1230 CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)

Antituberculotic and Activities of Primin, a Natural Benzoquinone: In vitro and in vivo Studies

by Deniz Tasdemir*a), Reto Brunb), Vanessa Yardleyc), Scott G. Franzblaud), and Peter Rüedie) a) Centre for Pharmacognosy and Phytotherapy, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK (phone: þ44-20-77535845; fax: þ44-20-77535909; e-mail: [email protected]) b) Department of Medical Parasitology and Biology, Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel c) Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7 HT, UK d) Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA e) Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich

Primin (¼2-methoxy-6-pentylcyclohexa-2,5-diene-1,4-dione), a natural benzoquinone synthesized in our laboratory, was investigated for its in vitro antiprotozoal, antimycobacterial, and cytotoxic m potential. Primin showed very potent activity against Trypanosoma brucei rhodesiense (IC50 0.144 m ) m m and Leishmania donovani (IC50 0.711 m ), and revealed low cytotoxicity (IC50 15.4 m ) on mammalian cells. Only moderate inhibitory activity was observed against Mycobacterium tuberculosis, Trypanosoma cruzi, and Plasmodium falciparum. When tested for in vivo efficacy in a Trypanosoma b. brucei rodent model, primin failed to cure the infection at 20 mg/kg given intraperitoneally. Primin was too toxic in vivo at a higher concentration (30 mg/kg, injected i.p. route) in mice infected with L. donovani. Taken together, primin can serve as a lead compound for the rational design of more potent and less toxic antiprotozoal agents.

Introduction. – Infectious diseases remain a major problem, particularly in tropical and subtropical countries. Current epidemiological evidence suggests that one-third of the worlds population is infected with Mycobacterium tuberculosis with 8 million new cases, and 2 million deaths per year are attributable to infection with this bacillus [1]. The malaria parasite Plasmodium falciparum is responsible for 1–2 million deaths and 500 million new cases annually, primarily in Africa [2]. African sleeping sickness caused by Trypanosoma brucei is endemic in 36 countries in sub-Saharan Africa and threatens both humans and animals. The global prevalence for this disease is currently estimated at 500,000. American Trypanosoma cruzi, the causative agent of Chagas disease, is widespread throughout South and Central America, with an estimated 18 million people infected. Approximately 12 million people worldwide are infected with different Leishmania species, and some 350 million people live at risk of acquiring leishmaniasis [3]. The high toxicity and inefficacy of the currently used for the treatment of these infectious diseases, as well as the emergence of -resistant strains of the causative organisms, led to an increased pressure on current regimes.

 2006 Verlag Helvetica Chimica Acta AG, Zürich CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 1231

Primin (¼2-methoxy-6-pentylcyclohexa-2,5-diene-1,4-dione), a natural compound occuring in Primula obconica and some other Primula plant species (Primulaceae), is known to be a sensitizer and, thus, the source of allergic contact dermatitis, the so- called primrose dermatitis in some humans [4][5]. This compound is not restricted to Primula sp., and has also been isolated from several other plants, e.g., Miconia (Melastomaceae) [6–8] and Iris (Iridaceae) species [9]. The sensitizing effect of primin and other p-benzoquinones has been ascribed to the covalent binding of a receptor protein of epidermal cells to C(3) and C(5) of the quinone ring system, which could be attacked by nucleophiles of the receptor protein [10][11]. We previously undertook several binding experiments with primin and 2-phenylethylamine and amino acids to investigate the mechanistic outcome of this addition reaction [12]. Besides the cell- mediated allergen effect, a number of other biological activities, such as [6], insect antifeedant [13], molluscicidal [14], and antineoplastic [6][7][15], of primin have been investigated. Some earlier reports point out the potential of primin and related benzoquinones against a few protozoa such as Leptomonas seymouri, Crithidia fasciculata, and Trypanosoma mega [16][17]; however, a broad spectrum antiparasitic and/or antimycobacterial potential of primin has remained uninvestigated. Therefore, we decided to assess in vitro growth inhibitory activity of this compound against a broad panel of parasitic protozoa (T. brucei rhodesiense, T. cruzi, L. donovani, and P. falciparum), as well as the causative agent of human tuberculosis, M. tuberculosis. We used bloodstream life-cycle forms of cultured parasites, which are the clinically most relevant forms. The compound was also tested on primary mammalian (rat skeletal myoblasts, L6) cells in order to determine its therapeutic (selectivity) index. The in vivo trypanocidal and leishmanicidal activities of the compound have also been studied in mouse models.

Results. –TheScheme briefly depicts the general route for the synthesis of the compound, which was described in detail by Liu [18]. A modified method of Schildknecht and Schmidt [19] has been employed, and our procedure [18] was two steps shorter with a higher overall yield (34%, instead of 5–23% in [19]): 1-(1- hydroxypentyl)-3,5-dimethoxybenzene (2) was obtained by a Grignard reaction starting from 3,5-dimethoxybenzaldehyde (1). Hydrogenolysis of 2 in glacial AcOH catalyzed by Pd on activated charcoal and a trace of 60% HClO4 [20] gave rise to 1,3- dimethoxy-5-pentylbenzene (3). Compound 3 was monodemethylated with EtSH and NaH [21] to generate 3-methoxy-5-pentylphenol (4). The oxidation of 4 was achieved with FREMYs salt [22] to afford primin. As given in Table 1, primin exhibited very potent in vitro trypanocidal activity m against African trypanosomes (T. b. rhodesiense) with an IC50 value of 144 n . However, its growth inhibitory activity against American T. cruzi and the malaria m parasite P. falciparum was ca. 70-fold less (IC50 values of 10.1 and 10.9 m , resp.). The 1232 CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)

Scheme. Synthesis of Primin According to [18]

a) Me(CH2)4Br, Mg/Et2O; 94%. b)H2 , Pd/C, AcOH, 60% HClO4 ; 83%. c) EtSH, NaH, DMF, reflux,

87%. d) FREMYs salt, KH2PO4 ,H2O (pH 8), 50%; overall yield a)–d): 34%. minimum inhibitory concentration (MIC) value of primin towards M. tuberculosis was m also low (60.3 m ). Primin revealed significant in vitro leishmanicidal potential (IC50 711 nm), which was comparable to that of miltefosine, the reference drug used for the m antileishmanial assay (IC50 373 n ). On the other hand, primin displayed only m moderate cytotoxicity on mammalian L6 cells (IC50 15.4 m ), similar to that reported m previously for some tumor cell lines (e.g., M109 lung carcinoma cell line: IC50 48.1 m ; m A2780 human ovarian cell line: IC50 13.9 m ) [7]. The potent in vitro activity and high selectivity indices observed against T. b. rhodesiense and L. donovani (SI values of 107 and 22, resp.) prompted us to evaluate the in vivo efficacy of primin. A 20 mg/kg (i.p.) dose was employed to treat mice infected with Trypanosoma b. brucei STIB795. Unfortunately, the treatment did not repress the parasitaemia, and the mice died on day 5.25 (average of four animals) as compared to 6 days for the untreated control animals (Table 2). No acute toxicity has been observed in mice dosed with primin during this in vivo experiment. Considering the higher in vitro IC50 value (lower activity) of primin towards L. donovani than that observed for T. b. rhodesiense, and the absence of bioactivity at 20 mg/kg in trypanosoma model, a higher dose regimen was

Table 1. In vitro Antimycobacterial, Antiprotozoal, and Cytotoxic Activities of Primina)b)

Compound Mycobacterium Trypanosoma Trypanosoma Leishmania Plasmodium L6 cell tuberculosis b. rhodesiense cruzi donovani falciparum toxicity

MIC IC50 IC50 IC50 IC50 IC50 Reference 0.1c) 0.0055d) 1.19e) 0.373 f) 0.016g) 0.018h) Primin 60.3 0.144 (107) 10.1 (1.5) 0.711 (22) 10.9 (1.4) 15.4 a m ) MIC and IC50 values are in m and are mean values from two to three replicates (the variation is max. 20%). b) The selectivity index (SI) of primin for each parasite is shown in the parentheses c d (SI¼IC50 value for cytotoxicity/IC50 value for antiprotozoal activity). ) . ) Melarsoprol. e) Benznidazole. f) Miltefosine. g) Artemisinin. h) Phodophyllotoxin. CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 1233

Table 2. In vivo Activity of Primin Against T. brucei brucei STIB795 Mouse Model

Compound tested Dosing regimen Injection Total dose Cured/infected Mean survival [d] [mg/kg] Controla) – – – 0/4 6.0 Pentamidineb) 5 mg/kg4 d i.p. 20 mg/kg 4/4 >30 Melarsoprolb) 2 mg/kg4 d i.p. 8 mg/kg 4/4 >30 Primin 20 mg/kg4 d i.p. 80 mg/kg 0/4 5.25 a) Untreated control to determine the mean survival. b) Standard drugs. selected for in vivo antileishmanial-activity assessments. Thus, all mice were dosed at 30 mg/kg, i.p. on day 1 (d1). Already in the following day (d2), all mice looked very unwell and were not dosed. The next day (d3), only one healthy looking mouse was fully dosed. The remaining four mice either died or were euthanatized as they were behaving extremely unwell. On d4, the last remaining mouse looked very unwell again, thus was not dosed. On the final day 5, the last mouse looked extremely unwell and so was euthanatized. Thus, primin proved to show strong acute toxicity to mice infected with Leishmania at 30 mg/kg concentration (Table 3).

Table 3. In vivo Activity of Primin Against L. donovani in HU3 in BALB/c mice

Compound Dosing regimen Injection Total dose % Inhibition Standard tested [mg/kg] 95% C. L. Errror of mean SbVa)b)15mgSbV/kg5 d s.c. 75 mg/kg 63.6521.45 10.9 Miltefosineb) 30 mg/kg5 d p.o. 150 mg/kg 96.620.88 0.4 Primin 30 mg/kg5 d i.p. 60 mg/kg All mice died – a)SbV: Pentavalent antimony (active ingredient of sodium stibogluconate). b) Standard drugs.

Discussion. – In this study, we report on the in vitro inhibitory activity of primin against a panel of pathogens with clinical importance for man. We believe, this is the first detailed study assessing the antituberculotic and broad spectrum antiparasitic potential of the compound. With an IC50 value in the ng range, primin appears to be a promising lead molecule for the treatment of African trypanosomiasis. Primin also possessed pronounced leishmanicidal potential comparable to the standard compound, miltefosine, which is used in the tretament of visceral leishmaniasis clinically. The moderate growth inhibitory effect of primin on two other parasites, T. cruzi and P. falciparum, and the bacillus M. tuberculosis, together with the reasonable therapeutic indices observed for T. b. rhodesiense and L. donovani suggested a selectivity for these protozoan parasites. This encouraged us to establish its in vivo efficacy on rodent models. Unfortunately, primin failed to show any significant effect in vivo towards Trypanosoma brucei at a low dose, and at a higher concentration it showed an acute toxicity to mice infected with L. donovani. Quinone metabolites are found in nearly all forms of life [23]. The primary metabolites, e.g., ubiquinone and plastoquinone, are involved in a wide variety of 1234 CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) biological and chemical processes, including photosynthesis, electron transport, protein modification, and cell signaling, and may act as potent antioxidants [24]. Secondary quinones are also ubiquitous in nature and found naturally in plants, fungi, and bacteria. They encompass a wide array of structurally diverse compounds which are thought to play defensive roles for many organisms [23]. Quinones exert a number of biological activities, such as antibacterial, , antimalarial, or anticancer. Although the exact mechanism(s) of the action(s) of these compounds have yet to be fully understood, at least some of their effects can be attributed to redox cycling that generates reactive oxygen species (ROS). Production of ROS can directly combat infection by causing severe oxidative stress within cells or can act in signaling cascades to elicit other protective cellular measures, such as apoptosis. However, ROS can also create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotox- icity, and carcinogenesis [25][26]. In general, the molecular basis for the initiation of quinone cytotoxicity in resting or non-dividing cells has been attributed to the oxidation of essential proteins by ROS and/or the alkylation (see below). However, cytotoxicity of quinones in rapidly dividing cells, e.g., tumor cells, is attributed to DNA modification [27]. It has been hypothesized that the cytotoxic potential of primin and related p-benzoquinones towards human leukemia (KG-1a) cells results from the formation of free radicals and subsequent DNA-strand breaks [15]. Further studies are necessary to identify the exact mechanism(s) of primin on cancerous and non-cancerous human cells. Alternatively to ROS generation, some quinones act as Michael acceptors and function as alkylation agents. Such quinones react with cellular nucleophiles (e.g., cysteinyl thiols or amine groups) of crucial cellular proteins, forming covalently linked Michael adducts that retain the ability to damage the cells [25][27]. In contrast to well- studied ROS generation and consequent oxidative stress in living cells, the role of Michael-adduct formation in quinone toxicity is not well-understood. Interestingly, sensitizing capacity of substituted p-benzoquinones, including primin, has been discussed, and the positions C(3) and C(5) (of the quinone) for a covalent binding to a receptor protein of epidermal cells have been studied. These positions can be attacked by nucleophiles of the receptor protein, e.g., SH and NH2 groups of amino acids [10][12]. Although the mechanisms appear to be essentially related, no study has been performed to understand underlying relationships between the allergenic and alkylation-based toxicity of primin. A number of natural and synthetic quinones show antiprotozoal activity [28][29]. The best example is probably atovaquone, a hydroxynapthaquinone, which is currently being clinically evaluated against malaria. Antimalarial activity of atovaquone has been attributed to its interference with the cytochrome c reductase complex [30]. Atovaquone has also antileishmanial effects [31]. Some hydroquinones inhibit Trypanosoma sp. by blocking the mitochondrial electron transport chain before cytochrome b [32]. Trypanothione reductase (TR), an essential enzyme for the thiol of tryponasomatids, is responsible for the protection of trypanosomes against oxidative stress. Some redox cyclers interact specifically with TR as subversive substrates [33]. Interestingly, a number of quinones have been shown to target TR from Trypanosoma congolense [34]. Thus, it is possible that antiparasitic activity of primin might involve inhibition of this enzyme. Consequently, the exact mechanism(s) by which primin cause CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 1235 antiparasitic and toxic effects can be quite complex and warrants further investigations. After uncovering the interaction of this compound with biological systems of parasites and humans, primin may serve as a lead for a medicinal-chemistry approach to design less toxic–allergenic derivatives with higher antiparasitic potency.

Experimental Part General. M.p.: Mettler FP5/52 apparatus; uncorrected. UV and IR spectra: Perkin-Elmer Lambda 9 UV/VIS/NIR and Perkin-Elmer 297 instruments, resp. NMR Spectra: Bruker ARX 300 spectrometer operating at 300 (1H) and 75 MHz (13C); the chemical shift values as ppm relative to TMS. CI-MS: Varian MAT 711 mass spectrometer; EI-MS: Finnigan MAT95 (70 eV) spectrometer. Synthesis of Primin. The title compound was synthesized as depicted in the Scheme; a detailed procedure can be found in [18]. Its purity was determined to be >99% by TLC, CI- and EI-MS, and 1H-NMR analyses.

Data of Primin. Yellow needles. M.p. 62–638. UV (MeOH): 267 (4.1), 361 (2.9). IR (CHCl3): 3020, 1 2960, 2930, 2860, 1680, 1650, 1602, 1458, 1320, 1235, 1180, 1052, 901, 835. H-NMR (300 MHz, CDCl3): 6.48 (dt, 4J(5,3) ¼ 2.3, 4J(1’,5)¼1.3, HC(5)); 5.87 (d, 4J(3,5)¼2.3, HC(3)); 3.81 (s, MeO); 2.42 (dt, 3 4 3 3 J(1’,2’)¼7.5, J(1’,5)¼1.3, CH2(1’); 1.50 (quint., J(2’,1’) ¼ J(2’,3’)¼7.5 , CH 2(2’)); 1.30 –1.34 (m, 3 13 CH2(3’), CH2(4’)); 0.87 (t, J(5’,4’) ¼ 6.8, Me(5’)). C-NMR (75 MHz, CDCl3): 187.5 (C(4)); 182.0 (C(1)); 158.7 (C(2)); 147.5 (C(6)); 132 (C(5)); 107 (C(3)); 56.1 (MeO); 31.2 (C(3’)); 28.6 (C(1’)); 27.3 (C(2’)); 22.2 (C(4’)); 13.8 (Me(5’)). CI-MS (isobutane): 209 (100, [MþH]þ ). EI-MS (70 eV): 208 (42, þ M ), 153 (100), 139 (13), 124 (35), 109 (19). HR-EI-MS: 208.11206 (0.00023, C12H16O3). The spectral data of primin were identical with those reported in [7]. Biological Tests. For bioassays, primin was dissolved in DMSO. The final concentration of DMSO was <1%, which had no significant effect on test organisms.

In vitro Assay for M. tuberculosis. H37Rv strain of M. tuberculosis (ATCC 27294, Rockville, MD) was grown to late log phase in Middlebrook7 H9 broth supplemented with 0.2% (v/v) glycerol, 0.05% Tween 80, and 10% (v/v) OADC. Cultures were centrifuged 15 min. at 48, washed twice, and resuspended in PBS (phosphate-buffered saline). Suspensions were then passed through an 8-mm filter to remove clumps, and aliquots were frozen at 808. The CFU (colony-forming unit) was determined by plating on 7 H11 agar plates. The MIC for Mycobacterium tuberculosis was assessed by the microplate alamar blue assay (MABA) as described in [35]. The compound was dissolved in DMSO at 12.8-mm concentration and added to culture media to produce a maximum final concentration of 128 mm. Eight two-fold serial dilutions of the test compound were prepared in Middlebrook7 H12 medium and added to 96-well microplates in a volume of 100 ml. M. tuberculosis (100 ml continuing 2104 CFU) was added to yield a final testing volume of 200 ml. Cultures were incubated for 7 days at 378 after which 12.5 ml of 20% Tween 80, and 20 mlofAlamar BlueTM were added to cultures. After incubation at 378 for 24 h, fluorescence was determined (excitation 530 nm, emission 590 nm). The MIC value was defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to the mean of replicate bacteria-only controls. In vitro Assays for T. brucei rhodesiense and L6 Cell Cytotoxicity. Minimum essential medium (50 ml) supplemented with 2-sulfanylethanol and 15% heat-inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions were prepared. Then, 104 bloodstream forms of Trypanosoma b. rhodesiense STIB 900 in 50 ml were added to each well, and the plate was incubated at TM 378 under a 5% CO2 atmosphere for 72 h. After addition of 10 mlofAlamar Blue to each well, the plates were incubated for another 2–4 h, and read in a Spectramax Gemini XS microplate fluorometer using an excitation wavelength of 536 nm and emission wavelength of 588 nm [36]. Fluorescence development was expressed as percentage of the control, and IC50 values were determined. Cytotoxicity was assessed using the same assay on rat skeletal myoblasts (L6 cells). In vitro T. cruzi Assay. Rat skeletal myoblasts (L6 cells) were seeded in 96-well microtiter plates at 2000 cells/well in 100 mlofRPMI 1640 medium with 10% FBS and 2 mml-glutamine. After 24 h, the medium was removed and replaced by 100 ml per well containing 5000 trypomastigote forms of T. cruzi 1236 CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)

Tulahuen strain C2C4 containing the b-galactosidase (Lac Z) gene. After 48 h, the medium was removed from the wells and replaced by 100 ml of fresh medium with or without a serial drug dilution. After 96 h of incubation, the substrate CPRG/Nonidet (50 ml) was added to all wells. A color reaction developed within 2–6 h was read photometrically at 540 nm. Data were transferred into a graphic program (MS TM Excel ), sigmoidal inhibition curves were determined, and IC50 values were calculated. In vitro L. donovani Assay. Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 378 in SM medium [37], at pH 5.4, supplemented with 10% heat-inactivated fetal bovine 5 serum under an atmosphere of 5% CO2 in air. A 100 ml of culture medium with 110 amastigotes from axenic culture with or without a serial drug dilution were seeded in 96-well microtiter plates. After 72 h of incubation, 10 mlofAlamar BlueTM was added to each well. The plates were incubated for another 2 h and read with a Spectramax Gemini XS microplate fluorometer using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Data were analyzed using the software Softmax Pro. Decrease of fluorescence was expressed as percentage of the fluorescence of control cultures and plotted against the drug concentrations. From the sigmoidal inhibition curves, the IC50 values were calculated. In vitro P. falciparum Assay. Antiplasmodial activity was determined using the chloroquine resistant strain K1. A modified [3H]hypoxanthine incorporation assay was applied as described in [38][39]. Briefly, infected human red cells cultivated in RPMI 1640 medium with 5% Albumax II were exposed to serial drug dilutions in microtiter plates. After 48 h of incubation at 378 in a reduced O2 atmosphere, 0.5 mCi [3H]hypoxanthine was added to each well. Viability was determined by measuring [3H]hypoxanthine incorporation by means of liquid scintillation counting after further 24-h incubation time. The results were recorded as counts per min per well at each drug concentration and expressed as percentage of the untreated controls. From the sigmoidal inhibition curves, IC50 values were calculated. Artemisinin served as positive control. In vivo Trypanosoma brucei brucei Assay. Groups of four female NMRI mice weighing 20–25 g were infected intraperitoneally (i.p.) on day 0 (d0) with 105 bloodstream forms of Trypanosoma brucei brucei STIB 795, which is a derivative of strain 427. Mice were treated on four consecutive days (d3 to d6 post- infection) with 50 mg/kg by the i.p. route. One group served as untreated controls, and two other groups were treated with the standard drugs pentamidine (5 mg/kg4 d) and melarsoprol (2 mg/kg4d). Parasitaemia of the mice was checked by tail blood examination on day 7 and thereafter 2/week. The day of death of the mice was recorded. In vivo L. donovani Assay. Female BALB/c mice (Charles Rivers, UK; 20 g) were infected with 2 107 amastigotes, in a 0.2 ml bolus via a lateral tail vein. The mice were then randomly sorted into groups of five. L. donovani HU3 amastigotes were harvested from a passage animal immediately prior to infection [40]. Seven days post infection mice were dosed intraperitoneally 30 mg/kg/day on day 1 (d1). In the second day, all mice looked very unwell and were not dosed. On d3, three mice were found dead, and one mouse was killed as it appeared extremely unwell. One mouse that looked healthy was fully dosed. On the 4th day, last remaining mouse looked very unwell again, thus, it was not dosed. On the final day 5, the last remaining mouse looked extremely unwell and so was killed. The standard drugs, PentostamTM (SbV: sodium stibogluconate, 15 mg/kg s.c.) and oral miltefosine (30 mg/kg) were dosed at the same time with primin for 5 consecutive days. Fourteen days post infection all mice were killed, the livers were weighed, and impression smears were made on glass slides, which were then fixed and stained. The antileishmanial activity of the standards was evaluated by counting the number of amastigotes per 500 host cell nucleiweight of the liver (mg), and comparing this value to untreated control values.

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Received August 15, 2006