Synthesis and anticoccidial activity of 4-(2-methoxyphenyl)-2- oxobutylquinazolinone derivatives

A series of 4-(2-methoxyphenyl)-2-oxo-butyl-quinazolinones were designed and synthesized based on the structure of febrifugine. The structures of the new compounds were confirmed by 1 H NMR, 13 C NMR, IR spectra and HRMS. The biological activity test results indicated that they exhibited anticoccidial activities against Eimeria tenella in the chicken diet with a dose of 9 mg/kg. Compared with halofuginone, these compounds have the advantages of shorter synthetic routes and lower cost.


Introduction
Coccidiosis is an intestinal infection caused by protozoan parasites of the genus Eimeria, especially for broilers and turkeys, which occurs all over the world and leads to extensive loss in the poultry industry. 1Anticoccidial drugs play a very important role in controlling chicken coccidiosis.But the drug resistance of Eimeria against anticoccidial drugs is increasing because many drugs have been used for a long time. 2 Thus, the discovery of new and effective anticoccidial drugs is urgently needed.
In China, Dichroa febrifuga is a well-known medicine for the treatment of malaria.Febrifugine (Figure 1, Compound 1) and its stereoisomer isofebrifugine (Figure 1, Compound 2) have been identified as the active components. 3The potent anticoccidial activity of febrifugine in poultry was discovered in the 1960's.Because of side effects, such as diarrhea, vomiting, 4 and liver toxicity, 5 it has been precluded as an anticoccidial drug.In 1967 halofuginone (Figure 1, Compound 3) (commercial name Stenorol) was designed and synthesized based on the structure of febrifugine by the American Cyanamid Company. 6,7Halofuginone is a broad-spectrum anticoccidial medicine with low toxicity and no cross-resistance. 8Compared to other anticoccidial drugs, halofuginone has a higher anticoccidial activity when administered to chickens in the diet with a concentration of 3 mg/kg.However, the synthetic process of to produce halofuginone is complicated (Scheme 1), 9 which leads to a high cost and limits its application.In order to find new and effective anticoccidial drugs with a simpler structure and lower cost, eight 4-(2-methoxyphenyl)-2-oxobutylquinazolinones were designed and synthesized according to the structure of febrifugine (Figure 2).Compared with the structure of halofuginone, the 4-(2'methoxyphenyl)-2-oxo-butyl moiety in the target compounds was easier to be introduced from salicylaldehyde and acetone.The 6' and 8' substitution of quinazolines is easier than the 6' and 7' substitution because the 3' and 5'-substitution of anthranilic acid can be performed more easily.Instead of a hydroxyl function in halofuginone with a methoxy at C-2″, the stability of the target compounds was improved.The anticoccidial activities of the new compounds were also evaluated according to the Anticoccidial Index (ACI) method. 10  A lesion value (0 -40) could reflect the effect of Eimeria to chicken's cecum.The lesion values in the groups of compounds 7b (7) and 7h (11) were lower than the infected nonmedicated group (15).This exhibited that compounds 7b and 7h could effectively reduce the effect of Eimeria to chickens' cecum.The oocyst value (0 -40) reflected the amount of oocysts produced by the elder generation of coccidia.The oocyst values in the groups treated with compounds 7b (20), 7c (20), 7e (20) and 7g (20) were lower than the infected non-medicated group (30).This shows that compounds 7b, 7c, 7e and 7g could effectively suppress the generation of oocysts.Compared with the infected non-medicated group, the lesion and oocyst value of 7b was reduced from 15 to 7 and 30 to 20, respectively.This indicates that compound 7b has an obvious anticoccidial efficacy against E. tenella.In the safe concentration for chickens, all the activity parameters might be improved with the increase of the concentration.
The weight gain and survival rate reflected the affection of drugs or coccidia on the growth of chickens.Compared with the infected non-medicated group, the weight gain was obviously improved from 69.9 to 100, 99, 107 and 101.9 by using compounds 7b, 7d, 7e and 7h, respectively.But the weight gain of chicken by adding compound 7c was 15.5, which was lower than the infected non-medicated group.The reason might be that compound 7c was toxic to chickens, which influenced their growth.The survival rates were 100 % in the test groups except 7a (95) and 7h (75).This result indicates that with a dose of 9 mg/kg in the chicken diet, the application of compounds 7b, 7d, 7e and 7h didn't affect the growth of chickens.
The anticoccidial index (ACI) could reflect the comprehensive anticoccidial ability of drugs, which was calculated from the growth rate, the survival rate, the lesion value and the oocyst value.

ACI = (weight gain + survival rate) × 100 -(Lesion value + oocyst value)
The ACI indicated that the compound 7b (173) and 7e (170) have obvious anticoccidial efficacy against E. tenella and compound 7d (154.0) and 7h (160.9)exhibited certain anticoccidial activities.The anticoccidial activity of the other compounds was not obvious.We selected only one concentration at random to test whether the synthesized compounds had anticoccidial activity without considering selecting the optimal concentration to obtain the best anti-coccidia index.
Analyzing the structures and activity of the 4-(2-methoxyphenyl)-2-oxo-butyl-quinazolinone derivatives, febrifugine and halofuginone, we found that (1) the quinazoline ring might play a vital role in the anticoccidial activity; (2) the introduction of halogen groups might change the anticoccidial activity; (3) the anticoccidial activity of these compounds probably have a relationship with the 2´-carbonyl and 3″-hydroxyl or 2″-methoxy.

Experimental Section
General Procedures.4][15][16][17] (E)-4-(2-hydroxyphenyl) but-3-en-2-one (3) was prepared according to the literature. 18Other solvents and reagents were obtained from commercial sources and used without further purification.Melting Points were recorded on a XRC-1 apparatus (Sichuan University Instrument Inc, Chengdu, China) and the values are uncorrected.Proton NMR spectra and 13 C NMR spectra were recorded on Bruker AV -400 MHz spectrometer (Bruker company, Switzerland) with CDCl 3 or d 6 -dimethyl sulfoxide as the solvent.Chemical shift values (δ) were given in ppm and were downfield from internal tetramethylsilane.Mass spectra were recorded with Agilent 6210 (DOF-MAS) spectrometer (Agilent Inc, Santa Clara, CA, USA) using the EI method.IR spectra were recorded with a Perkin-Elmer 16PC-FT instrument (Perkin-Elmer Inc, Norwalk Conn, CA, USA).Analytical TLC was performed on silica gel GF 254 , and spots were visualized with ultraviolet light.

Synthesis of quinazoline and its derivatives (2a -2h). General Procedure
A mixture of 1a -1h (0.06 mo1) and formamide 10.8 g (0.24 mo1) was stirred at 135 -165 °C for 4 h, then 15 ml water was added and a lot of solid appeared immediately.The mixture was cooled to 60 °C slowly, and then 30 ml water was added again.The mixture was stirred for another 30 minutes.The resulting precipitate was filtered and recrystallized with ethanol to give 2a -2h.Quinazolin-4(3H)-one (2a

Synthesis of 4-(2-Hydroxyphenyl) butan-2-one (4)
7.2 g Pd/C was added to a water-warmed solution (50 °C) of 3 (58.50g, 0.36 mol) in ethyl acetate (450 ml), the mixture was charged with hydrogen under stirring at 50 °C.When the absorbing of hydrogen was stopped, the reaction was quenched and cooled to RT.The solid was filtered out; the organic layer was dried with anhydrous Na 2 SO 4 and concentrated to give 4 as colorless oil.Yield: 98 %. 1

Synthesis of 4-(2-methoxyphenyl) butan-2-one (5)
A solution of 21.9 g (0.55 mol) of sodium hydroxide in 150 ml water was cooled to 0 °C and 54.0 g (0.33 mol) 4 was added under stirring.Then 45.8 g (0.36 mol) dimethyl sulfate was slowly added and the mixture was kept at 35 -45 °C.The reaction mixture was allowed to stir at this temperature for another 0.5 h and was heated to 95 -100 °C for 2 h, then cooled to room temperature and 80 ml water was added.The mixture was extracted with ethyl acetate (3 × 50 mL).The combined organic phase was washed with 3 % NaOH until the aqueous layer was colorless.The organic layer was separated out, and dried over anhydrous Na 2 SO 4 , concentrated and distilled under reduced pressure (8 mmHg, 110 °C) to give 29.82 g of 5 as colorless oil.Yield: 50.7 %. 1

Synthesis of 4-(2-methoxyphenyl)-2-oxo-butyl-Quinazolinone derivatives (7a -7h) General procedure
A solution of 4.94 g 6 (0.0192 mol) in 10 mL DMSO was added dropwise to a mixture of 2a -2h (0.016 mol), 1.0 g KI, 0.45 g (0.018 mol) NaH and 30 ml DMSO under stirring at 75 °C.After 1 h the mixture was cooled to RT, and then 100 ml water and 70 ml ethyl acetate was added.The organic layer was separated, washed with 70 ml water, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to give a crude product, which was chromatographed on a silica gel column using petroleum ether-ethyl acetate (8:1 v/v) as the mobile phase to give 7a -7h.

Biological assay
The anticoccidial activities of the compounds 7a -7h were evaluated according to the ACI method.Briefly, the chickens used to test the anticoccidial activity of compounds were 12-dayold broiler chickens bought from the Institute of Animal Science, Guangdong Province Academy of Agricultural Sciences.All chickens were fed by the feedstuff without any anticoccidial drugs and drank clean water.
Groups of these chickens were randomly housed in 11 cages with 20 in each.Groups 1 -8 of 13-day-old chickens were fed the basal starter diet with the compounds 7a-7h in 9 mg/Kg until the end of the test.Chicks in group 9 were fed the basal starter diet with decoquinte in 27 mg/Kg until the end of the test.Groups 1 -10 of 14-day-old chickens were infected artificially with the Eimeria tenella spores of the oocysts 100 000.Held on observation for 7 days after infection, recorded the weight gain, survival rate, lesion scores, and oocysts scores of the chicken, and calculated the ACI.