Environmental biotoxicity screening of some pyrrole and 1 , 4-dihydropyridine heterocyclic derivatives

A. Idhayadhulla, Aseer Manilal, Behailu Merdekios and R. Surendra Kumar Department of Chemistry, Sri Vinayaga College of Arts and Science, (Affiliated to Thiruvalluvar University), Ulundurpet – 606 107, Villupuram(Dt), Tamil Nadu, India. Department of Medical Laboratory Sciences, College of Medicine and Health sciences, Arba Minch University, Arba Minch, Ethiopia. Department of chemistry, Shivani Engineering College, Trichy, Tamil Nadu, India.


INTRODUCTION
The nematicide and larvicidal use is slate for reduction due to environmental problems, human and animal health concerns.For example, effective nematicides such as dibromochloropropane (DBCP) and ethylenedibromide (EDB) have been withdrawn from the market due to their deleterious effects on humans and the environment.According to World Health Organization (WHO), the one of strategies is to destroy their vectors or intermediate hosts.The best method is control of mosquito larvae using insecticides (Sun et al., 2010;Talontsi et al., 2011) such as organo-phosphates, natural products and heterocyclic types.It is an urgent need to develop new insecticides which are more environmentally safe and also biodegradable molecules.The pyrrole and 1,4-dihydropyridine derivatives have received considerable attention of synthetic and biological important such as anticoagulant (Idhayadhulla et al., .2012), anticonvulsant (Idhayadhulla et al. 2012) particularly 1,4dihydropyridine with thiosemicarbazide also biological importance such as anticonvulsant (Surendra Kumar et al., 2010), anticoagulant (Surendra Kumar et al., 2011b), anticancer (Surendra Kumar et al., 2011c).Our Previous work Brine shrimp cytotoxicity, Larvicidal, Nematicidial and Antifeedant activity were screening by using natural and marine based natural products (Manilal et al., 2011;Manilal et al., 2009), although first time we are focused by drug molecules screened for Brine shrimp cytotoxicity, Larvicidal, Nematicidial and Antifeedant (Deepa et al.,2010) activities.Insecticidal, nematicidal and acaricidal activities of pyrrole compounds were previously reported in (United States Patent 7186722 and Patent 7, 783/739, October 28, 1991).Synthesis and Larvicidal activities, antifungal activities of novel Chlorantraniliprole derivatives and their target in the Ryanodine Receptor reported in (Qichao et al., 2015;Xudong et al., 2007;Drissa et al., 2011).Insecticidal, nematicidal and acaricidal activities of pyrrole compounds were previously reported in (United States Patent 7186722 and Patent 7, 783 / 739, October 28, 1991).
Synthesis and Larvicidal activities, antifungal activities of novel Chlorantraniliprole derivatives and their target in the Ryanodine Receptor reported in (Qichao et al., 2015;Xudong et al.,2007;Drissa et al., 2011).In our present work, we are report here the synthesis of pyrrole and 1,4-dihyropyridine derivatives and their biotoxicity screening against Brine shrimp, ichthyotoxic toxicity larvicidal, and nematicidial activity screening.

Cytotoxic activity
The cytotoxic activity of the newly synthesized test compounds 1-6 was conducted according to the methodology described elsewhere (Manilal et al., 2009), The freshly hatched free-swimming nauplii of Artemia salina (Linnaeus) (Artemia salina, Sanders Great Salt Lake Brine Shrimp Company L.C., U.S.A.) was used as the test organism.The assay system was prepared with 2 mL of filtered seawater containing chosen concentration of synthesized chemicals (10, 20, 30 40 µg/mL) in cavity blocks (embryo cup).
Parallel vehicle control (using 2 % methanol) and negative control (without vehicle) wells were also kept.In each cavity blocks, 20 nauplii were transferred and the setup was allowed to remain for 24 h, under constant illumination.After 24 h, the dead nauplii were counted with a hand lens.Based on the percent mortality, the LD 50 of the test compounds was determined using probit scale (Wardlaw, 1985).

Ichthyotoxicity
The icthyotoxic activity of synthesized test compounds1-6 was carried out following the methodology of (Manilal et al., 2010).Briefly, five fingerlings of Oreochromis mossambicus (Peters) (1.5±1 cm) were introduced in each experimental and control glass bowls containing 1,000 mL of freshwater dissolved with chosen concentrations (10, 20, 30 and 40 µg/mL) of synthesized compounds 1-6.Immediate reflex changes and mortality were observed continuously for six hours at 1h interval for the next 12 h.After 24 h of exposure, the number dead and live fishes were counted.

Larvicidal activity
The assessment of larvicidal activity of synthesized test compounds 1-6 was tested against the urban mosquitoes C. quinquefasciatus using standard bioassay protocol (Manilal et al., 2011).Egg rafts of mosquito were obtained from drainage system.Eggs were reared under standard insectary conditions at ambient temperature (29±3°C), relative humidity 80±5%, 12:12 light: dark photoperiod and fed with ground shrimp feed daily.Larval development was monitored for seven days.The second and third stage larvae were collected at the tip of a pasture pipette and placed in cotton bud to remove excess water and transferred gently to the test vial (10 / vial) by tapping.The larval mortality was observed using various concentrations of synthesized compounds1-6(10,20,30,40 µg/mL).

Nematicidial activity
For the determination of nematicidal activity, juveniles of Meloidogyne javanica were used as test organism (Manilal et al., 2009).Assay system was prepared with 2 ml Milli Q water containing different concentrations (10, 20, 30 and 40 µg/mL) of synthesized test compounds 1-6 in glass tubes.Ten juveniles of M.javanica were transferred in test, positive (with 2% methanol) and negative(without vehicle) control tubes.Mortality was observed under a zoom stereomicroscope after 24 h of exposure.
Acute toxicity of synthesized compounds can be determined by the calculation of LD 50 , i.e., the dose that will kill 50% of animals of a particular species.The LD 50 method and calculation is described from the literature of (Miller and Tainter et al.,1944).

STRUCTURE ACTIVITY RELATIONSHIPS (SAR)
From the results of biotoxicity screening of pyrrole and 1,4-dihydropyridine derivatives, the following structure activity relationships figure.1 can be derived.The compounds 1-4 show that 100 % mortality at concentration 40 µg/mL.The compound 6 containing sulphur group and it shows that 100% mortality at concentration 30µg/mL against Brain shrimp cytotoxic.The compound 1, 2, 4 shows that 100 % mortality at concentration 40 µg/mL.The compounds 3,6 containing -NH-and methyl group and it show that 100 % mortality at concentration 30 µg/mL against Larvicidal activity.

Synthesis
We are reported previously above synthesized title compounds in international journal of biological chemistry (Idhayadhulla, et al., 2013), it is representing in Scheme 1.The characterization of synthesized compounds are summarized in Table1.

Bio-toxicity screening Brain shrimp cytotoxic activity
Brain shrimp cytotoxic activity was screened for compounds 1-6.The test was carried out by room temperature and measured the toxicity influence of the compounds.The compounds 1, 2, 4 and 5 were reduced to 100% mortality at (40 µg/mL).The compounds 3, 6 have very low LD 50 value compared with other compounds.The compound 1-6 shows that LD 50 values 19.96, 21.79, 8.72, 25.87, 26.22 and 12.30μg/mL respectively.The values are summarized in Table 2. Brain shrimp cytotoxic activity of the compounds 1-6 shows in figure 2 at concentration (10-40) μg /mL. (5,6) Scheme 2. Synthetic route of the compound (5 and 6).

CONCLUSION
The synthesized compounds 1-6 were screening the environmental biotoxicity of Brine shrimp cytotoxicity, Ichthyotoxicity profile and insecticidal activity of Larvicidal, Nematicidal activity.The compounds 3, 6 was highly toxic against all bioassays against Brain shrimp cytotoxic, Antifeedant, Larvicidal and Nematicidal screening.Such observation was consistent with the results about the sulfur containing heterocyclic compounds 3 and 6 are highly toxic and good activity against all bioassays.These findings demonstrate that the environmental biotoxicity represent a new template for future studies.
Value were the means of three replicates ± SD.