Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma cruzi - inhibitors bearing benzofuroxan and benzimidazole 1,3-dioxide core scaffolds

Alicia Merlino; Diego Benitez; Santiago Chavez; Jonathan Da Cunha; Paola Hernández; Luzineide W. Tinoco; Nuria E. Campillo; Juan A. Páez; Hugo Cerecetto; Mercedes González

doi:10.1039/C0MD00085J

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DOI: 10.1039/C0MD00085J (Concise Article) Med. Chem. Commun., 2010, 1, 216-228

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Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma cruzi-inhibitors bearing benzofuroxan and benzimidazole 1,3-dioxide core scaffolds

Alicia Merlino ^a, Diego Benitez ^a, Santiago Chavez ^a, Jonathan Da Cunha ^a, Paola Hernández ^a, Luzineide W. Tinoco ^b, Nuria E. Campillo ^c, Juan A. Páez ^c, Hugo Cerecetto *^a and Mercedes González *^a
^aGrupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, , 11400 Montevideo, Uruguay. E-mail: megonzal@fq.edu.uy; hcerecet@fq.edu.uy; Fax: +598 2 5250749; Tel: 598 2 5258618 (ext. 216)
^bLaboratory of Analysis and Development of Enzyme Inhibitors - LADIE, NPPN, Federal University of Rio de Janeiro,
^cInstituto de Química Médica, CSIC, Madrid, Spain

Received 19th June 2010 , Accepted 19th July 2010

First published on 11th August 2010

Abstract

Trypanosoma cruzi is the causative agent of Chagas' disease. The thiosemicarbazone moiety as a pharmacophore has been described for inhibition of the essential cysteine protease, cruzipain, of this parasite. Our recent study identified an amidinohydrazone containing benzofuroxan as a hit compound for cruzipain inhibition with trypanosomicidal activity. Structural modification of the amidinohydrazone, thio- and semicarbazone motifs, using benzofuroxan and including a benzimidazole 1,3-dioxide system as new core scaffolds are described. These changes allowed for the identification of new structural motifs with desired antitrypanosomal activity. The new amidinohydrazone, thio-, and semicarbazone derivatives had excellent anti-trypanosomal activity without improved cruzipain-inhibitory activity compared with the parent compounds. Relevant structural features of these derivatives for further modification have also been determined.

Introduction

Chagas' disease is the third most neglected disease in Latin America after malaria and schistosomiasis, and affects at least 15 million people with more than 25 million at risk of infection.¹ The infectious agent is the protozoan parasite Trypanosoma cruzi (T. cruzi), that causes symptoms progressing from mild swelling to intestinal disease and ultimately heart failure. Currently, there are two nitroaromatic-based drugs for treatment of this disease, Nifurtimox (Nfx, Lampit®, Scheme 1a) and Benznidazole (Bnz, Rochagan®, Scheme 1a).^2,3 Treatment with these drugs is inadequate, since the parasite is often not completely eliminated despite chronic administration, and there are unacceptable side effects.^4,5 Moreover, resistance to these agents has emerged.⁶ For these reasons the development of safer and more effective drugs against Chagas' disease is urgently needed.⁷


	Scheme 1 a) Drugs used clinically as anti-T. cruzi agents. b) Hybrid parent compounds with antitrypanosomal activity. c) Structural modifications described.

The use of multitarget-directed ligands (MTDLs) has emerged as a strategy for the development of new drugs to treat Chagas' disease.⁸ The approach is based on the combination of two or more pharmacophores into a new chemical entity, also defined as a hybrid-drug, and is only just beginning to be used in drug design for treatment of this disease. A recent focus of our attention has been on developing MTDLs as anti-T. cruzi agents using free radical-releasing moieties linked to DNA-interacting, sterol-biosynthesis-inhibitor and cruzipain (CP)-inhibitor pharmacophores.⁹

Using this strategy, we hybridized benzofuroxan heterocycle and amidinohydrazone, thio- and semicarbazone moieties¹⁰ to generate compounds with trypanosomicidal activity involving at least two mechanism of action. In addition,¹¹ we provided evidence that benzofuroxan derivatives were able to modify parasite dehydrogenase activity and to affect mitochondrial membrane potential, whereas amidinohydrazone and thiosemicarbazone moieties had already been described as pharmacophores for trypanothione reductase and CP inhibitors, respectively.¹² In this study we identified parent compounds 1–5 (Scheme 1b) with good in vitro trypanosomicidal activity.¹⁰ Compounds 1–5 had modest CP inhibitory activity, with the amidinohydrazone 4 having some trypanothione reductase inhibitory activity. Semicarbazone analogue 6 was a poor CP inhibitor and anti-T. cruzi agent. Another heterocycle system that we used as a scaffold for the development of new trypanosomicidal agents was the benzimidazole 1,3-dioxide system (i.e.7, Scheme 1b).⁹ We hypothesized that this heterocycle might act as a free radical-releasing pharmacophore; however, it was recently reported that this mode of action was not observed in the parasite.¹¹ Derivatives of this heterocycle have the advantage of being water soluble.

In our efforts to develop selective and novel trypanosomicidal compounds, we selected parent compounds 3–7 as molecular templates and the structural modifications we sought to investigate are shown in Scheme 1c. Thus, we used benzofuroxan and benzimidazole 1,3-dioxide as core scaffolds linked to amidinohydrazone, thio- and semicarbazone pharmacophores aiming to produce new anti-T. cruzi agents. The derivatives synthesized were examined for antiproliferative in vitro activity against the T. cruzi, Tulahuen 2 strain, for non-specific cytotoxicity on human red blood cells and J-774 mouse macrophages, and inhibition of CP. The ability of the compounds to interact with CP was examined by docking analyses.

Results and discussion

The starting point for the synthesis of the new hybrid derivatives was the preparation of the corresponding carbonylphenyloxymethylbenzofuroxan (8–11) as shown in Scheme 2. Aldehydes 8 and 9 were prepared according to a previous report¹⁰ with minor modification, using 5-bromomethylbenzofuroxan¹³ as starting material. Due to the unusual stability that the intramolecular hydrogen bond confers to salicylaldehyde and 2-hydroxyacetophenone, the alkylation reaction could not be done using the aforementioned strategy to synthesize derivatives 10 and 11. Consequently, these carbonyl derivatives, 10 and 11, were obtained using solvent-free microwave irradiation with K₂CO₃ and tetrabutylammonium iodide (TBAI) as phase transfer catalyst (Scheme 2). Carbonyl-containing benzimidazole 1,3-dioxide derivatives (12–14) were prepared by treatment of the corresponding benzofuroxan with 2-nitropropane and piperidine as base in THF at room temperature (Scheme 2). Amidinohydrazone, thio- and semicarbazone containing benzofuroxans 15–25 were prepared by condensation of the corresponding carbonyl derivative and hydrazones under acidic conditions as shown in Scheme 2. The same heterocycle transformation conditions used for the synthesis of derivatives 12–14 was used to prepare benzimidazole 1,3-dioxides 26–37 (Scheme 2). Compounds 4–6 (Scheme 1) were used as starting materials for the preparation of derivatives 26–28. We were unable to obtain the desired amidinohydrazones 26 and 31 because in both cases the starting material decomposed under the reaction conditions (piperidine/THF/r.t.). All the structures were determined by ¹H NMR, ¹³C NMR, NOE-diff, COSY, HSQC, and HMBC experiments, IR and MS. The purity of products 9–25, 27–30, and 32–37 was determined by TLC and microanalysis.


	Scheme 2 Synthetic procedure used for the preparation of hybrid compounds and precursors.

The new benzofuroxan and benzimidazole 1,3-dioxide derivatives, 9–25, 27–30, and 32–37, were assessed in vitro for antiproliferative activity against the epimastigote form of the T. cruzi, Tulahuen 2 strain.^9,10 The occurrence of the epimastigote form of T. cruzi as an obligate mammalian intracellular stage has been reevaluated and confirmed.¹⁴ Furthermore, it should be noted that a good correlation between antiproliferative epimastigote activity and in vivo anti-T. cruzi activity was observed with compounds from our chemical library.^9,15 Compounds were incorporated into the growth medium at 25.0 μM and the ability to inhibit the growth of the parasite was evaluated by comparison with untreated controls on day 5. The ID₅₀ doses (50% inhibitory dose) were determined for the most active compounds, and Nfx and Bnz were used as reference trypanosomicidal drugs. The starting carbonyl derivatives, 8–14, exhibited in some cases relevant anti-T. cruzi activities, the most active being the 2-substituted aldehyde 10 (Table 1). The semicarbazones and the amidinohydrazones, except for the 2-substituted amidinohydrazone 25, were not active against T. cruzi in culture. Derivative 25 showed higher activity than parent compound 4 (Table 1). The new N⁴-unsubstituted thiosemicarbazones, 18, 21, 27, 32 and 34, were less or as active as parent compound 5 (Table 1), whereas benzofuroxan 18 and benzimidazole 1,3-oxide derivatives 27 and 34 were the most active. However, when the thiosemicarbazone N⁴-position was substituted, and in particular by a phenyl moiety (i.e.16, 23 and 36, Table 1), higher anti-T. cruzi activities were observed compared with N⁴-unsubstituted derivatives. The semicarbazone, N⁴-allyl and N⁴-phenyl thiosemicarbazones derived from 2-formyl benzofuroxane and benzimidazole 1,3-dioxide, 35, 23 and 37, respectively, were the most active compounds obtained, having higher activities than references Nfx and Bnz.

Table 1 Biological characterization of hybrid derivatives using T. cruzi, and human red blood cells

Series^a		Compd	ID₅₀/μM		Compd	ID₅₀/μM		Compd	ID₅₀/μM		Compd	ID₅₀/μM
Series^a		Compd	T2^b^,^c	rbc^c^,^d	Compd	T2^b^,^c	rbc^c^,^d	Compd	T2^b^,^c	rbc^c^,^d	Compd	T2^b^,^c	rbc^c^,^d
a bfx: benzofuroxan; bz: benzimidazole 1,3-dioxide; ca: carbonyls; ah: amidinohydrazones; th: thiosemicarbazones; at: N⁴-allylthiosemicarbazones; pt: N⁴-phenylthiosemicarbazones. b T2: Tulahuen 2 strain. c The results are the means of three independent experiments. d rbc: Human red blood cells. e From ref. 10. f Problems with compound solubility were observed at higher doses. g “—”: compound was not obtained. h ag: the compound formed aggregates in the buffer at all doses assayed. i ns: not studied.
bfx	ca	8	8.7^e	< 50	9	28 ± 2	50 ± 5	10	6.3 ± 0.2	> 100	11	7.0 ± 1.2	> 200
bz	ca	12	17.0 ± 1.9	65 ± 3	13	> 25	< 50				14	> 25	< 50
bfx	ah	4	> 25 ^e	> 200	17	> 25	> 100^f	20	> 25	58 ± 3	25	16.6 ± 2.3	> 200
bz	ah	26	—^g	—	31	—	—	33	> 25.0	114 ± 3
bfx	th	5	15.0 ^e	> 200	18	15.0 ± 0.4	ag^h	21	> 25	189 ± 1
bz	th	27	17.0 ± 0.8	ag	32	24 ± 2	141 ± 5	34	14 ± 2	> 200
bfx	at	15	> 25.0	ag	19	13 ± 2	ag	22	23 ± 1	139 ± 2
bz	at	28	23.0 ± 1.5	> 100^h				35	2.7 ± 1.4	ag
bfx	pt	16	7 ± 3	ag				23	3.6 ± 0.4	> 200
bz	pt	29	> 25	< 50				36	7.7 ± 0.3	66.0 ± 0.8	Nfx	7.7^e	> 100^f
bfx	se	6	> 25.0 ^e	> 200				24	> 25	> 100^h	Bnz	7.4 ^e	nsⁱ
bz	se	30	> 25	> 200				37	4.7 ± 1.2	50.0 ± 0.4	AmpB	0.152 ± 0.006	1.5 ± 0.2

Two different biological systems, erythrocytes and macrophages, were used to estimate the potential toxicity of the hybrid derivatives (Tables 1 and 2), ^9,10,16 Erythrocytes served as a biological model for mammalian cells in direct contact with the trypomastigote, and macrophages a related model for the amastigote stage of the parasite. The compounds were added to cell suspensions at doses of 50.0–200.0 μM for erythrocytes and at doses of 100.0–400.0 μM for macrophages, toxicities were evaluated by comparison with untreated controls after 24 or 48 h and the ID₅₀ doses (50% cytotoxic dose) were determined. Nfx was used as the reference trypanosomicidal drug and amphotericin B (AmpB), used experimentally as an anti-T. cruzi agent,¹⁷ was used as a positive control in that it is a haemolytic agent (Table 1). Only soluble compounds were included in the macrophage-cytotoxicity assays (Table 2). A general relationship between the type of substituent (carbonyl, amidinohydrazone, thio- or semicarbazone) or type of core scaffold (benzofuroxan or benzimidazole 1,3-dioxide) and cytotoxicity was not evident. However, like anti-T. cruzi activities, a correlation between cytotoxicity and the substituent's position on the phenoxy moiety was apparent. This was particularly evident in the case of 4- and 2-substituted derivatives, the latter being less cytotoxic than the former (compare rbc cytotoxicities of 8 to 10 and 11, as well as 29 to 36). One of the best anti-T. cruzi agents, 2-thiosemicarbazone 23, had excellent selectivity indexes. The selectivity indexes for derivative 23 were SI_{erythrocyte/T.cruzi} > 55.6 and SI_{macrophage/T.cruzi} > 111.1, and in contrast to the thiosemicarbazone parent compound 5 (SI_{erythrocyte/T.cruzi} > 13.3 and SI_{macrophage/T.cruzi} = 26.7) and the carbonyl parent compound 10 (SI_{erythrocyte/T.cruzi} > 15.9 and SI_{macrophage/T.cruzi} = 12.4), and Nfx (SI_{erythrocyte/T.cruzi} = 13.0) and AmpB (SI_{erythrocyte/T.cruzi} ∼ 10.0) (Tables 1 and 2). Thus, derivative 23 had far better selectivity indexes than parent derivatives 5 and 10 and reference compounds.

Table 2 Biological characterization of hybrid derivatives using mammalian macrophage line J-774

Series^a		Compd	ID₅₀/μM^b	Compd	ID₅₀/μM^b
a bfx: benzofuroxan; bz: benzimidazole 1,3-dioxide; ca: carbonyls; ah: amidinohydrazones; th: thiosemicarbazones; at: N⁴-allylthiosemicarbazones; pt: N⁴-phenylthiosemicarbazones. b Results shown are the means of three independent experiments. c From ref. 10.
bfx	ca	8	60.0^c	10	78 ± 2
bz	ca	12	< 100
bfx	ah	4	< 50 ^c	20	< 100
bfx	th	5	400.0^c	21	< 100
bfx	at			22	138 ± 2
bfx	pt			23	> 400
bz	pt			36	< 100
bfx	se			24	< 100

We investigated the ability of the hybrid derivatives to inhibit CP to obtain information on their mechanism of trypanosomicidal activity. The compounds were tested at 25.0, 50.0, and 100.0 μM for the ability to inhibit CP. The compounds were assayed using Z-phenyl-arginine-7-amido-4-methylcoumarin hydrochloride (Z-Phe-Arg-AMC) as fluorogenic CP substrate, and compared with untreated control assays (Table 3). As non-specific compound aggregation could interfere with the assay or promote promiscuous CP inhibition, the assays were also done, for compounds 17 and 19, at acidic pH and in the presence of Triton X-100 (Table 4). The acidic pH is especially relevant for the protonation of the amidinohydrazone moiety¹⁸ that could change the interaction in the active site, and the use of the non-ionic surfactant is relevant to reduce aggregation and false inhibition.¹⁹ In spite of the fact that the new hybrid compounds were more active and selective against T. cruzi than the parent compounds, the CP inhibitory properties were less than the parent compounds. The best CP inhibitors were the 4- and 3-thiosemicarbazonyl containing benzofuroxans 16, 18 and 19, and the 4- and 3-thiosemicarbazonyl containing benzimidazole 1,3-dioxides 29 and 32. The thiosemicarbazone moiety was previously implicated as a CP inhibitor pharmacophore.²⁰ None of the 2-substituted derivatives, 10, 11, 14, 20–25 and 33–37, had significant activity in this assay. Although the pH change did not improve the activity of amidinohydrazone 17 (Table 4), thiosemicarbazone 19 tested in presence of Triton X-100, and at pH 7.3, demonstrated that the effect against CP was not the result of an unrelated inhibitory activity.

Table 3 Inhibition of CP activity by hybrid derivatives

Compd.	% of inhibition at 100.0 μM^a^,^b	ID₅₀/μM^a^,^b	Compd.	% of inhibition at 100.0 μM^a^,^b	ID₅₀/μM^a^,^b
a In buffer, pH 7.3, and absence of Triton X-100 (see Experimental Section). b The results are the means of three independent experiments. c mbth: (m-bromophenyl)ethylketone thiosemicarbazone (reference compound).
8	6.3 ± 0.6	>100.0	12	35.5 ± 1.5	>100.0
9	43.0 ± 0.8	∼ 100.0	13	45.0 ± 1.0	∼ 100.0
10	0.0	>100.0	14	0.0	>100.0
11	12.0 ± 0.7	>100.0
			27	13.7 ± 1.9	>100.0
15	38.0 ± 2.0	>100.0	28	51.0 ± 0.7	100.0 ± 2.0
16	95 ± 2	75.0 ± 2.1	29	72 ± 4	85.0 ± 1.3
			30	0.0	>100.0
17	42.0 ± 1.8	∼ 100.0
18	80 ± 3	78.0 ± 1.6	32	95 ± 3	64.0 ± 1.9
19	100 ± 2	55.0 ± 2.0
20	39.0 ± 1.5	>100.0	33	2.5 ± 0.5	>100.0
21	49.0 ± 0.8	100.0 ± 2.0	34	35.0 ± 1.6	>100.0
22	11.0 ± 2.1	>100.0	35	6.6 ± 0.9	>100.0
23	0.0	>100.0	36	0.0	>100.0
24	7.0 ± 1.3	>100.0	37	0.0	>100.0
25	21.0 ± 0.8	>100.0
			mbth	100.0 ± 0.5	0.10 ± 0.08

Table 4 Inhibition of CP activities of hybrid derivatives 17 and 18 under different experimental conditions

Compd.	pH	Triton X-100 addition	% of inhibition at 100.0 μM^a
a The results are the means of three independent experiments.
17	7.3	—	42.0 ± 1.8
17	5.3	—	28.0 ± 0.9
19	7.3	—	100.0
	7.3	+	90.0 ± 1.5
	5.3	—	79 ± 1

To gain insight into CP–ligand interactions, NMR and docking analyses were done. When a small molecule interacts with a biomolecule, a decrease in T1 is observed due to the slowing of ligand molecular motions as it binds to the macromolecule.²¹ Unfortunately, we were unable to obtain information using derivatives 15, 16, 18, 19 and 32 as ligands due to the low solubility of these derivatives under the assay conditions. Attempts to work with other conditions, pH and amount of DMSO were unsuccessful due to the relatively low stability of CP. Docking analysis using parent compound 4, the hybrid derivatives 16–19, 21, 23, 29 and 34, and (m-bromophenyl)ethylketone thiosemicarbazone (mbth) (Table 3) were in agreement with the experimental results (Table 5, Fig. 1). The CP–ligand complexes were analyzed using FlexiDock, a program that performs docking of conformationally flexible ligands into receptor binding sites and provides control of ligand binding characteristics, taking into account rigid, partially flexible, or fully flexible receptor side chains. FlexiDock incorporates the van der Waals, electrostatic, torsional and constraint energy terms of the Tripos force field, and uses a genetic algorithm (GA) to determine the optimum ligand geometry. GA borrows methodology and terminology from biological (or Darwinian) evolution, in that an iterative process is used in which the most fit members of a population will have the best chance of propagating themselves in future generations of analysis. The reference compound mbth showed small differences in the type of interaction compared with our derivatives (Table 6), the thiocarbonyl carbon was favorably positioned to form a covalent bond with the sulfur atom of the catalytic Cys25. Moreover, the His159 was properly oriented for protonating the resulting anion via the thiocarbonyl sulfur. So, as previously described, the potent inhibitory capacity of this compound could be explained by the possibility of forming a reversible covalent interaction with the active-site cysteine (Cys25). In the cases of our new hybrid derivatives, some relevant interactions could be identified (Table 6). Except for compound 23, all derivatives interacted by hydrogen bonding and/or hydrophobic interactions with one or more residues present in the substrate-binding cleft.

Table 5 Theoretical K_d, ΔG binding, and experimental ID₅₀ values of the CP–ligand complexes analyzed

Compd.	K _d/M^a	ΔG/kcal mol⁻¹^a	ID_50,CP
a Values were estimated from the most stable conformers.
4	9.7 × 10⁻⁴	−4.11	32.0
16	4.4 × 10⁻³	−3.21	75.0
17	6.0 × 10⁻³	−3.03	> 100.0
18	1.2 × 10⁻³	−3.92	78.0
19	2.9 × 10⁻³	−3.46	55.0
21	5.7 × 10⁻³	−3.06	100.0
23	3.6 × 10⁻¹	−0.61	> 100.0
29	5.4 × 10⁻³	−3.09	85.0
34	9.6 × 10⁻³	−2.75	> 100.0
mbth	6.2 × 10⁻⁴	−4.37	0.10

Table 6 Principal CP residues in contact with compounds analyzed


	Cys25 (S)	Gly66 (N)	Gly66 (O)	Leu67	Leu157	Asp158 (O)	His159 (Nδ1)	Glu205 (Oε2)
a HB: hydrogen bond. b According to labelled structure. c Hph: hydrophobic interactions. d Bfx: benzofuroxan ring. e no interaction. f all: allyl moiety. g The same atomic labels at the thiosemicarbazone moiety as those of the hybrid derivatives analyzed.
4	HB^a (N⁶-Bfx)^b	HB (Y = –O–)	HB (N⁵-Bfx)	Hph^c (Bfx^d)	—^e	HB/N²	—	HB/N³
16	—	—	—	—	Hph (Bfx)	—	—	—
17	—	—	HB/N⁴, HB (N²)	—	—	HB/N³	—	—
18	—	HB (O¹)	—	—	—	—	—	HB/N⁴
19	—	HB/N¹	HB/N⁴	Hph (all^f)	Hph (Bfx)	—	—	—
21	—	—	—	—	—	HB/N²	HB/N⁴
23	—	—	—	—	—	—	—	—
29	—	—	—	—	Hph	—	—	—
34	—	—	HB/N⁴	--	—	HB/N²	—	—
mbth	—	—	HB/N²	Hph	—	—	—	—


	Fig. 1 Theoretical K_dvs. experimental ID₅₀ values for CP–ligand complexes analyzed. Notes: ID₅₀ for derivative 23 was inferred by extrapolation as ca. 400 μM; the line shows tendency.

In the CP–19 complex (Fig. 2a) Gly66 formed stable hydrogen bonds with the N¹ (3.5 Å) and N⁴ (2.8 Å) thiosemicarbazone atoms of the ligand (Table 6), this interaction being a common stabilizing feature in CP–small molecule inhibitor complexes.^9,22–26 In addition, binding was assisted by hydrophobic interactions between the benzofuroxan rings of the ligand and Leu157 and between the allyl moiety and Leu67. The stabilizing effect of these hydrophobic interactions became evident after examination of the CP–16 complex (Fig. 2b). Compound 16, with an ID₅₀ of 75 μM, was stabilized in the active cleft only by Leu157 and the non-polar regions of Ala133 oriented toward the benzofuroxan ring of the inhibitor. This amino acid residue at the bottom of the S2 subsite was of particular importance as it was crucial in determining the substrate specificity of the enzyme.²⁶ Together with flexible Glu205, CP has an S2 subsite that is able to accept both basic and hydrophobic residues. In fact, when comparing the complexes of the amidinohydrazones 17 and 4 (as the protonated forms), CP–17 and CP–4, respectively, it could be seen that when the molecule occupies the active-cleft with the benzofuroxan moiety directed to the bottom of the S2 subsite, in the case of 17, the Glu205 sidechain adopted a conformation oriented toward the solvent (Fig. 2c). However, when the parent ligand 4 was docked at that site the Glu205 sidechain was oriented toward the inhibitor and interacted with a guanidine nitrogen (Fig. 2d, Table 6). The ability of parent compound 4 to adopt this conformation in the active site of the enzyme could explain its high performance as a CP inhibitor. These preferential orientations also explained why there was no relative improvement in the inhibitory activities of amidinohydrazone 17 and thiosemicarbazone 19 at pH 5.3.


	Fig. 2 Position of some representative derivatives in the binding-cleft of CP determined by docking. a. CP–19. b. CP–16. c. CP–17. d. CP–4.

Experimental section

Chemistry

General methods. Compounds 4–6, 8 and 5-bromomethylbenzofuroxan were prepared according to a procedure previously described.^10,13 Melting points were determined with an electrothermal melting point apparatus (Electrothermal 9100) and were uncorrected. Proton and carbon NMR spectra were recorded on a Bruker DPX-400 spectrometer at 298 K. The chemical shifts values are expressed in δ relative to tetramethylsilane as internal standard. At 298 K the benzofuroxan carbon signals appeared as very broad peaks or they did not appear. Mass spectra were determined either in a MSD 5973 Hewlett Packard or LC/MSD-Series100 Hewlett Packard spectrometer using electron impact (EI) or electrospray ionization (ESI), respectively. Infrared spectra were recorded on a Perkin-Elmer 1310 apparatus, using potassium bromide tablets. Microanalyses were done with a Fisons EA 1108 CHNS–O instrument and were within 0.4% of the values obtained by calculated compositions. Column chromatography was done using Merck silica gel (60–230 mesh). Most chemicals and solvents were analytical grade and used without further purification.
5-(3-Formylphenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (9). 5-Bromomethylbenzofuroxan (1 eq.) was added to a solution of 3-hydroxybenzaldehyde (200 mg, 1.64 mmol) in dry CH₃CN and K₂CO₃ (1 eq.). The reaction mixture was stirred at room temperature under nitrogen atmosphere and checked by TLC (Al₂O₃, petroleum ether–AcOEt, 7 [thin space (1/6-em)]

3) until the disappearance of the reactants. The solvent was evaporated in vacuo and the reaction mixture was partitioned between H₂O (50.0 mL) and EtOAc (3 × 50 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to obtain the desired product. Yellow solid (80%), mp 145.8 °C. ¹H NMR (CDCl₃, 400 MHz) δ (ppm): 5.16 (s, 2H), 7.31 (m, 1H), 7.36–7.69 (bs, 3H), 7.49 (s, 1H), 7.53 (d, 1H, J = 7.6 Hz), 7.55 (t, 1H, J₁ = 2.0 Hz, J₂ = 1.2 Hz), 10.00 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm): 68.7 (CH₂), 112.4 (Ar), 122.2 (Ar), 124.9 (Ar), 130.5 (Ar), 137.9 (C–C = N-NH-), 158.4 (C-O-CH₂), 191.8 (CHO). MS (EI), m/z (abundance, %): 270 (M^+•, 2), 254 (10), 149 (19), 133 (76), 121 (15), 77 (27). (Found: C, 61.9; H, 3.5; N, 10.2. C₁₄H₁₀N₂O₄ requires C, 62.2; H, 3.7; N, 10.4%)

General procedure for the synthesis of compounds 10 and 11. A mixture of 5-bromomethylbenzofuroxan (300 mg, 1.31 mmol), 2-hydroxybenzaldehyde or 2-hydroxyacetophenone (1.2 eq.) and TBAI was heated in a microwave oven (Microwave Digestion System, WX-4000, Shanghai EU Chemical Instrumetns) at 300 Watts in an open vessel flask for 1.30 min.²⁷ The reaction mixture was allowed to cool, and then extracted with EtOAc (2 × 50 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash chromatography (petroleum ether–EtOAc, 7 [thin space (1/6-em)]

3, as eluent).
5-(2-Formylphenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (10). Yellow solid (65%), mp 146.0 °C (d). ¹H NMR (CDCl₃, 400 MHz) δ (ppm): 5.37 (s, 2H), 7.15 (t, 1H, J = 7.5 Hz), 7.33 (d, 1H, J = 8.4 Hz), 7.69 (t, 1H, J₁ = 8.6 Hz, J₂ = 7.4 Hz), 7.57–7.77 (bs, 3H), 7.76 (d, 1H, J = 7.5 Hz), 10.50 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm): 69.5 (CH₂), 114.8 (Ar), 122.2 (Ar), 125.5 (C–C = N–NH–), 129.3 (Ar), 137.2 (Ar), 160.7 (C-O-CH₂), 190.2 (CHO). MS (EI), m/z (abundance, %): 270 (M^+•, 2), 253 (30), 149 (100), 133 (49), 121 (38), 105 (11), 89 (47), 77 (12). (Found: C, 62.0; H, 3.3; N, 10.1. C₁₄H₁₀N₂O₄ requires C, 62.2; H, 3.7; N, 10.4%).
5-(2-Acetylphenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (11). Yellow solid (32%), mp 125.7–126.4 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 2.59 (s, 3H), 5.41 (s, 2H), 7.09 (dt, 1H, J₁ = 7.2 Hz, J₂ = 7.8 Hz, J₁ = 0.8 Hz, J₂ = 0.4 Hz), 7.29 (d, 1H, J = 8.4 Hz), 7.54 (m, 1H), 7.58–7.68 (bs, 3H), 7.71 (dd, 1H, J = 7.6 Hz, J = 1.6 Hz). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 31.1 (CH₃), 69.2 (CH₂), 113.4 (Ar), 121.1 (Ar), 128.9 (C–C = N–NH–), 130.0 (Ar), 133.5 (Ar), 157.3 (C-O-CH₂), 198.1 (C [double bond, length as m-dash]

O). MS (ESI), m/z: 307 (M^+• + Na⁺). (Found: C, 63.7; H, 4.2; N, 9.5. C₁₅H₁₂N₂O₄ requires C, 63.4; H, 4.2; N, 9.8%).

General procedure for the synthesis of compounds 17, 20 and 25.²⁸. Aminoguanidine bicarbonate (1 eq.) and AcONa (2 eq.) was added to a solution of the corresponding carbonyl reactant (9–11) (1 eq.) in dry ethanol. The mixture was stirred at room temperature under nitrogen atmosphere until disappearance of the aldehyde was observed by TLC (SiO₂, petroleum ether–EtOAc, 6 [thin space (1/6-em)]

4). The precipitate was filtered and washed with ethanol.
5-(3-Amidinohydrazonophenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (17). Pale brown solid (85%), mp 155.2 °C (d). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 5.23 (s, 2H), 7.11 (s, 1H), 7.37 (m, 2H), 7.46–7.89 (bs, 3H), 7.61 (m, 2H), 7.96 (s, 1H), 8.08 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm): 68.44 (CH₂), 113.1 (Ar), 117.9 (Ar), 121.9 (Ar), 130.4 (Ar), 147.0 (CH), 135.3 (C–C = N–NH–), 155.7 (C = NH), 158.7 (C-O-CH₂). MS (EI), m/z (abundance, %): 326 (M^+•, 5), 310 (7), 252 (10), 237(9), 221 (12), 177 (26), 161 (75), 133 (46), 119 (60), 107(30), 73 (100). (Found: C, 54.9; H, 4.0; N, 25.4. C₁₅H₁₄N₆O₃ requires C, 55.2; H, 4.3; N, 25.7%).
5-(2-Amidinohydrazonophenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (20). Yellow solid (77%), 212.2 °C (d). ¹H NMR (CDCl₃, 400 MHz) δ (ppm): 5.23 (s, 2H), 5.31 (s, 2H), 5.89 (s, 2H), 6.97 (t, 1H, J₁ = 7.6 Hz, J₂ = 7.2 Hz), 7.12 (t, 1H, J₁ = 8.4 Hz, J₂ = 9.2 Hz), 7.27 (t, 1H, J₁ = 7.2 Hz, J₂ = 8.0 Hz), 7.49–7.78 (bs, 3H), 8.00 (d, 1H, J = 7.6 Hz), 8.39 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm): 69.2 (CH₂), 113.7 (Ar), 122.0 (Ar), 126.3 (C–C = N–NH–), 126.5 (Ar), 129.8 (Ar), 139.0 (Ar), 156.0 (C-O-CH₂), 161.3 (C = NH). MS (EI), m/z (abundance, %): 326 (M+•, 5), 310 (23), 252 (14), 237(9), 221 (11), 177 (34), 161 (82), 133 (41), 119 (64), 107(28), 73 (100). (Found: C, 55.3; H, 4.6; N, 25.3. C₁₅H₁₄N₆O₃ requires C, 55.2; H, 4.3; N, 25.7%).
5-(2-Methylamidinohydrazonophenyloxymethyl)benzo [1,2-c]1,2,5-oxadiazole N-oxide (25). Pale yellow solid (67%), 129.0 °C (d). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.56 (s, 3H), 5.32 (s, 2H), 7.08 (t, 3H, J₁ = 7.2 Hz, J₂ = 7.6 Hz), 7.27 (d, 1H, J = 8.4 Hz), 7.52–7.89 (bs, 3H), 7.57 (dt, 1H, J₁ = 7.2 Hz, J₂ = 8.4 Hz, J = 2.0 Hz), 7.65 (dd, 1H, J = 7.6 Hz, J = 1.6 Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 40.2 (CH₃), 69.2 (CH₂), 114.1 (Ar), 121.5 (Ar), 128.6 (C–C = N–NH–), 130.2 (Ar), 134.3 (Ar), 155.8 (HNC=NH), 157.2 (C–C = N-NH-), 167.2 (CH₃C=N). MS (EI), m/z (abundance, %): 267 (M^+• - O - CH₃N₃, 54), 250 (7), 205 (9), 149 (61), 133 (100), 121 (50), 103 (21), 89 (48), 77 (26). IR, ν/cm⁻¹: 3355, 3129, 3045, 2907, 1668, 1591, 1539, 1491, 1356, 1300, 1111, 774, 616. (Found: C, 56.3; H, 5.0; N, 24.6. C₁₆H₁₆N₆O₃ requires C, 56.5; H, 4.7; N, 24.7%).

General procedure for the synthesis of compounds 15, 16, 18, 19 and 21–24. N ⁴-Phenylthiosemicarbazide or N⁴-allylthiosemicarbazide (1 eq.) was added to a solution of the corresponding carbonyl reactant (8–11) (50 mg, 0.185 mmol) in dry ethanol (10.0 mL) acetic acid (0.1%) and semicarbazide, thiosemicarbazide. The reaction mixture was stirred at room temperature under nitrogen atmosphere until disappearance of the aldehyde was observed by TLC (SiO₂, petroleum ether–EtOAc, 6 [thin space (1/6-em)]

4). The precipitate was filtered and washed with ethanol.
5-[4-(N⁴-Allylthiosemicarbazono)phenyloxymethyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide, (15). Pale brown solid (73%), mp 176.7 °C (d). ¹H NMR (DMSO-d₆), 400 MHz) δ (ppm): 4.21 (t, 2H, J₁ = 5.2 Hz, J₂ = 5.6 Hz), 5.10 (dd, 1H, J_gem = 1.6 Hz, J_cis = 10.2 Hz), 5.15 (dd, 1H, J_gem = 1.6 Hz, J_trans = 17.2 Hz), 5.25 (s, 2H), 5.91 (m, 1H), 7.11 (d, 1H, J = 8.8 Hz), 7.42–7.68 (bs, 3H), 7.79 (d, 1H, J = 8.8 Hz), 8.02 (s, 1H), 8.65 (t, 1H, J = 6.0 Hz), 11.40 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 46.2 (CH₂), 69.6 (CH₂), 115.5 (Ar), 115.9 (C [double bond, length as m-dash]

C), 127.9 (C–C = N–NH–), 129.4 (Ar), 135.6 (C = C), 142.2, (C = NH), 159.6 (C-O-CH₂), 177.4 (C = S). MS (ESI), m/z: 429 (M + 2Na⁺). IR, ν/cm⁻¹: 3300, 3185, 3008, 2914, 1603, 1536, 1418, 1236, 1039, 927, 852, 743, 574. (Found: C, 56.6; H, 4.1; N, 18.0. C₁₈H₁₇N₅O₃S requires C, 56.4; H, 4.5; N, 18.3%).
5-[4-(N⁴-Phenylthiosemicarbazono)phenyloxymethyl] benzo[1,2-c]1,2,5-oxadiazole N-oxide (16). Yellow solid (51%), mp 180.0 °C (d). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 5.26 (s, 2H), 7.13 (d, 2H, J = 8.8 Hz), 7.21 (t, 1H, J₁ = 7.6 Hz, J₂ = 7.2 Hz), 7.37 (t, 2H, J₁ = 7.6 Hz, J₂ = 8.4 Hz), 7.56 (d, 2H, J = 7.6 Hz), 7.60–7.84 (bs, 3H), 7.89 (d, 2H, J = 8.8 Hz), 8.11 (s, 1H), 10.10 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 68.5 (CH₂), 115.5 (Ar), 125.7 (Ar), 126.3 (Ar), 127.7 (C–C = N–NH–), 128.5 (Ar), 129.8 (Ar), 139.6 (Ar), 143.1 (C = NH), 159.8 (C-O-CH₂), 176.1 (C = S). MS (EI), m/z (abundance, %): 401 (M^+• - H₂O, 13), 268 (17), 252 (21), 237 (40), 151 (33), 133 (100), 119 (39), 93 (90). IR, ν/cm⁻¹: 3246, 3141, 1689, 1600, 1539, 1369, 1247, 851, 743, 576. (Found: C, 59.9; H, 3.9; N, 16.5. C₂₁H₁₇N₅O₃S requires C, 60.1; H, 4.1; N, 16.7%).
5-(3-Thiosemicarbazonophenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (18). Pale brown solid (91%), mp 188.7 °C (d). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 5.24 (s, 2H), 7.10 (d, 1H, J = 7.4 Hz), 7.34 (m, 2H), 7.50–7.82 (bs, 3H), 7.62 (s, 1H), 7.95 (s, 1H), 8.10 (s, 1H), 8.28 (s, 1H), 11.50 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 68.6 (CH₂), 112.5 (Ar), 117.3 (Ar), 121.7 (Ar), 130.4 (Ar), 136.2 (C–C = N–NH–), 142.3 (C = NH), 158.6 (C-O-CH₂), 162.8 (Bfx), 178.5 (C = S). MS (EI), m/z (abundance, %): 327 (M^+• - 16, 8), 311 (3), 269 (10), 252 (21), 237 (25), 151 (30), 133 (100), 121 (39), 103 (20). IR ν: 3441, 3293, 3163, 3025, 1586, 1538, 1279, 1059, 941, 859, 790, 689. (Found: C, 52.5; H, 3.6; N, 20.2. C₁₅H₁₃N₅O₃S requires C, 52.5; H, 3.8; N, 20.4%).
5-[3-(N⁴-Allylthiosemicarbazono)phenyloxymethyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide (19). Pale brown solid (80%), mp 149.6 °C (d). ¹H NMR (DMSO-d₆), 400 MHz) δ (ppm): 4.23 (t, 2H, J₁ = 4.8 Hz, J₂ = 5.2 Hz), 5.10 (dd, 1H, J_gem = 1.2 Hz, J_cis = 12 Hz), 5.16 (dd, 1H, J_gem = 1.2 Hz, J_trans = 18 Hz), 5.24 (s, 2H), 5.93 (m, 1H), 7.12 (d, 1H, J = 6.8 Hz), 7.38 (d, 2H, J = 6.8 Hz), 7.56 (s, 1H), 7.64–7.88 (bs, 3H), 8.04 (s, 1H), 8.74 (t, 1H, J = 5.2 Hz), 11.60 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 46.2 (CH₂), 68.6 (CH₂), 113.5 (Ar), 116.0 (C [double bond, length as m-dash]

C), 116.6 (Ar), 121.3 (Ar), 130.4 (Ar), 135.5 (C [double bond, length as m-dash]

C), 136.2 (C–C = N–NH–), 142.2, (C = NH), 158.6 (C-O-CH₂), 177.7 (C = S). MS (ESI), m/z: 429 (M + 2Na⁺). (Found: C, 56.4; H, 4.9; N, 18.1. C₁₈H₁₇N₅O₃S requires C, 56.4; H, 4.5; N, 18.3%).
5-(2-Thiosemicarbazonophenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (21). Yellow solid (79%), mp 215.0 °C (d). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 5.27 (s, 2H), 7.03 (dd, 1H, J₁ = 7.6 Hz, J₂ = 7.4 Hz), 7.20 (d, 1H, J₁ = 8.4 Hz), 7.42 (dd, 1H, J₁ = 7.4 Hz, J₂ = 8.2 Hz), 7.49–7.79 (bs, 3H), 7.96 (s, 1H), 8.15 (s, 1H), 8.16 (d, 1H, ³J = 7.6 Hz), 8.62 (s, 1H), 11.50 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 69.3 (CH₂), 114.0 (Ar), 122.2 (Ar), 123.6 (C–C = N-NH-), 127.1 (Ar), 132.2 (Ar), 138.9 (CH), 157.1 (C-O-CH₂), 163.2 (Bfx), 178.7 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 327 (M^+• - 16, 12), 311 (2), 269 (12), 252 (21), 237 (26), 151 (36), 133 (100), 121 (39), 103 (26). (Found: C, 52.2; H, 3.5; N, 20.1. C₁₅H₁₃N₅O₃S requires C, 52.5; H, 3.8; N, 20.4%).
5-[2-(N⁴-Allylthiosemicarbazono)phenyloxymethyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide (22). Pale brown solid (73%), mp 191.4 °C (d). ¹H NMR (MeOH-d₄, 400 MHz) δ (ppm): 4.36 (m, 2H), 5.11 (dd, 1H, J_gem = 1.6 Hz, J_cis = 10.0 Hz), 5.23 (dd, 1H, J_gem = 1.6 Hz, J_trans = 17.2 Hz), 5.36 (s, 2H), 6.00 (m, 1H), 7.05 (t, 1H, J = 7.6 Hz), 7.24 (d, 1H, J = 8.0 Hz), 7.43 (dt, 1H, J₁ = 7.2 Hz, J₂ = 8.4 Hz, J = 1.6 Hz), 7.56–7.78 (bs, 3H), 8.06 (dd, 1H, J = 7.6 Hz, J = 1.6 Hz), 8.38 (s, 1H), 8.77 (s, 1H), 10.50 (s, 1H). ¹³C NMR (MeOH-d₄, 100 MHz) δ (ppm): 45.9 (CH₂), 69.3 (CH₂), 113.5 (Ar), 115.6 (C [double bond, length as m-dash]

C), 121.9 (Ar), 123.6 (C–C = N–NH–), 126.5 (Ar), 131.7 (Ar), 135.2 (C [double bond, length as m-dash]

C), 138.0, (CH), 157.2 (C-O-CH₂), 176.7 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 383 (M^+•, 0.3), 366 (5), 268 (19), 254 (21), 237 (10), 133 (36), 115 (100). (Found: C, 56.1; H, 4.3; N, 17.9. C₁₈H₁₇N₅O₃S requires C, 56.4; H, 4.5; N, 18.3%).
5-[2-(N⁴-Phenylthiosemicarbazono)phenyloxymethyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide (23). Pale brown solid (84%), mp 164.3 °C (d). ¹H NMR (MeOH-d₄, 400 MHz) δ (ppm): 5.38 (s, 2H), 7.08 (t, 1H, J = 7.6 Hz), 7.20 (t, 1H, J₁ = 7.6 Hz, J₂ = 7.2 Hz), 7.27 (d, 1H, J = 8.4 Hz), 7.37 (t, 2H, J = 8.0 Hz), 7.46 (dt, 1H, J = 7.6 Hz, J = 1.6 Hz), 7.52–7.72 (bs, 3H), 7.77 (t, 1H, J = 7.6 Hz), 8.20 (dd, 1H, J = 7.6 Hz, J = 1.6 Hz), 8.86 (s, 1H), 9.88 (s, 1H), 10.70 (s, 1H). ¹³C NMR (MeOH-d₄, 100 MHz) δ (ppm): 69.4 (CH₂), 113.5 (Ar), 122.0 (Ar), 123.4 (C–C = N–NH–), 125.1 (Ar), 125.5 (Ar), 127.0 (Ar), 128.5 (Ar), 131.8 (Ar), 138.7 (CH), 139.4 (Ar), 157.4 (C-O-CH₂), 176.90 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 419 (M^+•, 0.2), 401 (10), 268 (19), 252 (17), 237 (44), 151 (33), 133 (94), 119 (29), 93 (100). (Found: C, 60.3; H, 4.4; N, 16.4. C₂₁H₁₇N₅O₃S requires C, 60.1; H, 4.1; N, 16.7%).
5-(2-Semicarbazonophenyloxymethyl)benzo[1,2-c]1,2,5-oxadiazole N-oxide (24). Pale brown solid (56%), mp 237.6 °C (d). ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 5.26 (s, 2H), 6.44 (s, 2H), 7.02 (t, 1H, J₁ = 7.2 Hz, J₂ = 7.6 Hz), 7.17 (d, 1H, J = 8.8 Hz), 7.36 (t, 1H, J₁ = 8.6 Hz, J₂ = 7.6 Hz), 7.48–7.83 (bs, 3H), 7.49 (s, 1H), 8.04 (d, 1H, J₁ = 7.2 Hz), 8.36 (s, 1H), 10.32 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 69.3 (CH₂), 114.0 (Ar), 122.2 (Ar), 124.3 (C–C = N–NH–), 126.6 (Ar), 131.2 (Ar), 135.6 (C = NH), 156.5 (C-O-CH₂), 157.6 (C [double bond, length as m-dash]

O). MS (EI), m/z (abundance, %): 311 (5), 252 (13), 237 (69), 221 (10), 133 (100), 121 (26), 107 (11). (Found: C, 54.8; H, 3.8; N, 21.1. C₁₅H₁₃N₅O₄ requires C, 55.0; H, 4.0; N, 21.4%).

General procedure for the synthesis of compounds 12–14, 27–30 and 32–37. 2-Nitropropane (1.2 eq.) and piperidine (1.2 eq.) was added to a solution of the corresponding benzofuroxane derivative (50 mg) in dry THF (5 mL). The mixture was stirred at room temperature under nitrogen atmosphere for 24–72 h. The solvent was distilled in vacuo and the residue was purified by preparative TLC (Al₂O₃, petroleum ether–EtOAc, 6 [thin space (1/6-em)]

4).
5-(4-Formylphenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (12). Bordeaux solid (15%), mp 139.4–139.9 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 5.19 (s, 2H), 7.10 (d, 1H, J = 9.6 Hz), 7.28 (m, 4H), 7.93 (d, 2H, J = 8.8 Hz), 9.94 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.5 (CH₃), 68.6 (CH₂), 97.0 (C(CH₃)₂), 112.9 (Ar), 115.2 (Ar), 115.8 (Ar), 130.2 (Ar), 130.8 (Ar), 131.7 (Ar), 135.8 (C–C = N-NH-), 139.9 (Bfx), 163.1 (C-O-CH₂), 190.4 (CHO). MS (EI), m/z (abundance, %): 312 (M^+•, 37), 296 (40), 191 (32), 175 (61), 144 (52), 133 (100), 121 (7). (Found: C, 65.5; H, 5.1; N, 8.9. C₁₇H₁₆N₂O₄ requires C, 65.4; H, 5.2; N, 9.0%).
5-(3-Formylphenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (13). Bordeaux solid (7%), mp 129.5–130.7 °C. MS (EI), m/z (abundance, %): 312 (M^+•, 27), 296 (53), 265 (5), 191 (30), 175 (85), 144 (76), 133 (100), 121 (12). (Found: C, 65.2; H, 4.8; N, 8.7. C₁₇H₁₆N₂O₄ requires C, 65.4; H, 5.2; N, 9.0%).
5-(2-Acetylphenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (14). Bordeaux solid (8%), mp 112.5–113.4 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 1.57 (s, 3H), 5.64 (s, 2H), 6.73 (d, 1H, J = 9.6 Hz), 7.30 (d, 1H, J = 9.6 Hz), 7.43 (t, 1H, J₁ = 6.2, J₂ = 7.4), 7.55 (dd, 2H, J = 8.4 Hz, J₁ = 6.0 Hz, J₂ = 4.4 Hz), 7.87 (s, 1H), 7.93 (d, 1H, J = 9.2 Hz). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.6 (CH₃), 68.5 (CH₂), 96.7 (C(CH₃)₂), 112.2 (Ar), 113.2 (Ar), 116.1 (Ar), 121.3 (Ar), 123.9 (Ar), 128.7 (C–C = N-NH-), 130.7 (Ar), 136.2 (Ar), 140.6 (Bfx), 157.4 (C-O-CH₂), 198.0 (C [double bond, length as m-dash]

O). (Found: C, 65.9; H, 5.4; N, 8.4. C₁₈H₁₈N₂O₄ requires C, 66.2; H, 5.6; N, 8.6%).
5-(4-Thiosemicarbazonophenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (27). Bordeaux solid (34%), mp 122.2–123.0 °C. ¹H NMR (CDCl₃, 400 MHz) δ (ppm): 1.57 (s, 6H), 5.06 (s, 2H), 7.07 (m, 3H), 7.27 (d, 2H, J = 8.8 Hz), 7.77 (d, 2H, J = 8.8 Hz), 7.91 (s, 1H), 8.00 (s, 1H), 8.09 (s, 1H), 11.31 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 24.5 (CH₃), 68.8 (CH₂), 97.6 (C(CH₃)₂), 113.5 (Ar), 115.9 (Ar), 116.6 (Ar), 128.3 (Ar), 129.8 (Ar), 131.7 (Ar), 136.4 (C–C = N-NH-), 141.5 (Bfx), 142.9 (CH), 160.0 (C-O-CH₂), 178.61(C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 354 (M^+• - 16 – 15, 3), 333 (10), 322 (16), 195 (40), 149 (73), 131(20), 121 (75), 57 (100). (Found: C, 55.8; H, 4.9; N, 18.0. C₁₈H₁₉N₅O₃S requires C, 56.1; H, 5.0; N, 18.2%).
5-[4-(N⁴-Allylthiosemicarbazono)phenyloxymethyl]-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (28). Bordeaux solid (31%), mp 121.2–122.1 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 4.35 (t, 2H, J₁ = 4.6 Hz, J₂ = 5.0 Hz), 5.10 (s, 2H), 5.11 (dd, 1H, J_gem = 1.6 Hz, J_cis = 10.0 Hz), 5.22 (dd, 1H, J_gem = 1.6 Hz, J_trans = 17.2 Hz), 5.98 (m, 1H), 6.90 (d, 1H, J = 8.8 Hz), 7.11 (dd, 2H, J = 6.8 Hz, J = 2.0 Hz), 7.23 (d, 1H, J = 9.6 Hz), 7.63 (d, 1H, J = 8.8 Hz), 7.76 (d, 2H, J = 8.8 Hz), 8.15 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.5 (CH₃), 46.0 (CH₂), 68.3 (CH₂), 96.9 (C(CH₃)₂), 112.7 (Ar), 115.1 (Ar), 115.6 (C [double bond, length as m-dash]

C), 115.7 (Ar), 125.9 (Ar), 127.7 (C–C = N-NH-), 128.8 (Ar), 128.9 (Ar), 130.2 (Ar), 134.9 (C [double bond, length as m-dash]

C), 140.3 (Bfx), 142.4 (CH), 159.8 (C-O-CH₂), 176.7 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 396 (M^+• - C₂H₅, 4), 333 (10), 278 (17), 249 (3), 219 (15), 193 (12), 149 (62), 131(36), 57 (100). (Found: C, 59.1; H, 5.1; N, 16.2. C₂₁H₂₃N₅O₃S requires C, 59.3; H, 5.4; N, 16.5%).
5-[4-(N⁴-Phenylthiosemicarbazono)phenyloxymethyl]-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (29). Bordeaux solid (13%), mp 118.3–119.2 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 5.12 (s, 2H), 7.08 (d, 1H, J = 8.0 Hz), 7.14 (dd, 2H, J = 8.4 Hz, J₁ = 2.0 Hz, J₂ = 1.6 Hz), 7.22 (t, 2H, J₁ = 9.0 Hz, J₂ = 11 Hz), 7.29 (s, 1H), 7.37 (t, 2H, J₁ = 7.6 Hz, J₂ = 8.0 Hz), 7.76 (d, 2H, J = 7.6 Hz), 7.86 (d, 2H, J = 8.8 Hz), 8.23 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.5 (CH₃), 68.3 (CH₂), 96.9 (C(CH₃)₂), 112.7 (Ar), 115.1 (Ar), 115.7 (Ar), 124.6 (Ar), 125.0 (Ar), 128.1 (Ar), 129.2 (Ar), 130.2 (Ar), 135.8 (C–C = N-NH-), 140.4 (Bfx), 142.4 (CH), 159.9 (C-O-CH₂), 176.6 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 412 (M^+• - 2 × 17 – 15, 5), 283 (3), 271(20), 207 (10), 177 (5), 145 (100), 135 (27), 93 (52). (Found: C, 62.2; H, 5.2; N, 14.9. C₂₄H₂₃N₅O₃S requires C, 62.5; H, 5.0; N, 15.2%).
5-(4-Semicarbazonophenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (30). Bordeaux solid (25%), mp 123.3-124.1 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 1.57 (s, 6H), 5.05 (s, 2H), 6.39 (s, 2H), 7.04 (m, 3H), 7.26 (d, 2H, J = 8.8 Hz), 7.68 (d, 2H, J = 8.8 Hz), 7.80 (s, 1H), 10.08 (s, 1H). (Found: C, 58.2; H, 4.9; N, 18.9. C₁₈H₁₉N₅O₄ requires C, 58.5; H, 5.2; N, 19.0%).
5-(3-Thiosemicarbazonophenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (32). Bordeaux solid (20%), mp 116.4–117.2 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 5.09 (s, 2H), 7.08 (d, 1H, J = 9.6 Hz), 7.13 (m, 1H), 7.24 (2, 1H, J = 9.2), 7.55 (t, 1H, J = 1.2 Hz), 7.39 (m, 2H), 7.59 (d, 1H, J = 2.0 Hz), 8.17 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.5 (CH₃), 68.3 (CH₂), 96.9 (C(CH₃)₂), 112.5 (Ar), 112.6 (Ar), 115.7 (Ar), 116.7 (Ar), 121.1 (Ar), 129.9 (Ar), 130.3 (Ar), 136.0 (C–C = N-NH-), 140.5 (Bfx), 142.1 (C [double bond, length as m-dash]

NH), 158.7 (C-O-CH₂), 178.7 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 354 (M^+• - 16 – 15, 6), 333 (13), 195 (29), 177 (8), 149 (100), 131 (10), 121 (32), 57 (11). (Found: C, 56.0; H, 5.0; N, 17.8. C₁₈H₁₉N₅O₃S requires C, 56.1; H, 5.0; N, 18.2%).
5-(2-Amidinohydrazonophenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (33). Bordeaux solid (22%), mp 125.7–126.6 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 5.24 (s, 2H), 7.16 (m, 2H), 7.25 (d, 1H, J = 9.6 Hz), 7.34 (m, 2H), 7.67 (dt, 1H, J₁ = 7.6 Hz, J₂ = 8.2 Hz, J₁ = 1.6 Hz, J₂ = 2.0 Hz), 7.81(dd, 1H, J = 7.6 Hz, J = 1.6 Hz), 7.97 (bs, 4H), 10.6 (s, 1H). MS (EI), m/z (abundance, %): 368 (M^+•, 3), 354 (M^+• −14, 7), 296 (18), 278 (24), 175 (77), 145 (83), 133 (84), 121 (100), 69 (91). (Found: C, 58.6; H, 5.2; N, 22.7. C₁₈H₂₀N₆O₃ requires C, 58.7; H, 5.5; N, 22.8%).
5-(2-Thiosemicarbazonophenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (34). Bordeaux solid (30%), 124.5–125.8 °C. ¹H NMR (CDCl₃, 400 MHz) δ (ppm): 1.63 (s, 6H), 5.15 (s, 2H), 7.04 (s, 2H), 7.12 (d, 1H, J = 9.6 Hz), 7.22 (m, 2H), 7.40 (m, 3H), 7.98 (s, 1H), 8.76 (s, 1H). MS (EI), m/z (abundance, %): 354 (M^+• - 16 – 15, 5), 333 (25), 195 (7), 177 (7), 149 (85), 131(20), 121 (100), 57 (69). (Found: C, 55.7; H, 4.7; N, 18.3. C₁₈H₁₉N₅O₃S requires C, 56.1; H, 5.0; N, 18.2%).
5-[2-(N⁴-Allylthiosemicarbazono)phenyloxymethyl]-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (35). Bordeaux solid (20%), mp 114.7–115.0 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 4.37 (t, 2H, J = 5.6 Hz), 5.11 (dd, 1H, J_gem = 1.6 Hz, J_cis = 10.0 Hz), 5.14 (s, 2H), 5.24 (dd, 1H, J_gem = 1.6 Hz, J_trans = 17.2 Hz), 6.00 (m, 1H), 7.03 (t, 1H, J₁ = 7.2 Hz, J₂ = 8.0 Hz), 7.12 (d, 1H, J = 9.6 Hz), 7.21(dd, 2H, J = 11.8 Hz, J = 3.6 Hz), 7.31(s, 1H), 7.41(dt, 1H, J₁ = 7.6 Hz, J₂ = 8.0 Hz, J = 1.6 Hz), 8.04 (dd, 1H, J = 8.0 Hz, J = 1.6 Hz), 8.37 (s, 1H), 8.72 (s, 1H), 10.60 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.9 (CH₃), 45.1 (CH₂), 69.4 (CH₂), 97.3 (C(CH₃)₂), 113.1 (Ar), 113.5 (Ar), 115.6 (C [double bond, length as m-dash]

C), 116.1 (Ar), 121.8 (Ar), 123.7 (Ar), 126.5 (Ar), 130.7 (Ar), 131.6 (Ar), 135.2 (C [double bond, length as m-dash]

C), 138.0 (CH), 140.7 (Bfx), 157.30 (C-O-CH₂), 176.6 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 396 (M^+• - C₂H₅, 3), 333 (8), 278 (15), 249 (5), 219 (10), 193 (10), 149 (57), 131(35), 57 (100). (Found: C, 58.9; H, 5.2; N, 16.1. C₂₁H₂₃N₅O₃S requires C, 59.3; H, 5.4; N, 16.5%).
5-[2-(N⁴-Phenylthiosemicarbazono)formylphenyloxy methyl]-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (36). Bordeaux solid (14%), mp 122.8–123.3 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.62 (s, 6H), 5.17 (s, 2H), 7.19 (m, 1H), 7.24 (m, 5H), 7.36 (t, 2H, J₁ = 7.6 Hz, J₂ = 8.0 Hz), 7.44 (m, 1H), 7.78 (d, 2H, J₁ = 8.0 Hz), 8.20 (dt, 1H, J₁ = 7.2 Hz, J₂ = 8.8 Hz, J = 1.6 Hz), 8.88 (s, 1H), 9.89 (s, 1H), 10.90 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.6 (CH₃), 68.9 (CH₂), 97.6 (C(CH₃)₂), 112.6 (Ar), 113.0 (Ar), 115.7 (Ar), 121.5 (Ar), 122.9 (C–C = N-NH-), 124.6 (Ar), 125.1 (Ar), 126.5 (Ar), 128.1 (Ar), 128.7 (Ar), 130.3 (Ar), 131.1 (Ar), 131.6 (Ar), 138.3, (C = NH), 139.4 (Ar), 140.2 (Bfx), 157.2 (C-O-CH₂), 176.8 (C [double bond, length as m-dash]

S). MS (EI), m/z (abundance, %): 412 (M^+• - 2 × 17 – 15, 13), 283 (5), 271(16), 207 (2), 177 (12), 145 (100), 135 (21), 93 (49). (Found: C, 62.3; H, 4.8; N, 15.0. C₂₄H₂₃N₅O₃S requires C, 62.5; H, 5.0; N, 15.2%).
5-(2-Semicarbazonophenyloxymethyl)-2,2-dimethyl-2H-benzimidazole 1,3-di-N-oxide (37). Bordeaux solid (10%), mp 125.5–126.3 °C. ¹H NMR (acetone-d₆, 400 MHz) δ (ppm): 1.63 (s, 6H), 5.13 (s, 2H), 6.91 (d, 2H, J = 8.4 Hz), 7.24 (d, 2H, J₁ = 8.0 Hz), 7.32 (s, 1H), 7.45 (d, 1H, J = 8.0 Hz), 8.04 (dd, 1H, J = 7.6 Hz, J = 1.6 Hz), 8.50 (s, 1H). ¹³C NMR (acetone-d₆, 100 MHz) δ (ppm): 23.0 (CH₃), 68.9 (CH₂), 96.6 (C(CH₃)₂), 112.3 (Ar), 116.0 (Ar), 121.1 (Ar), 124.1 (C–C = N–NH–), 125.3 (Ar), 129.3 (Ar), 130.0 (Ar), 136.0 (C [double bond, length as m-dash]

NH), 140.6 (C-O-CH₂), 141.8 (Ar), 156.2 (Bfx), 158.0 (C [double bond, length as m-dash]

O). MS (EI), m/z (abundance, %): 369 (M^+•, 3), 336 (11), 279 (18), 237 (44), 175 (47), 145 (100), 133 (59). (Found: C, 58.4; H, 5.0; N, 18.8. C₁₈H₁₉N₅O₄ requires C, 58.5; H, 5.2; N, 19.0%).

Biology

In vitro anti-trypanosomal activity. T. cruzi epimastigotes (Tulahuen 2 strain) were grown axenically at 28 °C in BHI-Tryptose as previously described,^9–11 complemented with 5% fetal calf serum. Cells were harvested in late log phase, suspended in fresh medium, counted in a Neubauer's chamber and placed in 24-well plates (2 × 10⁶ mL⁻¹). Cell growth was measured as the absorbance of the culture at 590 nm, which was found to be proportional to the number of cells. Before inoculation, the medium was supplemented with the indicated amount of the compound to be analyzed from a stock solution in DMSO. The final concentration of DMSO in the culture media never exceeded 1% and a control was run with 1% DMSO and the absence of any compound. No effect on epimastigote growth was observed in the presence of up to 1% DMSO in the culture media. Nfx and Bnz were used as the reference trypanocidal drugs. The percentage of growth inhibition was calculated as follows {1 − [(A_p − A_0p)/(A_c − A_0c)]} × 100, where A_p = A₅₉₀ of the culture containing the compound to be analyzed at day 5; A0p = A₅₉₀ of the culture containing the compound to be analyzed just after addition of the inocula (day 0); A_c = A₅₉₀ of the culture in the absence of any compound (control) at day 5; A_0c = A₅₉₀ in the absence of the compound at day 0. To determine ID₅₀ values, parasite growth was followed in the absence (control) and presence of increasing concentrations of the corresponding compound. The ID₅₀ values were determined as the drug concentrations required to reduce the absorbance by half that measured for untreated controls.

Unspecific mammalian cytotoxicity.^9,10. J-774 murine macrophage-like cells (ATCC, USA) were maintained by passage in Dulbecco's modified Eagle's medium (DMEM) containing 4 mM L-glutamine, and supplemented with 10% heat-inactivated fetal calf serum. J-774 cells were seeded (1 × 10⁵ cells/well) in 96-well microplates with 200 μL RPMI 1640 medium supplemented with 20% heat inactivated foetal calf serum. Cells were allowed to attach for 48 h in a humidified 5% CO₂/95% air atmosphere at 37 °C and then exposed to compounds (100.0–400.0 μM) for 48 h. Afterwards, cell viability was assessed by measuring the mitochondrial-dependent reduction of MTT (Sigma) to formazan. For that purpose, MTT was added to cells to a final concentration 0.4 mg mL⁻¹ and cells were incubated at 37 °C for 3 h. After removing the media, formazan crystals were dissolved in DMSO (180 μL), and absorbance at 595 nm was determined using a microplate spectrophotometer. Results are expressed as ID₅₀ (compound concentration that reduced 50% control absorbance at 595 nm). Every ID₅₀ is the average of three different experiments.

Red blood cell lysis assay.²⁹. Human blood collected in sodium citrate solution (3.8%) was centrifuged at 200 g for 10 min at 4 °C. The plasma supernatant was removed and erythrocytes were suspended in ice cold PBS. The cells were again centrifuged at 200 g for 10 min at 4 °C. This procedure was repeated two more times to ensure the removal of any released haemoglobin. Once the supernatant was removed after the last wash, the cells were suspended in PBS to 2% w/v red blood cell solution. A volume of 400 μL of compound to be analyzed, in PBS (final doses 50, 100 and 200 μM), negative control (solution of PBS), or AmpB (final dose 1.5 μM) were added in 400 μL to the 2% w/v red blood cell solution. Ten replicates for each concentration were done (see below), and were incubated for 24 h at 37 °C prior to analysis. Complete haemolysis was attained using neat water yielding the 100% control value (positive control). After incubation, the tubes were centrifuged and the supernatants were transferred to new tubes. The release of haemoglobin into the supernatant was determined spectrophotometrically at 405 nm using an EL 301 MICROWELL STRIP READER. Results are expressed as percentage of total haemoglobin released in the presence of the compounds. This percentage was calculated using the equation percentage haemolysis (%) = [(A₁ − A₀)/A₁_water] × 100, where A₁ is the absorbance at 405 nm of the test sample at t = 24 h, A₀ is the absorbance at 405 nm of the test sample at t = 0 h, and A₁_water is the absorbance at 405 nm of the positive control (water) at t = 24 h. The experiments were done by quintuplicate.

Cruzipain inhibitory activity³⁰. CP (6 μM) was incubated in a reaction mixture containing 50 mM PBS, pH 7.3, or acetate buffer, pH 5.3, 5 mM DTT and 25, 50 or 100 μM compound for 5 min at room temperature. Fluorogenic substrate Z-Phe-Arg-AMC (K_M = 1.8 μM) was added to a concentration of 10 μM, and the increase in fluorescence (excitation 380 nm and emission 460 nm) was monitored for 10 min at room temperature in a 96-well microplate Varioskan spectrofluorometer and spectrophotometer. Compounds were added as solutions in DMSO and positive controls contained only buffered solvent. The final assay volume was 100 μL and the final DMSO concentration never exceeded 10%. ID₅₀ values were independently determined from the three inhibitor concentrations. The CP-inhibitor reference compound mbth was included in the analysis as a control. The values represent means of at least three experiments.

Non-specific inhibition was evaluated in the presence of Triton X-100. A stock solution 0.02% (v/v) of the detergent was freshly prepared in 100 mM PBS or acetate buffer (pH 7.3 or 5.3, respectively) with EDTA and DTT to achieve the same final concentrations as in the standard assay. 50 μL of this solution was then added to 50 μL reaction mix to obtain 0.01% of Triton X-100 in the final reaction mix. This concentration of Triton X-100 was found not to interfere with CP activity.

Docking studies

The structures of the ligands, as protonated form for the amidinohydrazones and as neutral form for the thio- and semicarbazones, were built with standard bond lengths and angles using the molecular modeling package SYBYL 8.1³¹ and their energies were minimized using the Conjugate Gradient algorithm with a conjugated gradient of <0.001 kcal mol⁻¹ convergent criteria provided by the MMFF94 force field³² and MMFF94 electrostatic charges. The ligands considered were superimposed onto the inhibitor present in the reference structure (pdb code 1f29) but without forming any covalent bond with the enzyme. The ligand–receptor complex was subjected to energy minimization using the MMFF94 force field and MMFF94 electrostatic charges and their energies were minimized using the protocol previously indicated with a conjugate gradient of <0.1 kcal mol⁻¹ convergent criteria. These complexes were the input structure for docking using the FlexiDock command.³³ During the flexible docking analysis, the ligands and a sphere of 6 Å around the corresponding ligand were considered flexible. For each complex three flexible docking analyses were run. The default SYBYL FlexiDock parameters were utilized in all cases, with maximum and minimum iterations (MI) set for each complex according to MI = [N° of rotable bonds in the protein + N° of rotable bonds in the ligand + 6 × 1000 − 500, obtaining a series of model complexes. All conformations obtained with FlexiDock were clustered using a hierarchical cluster analysis taking into account the score and the distances of ligand moieties to key amino acids present in the active site of the enzyme. We chose the conformation with highest FlexiDock score (better interactions) and refined the minimization energy step using a conjugate gradient of <0.01 kcal mol⁻¹ convergent criteria. Analysis of the refined receptor–ligand complex models was based on hydrogen bond, aromatic and hydrophobic interactions predicted with the LPC (Ligand Protein Contact) program³⁴ and the values of ΔG binding and dissociation constants obtained from the difference accessible surface area method using the STC (Structural Thermodynamics Calculations) program.³⁵

Discussion and conclusions

We identified new benzofuroxan and benzimidazole 1,3-dioxide derivatives with interesting trypanosomicidal activities. Compounds 23, 35 and 37 exhibited relevant anti-T. cruzi activity, suggesting that a 2-substituted structural motif played a role that resulted in altering activities relative to those of the corresponding 4-substituted analogues, i.e., 16, 28 and 30, respectively (Table 1 and Fig. 3a). Furthermore, the erythrocyte cytotoxicity of derivative 23 was lower than that of either Nfx or AmpB (Table 1).


	Fig. 3 a) In vitro anti-T. cruzi active moieties described here. b) A previous CP inhibitor and the best ones described here.

The anti-T. cruzi profile of these derivatives did not appear to be highly correlated with CP inhibition, i.e. derivative 23 was unable to inhibit CP in the assay conditions (Table 3), and was the most active and selective anti-T. cruzi agent. However, some derivatives displayed interesting CP-inhibitory activities that allow us to corroborate some structure–activity features. On the one hand, no clear relationship between core scaffold, benzofuroxan or benzimidazole 1,3-dioxide, and CP inhibitory activity was observed, i.e., comparing inhibition of the pair 15 and 28 with that of the pair 21 and 34 (Table 3). For the first pair the benzimidazole 1,3-oxide derivative was the most active, whereas the opposite was the case for the second pair. On the other hand, there appeared to be a consistent relationship between both substituents’ type and position and CP inhibitory activity. In general, the thiosemicarbazones were the best CP inhibitors such that the order thiosemicarbazonyl > amidinohydrazonyl ∼ carbonyl > semicarbazonyl summarized the relative inhibitory activities. In reference to the position of these moieties, the 3-substituted derivatives exhibited better inhibitory activities, i.e., when comparing activities of 3-substituted derivatives (18, 19 and 32) the 4-substituted (15, 27 and 28) and 2-substituted derivatives (21, 22, 34 and 35) (Table 3). These results were consistent with previous reports describing other thiosemicarbazones (Fig. 3b).^20,36 Theoretical results clearly indicated that 3-substituted derivatives were able to interact via hydrogen bonding and hydrophobic interactions with CP consistent with the more negative ΔG for the most stable conformers (Table 5) and our experimental data.

The most lipophilic derivatives were the most active against the whole parasite, i.e. hybrid compounds 16, 23, 35 and 36, with values of miLogP³⁷ of 4.86, 4.82, 3.93 and 4.35 (Table 7), respectively, while the most hydrophilic derivatives were inactive, i.e., hybrid compounds 17, 24, 30 and 33, with values of miLogP of 2.67, 2.84, 2.42 and 2.17, respectively. When the molecular lipophilicity potential (MLP), virtual LogP,³⁸ was calculated for the most active benzimidazole 1,3-dioxide derivative, 35, and the least active, 33, the difference in the lipophilicities was correlated with the relative inhibitory activities. The MLP analysis showed that the main differences were localized to the 2-phenyl substituents (Fig. 4). In addition, no relationship between miLogPs and CP inhibitory activity was found.

Table 7 Estimated properties of compounds investigated.^a,³⁷

Compd	miLogP	nON	nOHNH	MW	nviolations	TPSA
a miLogP, logarithm of compound partition coefficient between n-octanol and water; nON, number of hydrogen bond acceptors; nOHNH, number of hydrogen bond donors; MW, molecular weight; TPSA, topological polar surface area (Å²).
8	3.28	6	0	270.2	0	77.8
10	3.23	6	0	270.2	0	77.8
11	3.33	6	0	284.3	0	77.8
16	4.86	8	2	419.5	0	97.1
18	3.40	8	3	343.4	0	111.1
19	4.43	8	2	383.4	0	97.1
23	4.82	8	2	419.5	0	97.1
32	2.94	8	3	385.4	0	117.1
35	3.93	8	2	425.5	0	103.2
36	4.35	8	2	461.5	0	103.2
37	2.37	9	3	369.4	0	134.2
Nfx	0.71	8	0	287.30	0	108.71


	Fig. 4 MLP maps, calculated projections of Broto-Moreau lipophilicity atomic constants on the molecular surface.³⁸ Molecules 33 and 35 are represented as tubes with the traditional atom colors. MLP colors: red/yellow for hydrophilic regions; violet/blue for lipophilic regions; green for intermediate regions. The arrows show the polar substituent for 33 (a) and the lipophilic substituent for 35 (b).

In terms of the hybrid derivatives drug-likeness properties, their conformity with the criteria of Lipinski's rule was analyzed.³⁹ In this sense, we determined some properties, i.e., miLogP, number of donor and acceptor hydrogen bonds and molecular weight (Table 7),³⁷ which determine whether these derivatives are similar to known drugs. All the most active derivatives, against whole parasite or CP, had properties in compliance with Lipinski's rule and some had better polar topological surface areas (TPSA,⁴⁰ Table 7) than the value for Nfx, distinguishing them as promising candidates for further drug development.

Further biological studies, QSAR studies, and in vivo activities are currently underway.

Acknowledgements

Financial support from RIDIMEDCHAG network (CYTED), from Collaborative Project UdelaR (Uruguay) – CSIC (Spain) (#2007UY0004) and from REDCLARA-AECID is acknowledged. We thank PEDECIBA-ANII for scholarships to AM, DB, and PH and PEDECIBA for a fellowship to AM. We thank Dr Graciela Mahler for the gift of the mbth reference compound.

Notes and references

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