virus PA N endonuclease inhibitors
1.4 PA N ENDONUCLEASE INHIBITORS
1.4.3 N-hydroxamic acids family and catechins
During a high-throughput in vitro screening, it has been highlighted the inhibitory activity of N-hydroxamic acid derivatives on the capped RNA-dependent transcription of influenza A and B with the hit compound 1 (Figure 13) having an IC50 value of about 40 M on both types. SAR studies of this derivative have revealed that the hydroxamic moiety, together with the phenolic hydroxyl and the nitrogen atom of quinoline, are essential for activity; moreover, the insertion of the hydroxamic acid on a cyclic ring scaffold can enhance the activity, as verified for 2 and 3, which mimic the binding motif of Flutimide. However, these compounds resulted to be toxic, then no further investigation of antiviral in vivo activity has been developed.
309Tomassini J.E. et al.; Antimicrob. Agents Chemother. 40; 1189–1193 (1996).
310Singh S.B. and Tomassini J.E.; J. Org. Chem. 66; 5504–5516 (2001).
311Baranov M.S. et al.; Cheminform 70; 3714–3719 (2014).
Figure 13. N-hydroxamic acid analogues and the relative IC50 values.
Catechins, too, were found to impair influenza A replication and viral RNA synthesis: this has been attributed to the alteration of the physical properties of the viral membrane312. Recent studies have revealed that ECG ((-)-epicatechin gallate) and EGCG ((-)-epigallocatechin gallate) have an inhibitory activity on PA endonuclease: docking studies confirmed the hypothesis that the galloyl group chelates both metal ions by the three adjacent oxygen atoms313. By a screening of several types of phytochemicals, three of them have been identified as PA endonuclease inhibitors: marchantins (Ms), perrottetins (Pes) and plagiochins (Pls)314.
1.4.4 3-hydroxypyridinone and analogues
X-ray crystallography fragment screening first, and, then, FRET-based endonuclease inhibition assays have led to the discovery of compound 9 as a hit derivative315,316. Phenyl and substituted-phenyl have been introduced on the 3-hydroxypyridin-2(1H)-one at the 4-,5- and 6-position to try to make more efficient interactions with the diverse hydrophobic pockets that surround the active site. Among all, compounds that are bis-substituted at 5- and 6-position, like compounds 10 and 11 (Figure 14), resulted to be the most active, while the 4-position substituted compounds resulted to have almost lost their activity.
312Song J.M. et al.; Antiviral Res. 68; 66–74 (2005).
313Kuzuhara T. et al.; PLoS Curr. 1; RRN1052 (2009).
314Iwai Y. et al.; PLoS One 6; e19825 (2011).
315Bauman J.D. et al.; ACS Chem. Biol. 8; 2501–2508 (2011).
316Parhi A.K. et al.; Bioorg. Med. Chem. 21; 6435–6446 (2013).
Figure 14 .3-hydroxypyridinone analogues molecular structures.
A step forward has been made by trying to change the ring skeleton by the introduction of nitrogen heterocycles: 5-hydroxypyrimidin-4(3H)-one analogues have been synthesized and analyzed in enzymatic assay317,318. Modifications on the 3-hydroxyquinolin-2(1H)-one have led to two potent derivatives, 12 and 13 (IC50= 0.5 M, both) which have a p-fluorophenyl group at 6-and7-position respectively. From X-ray data it has been possible to elucidate the chelating motif of 13: the ketone group at 2-position and the 3-hydroxyl are involved into metal coordination, while the NH group of quinoline core coordinates the water molecule that chelates a metal ion.
Nowadays, none of the worldwide approved antiviral drugs acts by impairing the activity of PAN
endonuclease; recently, two PA inhibitors (i.e. Baloxavir marboxil and AL-794, S-033188) have reached out advanced steps in clinical trials, highlighting the validity of this innovative molecular target319. Moreover, Baloxavir marboxil has reached out phase III clinical trials320 and it has been approved in 2008 in Japan as antiviral therapeutic agent321. Even if steps forward in this field have been made in the past years, there is still an urgent need of new antiviral compounds possible based on novel molecular scaffold: this is mainly due to the resistance that viruses have developed against the most common therapy.
317Sagong H.Y. et al.; ACS Med. Chem. Lett. 4; 547–550 (2013).
318Sagong H.Y. et al.; J. Med. Chem. 57; 8086–8098 (2014).
319Wu X., Sun Q., Zhang C., Yang S., Li L., Jia Z.; Theranostics 7; 826-845 (2017).
320Portsmouth S., Kawaguchi K., Arai M., Tsuchiya K., Uehara T.; Open Forum Infect. Dis. 4(Suppl. 1); S734(2017).
321Heo Y.A.; Drugs 78; 693-697 (2018).
Figure 15. Chemical structure of Baloxavir marboxil.
AIM OF THE PROJECT. The research has moved forward, and interesting results are reported by Zhao and co-workers322: they’ve have explored a novel molecular scaffold, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one one, as inhibitor for the HIV Integrase in order to impair its activity, then the viral replication process. From preliminary enzymatic assay results seem to be really promising, revealing IC50 values in the low micromolar range. From data reported, it is possible to infer that the use of a 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold could be a good strategy to simultaneously chelate both the metal ions present in the active site of a magnesium-dependent enzyme. We have, then, decide to use the same scaffold modifying it in order to increase the affinity for Influenza virus PA endonuclease active center. The panel shown in Figure 16 has been synthesized and preliminary IC50 values for PA endonuclease inhibition have been estimated by a FRET-based assay.
322Zhao X.Z. et al.; J. Med. Chem. 51; 251-259 (2008).
Figure 16. 2,3-Dihydro-6,7-dihydroxy-1H-isoindol-1-one derivatives as Mg2+ targeting PAN endonuclease inhibitors.
On the basis of enzymatic in vitro assays, a second panel of molecules has been, then, synthesized in order to highlight the role of the substituent at the 4-position on the aromatic ring as well as the length of the spacer between the isoindol-1-one moiety and the benzylic one. Biological assays on this second panel of molecules are still ongoing at KU Leuven University (Prof. Lieve Naesens).
2. EXPERIMENTAL SECTION
2.1 MATERIALS AND METHODS
All reagents of commercial quality were purchased from Sigma-Aldrich and used without further purification. The purity of the synthesized compounds was determined by elemental analysis and verified to be ≥ 95%. 1H-NMR spectra were recorded at 25 °C on a Bruker Avance 500 FT spectrophotometer. The ATR-IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by using a diamond crystal plate in the range of 4000-400 cm-1. Elemental analyses were performed by using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation. The purity of all compounds used in assays was determined to be ≥95% by 1H NMR spectroscopy and confirmed by high-resolution mass spectrometry (HRMS) experiments using an Agilent 6230 Accurate-Mass LC-TOFMS at the U.C. San Diego Molecular Mass Spectrometry Facility (MMSF). Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI) time-of-flight Micromass 4LCZ spectrometer.
Samples were dissolved in methanol. MS spectra were acquired with a DSQII Thermo Fisher apparatus, equipped with a single quadrupole analyzer in positive EI mode, by means of a DEP-probe (Direct Exposure Probe) equipped with a Re-filament. The UV-vis spectra were collected using a Thermo Evolution 260 Bio spectrophotometer provided with a thermostatting Peltier device, and quartz cuvettes with 1 cm path length.