Synthesis and study of new 4-quinazolinone inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP)

4-Quinazolinones modified with 2,2,5,5-tetramethyl-2,5-dihydro-1 H -pyrrole or 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine rings and their N -oxyl derivatives were synthesized and some of them evaluated for protecting activity against H 2 O 2 induced cell death on WRL-68 human liver cell line. Compounds 15a, 15c and 15d exhibited remarkably inhibitory effects on PARP enzyme in vitro .


Introduction
Poly(ADP-ribose) polymerase is an abundant eucariotic nuclear zinc-finger DNA-binding protein with a role of genomic repair. 1,2DNA strand breaks generated either directly by genotoxic agents (alkylating agents, reactive oxygen species (ROS), ionizing radiation) or indirectly, following enzymatic incision of a DNA-base lesion, activate PARP enzyme.PARP catalyzes the biochemical conversion of the respiratory coenzyme, nicotinamide dinucleotide (NAD) to poly(ADP-ribose) and nicotinamide, therefore the latter is a weak feedback inhibitor of this enzyme.Although PARP has an important role in genomic repair mechanism, the massive activation of PARP results in extended ADP ribosylation and depletion of NAD.In the next step this leads to ATP depletion in an effort to resynthesize NAD and ultimately the lack of ATP and NAD causes mitochondrial dysfunction and cell death.The first PARP inhibitors were based on mimicking the structure of nicotinamide and benzamide analogs. 3Recent research aimed at synthesizing more potent and selective PARP inhibitors based on the crystal structure of the catalytic domain of PARP, leading to polycyclic amides and lactams. 4,5One of the most potent compounds evaluated recently is the 8-hydroxy-2-methylquinazolin-4(3H)-one (NU-1025, IC 50 = 400 nM). 6,7In our laboratory we have synthesized 4-quinazolinone derivative, H-2641 as an antiarrhythmic drug candidate. 8It was a real challenge to test and compare this compound with basic quinazolines 1-3 (Figure 1) on PARP inhibitory activity assay as well as synthesizing new quinazolinone derivatives attached to 1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole or 1oxyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine and their amine precursors, because these rings exhibited radical scavenging activity and protected against oxidative stress.This fact can be a significant contribution in designing new PARP inhibitors against cell damage induced by antiviral 11 and anticancer drugs.Application of anticancer and antiviral compounds in the therapy is also mostly inevitably complemented by ROS formation whose in statu nascendi scavenging may moderate further the side-effects of these drugs compared to regular PARP inhibitors.

Issue in
Finally, our attention turned to such quinazolinone derivatives in which pyrroline and tetrahydropyridine rings were not linked via heteroatom.Considering that anthranilic acid and βamino acid derivatives are readily available key starting materials for quinazoline synthesis, 18 this problem could be solved by acylation of 2-aminobenzonitrile with paramagnetic acid chlorides 17 19   Regarding the structure-activity relationship (SAR), the principal ring compound protecting activity greatly varied depending on the substituent.Compounds 1, 2 exhibited good activity, compound 3 exhibited limited activity, 4, 5 and H-2641 with cardioprotective activity 8 were inactive in PARP inhibitory assay.The ω-amino S-alkylated compounds 15a-d exhibited better protection activity than the S-ethyl one (4).The piperidine compounds 15c and 15d did not have any significant difference in activity, the preliminary toxicity was also similar.Among the dimethylamino containing side-chain compounds 15a exhibited better activity than 15b.Compounds substituted on the lactam NH (8, H-2641) were inactive, while compound 9 exhibited some moderate activity.Quinazolinones substituted at the 2-position with tetrahydropyridine rings showed good activity, however the amino precursor 22 had slightly better activity than the corresponding N-oxyl derivative 21.The N-hydroxy salt of radical 19 was inactive (Table 1 and 2 In conclusion, series of 4(3H)-quinazolinone derivatives were synthesized by a convenient alkylation of 4(3H)-quinazolinones (2, 3) and acylation of anthranilonitrile followed by cyclisation.Among the synthesized and studied compounds 4(3H)-quinazolinone derivatives substituted at the 2-position with tertiary amines containing S-ethyl spacer were found to be effective PARP inhibitors.Nitroxides or its amino precursor has lower inhibitory activity than tertiary amines, however nitroxides and its amino precursor may have a potential advantage as a possible radical scavenger analogously to the previous results. 9,10,21perimental Section General Procedures.Melting points were determined with a Boetius micro melting point apparatus and are uncorrected.Elemental analyses (C, H, N, S) were performed on Fisons EA 1110 CHNS elemental analyser.The IR (Specord 75) spectra were in each case consistent with the assigned structure.Mass spectra were recorded on a VG TRIO-2 instrument in the EI mode. 1 H NMR spectra were recorded with Varian Unity Inova 400 WB spectrometer; chemical shifts were referenced to TMS.Flash column chromatography was performed on Merck Kieselgel 60 (0.040-0.063 mm).The HPLC analyses were performed on HP1100, UV detection monitored at 250 nm using a Hypersyl BDS-C18 column.Qualitative TLC was carried out on commercially prepared plates (20 x 20 x 0.02 cm) coated with Merck Kieselgel GF 254 .Compounds H-2641, 8 4, 12 5, 13 6, 14 7, 16 15d, 16 17, 18 18 18 were prepared according to published procedures, compound 1, chloroethyl)piperidine and anthranilonitrile were purchased from Aldrich.

Protecting effect of 4-quinazolinone derivatives against H 2 O 2 induced cell death determined in WRL-68 human liver cell line
Cell culture.WRL-68 human liver cell line was from American Type Culture Collection (Rockville, MD).Cell lines were grown in humidified 5 % CO 2 atmosphere at 37 °C and maintained in culture as monolayer adherent cells in Dulbecco's Modified Eagle's Medium containing 1% antibiotic-antimycotic solution (Sigma, St. Louis, MO) and 10 % fetal calf serum.Cells were passaged at intervals of 3 days.Detection of cell survival.Cells were seeded into 96well plates at a starting density of 2.5 x 10 4 cell/well and cultured overnight in humidified 5 % CO 2 atmosphere at 37 °C.The following day, 0.3 mM H 2 O 2 was added to the medium either alone or in the presence of protecting agent.Three hours later, the medium was removed and 0.5 % of the water soluble mitochondrial dye, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT + ) was added.Incubation was continued for 3 more hours, the medium was removed, and the metabolically reduced water insoluble blue formasan dye was solubilised by acidic isopropanol.Optical densities were determined by an Anthos Labtech 2010 ELISA reader (Wien, Austria) at 550 nm wavelength.All experiments were run in at least 6 parallels and repeated 3 times.Data showed in Table 1 are the concentration in µM at which the rate of H 2 O 2 induced cell death is inhibited with 50 %.

Inhibitory effects of 4-quinazolinone derivatives on PARP enzyme in vitro
The poly-ADP-ribose polymerase was isolated from rat liver based on the method described before. 22,23The potential inhibitory effects of 4-quinazolinone derivatives were tested in this assay system.The PARP activity was determined in 130 µl reaction mixture contained 100 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl 2 , 10 % glycerol, 1.5 mM DTT, 1 mM [Adenine-2,8-3 H] NAD + (4.500 cpm/nmol), 10 µg activated DNA and 10 µg histones.The incubation time was 15 minutes in the presence or in the absence of 4-quinazolinone derivatives, and the reaction was stopped by the addition of trichloroacetic acid (8 %).After addition of 0.5 mg albumin, precipitation was allowed to proceed for at least 20 minutes on ice, and the insoluble material was collected on a glass filter washed five times with 5 % perchloric acid.The protein-bound radioactivity was determined by a LS-200 Beckman scintillation counter, data showed in Table 2 are the concentration in µM.
and 18 in CH 2 Cl 2 in the presence of triethylamine.The crude amides could be cyclised in aqueous dioxane with NaBO 3 20 to give compounds 19 and 21, respectively.Reduction of compounds 19 and 21 with Fe powder in AcOH gave the corresponding amines 20 and 22 (Scheme 3).

Table 2 .
Inhibitory effects of compounds 15a-d on PARP enzyme in vitro