Synthesis and antiviral activity of anthracene derivatives of isoxazolino-carbocyclic nucleoside analogues

Tetrahedron Letters 56 (2015) 1986–1990

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Synthesis and antiviral activity of anthracene derivatives of isoxazolino-carbocyclic nucleoside analogues Misal Giuseppe Memeo a,⇑, Francesco Lapolla a, Giovanni Maga b, Paolo Quadrelli a,⇑ a b

Department of Chemistry, University of Pavia, Viale Taramelli 12, 27110 Pavia, Italy Institute of Molecular Genetics IGM-CNR, University of Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy

a r t i c l e

i n f o

Article history: Received 2 January 2015 Revised 18 February 2015 Accepted 20 February 2015 Available online 26 February 2015 Keywords: 1,3-Dipolar cycloadditions Nitrile oxides Nitrosocarbonyls Nucleoside analogues Antivirals

a b s t r a c t Isoxazolino-carbocyclic anthracene nor-nucleosides were prepared through nitrosocarbonyl chemistry and tested for their inhibitory activity against some viruses, such as Herpes simplex viruses of type 1 and 2, Zoster virus and Hepatitis B and C. The activities were almost negligible in most of the cases. A remarkable antiviral activity was found for a specific regioisomer with no cellular toxicity at 1–100 lM dose concentration in the case of Human Papilloma virus. Ó 2015 Elsevier Ltd. All rights reserved.

In recent years some isoxazolidinyl polycyclic aromatic hydrocarbons were proposed as DNA-intercalating antitumor agents1 and modelling studies2 on these structures confirm the degree of binding when a polycyclic aromatic residue is linked to an isoxazoline heterocyclic ring. In order to have further insights on the structure–activity relationship (SAR) and in particular to confirm the specific role of the aromatic residues in relationship with the type of heterobases inserted on a isoxazolino-carbocycle nucleosidic structure, we have investigated new chemical compounds belonging to the nucleoside analogue family whose synthesis relies upon the synthetic protocol based on the nitrosocarbonyl chemistry and 1,3-dipolar cycloaddition of the stable anthracenenitrile oxide.3 In this strategy there are two pivotal steps. First, nitrosocarbonyls (R–CO–NO) 1 are fleeting intermediates and highly reactive p2 components in Hetero Diels–Alder (HDA) cycloadditions.4 Typically, nitrosocarbonyls 1 are generated through periodate oxidation of hydroxamic acids or under other oxidative conditions based on transition metal catalysed reactions5 or PhI(OAc)2 oxidations.6 An alternative methodology to nitrosocarbonyls is represented by the mild oxidation of nitrile oxides with N-methylmorpholine-N-oxide (NMO).7,8 Second, we used a stable and conveniently prepared anthracenenitrile oxide 3 as 1,3-dipole, bearing a fluorophore polycyclic aromatic substituent (Scheme 1).9 We wish to present a study aiming to find out the scope and eventual limitations of this chemistry upon variation of the ⇑ Corresponding authors. Tel.: +39 0382 987315; fax: +39 0382 987323. E-mail addresses: [email protected] [email protected] (P. Quadrelli). http://dx.doi.org/10.1016/j.tetlet.2015.02.114 0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

(M.G.

Memeo),

structural features of the heterobase, fixing the 1,3-dipole structure and trying to suggest the role of heterobases and aromatic residues in determining shape and biological activity of the synthesized new structures. The N-benzoyl-2,3-oxazanorborn-5-ene 2 was allowed to react with a slight excess (1.2 equiv) of anthracenenitrile oxide 3, prepared according to the published procedure,10 in dichloromethane (DCM) solution for 2 days (Scheme 1). After this period of time, the residue, obtained after evaporation of the solvent, was submitted to chromatographic separation to isolate the two regioisomeric exo-cycloadducts 4a and 4b in very good yields (39% and 47%, respectively). The structures of regioisomeric compounds 4 were consistent with the published analytical and spectroscopic data.11 Alkaline hydrolysis3,11 of cycloadducts 4a,b takes place smoothly in the presence of 1.1 equiv of NaOH in methanol, stirring at room temperature for 12 h (Scheme 2). The hydroxylamine derivatives 5a,b were obtained in quantitative yields and found identical with authentic samples previously obtained. Hydrogenolysis11 of the derivatives 5a,b under standard conditions (H2, Pd/C 10%, AcOEt), gave quantitatively the desired aminols 6a,b whose structures were confirmed from their spectroscopic data.3 The regioisomeric aminols12 6a,b were then converted into the uracil nucleosides13 through the linear construction of these heterocycles.14 This highly reliable synthetic route to uracil nucleosides involves the steps illustrated in Scheme 2 and started with the preparation of the suitable isocyanate, whose structure is sketched in the inset (c) of Scheme 2.

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M. G. Memeo et al. / Tetrahedron Letters 56 (2015) 1986–1990

Scheme 1. Synthesis of regioisomeric cycloadducts 4a,b.

The 3-methoxy-2-propenoyl isocyanate was easily prepared from the commercially available methyl 3-methoxy-2-propenoate through basic hydrolysis to the acid,14 conversion to the chloride with thionyl chloride15 and coupling with silver cyanate in benzene.14 Aminols 6a,b, dissolved in DMF, were added to the benzene solutions of isocyanate, according to the procedure reported in the literature,14 by performing the reactions at 20 °C for 12 h in the presence of MS 4 Å. After chromatographic purification, the urea adducts 7a,b were obtained in fair yields (40% and 42%, respectively). Their structures rely upon the analytical and spectroscopic data. Neat distinctive bands corresponding to the OH (3529, 3283 cm 1) and the two NH (3248, 3283 cm 1) groups were evident in the IR spectra as well as the bands corresponding to the C@N double bonds (1673, 1666 cm 1). The NMR spectra showed the signals of the methoxy propenoyl chains as well as those of the carbocyclic moiety in the usual ranges (for details, see Supplementary material, SM). Cyclization of the ureas 7a,b took place smoothly upon heating in dioxane solutions for 2 h in the presence of catalytic amounts of p-toluenesulfonic acid (p-TsOH). The uracil nucleosides 8a,b were isolated from these solutions after pH adjustment to 7 and extraction with dichloromethane. The yields of the cyclization steps were satisfactory (85% and 86%, respectively) and the structures of the nucleosides 8a,b rely upon their analytical and spectroscopic data. The IR spectra of nucleosides 8a,b showed OH and NH bands in the usual range (3348–3565 cm 1), along with the C@N double bonds at 1673 cm 1. The 1H NMR spectra of the uracil nucleosides 8a,b showed the characteristic coupled vinyl protons of the uracil unit as doublets at d 5.55 and 7.55 (J = 12.0 Hz) and at d 5.34 and 7.47 (J = 12.0 Hz), respectively for 8a and 8b. The NH groups of the uracil ring were also found, respectively, at d 11.40 and 10.81 for 8a and 8b. The other signals are in the expected range (see SM). The reported synthetic pathway towards new nucleoside analogues was found to be robust and reliable and easily applicable to the synthesis of pyrimidine derivatives. In terms of yields, all the synthetic steps gave fair to very good results, and the quality of the products was found to be excellent. This rapid access to the target molecules allowed for their preparation in large amounts suitable for biological tests. We submitted samples of compounds 8a,b to the NIH/NIAID (USA) for a first antiviral evaluation.16 Samples of compounds 8a,b were tested for their inhibitory activity against the Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), Varicella-Zoster virus (VZV), Vaccinia virus (VV), Punta Toro virus (PTV), Hepatitis B virus (HBV),

Hepatitis C virus (HCV), Neuroamidase (NA), Adenovirus (AV) and Coxsackie virus (CV). The antiviral activity of the above-reported compounds was tested in vitro in cell line HFF (strain E-377 for HSV-1, strain G for HSV-2, strain Ellen for VZV, strain Copenhagen for VV, strain NA for NA), in cell line Vero 76 (strain Adames) for PTV, cell line 2.2.15 (strain ayw) for HBV, in cell line Huh-Luc/Neo ET (strain CON-1) for HCV, in cell line HeLa (strain Type 5) for AV and in cell line LLC-MK2 (strain Nancy) for CV. The tested products were found inactive against all the tested viruses, independently from their type, Herpesviridae, Bunyaviridae, Respiratory, Hepatitis or other. The compounds were also screened against different enzymatic targets, such as viral HIV-1 reverse transcriptase and human DNA repair ‘polymerases 1 and b’. The compounds were not active in these assays. The complete antiviral activity results are reported in the SM. However, compounds 8a,b displayed some interesting activities against HPV (cell line HEK 293, strain HPV-11) and notably differences with respect to their regioisomeric structures as shown in Table 1. Regioisomer 8a showed moderate antiviral activity at one single-concentration. An interesting and quite high antiviral activity was displayed by compound 8b and low cellular toxicity at the dose tested. The complex HPV replication machinery begins with the specific binding of E2 to the viral DNA and recruitment of protein E1, which rapidly oligomerizes to an hexamer (around the nucleic acid) displaying its helicase activity. With this mechanism in mind, we first evaluated the binding affinity of 8b to the HPV11 E2 protein (Fig. 1). In the literature, the crystal structure of this protein is reported along with an indandione derivative as specific inhibitors of the E2–E1 interaction.17 Flexible molecular docking of 8b on this receptor gives a docking score that is comparable and even higher than the ones obtained through the self-docking of the crystallographic inhibitor. As it can be seen from Figure 1, 8b is anchored at the receptor pocket involved in the protein– protein interaction (PPI) through the isoxazoline ring and the

Table 1 Primary antiviral evaluation for compound 8a,b against HPV

8a 8b Cidofovir

EC50

EC90

CC50

SI50

SI90

>100 6 148

>100 57 >200

>100 >100 >200

1 >17 >1

1 >2 1

Drug concn. range: 1–100 lM; control concn. range: 50–200 lM. Control assay name: quantitative polymerase chain reaction (DNA)/trypan blue (toxicity).

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M. G. Memeo et al. / Tetrahedron Letters 56 (2015) 1986–1990

9-Anthryl

O

N N

N

O

N

COPh O

9-Anthryl

O

4a

4b

a

a

a

NaOH/MeOH r.t., 12 h

9-Anthryl

N

N

O NH

NH

O

5b b

b

9-Anthryl

O

9-Anthryl

O

5a

b

H2, Pd/C 10%, AcOEt r.t., 3 h

O

N

N

NH2

NH2

O

OH

9-Anthryl

OH

c

6a

6b

O

c

c

NH

OMe

OMe OCN

MeO 9-Anthryl

O

1.2 equivs. DMF, -20 °C, 18 h

O

O N

N

N H

O

COPh

N H

O N H

O

O

OH

9-Anthryl

OH

N H

7b

7a d d O

HO H

H

Dioxane p-TsOH 80 °C, 2 h

d O

HO

NH N

H

H

H

NH N

H

O H

O N

O 9-Anthryl N

9-Anthryl

8a

H

O

8b Scheme 2. Synthesis of regioisomeric nucleoside analogues 8a,b.

histidine His32 residue. The anthracene and the uracil substituted cyclopentanol moieties are perfectly complementary to the pocket involved in the PPI, and, in particular, the anthracene substituent shades a hydrophobic pocket involved in the specific E2–E1 interaction. A second round of molecular docking calculations was conducted on the specific sequence of DNA recognized by viral E2. Hegde and co-workers co-crystallized the E2–DNA complex (HPV 18) pointing out a strong distortion of the DNA double helix due to the interaction with E2.18 Using this structure, we obtained strong

indications that 8b can specifically bind the DNA minor groove (Fig. 2). The anti configuration between the uracil and anthracene substituents allows for the 8b backbone adaptations on the minor DNA groove. The interactions of the uracil ring as well as the cyclopentane hydroxyl group with the viral DNA are established with the heterobases of the nucleic acid, reasonably inhibiting in this way the DNA replication machinery. The last docking calculations were carried out on the viral helicase protein E1. In this case low docking scores along with lack of specific interactions suggests the low probability that E1

M. G. Memeo et al. / Tetrahedron Letters 56 (2015) 1986–1990

Figure 1. Most representative binding mode of compound 8b after 10 ns of MD simulation, starting from the crystallized structure of HPV11 E2 protein.

2.21 Å

2.12 Å

1989

these mechanisms are in action in different cases depending upon the biological targets. The primary antiviral tests seem to suggest the relevance of the role of the anti geometry between heterobase and anthracene residue as a key structural feature. The heterobases, both a purine and a pyrimidine, must be located in that configuration with respect to the anthracene ring. The regioisomer 8b is in fact the most active compound. Even though the mechanism of action of these novel compounds still remains an open question, we are actively pursuing the synthesis of novel nor- and classical nucleoside analogues bearing other polycyclic aromatic substituents in order to produce further insights on this topic. In conclusion, the synthesis of isoxazolino-carbocyclic nornucleosides having anthracene substituents was properly tuned through the nitrosocarbonyl chemistry, starting from the stereodefined heterocyclic aminols 6a,b. The latter are readily available through exo selective 1,3-dipolar cycloadditions of anthracenenitrile oxide to N-benzoyl-2,3-oxazanorborn-5-ene 2 and simple elaborations of the cycloadducts 4a,b. The stereodefined heterocyclic aminols 6a,b afford the carbocyclic skeleton for the linear construction of the desired heterobase moieties. The nucleoside derivatives 8a,b were prepared and evaluated for their inhibitory activity against a variety of viruses, such as the Herpes simplex viruses 1 and 2, Varicella-Zoster virus (VZV), Vaccinia virus (VV), Punta Toro virus (PTV), Hepatitis B and C viruses, Neuroamidase (NA), Adenovirus (AV) and Coxsackie virus (CV). In all these cases they were found to be inactive. A remarkable specificity was observed for the Human Papilloma virus (HPV11). Good antiviral activity was found for compound 8b with low cellular toxicity at the dose tested. These results are part of larger project having HPV as target of our synthetic efforts, including the preparation of a new library of compounds. Acknowledgments

Figure 2. Binding of the compound 8b with DNA of HPV-18 from the cocrystallized the E2–DNA complex.

represents the biological target. To summarize, we can hypothesize a double activity of 8b, both on E2 and the DNA. Furthermore, the anthracene moiety could eventually intercalate the ds since it is well known its behaviour as an intercalating agent.1,2,19 In order to dive into the mechanism of action of 8b, we are pursuing binding assays on isolated E2 protein and dsDNA chains on both the synthesized regioisomeric compounds as well as on the starting aminols and anthracene derivatives for the sake of comparison. This preliminary screening in HPV tests and the modelling evaluations do not allow for a firm attribution of an action mechanism against HPV, since other data such as complete toxicity evaluation must confirm initial assumptions. The role as a therapeutic agent must be further investigated in order to verify the possibility for compound 8b to be incorporated into an oligonucleotide chain or to intercalate into a polymerase as well as a whole host of other proteins.20 Noteworthy, the fluorescent properties of these compounds add further tools for our future studies. The use of fluorescent heterobases in the nucleoside synthesis has attracted the interest of various research groups as marker molecules or in the field of imaging to follow their path inside the cells in order to achieve a better understanding of the ‘in vitro’ and ‘in vivo’ mechanisms.21 Fluorescent heterobases can be considered ‘molecular labels’22 to be assembled in specific oligofluorosides, as sensors23 or indicators of the DNA behaviour.24 New fluorophores are constantly developed as well as their applications.25 Fluorescent polyaromatic groups may be active through their ability to establish p–p stacking interactions with themselves26 as well as DNA intercalators;27

Financial support by the University of Pavia, MIUR (PRIN 2011, CUP: F11J12000210001) is gratefully acknowledged. We also thank the Fondazione Banca del Monte di Lombardia (Progetto Professionalità I. Becchi 2013) for a grant to MGM. We warmly thank Dr. M. Prichard (University of Alabama at Birmingham) for Herpes, Varicella and HPV tests, Prof. D. Smee (Utah State University) for Punta Toro tests, Prof. M. Murray (South. Res. Inst.) for Hepatitis, Adeno and Coxsackie tests and Prof. B. Korba (Georgetown University) for Hepatitis virus tests. COST Action CM1004 ‘Synthetic Probes for Chemical Proteomics and Elucidation of Biosynthetic Pathways’ is gratefully acknowledged. The results here reported were communicated at the COST Action CM1004 Scientific Meeting, Trinity College, Cambridge (UK), 24-25 March, 2014. Supplementary data Supplementary data (complete antiviral activity, experimental section and copies of 1H and 13C NMR spectra) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.tetlet.2015.02.114. References and notes 1. Rescifina, A.; Chiacchio, U.; Corsaro, A.; Piperno, A.; Romeo, R. Eur. J. Med. Chem. 2011, 46, 129–136. 2. Rescifina, A.; Chiacchio, U.; Piperno, A.; Sortino, S. New J. Chem. 2006, 30, 554– 561. 3. Moggio, Y.; Legnani, L.; Bovio, B.; Memeo, M. G.; Quadrelli, P. Tetrahedron 2012, 68, 1384–1392. 4. (a) Kirby, G. W. Chem. Soc. Rev. 1977, 6, 1–24; (b) Carruthers, W. Cycloaddition Reactions in Organic Synthesis; Pergamon: Oxford, 1990; (c) Boger, D. L.;

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