Abstract
Treatment of leishmaniasis by chemotherapy remains a challenge because of limited efficacy, toxic side effects, and drug resistance. We previously reported that synthetic flavonoid dimers have potent antipromastigote and antiamastigote activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Here, we further investigate their leishmanicidal activities against cutaneous Leishmania species. One of the flavonoid dimers (compound 39) has marked antipromastigote (50% inhibitory concentrations [IC50s], 0.19 to 0.69 μM) and antiamastigote (IC50s, 0.17 to 2.2 μM) activities toward different species of Leishmania that cause cutaneous leishmaniasis, including Leishmania amazonensis, Leishmania braziliensis, Leishmania tropica, and Leishmania major. Compound 39 is not toxic to peritoneal elicited macrophages, with IC50 values higher than 88 μM. In the mouse model of cutaneous leishmaniasis induced by subcutaneous inoculation of L. amazonensis in mouse footpads, intralesional administration of 2.5 mg/kg of body weight of compound 39.HCl can reduce footpad thickness by 36%, compared with that of controls values. The amastigote load in the lesions was reduced 20-fold. The present study suggests that flavonoid dimer 39 represents a new class of safe and effective leishmanicidal agent against visceral and cutaneous leishmaniasis.
INTRODUCTION
Leishmaniasis is a serious parasitic disease found in 98 countries in 5 continents, causing 20,000 to 40,000 deaths per year (1). The disease has three main types of manifestations, i.e., visceral, cutaneous, and mucocutaneous. Cutaneous leishmaniasis (CL) has an occurrence rate of 0.7 to 1.2 million cases per year (1). The most affected countries include Afghanistan, Algeria, Iran, and Brazil. CL usually induces skin ulcers on exposed parts of the body. If left untreated, CL leaves permanent scars and may cause disfigurement. This disease is widespread in both the Old World and the New World (2). Currently, there is no human vaccine available, and chemotherapy is the major approach for treatment of leishmaniasis (3). Pentavalent antimonial compounds such as Pentostam or Glucantime are first-line antileishmanial agents that have been used clinically for over 50 years (4, 5). Antimonial treatment is far from satisfactory, however, due to the need for intramuscular administration and long treatment periods, side effects, and the emergence of antimonial-resistant cases (4, 6, 7). Second-line drugs such as amphotericin B and pentamidine may be toxic and expensive (5). The new orally administered drug miltefosine is highly efficacious but suffers from problems such as the need for long treatment periods, potential teratogenicity, and long residence times in patients, which can potentially lead to drug resistance. There is an urgent need for novel, cheaper, potent, and safe antileishmanial compounds for treatment of leishmaniasis.
Plant-derived natural products such as flavonoids have been reported to have a wide range of biological activities, such as antioxidative and anticancer effects (8, 9). They have been a good source for discovering new antileishmanial agents (10–12). Previously, we demonstrated that synthetic flavonoid dimers can inhibit the pumping activity of ATP-binding cassette (ABC) transporters, resulting in increases in intracellular drug accumulation and thereby reversing drug resistance in both cancer and Leishmania (13–18). Furthermore, some of the flavonoid dimers were found to have potent antipromastigote and antiamastigote activity toward Leishmania donovani, which causes a fatal visceral form of leishmaniasis. The 50% inhibitory concentration (IC50) values were around 0.2 μM for promastigotes and 0.6 μM for amastigotes (19).
In the present study, we further demonstrate that flavonoid dimers have significant antileishmanial activity in vitro against several species of Leishmania that cause CL. For one particularly active compound, flavonoid dimer 39, antipromastigote and antiamastigote activities were studied and results were compared with those for known antileishmanial agents in terms of therapeutic index values. Finally, the in vivo efficacy of compound 39.HCl against cutaneous leishmaniasis in a mouse model was also demonstrated.
MATERIALS AND METHODS
Chemicals.
The flavonoid dimers 1 to 3 and 5 to 30 and the aminoethylene glycol-linked flavonoid dimers 31 to 50, 53, 56 to 61, and 68 to 73 were prepared according to the reported procedures, and their chemical structures are shown in Fig. 1 (13–15, 19). The HCl salt of compound 39 was prepared by adding excess 35% concentrated hydrochloric acid to a solution of compound 39 in chloroform. After stirring for 30 min, the mixture was evaporated to dryness under high vacuum to afford compound 39.HCl salt as a brownish solid. The purity of tested compounds was determined by high-performance liquid chromatography (HPLC), which was performed by using an Agilent 1100 series HPLC system with an analytical Agilent Prep-Sil Scalar column (4.6 mm by 250 mm; pore size, 5 μm) with isocratic elution with hexane (50%)-ethyl acetate (25%)-methanol (25%), at a flow rate of 1 ml/min, and UV detection at 320 nm (reference at 450 nm). All tested compounds were shown to have >95% purity according to HPLC. Pentamidine and amphotericin B were purchased from Sigma. Miltefosine was from Cayman (Ann Arbor, MI). Sodium stibogluconate (SSG) was a generous gift from GlaxoSmithKline (United Kingdom).
FIG 1.
Chemical structures of previously reported flavonoid dimers 1 to 73 and other antileishmanial compounds used in this study. PEG 600, with an average number of EG units of 13, was used to synthesize compound 15.
Cell lines and cell culture.
Promastigotes of cutaneous Leishmania (Leishmania major 50122 [MHOM/IL/67/JERICHO II (Lm50122)] from ATCC, Leishmania amazonensis LV78, Leishmania braziliensis UA847, L. major Friedlin V1 [FV1], and Leishmania tropica EP41) were employed in this study. The last four species were kindly provided by K. P. Chang. All species were cultured at 27°C for 4 days in Schneider's Drosophila medium (pH 6.9) (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) (HyClone) with 4 mM glutamine (Sigma) and 25 μg/ml gentamicin (Invitrogen) (20).
In vitro antipromastigote activity.
Antipromastigote activity was determined according to procedures described previously (16), with the CellTiter 96 AQueous assay (Promega) employing a tetrazolium compound. Promastigotes were seeded into 96-well flat-bottomed microtiter plates at 1 × 105 cells per well, in a final volume of 100 μl medium, and were incubated with a series of concentrations of synthetic flavonoid dimers or known antileishmanial agents. Parasites were incubated at 27°C for 72 h. After 72 h of incubation, 10 μl of a mixture of MTS [2-(4,5-dimethylthiazol-2-yl)-5-[3-(carboxymethoxy)phenyl]-2-(4-sulfophenyl)-2H-tetrazolium] and PMS (phenazine methosulfate) (both purchased from Sigma) was added to each well of the microtiter plate. The plate was then incubated at 27°C for 4 h for color development. After 4 h of incubation, the optical density (OD) values were determined at 490 nm using an automated microtiter plate reader (Bio-Rad).
In vitro antiamastigote activity.
Mouse peritoneal elicited macrophages (PEMs) were obtained as described previously (18). A round coverslip (12 mm in diameter) was placed into each well of a 24-well culture plate. Mouse PEMs were resuspended in supplemented Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated FBS (vol/vol), 100 U/ml penicillin, and 100 μg/ml streptomycin and were seeded into each well at a cell density of 1 × 105 cells per 500 μl. Macrophages were allowed to attach overnight. Nonadherent cells were removed by two gentle washes with unsupplemented DMEM. Adherent macrophages were infected with late-log-phase promastigotes, at a parasite/macrophage ratio of 20:1, overnight at 37°C in 5% CO2. Noninternalized promastigotes were removed by two washes with unsupplemented DMEM. Infected macrophages were further incubated in 500 μl of supplemented DMEM, in the presence or absence of flavonoid dimers or known antileishmanial agents, for 72 h at 37°C. After incubation, coverslips were stained with Giemsa, and the percentages of macrophages infected and the numbers of amastigotes per 100 macrophages were enumerated.
In vivo cutaneous leishmaniasis mouse model.
The CL animal model was established using highly susceptible 3- to 4-week-old female BALB/c mice weighing about 20 g. Animals were housed in groups of five in cages under controlled environmental conditions (12-h/12-h light/dark cycle and room temperature).
On day 0, BALB/c mice were infected by subcutaneous inoculation of 1 × 107 infective promastigotes of L. amazonensis LV78 into the left hind footpad. The thickness of the footpad lesion was measured using a dial caliper. Treatments were initiated when the lesion thickness reached approximately 0.5 mm, on postinfection day 16. Fifty microliters of either antileishmanial agent or control solvent was administered intralesionally every 4 days. The treatment groups received either 0.5 or 2.5 mg/kg of body weight of compound 39.HCl dissolved in 50% polyethylene glycol (PEG) (n = 12 mice). The positive-control group received 28 mg/kg SSG dissolved in 0.9% NaCl (n = 11 mice). The solvent-control group received 50% PEG (n = 11 mice). The untreated control group received 0.9% NaCl (n = 5 mice). A total of eight injections, on days 16, 20, 24, 28, 32, 36, 40, and 44, were given. The experiment was completed on day 48. Lesion sizes and mouse weights were measured after each treatment throughout the experiment. Lesion sizes were determined by subtracting the thickness of the left lesion-bearing footpad from that of the right uninfected control footpad. All mice from different groups were then sacrificed for determination of lesion weights and parasite burdens.
All animals used in this work were maintained using the guidelines provided in Laboratory Animal Use and Care from the Chinese CDC and the Rules for Medical Laboratory Animals (1998) from the Ministry of Health, China. All protocols for animal use in this work were approved by the Laboratory Animal Use and Care Committee of Sun Yat-Sen University (under license no. 2012CB53000).
Parasite burden by limiting dilution assay.
Forty-eight days postinfection, four mice from each group were picked randomly for assessment of the parasite number in each lesion. Parasite numbers in the lesions were determined by limiting dilution assay. Under sterile conditions, the lesion-bearing footpad was removed, cut into small pieces, and resuspended in 5 ml of supplemented Schneider's Drosophila medium. The footpad was homogenized in a glass homogenizer by upward and downward pulling 10 times. The cell suspensions were serially diluted in 10-fold dilutions (from 1:10 to 1:1 × 107), and 1.2 ml of the diluted suspension was aliquoted into a 96-well plate, with 100 μl per well. The plate was incubated at 27°C for 2 weeks and then examined under an inverted microscope to determine the presence or absence of mobile promastigotes. The final titer was defined as the last dilution at which at least one well contained no parasite.
RESULTS
In vitro antipromastigote activity. (i) Compounds and Leishmania species.
In our previous study, we reported the activities of flavonoid dimers toward different species of L. donovani promastigotes and amastigotes, including wild-type LdAG83, sodium stibogluconate (SSG)-resistant Ld39, and pentamidine-resistant LdAG83PentR50 (18). In this study, five CL-causing Leishmania species, including L. amazonensis LV78, L. braziliensis UA847, L. tropica EP41, L. major FV1, and L. major Lm50122, were studied. Flavonoid dimers 1 to 3 and 5 to 30 and aminoethylene glycol-linked flavonoid dimers 31 to 50, 53, 56 to 61, and 68 to 73 were prepared according to the reported procedures, and their chemical structures are shown in Fig. 1 (13–15, 19).
(ii) PEG-linked flavonoid dimers 1 to 3 and 5 to 30.
All flavonoid dimers in series A contain hydroxyl groups in ring A or ring C, with different linker lengths of 3 to 5 ethylene glycol (EG) units (Fig. 1). Compounds 1 and 2, with 3 or 4 EG units, displayed antipromastigote activity toward L. braziliensis UA847, L. tropica EP41, and L. major Lm50122, with IC50 values ranging from 0.8 to 13.7 μM, but not L. amazonensis LV78 and L. major FV1 (Table 1). The compounds did not show toxicity toward RAW264.7 macrophages, with IC50 values of >200 μM.
TABLE 1.
Antipromastigote activities of synthetic flavonoid dimers with PEG linkers
| Series and compounda | IC50 (mean ± SEM) (μM) forb: |
|||||
|---|---|---|---|---|---|---|
| CL promastigotes |
RAW264.7 macrophagesc | |||||
| L. amazonensis LV78 | L. braziliensis UA847 | L. tropica EP41 | L. major FV1 | L. major Lm50122 | ||
| A | ||||||
| 1 | >100 | 2.4 ± 1.6 | 3.0 ± 1.6 | >100 | 0.8 ± 0.4 | >200 |
| 2 | >100 | 5.0 ± 2.0 | 13.7 ± 8.6 | >100 | 0.9 ± 0.0 | >200 |
| 3 | >100 | >100 | >100 | >100 | ND | >200 |
| 5 | >100 | >100 | >100 | >100 | ND | ND |
| 6 | >100 | >100 | >100 | >100 | ND | ND |
| B | ||||||
| 7 | >100 | >100 | >100 | >100 | ND | >100 |
| 8 | >100 | >100 | >100 | >100 | ND | >95 |
| 9 | >100 | >100 | >100 | >100 | ND | >100 |
| 10 | 12.1 ± 4.9 | 5.2 ± 1.6 | 11.4 ± 4.3 | 29.5 | ND | 8.9 ± 2.3 |
| 11 | 6.2 ± 1.8 | 2.6 ± 1.0 | 3.9 ± 0.4 | 9.5 | ND | 7.1 ± 1.2 |
| 12 | 4.9 ± 0.9 | 3.6 ± 0.6 | 4.9 ± 0.3 | 7.1 | ND | 4.8 ± 0.3 |
| 13 | 6.2 ± 1.7 | 3.3 ± 0.4 | 4.5 ± 0.5 | 7.9 | ND | 4.4 ± 0.4 |
| 14 | 5.1 ± 0.6 | 5.6 ± 1.9 | 5.7 ± 0.4 | 7.1 | ND | 8.1 ± 1.2 |
| 15 | 22.6 ± 4.3 | 31.0 ± 8.9 | 35.5 ± 9.1 | 29.4 | ND | 8.4 ± 2.4 |
| C | ||||||
| 16 | >100 | >100 | >100 | >100 | ND | >100 |
| 17 | >100 | >100 | >100 | >100 | ND | >100 |
| 18 | >100 | >100 | 13.3 ± 4.4 | >100 | ND | >100 |
| 19 | >100 | >100 | >100 | >100 | ND | >100 |
| 20 | 7.4 ± 3.7 | 3.3 ± 1.3 | 1.9 ± 0.2 | >100 | 3.0 ± 0.1 | >200 |
| 21 | 9.4 ± 1.9 | 2.9 ± 1.1 | 2.3 ± 0.2 | 11.1 | ND | 5.5 ± 0.7 |
| 22 | 9.4 ± 3.3 | 6.8 ± 4.8 | 3.1 ± 0.1 | >100 | ND | 8.1 ± 0.6 |
| 23 | 7.9 ± 3.7 | 1.6 ± 0.5 | 1.7 ± 0.2 | 6.0 | ND | 3.1 ± 1.3 |
| 24 | 2.0 ± 0.2 | 1.8 ± 0.7 | 1.7 ± 0.1 | 3.9 | ND | 2.3 ± 0.4 |
| 25 | >100 | 2.8 ± 1.5 | >100 | >100 | ND | 88.6 ± 4.5 |
| 26 | 4.2 ± 1.2 | 2.1 ± 0.5 | 3.1 ± 0.4 | 6.2 | 2.4 ± 0.2 | >80 |
| 27 | 2.9 ± 0.6 | 2.1 ± 0.7 | 1.9 ± 0.2 | 3.9 | ND | 5.3 ± 2.1 |
| 28 | 41.3 ± 13.7 | 12.4 ± 3.8 | 22.8 ± 5.9 | >100 | 1.0 ± 0.0 | >200 |
| 29 | >100 | 4.0 ± 0.5 | >100 | >100 | 5.6 ± 2.0 | >200 |
| 30 | 6.1 ± 1.7 | 2.8 ± 0.2 | 4.6 ± 1.2 | 16.8 | ND | 7.8 ± 1.1 |
Synthetic flavonoid dimers were grouped into three series (series A to C) with various linker lengths containing different numbers of EG units. Substitutions were made at the flavone A ring.
IC50 values for antipromastigote activity toward L. amazonensis LV78, L. braziliensis UA847, L. tropica EP41, L. major FV1, and L. major Lm50122 are shown as mean ± standard error of the mean (SEM) for 1 to 4 independent experiments. All compounds were dissolved in DMSO and the highest percentage of dimethyl sulfoxide (DMSO) used was 1%, at which no toxicity for promastigotes and RAW 264.7 cells was observed. ND, not determined.
The data on compound toxicity toward the macrophage RAW 264.7 cell line have been published (19).
In series B, all hydroxyl groups in ring A have been removed from the flavonoid moieties. Flavonoid dimers with shorter linker lengths (compounds 7 to 9, with 2 to 4 EG units) displayed no significant antipromastigote activity. This result suggests that a polar group in ring A is a favorable functional group for shorter flavonoid dimers. In contrast, flavonoid dimers with longer linker lengths (compounds 10 to 15, with 5 to 13 EG units) remained active toward CL promastigotes, with IC50 values ranging from 2.6 to 36 μM. Unfortunately, compounds 10 to 15 are toxic toward macrophages and therefore not useful.
In series C, hydrophobic substitutions at position 3, 6, or 7, such as a methyl group (compounds 16 to 18 and 22 to 27), ethyl group (compounds 19 to 21), or fluoro group (compounds 28 to 30), were introduced into ring A or C. Compounds 20 and 26 showed potent antipromastigote activity toward CL promastigotes (IC50 values of 1.9 to 7.4 μM), with no toxicity toward macrophages.
(iii) Amine-linked flavonoid dimers.
Although flavonoid dimers 20 and 26 have significant antipromastigote activity without toxicity toward RAW 264.7 macrophages, they are only sparingly soluble in aqueous medium, possibly due to their hydrophobic properties. Attempts to use them in animal experiments were impractical. We previously synthesized a new class of flavonoid dimers with an amine group in the middle of the PEG linker (3). The amino group generally confers better aqueous solubility and thus better physicochemical properties for potential drug development. Compounds 31 to 50, with different substituents on the amine nitrogen, were tested for their antipromastigote activities (Table 2). Compound 31 (with R = H) was cytotoxic to both promastigotes and RAW 264.7 macrophages, with IC50 values of 1.9 to 14.7 μM. Comparing compounds 31 and 9, it is clear that the replacement of the central oxygen by an amine in the linker changed the activity dramatically. Replacing the hydrogen with an ethyl group (compound 32), hydroxyethyl group (compound 33), or ethyl propanoate group (compound 34) on the amine group maintained cytotoxicity toward both promastigotes (IC50 values for CL promastigotes of 1.3 to 11.0 μM) and RAW 264.7 cells (IC50 values for RAW 264.7 cells of 6.4 to 16.0 μM).
TABLE 2.
Antipromastigote activities of amine-linked and compound 39-derived flavonoid dimers
| Compounda | IC50 (mean ± SEM) (μM) forb: |
||||||
|---|---|---|---|---|---|---|---|
| CL promastigotes |
Macrophagesc |
||||||
| L. amazonensis LV78 | L. braziliensis UA847 | L. tropica EP41 | L. major FV1 | L. major Lm50122 | RAW264.7 | PEMs | |
| 31 | 14.7 ± 5.5 | 1.9 ± 0.3 | 9.1 ± 3.1 | ND | ND | 2.1 ± 0.9 | ND |
| 32 | 8.1 ± 1.6 | 1.3 ± 0.1 | 1.9 ± 0.6 | ND | ND | 8.3 ± 1.0 | ND |
| 33 | 5.9 ± 1.1 | 2.5 ± 1.1 | 3.2 ± 1.7 | ND | ND | 6.4 ± 1.5 | ND |
| 34 | 11.0 ± 2.0 | 2.4 ± 0.4 | 5.2 ± 1.6 | ND | ND | 16.0 ± 6.2 | ND |
| 35 | 4.4 ± 1.0 | 2.3 ± 0.2 | 4.7 ± 2.3 | ND | ND | >100 | ND |
| 36 | >100 | >100 | 99.3 | ND | ND | >79 | ND |
| 37 | >100 | >100 | 40.0 ± 2.4 | ND | >100 | 70.3 ± 9.7 | ND |
| 38 | >100 | >100 | >57 | ND | >100 | 45.7 ± 19.1 | ND |
| 39 | 0.43 ± 0.09 | 0.43 ± 0.11 | 0.19 ± 0.03 | 0.69 ± 0.04 | 0.38 ± 0.03 | >100 | >88 |
| 40 | 39.2 ± 11.8 | 33.5 ± 14.8 | 5.1 ± 0.9 | >100 | 21.8 ± 6.3 | 53.0 ± 12.7 | 22.7 ± 4.7 |
| 41 | >100 | >100 | >88 | ND | ND | 65.0 | ND |
| 42 | >100 | 0.8 ± 0.3 | 0.4 ± 0.0 | ND | ND | 96.0 ± 4.0 | ND |
| 43 | >100 | >100 | >100 | ND | ND | >100 | ND |
| 44 | >100 | 48.2 ± 0.2 | >79 | ND | ND | 85.0 | ND |
| 45 | >100 | >100 | >100 | ND | ND | ND | ND |
| 46 | >100 | >100 | >100 | ND | ND | ND | ND |
| 47 | >100 | >100 | >100 | ND | ND | ND | ND |
| 48 | >100 | >100 | >100 | ND | ND | ND | ND |
| 49 | >100 | >100 | >100 | ND | ND | ND | ND |
| 50 | 35.3 ± 14.2 | 9.0 ± 3.2 | 12.9 ± 7.8 | ND | ND | ND | ND |
| 53 | >100 | >100 | 0.8 ± 0.1 | >100 | ND | ND | >58 |
| 56 | 1.5 ± 0.8 | 0.8 ± 0.1 | 0.3 ± 0.0 | 1.6 ± 0.3 | 3.4 ± 1.8 | ND | >50 |
| 57 | ND | ND | ND | ND | 5.3 ± 1.8 | ND | >11 |
| 58 | 4.3 | 4.3 | 4.5 | 29.1 | 7.4 ± 4.9 | ND | >33 |
| 59 | 1.5 ± 0.2 | 1.4 ± 0.3 | 0.6 ± 0.1 | 1.7 ± 0.6 | 2.4 ± 0.9 | ND | 2.7 ± 0.3 |
| 60 | 1.8 ± 0.4 | 1.6 ± 0.2 | 0.7 ± 0.1 | 3.0 ± 0.2 | 4.6 ± 2.3 | ND | >50 |
| 61 | 0.6 ± 0.1 | 0.7 ± 0.1 | 0.3 ± 0.0 | 0.8 ± 0.2 | 3.1 ± 0.2 | ND | >100 |
| 68 | 7.7 ± 1.8 | 6.3 ± 1.1 | 0.5 ± 0.1 | 5.6 ± 2.8 | 8.0 | ND | 33.1 ± 18.9 |
| 69 | >100 | >100 | 1.0 ± 0.3 | >100 | >50 | ND | >100 |
| 70 | >100 | >100 | 33.3 ± 12.5 | >100 | >50 | ND | >92 |
| 71 | 2.4 ± 0.7 | >100 | 0.4 ± 0.0 | >100 | >50 | ND | >100 |
| 72 | 3.2 ± 1.2 | 1.7 ± 0.5 | 0.4 ± 0.1 | 3.2 ± 0.4 | 4.3 ± 1.6 | ND | >33 |
| 73 | >100 | >100 | 7.7 ± 3.9 | >100 | ND | ND | ND |
Flavonoid dimers containing an amino PEG linker (compounds 31 to 50, 53, 56 to 61, and 68 to 73) with different substitutions on the linker were synthesized and tested for their cytotoxicity toward different cutaneous promastigotes, RAW264.7 cells, and PEM cells.
IC50 values are shown as mean ± standard error of the mean (SEM) for 1 to 4 independent experiments. All compounds were dissolved in DMSO and the highest percentage of DMSO used was 1%, at which no toxicity for promastigotes, RAW 264.7 cells, and PEMs was observed. ND, not determined.
The data on compound toxicity toward macrophage RAW 264.7 and PEM cells have been published (19).
Interestingly, when bulkier R groups were introduced at the amine nitrogen, the flavonoid dimers thus generated (compounds 35 to 44) were generally nontoxic to RAW 264.7 cells, with IC50 values ranging from 45.7 μM to >100 μM (Table 2). For example, the compound with a tert-butoxycarbonyl (Boc) group (compound 35) displayed marked antipromastigote activity (IC50, 2.3 to 4.7 μM) without toxicity toward RAW 264.7 cells. Introduction of a benzyl group into the amino linker (compound 36) generated a compound with no antipromastigote activity and toxic effects on RAW 264.7 cells. When the benzyl group contained a polar nitro group (compound 37) or carboxylic ester group (compound 38) at the C4 position, the antipromastigote activity was completely lost. In addition, the benzyl group containing a fluorine atom at position C2 (compound 45), C3 (compound 46), or C4 (compound 47), two fluorine atoms at positions C3 and C4 (compound 48), or three fluorine atoms at positions C2, C3, and C4 (compound 49) also resulted in complete loss of antipromastigote activity. A benzyl group with a trifluoromethyl group at the C4 position (compound 50) improved antipromastigote activity slightly in comparison with compounds 45 to 49. Placing the phenyl group further away from the nitrogen (compound 44) and replacing the H atom with N-succinimide (compound 41) or N-tosylate (compound 43) resulted in low levels of antipromastigote activity. Interestingly, replacing N-tosylate with N-mesylate (compound 42) caused marked antipromastigote activity (IC50 values for L. braziliensis UA847 and L. tropica EP41 of 0.4 to 0.8 μM).
(iv) Amine-linked compound 39-derived flavonoid dimers.
Flavonoid dimers 39 and 40 are of particular interest. Introduction of a pyridine ring as part of R resulted in very strong selective antipromastigote activity. Compound 39, with nitrogen at position 4 of the pyridine ring, had the highest level of antipromastigote activity among all flavonoid dimers. No toxicity to RAW 264.7 cells was observed. Compound 40, containing nitrogen at position 2 of the pyridine ring, displayed at least 27-fold lower levels of antipromastigote activity than did compound 39, indicating that the position of the nitrogen atom on the pyridine ring is of critical importance for antipromastigote activity.
It was found that compound 39, which possesses a pyridine ring at the amine linker, showed the most potent antipromastigote and antiamastigote activity toward visceral L. donovani (19). Here, compound 39 also displayed significant antipromastigote activity toward all species of CL promastigotes, with IC50 values ranging from 0.19 to 0.69 μM (Table 2). The position of the nitrogen atom in the pyridine ring was important in determining the antipromastigote activity. The rank order of antipromastigote activity was as follows: para-position (compound 39; IC50 values ranged from 0.19 to 0.69 μM) > meta-position (compound 68; IC50 values ranged from 0.5 to 8.0 μM) ≫ ortho-position (compound 40; IC50 values ranged from 5.1 to >100 μM). In general, a bromo substitution at the meta-position (compound 69) or ortho-position (compound 71) and a cyano group at the meta-position (compound 70) of the pyridine ring reduced or completely destroyed the antipromastigote activity. Moreover, replacement of the pyridine ring by a pyrimidine ring (compound 72) yielded at least 2-fold lower levels of antipromastigote activity, in comparison with the parent compound 39. Finally, compound 73 completely lost the antipromastigote activity, probably due to the bulkiness of the quinidine ring.
The linker length and position of attachment of the two flavones to the amino PEG linker are also important factors in determining the antileishmanial activity. Compound 53 (with a shorter amino PEG linker than in compound 39) lost the antipromastigote activity. Attachment at the C3′ position of the B ring (compound 56) did not change the antipromastigote activity significantly in comparison with compound 39, which has the linker attached at the C4′ position. However, attachment at the C2′ position of the B ring (compound 57) or the C3 position of the C ring (compound 58) resulted in at least 10-fold reductions in antipromastigote activity, in comparison with parent compound 39. C3′-methoxy substitution in the B ring (compound 59) caused remarkable cytotoxicity toward both promastigotes and host PEM cells. In contrast, C3-methoxy substitution in the C ring (compound 60) or addition of a fluorine atom at the C6 position of the A ring (compound 61) did not cause any toxic effect.
(v) Comparison of antipromastigote activities of compound 39 and other antileishmanial agents.
We compared compound 39 with other antileishmanials, namely, pentamidine, amphotericin B, and miltefosine (Table 3). The second-line antileishmanials pentamidine and miltefosine displayed moderate antipromastigote activity (IC50 values ranging from 1.1 to 17.8 μM and from 5.3 to 32.7 μM, respectively) and moderate cytotoxicity toward PEM cells (IC50 values of 30.4 μM and 75.3 μM, respectively). Amphotericin B had the highest level of antipromastigote activity (IC50 values ranging from 0.13 to 0.34 μM). However, it was also the most toxic compound for PEM cells (IC50 of 7.4 μM) (Table 3). In comparison, compound 39 was as potent as amphotericin B in killing promastigotes, with IC50 values ranging from 0.19 to 0.69 μM (Table 3). Importantly, compound 39 was not toxic to PEM cells (IC50 of >88 μM) (Table 3).
TABLE 3.
Antipromastigote activities and therapeutic index values of standard antileishmanials versus compound 39
| Compound | Promastigotesa |
PEMs, IC50 (mean ± SEM) (μM)b | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
L. amazonensis LV78 |
L. braziliensis UA847 |
L. tropica EP41 |
L. major FV1 |
L. major Lm50122 |
|||||||
| IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | ||
| Pentamidinec | 15.7 ± 5.2 | 1.9 | 1.1 ± 0.1 | 27.6 | 14.1 ± 5.4 | 2.2 | 17.8 ± 2.2 | 1.7 | 7.7 ± 1.0 | 3.9 | 30.4 ± 10.5 |
| Miltefosinec | 32.7 ± 26.9 | 2.3 | 13.0 ± 1.3 | 5.8 | 5.3 ± 1.0 | 14.2 | 9.7 ± 1.1 | 7.8 | 9.8 ± 2.3 | 7.7 | 75.3 ± 9.4 |
| Amphotericin Bd | 0.24 ± 0.03 | 30.8 | 0.17 ± 0.03 | 43.5 | 0.13 ± 0.01 | 56.9 | 0.29 ± 0.05 | 25.5 | 0.34 ± 0.06 | 21.8 | 7.4 ± 0.4 |
| 39d | 0.43 ± 0.09 | >204.7 | 0.43 ± 0.11 | >204.7 | 0.19 ± 0.03 | >463.2 | 0.69 ± 0.04 | >127.5 | 0.38 ± 0.03 | >231.6 | >88 |
IC50 values of current antileishmanials and flavonoid dimer 39 toward promastigotes (L. amazonensis LV78, L. braziliensis UA847, L. tropica EP41, L. major FV1, and L. major Lm50122) and macrophages (PEMs) were determined. IC50 values are presented as mean ± standard error of the mean (SEM) for 2 to 4 independent experiments. The therapeutic index was defined as the ratio of the IC50 of the antileishmanial against PEMs to the IC50 against promastigotes.
The data on compound toxicity toward PEMs have been published (19).
Compounds were dissolved in sterile H2O.
Compounds were dissolved in DMSO. No toxicity to PEM cells was observed at 1% DMSO.
We also determined the therapeutic index values, defined as the ratio of the IC50 against PEM cells to the IC50 against promastigotes, for these antileishmanial agents. A therapeutic index less than 10 would indicate probable nonselective cytotoxicity for the tested compounds (21). As shown in Table 3, compound 39 had the highest therapeutic index (>127.5 to >463.2), followed by amphotericin B (21.8 to 56.9). Other compounds displayed significantly lower therapeutic index values, ranging from 1.7 to 27.6.
In vitro antiamastigote activity of flavonoid dimer 39 and its derivatives.
We investigated whether compound 39 had antiamastigote activity against L. amazonensis LV78 and L. braziliensis UA847. Intracellular amastigotes were obtained by infecting PEM cells with late-log-phase L. amazonensis LV78 and L. braziliensis UA847 promastigotes. Very pronounced antiamastigote activity was noted for compound 39, in comparison with solvent control. Numerous amastigotes were observed in control host macrophages (Fig. 2A and D), whereas only a few amastigotes or none were noted for compound 39-treated L. amazonensis LV78 (2 μM) (Fig. 2B) and L. braziliensis UA847 (2 μM) (Fig. 2E). The percentages of infected macrophages were decreased from 81.0% in the solvent control to 2.0% for compound 39-treated L. amazonensis LV78 (Fig. 2C) and from 51.0% to 0.6% for compound 39-treated L. braziliensis UA847 (Fig. 2F); the numbers of amastigotes per 100 macrophages were reduced from 324 amastigotes to 3 amastigotes per 100 macrophages (Fig. 2C) and from 99 amastigotes to 1 amastigote per 100 macrophages (Fig. 2F), respectively, after the addition of 2 μM compound 39. These results suggest that compound 39 has potent antiamastigote activity.
FIG 2.
Antiamastigote activity of compound 39 against L. amazonensis LV78 and L. braziliensis UA847 amastigotes grown in PEM cells. PEM cells were infected with late-log-phase promastigotes for 24 h at 37°C. Infected macrophages were then treated for 3 days at 37°C with either 0.1% dimethyl sulfoxide (DMSO) or 2 μM compound 39. After 3 days, the coverslips were stained with Giemsa. (A, B, D, and E) Micrographs of PEM cells with L. amazonensis LV78 treated with 0.1% DMSO (A), L. amazonensis LV78 treated with 2 μM compound 39 (B), L. braziliensis UA847 treated with 0.1% DMSO (D), and L. braziliensis UA847 treated with 2 μM compound 39 (E). Three representative microscopic pictures are shown for each treatment group. Magnification, ×1,000. (C and F) Percentages of macrophages infected and numbers of amastigotes per 100 macrophages determined for L. amazonensis LV78 (C) and L. braziliensis UA847 (F).
We compared compound 39 and its derivatives (compounds 39, 40, 42, 60, 61, 68, and 72) with amphotericin B and SSG in terms of their antiamastigote activity and therapeutic index values. All flavonoid dimers (except compound 61) showed promising antiamastigote activity toward L. amazonensis and L. braziliensis, with IC50 values below 10 μM. Compound 39 was most potent, with IC50 values below 2.2 μM (Table 4). As noted for antipromastigote activity, the position of the nitrogen atom in the pyridine was important in determining antiamastigote activity. The compound with a nitrogen atom in the para-position (compound 39) had stronger leishmanicidal activity than did those with a nitrogen atom in the meta-position (compound 68) or ortho-position (compound 40). The addition of a fluorine atom at the C6 position of the A ring (compound 61) and replacement of pyridine by pyrimidine (compound 72) resulted in the reduction of antiamastigote activity. Amphotericin B had the highest level of antiamastigote activity (IC50 values of 0.049 to 0.360 μM), followed by compound 39 (IC50 values of 0.17 to 2.2 μM). Compound 42, with a mesylate group at the amine linker, exhibited moderate antiamastigote activity (IC50 values of 6.5 to 9.0 μM toward L. amazonensis and L. braziliensis) (Table 4). SSG exhibited the weakest antiamastigote activity, with IC50 values ranging from 18.5 to 42.0 μM. Amphotericin B, however, was also the most toxic compound toward PEM cells (IC50 of 7.4 μM). In contrast, compound 39 was almost nontoxic to PEM cells (IC50 of >88 μM). Therefore, the therapeutic index for compound 39 (therapeutic index values ranged from >40.0 to >517.6) was slightly higher than that for amphotericin B (therapeutic index values ranged from 20.6 to 151.0), suggesting that both compounds possess very high selective cytotoxicity for Leishmania amastigotes.
TABLE 4.
Antiamastigote activities and therapeutic index values of SSG and amphotericin B versus compound 39 and its derivatives
| Compound | Amastigotesa |
Macrophages |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
L. amazonensis LV78 |
L. braziliensis UA847 |
L. tropica EP41 |
L. major FV1 |
RAW264.7, IC50 (mean ± SEM) (μM) | PEMs, IC50 (mean ± SEM) (μM) | |||||
| IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | IC50 (mean ± SEM) (μM) | Therapeutic index | |||
| Amphotericin B | 0.055 ± 0.029 | 134.5 | 0.049 ± 0.011 | 151.0 | 0.076 ± 0.023 | 97.4 | 0.360 ± 0.06 | 20.6 | ND | 7.4 ± 0.4 |
| SSG | 32.5 ± 17.6 | >338.5 | 35.6 ± 19.9 | >308.9 | 18.5 ± 0.5 | >594.6 | 42.0 | >261.9 | ND | >11000 |
| 39 | 0.37 ± 0.07 | >237.8b | 0.17 ± 0.02 | >517.6b | 1.8 ± 0.7 | >48.9b | 2.2 ± 1.1 | >40.0b | >100 | >88 |
| 40 | 1.1 ± 0.5 | 20.6b | 4.4 ± 1.7 | 5.2b | >10 | <2.3b | ND | ND | 53.0 ± 12.7 | 22.7 ± 4.7 |
| 42 | 6.5 ± 1.0 | 14.8c | 9.0 | 10.7c | ND | ND | ND | ND | 96.0 ± 4.0 | ND |
| 60 | 2.1 ± 0.4 | >23.8 | 3.3 ± 1.1 | >15.2 | >10 | <5.0 | ND | ND | ND | >50 |
| 61 | >10 | <10.0 | >10 | <10.0 | >10 | <10.0 | ND | ND | ND | >100 |
| 68 | 1.2 ± 0.1 | 27.6 | 0.90 | 36.8 | >10 | <3.3 | ND | ND | ND | 33.1 ± 18.9 |
| 72 | 2.9 ± 0.4 | >11.4 | 5.0 ± 1.8 | >6.6 | >10 | <3.3 | ND | ND | ND | >33 |
PEM cells were infected for 24 h at 37°C with late-log-phase L. amazonensis LV78, L. braziliensis UA847, L. tropica EP41, or L. major FV1. Infected macrophages were then treated with various antileishmanials and incubated for 3 days at 37°C. After 3 days, the coverslips were stained with Giemsa. The numbers of amastigotes per 100 macrophages were determined and used to calculate IC50 values. The IC50 values are presented as mean ± standard error of mean (SEM) for 1 to 3 independent experiments. The therapeutic index was determined as the ratio of the IC50 against RAW264.7 or PEM cells to the IC50 against cutaneous amastigotes. ND, not determined.
Therapeutic indexes are normalized to PEM cells.
Therapeutic indexes are normalized to RAW264.7 cells.
In vivo antileishmanial activity of flavonoid dimer 39.
Compound 39 was tested for its antileishmanial efficacy in a murine model of cutaneous leishmaniasis, i.e., BALB/c mice infected with L. amazonensis LV78 in the footpad. Measurement of lesion thickness was used as an indicator of disease progression. Intralesional administration of the hydrochloride salt of compound 39 (2.5 mg/kg [3 mM] every 4 days 8 times) could inhibit lesion growth as efficiently as administration of SSG (28 mg/kg [31 mM] every 4 days 8 times) (Fig. 3A). Forty-eight days postinfection, the lesion thicknesses for compound 39.HCl- and SSG-treated mice were reduced by 32% (P < 0.05) and 36% (P < 0.05), respectively, in comparison with the PEG control (Fig. 3B), whereas footpad weights were reduced to 67% (P < 0.05) and 47% (P < 0.005) of the PEG control value, respectively (Fig. 3C). Parasite burdens in the footpads of compound 39.HCl- and SSG-treated mice, as measured by the number of amastigotes recovered from infected footpads after in vitro culture, were reduced to 6% (P < 0.001) and 5% (P < 0.001) of PEG control values, respectively (Fig. 3D). The efficacy of compound 39.HCl was dose dependent, because 2.5 mg/kg, but not 0.5 mg/kg, of compound 39.HCl was effective in reducing lesion thickness/weight or parasite burden (Fig. 3B and C). Throughout the whole period of the experiment, there was no significant difference in the body weights of mice from different groups (data not shown), suggesting that neither compound 39.HCl nor SSG exhibited any significant toxicity to the animals at the indicated dosages.
FIG 3.
In vivo antileishmanial efficacy of flavonoid dimer 39.HCl. BALB/c mice were infected in the footpads by subcutaneous infection with 1 × 107 log-phase promastigotes of cutaneous L. amazonensis LV78. The treatments included 0.9% NaCl, 50% PEG, 28 mg/kg SSG, 2.5 mg/kg compound 39.HCl, and 0.5 mg/kg compound 39.HCl. (A) Growth rates of lesions. The drugs were intralesionally injected every 4 days 8 times, starting 16 days postinfection. The lesion thickness was plotted as an indicator of disease progression. (B) Lesion thicknesses 48 days postinfection. (C) Lesion weights 48 days postinfection. (D) Parasite burdens of mice 48 days postinfection. P values for comparisons between the solvent group (50% PEG) and treatment groups (28 mg/kg SSG, 2.5 mg/kg compound 39.HCl, or 0.5 mg/kg compound 39.HCl) were calculated using Student's t test for two paired samples. The experimental value was considered significantly different when the P value was <0.05. The values are presented as mean ± standard error of mean.
In summary, we found that flavonoid dimer 39 was potent against both cutaneous promastigotes (IC50 values of 0.19 to 0.69 μM) and amastigotes (IC50 values of 0.17 to 2.2 μM). Its activity was comparable to or slightly lower than that of amphotericin B (IC50 values of 0.13 to 0.34 μM for promastigotes and 0.049 to 0.360 μM for amastigotes) and better than that of SSG (Tables 3 and 4). Compound 39.HCl at 2.5 mg/kg every 4 days 8 times could reduce the parasite burden in a mouse model of CL by 20-fold, to 5 to 6% of control values. With its low toxicity against macrophages, compound 39 is a potential candidate compound for further development as an antileishmanial drug for treatment of cutaneous leishmaniasis.
DISCUSSION
The use of medicinal plants against parasitic diseases has been studied for a long time (10). Flavonoids, which are abundantly present in fruits, vegetables, and tea, can provide a good source of compounds for the design of antileishmanial drugs (11, 22). Two important members of the flavonoid family, namely, quercetin and luteolin, have been reported to have marked antileishmanial activity (23–26). Based on monomeric flavonoids, we previously synthesized a library of flavonoid dimers and demonstrated that they represent a new class of potent safe antileishmanial agents against L. donovani promastigotes and amastigotes (19). Of particular interest is compound 39, which contains a pyridine ring at the amine PEG linker. It is more potent than monomeric quercetin, luteolin, and other bioflavonoids, and its activity is comparable to that of amphotericin B (19).
Here, by screening the flavonoid dimer library we previously synthesized, we found that flavonoid dimer is active against different CL-causing Leishmania species, including L. amazonensis, L. braziliensis, L. tropica, and L. major. Among the flavonoid dimers screened, compound 39 had the highest antipromastigote activity (IC50, 0.19 to 0.69 μM). Although the antiamastigote activity of compound 39 (IC50, 0.17 to 2.2 μM) was lower than that of amphotericin B (IC50, 0.05 to 0.36 μM), its therapeutic index (>40 to >518) was higher than that of amphotericin B (21 to 151) due to its low toxicity toward macrophages (IC50, >88 μM) (Table 4).
The salt form of compound 39, compound 39.HCl, was as potent as SSG in reducing the lesion thickness/weight in a cutaneous leishmaniasis animal model, even when 10-fold less compound 39.HCl was used. When used at one intralesional injection of 2.5 mg/kg every 4 days 8 times, compound 39.HCl could reduce the parasite burden in the infected footpads to 6% of the solvent control values (Fig. 3D). Reduction of the dose to 0.5 mg/kg abolished the activity of compound 39.HCl (Fig. 3D). We did not observe any obvious toxicity symptoms, such as death or significant weight loss, in the compound 39.HCl-treated animals. Compared with SSG, which has many drawbacks including toxic side effects, a need for intramuscular administration, and the emergence of drug resistance (5), compound 39.HCl seems to be safer to use.
One potential drawback of compound 39 is its relatively low solubility in aqueous solutions. Introduction of an amine group into flavonoid dimers, together with the use of the salt form (compound 39.HCl) instead of the free base (compound 39), did increase the water solubility. Despite these steps, compound 39.HCl still has relatively low water solubility and requires the use of 50% PEG as a solvent in animal studies. Formulation studies to search for better solvents are ongoing. Future studies should include trials of different administration routes (topical, intravenous, intraperitoneal, or oral approaches) in animal studies. Determination of the pharmacokinetic profile and extensive toxicity studies will also be necessary. Here, intralesional administration of compound 39.HCl at the dosage applied did not show any observable signs of toxicity in mice.
It has been reported that quercetin can induce the death of L. amazonensis by increasing the production of reactive oxygen species and collapsing the mitochondrial potential (24). Quercetin has also been reported to have multiple targets, including arginase, which is an important enzyme in the polyamine biosynthesis pathway (23), topoisomerase II in kinetoplasts, which induces DNA cleavage leading to apoptosis (25), and iron metal, which is important for growth and replication of the parasite (26). The mechanisms of action and molecular targets of compound 39 remain to be studied.
In summary, we have discovered that flavonoid dimers, particularly compound 39 and its salt form compound 39.HCl, are effective against different CL-causing Leishmania species in both in vitro and in vivo studies. With low levels of toxicity toward macrophages, we hope that flavonoid dimers can be further developed in the future to be used clinically against CL.
ACKNOWLEDGMENTS
The work described in this paper was supported by the Hong Kong Research Grant Council General Research Fund (grant PolyU 5609/10M) and the Hong Kong Polytechnic University (grant G-U974).
Footnotes
Published ahead of print 31 March 2014
REFERENCES
- 1.Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M. 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7:e35671. 10.1371/journal.pone.0035671 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Reithinger R, Dujardin J-C, Louzir H, Pirmez C, Alexander B, Brooker S. 2007. Cutaneous leishmaniasis. Lancet Infect. Dis. 7:581–596. 10.1016/S1473-3099(07)70209-8 [DOI] [PubMed] [Google Scholar]
- 3.González U, Pinart M, Reveiz L, Rengifo-Pardo M, Tweed J, Macaya A, Alvar J. 2010. Designing and reporting clinical trials on treatments for cutaneous leishmaniasis. Clin. Infect. Dis. 51:409–419. 10.1086/655134 [DOI] [PubMed] [Google Scholar]
- 4.Sundar S, Chakravarty J. 2013. Leishmaniasis: an update of current pharmacotherapy. Expert Opin. Pharmacother. 14:53–63. 10.1517/14656566.2013.755515 [DOI] [PubMed] [Google Scholar]
- 5.Berman JD. 1997. Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin. Infect. Dis. 24:684–703. 10.1093/clind/24.4.684 [DOI] [PubMed] [Google Scholar]
- 6.Desjeux P. 2004. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. 27:305–318. 10.1016/j.cimid.2004.03.004 [DOI] [PubMed] [Google Scholar]
- 7.Mishra J, Saxena A, Singh S. 2007. Chemotherapy of leishmaniasis: past, present and future. Curr. Med. Chem. 14:1153–1169. 10.2174/092986707780362862 [DOI] [PubMed] [Google Scholar]
- 8.Mishra BB, Tiwari VK. 2011. Natural products: an evolving role in future drug discovery. Eur. J. Med. Chem. 46:4769–4807. 10.1016/j.ejmech.2011.07.057 [DOI] [PubMed] [Google Scholar]
- 9.Harborne JB, Williams CA. 2000. Advances in flavonoid research since 1992. Phytochemistry 55:481–504. 10.1016/S0031-9422(00)00235-1 [DOI] [PubMed] [Google Scholar]
- 10.Kayser O, Kiderlen AF, Croft SL. 2003. Natural products as antiparasitic drugs. Parasitol. Res. 90(Suppl 2):S55–S62. 10.1007/s00436-002-0768-3 [DOI] [PubMed] [Google Scholar]
- 11.Salem MM, Werbovetz KA. 2006. Natural products from plants as drug candidates and lead compounds against leishmaniasis and trypanosomiasis. Curr. Med. Chem. 13:2571–2598. 10.2174/092986706778201611 [DOI] [PubMed] [Google Scholar]
- 12.Singh N, Mishra BB, Bajpai S, Singh RK, Tiwari VK. 2014. Natural product based leads to fight against leishmaniasis. Bioorg. Med. Chem. 22:18–45. 10.1016/j.bmc.2013.11.048 [DOI] [PubMed] [Google Scholar]
- 13.Chan KF, Wong IL, Kan JW, Yan CS, Chow LM, Chan TH. 2012. Amine linked flavonoid dimers as modulators for P-glycoprotein-based multidrug resistance: structure-activity relationship and mechanism of modulation. J. Med. Chem. 55:1999–2014. 10.1021/jm201121b [DOI] [PubMed] [Google Scholar]
- 14.Chan KF, Zhao Y, Burkett BA, Wong IL, Chow LM, Chan TH. 2006. Flavonoid dimers as bivalent modulators for P-glycoprotein-based multidrug resistance: synthetic apigenin homodimers linked with defined-length poly(ethylene glycol) spacers increase drug retention and enhance chemosensitivity in resistant cancer cells. J. Med. Chem. 49:6742–6759. 10.1021/jm060593+ [DOI] [PubMed] [Google Scholar]
- 15.Chan KF, Zhao Y, Chow TW, Yan CS, Ma DL, Burkett BA, Wong IL, Chow LM, Chan TH. 2009. Flavonoid dimers as bivalent modulators for P-glycoprotein-based multidrug resistance: structure-activity relationships. ChemMedChem 4:594–614. 10.1002/cmdc.200800413 [DOI] [PubMed] [Google Scholar]
- 16.Wong IL, Chan KF, Burkett BA, Zhao Y, Chai Y, Sun H, Chan TH, Chow LM. 2007. Flavonoid dimers as bivalent modulators for pentamidine and sodium stiboglucanate resistance in Leishmania. Antimicrob. Agents Chemother. 51:930–940. 10.1128/AAC.00998-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wong IL, Chan KF, Tsang KH, Lam CY, Zhao Y, Chan TH, Chow LM. 2009. Modulation of multidrug resistance protein 1 (MRP1/ABCC1)-mediated multidrug resistance by bivalent apigenin homodimers and their derivatives. J. Med. Chem. 52:5311–5322. 10.1021/jm900194w [DOI] [PubMed] [Google Scholar]
- 18.Wong IL, Chan KF, Zhao Y, Chan TH, Chow LM. 2009. Quinacrine and a novel apigenin dimer can synergistically increase the pentamidine susceptibility of the protozoan parasite Leishmania. J. Antimicrob. Chemother. 63:1179–1190. 10.1093/jac/dkp130 [DOI] [PubMed] [Google Scholar]
- 19.Wong IL, Chan KF, Chan TH, Chow LM. 2012. Flavonoid dimers as novel, potent antileishmanial agents. J. Med. Chem. 55:8891–8902. 10.1021/jm301172v [DOI] [PubMed] [Google Scholar]
- 20.Chow LM, Wong AK, Ullman B, Wirth DF. 1993. Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii. Mol. Biochem. Parasitol. 60:195–208. 10.1016/0166-6851(93)90131-G [DOI] [PubMed] [Google Scholar]
- 21.Weniger B, Vonthron-Senecheau C, Kaiser M, Brun R, Anton R. 2006. Comparative antiplasmodial, leishmanicidal and antitrypanosomal activities of several biflavonoids. Phytomedicine 13:176–180. 10.1016/j.phymed.2004.10.008 [DOI] [PubMed] [Google Scholar]
- 22.Tasdemir D, Kaiser M, Brun R, Yardley V, Schmidt TJ, Tosun F, Ruedi P. 2006. Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrob. Agents Chemother. 50:1352–1364. 10.1128/AAC.50.4.1352-1364.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.da Silva ER, Maquiaveli Cdo C, Magalhaes PP. 2012. The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase. Exp. Parasitol. 130:183–188. 10.1016/j.exppara.2012.01.015 [DOI] [PubMed] [Google Scholar]
- 24.Fonseca-Silva F, Inacio JD, Canto-Cavalheiro MM, Almeida-Amaral EE. 2011. Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis. PLoS One 6:e14666. 10.1371/journal.pone.0014666 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Mittra B, Saha A, Chowdhury AR, Pal C, Mandal S, Mukhopadhyay S, Bandyopadhyay S, Majumder HK. 2000. Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis. Mol. Med. 6:527–541. 10.1007/s0089400060527 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Sen G, Mukhopadhyay S, Ray M, Biswas T. 2008. Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity. J. Antimicrob. Chemother. 61:1066–1075. 10.1093/jac/dkn053 [DOI] [PubMed] [Google Scholar]