclic adenosine analogues, 6'-ß-fluoro-homoaristeromycin (FHA) and 6'-fluoro-homoneplanocin A (FHNA), that displayed potent activity against CHIKV and Semliki Forest virus (SFV) with 50% effective concentrations in the nanomolar range at nontoxic concentrations. The compounds, designed as inhibitors of the host enzyme S-adenosylhomocysteine (SAH) hydrolase, impeded postentry steps in CHIKV and SFV replication. Selection of FHNA-resistant mutants and reverse genetics studies demonstrated that the combination of mutations G230R and K299E in CHIKV nonstructural protein 1 (nsP1) conferred resistance to the compounds. Enzymatic assays with purified wild-type (wt) SFV nsP1 suggested that an oxidized (3'-keto) form, rather than FHNA itself, directly inhibited the MTase activity, while a mutant protein with the K231R and K299E substitutions was insensitive to the compound. Both wt nsP1 and the resistant mutant were equally sensitive to the inhibitory effect of SAH. Our combined data suggest that FHA and FHNA inhibit CHIKV and SFV replication by directly targeting the MTase activity of nsP1, rather than through an indirect effect on host SAH hydrolase. The high potency and selectivity of these novel alphavirus mRNA capping inhibitors warrant further preclinical investigation of these compounds.

Identification of 6'-ß-fluoro-homoaristeromycin as a potent inhibitor of chikungunya virus replication.

We have reported on aristeromycin (1) and 6'-fluorinated-aristeromycin analogues (2), which are active against RNA viruses such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). However, these exhibit substantial cytotoxicity. As this cytotoxicity may be attributed to 5'-phosphorylation, we designed and synthesized one-carbon homologated 6'-fluorinated-aristeromycin analogues. This modification prevents 5'-phosphorlyation by cellular kinases, whereas the inhibitory activity towards S-adenosyl-l-homocysteine (SAH) hydrolase will be retained. The enantiomerically pure 6'-fluorinated-5'-homoaristeromycin analogues 3a-e were synthesized via the electrophilic fluorination of the silyl enol ether with Selectfluor, using a base-build up approach as the key steps. All synthesized compounds exhibited potent inhibitory activity towards SAH hydrolase, among which 6'-ß-fluoroadenosine analogue 3a was the most potent (IC50 = 0.36 µM). Among the compounds tested, 6'-ß-fluoro-homoaristeromycin 3a showed potent antiviral activity (EC50 = 0.12 µM) against the CHIKV, without noticeable cytotoxicity up to 250 µM. Only 3a displayed anti-CHIKV activity, whereas both3a and 3b inhibited SAH hydrolase with similar IC50 values (0.36 and 0.37 µM, respectively), which suggested that 3a's antiviral activity did not merely depend on the inhibition of SAH hydrolase. This is further supported by the fact that the antiviral effect was specific for CHIKV and some other alphaviruses and none of the homologated analogues inhibited other RNA viruses, such as SARS-CoV, MERS-CoV, and ZIKV. The potent inhibition and high selectivity index make 6'-ß-fluoro-homoaristeromycin (3a) a promising new template for the development of antivirals against CHIKV, a serious re-emerging pathogen that has infected millions of people over the past 15 years.

Design, Synthesis, and Anti-RNA Virus Activity of 6'-Fluorinated-Aristeromycin Analogues.

The 6'-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6'-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and N6-methyladenosine analogues 2a-e showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a-c exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6',6'-Difluoroaristeromycin (2c) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6'-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.

Synthesis and anti-HIV activity of L-2',3'-Dideoxy-4'-selenonucleosides (L-4'-Se-ddNs).

Based on the potent anti-HIV activity of L-2',3'-dideoxycytidine (L-ddC), L-2',3'-dideoxy-4'-selenonucleosides (L-4'-Se-ddNs) have been synthesized from natural chiral template, L-glutamic acid, using Pummerer-type condensation as a key step. All synthesized compounds were assayed for anti-HIV-1 activity, but none of them did show any significant antiviral activity up to 100 µM, probably due to conformational differences between L-ddC and L-4'-Se-ddC, induced by the bulky selenium atom, which might play an important role in phosphorylation by cellular kinase.

Correlation study between A3 adenosine receptor binding affinity and anti-renal interstitial fibrosis activity of truncated adenosine derivatives.

Truncated 4'-thionucleosides 1-4 and 4'-oxonucleosides 5-8 as potent and selective A3AR antagonists were synthesized from D-mannose and D-erythronic acid γ-lactone, respectively. These nucleosides were evaluated for their anti-fibrotic renoprotective activity in TGF-ß1-treated murine proximal tubular (mProx) cells. Their antagonistic activities for A3AR were proportional to their inhibitory activities against TGF-ß1-induced collagen I upregulation in mProx cells. This result suggests that the binding affinity of A3AR antagonists is closely correlated with their anti-fibrotic activity. Thus, A3AR antagonists might be novel therapeutic candidates for treating chronic kidney disease.

Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) Inhibitors as Potential Therapeutics.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a member of an evolutionarily conserved family of protein kinases that belongs to the CMGC group of kinases. DYRK1A, encoded by a gene located in the human chromosome 21q22.2 region, has attracted attention due to its association with both neuropathological phenotypes and cancer susceptibility in patients with Down syndrome (DS). Inhibition of DYRK1A attenuates cognitive dysfunctions in animal models for both DS and Alzheimer's disease (AD). Furthermore, DYRK1A has been studied as a potential cancer therapeutic target because of its role in the regulation of cell cycle progression by affecting both tumor suppressors and oncogenes. Consequently, selective synthetic inhibitors have been developed to determine the role of DYRK1A in various human diseases. Our perspective includes a comprehensive review of potent and selective DYRK1A inhibitors and their forthcoming therapeutic applications.

Design, synthesis and anticancer activity of fluorocyclopentenyl-purines and - pyrimidines.

Based on the potent anticancer activity of 6'-fluorocyclopentenyl-cytosine 2b in phase IIa clinical trials for the treatment of gemcitabine-resistant pancreatic cancer, we carried out a systematic structure-activity relationship study of 6'-fluorocyclopentenyl-pyrimidines 3a-i and -purines 3j-o to discover novel anticancer agents. We also synthesized the phosphoramidate prodrug 3p of adenine derivative 1b to determine if the anticancer activity depended on the inhibition of DNA and/or RNA polymerase in cancer cells and/or on the inhibition of S-adenosylhomocysteine (SAH) hydrolase. All of the synthesized pyrimidine nucleosides exhibited much less potent anticancer activity in vitro than the cytosine derivative 2b, acting as RNA and/or DNA polymerase inhibitor, indicating that they could not be efficiently converted to their triphosphates for anticancer activity. Among all the synthesized purine nucleosides, adenine derivative 1b and N6-methyladenine derivative 3k showed potent anticancer activity, showing equipotent inhibitory activity as the positive control, neplanocin A (1a) or Ara-C. However, the phosphoramidate prodrug 3p showed less anticancer activity than 1b, indicating that it did not act as a RNA and/or DNA polymerase inhibitor like 2b. This result also demonstrates that the anticancer activity of 1b largely depends on the inhibition of histone methyltransferase, resulting from strong inhibition of SAH hydrolase. The deamination of the N6-amino group, the addition of the bulky alkyl group at the N6-amino group, or the introduction of the amino group at the C2 position almost abolished the anticancer activity.

Asymmetric Synthesis of (-)-6'-ß-Fluoro-aristeromycin via Stereoselective Electrophilic Fluorination.

(-)-6'-ß-Fluoro-aristeromycin (2), a potent inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase, has been synthesized via stereoselective electrophilic fluorination followed by a purine base build-up approach. Interestingly, purine base condensation using a cyclic sulfate resulted in a synthesis of (+)-5'-ß-fluoro-isoaristeromycin (2a). Computational analysis indicates that the fluorine atom controlled the regioselectivity of the purine base substitution.

Stereoselective Synthesis of 4'-Selenonucleosides via the Seleno-Michael Reaction.

5'-Homo-4'-selenonucleosides, a class of next-generation nucleosides, are synthesized from D-ribose via a 4-selenosugar intermediate. The key step in synthesizing this intermediate is a seleno-Michael reaction. 5'-Homo-4'-selenouridine and -adenosine are prepared using Pummerer-type and Vorbrüggen condensation, respectively. © 2017 by John Wiley & Sons, Inc.