#Nitrosocarbonyls 1: Antiviral Activity of N-(4-Hydroxycyclohex-2-en-1-yl)quinoline-2-carboxamide against the Influenza A Virus H1N1

Influenza virus flu A H1N1 still remains a target for its inhibition with small molecules. Fleeting nitrosocarbonyl intermediates are at work in a short-cut synthesis of carbocyclic nucleoside analogues. The strategy of the synthetic approaches is presented along with the in vitro antiviral tests. The nucleoside derivatives were tested for their inhibitory activity against a variety of viruses. Promising antiviral activities were found for specific compounds in the case of flu A H1N1.


Introduction
Influenza virus flu A H1N1 still remains a target for its inhibition with small molecules. The outbreak of the human pandemic influenza A (H1N1) caused a considerable public concern. Recent studies focused the attention on the understanding of novel viruses by analyzing the relationship between molecular characteristics and pathogenic properties [1]. Results of this analysis indicated that the human pandemic influenza A (H1N1) virus was a new reassorted virus combining genetic materials from the avian flu (H1N1) virus, classical swine flu (H1N1) virus, human flu (H3N2) virus, and Eurasian swine flu (H1N1) virus. The analysis of the sequences for receptor-binding and cleavage sites of hemagglutinin (HA) allowed for determining that replication could be inhibited by molecules such as oseltamivir (Tamiflu), a diamine derivative of the shikimic acid [2], and zanamivir (Relenza), a derivative of the 3,4-diamino-2-(hydroxymethyl)-3,4-dihydro-2Hpyran-6-carboxylic acid [3][4][5].
The influenza A virus is an orthomyxovirus, constituted by receptor-binding complexes containing two primary structural proteins: hemagglutinin (HA) and neuraminidase (NA). HA is the primary protein responsible for binding to receptor sites on the cell membrane, allowing the virion to enter the cell [4][5][6][7][8].
On pursuing our studies and in the search of novel small molecules with potential antiviral activity, we prepared some nucleoside derivatives, found moderately active against human herpes and varicella viruses [9] or strongly active against the human papilloma virus (HPV) [10] as well as influenza A virus H1N1 [11]. The synthetic protocol relied upon the nitrosocarbonyl chemistry.
Nitrosocarbonyls are fleeting intermediates whose generation as well as synthetic applications represents a remarkable piece of organic chemistry [12]. The nitrosocarbonyl intermediates 1 (R-CONO) are highly reactive species discovered at the beginning of the seventies by Kirby and their powerful dienophilic and enophilic activities were reported in 1973 in his seminal paper [13].
Nitrosocarbonyl or acylnitroso intermediates attracted a great deal of attention in the area during the last decades since highly functionalized molecules can be readily achieved [13][14][15][16]. The generation of nitrosocarbonyls 1 can be easily performed according to two main synthetic approaches summarized in the two working packages (WP1 and WP2) reported in Scheme 1. WP1 shows the use of hydroxamic acids 2 as starting compounds whose oxidation is performed either with the more soluble tetraalkylammonium periodate salts or PhI(OAc) 2 (path a) [17]. Other oxidative conditions were developed such as the use of transition metal catalyzed reactions [18][19][20][21] and peroxides [22,23] (paths b and c). Recently, the oxidation with copper salts in the presence of air [24,25] (path d) or photochemical sensibilized oxidation processes [26] (path e) became valuable mild methods for nitrosocarbonyl generation [27].
WP2 shows the use of nitrile oxides 5 for the case at hand; these 1,3-dipoles find in the wide family of the aldehydes the main and most convenient source with the possibility of proposing a variety of substituents. This valuable alternative towards nitrosocarbonyls 1 relies upon the mild oxidation of nitrile oxides 5 with N-methylmorpholine-N-oxide (path f, NMO) [28] and the clean thermolysis (path g) and photolysis (path h) of 1,2,4-oxadiazole-4-oxides 6, the most reactive dimer of nitrile oxides 5 [29,30].
During our studies, we developed the synthesis of a new class of isoxazoline-carbocyclic nucleosides 8 starting from the regioisomeric aminols 7 (Scheme 2) through elaboration of the HDA cycloadducts 3 [31][32][33][34]. From the ene adduct of nitrosocarbonyls and the 3-methyl-2-buten-1-ol, the isoxazolidine 9, the preparation of new N,O-nucleoside pyrimidine analogues 10 was achieved through the Vorbrüggen protocol [35,36]. We wish to report here the application of the WP2 protocol for the rapid and easy synthesis of new promising active nucleoside analogues. This simplified synthetic strategy implies the replacement of traditional heterobases, such as purines or pyrimidines, with selected and easy available heterocycles containing one nitrogen atom; once prepared, the products are submitted for in vitro tests to determine eventual activity and as a consequence further structural modifications. The required heterocyclic aldehydes containing one nitrogen atom are converted into the corresponding nitrile oxides as convenient source for the required nitrosocarbonyl intermediates.

Oxidation of Nitrile Oxides and Cleavage of N-O Bonds.
Pyridine and quinoline hydroximoyl chlorides were prepared according to the literature procedures (Scheme 3) [37][38][39]. The selected aldehydes were converted in high yields into the corresponding oximes following the classical methods [37][38][39] and from the latter the desired hydroximoyl chlorides were obtained upon chlorination with chlorine gas in chloroform as solvent at −20 ∘ C for 1.5 h. The in situ generation of the nitrile oxides 5a-d (Et 3 N, 1.2 equiv.) is required in the present cases since none of the nitrile oxides at hand displayed any stability at room temperature, possibly in the solid state, for a long time. The mild oxidation of these 1,3-dipoles with Nmethyl-morpholine N-oxide (NMO, 1.3 equiv.) is conducted one-pot in the presence of the required trapping diene (freshly distilled cyclopentadiene or 1,3-cyclohexadiene, 2 equiv.) to afford the nitrosocarbonyl HDA cycloadducts 11ad and 12a-d.
Compounds 11a-d and 12a-d were isolated from good to high yields (45-65%) and fully characterized from their analytical and spectroscopic data, except for 11d, already reported [11]. For the new products, the spectroscopic data are consistent with the reported structures; we wish to point out the most relevant and diagnostic 1 H NMR (CDCl 3 ) signals relative to the 2,3-oxazanorborn-5-ene moieties in the products 11 at hand. The olefinic protons and the deshielded bridge-head protons were found in the range = 6.37-6.67 and = 5.37-6.16, respectively. Similarly, the 2,3oxazabicyclo[2.2.2]oct-5-ene moieties of the products 12 are clearly observed in the 1 H NMR (CDCl 3 ) spectra. The olefinic protons and the deshielded bridge-head protons were found, respectively, in the ranges = 5.74-6.71 and = 4.79-5.55.
Compounds 11a-d and 12a-d were then submitted to the mild reductive cleavage of the N-O bond using the Al(Hg) amalgam in THF/H 2 O as solvents at 0 ∘ C [40]. The 4-hydroxycyclopent-2-enyl heterocyclic amides 13a-d and 14a-d were obtained in very high yields and the structures were attributed on the basis of their fully consistent analytical and spectroscopic data (Scheme 4).
The most relevant and diagnostic 1 H NMR signals are those relative to the olefinic protons of the cyclopentene and cyclohexene moieties found at = 5.94-6.15 (dd, = 5 Hz in CDCl 3 ) and at = 5.66-6.05 (dd, = 10 Hz in DMSO), respectively. All the other signals are found in the expected range for the given attributions.
Samples of the compounds 13a-d and 14a-d were straight submitted for a primary in vitro antiviral screening in collaboration with the NIH/NIAID (USA).

Antiviral
Activity. This rapid access to the target molecules allowed for their preparation in good amounts suitable for several biological tests. Compounds 13a-d and 14a-d were tested for their inhibitory activity against herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), vaccinia virus (VV), and hepatitis B virus (HBV). The antiviral activity of the above-reported compounds was tested in vitro in cell line HFF (strain E-377 for HSV-1, strain MS for HSV-2, and strain Copenhagen for VV) and cell line HepG 2. Both compounds 13a-d and 14a-d were found inactive against HSV-1 and -2; EC 50 and CC 50 values were >300 with a SI 50 = 1 (drug concentration range 0.096-300 M; acyclovir control concentration range 0.032-100 M). Compounds 13a-d and 14a-d also displayed a complete inactivity against the VV; EC 50 and CC 50 values were >300 with a SI 50 = 1 (drug concentration range 0.096-300 M; cidofovir control concentration range 0.096-300 M).
The in vitro tests against HBV revealed a substantial inactivity of compounds 13a-d and of compounds 14a, b, and d since the EC 50 and CC 50 values were >20 with a SI 50 = 1 (drug concentration range 0.063-20 M; 3TC control concentration range 0.00032-0.1 M). Compound 14c only gave a different value of EC 50 = 9.64 even though the CC 50 value was found >100 with a SI 50 = 1, while the cidofovir used as reference data gave EC 50 = 180, CC 50 > 200 with a SI 50 > 1.
The in vitro tests against HPV revealed a complete inactivity of compounds 13a-d and 14a-d since the EC 50 and CC 50 values were >20 with a SI 50 = 1 (drug concentration range 0.063-20 M; 3TC control concentration range 0.00032-0.1 M). The NA tests gave for compounds 13b, c and 14ac comparable results (CC 50 > 300) with those of cidofovir used as control drug (drug assay: neutral red (toxicity); drug concentration range 0.096-300 mM; cidofovir control concentration range 0.096-300 mM).
The derivatives 13a-d and 14a-d were also tested against the respiratory influenza A H1N1 virus and Table 1 reports the primary antiviral activities of the tested compounds and control experiments on the specified drug. The majority of the compounds were found to be inactive, but in a single case 14d (Table 1, entries 15 and 16) the EC 50 values are just 10 times those of the reference compound while the SI 50 values require to be increased by the same factor.

Discussion and Conclusions
These primary antiviral activity data represent somewhat the prelude to possible developments for more active compounds through modifications of the synthetic pathway, chosen on the basis of previous results [11]. Indeed the synthesis is suitable for convenient, easy, and straightforward structural modifications. The larger activity shown by those compounds, which bear a naphthyl-like residue, that is, the quinolone ring, could suggest a possible mechanism based on DNA intercalation. Polycyclic aromatic hydrocarbons are known as DNA-intercalating agents and molecular modeling studies on these structures confirm the degree of binding when a polycyclic aromatic residue is linked to an heterocyclic ring [41,42].
Polyaromatic groups may be active through their ability to establish -stacking interactions with themselves as well as DNA intercalators; these mechanisms are in action in different cases depending upon the biological targets [43].
An heterocyclic ring bearing a single heteroatom (the pyridine ring) seems to be somewhat inefficient from the biological point of view even though the nitrogen atom orientation can be easily changed in compound 14 from a to c (Figure 1).
On the other hand, the quinoline ring possesses the structural features to display this type of action mechanism as shown in many cases in the recent literature [44,45]. The quinoline is a naphthalene-like heterocycle and assumes different orientations suitable to adapt the structure during the interaction with DNA or active sites of proteins involved into the virus replication (DFT conformational studies at the B3LYP/6-31G(d) [46] level; Figure 2).
Docking studies are pursued to determine the key point concerning the SAR able to orientate positively the synthesis towards a specific target. Moreover, the fragment-based drug design (FBDD) represents a challenging approach in the case at hand; the reported structures seem to fit nicely with the application of computational FBDD for selecting the structural modification to increase the biological activity [47,48].
The simplified syntheses described rely upon the valuable chemistry of nitrosocarbonyl intermediates and the easy synthetic elaboration of the corresponding hetero-Diels-Alder cycloadducts. The replacement of traditional heterobases, such as purines or pyrimidines, with selected and easy available heterocycles containing one nitrogen atom, although simple or elemental, allows for a viral target oriented production of small molecules able to display some inhibition activity and suitable for further development under structural modification suggested by docking as well as FBDD analyses.

Experimental Section
4.1. General. All melting points are uncorrected. Elemental analyses were done on a C. Erba 1106 elemental analyzer. IR spectra (Nujol mulls) were recorded on an FT-IR Perkin-Elmer RX-1. 1 H-and 13 C-NMR spectra were recorded on a Bruker AVANCE 300 in the specified deuterated solvents. Chemical shifts are expressed in ppm from internal tetramethylsilane ( ). Column chromatography and tlc: silica gel 60 (0.063-0.200 mm) (Merck); eluant cyclohexane/ethyl acetate from 9 : 1 to 5 : 5. The identification of samples from different experiments was secured by mixed mps and superimposable IR spectra. Compound purification: all the products described were suitably purified; solid compounds were recrystallized from proper solvents and oily compounds were purified through bulb-to-bulb distillation (Kugelrohr distillation, Buchi GKR-51).

Materials.
Cyclopentadiene was freshly distilled from the dimer purchased from Sigma-Aldrich. 1,3-Cyclohexadiene, pyridine-carbaldehyde, 2-quinoline-carbaldehyde, and 4-bromothiazole-2-carbaldehyde were purchased from Sigma-Aldrich. All other reagents and solvents were purchased from Sigma-Aldrich and Alfa-Aesar and used without any further purification. The heteroaromatic hydroximoyl chlorides required for the in situ generation of the nitrile oxides 5 were prepared according to the literature reported procedures [26]. 11a-d and 12a- 13a-d and 14a-d. To a stirred solution of the cycloadducts 11a-d and 12a-d (1 g) in 200 mL of THF/H 2 O 10/1 under nitrogen and at 0 ∘ C, 1 g of Al(Hg) is added portionwise and the reaction is conducted until complete consumption of the starting materials (monitored by TLC). After completion, the solutions were diluted with THF and filtered over celite. The organic phases were then evaporated to dryness to leave the corresponding aminols 13a-d and 14ad in the reported yield and were fully characterized.

Computational Methods.
All calculations were carried out using the Gaussian 09 [46] program package through optimizations in the gas phase at the B3LYP/6-31G(d) level. Vibrational frequencies were computed to verify that the optimized structures were minima.

Antiviral
Assays. The National Institute of Allergy and Infectious Diseases (NIAID) established the AACF under a contract with Southern Research Institute. The NIAID, through the AACF, provides free and confidential services for suppliers, who are interested in submitting compounds to be evaluated for antiviral activity. Tested compounds were delivered in standard DMSO solutions. The methods applied for the different assays can be found at the URL via the internet at http://niaid-aacf.org/.
The antiviral tests were conducted on the derivatives 13ad and 14a-d suitably purified; compounds 13b-d and 14bd were recrystallized from proper solvents and compounds 13a and 14a were purified through bulb-to-bulb distillation (Kugelrohr distillation).