One-Pot Microwave Synthesis of Pyrimido [ 4 , 5b ] quinoline and its Cand S-Glycosides with Anti-Inflammatory and Anticancer Activities

An efficient one-pot synthesis of 2-thioxopyrimido[4,5-b]quinoline 3a,b has been accomplished from a three-component reaction of 6-aminothiouracil, cyclohexanone and aromatic aldehyde under microwave irradiation. Compound 3a,b was used as a key intermediate for the synthesis of Sand C-nucleoside analogs of types, 5-(4-fluorophenyl / 4-anisyl)-2-S-(-D-ribofuranosyl / arabinofuranosyl)-6,7,8,9-tetrahydro-3Hpyrimido[4,5-b]quinolin-4-one (6a–d) and 5-(4-fluorophenyl / 4-anisyl)-2-S-(-D-gluco / galactopyranosyl)-6,7,8,9-tetrahydro-3H-pyrimido[4,5b]quinolin-4-one (8a–d). Also. the 2-hydrazino compounds 9a,b were used for the synthesis of 3-(glycosyl)-6-(4-substituted phenyl)-7,8,9,10tetrahydro[1,2,4]triazolo[4',3':1,2]pyrimido[4,5-b]quinoline-5-(1H)-one (11a–d and 13a–d). The title compounds were investigated for antiinflammatory and anticancer activities. Compounds 11a exhibited the comparable anti-inflammatory activity (83.4 %) to the standard drug Indomethacin (85.2 %). 5-(4-Fluorophenyl)-2-S-(-D-ribofuranosyl)-6,7,8,9-tetrahydro-3H-pyrimido[4,5-b]quinolin-4-one 6a and 3-(ribosyl)-5(4-fluorophenyl)-7,8,9,10-tetrahydro[1,2,4]triazolo[4',3':1,2]pyrimido[4,5-b]quinolin-5-one (13a) exhibited the maximum cytotoxic effect against the three human cancer cell lines with inhibitory effects higher than the reference doxorubicin.


INTRODUCTION
ANCER disease is a major worldwide problem.In the new millennium, rapid progress has been made in the area of a cancer cell, it has become clear that inflammation has an essential role in increased cancer risk. [1,2]The process of development of cancers may be due to inflammatory cells, in addition to a variety of mediators, like cytokines, chemokines and enzymes. [3]xidative stress is an important mechanism in the pathogenesis of many diseases including cancer.The generation of reactive oxygen species (ROS) with consecutive DNA damage is an initial step in carcinogenesis induced by inflammatory processes. [4]ROS is generated either via inflammatory cytokines or via cytochrome P-450 2E1 induction and may lead to lipid peroxidation.Chemokines and pro-inflammatory cytokines as interleukin(IL)-6 and IL-1α can favor the growth of tumor while the treatment with NSAIDS can minimize cancer incidence, [5] so there is a strong relation between cancer and inflammation.Some of the pro-inflammatory factors such as reactive oxygen species, prostaglandin E2(PGE2) and tumor necrosis factor α (TNF α) are among molecules that play a major role in suppressing inflammation. [6]Nonsteroidal anti-inflammatory drugs (NSAIDS) have inhibitory activity toward cyclooxygenase-1(COX-1) and cyclooxygenase-2 (COX-2). [7]NSAIDS suppress transcription factor NF-kB which regulates COX-2 and inhibits the tumor cell. [8]uinoline occupies the catalytic split of human DNA repair O 6 -alkylguanine DNA alkyltransferase, by acting as analogs of the O 6 -guanine moiety in the natural substrate, and reaching the catalytic residue Cys145. [9]Furthermore, pyrimidine and fused heterocyclic pyrimidine derivatives show anti-inflammatory and anticancer activities, [10] so the fused ring of quinoline and pyrimidine skeletons C pyrimidoquinolines are considered to be promising nuclei for anticancer drug development.In addition to the wide range of biological activity of quinoline and pyrimidoquinoline derivatives, these compounds have attracted a great deal of attention in the field of medicinal chemistry.Quinoline and pyrimidoquinoline derivatives are an important class of therapeutically useful antibacterial drugs, [11][12][13][14] anticancer, [15,16] antioxidant, analgesic and antiinflammatory activities, [17][18][19] antiallergic, [20] microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor. [21]Also, some of these derivatives showed antimalarial activity. [22]icrowave mediated multi-component reactions constitute an especially attractive synthetic strategy for rapid and efficient library generation because products are formed in a single step and diversity can be achieved by varying the reacting components.In continuation of our efforts towards multi-component reactions, [12] we report herein a conventional and microwave rapid synthesis of pyrimido [4,5-b]quinoline from a threecomponent reaction.

Synthesis of 5-Aryl-2-methylthio-6,7,8,9tetrahydro-3H-pyrimido[4,5-b]quinolin-4-one (4a,b)
To a warm ethanolic potassium hydroxide solution (prepared by dissolving 0.01 mol of potassium hydroxide in 30 mL absolute ethanol) was added compound 3a,b (0.01 mol), the heating was continued for 30 min, the mixture was allowed to cool to room temperature and methyl iodide (0.12 mol) was added.The mixture was stirred under reflux for 3 h, cooled to room temperature, and poured onto cold water (100 mL).The solid precipitated was filtered off, washed with water and dried, crystallized from DMF.

Animals
Adult male albino rats (Harlan Sprague-Dawley), weighing 150-180 g, were used for the evaluation of antiinflammatory activity.Animals were fasted for 12 hours before the assay.International principle and local regulations concerning the care of used laboratory animals was taken into account. [23]All animals were obtained from the animal house colony of the National Research Centre, Cairo, Egypt.The animals were acclimatized to the experimental room having temperature 22 ± 1 °C, controlled humidity conditions, and 14 : 10 h light and dark cycle.The rats were fed on autoclaved standard mice food pellets (Hindustan Lever Ltd., New Delhi) and water ad libitum.

Anti-Inflammatory Activity
Carrageenin-induced paw edema test was performed on male albino rats by using the method of Winter et al. [24] The animals were weighed, marked for identification and divided into 14 groups, each group containing 6 animals. 1 % carboxymethyl cellulose (CMC) was selected as vehicle to suspend the standard drug and test compounds.The 1 st group was kept as control and was given the respective volume of vehicle (1 % CMC, oral) only.The 2 nd to 13 th groups were given a 100 mg kg -1 body mass oral dose of test compounds.One hour later, 0.2 mL of 1 % carrageenan suspension in 0.9 % NaCl solution was injected subcutaneously, into the subplantar tissue of the right hind paw of each mouse and the paw volume was measured with a plethysmometer (UGO Basile 7140, model-7141, Biological research apparatus, Italy) .The initial paw volume was measured within 30 s of the injection and remeasured again 1 h, 2 h, 3 h and 4 h after administration of Carrageenan.The last group was administered indomethacin in a dose of 10 mg kg -1 orally as a standard reference. [25]The mean increase in paw volume was compared with that of control group and percent inhibition values were calculated by the formula given below: % anti-inflammatory activity = (Vc -Vt / Vc) × 100.Where Vt represents the paw volume in drug treated animals and Vc represents the paw volume of control group of animals.

CELL CULTURES
Some of the synthesized compounds (3a,b), (6a,b), (8a,b), (9a,b), (11a,b) and (13a,b) were tested for in vitro anticancer activity against three human tumor cell lines, HepG2 (human liver carcinoma), NCI-H460 (non-small cell lung cancer) and MCF-7 (breast adenocarcinoma) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. [26]HepG2 and NCI-H460 were kindly provided by the National Cancer Institute (Cairo, Egypt) and MCF-7 was obtained from the European Collection of Cell Cultures (Salisbury, UK).They grew as monolayers and were routinely maintained in RPMI-1640 medium supplemented with 5 % heat inactivated FBS, 2 mmol L -1 glutamine and antibiotics (penicillin 100 U mL -1 , streptomycin 100 µg mL -1 ), at 37 °C in a humidified atmosphere containing 5 % CO2.Exponentially growing cells were obtained by plating 1.5 × 10 5 cells mL -1 , followed by 24 h incubation.The effect of the vehicle solvent DMSO on the growth of these cell lines was evaluated by exposing untreated control cells to the maximum concentration (0.5 %) of DMSO used in each assay.The effect of compounds on in vitro growth of human tumor cell lines was evaluated according to the procedure adopted by the national cancer institute (NCI, USA) by using sulforhodamine B as protein binding dye to assess cell growth. [27]Cells growing exponentially in 96-well plates were then exposed for 48 h to five different concentrations of each test compound (5, 12, 25, 50 and 100 µmol L -1 ).After this exposure period, adherent cells were fixed, washed and stained.The bound stain was solubilized and the optical density (absorbance) was measured, and the growth inhibition of 50 % (GI50) was calculated. [28]Doxorubicin was used as a reference compound (Table 2).

RESULTS AND DISCUSSION
In continuation of our drug research program, and on the basis of the above considerations, original nucleoside analogs directed upon reverse transcriptase still aroused considerable interest. [29]In this study the synthetic pathways depicted in Schemes 1 and 2 outlines the chemistry of the present study.Thus, pyrimido- [4,5b]quinoline as the starting materials 3a,b are easily prepared following the well established procedure reported in the literature. [12]Treatment of 6  13 C NMR spectrum of the 2-thioxo-(4a) and 2-methylthiopyrimidine in the literature [30] indicated that the site of the alkylation is the sulfur atom rather than the nitrogen atom (Scheme 1).

Table 2 .
Effects of synthesized compounds on the growth of the three human tumor cell lines.Results are given as concentrations that were able to cause 50 % cell growth inhibition (GI50) after continuous exposure for 48 h.Mean ± SEM of three independent experiments performed in duplicate.