Effect of Some Synthesized Pyrrolidines in Growth of L. infantum Promastigotes

I n this research, three pyrrolidine compounds (P1-P3) were synthesized and then tested for efficacy against L. infantum promastigotes in vitro. The study included preparation of some chalcones and schiff bases then the condensation of both to get the pyrrolidines and studying the effect of pyrrolidine compounds in growth, generation number and time of parasites. They determined using concentrations between (5-30) μg/ml. The effect of the compounds was found to be important to the parasite growth as the more the concentration the less the growth, generation number and increased the generation time. At the logarithmic phase, the LD 50 were (10), (20) and (30)μg/ml for (P1-P3) compounds, respectively.


Introduction
Leishmaniasis is a poverty-associated disease with several different forms, of which the two visceral and cutaneous leishmaniasis are most common.Visceral form is fatal without treatment while the cutaneous infections has a spectrum of presentations, typically with self-healing or chronic lesions on the skin.Leishmaniasis results from infection by various species of Leishmania, approximately 30 species have been described and at least 20 of these organisms are pathogenic for mammals.Human visceral leishmaniasis is primarily caused by L. donovani, L. archibaldi, L. infantum, and L. chagasi.L. infantum that cause cutaneous and visceral infections it, which mainly causes visceral leishmaniasis in people, can cause both visceral and cutaneous disease in dogs, and primarily causes skin lesions in cats and horses [1].Leishmania spp. is digenetic organisms shuttling between a flagellated-promastigote in the mid-gut of the sand fly vector and an intracellular-amastigote in the mammalian host.Sand flies are blood feeders and the infectious promastigotes are transmitted during a blood feeding meal.Promastigotes attach to mononuclear phagocytes and are taken up by phagocytosis into a phagosome, which fuse with lysosomes to form the phagolysosome.Once inside the macrophage, promastigotes undergoes biochemical and metabolic changes and differentiate to the obligatory intracellular form of the parasite.Amastigotes are released from macrophages and can re-invade dendritic and fibroblasts cells as well as new macrophages [2].Leishmania parasites are spread in Iraq, observed in several provinces and considered one of the areas affected by visceral and cutaneous leishmaniasis [3].From decades ago, there is no effective drug to treat leishmaniasis, there are 25 compounds or chemical formula showed effectiveness against leishmania but a few of these compounds proved its worth.None of the available drugs can be considered ideal due to their high toxicity, higher prices, long duration of treatment, and severe adverse reactions, which often lead to treatment abandonment.Pentavalent antimony compounds have been the firstline drugs for treatment of leishmaniasis, which are available in two formulations (Pentostam, C 12 H 38 O 26 Sb) and (Glucantime, C 14 H 29 O 10 N 2 Sb).The second line drug for the treatment is Amphotericin B (C 47 H 73 NO 17 ) as well Miltefosine, Paramomycine and Pentamidine but none of them are effectiveness [4].Therefore, there is a need to develop or use new compounds.The compounds that have attracted the attention of many researchers are heterocyclic compounds, which are among the most important types of organic chemistry.They are characterized by containing one or several atoms in their molecular structure, at least one atom other than carbon.Heterocyclic compounds build up by 3 to 6 rings, the most common consist of 3 or 6 rings which include heteroatoms such as nitrogen, oxygen or sulfur.They synthesize pyrrolidine compounds with formula C 5 H 9 N.These compounds are cyclic secondary amine containing five heterogeneous rings, four carbon atoms and one nitrogen.The five-heterogeneous system is considered the most important part of these compounds, many of them have been shown biological activity as antibacterial [5], antifungal [6], anticancer [7], antitumor [8] and antiviral [9].Thus, the current research drives at the synthesis of some five rings compounds (pyrrolidines) and study their effects in the growth of L. infantum promastigote.

Preparation of Chalcones (1,3-Diphenyl prop-2ene-1-one)
Chalcones were prepared according to [10].A mixture of 2.2 gm (0.05 mole) of sodium hydroxide pellets, (20) ml of water and (12.5) ml of ethanol was magnetically stirred in a (100) ml round-bottomed flask which immersed in an ice-bath.A (0.043 mole) of freshly distilled of appropriate acetophenone was poured on the stirred mixture followed by (0.043 mole) of substituted benzaldehyde.The temperature of the mixture was kept at (20-25) C° with a vigorous stirring for three hrs.until they become thick, and then kept in a refrigerator overnight.The product was filtered and washed until the neutralization of filtrates, then washed with (20) ml of cold-ethanol.After drying the crude chalcone in air, it was recrystallized from ethanol.Some physical properties data were illustrated in Table (1).

Synthesis of pyrrolidines
These compounds were prepared from a mixture of (0.01) mole schiff-base with (0.01) mole of chalcone.The mixture was magnetically stirred then, (10) ml of ethanol, (3) ml 50% sodium hydroxide added `and the stirring continues for one hour at room temperature.The mixture was kept overnight, and then add (100) ml of water.The product filtered and washed, after drying it was recrystallized by methanol-ethyl acetate [12] as shown in Table (3).

Comp
No.

B. Cultivation of parasites:
1.9 ml of liquid phase was added to McCantry vials that contained 5 ml solid phase slants, then 0.1 ml of promastigotes inoculums was taken from stock culture during log phase, so that the initial density of the organism was 2 x 10 5 / ml, then the organisms of new culture incubated at 25 C o for 5 days, was counted using a haemocytometer.

C. Effect of the pyrrolidines on growth, number of generation and generation time of L. infantum promastigotes:
Effect of the pyrrolidines on growth was studied in vitro in comparison with untreated groups.The compound was dissolved in 2% DMSO.Five concentrations (5,10,15,20,30) μg / ml were used to determine the (LD 50 ) of the cultivated organisms.Numbers of promastigotes were determined at (72,120) hrs.time interval, then generation number and time were estimated according to [14].

Statistical Analysis
The statistical program (SPSS) version (21) was used to analyze the data [15].
The previous discussion of UV, IR and NMR data was applied on the chains (schiff bases and pyrrolidines) as shows in Tables ( 6), ( 7), ( 8) and (9).Spectral data was mentioned to the compatible with the suggested structures of chalcones, schiff bases and Pyrrolidines.
No. shows in Tables ( 6) and ( 7) by nucleophilic addition of primary amine on aldehyde or ketone carbonyl atom to form carbinol amine with loses of H 2 O molecule [20].

Biological
Effect of synthesized pyrrolidines on parasite growth Table (10), (11) and (12) show the inhibitory effect of different concentrations of the synthesized pyrrolidines on L. infantum growth in comparison with control group during different (72) and (120) intervals.( 10), ( 20) and (30) µg / ml seemed to be the 50% inhibitory-concentration of the promastigotes (LD 50 ) at the log phase (120) hrs. of the compounds P1, P2 and P3 respectively.On the other hand, the highest concentrations inhibit more than 80% of growth.4.60 ± 0.05 a 64 12.7 ± 0.15 a 73 * 3-replicates were used for each treatment.Mean and Standard Error were multiplied x 10 6 .** According to Duncan-test, different letter refers to presence of significant differences between treatments at P ≤ 0.05.

Table (12): Effect of different concentrations of P3 on L. infantum promastigote numbers
* 3-replicates were used for each treatment.Mean and Standard Error were multiplied x 10 6 .** According to Duncan-test, different letter refers to presence of significant differences between treatments at P ≤ 0.05.These results which is agreement with [21] used the pyridine derivatives at the concentration that kills 50% of L. tropica parasites, they demonstrated that some of these compounds had an inhibitory effect in concentration lower than used with Glucantime which was used to treatment these parasites.Other researchers used low molecular weight compounds as antileishmanial agents.For example, [22] which indicated an inhibitory effect of amide compounds, that extracted from Paper amalago and their synthetic analogs on L. amazonensis in LD 50 at logarithmic phase.Also [23] used both piperzine and pyrrolidine compounds and their derivatives, they explained that five of these compounds at (10) mM concentration had given an effective inhibitory in reducing growth of Plasmodium falciparum parasites by killing 50% of these parasites, as well as they mentioned that these compounds are easily intestinal absorbed.In addition, other researchers tried to use the five ring thiohydantions derivatives as antitrypanosomal, [24] indicated that these compounds gave a disincentive effect on growth and gave 100% cure rate in mice infected with parasites after 4 days of oral treatment at 50 mg/kg twice per day.It is worthy to mention [25,5] confirmed that some pyrrolidines synthesis and their derivatives are effective as antibacterial.

Effect of synthesized pyrrolidines on parasite generation number and time
On the light of the previously growth indices, effect of the pyrrolidines on generation number and generation time of L. infantum promastigotes were estimated.As for the effect of different concentrations of the pyrrolidines on generation number Table (13) inverse correlation between generation number and concentration were observed, generation number at log phase ranged from (5.92) at 5.0 µg to (2.59) at 30 µg, when compared with control (6.36 generations) of P1, also ranged from (7.54) at 5.0 µg to (5.98) at 30 µg, when compared with control (7.89 generations) of P2 and ranged from (7.60) at 5.0 µg to (6.83) at 30 µg, when compared with control (7.82 generations) of P3.  32.5 ± 0.01 a 46.3 ± 0.04 a 15.9 ± 0.05 a 20.0 ± 0.05 a 15.0 ± 0.08 a 17.5 ± 0.17 a * 3-replicates were used for each treatment.Mean and Standard Error were multiplied x 10 6 .** According to Duncan-test, different letter refers to presence of significant differences between treatments at P ≤ 0.05.This results agree with [26] they proved that L. tropica parasites when treated with 2.5 μg / ml of 3,4dihydro-4-(p-anisyl)-6-phenyl pyrimidine -2(1H)-one at the lethal concentration 50% reduced one generation number and increased generation time by 2.5 hrs., and the use of the lethal concentration 90% led to reduction 5 generations and increased the generation time to about 40 hrs.[27] used some synthesized pyrazoline and pyrimidine at IC 50 concentration, which resulted in a reduction of 0.5-1.5 generation and increasing generation time by about 1-5 hrs.The synthesized pyrrolidines exhibited relatively high antileishmanial activity.However, the mechanism by which the pyrrolidines killed the parasite is not known.The mechanism of kills may be due to the affected of compounds on cell membrane through effects on osmosis, this will alter the permeability of the cell and may be effect on protein and nucleic acids metabolism.The difference in effect between the compounds might be due to substituted group, for example: nitro group, dimethyl and methylene as well the aromatic ring effect.Thus there is a need for test the toxicity of the compounds and subsequent biochemical studies to determinate the molecular location that may targeted by this antileishmanial agent.

Table ( 10): Effect of different concentrations of P1 on L. infantum promastigote numbers
-replicates were used for each treatment.Mean and Standard Error were multiplied x 10 6 .** According to Duncan-test, different letter refers to presence of significant differences between treatments at P ≤ 0.05.
** According to Duncan-test, different letter refers to presence of significant differences between treatments at P ≤ 0.05.