Fluorescent Diethylcarbamazine Analogues: Sites of Accumulation in

Oct 10, 2007 - ... rhodamine B. Confocal microscopy is used to show that the drug accumulates in the adult filarial worms in the pharynx, esophagus, a...
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Bioconjugate Chem. 2007, 18, 1818–1823

Fluorescent Diethylcarbamazine Analogues: Sites of Accumulation in Brugia malayi Amy Junnila,† D. Scott Bohle,*,‡ Roger Prichard,*,† Inna Perepichka,‡ and Carla Spina‡ Institute of Parasitology and Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, PQ H3A 2K6, Canada. Received March 23, 2007; Revised Manuscript Received July 18, 2007

New fluorescein and rhodamine B-labeled antifilarial drug DEC analogues for use in drug localization studies with confocal microscopy have been prepared by a high-yield three-step synthesis. The resulting β-amine-substituted DEC analogue has a single ethyl substituent which is β-aminated to accommodate the fluorophore of either fluorescein isothiocyananate or rhodamine B. Confocal microscopy is used to show that the drug accumulates in the adult filarial worms in the pharynx, esophagus, and near the nerve ring of all adults, as well as in the uteri and vulva and the testes of the females and males.

INTRODUCTION

Scheme 1. Structure of DEC

Lymphatic filariasis (LF) is a debilitating disease caused by infection with the filarial worms Wuchereria bancrofti or Brugia malayi. It remains an important disease in tropical areas of Asia, Africa, and the Americas with an estimated 120 million people infected worldwide. LF is caused by the spread of the nematodes by mosquito bites between infected individuals, and its most remarkable symptom is known as elephantiasis which results from the swelling of tissues above infected lymphatic ducts. Diethylcarbamazine (DEC) has been the drug of choice in treating LF since its discovery in 1947. (1–3) It very rapidly kills circulating first-stage larvae (microfilariae, mf) and may also kill or sterilize adult worms. However, there has been relatively little sustained effort with modern tools to understand its localization and drug targets. Furthermore, there are published (4) and preliminary reports of a lack of adequate response to DEC treatment, and drug resistance may be developing. In order to understand the possible mechanisms of resistance to a drug, it is critically important to understand how it works. Fluorescent analogues which share the biological activity of the pharmaceutical would be very useful tools for understanding how DEC works. Early medicinal chemistry studies identified the fundamental importance of the piperazine ring and the 4-N-methyl substituent on the ring (Scheme 1). DEC analogues with a range of substituents for the dialkylcarbamyl group show antifilarial activity, but substitution of the methyl and modification of the ring leads to drug inactivity. (5, 6) In this study, we have synthesized new fluorescein and rhodamine B-labeled DEC analogues for use in drug localization studies with confocal microscopy. A high-yield three-step synthesis (Scheme 2) results in the β-amine-substituted DEC analogue 3 in which only a single ethyl substituent is altered, thus preserving the crucial pharmacophore of DEC. (5, 6) Compound 3, by attachment to its reactive amine group, can be readily conjugated to fluorescein isothiocyanate (FITC) to give 4 or rhodamine B (RHB) to give 5 (Scheme 3). As 3 is the key intermediate of the new fluorescent probes, it was tested for its antifilarial action in Mongolian gerbils (Meriones unguiculatus) infected with B. malayi prior to use in drug localization experiments. Solutions of compound 3 † ‡

Institute of Parasitology. Department of Chemistry.

which would produce dose rates equivalent to standard dosages of DEC had antifilarial activity comparable to that of DEC, so we proceeded to conjugate 3 to FITC and RHB in order to localize drug accumulation in B. malayi in Vitro.

EXPERIMENTAL PROCEDURES General Experimental. All reagents and solvents were used as supplied commercially, except for tetrahydrofuran (THF) and methylene chloride, which were distilled from Na/Ph2CO and CaH2, respectively. N-ethylethylenediamine, 4-methyl-1-piperazinecarbonyl chloride hydrochloride, fluorescein isothiocyanate (90%), and rhodamine B isothiocyanate mixture of isomers were purchased from Sigma Chemical Company.1H and 13C NMR spectra were measured on a Varian Mercury 300 or 400 MHz spectrometer. All chemical shifts were recorded in δ (ppm) relative to tetramethylsilane (TMS). Melting points were measured with a 2010 differential scanning calorimeter. The IR spectra were taken in KBr disks or as a thin film on a NaCl plate using an ABB Bomem MB Series spectrometer with spectral resolution of 2 cm-1. UV/visible spectra were measured using a Cary 300 Bio UV–visible spectrophotometer from Varian. Fluorescence spectra were recorded on a Varian CaryEclipse fluorometer or a FluoroMax 2 (ISA) Jobin Yvon-SPEX spectrofluorometer. The pH values were monitored with Cole Palmer pH meter (model 05669-20) using a Ag/AgCl electrode. The mass spectra analyses were obtained on a Finnigan LCQDuo spectrometer operating at 70 eV. Aluminum-backed silica plates with UV indicator and silica gel 60 Å (70–270 mesh) were used in analytical thin-layer chromatography (TLC) and silica gel column chromatography, respectively. Elemental analyses were performed by Quantitative Technologies, Inc. Synthesis of New DEC Analogues. tert-Butyl 2-(Ethylamino)ethylcarbamate 1. The protected N-ethylethylenediamine 1 was prepared by literature methods. (7) Recrystallization from pentane with cooling in the freezer led to large crystals of 1

10.1021/bc0701040 CCC: $37.00  2007 American Chemical Society Published on Web 10/10/2007

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Scheme 2. Synthesis of 3 Intermediate Compound

Figure 1. Microfilarial recovery 24 h after treatment. N ) 4 gerbils per treatment group. Dose levels are shown under each column.

Scheme 3. Synthesis of 4, DEC-FITC and 5, DEC-Rhodamine B

Figure 2. Microfilarial recovery 10 days after treatment. N ) 4 gerbils per treatment group. Dose levels are shown under each column.

(67%), mp 54–55 °C. 1H NMR (CDCl3, 300 MHz) δ: 1.05 (t, 3H, J ) 7 Hz), 1.39 (s, 9H), 2.60 (q, 2H, J ) 7 Hz), 2.67 (t, 2H, J ) 6 Hz), 3.16 (q, 2H, J ) 6 Hz), 5.09 (brs, NH). 13C NMR (CDCl3, 300 MHz) δ: 15.7, 28.7, 40.7, 44.1, 49.3, 79.2, 156.2. MS (EI) m/z: 188 (1.85%), 115 (29.9%), 99 (83.2%), 58 (100%). Anal. Calcd for C9H20N2O2 (188.15 g/mol): C, 57.42; H, 10.71; N, 14.88. Found: C, 57.42; H, 10.94; N, 14.81. tert-Butyl 2-(N-Ethyl-4-methylpiperzaine-1-carboxamido)ethylcarbamate 2. To a solution of 4-methyl-1-piperazinecarbonyl chloride hydrochloride (2.39 g, 12 mmol) and well-powdered, anhydrous potassium carbonate (4.62 g, 33 mmol) in dry acetonitrile was added 1 (0.90 g, 4.78 mmol) under nitrogen at room temperature. The reaction mixture was stirred overnight, then heated at 60 °C for 3 h. After cooling to room temperature, the precipitate was filtered and washed with acetonitrile (2 × 25 mL). The filtrate was concentrated, and the residue was purified by flash chromatography on silica gel with CH2Cl2/ methanol (1:0.1 v/v) as an eluent followed by recrystallization from hexane to give 2 as a white solid (1.00 g, 66.5%), mp 70–71 °C. 1H NMR (CDCl3, 400 MHz) δ: 1.14 (t, 3H, J ) 7 Hz), 1.42 (s, 9H), 2.29 (s, 3H), 2.39 (t, 3H, J ) 5 Hz), 3.19–3.29 (m, 10H), 5.12 (brs, NH). 13C NMR (CDCl3, 300 MHz) δ: 13.4, 28.4, 38.9, 43.5, 45.7, 46.2, 46.9, 54.7, 79.1, 155.9, 164.6. IR (cm-1): 3330s, 3002m, 2973m, 2942m, 2846m, 2799m, 1705s, 1627s, 1522s, 1448s, 1390m, 1362m, 1283m, 1258m, 1184m, 1164m, 1007m, 976m, 958m, 885m, 774m, 650m, 557m, 529m. MS (EI) m/z: 314 (2.77%), 240 (17.7%), 126 (100%). Anal. Calcd for C14H30N4O3 (314.42 g/mol): C, 57.29; H, 9.61; N, 17.81. Found: C, 57.33; H, 9.53; N, 17.76.

N-(2-Aminoethyl)-N-ethyl-4-methylpiperzaine-1-carboxamide Dihydrochloride 3. The tert-butoxycarbonyl group of 2 was removed by the following procedure: HCl (2.0 M in methanol, 5 mL) was added to a stirred solution of 2 (1.00 g, 3.19 mmol) in methanol (5 mL) at ambient temperature for an additional 3 h. The solvent was evaporated, yielding oil which crystallized after drying in Vacuo (0.88 g, 97%). 1H NMR (CH3OH-d4, 400 MHz) δ: 1.22 (t, 3H, J ) 7 Hz), 2.94 (s, 3H), 3.12–3.33 (m, 10H), 3.48–3.52 (m, 4H). 13C NMR (CDCl3, 400 MHz) δ: 13.4, 43.6, 45.4, 46.1, 46.8, 49.7, 54.8, 164.4. The resultant product 3 can be stored, at 4 °C, for at least 3 months without decomposition. 1-(Fluorescein-5-yl)-3-(2-N-ethyl-4-methylpiperzaine-1carboxamido)ethylthiourea 4. To a solution of 3 (0.187 g, 0.652 mmol) and triethylamine (0.132 g, 1.30 mmol) in anhydrous dimethylformamide was added FITC (0.127 g, 0.326 mmol). The reaction was stirred at room temperature under nitrogen atmosphere with protection from light overnight. The complete removal of FITC was monitored by TLC (eluent CH2CL2/ methanol 1:0.3 v/v). The solvent was then removed in Vacuo and the product was purified by flash column chromatography using CH2Cl2/methanol (1:0.3 v/v) as an eluent to give 4, which is isolated as an intense orange powder (0.12 g, 61%). 1H NMR (DMSO-d6, 400 MHz) δ: 1.06 (t, 3H, J ) 7 Hz), 2.12 (s, 3H), 2.24 (t, 3H, J ) 5 Hz), 3.08–3.19 (m, 10H), 6.47–6.63 (m, 6H), 7.11 (d, 1H, J ) 8 Hz), 7.62 (d, 1H, J ) 8 Hz), 8.14 (s, 1H), 10.3 (br s, 2H). IR (KBr, cm-1): 3306br, 3146br, 2984br, 1633s, 1581s, 1565s, 1499m, 1465s, 1424m, 1384m, 1366w, 1319s, 1288s, 1255m, 1208m, 1172w, 1111m, 1030w, 966w, 909w, 843w, 802w, 574w, 484w. MS (ESI) C31H33O6N5S, 603 g/mol) m/z: found [M + H]+ 604 (29%), [M + Na]+ 626 (35%). Fluorescence (in water): λexc ) 498 nm, λem ) 520 nm. 1-(Rhodamine-5(6)-yl)-3-(2-N-ethyl-4-methylpiperzaine-1carboxamido)ethylthiourea 5. To a solution of 3 (0.157 g, 0.548 mmol) and triethylamine (0.06 g, 0.60 mmol) in anhydrous

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Figure 3. Adult parasite recovery 24 h after treatment. N ) 4 gerbils per treatment group, with the same dose levels as used in Figures 1 and 2, shown under each column.

dimethylformamide (5 mL) was added rhodamine B isothiocyanate (mixture of isomers, 0.18 g, 0.336 mmol), and the mixture was stirred at room temperature under nitrogen atmosphere with protection from light overnight. Then, the temperature was raised to 70 °C for 10 h (reaction progress was monitored by TLC, eluent CH2Cl2/methanol 1:0.3 v/v). The final product 5 was reprecipitated several times from methanol/diethyl ether as a magenta powder (0.11 g, 43.7%) and used without purification for the next analyses. Fluorescence (in methanol): λexc ) 548 nm, λem ) 570 nm. 1-(Fluorescein-5-yl)-3-butylthiourea Control 1. Under nitrogen, butylamine (0.154 mL, 0.5 M solution in ethanol, 0.077 mmol) was added to a solution of FITC (0.021 g, 0.054 mmol) in dry ethanol (0.1 mL) using a gas-tight microsyringe, and the mixture was stirred at room temperature with protection from light overnight. Progress of the reaction was monitored by TLC (eluent CH2Cl2/methanol, 1:0.1 v/v, Rf ) 0.5). After removal of the solvent, the crude product was purified by column chromatography on silica gel with CH2Cl2/methanol (1:0.05 f 1:0.3 v/v) as an eluent to give a deep yellow–orange powder of control 1 (0.017 g, 68%) 1H NMR (CH3OH-d4, 400 MHz) δ: 1.01 (t, 3H, J ) 10 Hz), 1.44 (m, 2H), 1.67 (m, 2H), 2.03 (s, 1H), 3.63 (m, 2H), 3.98 (s, 1H), 6.72–7.02 (m, 6H), 7.23 (d, 1H, J ) 8 Hz), 7.86 (d, 1H, J ) 6 Hz), 8.25 (s, 1H). IR (KBr, cm-1): 3372br, 3253br, 3065br, 2960s, 2929s, 2873m, 2858m, 1685s, 1593s, 1507s, 1465s, 1384s, 1320s, 1261s, 1208s, 1182s,

Junnila et al.

1112s, 1026w, 995w, 914w, 850s, 804w, 724w, 602w. Fluorescence (in water): λexc ) 505 nm, λem ) 526 nm. 1-(Rhodamine-5(6)-yl)-3-butylthiourea Control 2. Under nitrogen atmosphere, butylamine (0.106 mL, 0.5 M in EtOH, 0.053 mmol) was added to the solution of Rhodamine B isothiocyanate (21 mg, 0.039 mmol) in dry ethanol (0.1 mL) using a gas-tight microsyringe. The mixture was stirred at room temperature with protection from light overnight, then heated at 70 °C for 6 h. Progress of the reaction was monitored by TLC (eluent CH2Cl2/methanol, 4:0.3 v/v). After removal of the solvent, the final product was achieved by reprecipitation of the reaction mixture dissolved in minimal methanol with the addition of diethyl ether to give a deep purple powder of control 2 (8.3 mg, 35%) 1H NMR (CH3OH-d4, 400 MHz) δ: 0.92 (t, 3H, J ) 7 Hz), 1.30–1.43 (m, 14H), 1.75 (m, 2H), 2.00 (s, 1H), 2.97 (m, 2H), 3.60 (m, 8H), 6.67–6.93 (m, 6H), (d, 7.17 1H, J ) 10 Hz), (d, 7.47 1H, J ) 10 Hz), 8.23 (s, 1H). IR (KBr, cm-1): 3422br, 3213br, 2967s, 2936w, 2878w, 2793m, 2527w, 2416w, 1645ws, 1590s, 1528s, 1466s, 1412s, 1338s, 1274s, 1247s, 1181s, 1133s, 1075s, 1012w, 977w, 923w, 825w, 685w. Fluorescence (in water): λexc ) 553 nm, λem ) 580 nm. Parasite Material. Male Mongolian gerbils (Meriones unguiculatus) infected intraperitoneally with 400 L3 infective larvae of B. malayi were obtained from Dr. J. McCall, University of Georgia. Microfilariae were obtained from the peritoneal cavities of gerbils by lavage with 5 mL phosphate buffered saline; adults were recovered during the subsequent necropsy. Adult worms were washed 3 times in RPMI 1640 containing 20 mM Hepes pH 7.3 to remove any adhering host cells; this was verified by light microscopy. Microfilariae were concentrated by centrifugation of the peritoneal exudates for 5 min at 12 000 rpm; the supernatant was discarded. The pellet containing microfilariae was washed 3 times by adding 1 mL Hepesbuffered RMPI 1640 followed by gentle agitation and centrifugation. Microfilariae were further separated from host cells by the percoll gradient method. Antifilarial Activity of 3 in Gerbils. Antifilarial activity of the intermediate compound 3 was assessed by in ViVo comparison with DEC at two dose rates. Gerbils (N ) 4 for each treatment group) received one of the following combinations: 200 mg/kg or 50 mg/kg DEC in 200 µL normal saline, or 160 mg/kg or 38 mg/kg compound 3 in 200 µL normal saline, or 200 µL normal saline alone for control. All gerbils received

Figure 4. Representative controls. (A) Adult female anterior end stained with unconjugated FITC; (B) adult female posterior end stained with unconjugated RHB; (C) adult female anterior end stained with modified control FITC (control 1); (D) light microscope view of C; (E) adult male anterior end stained with modified control RHB (control 2); (F) light microscope view of E. All worms were treated for 8 h, either in medium alone in the case of the controls or with the probe at a concentration of 5 µg/mL.

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Scheme 4. Synthesis of FITC-Control and Rhodamine-Control 2 Fluorophores

intraperitoneal injections of the specified dose or control for three consecutive days. After necropsy on the third day, adult worms were counted under a dissecting microscope, and larvae were counted using the hemecytometer method. Means +/standard deviation were calculated, and the difference between the parasites recovered from the treatment groups and control untreated group was assessed statistically. Statistical Analysis. Data were analyzed using SPSS (version 10.0) statistical package (Chicago, IL). Samples were compared with the Kruskal–Wallis rank test. Significance was assessed at P e 0.05. In Witro Culture of Brugia malayi with Experimental Compounds. Individual adult worms or groups of approximately 10 microfilariae were incubated in 2 mL of standard medium (without fetal calf serum) as previously described in detail. (8) Briefly, RPMI 1640 was supplemented with 25 mM Hepes buffer, 2 mM glutamine, 100 U/mL streptomycin, 100 µg/mL penicillin, and 0.25 µg/mL amphotericin B. Cultures were set up in triplicate, and incubated at 37 °C in a 95% air/ 5% CO2 atmosphere. An optimal concentration of 4 or 5 (5 µg/mL) was determined by examining worms incubated in concentrations ranging from 0.1 to 20 µg/mL. The optimal concentration was defined as the concentration in which uptake could be detected with the lowest possible amount of background fluorescence. For all subsequent studies, worms received 4 (5 µg/mL), 5 (5 µg/mL), 5 µg/mL native DEC, 5 µg/mL RHB unconjugated mixed isomers, or unconjugated FITC isomer 1 (5 µg/mL) for 8 h. Live stained worms were then washed for 4 h in standard medium (4 changes—1 every hour). Worms were mounted with pro-long antifade kit (Molecular Probes) according to the manufacturer’s instructions. Confocal Microscopy. Confocal images were taken on a Carl Zeiss LSM 510 Laser Scanning System. Images were processed with Zeiss LSM 5 Image Browser (version 3.1.0.99).

RESULTS Synthesis of Fluorescent DEC Analogues. The design and synthesis of the DEC fluorescent analogues 4 and 5, utilized the only site with any flexibility in the molecule, one of the terminal methyls of the diethylamino group. From prior structure–activity studies, it was known that the antifilarial activity of DEC was retained when one of the ethyls was replaced with smaller substituents and the other is larger. (3) To build the terminal amino substitutent in 3, the selective protection of the primary amine with a t-boc group was used before condensation with the piperazinecarbonyl chloride to give 2 after two steps. Removal of t-boc with acid restored the primary amine, which was then immediately condensed with the fluorescent isothio-

Figure 5. Sites of fluorescent DEC analogue accumulation in adult worms. (A) Adult female esophagus stained with DEC-FITC 4; (B) light microscopy view of A; (C) adult male pharynx stained with DECRHB 5; (D) light microscopy view of C. Times and probe concentrations used correspond to those used in Figure 4.

Figure 6. Sites of fluorescent DEC analogue accumulation in adult female worms. (A) Adult female uterus stained with DEC-FITC 4 (midbody); (B) light microscopy view of A; (C) adult female uterus and vulva stained with DEC-FITC 4 (anterior end); (D) light microscopy view of C. Times and probe concentrations used correspond to those used in Figure 4.

cyanate FITC or Rhodamine B to give the final fluorescentlabeled DEC analogue 4 or 5. The four step sequence is shown in Schemes 2 and 32 and 3. Microfilarial Recovery. Within 24 h after treatment, both concentrations of DEC and 3 resulted in a significant increase of microfilarial levels compared to the control (P < 0.05). The low dose rate of both compounds demonstrated a significantly higher microfilarial increase than the high dose rate (P < 0.05) (Figure 1). To determine if 4 and 5 required more time to act on mf, the experiment was carried out again with the sacrifice taking place at 10 days posttreatment instead of 24 h. Ten days after DEC or 3 treatment, microfilarial levels were significantly lower (P

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Figure 7. Sites of fluorescent DEC analogue accumulation in adult male worms. (A) Adult male testis stained with DEC-RHB 5 (midbody), spermatozoa visible; (B) light microscopy view of A; (C) adult male testis stained with DEC-FITC 5 (mid-body), spermatozoa visible; (D) light microscopy view of C. Times and probe concentrations used correspond to those used in Figure 4.

< 0.05) in the treated animals compared with the control, for both compounds and dose rates (Figure 2). Adult Recovery. At 24 h after treatment, DEC and 3, at both high and low dose rates, showed adulticidal activity, and adult worm numbers were significantly lower than in the control gerbils (P < 0.05). The higher dose rate of both compounds was more effective than the lower dose rate of both compounds (P < 0.05) (Figure 3). At 10 days posttreatment, there were very few adults alive, and these were difficult to count, as only parts of worms were visible in clusters of nodules. Adult counts at 10 days are not shown. Confocal Fluorescence Microscopy. Because 3 and DEC behaved similarly, 3 was conjugated to fluorescent molecules FITC and RHB for further use in confocal fluorescence microscopy. The fluorophores FITC and RHB (used unconjugated as controls) were found to be highly reactive, producing bright and nonspecific staining in the parasites (Figure 4) and were therefore unsuitable as controls. The reactive groups of these fluorophores were modified (Scheme 4) to closely resemble the conjugates used in this study. Control worms treated in media alone (8 h) and worms treated with modified control fluorophores (5 µg/mL for 8 h) showed no significant staining. At the same concentration (5 µg/mL) and incubation time (8 h), 4 and 5 accumulated in the pharynx, esophagus, and near the nerve ring of all adults (Figure 5), uteri and vulva (Figure 6), and testes (Figure 7). When adult worms were examined by confocal microscopy, the intense staining observed in the uterus, testis, and intestine appeared to be localized to the lining (epithelium) of these organs. Additionally, in a few distinct sections of male testis, the contents of this organ were also stained (Figure 7). Microfilariae accumulated stain in three distinct body areas (Figure 8); area 1 consisted of the excretory pore and the excretory cell, area 2 was the innerbody, and area 3 consisted of the G2, 3, and 4 cells and possibly the anal pore. As well, it appeared that somatic cell nuclei of microfilaria accumulated stain.

DISCUSSION Conjugation of DEC to fluorescent molecules required a β-amine substitution. It was therefore necessary to determine

Figure 8. Sites of fluorescent DEC analogue accumulation in microfilariae. (A) Microfilaria stained with 4 DEC-FITC; (B) light microscopy view of A; (C) microfilaria stained with DEC-RHB 5; (D) light microscopy view of C; (E) reprinted from Noble and Noble (1976) Parasitology: The Biology of Animal Parasites, 4th ed., Lea and Febiger, Philadelphia. Times and probe concentrations used correspond to those used in Figure 4.

whether compound 3 would act similarly to DEC in its filaricidal abilities. Similar strategies have been used to determine the subcellular localization of the adrenoceptor inhibitor prazosin when conjugated to BODIPY. (9) In survival assays carried out in the gerbil/Brugia malayi model, both DEC and 3 caused a surprising initial increase in peritoneal microfilariae 24 h posttreatment. This may be due to toxic effects of these compounds on the adult worms, causing them to release larvae from the uterus. In this particular host/parasite model, DEC and related compounds required several days to effectively reduce microfilariae, so the experiment was carried out again but animals were sacrificed at 10 days posttreatment instead of 24 h. This time, treatment with both DEC and 3 significantly reduced the number of peritoneal microfilariae and further reduced the number of adult worms. In fact, only parts of worms were visible, as they were being broken down in nodules found on the spleen and the liver and free-floating in the peritoneal cavity. Since 3 produced similar antiparasitic activity to DEC, it was conjugated to FITC and RHB for further use in confocal fluorescence microscopy. In this study, no fluorescence was seen in the muscular system and very little fluorescence was seen in the nervous system (confined only to the area around the anterior nerve ring), which tends to exclude a strictly neuromuscular mode of action of DEC. Adult Brugia malayi did, however, strongly accumulate 4 and 5 in the pharynx, esophagus, intestine, and reproductive organs. The sites of accumulation seen here in adult Brugia exposed to the fluorescent DEC analogue together with previous studies

Fluorescent Diethylcarbamazine Analogues

that propose DEC acts by “unmasking previously hidden antigens” could suggest that the drug interferes with either excretion/secretion of a key immunomodulatory factor or with uptake/adsorption of host proteins, for example, serum albumin, which is believed to hide the worms from the immune system. Clearly the drug is accumulating at these sites and this is likely to occur where the drug receptors are localized. What is not observed is a random or diffuse distribution of 3, which would suggest that it has little specific activity. Chen and Howells were unable to demonstrate an oral (i.e., pharangeal pumping) uptake in Vitro of trypan blue, ferritin, or fluorescein conjugated serum proteins by larvae and adults of Brugia pahangi. (10) It was later demonstrated that the gut of filarial worms is largely nonfunctional and that the cuticle serves as an absorptive as well as excretory surface. (11) As no significant fluorescence was detected in the cuticle, it is likely that DEC and DEC analogues are taken up transcuticularly and subsequently accumulate in target tissues. A study conducted in 1996 provided the first evidence that suggested the existence of an excretory organ in filarial worms in the region of the anterior nerve ring (12), and it is believed that the uterus, vas deferens/testis, and intestine are the major organs where excretory proteins, such as superoxide dismutase (13) and gluthaione S-transferase (14) among others, are synthesized and exported. Our data show fluorescent DEC analogues accumulated in major sites of synthesis of secreted protein (13), which lends new evidence to the idea that DEC may be interfering with a key excretory/secretory product. Microfilariae treated with fluorescent DEC analogues accumulated fluorophore in the inner body, the excretory pore, and the excretory cell, as well as the G-cells. Slight fluorescence was also seen in the pharyngeal thread and some somatic cell nuclei. The microfilariae have a nonfunctional gut (15, 16), but they do contain metabolically active sites within the body. (15) Feng (1936) demonstrated that the inner body of Brugia microfilaria occupies the space that later becomes the intestine in developing larvae. It is interesting to note that DEC analogues accumulate in tissues destined to become those which also accumulate these compounds in the adult worms. The biological significance of these findings is that the drug receptors are most likely localized at the accummulation sites of 3, and the question we are now determining is the specific protein or receptor target for these drugs. Advancing knowledge of drug delivery and activity requires an understanding of drug design and drug stability while taking into account the complexities imposed by the biological system (17) including cell/tissue penetration, drug-target interaction, and the pharmacodynamic consequences of alterations to native drugs. (18) In this study, we developed and synthesized fluorescent drug analogues that helped to address these complexities. The fluorescent DEC analogues synthesized here are small enough to penetrate the filarial cuticle, the crucial pharmacophore is accessible to potential receptors, and the major compound to be attached to the fluorophore 3 retains filaricidal activity. The use of fluorescent drugs to investigate their targets in cells and tissues continues to expand at a rapid pace. Fluorescence imaging of protein–ligand interactions could provide an important biological context for drugs under study that might be otherwise overlooked by traditional biochemical techniques.

ACKNOWLEDGMENT The authors gratefully acknowledge support from the FQRNT and the Centre for Host-Parasite Interactions for support in the

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form of a Centre New Iniative grant. Support from NSERC (to R.P. and D.S.B.) is gratefully acknowledged. In addition, fellowship support for I.P. and C.S. from the CIHR Chemical Biology Traineeship Program is gratefully acknowledged.

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