key: cord-327946-mqakaisa
authors: Massari, Serena; Bertagnin, Chiara; Pismataro, Maria Chiara; Donnadio, Anna; Nannetti, Giulio; Felicetti, Tommaso; Di Bona, Stefano; Nizi, Maria Giulia; Tensi, Leonardo; Manfroni, Giuseppe; Loza, Maria Isabel; Sabatini, Stefano; Cecchetti, Violetta; Brea, Jose; Goracci, Laura; Loregian, Arianna; Tabarrini, Oriana
title: Synthesis and Characterization of 1,2,4-Triazolo[1,5-a]pyrimidine-2-carboxamide-based Compounds Targeting the PA-PB1 Interface of Influenza A Virus Polymerase
date: 2020-10-16
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2020.112944
sha: 
doc_id: 327946
cord_uid: mqakaisa

Influenza viruses (Flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. Antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-Flu armamentarium still remains inadequate. In search for new anti-Flu drugs, our group has focused on viral RNA-dependent RNA polymerase (RdRP) developing disruptors of PA-PB1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. By merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. In particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important SAR insight and identify new active compounds showing both the ability to inhibit PA-PB1 interaction and viral replication in the micromolar range and at non-toxic concentrations. For few compounds, the ability to efficiently inhibit PA-PB1 subunits interaction did not translate into anti-Flu activity. Chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. Finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, H-bonds enhanced its binding within the PA(C) cavity.

Influenza (Flu) viruses are responsible for seasonal epidemics resulting in about 3 to 5 million cases of severe respiratory illness and 290,000 to 650,000 deaths each year all over the world [1] . Moreover, they are also able to generate pandemic outbreaks that occur when new highly virulent FluA subtypes generated by antigenic shift are transmitted from animals to humans and sustainably spread among people.

In 1918, humanity experienced the "Spanish Flu" caused by FluA(H1N1) [2] , an influenza pandemic that intensively and speedily struck world population infecting about 500 million people and killing from 20 to 40 million people globally [3] . Other two pandemics occurred in 20 th century: the "Asian Flu" caused by Flu A(H2N2) virus [4] , which started in China in 1957 and spread globally causing about one to four million deaths, and the "1968 Flu pandemic" caused by Flu A(H3N2) virus, which started in Hong Kong and spread to the United States causing one million deaths. In 2003, the avian Flu A(H5N1) re-emerged passing the species barrier but fortunately not spreading sustainably from person to person, while, in 2009, the "swine Flu" A(H1N1) virus was responsible for the first pandemic of the 21 st century [5] ; it started in Mexico and spread rapidly around the world causing from 150,000 to 600,000 deaths [6] . To date, the avian Flu A(H5N1) and

Flu A(H7N9) are of particular concern to public health due to their potential to cause an influenza pandemic [7] . [32] .

Computational studies performed on the positional isomers 3 and 4 within the PA C cavity suggested that the molecules have a different orientation and extents of hydrophobic and H-bond interactions within the cavity [32] . Thus, while the elongated shape of 4 allows it to recognize all the three hydrophobic regions described by Liu and Yao [35] within the PB1 binding site, compound 3 is J o u r n a l P r e -p r o o f shifted toward the opposite side of the cavity and matches only the first hydrophobic region, generated by W706. Nevertheless, compound 3 establishes a very favorable H-bond between its C-2 amidic carbonyl group and the Q408 residue, which could be the reason for its efficient inhibition of PA-PB1 heterodimerization.

In the present study, additional hybrid compounds were synthesized as analogues of compound 3 (compounds 5-18, Figure 2 ) and compound 4 (compounds 19-26, Figure 2 ). By mainly focusing on the cHTC core, various structural modifications were undertaken investigating the cycloheptane, the thiophene and the 2-carboxamide moieties. From the antiviral activity, SAR insights were obtained with the main indication entailing the favorable replacement of the cHTC core by a simpler 2carbamoylphenyl moiety. In depth studies were pursued to determine the ability of the compounds to interfere with RdRP functions, their metabolic stability as well as to predict their binding mode within the PA C cavity. Further studies were also performed on a couple of regioisomers to investigate the different behavior in inhibiting PA-PB1 interaction and viral growth. 

The synthesis of all the target compounds 5-13, 15-22 and 24-29 was accomplished, as reported in Schemes 2-8, by coupling reaction of the appropriate reagent with 5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carbonyl chloride 30 [32] or 7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carbonyl chloride 31 [32] . As shown in Scheme 1, derivatives 30 and 31 were obtained by chlorination of the corresponding carboxylic acids [32] , which were in turn prepared by cyclocondensation of ethyl 5-amino-1,2,4-triazole-3-carboxylate [36] and 1phenylbutane-1,3-dione in acetic acid at reflux furnishing ethyl 5-methyl-7-phenyl- interaction. Of note, derivative 8 emerged as the most active anti-Flu compound herein reported, even more than hit compound 3. These results suggested that the presence of a cycloalkyl moiety fused to the thiophene ring is important to obtain anti-Flu activity, although its aromatization is still tolerated but detrimental for anti-PA-PB1 activity.

Then, a set of four derivatives was synthesized in which the thiophene-based core was linked to the TZP moiety by the C-3 position. The thienopyridine derivative 12 was the most potent PA-PB1

inhibitor among the compounds herein reported (IC 50 of 3.3 μM), but at the expense of the antiviral activity (EC 50 > 100 μM), while derivative 13 exhibited a balanced biological profile (IC 50 of 31

μM and EC 50 of 43 μM). These results indicated that, in this series of compounds, the lack of a cycloalkyl ring fused to the thiophene does not impair the activity.

No anti-PA-PB1 activity was shown by cycloheptathiophene compound 10 and benzothiophene compound 11, the strict analogs of compounds 3 and 9, respectively, although the latter showed All the compounds were non-toxic up to 250 μM concentration, with the exception of derivatives 10 and 18, which, however, showed a mild cytotoxic effect (CC 50 = 90 and 101 μM, respectively). In summary, the synthesis of novel hybrid compounds, aimed at clarifying the best substitution pattern for the C-2 position of the TZP ring, has led to the identification of some interesting compounds able to inhibit the PA-PB1 interaction and the viral growth. Interesting SAR insights have been outlined. In particular, the cHTC portion can be replaced by a benzene ring (as in 

Metabolic stability in human liver microsomes (HLM) was studied by monitoring the percentage of 

Although the MS/MS fragmentation is not sufficient to identify the exact position of the site of metabolism in the aromatic ring, the most probable site for hydroxylation appears to be at C-4 position, according to MetaSite [64] predictions within the WebMetabase analysis tool (Table S3 and Figure S3 , supporting information).

Regarding the human plasma protein binding, samples were analyzed by employing a rapid equilibrium system device and adding them in buffer and measuring the compound presence in the human plasma compartment after 4 h of incubation by means of UPLC/MS/MS. It was observed that compound 23 showed an unbound fraction of 22.45%, which together with the good metabolic stability predicts a good bioavailability of the compound in human plasma.

Finally, with the aim to gain information on how two different moieties, such as the cHTC and the 2-carbamoylphenyl, showed a favorable ability to disrupt PA-PB1 interaction, computational studies were performed to predict the binding mode of benzamide derivative 23 within the PA cavity with respect to hit compound 4. For comparative purpose, also its isomer 14, which was active in ELISA-based assay but devoid of any antiviral activity, was studied in comparison to hit compound 3.

The study was conducted using the same method applied for the evaluation of the binding mode of compounds 3 and 4 using the FLAP software [65] , and the most probable binding poses for the two isomers are illustrated in Figure 4 . In agreement with the biological results, both compounds showed an efficient binding within the PA C , although through different orientations and interactions. In particular, both compounds were found to efficiently interact with W706 through hydrophobic interactions, as observed for all the inhibitors of the PA-PB1 interaction we have studied so far [24] .

Similarly to 4, compound 23 displayed a favorable hydrophobic interaction with all the three hydrophobic regions previously described by Liu and Yao [35] . Indeed, according to the proposed J o u r n a l P r e -p r o o f description of the protein cavity, the first hydrophobic region is centered on residues W706 and F411, the second one is defined by F710 and L666, and the third one includes L640, V636, M595, and W619. However, differently from 4, 23 seems to be involved in the formation of a favorable Hbond between the 2-carboxamide NH group and the hydroxyl group of T639. This amino acid was not found to be involved in such kind of interaction in other compounds studied so far by us. interaction. This data seems to confirm the importance of an efficient interaction with W706 for inhibition of PA-PB1 heterodimerization.

In conclusion, the computational study confirmed the central interaction between the inhibitors and W706, and the 2-carboxyamide group of 23 and 14 was pivotal in the interaction with PA by establishing favorable H-bonds. This is also experimentally confirmed, since the analogues lacking the 2-carboxamide substituent showed a very weak activity in the ELISA PA-PB1 interaction assays [32] . 

The Flu RdRP is emerging as a privileged drug-target as demonstrated by the most recently approved compounds or in the pipeline such as favipiravir, baloxavir marboxil and pimodivir, which act by inhibiting each of the three RdRP subunits.

By applying an alternative approach to inhibit Flu RdRP functions, since many years we have been working on the development of compounds able to interfere with RdRP subunits interaction and in 

Commercially available starting materials, reagents, and solvents were used as supplied. All After cooling, the reaction mixture was filtered over celite and the filtrate was evaporated to dryness, to give a residue that was stirred with saturated NaHCO 3 solution for 15 min. The solution was then extracted with CH 2 Cl 2 and the organic layers were evaporated to dryness to give a grey solid, which was purified by flash chromatography eluting with MeOH/CH 2 Cl 2 (2%), to give 9 [37] (1.0 equiv) in well dry CH 2 Cl 2 , oxalyl chloride (3 equiv) was added and after 30 min dry DMF (2 drops) was added. After 2h, the reaction mixture was evaporated to dryness to give 30 [37] or 31 [37] that was dissolved in well dry CH 2 Cl 2 and added of the appropriate amine (1.0 equiv) and DIPEA (1.0 equiv). The reaction mixture was maintained at r.t. until no starting material was detected by TLC. Then, it was worked up through two procedures: (procedure 1) the reaction mixture was evaporated to dryness to give a residue that was poured into ice/water providing a precipitate which was filtered and purified as reported in the description of the compounds; or (procedure 2) the precipitate formed in the reaction mixture was filtered and purified as reported in the description of the compounds. 

The title compound was prepared starting from 36 [38] and 30 [37] ( N-(3-carbamoyl-6-ethyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-methyl-7- (9) . The title compound was prepared starting from 40 [41] and 30 [37] (11) . The title compound was prepared starting from 48 [48] and 30 [37] through 

. [49] The title compound was prepared starting from methyl 3-aminothiophene-2-carboxylate through Method B providing 49 [47] in 100% yield, 

carboxamide (13). The title compound was prepared starting from 50 [49] and 30 [37] through 

carboxamide (22) . The title compound was prepared starting from 50 [49] and 31 [37] through 

The minireplicon assay was performed as described [27] , with some modifications. Briefly, HEK

293T cells (2 × 10 5 cells per well) were plated into 24-well plates and incubated overnight at 37 °C.

The next day, cells were transfected using calcium phosphate co-precipitation method with pcDNA- 

The stock solutions (10-2 M) of the assayed compounds were diluted to decreased molarity, from 300 μM to 0.1μM, in 384 well transparent plate (Greiner 781801) with 1% DMSO: 99% PBS buffer. Then, they were incubate at 37 ºC and read after 2 hours in a NEPHELOstar Plus (BMG LABTECH). The results were adjusted to a segmented regression to obtain the maximum concentration in which compounds are soluble. Digossin, prazosin and progesterone were used as reference compounds (equilibrium solubility = 84.0, 62.8 and 6.5 μM, respectively) [71] .

J o u r n a l P r e -p r o o f X-Ray diffraction patterns of the crystals were recorded using a Bruker D8 Venture diffractometer equipped with an Incoatec ImuS3.0 microfocus sealed-tube Mo Kα (λ = 0.71073 Å) source and a CCD Photon II detector. The analyses were carried out at 120 (2) K using an Oxford Cryosystems 800 cooler. The data collected through generic φ and ω were integrated and reduced using the Bruker AXS V8 Saint Software. The structures were solved and all the thermal parameters were anisotropically refined using the SHELXT and SHELXL packages of the Bruker APEX3 software.

The assay was carried out by employing Rapid Equilibrium Dyalisis (RED) from Thermo Flow: 0.6 ml/min. The chromatographic equipment employed was an UPLC QSM Waters Acquity.

Compound concentrations were calculated from the MS peak areas.

Test compounds (10 μM www.moldiscovery.com).

C 23 H 19 N 5 O 2 S 430.1338 (M+H) + , found 430.133652 (M+H) + . HPLC, ret. time: 3.652, peak area: 97

The title compound was prepared starting from 2-aminobenzothiazole and 30

36% yield as yellow solid; 1 H NMR (DMSOd 6 , 400 MHz): δ 2.70 (s, 3H, CH 3 ), 7.30 (t, J = 7.4 Hz, 1H, aromatic CH), 7.40 (t, J = 7.3 Hz, 1H, aromatic CH), 7.55-7.70 (m, 4H, aromatic CH and H-6), 7.80 (d, J = 7.9 Hz, 1H, aromatic CH), 8.10 (d, J = 7.7 Hz, 1H, aromatic CH)

102196 (2M+H) + . HPLC, ret. time: 3.278 min, peak area: 98

The title compound was prepared starting from 2-aminobenzothiazole and 31

worked up through procedure 2, and purified by crystallization by DMF, in 57% yield as yellow solid; 1 H NMR (DMSO-d 6 , 400 MHz): δ 2.85 (s, 1H

10 (s, 1H, H-6), 8.25-8.30 (m, 2H, aromatic CH)

HRMS: m/z calcd for C 20 H 11 N 6 OS 387.1029 (M+H) + , found 387.1022 (M+H) + . HPLC, ret. time: 2.898 min, peak area: organization WHO, Influenza

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