Synthesis and Evaluation of Dinitroanilines for Treatment of Cryptosporidiosis

Abstract

The efficacy of a series of dinitroaniline herbicide derivatives for the treatment of Cryptosporidium parvum infections has been studied. The lead compounds oryzalin (compound 1) and trifluralin (compound 2) have low water solubility (<3 ppm) which was alleged to be a major contributor to their poor pharmacokinetic availability. Derivatives of compounds 1 and 2 were synthesized. In these derivatives the functionality at the C-1 amine position or the C-4 position was substituted with groups with various hydrophilicities to determine if a direct relation existed between water solubility and overall activity. The chlorinated precursors of these derivatives were also examined and were found to be less active in the C. parvum assays, a result in direct contrast to earlier work with Leishmania. Enhanced water solubility alone did not overcome the drug availability problem; however, several candidates with similar activities but with toxicities lower than those of the lead compounds were produced.

Opportunistic infections caused by protozoan parasites are a major concern for immunocompromised patients, especially those with AIDS (19). These obligate, intracellular parasites infect a variety of tissues, resulting in chronic disease states including bronchial dysfunction, blindness, encephalitis, and severe diarrhea. The last malady, which can be caused by the apicomplexan Cryptosporidium parvum, is often protracted and may be life-threatening in immunodeficient individuals (13, 29). Adequate therapies for clearing the host of these parasites are lacking, and the few agents demonstrating efficacy may be best suited for prophylaxis. While the in vivo and in vitro activities of several agents (spiramycin, halofunginone lactate, eflornithine, sinefungin, and paromomycin) have provided some promising results (3, 5, 10, 20, 21, 23), as has the hyperimmune bovine colostrum (27), thorough clinical and toxicological studies have not been completed. The prevalence and the recurring nature of these infections underscore the need for selective, nontoxic treatments.

Antimicrotubule compounds have shown promise as chemotherapeutic agents by reducing infections in vitro with a human cell line (28). The drugs used in that study, colchicine and vinblastine, disrupt both parasitic and mammalian microtubule formation and therefore would not be selective for the treatment of infectious states in mammalian hosts. However, the discovery that the antimicrotubule herbicides oryzalin (compound 1) and trifluralin (compound 2) (Fig. 1) were selective toward Leishmania (6–8) and Trypanosoma parasites (9) in mammalian hosts provided an exciting breakthrough for parasitic chemotherapy; selective eradication of the parasitic organism occurred with minimal damage to the host. Useful levels of activity against other apicomplexan parasites with negligible toxicity to the mammalian host have also been seen with these agents (1, 12, 16).

FIG. 1.

Anti-microtubule agents oryzalin (compound 1) and trifluralin (compound 2).

These dinitroaniline herbicides have a low water solubility and an unusually low vapor pressure, two features that have created problems in their development as antiparasitic agents; the results of heterogeneous biological assays are difficult to interpret, inordinately large dosages are necessary for in vivo studies, and the exogenous solvents frequently used to increase solubility lead to undesirable side effects. The successful drug candidate should be appreciably soluble in aqueous medium to allow effective transport, cellular recognition, and a residence time at the target site and should also be capable of membrane permeation; the parasites are encased in a parasitophorous vacuole within the host cell. The incorporation of more hydrophilic groups into the molecular structure will alter the water solubility, although a compound’s motility in lipophilic membranes is delicately balanced between hydrophilic and hydrophobic groups. The Hansch parameter [log(P) or π value] is useful for predicting this motility, and a log(P) value of 2.00 to 2.40 is considered optimum for a substance that must traverse cellular membranes (26). The log(P) value may be calculated for any functionalized compound by summing the π values of the various components from a standard table (15).

We have synthesized derivatives of dinitroanilines 1 and 2 (Fig. 2) in which the amine substituent has been altered to (i) increase the hydrophilicity of the molecule, (ii) restrict the degrees of freedom in the alkyl chains (cyclic structures), or (iii) increase the hydrogen-bonding capabilities (secondary amines). The substituents at C-4 were also replaced with isosteric functional groups (CONHR and CO₂H) to impart different solubility properties. The relative hydrophilicities of these derivatives were determined by correlating the calculated Hansch parameters with water solubility data from a UV analysis. These new compounds were also assayed for their parasiticidal activity by an improved chemiluminescence method (30).

FIG. 2.

Synthesis of dinitroaniline derivatives.

We have also investigated the chlorinated precursors (compounds 3 to 6) to determine their activities against the C. parvum organism. This analysis was stimulated by a report by Callahan et al. (4) that showed that chloralin (compound 3B) is 10² more active than trifluralin against Leishmania. They proposed a mechanism of action that suggested that a nucleophilic aromatic substitution between cysteine residues and the dinitroanilines was responsible for the observed activity. If C. parvum is susceptible to the chlorinated derivatives and incorporates these compounds through the formation of covalent bonds, then the specific interaction could be located and a better understanding of dinitroaniline efficacy would result. Herein we describe the synthesis of these derivatives and the results of the biological screenings.

MATERIALS AND METHODS

Melting points have not been calibrated and as such are uncorrected. Infrared (IR) spectra were recorded as thin films on NaCl plates for liquid samples or KBr pellets for solids and are calibrated to a polystyrene film standard. The ¹H nuclear magnetic resonance (NMR) and ¹³C NMR spectra were recorded at 360 and 90 MHz, respectively, and the chemical shifts are reported in parts per million relative to an internal tetramethylsilane signal. Elemental analyses were performed by either Midwest Microlab, Indianapolis, Ind., or Supersun Technology Analytical Lab, Stonybrook, N.Y.

Chemicals and solvents were purchased from Aldrich Chemical Company, VWR Scientific, and J. T. Baker and were used as received. The 4-chloro-3,5-dinitrobenzenesulfonamide (compound 3a) was prepared as described previously (25), and N,N-bis-(methoxyethyl)amine was synthesized by the procedure of Remizov (22). All chromatography solvents were distilled from glass prior to use. Silica gel 60-H (E. Merck) was used for flash chromatography, and precoated glass plates 60F-254 (thickness, 0.25 mm; E. Merck) were used for thin-layer chromatography.

General procedure for aryl chloride displacement.

A suspension of the aryl chloride (compound 3, 4, 5, or 6) (5.30 mmol) in 95% ethanol (25 ml) was stirred as the amine (5.80 mmol) and triethylamine (5.80 mmol) were added simultaneously via a syringe. The orange mixture was refluxed for 1 h, cooled to room temperature, and poured over ice (200 g). After stirring, the orange precipitate that formed was removed by filtration or the yellow-orange mixture was extracted with ethyl acetate (EtOAc) (three times with 50 ml each time). The organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo to provide an orange oil. The crude reaction products were further purified by flash chromatography (120 g of SiO₂, 33% EtOAc-hexanes) to afford oils that solidified upon prolonged standing at 1.0 torr (Table 1, compounds 1a to 1g, and 2a to 2g; Table 2, compounds 7a to 7f).

TABLE 1.

Analytical data for derivatives of oryzalin (compounds 1a to g) and trifluralin (compounds 2a to g)

Compound	% Yield	mp (°C)	1H NMR (DMSO-d6)	Elemental analysis

TABLE 2.

Analytical data for C-1 derivatives (compounds 7a to f)

Compound	% Yield	mp (°C)	1H NMR (DMSO-d6)	Elemental analysis

Water solubility measurement.

The water solubilities of the new compounds were determined from UV data collected with two independent samples of the derivatives. These samples were prepared by suspending 10-mg samples of the material in H₂O (10 ml) and then stirring one at 0°C and the other at 60°C for 2 h, followed by equilibration at room temperature (24°C) for 12 h. These samples were passed through a glass wool filter, and the UV spectra were recorded. A standard solution of known concentration was prepared by dissolving 20 mg of the compound in methanol (50 ml) and then diluting this solution with H₂O (20 or 50 times), followed by recording of the UV spectrum. From the standard of known concentration the absorptivity could be calculated, and the absorptivity was then used to determine the concentration of substrate in the pure H₂O sample. The solubilities are reported in parts per million, and the results are averages of duplicate runs.

Methyl 4-chloro-3,5-dinitrobenzoate (compound 5).

To a solution of 10.0 g (41 mmol) of compound 4 in diethyl ether (75 ml) at 0°C was added an excess of ethereal diazomethane. After stirring for 12 h, the excess diazomethane was quenched by the dropwise addition of acetic acid (0.2 ml). The solvent was removed in vacuo and the residue was recrystallized from diethyl ether to provide 8.2 g (78%) of the ester as a white solid. mp 102 to 103°C; R_f, 49 (50% EtOAc-hexanes); IR (film) ν 3083, 1728, 1536, 1435, 1348, 1308, 1290, 922, 718, 670, 665 cm⁻¹; ¹H NMR (CDCl₃) δ 8.60 (s, 2H), 4.02 (s, 3H); ¹³C NMR (CDCl₃) δ 162.4, 149.6, 130.8, 128.2, 124.8, 53.7.

4-Chloro-3,5-dinitro-N-methylbenzamide (compound 6).

A flask containing 6.00 g (24.4 mmol) of compound 4 and 8.10 g (38.9 mmol) of phosphorous pentachloride was thoroughly mixed by shaking vigorously and, after the fitting of a condenser, was heated to 130°C. The resultant orange solution was heated for 2 h and cooled to 70°C, at which point the phosphoryl chloride by-product was removed by aspirator distillation. The solid acid chloride product was dissolved in acetone (50 ml), and the solution was cooled to −60°C prior to the dropwise addition of 5.50 ml (63.0 mmol) of a 40% aqueous solution of methylamine. It is imperative that the addition be performed at −60°C to avoid competitive displacement of the aryl chloride. The addition took 30 min, and the formed precipitate became very thick such that mixing could be achieved only by swirling. After the addition was complete, the slurry was poured into ice water (200 ml), and the yellow precipitate was removed by filtration, washed with H₂O (100 ml), and dried. This provided 5.14 g (81%) of the N-methylbenzamide as a yellow solid. mp 188 to 189°C; R_f, 26 (50% EtOAc-hexanes); IR (film) ν 3314, 3070, 1638, 1556, 1541, 1407, 1368, 1340, 1315, 921, 717, 671, 665 cm⁻¹; ¹H NMR (dimethyl sulfoxide [DMSO]-d₆) δ 8.65 (br s, 1H), 8.43 (s, 2H), 2.50 (d, J = 4.5 Hz, 3H); ¹³C NMR (DMSO-d₆) δ 162.1, 148.7, 135.0, 128.6, 127.1, 26.5.

Antiparasitic activity.

The antiparasitic activities of the dinitroaniline derivatives were determined in vitro by measuring the growth of C. parvum in Madin-Darby canine kidney cells by a chemiluminescence immunoassay (30). The source for C. parvum oocysts was the IOWA bovine isolate produced by newborn Holstein bull calves, and this was originally obtained from the National Animal Disease Center, Ames, Iowa. The oocysts were collected, treated with NaIO₄ (10 mM in 0.1 M sodium acetate [NaOAc]-buffered saline), washed with 0.1 M NaOAc-buffered saline, and centrifuged. The pellets were suspended in Dulbecco’s modified Eagle medium base containing 0.75% sodium taurocholate, incubated for 10 min, and then diluted with Ultraculture medium (Biowhittaker, Inc., Walkersville, Ill.) prior to inoculation onto plates containing confluent Madin-Darby canine kidney (MDCK) cells. After an initial incubation period (3 h), the culture wells were washed with phosphate-buffered saline (PBS) and filled with fresh Ultraculture medium with or without drug. The dinitroanilines were dissolved in 100% DMSO before addition to the Ultraculture medium, and the final DMSO concentration in the cultures was less than 0.01%. Ultraculture medium with 0.01% DMSO was used for all controls. Culture plates were incubated at 37°C in a 5% CO₂ atmosphere for 48 h, washed with PBS, and then fixed with Bouin’s solution.

Fixed culture monolayers were decolorized by washing with 70% ethanol (five times), followed by washing with 20 mM Tris-HCl (pH 7.5)–150 mM NaCl–0.05% Tween-20 (TBST), and were finally blocked by shaking with 1% bovine serum albumin–TBST at room temperature for 30 min. After treatment with rabbit anti-C. parvum sera, the plates were sequentially incubated with biotin-labeled goat anti-rabbit immunoglobulin G, horseradish peroxidase-labeled streptavidin, and enhanced luminol as the substrate. The plates were read with an ML3000 Luminometer to determine the relative light units. The means were calculated from the readings for four replicate wells, and all experiments were repeated at least twice. The effective concentrations that caused the death of 50% of the parasite bodies present (EC₅₀s) are presented in Table 3. The median inhibitory effects were calculated by using Combo stat software (2).

TABLE 3.

Antiparasitic activities of the studied compounds against C. parvum

Series and compound	Log(P)	H2O solubility (ppm)	EC50 (μM [Ra])	TC50b (μM [R])
Series A
1	1.93	7.0	0.15 (0.99)	3.9 (0.99)
1a	−1.39	2,500.0	—c	—d
1b	−0.01	380.0	10.3 (0.97)	9.7 (0.87)
1c	−0.88	60.0	0.46 (0.87)	25.5 (0.96)
1d	1.05	11.0	0.90 (0.97)	—d
1e	1.98	18.0	0.74 (0.97)	85.9 (0.95)
1f	0.48	11.5	1.26 (0.76)	36.6 (0.94)
1g	1.15	9.0	71.4 (0.96)	153.0 (0.91)
Series B
2	4.41	5.0	0.13 (0.87)	—d
2a	0.84	1,290.0	11.3 (0.94)	65.9 (0.94)
2b	2.22	137.0	6.5 (0.89)	139.9 (0.88)
2c	1.60	10.0	6.16 (0.73)	—d
2d	3.53	2.4	2.1 (0.88)	—d
2e	4.46	2.6	0.67 (0.66)	185 (0.92)
2f	2.96	10.0	1.39 (0.75)	—d
2g	3.63	6.0	0.12 (0.93)	—d
Series C
7a	3.20	NAe	5.8 (0.98)	93.1 (0.06)
7b	0.87	NA	—c	—d
7c	0.93	NA	66.9 (0.86)	—d
7d	2.28	NA	7.2 (0.98)	157 (0.95)
7e	2.55	NA	4.7 (0.98)	96 (0.97)
7f	0.22	NA	49.5 (0.84)	96 (0.97)

^aR, confidence limits. 

^bTC₅₀, concentration at which 50% of the control cells are killed. 

^c—, no activity detected at a concentration of 10 μM or less. 

^d—, no toxic effects noted at the highest concentration tested (80 μM). 

^eNA, not available. 

Toxicity.

The toxicities of the derivatives toward MDCK cells were determined by a commercial tetrazolium dye reduction assay (Cell Titer 96; Promega, Madison, Wis.), with data collected over a range of concentrations (0.1 to 80 μM). The concentrations that caused toxic effects in 50% of the control cells were calculated as described above for the EC₅₀s and are listed in Table 3.

RESULTS AND DISCUSSION

Our derivatives had a wide range of solubilities and are stable, easily handled solids that can be divided into three main categories: the C-1 amine derivatives of oryzalin (compounds 1a to 1g; series A), trifluralin (compounds 2a to 2f; series B), and the C-4 isosteric derivatives (compounds 7a to 7g; series C). A correlation between the calculated Hansch parameter [log(P)] and the measured H₂O solubility for the compounds in series A and series B was quite good when the samples examined were either extremely hydrophilic [log(P), <0.0] or lipophilic [log(P), >3.0]. A similar correlation with the other compounds gave poor results, which indicates that other variables influence drug partitioning. For example, the oxygenated side chains (compounds a to c) showed excellent water solubility, but the cyclic ethers (compounds c) were less water soluble than that predicted by log(P) calculations. The increased lipophilicity of the secondary amine substrates (compounds d to g) could be ascribed to a decreased basicity of the lone nitrogen pair due to more effective conjugation with the nitro groups. Within this amine subset of compounds, the variances arise from a difference in the steric environment around the nitrogen; the most hydrophilic member of this group, compound f, has the least encumbered nitrogen atom. The use of these parameters for the evaluation of differences in solubility for the dinitroaniline class of compounds has its merits; however, the correlation is not perfect for the intermediate-range compounds [log(P), 0.5 to 2.0]. It is clear that the Hansch system, a measure of compound partitioning, cannot be used as a definitive judge of H₂O solubility.

The results from the assays (Table 3) indicate that an increase in the water solubility alone is not sufficient for enhancing parasiticidal activity. Impressive numbers were achieved for several of the derivatives. In particular, compounds 1c to 1e, 2e, and 2g provided EC₅₀s in the range of those of the lead compounds without detectable toxicity; however, these compounds showed no increased hydrophilicity. The most water soluble derivatives (compounds a and b) showed little or no activity (<100 times that of the lead compounds), which may be due to an inability to transverse cell membranes; the Hansch parameters predict that they would not be very motile. Those derivatives with log(P) values more appropriate for trans-membrane motility showed activity in the assay, and in some cases (compounds 1d, 2c, 2d, and 7c) the toxicities were significantly lower than those of the two lead compounds, but the EC₅₀s were also higher. These results are intriguing, for they suggest that while water solubility alone will not solve the drug availability problem, a viable drug delivery system may uncover some interesting drug profiles.

A prominent feature of the compounds in series C was that these compounds generally showed low toxicities. As with the previous series (A and B), an increase in hydrophilicity did not guarantee higher activity levels, and all of the morpholine derivatives (compounds 7b, 7c, and 7f) were less active than their amine counterparts (compounds 7d and 7e). The simple amine analogs showed some activity and low toxicity; however, their activities were far too low for further consideration.

Chlorinated derivatives 3a (EC₅₀ = 50 μM) and 4 (EC₅₀ = 106.6 μM) were not as active as the lead compounds against C. parvum, whereas derivatives 3b and 5 were toxic at 20 μM. These results are in contrast to those of the Leishmania studies of Callahan et al. (4), in which chloralin (compound 3b) showed a high level of activity against those parasites. Our findings suggest that a universal mode of interaction of dinitroanilines with apicomplexans is unlikely, and we are conducting labeling studies to determine how these compounds interact with C. parvum.

Several of the active compounds were extremely toxic, which raises the issue of whether the observed activity is a consequence of the toxic effects or whether there are concurrent antiparasitic responses and cytotoxic effects. It is clear that these compounds are capable of eradicating C. parvum with a minimal toxic response, but a derivative with a lower effective dose is necessary. Maintaining the generic composition and increasing the water solubility did not sufficiently improve availability. However, the promising activities of compounds 2c to 2g; coupled with their decreased toxicities and sustained antiparasitic activities, supports the development of a better drug delivery system.

The ultimate target for such a drug delivery system is the epithelium of the gut, so the appropriate vehicle must be recognized by the receptors on the surface of this organ. Carbohydrate receptors are abundant on these cells, and in recent years glycoconjugate chemistry has blossomed as an effective strategy for the delivery of molecules with poor transport profiles (11, 14, 24). The application of such a strategy will require the synthesis of dinitroaniline derivatives that can function as an acceptor in glycosylation reactions; i.e., the acceptor must have an accessible hydroxyl function, while still maintaining significant levels of activity. We are focusing on analogs in the trifluralin series (compound 2) because these are considerably less toxic than the sulfonamide congeners (compound 1). Derivatives of pendimethalin, another dinitroaniline with activity greater (10²) than those of the lead compounds tested in this work, are also being pursued (17, 18).