Evaluation of New Thiazolide/Thiadiazolide Derivatives Reveals Nitro Group-Independent Efficacy against In Vitro Development of Cryptosporidium parvum

Abstract

Thirty-nine new thiazolide/thiadiazolide compounds were compared with the nitrothiazole nitazoxanide for activity against Cryptosporidium parvum development in HCT-8 cells. Twenty-seven agents exerted ≥90% inhibition. Agents with a lower 50% inhibitory concentration (IC₅₀) than nitazoxanide were either NO₂ or halogen 5 substituted on the thiazole moiety. Other 5 substitutions such as methyl, C₃H₇, C₆H₁₁, H, SO₂CH₃, and SCH₃ negatively impacted activity. Five-substituted deacetylated analogues exhibited higher IC₅₀s than their acetylated counterparts. Halogeno-thiazolide/thiadiazolides may provide valuable nitro-free alternatives to nitazoxanide.

Human cryptosporidiosis is recognized as a threat to immunocompromised individuals and is responsible for diarrhea in immunocompetent children and adults (2). Few agents exhibited a suppressive effect on in vitro Cryptosporidium parvum growth, and none was effective in all situations in vivo (1, 19). The nitrothiazole nitazoxanide [2-acetyloxy-N-(5-nitro 2-thiazolyl)benzamide] (NTZ) has been approved for the treatment of diarrheas caused by C. parvum and Giardia duodenalis in immunocompetent adults and children from 12 months of age (7, 12, 18). Recently, NTZ-related thiazolide/thiadiazolide derivatives were reported to be active in vitro against anaerobic bacteria, influenza A virus, an extracellular protozoan, G. duodenalis, and two closely related apicomplexan protozoa, Neospora caninum and Sarcocystis neurona (6, 11, 14, 17, 20). In the present study, new thiazolide/thiadiazolide compounds were compared with NTZ for activity against C. parvum in vitro development.

Agents (Table 1 and Fig. 1) were synthesized at the Romark Center for Drug Discovery at the University of Liverpool, Liverpool, United Kingdom, and supplied by Romark Laboratories, L.C., Tampa, FL. Powder forms were solubilized in dimethyl sulfoxide (5 g/liter) and stored at −20°C until used. Final concentrations in cultures ranged from 0.1 to 10 mg/liter, corresponding to a maximum dimethyl sulfoxide concentration of 0.2% (vol/vol). HCT-8 cells (ATCC CRL 244; American Type Culture Collection, Manassas, VA) were cultured as previously described (8). Agent-induced effects on the viability of confluent HCT-8 cells were monitored by light microscopic inspection and a tetrazolium assay (CellTiter 96 AQ_ueous nonradioactive cell proliferation assay; Promega, Madison, WI). Absent, mild, moderate, and severe effects were rated as described previously (8, 22). C. parvum oocysts of the Nouzilly isolate (a kind gift from M. Naciri, INRA, Nouzilly, France) were purified from calf feces and permitted to excystate as previously described (10). After being sieved through a 5-μm Nuclepore filter, 2.5 × 10⁵ to 5 × 10⁵ sporozoites were added to each confluent HCT-8 monolayer well. Two hours later, supernatants were removed and replaced with agent-containing or agent-free medium. Forty-six hours later, all parasite stages of methanol-fixed cultures were counted in 20 microscopic fields (×1,250) using indirect immunofluorescence as described previously (9, 10). Each set of experiments was done at least twice. Inhibitory activity (percent) was calculated as follows: [(mean number of parasite forms in treated cultures − mean number of parasite forms in untreated cultures)/mean number of parasite forms in untreated wells] × 100. Results are expressed as 50% inhibitory concentrations (IC₅₀s); the IC₅₀ was defined as the concentration (wt/vol) of an agent which resulted in a mean 50% inhibitory activity. For agents exhibiting ≥90% inhibitory activity, IC₉₀s are similarly given. The significance of differences between the endpoint values of experimental and control cultures was determined by using Student's t test, thus assuming normal-like distributions of values. P values of <0.05 were considered statistically significant.

TABLE 1.

Chemical structures and in vitro inhibitory activities on C. parvum development of NTZ and 39 new thiazolide/thiadiazolide derivatives

Agent	Mol wt	Substitution						IC50(mg/liter)a	IC90(mg/liter)a
		Thiazole ring		Benzene ring
		X	Y	R1	R2	R3	R4
NTZ	307.3	NO2	H	OAcb	H	H	H	1.2	10
RM4801	279.3	NO2	H	OH	CH3	H	H	0.7	5.8e
RM4802	321.3	NO2	H	OAc	CH3	H	H	0.5	1e
RM4805	295.3	NO2	H	OH	OCH3	H	H	0.4	1e
RM4807	279.3	NO2	H	OH	H	H	CH3	0.5	0.9e
RM4809	279.3	NO2	H	OH	H	CH3	H	1.4	5e
RM4814	279.3	NO2	H	OMec	H	H	H	5.5	NAd
RM4815	279.3	NO2	CH3	OH	H	H	H	0.6	1e
RM4804	310.8	Cl	H	OAc	CH3	H	H	0..4	1e
RM4848	254.7	Cl	H	OH	H	H	H	2.6	6e
RM4850	268.7	Cl	H	OH	H	H	CH3	0.6	6.7e
RM4851	268.7	Cl	H	OH	CH3	H	H	2..3	4.6e
RM4852	268.7	Cl	H	OH	H	CH3	H	0.8	5.5e
RM4865	310.8	Cl	H	OAc	H	H	CH3	0.1	3.8e
RM4803	355.2	Br	H	OAc	CH3	H	H	0.6	5.6
RM4806	371.2	Br	H	OAc	OCH3	H	H	4.6	NA
RM4819	313.2	Br	H	OH	CH3	H	H	1.4	4.8e
RM4820	341.2	Br	H	OAc	H	H	H	0.6	4e
RM4821	355.2	Br	H	OAc	H	H	CH3	1.7	4.4e
RM4822	355.2	Br	H	OAc	H	CH3	H	0.7	3.7e
RM4823	355.2	Br	CH3	OAc	H	H	H	2.1	4.8e
RM4832	299.1	Br	H	OH	H	H	H	2.5	8
RM4847	313.2	Br	H	OH	H	CH3	H	5.2	9.3
RM4858	313.2	Br	CH3	OH	H	H	H	2.9	9.1
RM4859	327.2	Br	CH3	OH	CH3	H	H	0.1	1e
RM4860	389.3	Br	C6H5	OH	CH3	H	H	0.1	1e
RM4861	375.2	Br	C6H5	OH	H	H	H	0.4	1
RM4816f	331.3	CF3	H	OAc	H	H	H	0.7	6.6
RM4854	238.2	F	H	OH	H	H	H	1.8	8.6
RM4862	330.8	C6H4Cl	H	OH	H	H	H	6.1	NA
RM4856	302.4	C6H11	H	OH	H	H	H	>10	NA
RM4855	262.3	C3H7	H	OH	H	H	H	6.7	NA
RM4853	234.3	CH3	H	OH	H	H	H	>10	NA
RM4857	220.3	H	H	OH	H	H	H	>10	NA
RM4813f	263.3	H	H	OAc	H	H	H	5.0	NA
RM4817f	309.4	SCH3	H	OAc	H	H	H	6.4	9.2
RM4863	298.3	SO2CH3	H	OH	H	H	H	6.9	NA
RM4864	340.4	SO2CH3	H	OAc	H	H	H	5.0	NA
RM4818g	510.5	H	CH2COOCH2CH3	OAc	H	H	H	>10	NA
RM4824	348.4	H	CH2COOCH2CH3	OAc	H	H	H	8.4	NA

^aCompared with a control treated with the same volume of dimethyl sulfoxide at 48 h postinfection.

^bAc, acetyl.

^cMe, methyl.

^dNA, not applicable.

^eMaximal inhibitory activity of >99% (complete growth inhibition).

^fThiadiazolide agent.

^gBenzamide N substitution with COC₆H₄OCOCH₃.

FIG. 1.

Chemical structures of the thiazolides (A) and thiadiazolides (B) used in this study.

High-yield complete (asexual and sexual stages) parasite development was obtained (8, 9). Assays were performed at host cell confluence to limit the apoptosis-inducing activity recently reported for NTZ and RM4819 in human enterocytic Caco-2 cells (16). At 1- and 5-mg/liter concentrations of all of the agents (i.e., 1.96 to 22.70 μM), no alteration of HCT-8 cells was observed after 48 h of contact. At a 10-mg/liter (25.69 to 37.22 μM) concentration, moderate alteration of cell viability was noted for RM4802 to -4805, RM4807, RM4815 to -4817, RM4819, RM4821, RM4848, RM4851, RM4852, RM4854, RM4858 to -4862, and RM4865. Twenty-seven of 39 thiazolides/thiadiazolides exerted ≥90% dose-dependent parasite inhibition, with IC₉₀s ranging from 0.9 to 9.3 mg/liter (3.2 to 29.7 μM) (Table 1 and Fig. 2). For agents with both a lower IC₅₀ than that of NTZ (P < 0.05) and alteration of cell viability (RM4802 to -4805, RM4807, RM4815, RM4816, RM4852, RM4859, RM4860, and RM4865), the ratio of the concentration affecting cell viability (31.12 to 37.22 μM) to the IC₅₀ (0.32 to 2.98 μM) ranged from 13 to 100.

FIG. 2.

Inhibition curves of representative active compounds. RM4959 and RM4860 are 5-bromothiazolides; RM4850 (deacetylated metabolite of RM4865) and RM4865 are 5-chlorothiazolides; RM4816 is a thiadiazolide. Data, expressed as percent inhibition compared with untreated infected control cultures, represent the means of triplicate experiments. Error bars indicate standard deviations that were >5%.

Sixteen agents with IC₅₀s lower than that of NTZ (P < 0.05) have either an NO₂ or a halogen thiazole/thiadiazole X substitution (Table 1). Compared with NTZ, 5/7 NO₂ X-substituted agents exhibited a lower IC₅₀ (P < 0.05), 1/7 (RM4809) had an equivalent IC₅₀ (P > 0.05), and 1/7 (RM4814, which contains a methoxy group at position R1 of the benzene ring) was considered inefficient.

All chlorothiazolides were strong inhibitors, regardless of the substitution on the benzene ring (IC₅₀s ranging from 0.1 to 2.6 mg/liter, i.e., 0.3 to 10.2 μM), and four agents exhibited IC₅₀s lower than that of NTZ (Table 1, P < 0.01). Of two F X-substituted agents, one was very active (CF₃ substitution, RM4816), with an IC₅₀ lower than that of NTZ (P < 0.05), and the other exerted limited activity (RM4854). CH₃, C₃H₇, C₆H₁₁, H, SO₂CH₃, and SCH₃ X substitutions negatively impacted the inhibitory activity, producing IC₅₀s lower than that of NTZ (P < 0.05).

In the hydrophobic 5-bromothiazolide series, 12/13 agents exhibited ≥90% maximal inhibition, with IC₅₀s ranging from 0.4 to 2.8 mg/liter (0.3 to 16.6 μM) (Table 1). Compared with NTZ, six agents (RM4803, RM4820, RM4822, RM4859, RM4860, and RM4861) exhibited lower IC₅₀s (ranging from 0.1 to 0.7 mg/liter, i.e., 0.3 to 2 μM), one (RM4819) had a similar IC₅₀ (P = 0.5), and six (RM4806, RM4821, RM4823, RM4832, RM4847, and RM4858) had higher IC₅₀s (ranging from 1.7 to 5.2 mg/liter, i.e., 4.9 to 16.6 μM; P < 0.05).

Of three thiadiazolides, one (RM4816) was very active, with an IC₅₀ lower than that of NTZ (P < 0.01), and two were less active than NTZ (P < 0.05).

The biological activity of NTZ was previously reported to depend on the nitro group, possibly via inhibition of an oxidoreductase (3, 13, 21). A 5-nitro group was found to be a prerequisite for the in vitro efficiency of RM4805 and RM4807 against G. duodenalis, although it was not sufficient since further modification of the benzene moiety could reduce or even abrogate their activities (14). In contrast, consistent with previous N. caninum, Besnoitia besnoiti, and S. neurona in vitro studies, the present data suggest that the nitro group on the thiazole ring is not required for the activity of thiazolides against intracellular protozoa, implying differences between the intracellular and extracellular protozoal targets (4, 6, 11, 12, 15).

As previously shown for NTZ and its biologically active deacetylated metabolite tizoxanide, both acetylated (at R1 on the benzene ring) thiazolide derivatives and their deacetylated counterparts, which represent their most probable primary metabolites, displayed excellent inhibitory effects, although they were lower for deacetylated agents (P = 0.03), consistent with previous observations for N. caninum with RM4822 and RM4847 (5, 10). Substitution of a bromo or chloro group for the nitro group at the 5 position did not significantly impact the activity of deacetylated compounds, provided that position R2 (or R3 or R4) was substituted with a methyl group in the 5-bromo or the 5-chloro series, respectively. A methyl group at position R3 in the 5-bromo series and a polar methoxy group in position R1 in the 5-nitro series notably reduced the efficacy of deacetylated compounds. These data suggest that both the thiazole and benzene residues serve as key pharmacophores, as reported for N. caninum and S. neurona (5, 6, 11).

The present report provides the first evidence of in vitro inhibitory activities of new NTZ-derived thiazolides/thiadiazolides against C. parvum. It is noteworthy that agents where the nitro group was eliminated, as well as nitrothiazolide, were active. Nitro-free agents and especially halogeno compounds may provide a valuable alternative to NTZ, depending on future studies of their in vivo activities.