Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione

A series of twenty arylpiperazine derivatives of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione have been prepared. These derivatives were tested in vitro with the aim of identifying novel lead compounds active against emergent and re-emergent human and cattle infectious diseases (AIDS, hepatitis B and C, tuberculosis, bovine viral diarrhea). In particular, these compounds were evaluated in vitro against representatives of different virus classes, such as a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNA+ viruses Yellow fever virus (YFV) and Bovine viral diarrhea virus (BVDV), both belonging to the Flaviridae. Compounds 2c, 2g and 3d showed a modest activity against CVB-2. The molecular structures of the starting imide 1 and one of propyl-piperazine derivatives, 3b, have been determined by an X-ray crystallography study.


Introduction
x HCl x HCl , , ,
The N-propyl-piperazine derivative 3b adopts an extended conformation. In its protonated form it exists as a cation/anion pair ( Figure 1); the observed geometry of the N + -H...Clhydrogen bond is: N + ...Cldistance of 3.032(6) Å and ∠N + -H...Clangle of 175º.  The synthesized arylpiperazine derivatives were evaluated in vitro in parallel cell-based assays for cytotoxicity and antiviral activity (Table 3) against viruses representative of two of the three genera of the Flaviviridae family, i.e., Flaviviruses (YFV) and Pestiviruses (BVDV). The title compounds were also tested against representatives of other virus families. Among ssRNA+ were a Retrovirus (Human Immunodeficiency Virus type 1, HIV-1), two Picornaviruses (Coxsackie Virus type B2, CVB-2, and Poliovirus type-1, Sabin strain, Sb-1); among ssRNA-were a Paramyxoviridae (a Rhabdoviridae, Vesicular Stomatitis Virus, VSV) representative. Among double-stranded RNA (dsRNA) viruses was a Reoviridae representative (Respiratory Enteric Orphan Virus type-1, Reo-1). Two representatives of DNA virus families were also included: Herpes Symplex type 1, HSV-1 (Herpesviridae) and Vaccinia Virus, VV (Poxviridae). Although compound 1 was characterized by high cytotoxicity (Table 3), its derivatives possessed considerably low cytotoxicity. None of the compounds showed any activities against all viruses tested, with the exception of compounds 2c, 2g and 3d, that showed a modest activity against CVB-2-mediated diseases and potentiality as therapeutic targets (EC 50 range = 10-17 μM) ( Table 3).

General procedure for the preparation of hydrochloride salts of compounds 2a-2j and 3a-3j
The solid product was dissolved in methanol saturated with gaseous HCl. The hydrochloride was precipitated by addition of diethyl ether. The crude product was crystallized from methanol/ethyl ether. Characterization data is given in Table 1.

X-Ray crystal structure analysis of 1 and 3b diethyl ether hemisolvate
Measurements were carried out at 292 K with a KM4 diffractometer using graphite monochromated CuK α radiation (λ = 1.54178 Å) and ω/2θ scan mode. Intensities were corrected for absorption. Structure was solved by the SHELXS-97 program and refined by full-matrix least-squares on F 2 using the SHELXL-97 program [18]. The non-H atoms were refined anisotropically. The H-atom positions were calculated from geometry and the 'riding' model for the C-H bonds was used in the refinement. The N-bonded H-atoms were located in the difference maps. Crystal data are listed in Table 2. The experimental details and final atomic parameters for 1 and 3b have been deposited with the Cambridge Crystallographic Data Centre as supplementary material. Copies of the data can be obtained free of charge on application to The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-1223-336-033; E-Mail: deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk).

Cell-based assays
Compounds were dissolved in DMSO at 100 mM and then diluted in culture medium. Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Cell lines supporting the multiplication of RNA viruses were the following: CD4 + human T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK); Baby Hamster Kidney (BHK-21) and Monkey kidney (Vero 76) cells.

Cytotoxicity Assays
For cytotoxicity tests, run in parallel with antiviral assays, MDBK, BHK and Vero 76 cells were resuspended in 96 multiwell plates at an initial density of 6 × 10 5 , 1 × 10 6 and 5 × 10 5 cells/mL, respectively, in maintenance medium, without or with serial dilutions of test compounds. Cell viability was determined after 48-120 hrs at 37 °C in a humidified CO 2 (5%) atmosphere by the MTT method. The cell number of Vero 76 monolayers was determined by staining with the crystal violet dye.