Design , synthesis and evaluation of the molluscicidal activity of novel hydrocarbyl substituted salicylic acid derivatives

Based on the molluscicidal activity of ginkgolic acids (GAs) isolated from Ginkgo biloba L and synthetic GA analogues, a series of long chain, substituted at 4 and 5 position, salicylic acid derivatives, including the Z/E isomers and their saturated derivatives, were prepared. Their molluscicidal activities against the host snail Oncomelania hupensis were evaluated. The acute fish toxicity against the wildtype zebrafish of saturated derivatives was tested. The results and analysis of the structural-activity relationship revealed that the E-isomers and their saturated derivatives were better molluscicidal agents than their respective Z-isomers. Molluscicidal activities were increased with the growing of the alkyl chain lengths. Acute fish toxicity test indicated that the synthesized salicylic acid derivatives were much safer than niclosamide in field use.


INTRODUCTION
Schistosomiasis is one of the most prevalent of the tropical infectious diseases with more than 200 million people infected globally and close to 800 million at risk (Steinmann et al., 2006).In China, zoonotic schistosomiasis, caused by Schistosoma japonicum, is a chronic debilitating disease in the south, with 325,824 infected in 2010 (Lei et al., 2011) and 65 million people at risk of infection (Zhou et al., 2005;McManus et al., 2010).The snail, Oncomelania hupensis, is the main agent responsible for the transmission of this disease.In current years, China is facing a new challenge because schistosomiasis has reemerged in some regions due to an addition in habitats suitable for the snails, including increased flood areas and large water resource development projects (Yuan et al., 2005).
Snail control can be a powerful intervention tool as it essentially removes the intermediate host from the parasite's life cycle, thus breaking transmission (Fong, 2013).Currently, the niclosamide is the only commercially available molluscicide recommended by the World Health Organization (WHO, 1993); however, its use meets with many complications, notably environmental toxicity and high-cost implications (McManus et al., 2009(McManus et al., , 2010)).Due to this fact, much attention has been given to more environmentally friendly chemicals of natural products to identify novel molluscidal agents (Singh et al., 2010;Pereira et al., 2011;Miyasato et al., 2012;González-Cruz and San Martín, 2013;Rodrigues et al., 2013) and then the synthesis of new chemicals analogues with increased activities is ensuing (Fadda et al., 2009;EI Shehry et al., 2010;Hajare et al., 2011).
Ginkgolic acids (GAs) are natural salicylic acid derivatives of long alkyl or alkenyl substituents at the ortho position to the carboxyl group, which represent a new group of potential molluscicidal agents that have been discovered in recent years (Pan et al., 2007).Other 6-alkylsalicylic acids have also been identified in other plant species such as the anacardic acids (Ana) isolated from the cashew (Anacardium occidentale).Kubo and colleagues found these compounds to have molluscicidal activities against the South American snail Biomphalaria glabratus (Kubo et al., 1986).
In our previous study, five monomers of GAs were separated and purified from the sarcotesta of Ginkgo biloba L. The order for their molluscicidal activities were as follows: GA 13:0 >GA 15:1 >GA 15:0 >GA 17:1 >GA 17:2 (Mao et al., 2007).We also study the molluscicidal effects of a series of Z/E isomers of GA analogues with different chain lengths (5 to 13 carbon atoms) and phenyl rings and find that the E-isomers are better molluscicidal agents than their respective Z-isomers and molluscicidal activity is found to decrease regularly with the shortening of the alkyl chain lengths (Zhang et al., 2011).
In order to further understand the relationship of the substituted position of the long chain in salicylic acid and their molluscicidal activity, a series of Z/E isomers and their saturated products of 4-and 5-alkylsalicylic acid derivatives with different chain lengths (7-13 carbon atoms) have been synthesized from 4-and 5methylsalicylic acid.Their molluscicidal effects against the host snail O. hupensis were evaluated, and the structure-activity relationships were explored.

Synthesis of the compounds
The unhydrolyzed mixtures of Z and E isomers 6a-6c and 18a-18c were synthesized from 5-methylsalicylic and 4-methylsalicylic acid according to the reaction sequence delineated in Schemes 1 and 2 (Hamada et al., 1991).These were separated by column chromatography on silica gel.were obtained by ester hydrolysis and extracted from acidic solution with dichloromethane.To the Z and E isomers, based on the spin-spin coupling constants of the double bond protons, the isomer with smaller coupling constant (J=8-12 Hz) was assigned to the Z-configuration, and the larger coupling constant (J= 12-16 Hz) was assigned to the E configuration.
Methyl O-acetyl-6-bromomethylsalicylate (compound 4): To a stirred solution of compound 3 (11.45g, 55 mmol) dissolved in carbon tetrachloride (110 ml) were added a mixture of Nbromosuccinimide (11.23 g, 63 mmol) and azobisisobutyronitrile (78.5 mg, 0.05 mmol).This solution was then heated at reflux for 3 h.After cooling, the reaction mixture was filtered and the filtrate was condensed under reduced pressure to give the crude product.Chromatographic purification of the crude material (silica gel, petroleum ether-ethyl acetate = 30:1) gave the bromide 4 (13.19 g, 84%) as a white powder.This power was directly used for the next reaction without further identification.

4-Acetoxy
Scheme 1. Synthesis of 5-hydrocarbyl substituted salicylic acid derivatives from 5-methylsalicylic acid.Scheme 2. Synthesis of 4-hydrocarbyl substituted salicylic acid derivatives from 4-methylsalicylic acid.mmol) and triphenylphosphine (10.74 g, 41 mmol) was added to a magnetically stirred round bottomed flask at 0°C, and then slowly heated to 130°C.After all the power melted together, the reaction mixture was cooled to 70°C.A small amount of chloroform was added and refluxed until all the solid had dissolved.Anhydrous ether (35 ml) was then added and the reaction mixture was filtered to give the phosphonium salt as a slightly yellow powder (21.7 g, 97%).

General procedure for preparation of methyl O-acetyl-5-(1alkenyl) salicylates (compound 6a-6c):
The phosphonium salt 5 (548 mg, 1 mmol) was dissolved in DMSO (5 ml).Triethylamine (0.170 ml, 1.2 mmol) and different aldehydes were added.The reaction mixture was heated to 125°C for 21 to 26 h, then allowed to cool to ambient temperature, poured onto ice-cold water (25 ml), and extracted three times with ether (each 50 ml).The organic layer was washed with water (50 ml) and saturated brine (50 ml), and dried over Na 2 SO 4 .The solution was then filtered, and concentrated under reduced pressure to give the crude product as a mixture of geometric isomers.

General procedure for preparation of methyl O-acetyl-5-(1alkyl) salicylates (compound 11a-11c):
The mixture of geometric isomers was dissolved in acetic acid.10% Molecular quantities of 10% Pd/C were added.The reaction mixture was swung under 60 psi of H 2 for 9 to 11 h at room temperature, then filtered and the filtrate was poured onto water, and extracted three times with ethyl acetate.The organic layer was washed with saturated brine, and dried over Na 2 SO 4. The solution was then filtered, and concentrated under reduced pressure to give the crude product.Chromatographic purification of the crude material (silica gel mixture with 1 to 2% weight quantities of AgNO 3 , petroleum etherethyl acetate = 50:1) gave the 11a-11c.

General procedure for preparation of 5-(1-alkenyl) salicylic acids and 5-(1-alkyl) salicylic acids:
Triple molecular quantities of 1 mol/L NaOH and the ester 7a-7c, 8a-8c and 11a-11c, in EtOH were heated at reflux for 5 to 8 h, respectively.After removal of the solvent, the residue was acidified to pH 2 with 1 mol/L HCl, diluted with water, and extracted three times with dichloromethane.The organic layer was washed with water and saturated brine, dried over Na 2 SO 4 , and concentrated to give the products as yellowish or white powders.

Synthesis of 4-hydrocarbyl substituted salicylic acid derivatives
The general procedure for the synthesis of 4-(1-alkenyl)salicylic acids and 4-(1-alkyl)salicylic acids was similar as the synthesis of 5-(1-alkenyl)salicylic and 5-(1-alkyl)salicylic acids.So, the main intermediates and the details of the products were listed and the process of the total synthesis are as shown in Scheme 2.

Molluscicidal test
The O. hupensis specimens were collected from the endemic area of Schistosomiasis japonica in Anhui province, China.The test was carried out with the immersion method in accordance with WHO guide-line for laboratory molluscicidal tests (Lemma et al., 1984).The snails were placed in a dark wet and warm (about 25°C) environment for 2 days without providing food.Healthy adult snails (age: about 6 months; presence of 7 to 8 whorls) were used in all molluscicidal tests.Each compound was dissolved into a small amount of dimethyl sulfoxide (less than 0.1% final concentration) and then diluted with dechlorinated water to prepare for the solutions (50, 25, 12.5, 6.25, and 3.125 mg/L, respectively).Niclosamide (Nic) was used as a positive control (concentrations: 2, 1, 0.5, 0.25, 0.125, 0.0625 mg/L, respectively) and dechlorinated water with same concentration of dimethyl sulfoxide as the negative control.The molluscicidal test was carried out using the immersion method (WHO, 1984).Ten snails in one group were placed into beakers containing test compound at different concentrations for a period of 48 h at 25 ± 2°C.Each beaker was covered with nylon netting to prevent the snails from escaping.Snail mortality was carefully checked and all tests were carried out in duplicate.We got the test data in average of 4 dependent experiments.The LC 10 , LC 50 and LC 90 values were determined using Probit analysis (Finney, 1971).

Acute fish toxicity test
The wild-type zebrafish (AB* strain), Danio rerio, was obtained from the Zebrafish Breeding Base of the Key Laboratory of Molecular Medicine of the Ministry of Education, Fudan University.The test was carried out with the Fish Embryo Toxicity (FET) test in accordance with the OECD Guideline for the Testing of Chemicals (Braunbeck et al., 2005;OECD, 2006).Healthy adult fishes (age: 5 months; length: 4 to 6 cm; room temperature: 25°C) were used in all toxicity tests regardless of males or females.Each compound was dissolved into a small amount of dimethyl sulfoxide (less than 0.1% final concentration) and then diluted with dechlorinated water to prepare for the solutions (50, 25, 12.5, and 6.25 mg/L,  respectively).Niclosamide (Nic) was used as a positive control (concentrations: 2, 1, and 0.5 mg/L, respectively) and dechlorinated water with same concentration of dimethyl sulfoxide as the negative control.Seven fish in one group were placed into 500 ml beakers containing test compound at different concentrations for a period of 48 h at 25 ± 2°C.The dead fish were removed as soon as they were found dead in the beakers.Air was bubbled continuously in each beaker during the test.LC 50 values were also calculated using Probit analysis of the mortality data.

RESULTS AND DISCUSSION
In this study, a series of Z/E isomers and their corresponding saturated derivatives of 4-and 5hydrocarbyl substituted salicylic acid were prepared.The results of the molluscicidal assays and the representative LC 10 , LC 50 , and LC 90 values with the confidence limit were shown in Table 1.The results and analysis of the structuralactivity relationship revealed that the E-isomers and their saturated derivatives were better molluscicidal agents than their respective Zisomers.Activity was found to increase regularly with the growing of the alkyl chain lengths in the following sequence: c > b > a.The compounds containing 13 carbon atoms chain, 10c, 12, 22c, and 24 had the strongest activities among all of the analogues tested.Their molluscicidal activities are similar to ginkgolic acid analogues (Zhang et al., 2011).This means that the molluscicidal activity of hydrocarbyl substituted salicylic acid derivatives is concerned with the side chain lengths rather than the substituted position and maybe concerned with the postulate of physical rather than a chemical mechanism of toxicity (De Villiers et al., 1967).
The results of the acute fish toxicity assays and the representative LC 50 values with the confidence limit were shown in Table 2.In this test, we chose some analogues which had better molluscicidal activities to examine their acute fish toxicity.Results revealed that all of the hydrocarbyl substituted salicylic acid derivatives in the test exhibited low toxicity to zebrafish.According to the LC 50 values, 24c showed the strongest toxicity among all of the analogues tested.In the test, the negative control had no dead fish in 48 h, while the Niclosamide group had all fish dead in 2 h even in the lowest test concentration 0.5 mg/L.

Conclusions
Synthetic 4-and 5-hydrocarbyl substituted salicylic acid derivatives represent a new class of chemical entities with promising molluscicidal activities.The compounds containing 13 carbon atoms chain that had the strongest activities among all of the analogues tested.In consideration of the price and difficulty to prepare the 6-hydrocarbyl substituted salicylic acid derivatives, ginkgolic acid analogues, the synthesis of 4-and 5hydrocarbyl salicylic acid derivatives were more convenient.The acute fish toxicity test indicated that the synthesized salicylic acid derivatives were much safer than niclosamide in field use.

Table 1 .
Molluscicidal activity (mg/L) of the hydrocarbyl substituted salicylic acid derivatives against snail O. hupensis under laboratory conditions and 48 h exposure.
a Confidence limit.

Table 2 .
Acute fish toxicity (mg/L) of the hydrocarbyl substituted salicylic acid derivatives against zebrafish under laboratory conditions and 48 h exposure.
a Confidence limit.