Studies on Parasitologic and Haematologic Activities of an Enaminone Derivative of 4-Hydroxyquinolin-2(1H)-one Against Murine Schistosomiasis Mansoni

Amal M. El–Shennawy, MD; Amira H. Mohamed, MD; Mohamed Abass, MD

In This Article

Materials and Methods

Schistosome Infections

S mansoni cercariae were provided by the Malacology Lab of the Schistosomiasis Unit (SBSP), Theodor Bilharz Research Institute (TBRI), where laboratory–bred B alexandrina are maintained. Infection was performed by tail immersion using 100 S mansoni cercariae to each mouse. Laboratory–bred male albino mice of the CDI strain, each weighing 18–20 grams, were used in this study. Experimental animals were kept in an air–conditioned room at 21°C and received food containing 24% protein.

Pharmacologic Agents

Praziquantel. PZQ (EMBAY 8440) was purchased from Bayer (Leverkusen, Germany) and E. Merck (Darmstadt, Germany).

Synthesis of BDHQ

As outlined in Figure 1, condensation of N–butylaniline with excess diethyl malonate led to the formation of 6–butyl–4–hydroxypyranoquinolinedione BHPQ. Alkaline hydrolysis of the product BHPQ, using 15% sodium hydroxide, furnished the corresponding 3–acetyl–1–butyl–4–hydroxyquinolin–2(1H)–one (ABHQ).[8] Thermal condensation of ABHQ with dimethylformamide–dimethylacetal (DMF–DMA) afforded1–butyl–3–[2E–3–(dimethylamino)prop–2–enoyl]–4–hydroxy–quinoline–2(1H)–one (BDHQ), in 81% yield.[7] IR (frequency), mass, and elemental analyses supported the structure of BDHQ. Besides, 1H NMR revealed that BDHQ assumes an E–form where the coupling constant of the olefinic protons is 13.5 Hz.

Figure 1.

Reagents and conditions: (a) diethyl malonate, 220°C, 4 hrs; (b) aq. NaOH (15%), reflux, 2 hrs; (c) DMF–DMA, toluene, 110°C, 2 hrs.

Experimental Design


Melting point is uncorrected and was determined in an open capillary tube on a digital Gallen–kamp MFB–595 apparatus. Infrared spectrum was recorded on a Perkin–Elmer 1650 FT–IR spectrophotometer, using a sample in KBr pellet. 1H NMR spectrum was recorded on Varian Gemini (200 MHz), using TMS as an internal standard and CDCl3 as solvents. Mass spectrum was taken on a Shimadzu GCMS QP–1000EX instrument by direct inlet technique at beam energy 70 eV. Elemental microanalysis was performed on a Perkin–Elmer 2400 analyzer.

1–Butyl–3–[2E–3–(dimethylamino)prop–2–enoyl]–4–hydroxyquinolin–2(1H)–one (BDHQ)

DMF–DMA (1.4 mL, 10 mmol) was added to a solution of acetylquinolinone ABHQ, in dry toluene (50 mL), and the reaction mixture was heated under reflux for 2 hours. The excess solvent was evaporated in vacuum and the residual material was triturated with petroleum ether (60–80°C) (25 mL), filtered off and crystallized from benzene. Yield 2.54 g (81 %); mp 126–7°C. IR (KBr): νmax 3131, 3080, 2956, 2924, 2860, 1642 (C = O), 1622, 1526, 1498 cm–1; 1H NMR (200 MHz, CDCl3): δ0.96 (t, 3H, N–(CH2)3CH 3), 1.31 (m, 4H, N–CH2(CH 2)2CH3), 3.02 (s, 3H, N(CH3)2), 3.21 (s, 3H, N(CH3)2), 3.87 (t, 2H, N–CH 2(CH2)2CH3), 7.08 (d, J = 13.5 Hz, 1H, COCH=CH–N), 7.21–7.32 (m, 2H, Harom), 7.59 (t, 1H, 7–Hquinolinone), 8.09 (d, J = 13.5 Hz, 1H, COCH=CH–N), 8.22 (d, J = 8 Hz, 1H, 5–Hquinolinone); MS: m/z (I %) 314 (M+, 28), 270 (100). Anal. calculated for C18H22N2O3 (314.39): C, 68.77; H, 7.05; N, 8.91. Results: C, 68.68; H, 6.94; N, 8.56.


The mice were divided into 4 groups, 10 animals each. Group I (infected untreated controls): 5 mice were sacrificed after 3 weeks (so that the schistosomules could be counted) and the other 5 after 6 weeks (so the adult worms could be counted). Group II: 5 mice were treated at the third week post infection through oral use of an esophogeal tube with BDHQ (10 mg/mL) for 2 consecutive days (0.5 mL x 2), and the other 5 mice were treated at the sixth week post infection as above. Group III: Same as group II, but we used a higher dose of BDHQ (1 mL x 2). Group IV: 5 mice were treated at the third week post infection with PZQ for 2 consecutive days (2 x 500 mg/kg), and the other 5 mice were treated at the sixth week post infection as above. All groups were sacrificed 2 weeks after treatment.

Sample Collection and Preparation

After the mice were scarified by cervical dislocation, some of the blood was collected in tubes containing EDTA as anticoagulant and centrifuged to obtain the buffy coat, and some of the blood was collected in tubes without anticoagulant and centrifuged to obtain the serum, which was frozen at –20ºC until tested. The hepatic and portomesenteric vessels were perfused to study worm load and sex. Worms, sections of the livers and spleens, buffy coat, and bone marrow from the femur bones were fixed in 4% glutaraldehyde with sodium cacodylate for electron microscopic examination. The rest of the liver and intestinal fragment tissues were used for the recovery of eggs and determination of the percentage of eggs in each developmental stage.

Worm Burden and Distribution

Worm burden and sex were studied after perfusion of the hepatoportomesenteric vessels.[9]

Tissue Egg Load

The number of eggs per gram of tissue was studied by weighing 0.5 g of liver or small intestine, which was then digested and incubated overnight in 5% KOH. The hepatic and intestinal tissue egg loads were determined by multiplying the average number of eggs in each 1–mL sample by the total volume of KOH and then dividing that value by the weight of the sample to yield the number of eggs per gram of tissue.[10]

Electron Microscopic Examination

The collected samples were transferred after 2 hours from glutaraldehyde and sodium cacodylate to be fixed in 2% osmium tetraoxide, dehydrated with increasing concentrations of alcohol, and embedded in epoxy resin according to the technique of Grimaud and colleagues.[11]

Semi–thin and ultra–thin section sections were cut with a Leika ultramicrotome. Ultra–thin sections were contrasted with uranyl acetate and lead citrate stains and were then examined by Phillips EM–208 electron microscope.

IFN–gamma Assays by ELISA

The collected sera were tested with commercially available enzyme linked immunosorbent assay (ELISA) kits (Quantikine M mouse IFN–gamma, Diaclone Research USR, United Kingdom). The procedure was done according to the manufacturer's instruction. Standard curves were generated from known concentrations of cytokine provided by the manufacturer. Standard curves were then used to predict the quantity of cytokine based on the level of spectrophotometric absorbance for each sample using regression analysis.

Measurement of Serum Anti–SWAP IgE

Measurement of serum level of IgE by ELISA was based on the original method of Engvall and Perlman.[12] For assessment of serum Ig E, Immulon 4 (Dynatech Laboratories, Chantilly, Virginia, USA) microtiter plates were coated overnight at 4°C with SWAP (1 mcg in 50 mcl/well) diluted in PBS. Plates were blocked with 200 mcl 5% nonfat dry milk/PBS for 2 hours at 37°C. The blocking solution was aspirated and the wells washed 6 times with PBS/0.05% Tween–20 (Sigma) (St. Louis, Missouri, USA). Individual mouse serum was diluted 1/100 in 1% BSA/PBS, and 50 mcl was added to appropriate wells. Plates were incubated at 37°C for 90 minutes and then washed 6 times with PBS/0.05% Tween–20. Fifty microliters of isotype–specific horseradish peroxidase–conjugated rabbit anti–mouse Abs in 1% BSA/PBS diluted at 1/500 were added to the wells and incubated at 37°C for 2 hours. Wells were again washed 6 times with PBS/0.05% Tween–20 and 100 mcl of (2,2'–azino–di(3–ethylbenzthiazolinesulfonate) (ABTS: H2O2 substrate [Kirkegaard & Perry Laboratories, Gaithersburg, Maryland, USA]) was added and the reactions developed in the dark at room temperature for 20 to 30 minutes. Absorbance at 405 nm was determined using an ELISA reader (Bio–Rad Microplate Reader, Richmond, California, USA).

Statistical Analysis

The data are presented as mean ± standard error of mean (X ± SE). The means of the different groups were compared globally using the analysis of variance (ANOVA). The data were considered significant if P values were ≤ .05.


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