Indolylquinoline derivatives are cytotoxic to Leishmania donovani promastigotes and amastigotes in vitro and are effective in treating murine visceral leishmaniasis

Ganes Chakrabartia, Anirban Basua, Partha Pratim Mannaa, Sashi Bhusahan Mahatob, Nirup Bikash Mandalb and Santu Bandyopadhyaya,*

a Cellular Immunology Laboratory b Steroid and Terpenoid Chemistry Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A wide variety of biologically active compounds contain indole and quinoline nuclei. Some novel indolylquinoline derivatives were synthesized from indole by Friedel-Crafts acylation reaction. Out of the four derivatives tested, 2-(2''-acetamidobenzyl)-3-(3'-indolylquinoline) (C) had no effect on the promastigotes or amastigotes of Leishmania donovani in vitro. The remaining three analogues, 2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A), 2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), and 2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D), inhibited the growth of L. donovani promastigotes in vitro and were cytotoxic to both the promastigote and amastigote forms of the parasite. These three derivatives were also effective in eliminating L. donovani amastigotes from BALB/c mouse peritoneal macrophages in vitro. One indolylquinoline derivative [A] was used to treat established visceral leishmaniasis in BALB/c mice. This compound was significantly more effective than sodium antimony gluconate (SAG) in reducing the splenic parasite load at a much lower concentration (5% of SAG). Our results suggest that indolylquinoline derivatives may be exploited as antileishmanial agents.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Leishmaniasis is a global health problem. Infection by various species and strains of Leishmania causes a wide spectrum of disease in humans, with many different clinical presentations. The severity of the disease is largely dictated by the immunological status of the infected individual and by the species of Leishmania involved. Approximately 350 million people in 80 countries are estimated to be threatened by the disease. 1 The World Health Organization (WHO) estimated 12 million cases of leishmaniasis worldwide, with over 400,000 new cases each year. 2 Leishmania spp. exists in two morphologically distinct forms: a motile flagellated form (promastigotes) and an intracellular non-flagellated form (amastigotes). The promastigotes are ingested by mononuclear phagocytes of the host, where they transform into immotile amastigotes, multiply, rupture the host cell, and then invade other cells.3

The visceral form of leishmaniasis, commonly known as kala-azar, is caused by the parasite Leishmania donovani and is often fatal. Despite tremendous progress made in understanding the biochemistry and molecular biology of Leishmaniaspp., treatment by chemotherapy has seen very little progress in recent years. The toxic pentavalent antimonials remain the mainstay of treatment for leishmaniasis. The second line drugs, pentamidine and amphotericin B, although used clinically, have serious toxic side effects. 4 Therefore, improved drug therapy for leishmaniasis remains desirable.

The indole and quinoline nuclei are prevalent in a wide variety of biologically active compounds. Some indole and quinoline derivatives have been reported to possess antileishmanial activity.5,6,7,8,9 A one-step synthesis of some novel indolylquinoline derivatives has been developed using indole as the substrate under Friedel-Crafts acylation reaction conditions. 10 We investigated the antileishmanial activity of these indolylquinoline derivatives both in vitro and in vivo; the results are described in this communication.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of indolylquinoline derivatives

Four indolylquinoline derivatives, 2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A), 2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), 2-(2''- acetamidobenzyl)-3-(3'-indolylquinoline) (C) and 2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D) (Figure 1), were prepared as described.10 The reaction was carried out with indole (3–4 mol) as substrate, acylchloride (1 mol), and anhydrous aluminium chloride (AlCl3, 1.5 3–2 mol). The substrate was dissolved in nitrobenzene, cooled to 15–20°C, and the catalyst was added in gradual increments. The acylating agent was then added slowly with constant stirring. The reaction mixture was kept at 25°C for 1 h, warmed to 85°C for 4 h, and then kept overnight at room temperature. The reaction mixture was treated with an ice-HCl (1:1) mixture, neutralized with sodium bicarbonate (NaHCO3) solution, and extracted with chloroform (CHCl3). The extract was evaporated under reduced pressure. The concentrated gummy mass was adsorbed with silica gel and subjected to column chromatography. Stock solutions of indolylquinoline derivatives were made in dimethyl sulphoxide (DMSO) at 10,000 mg/L and further dilutions were made in medium containing 8% fetal bovine serum (FBS) immediately before use. In all experiments, the final concentration of DMSO was <=0.1% (v/v) and was nontoxic to promastigotes and amastigotes of L. donovani, and to human peripheral blood mononuclear cells (PBMC).



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Figure 1. Structure of indolylquinoline derivatives: 2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A), 2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), 2-(2''- acetamidobenzyl)-3-(3'-indolylquinoline) (C) and 2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D).

 
Parasite

L. donovani strain AG83 was originally obtained from an Indian kala-azar patient 11 and maintained in golden hamsters. Amastigotes were isolated from spleens of L. donovani infected golden hamsters as described. 12 The spleen was rinsed in ice cold phosphate-buffered saline (PBS)-glucose (55 mM)/EDTA (2 mM), then lightly homogenized, macroscopic particles were allowed to settle, and the turbid suspension was decanted. This suspension was centrifuged at 100g for 10 min at 4°C. The amastigote-enriched suspension was centrifuged at 800g for 10 min. The pellet was suspended in 45% Percoll (8.0 mL), and finally 25% Percoll (4.0 mL) was layered over the amastigote suspension and centrifuged at 5000g for 1 h. The band containing amastigotes was taken and washed with PBS (x3), and finally resuspended in Medium-199 (Gibco Laboratories, New York, NY, USA) supplemented with 20% FBS. Promastigotes were obtained by transforming amastigotes and were maintained in vitro in Medium-199 supplemented with 8% FBS.

Isolation of normal human peripheral blood mononuclear cells (PBMC)

PBMC were separated from heparinized whole blood of normal donors by Ficoll-Hypaque density gradient centrifugation as described.13

In-vitro growth of L. donovani promastigotes in the presence of indolylquinoline derivatives

Promastigotes (1 x 106) were incubated with or without various concentrations of indolylquinoline derivatives or standard antileishmanial drugs in Medium-199 (1.0 mL) supplemented with 8% FBS at 22°C. Growth of promastigotes was monitored by counting the number of motile promastigotes microscopically.

Cytotoxicity assays

In-vitro grown promastigotes, freshly purified amastigotes, or normal human PBMC (2 x 106 of each) were washed and resuspended in Medium-199 (1.0 mL) containing 8% FBS. Fifty microcuries of sodium chromate-51Cr (Na 251CrO4, BARC, Bombay, India) were added to promastigotes or purified amastigotes and incubated for 6 h at 22°C. Normal human PBMC were incubated with Na251CrO4 for 2 h at 37°C with gentle shaking every 15 min. Radiolabelled promastigotes, amastigotes, or normal human PBMC were washed (x4) and resuspended in Medium-199 supplemented with 8% FBS at a concentration of 1.0 x 106/mL. Indicated concentrations of indolylquinoline derivatives or sodium antimony gluconate were added to radiolabelled promastigotes or amastigotes (0.5 x 106) in a total volume of 1.0 mL in 5.0 mL tubes, and to radiolabelled normal human PBMC (0.2 x 105) in a total volume of 0.2 mL in microtitre plates. After 18 h of incubation at 37°C, promastigotes or amastigotes were centrifuged and 0.5 mL aliquots of cell-free supernatants were collected and counted in a gamma counter. For normal human PBMC, 0.1 mL of supernatants were collected from each well without disturbing the cell pellet. The percentage 51Cr release was calculated using the formula:

where CPM denotes counts per minute, the spontaneous release was that obtained from promastigotes, amastigotes, or normal PBMC incubated with medium alone, the experimental release was that obtained from these cultures incubated with indolylquinoline derivatives or sodium antimony gluconate, and the total release was that obtained from these cultures incubated with 1 N HCl. In 18 h assays, the spontaneous release never exceeded 30% of the total release from either form of the parasite or normal human PBMC.

In-vitro infection of BALB/c peritoneal macrophages

Thioglycolate-elicited peritoneal exudate was used as the source of macrophages for better recovery and easier isolation. Approximately 2.5 x 105 macrophages were allowed to adhere to glass coverslips (20 mm x 25 mm) in RPMI-1640 (Gibco Laboratories) supplemented with 10% FBS and cultured for 5–7 days at 37°C in 5% CO2 before in-vitro infection with L. donovani. Stationary phase L. donovani promastigotes (5.0 x 106) were added to each coverslip and incubated for 6 h at 37°C in 5% CO2.

Determination of antileishmanial activity of indolylquinoline derivatives on L. donovani infected BALB/c macrophages in vitro

Coverslips were washed (x3) with 10% FBS-supplemented RPMI-1640 to remove uningested parasites and incubated for 2 days in the presence or absence of graded concentrations of indolylquinoline derivatives, pentamidine, amphotericin B (Sigma Chemical Co., St Louis, MO, USA), and sodium antimony gluconate (Gluconate Health Ltd, Calcutta, India). Infected macrophage cultures were washed with PBS, fixed with prechilled methanol, stained with Giemsa, and examined microscopically under oil immersion. At least 400 target macrophages were examined for each coverslip. Antileishmanial activity was determined by calculating the number of amastigotes per 100 macrophages.

Determination of antileishmanial activity of indolylquinoline derivatives in vivo

BALB/c mice (45–50 days old) were injected in vivo with freshly transformed promastigotes of L. donovani (2 x 107/mouse). Therapy with indolylquinoline derivative A or sodium antimony gluconate started 1 month after infection. Indolylquinoline derivative was used orally (12.5 mg per kg body weight). Each mouse received a total of 21 oral administrations every day. Sodium antimony gluconate was injected im (250 mg/kg body weight). Each mouse received a total of ten injections every alternate day. Mice in the control group received PBS by oral feeding every day for 21 days. Mice in all groups were killed 2 weeks after the last treatment. The splenic parasite load was determined from impression smears after Giemsa staining. Results are expressed as the total parasite load per spleen, using the formula:


Statistical analysis

Statistical analyses were performed by Student's t-test with the program Tadpole III (written by T. H. Caradoc-Davies, Wakari Hospital, Dunedin, New Zealand; published and distributed by Biosoft, Cambridge, UK).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of indolylquinoline derivatives on the growth of L. donovani promastigotes in vitro

L. donovani promastigotes can be grown in vitro at 22°C. Growth of the promastigotes peaked within 4–6 days of culture (Figure 2). To determine whether any of the indolylquinoline derivatives had any effect on the promastigotes, various concentrations of these derivatives (dissolved in DMSO) were added to the promastigote cultures individually. DMSO had no effect on the growth of L. donovani promastigotes in vitro at a final concentration of 0.1% (v/v). Out of four indolylquinoline derivatives tested, derivative C[2-(2''-acetamidobenzyl)-3-(3'-indolylquinoline)] had no appreciable effect on the growth of L. donovani promastigotes in vitro at concentrations as high as 10 mg/L (Figure 2). This derivative was also ineffective at lower concentrations, i.e. 1.0 mg/L to 5.0 mg/L. By contrast, each of the remaining three indolylquinoline derivatives (derivatives A, B, D) was found to inhibit the growth of L. donovanipromastigotes in vitro. The degrees of inhibition by derivatives A, B and D were very similar. Each of these three derivatives at a concentration of 10 mg/L inhibited the growth of L. donovani promastigotes by approximately 98% on the second, fourth, or sixth day of culture (P < 0.0001 for each of the indicated derivatives). A lower concentration (5.0 mg/L) of these derivatives was also growth inhibitory to L. donovani promastigotes. This concentration of derivative A, B or D inhibited promastigote growth by approximately 70% on both the second and fourth days of culture (P < 0.001 for each comparison) and by 40% on the sixth day (P < 0.03 for each comparison). However, these indolylquinoline derivatives at a final concentration <=2.5 mg/L had no effect on the growth of L. donovani promastigotes (Figure 2 A–D). For comparison, two additional compounds (pentamidine and amphotericin B), usually used as second-line antileishmanial drugs, were included as positive controls. Both pentamidine and amphotericin B at a final concentration >=2.5 mg/L inhibited the in-vitro growth of L. donovani promastigotes almost completely (Figure 2). At a final concentration of 1.0 mg/L, pentamidine inhibited promastigote growth poorly, and the differences were not significant. However, amphotericin B at the same concentration (1.0 mg/L) inhibited promastigote growth by 28.8% on the second day, 67.4% on the fourth day (P < 0.001), and 43.8% on the sixth day of culture (P < 0.03).



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Figure 2. Effect of indolylquinoline derivatives A–D, pentamidine, and amphotericin B on the growth of L. donovani promastigotes in vitro. Data are show for one representative experiment out of three with similar results. ({circ}), 10.0 mg/L; ({square}), 5.0 mg/L; (•), 2.5 mg/L; ({blacksquare}), 1.0 mg/L; ({blacktriangleup}), 0.1% DMSO; ({triangleup}), control medium.

 
Indolylquinoline derivatives are cytotoxic to promastigotes and amastigotes of L. donovani in vitro

To examine whether indolylquinoline derivatives were cytotoxic to the parasite, 51Cr-release assays were performed with radiolabelled promastigotes and amastigotes of L. donovani in the presence of varying concentrations of these derivatives. The antileishmanial drug sodium antimony gluconate (SAG) was used for comparison and normal human PBMC were used as a control for nonspecific toxicity. As shown in Figure 3, DMSO up to a concentration of 0.1% (v/v) was nontoxic to human PBMC or L. donovani promastigotes. However, DMSO was found to be toxic to purified amastigotes of L. donovaniin a dose-dependent manner. Neither any of the indolylquinoline derivatives tested (A, B, C or D) nor SAG was toxic to human PBMC. The three derivatives other than indolylquinoline derivative C were significantly cytotoxic to both promastigotes and amastigotes of L. donovani. The cytotoxicity of derivative C was only marginal over DMSO on L. donovani amastigotes and was not tested on promastigotes. SAG was nontoxic to both promastigotes and amastigotes up to a concentration of 10 mg/L (not shown). However, higher concentrations of this drug (250–500 mg/L) were cytotoxic to both forms of the parasite (Figure 3).



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Figure 3. In-vitro cytotoxicity of indolylquinoline derivatives and sodium antimony gluconate on cultured promastigotes, purified amastigotes of L. donovani, and normal human peripheral blood mononuclear cells. 18 h 51Cr-release assays were performed as described in Materials and methods. Mean ± S.D. of triplicate determinations. *P<0.0001. **P<0.001. ***P<0.002.

 
Indolylquinoline derivatives are effective in destroying intracellular amastigotes of L. donovani in vitro

The protozoan parasite L. donovani survives and multiplies within mammalian macrophages. It is therefore of interest to test the efficacy of these indolylquinoline derivatives on intracellular amastigotes. Peritoneal macrophages of BALB/c mice were infected in vitro with L. donovani and then incubated with graded concentrations of these agents. SAG, pentamidine, and amphotericin B were used as positive controls. As shown in Figure 4, SAG was effective in reducing intracellular parasite burden only at a high concentration (500 mg/L). The other two known antileishmanial compounds tested, pentamidine and amphotericin B, were both effective at lower concentrations. At a concentration as low as 1.0 mg/L, pentamidine reduced the intracellular parasite load by 70%. Amphotericin B was even more effective at this concentration (90% reduction of intracellular parasite load). Of the four indolylquinoline derivatives, derivative C had no appreciable antileishmanial activity. Derivatives A and B were almost equally effective. Each of these derivatives had no appreciable effect on intracellular amastigotes at 1.0 mg/L but higher concentrations were effective. Antileishmanial activity of derivative D was not detectable up to 2.5 mg/L; however, higher concentrations of this derivative significantly reduced the intracellular amastigote load (Figure 4). Giemsa-stained micrographs of L. donovaniinfected BALB/c peritoneal macrophages incubated with indolylquinoline derivatives or sodium antimony gluconate are shown in Figure 5.



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Figure 4. In-vitro antileishmanial activity of indolylquinoline derivatives, sodium antimony gluconate, pentamidine, and amphotericin B on L. donovani infected BALB/c peritoneal macrophages. Mean ± S.D. Of three separate experiments. *P<0.001. **P<0.003. ***P<0.005. ****P<0.01.

 






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Figure 5. Giemsa-stained micrographs of L. donovani infected BALB/c peritoneal macrophages after incubation for 2 days at 37°C, 5% CO2 in medium containing 0.1% (V/v) DMSO (a), indolylquinoline derivative A (b), indolylquinoline derivatives B (c), indolylquinoline derivative D (d), or sodium antimony gluconate (e). Each of the indicated indolylquinoline derivatives was used at a final concentration of 5 mg/L. Sodium antimony gluconate was used at 500 mg/L. Magnification x1000. Note the presence of healthy intracellular amastigotes in cultures incubated with media (containing DMSO), and the degraded intracellular amastigotes in cultures incubated with indolylquinoline derivatives or sodium antimony gluconate.

 
Treatment of established murine visceral leishmaniasis with an indolylquinoline derivative

BALB/c mice were infected with L. donovani as described above. After 1 month, groups of four or five mice were treated with PBS, SAG, or indolylquinoline derivative A. The parasite load in the spleen was then determined as described. 14 As shown in the Table, indolylquinoline derivative A was significantly more effective than sodium antimony gluconate (P < 0.01) in reducing the splenic parasite load even at a much lower concentration. SAG therapy at a dose of 250 mg per kg body weight reduced the splenic parasite load by 56%. On the other hand, indolylquinoline derivative A reduced the parasite load by 86% at a concentration as low as 12.5 mg per kg body weight.


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Table. Treatment of established murine visceral leishmaniasis with indolylquinoline derivative A: comparison with sodium antimony gluconate
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Indolylquinoline derivatives synthesized in our laboratory were evaluated for antileishmanial activity both in vitro and in vivo. In-vitro activity was tested on cultured promastigotes, purified amastigotes of a pathogenic strain of L. donovani, and intracellular amastigotes of L. donovani infected mouse peritoneal macrophages. In-vitro studies were extended in vivo in L. donovani infected BALB/c mice with one indolylquinoline derivative (derivative A).

Of four indolylquinoline derivatives tested, three had significant antileishmanial activity on cultured promastigotes, purified amastigotes, and intracellular amastigotes of L. donovani in vitro. None of these indolylquinolines was toxic to normal human cells. These indolylquinolines were less active than pentamidine or amphotericin B but were significantly more effective antileishmanial agents than sodium antimony gluconate in vitro. Our in-vivo studies with one indolylquinoline derivative in L. donovani infected BALB/c mice are encouraging. Sodium antimony gluconate was used as a positive control for in-vivo studies, and this drug partially reduced the splenic parasite load. These results are in agreement with a previous report on Leishmania majorinfection. 15 The indolylquinoline derivative was significantly more effective than sodium antimony gluconate in reducing the splenic parasite load at a much lower concentration.

A wide variety of biologically active compounds contain indole or quinoline nuclei. Some indole or quinoline derivatives have been reported to possess antileishmanial activity. 5,6,7,8,9,16 We report here that some indolylquinoline derivatives have significant antileishmanial activity both in vitro and in vivo. A recent report indicated that indolylquinoline derivatives inhibit catalytic activities of both type I and type II topoisomerases of L. donovani and that Leishmania topoisomerases are more susceptible to these agents.17 Inhibition of Leishmania topoisomerases may explain, at least in part, the antileishmanial activity of indolylquinoline derivatives, and these compounds may be exploited as antileishmanial agents.


    Acknowledgments
 
This work was supported by the Council of Scientific and Industrial Research, Departments of Science and Technology and Biotechnology, Government of India. We also Thank H. N. Dutta, S. Sahu and D. Das for the art work.


    Notes
 
* Corresponding author. Fax: +91-33-473-5197/0284. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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15 . Nabors, G. S. & Farrell, J. P. (1994). Depletion of interleukin-4 in BALB/c mice with established Leishmania major infections increases the efficacy of antimony therapy and promotes Th1-like responses. Infection and Immunity 62, 5498–504.[Abstract]

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17 . Ray, S., Sadhukhan, P. K., Mandal, N. B., Mahato, S. B. & Majumder, H. K. (1997). Dual inhibition of DNA topoisomerases of Leishmania donovani by novel indolyl quinolines. Biochemical and Biophysical Research Communications 230, 171–5.[ISI][Medline]

Received 20 May 1998; returned 5 August 1998; revised 23 September 1998; accepted 12 October 1998