Sci Pharm www.scipharm.at Research article Open Access Synthesis and Biological Evaluation of New 3-Phenyl-1-[(4-arylpiperazin-1-yl)alkyl]piperidine-2,6-diones

A set of 13 alkyl derivatives of 3-phenylpiperidine-2,6-dione were synthesized. Newly obtained compounds were investigated in vitro against HIV-1 and other selected viruses. The benzyl 3f and fluorophenyl 3g derivatives showed moderate protection against CVB-2 and the compound 3g also against HSV-1. Derivatives were tested also for their antibacterial and antifungal activity. The molecular structures of 3a and 3d were determined by an X-ray analysis.

Title compounds were evaluated for antiviral activity against viruses representative of two of the three genera of Flaviviridae family, that is, Flaviviruses (Yellow Fever Virus, YFV) and Pestiviruses (Bovine Viral Diarrhoea Virus, BVDV), as Hepaciviruses can hardly be used in routine cell-based assays. Compounds were also tested against representatives of other virus families. Among ssRNA+ were a retrovirus (human immunodeficiency virus type 1, HIV-1) and two Picornaviruses (Coxsackie Virus type B2, CVB-2 and Poliovirus type-1, Sabin strain, Sb-1); among ssRNA-were a Rhabdoviridae (Vesicular Stomatitis Virus, VSV) representative. Among double-stranded RNA (dsRNA) viruses was a Reoviridae representative (Reo-1). Two representatives of DNA virus families were also included: Herpes Simplex type-1, HSV-1 (Herpesviridae) and Vaccinia Virus, VV (Poxviridae).
In addition to the antiviral activity, compounds were evaluated in vitro against representative strains of Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Pseudomonas aeruginosa), yeasts and moulds (Candida albicans and Aspergillus niger).

Tab. 2.
Antibacterial and antifungal activities of 3-phenylpiperidine-2,6-dione and its derivatives. Gram-negative rods as well as gram-positive strains and fungal organisms were resistant to all tested agents. Their minimal inhibitory concentration (MIC) values for all compounds were above 100 µM (Table 2). However, none of title compounds turned out to be active against HIV-1, BVDV or representatives of ssRNA-and dsRNA viruses.

C. albicans DSM 1386
Theoretical calculated lipophilicity (logP) of synthesized compounds ranged from 1.89 to 3.92. According to Clark and Lobell rules [21], all derivatives could cross the blood-brain barrier to act as ligands of receptors of central nervous system.

Chemistry
All chemicals and solvents were purchased from Aldrich (Vienna, Austria). Melting points were determined on an Electrothermal Digital Melting Point Apparatus (Essex, UK) and are uncorrected. The 1 H-NMR spectra were recorded on a Bruker (Rheinstetten, Germany) spectrometer, operating at 400 or 300 MHz. The chemical shift values are expressed in ppm relative to TMS as an internal standard. Elemental analyses were recorded on a CHN model 2400 Perkin-Elmer (Hitachi, Tokyo, Japan). TLC was carried out using silica gel 60 F 254 , layer thickness 0.25 mm (E. Merck, Darmstadt, Germany) and the results were visualized using UV lamp at 254 nm. Column chromatography was carried out using silica gel 60 (200-400 mesh, Merck).
The elemental analyses and 1 H-NMR spectra, as well as melting points are given for dihydrochlorides (except for compounds 3, 4, 3a and 3d). Yields are presented for crude products.
Molecular structure of 3a and 3d was confirmed by an X-ray crystallography ( Table 3). The intensity data were collected at room temperature with a KM4 diffractometer using graphite monochromated CuKα radiation (λ = 1.54178 Å) and ω -2θ scan mode. Both compounds crystallized as hemihydrates and their crystals were of poor quality. Structure was solved by the SHELXS-97 program and refined by full-matrix least-squares on F 2 using the SHELXL-97 program [18]. Non-hydrogen atoms were refined with anisotropic displacement parameters, except those of phenyl rings of 3a. The H-atoms of molecules 3a and 3d were positioned geometrically and 'riding' model was used in the refinement, while H-atoms of water molecules were located on difference maps. Crystallographic data had been deposited with the Cambridge Crystallographic Data Center.

3-Phenylpiperidine-2,6-dione (2)
A mixture of 2-phenylglutaric anhydride (0.0165 mol) and (NH 4 ) 2 CO 3 (0.125 mol) was reduced to powder and heated up to 185 °C using a metal bath of Wood's alloy. When CO 2 and NH 3 were completely liberated, 10 cm 3 of paraffin oil was added and whole mixture was heated until the evolution of gas ceased. The residue was crystallized from hexane and ethyl acetate; mp 143 °C (mp 142-143 from methanol [23]).
Obtained compounds were converted into their hydrochlorides. 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.

Compounds
Compounds were dissolved in DMSO at 100 mM and then diluted in culture medium.

Cells and Viruses
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.

Bacterial strains
The antibacterial activity of compounds was tested against collection strains representative of Gram-positive bacteria (Staphylococcus aureus DSM 2569) and Gram-negative bacteria (Pseudomonas aeruginosa DSM 1117). Antifungal activity was tested against collection strains representative of yeasts (Candida albicans DSM 1386) and moulds (Aspergillus niger DSM 1988).

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 x 10 5 , 1 x 10 6 and 5 x 10 5 cells/mL, respectively, in maintenance medium, without or with serial dilutions of tested 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.

Antiviral assay
Activity Activity of compounds against Yellow Fever Virus (YFV) and Reo virus type-1 (Reo-1) was based on inhibition of virus-induced cytopathogenicity in acutely infected BHK-21 cells. Activities against Bovine Viral Diarrhoea Virus (BVDV), in infected MDBK cells, were also based on inhibition of virus-induced cytopathogenicity.
BHK and MDBK cells were seeded in 96-well plates at a density of 5 x 10 4 and 3 x 10 4 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO 2 (5%) atmosphere.

Antibacterial and antifungal assays
The antibacterial and antifungal activities were evaluated by determining the Minimum inhibitory concentration (MIC) by the broth microdilution procedure.
Bacterial strains were grown on Tryptic soy agar at 37 °C for 1 day. Cell suspensions of these recent cultures were prepared in sterile 0.85% saline solution by 4-5 colonies. The turbidity of the suspensions was adjusted to the McFarland 0.5 standard. Suspensions were diluted in cation-supplemented Mueller-Hinton broth. For each microorganism, 100 µL of the fivefold serial dilutions of the compounds in cation-supplemented Mueller-Hinton broth and 100 µL of inoculum were added to each well of a microdilution plate (final titre 5 x 10 5 CFU/mL). The inoculated plates were incubated at 37 °C in non-CO 2 incubator and humid atmosphere. The MICs were determined after 16-20 h [25].
Fungal strains were grown on Sabouraud's dextrose agar at 35 °C for 1-5 days. Suspensions of these recent cultures were prepared in sterile saline solution (NaCl 0.85%). Suspensions were then diluted in Sabouraud's dextrose broth. 100 µL of the fivefold serial dilutions of the compounds in Sabouraud's dextrose broth and 100 µL of inoculum were added to each well of a microdilution plate (C. albicans 1 x 10 4 cell/mL; A. niger OD 600 0.05). The inoculated plates were incubated at 35 °C in non-CO 2 incubator and humid atmosphere. The MICs were determined after 24 and 48 h.
The concentration of each inoculum was confirmed by viable counts on agar plates by plating the appropriate dilution of the growth control well, immediately after inoculation, and incubating until visible growth. MIC corresponded to the lowest concentration of an antimicrobial compound that showed complete growth inhibition.

Linear regression analysis
Viral and cell growth at each drug concentration was expressed as percentage of untreated controls and the concentrations resulting in 50% (EC 50 , CC 50 ) growth inhibition were determined by linear regression analysis.