Synthesis and antiviral activity of 4-(7,7-dimethyl-4-[4-{N-aroyl/benzyl}1-piperazinyl]-5,6,7,8-tetrahydroquinazolin-2-yl)morpholine derivatives

The synthesis of a series of 4-(7,7-dimethyl-4-[4-{ N -aroyl/(het)aroyl/ benzyl}1-piperazinyl]-5,6,7,8-tetrahydroquinazolin-2-yl)morpholine derivatives has been described. The antiviral activity of these compounds against avian paramyxovirus (APMV-1), which is a Newcastle disease virus has also been screened.


Introduction
Heterocyclic chemistry is one of the largest classical divisions within organic chemistry, [1][2][3] and its importance is highlighted by the frequency that heterocycles appear in biologically active compounds.0][11] Piperazine is also an important heterocyclic compound present in many of the notable anti-helmintic, anti-depressant and antihistamines drugs. 12N-Aroyl-substituted piperazines attached to 4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones showed poly(ADP-ribose)polymerase-1 inhibition property. 13Recently, Wang et al. have reported that N-aroyl or N-benzyl-substituted piperazines attached to ubiquinones have antioxidant properties. 14Hence, it is presumed that molecules containing these heterocyclic hubs would exhibit promising biological activity.In the recent evaluation towards antiviral research, study on Newcastle disease virus (NDV) has been an attractive area for Virologists due to its economic importance.These viruses can infect more than 250 bird species and the disease onset is rapid with clinical signs appearing within 48 h. 15,16 urther, currently used vaccines are not 100% protective and there is a definite need to combat the disease through other strategies that include using antiviral drugs.As on date, there is no approved drug against NDV and Ribavirin, a well-known broad spectrum antiviral drug is approved for treatment of respiratory syncytial virus, a human paramyxovirus. 17However, Ribavirin is costly, there are concerns about its efficacy and it is shown to have potential toxic effects on exposed individuals when administered via aerosol. 18,19Since pyrimidine template is present in various commercial antiviral drug substances such as Abacavir, Idoxuridine, Valganciclovir and Zidovudine, novel compounds that contain pyrimidine template are designed and screened for antiviral activity.Therefore, the main objective of the present endeavor is to synthesize 4-(7,7-dimethyl-4-{piperazin-1-yl}-5,6,7,8tetrahydroquinazolin-2-yl)morpholine 1 and its N-aroyl/(het) aroyl 2 and N-benzyl-substituted piperizine derivatives 3 and to screen their antiviral activity against a NDV viz.avian paramyxovirus (APMV-1).
The protocol employed for the synthesis of N-aroyl 2a-g and N-benzyl-substituted piperizine 3a-f derivatives of 1 is depicted in Scheme 2. Several N-aroyl-substituted piperizine derivatives 2a-g were prepared by treating 1 with corresponding substituted benzoyl chloride or heterocyclic acyl chloride a-g.Similarly Nbenzyl-substituted piperizine 3a-f derivatives of 1 were synthesized via reductive amination of 1 by respective aryl aldehydes using sodium triacetoxyborohydride (STAB).The structures of these compounds 2a-g and 3a-f were confirmed by 1 H and 13 C NMR and LCMS spectra.Scheme 2. Synthesis of N-aroyl-substituted piperizines 2a-g and N-benzyl-substituted piperizines 3a-f from 1.

Antiviral activity
To test the antiviral activity of these compounds, they were initially screened by MTT assay 23 in African Green Monkey Kidney cell line, Vero cell line.The maximum non-cytotoxic concentration, at which no significant changes were detected in cellular morphology of Vero cells was used as the highest test dose for testing the antiviral activity of the compounds by viral plaque reduction assay using an avian paramyxovirus (APMV-1).The commercially available antiviral drug, Ribavirin, was used for comparing the antiviral potential of the test compounds.The maximum non-cytotoxic concentration (MNCC), at which no significant changes were detected in cellular morphology of Vero cells, of Ribavirin was 31.25 µg/mL.The 50% cytotoxic concentration (CC50: dose that inhibited the growth by 50% compared to untreated cells) of Ribavirin was determined to be 400 µg/mL and 32% viral plaque reduction was observed by Ribavirin at dose of 31.25 µg/mL.
In a typical experiment, monolayers of Vero cells in 24-well plate were incubated with five different concentrations of test compounds (0.1, 0.01, 0.001, 0.0001 and 0.00001 M) for 1 h.The cells were washed with PBS thrice and then infected with a known dose of Newcastle disease virus for 1 h.The cells were washed again with PBS thrice and overlaid with methyl cellulose media.The cells were incubated at 37°C with 5% CO2 for 5 days.During the incubation period the cells were observed every day.On the fifth day, the overlay media was removed, the cells were fixed with cold methanol for 30 min, then stained with 1% crystal violet and air dried.The number of plaques produced by viral infection was counted in each well.The percentage of plaque reduction was determined by calculating the reduction in the number of plaques upon compound treatment compared to untreated NDV infected cells which was defined as 100%.The results thus obtained are collected in Table 1.The results provided in the Table 1 indicated that the test compounds 2a and 2c-g showed antiviral activity by inhibiting the plaque formation by 22, 6, 25, 12, 28 and 17%, respectively at the minimum test dose when compared to infected untreated controls, while the compounds 2b and 3a-f did not show any antiviral activity at the tested concentrations.Also, the results suggested that N-aroyl/(het)aroyl derivatives showed antiviral activity, whereas N-benzyl analogues showed no such activity.It is evidently observed that presence of carbonyl group in N-aroyl/(het)aroyl derivatives may be responsible for the antiviral efficacy of these compounds.Among the compounds under investigation N-imidazolyl-substituted piperizine derivative 2f exhibited relatively a higher antiviral activity than the others.

Experimental Section
General.All the chemicals used in the study are commercially available high purity grade (Aldrich or Merck, India).Commercially available reagent grade solvents were used as received.TLC experiments were performed on alumina-backed silica gel 40F254 plates (Merck, Germany).The plates were illuminated under UV (254 nm) and KMnO4.Melting points were determined using a melting point apparatus (B-540 Buchi, Germany) without corrections.All 1 H and 13 C NMR spectra were recorded on a Bruker 300 or 400 MHz instrument.Molecular masses of unknown compounds were checked by LCMS 6200 series Agilent Technology instrument.Chemical shifts are reported in ppm (δ) with reference to internal standard TMS.The signals are designated as follows: singlet (s), doublet (d), triplet (t), doublet of doublet (dd), doublet of triplet (dt), multiplet (m), and broad singlet (bs).IR spectra were recorded using a FT-IR spectrometer (Bruker, Germany) using a diamond attenuated total reflectance (ATR) single reflectance module (24 scans).All reactions were carried out under a nitrogen / argon atmosphere unless otherwise stated.Elemental analysis was carried out with a Thermo Scientific, model Flash 1112EA apparatus and Eagar xperience software.

Table 1 .
Antiviral activity of the compounds against APMV-1