Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma...

CONCISE ARTICLE www.rsc.org/medchemcomm | MedChemComm

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online / Journal Homepage / Table of Contents for this issue

Development of second generation amidinohydrazones, thio- andsemicarbazones as Trypanosoma cruzi-inhibitors bearing benzofuroxan andbenzimidazole 1,3-dioxide core scaffolds

Alicia Merlino,a Diego Benitez,a Santiago Chavez,a Jonathan Da Cunha,a Paola Hern�andez,a

Luzineide W. Tinoco,b Nuria E. Campillo,c Juan A. P�aez,c Hugo Cerecetto*a and Mercedes Gonz�alez*a

Received 19th June 2010, Accepted 19th July 2010

DOI: 10.1039/c0md00085j

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 inad-

equate, 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

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

aGrupo de Qu�ımica Medicinal, Laboratorio de Qu�ımica Org�anica, Facultadde Ciencias-Facultad de Qu�ımica, Universidad de la Rep�ublica, Igu�a 422511400 Montevideo, Uruguay. E-mail: [email protected]; [email protected]; 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 Qu�ımica M�edica, CSIC, Madrid, Spain

216 | Med. Chem. Commun., 2010, 1, 216–228

least two mechanism of action. In addition,11 we provided evidence

that benzofuroxan derivatives were able to modify parasite dehy-

drogenase activity and to affect mitochondrial membrane poten-

tial, whereas amidinohydrazone and thiosemicarbazone moieties

had already been described as pharmacophores for trypanothione

reductase and CP inhibitors, respectively.12 In this study we iden-

tified parent compounds 1–5 (Scheme 1b) with good in vitro try-

panosomicidal 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 benzimid-

azole 1,3-dioxide system (i.e. 7, Scheme 1b).9 We hypothesized that

this heterocycle might act as a free radical-releasing pharmaco-

phore; 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 benz-

imidazole 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 carbonyl-

phenyloxymethylbenzofuroxan (8–11) as shown in Scheme 2.

This journal is ª The Royal Society of Chemistry 2010

http://dx.doi.org/10.1039/C0MD00085J

http://pubs.rsc.org/en/journals/journal/MD

http://pubs.rsc.org/en/journals/journal/MD?issueid=MD001003

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

described.

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

Aldehydes 8 and 9 were prepared according to a previous

report10 with minor modification, using 5-bromomethylbenzo-

furoxan13 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). Amidinohy-

drazone, 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 benz-

imidazole 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 amidi-

nohydrazones 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.

The new benzofuroxan and benzimidazole 1,3-dioxide deriv-

atives, 9–25, 27–30, and 32–37, were assessed in vitro for anti-

proliferative activity against the epimastigote form of the

T. cruzi, Tulahuen 2 strain.9,10 The occurrence of the epi-

mastigote form of T. cruzi as an obligate mammalian intracel-

lular 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

mM 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 semi-

carbazones 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

Med. Chem. Commun., 2010, 1, 216–228 | 217


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

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

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,

Table 1 Biological characterization of hybrid derivatives using T. cruzi, and

Seriesa Compd

ID50/mM

Compd

ID50/mM

T2b,c rbcc,d T2b,c rbcc,d

bfx ca 8 8.7e < 50 9 28 � 2 50 � 5bz 12 17.0 � 1.9 65 � 3 13 > 25 < 50bfx ah 4 > 25 e > 200 17 > 25 > 100f

bz 26 —g — 31 — —bfx th 5 15.0 e > 200 18 15.0 � 0.4 agh

bz 27 17.0 � 0.8 ag 32 24 � 2 141 � 5bfx at 15 > 25.0 ag 19 13 � 2 agbz 28 23.0 � 1.5 > 100h

bfx pt 16 7 � 3 agbz 29 > 25 < 50bfx se 6 > 25.0 e > 200bz 30 > 25 > 200

a bfx: benzofuroxan; bz: benzimidazole 1,3-dioxide; ca: carbonyls; ah: amidinN4-phenylthiosemicarbazones. b T2: Tulahuen 2 strain. c The results are thee From ref. 10. f Problems with compound solubility were observed at hiformed aggregates in the buffer at all doses assayed. i ns: not studied.

218 | Med. Chem. Commun., 2010, 1, 216–228

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.

Two different biological systems, erythrocytes and macro-

phages, were used to estimate the potential toxicity of the hybrid

derivatives (Tables 1 and 2),9,10,16 Erythrocytes served as

human red blood cells

Compd

ID50/mM

Compd

ID50/mM

T2b,c rbcc,d T2b,c rbcc,d

10 6.3 � 0.2 > 100 11 7.0 � 1.2 > 20014 > 25 < 50

20 > 25 58 � 3 25 16.6 � 2.3 > 20033 > 25.0 114 � 321 > 25 189 � 134 14 � 2 > 20022 23 � 1 139 � 235 2.7 � 1.4 ag23 3.6 � 0.4 > 20036 7.7 � 0.3 66.0 � 0.8 Nfx 7.7e > 100f

24 > 25 > 100h Bnz 7.4 e nsi

37 4.7 � 1.2 50.0 � 0.4 AmpB 0.152 � 0.006 1.5 � 0.2

ohydrazones; th: thiosemicarbazones; at: N4-allylthiosemicarbazones; pt:means of three independent experiments. d rbc: Human red blood cells.gher doses. g ‘‘—’’: compound was not obtained. h ag: the compound



Table 3 Inhibition of CP activity by hybrid derivatives

Compd.% of inhibitionat 100.0 mMa,b ID50/mMa,b Compd.

% of inhibitionat 100.0 mMa,b ID50/mMa,b

8 6.3 � 0.6 >100.0 12 35.5 � 1.5 >100.09 43.0 � 0.8 � 100.0 13 45.0 � 1.0 � 100.010 0.0 >100.0 14 0.0 >100.011 12.0 � 0.7 >100.0

27 13.7 � 1.9 >100.015 38.0 � 2.0 >100.0 28 51.0 � 0.7 100.0 � 2.016 95 � 2 75.0 � 2.1 29 72 � 4 85.0 � 1.3

30 0.0 >100.017 42.0 � 1.8 � 100.018 80 � 3 78.0 � 1.6 32 95 � 3 64.0 � 1.919 100 � 2 55.0 � 2.020 39.0 � 1.5 >100.0 33 2.5 � 0.5 >100.021 49.0 � 0.8 100.0 � 2.0 34 35.0 � 1.6 >100.022 11.0 � 2.1 >100.0 35 6.6 � 0.9 >100.023 0.0 >100.0 36 0.0 >100.024 7.0 � 1.3 >100.0 37 0.0 >100.025 21.0 � 0.8 >100.0

mbthc 100.0 � 0.5 0.10 � 0.08

a In buffer, pH 7.3, and absence of Triton X-100 (see ExperimentalSection). b The results are the means of three independent experiments.c mbth: (m-bromophenyl)ethylketone thiosemicarbazone (referencecompound).

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

Compd. pH Triton X-100 addition % of inhibition at 100.0 mMa

17 7.3 — 42.0 � 1.85.3 — 28.0 � 0.9

19 7.3 — 100.0

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

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 mM for erythrocytes and

at doses of 100.0–400.0 mM 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 ampho-

tericin 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-thio-

semicarbazone 23, had excellent selectivity indexes. The selec-

tivity indexes for derivative 23 were SIerythrocyte/T.cruzi > 55.6 and

SImacrophage/T.cruzi > 111.1, and in contrast to the thio-

semicarbazone parent compound 5 (SIerythrocyte/T.cruzi > 13.3 and

SImacrophage/T.cruzi ¼ 26.7) and the carbonyl parent compound

10 (SIerythrocyte/T.cruzi > 15.9 and SImacrophage/T.cruzi ¼ 12.4), and

Nfx (SIerythrocyte/T.cruzi ¼ 13.0) and AmpB (SIerythrocyte/T.cruzi �10.0) (Tables 1 and 2). Thus, derivative 23 had far better selec-

tivity indexes than parent derivatives 5 and 10 and reference

compounds.
+ 90.0 � 1.5
5.3 — 79 � 1

a The results are the means of three independent experiments.

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

Seriesa Compd ID50/mMb Compd ID50/mMb

bfx ca 8 60.0c 10 78 � 2bz 12 < 100bfx ah 4 < 50 c 20 < 100bfx th 5 400.0c 21 < 100bfx at 22 138 � 2bfx pt 23 > 400bz 36 < 100bfx se 24 < 100

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 threeindependent experiments. c From ref. 10.

We investigated the ability of the hybrid derivatives to inhibit

CP to obtain information on their mechanism of trypanosomi-

cidal activity. The compounds were tested at 25.0, 50.0, and 100.0

mM for the ability to inhibit CP. The compounds were assayed

using Z-phenyl-arginine-7-amido-4-methylcoumarin hydrochlo-

ride (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 previ-

ously 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), thio-

semicarbazone 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.

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 macro-

molecule.21 Unfortunately, we were unable to obtain information

using derivatives 15, 16, 18, 19 and 32 as ligands due to the low

Med. Chem. Commun., 2010, 1, 216–228 | 219


Table 5 Theoretical Kd, DG binding, and experimental ID50 values ofthe CP–ligand complexes analyzed

Compd. Kd/Ma DG/kcal mol�1a ID50,CP

4 9.7 � 10�4 �4.11 32.016 4.4 � 10�3 �3.21 75.017 6.0 � 10�3 �3.03 > 100.018 1.2 � 10�3 �3.92 78.019 2.9 � 10�3 �3.46 55.021 5.7 � 10�3 �3.06 100.023 3.6 � 10�1 �0.61 > 100.029 5.4 � 10�3 �3.09 85.034 9.6 � 10�3 �2.75 > 100.0mbth 6.2 � 10�4 �4.37 0.10

a Values were estimated from the most stable conformers.

Fig. 1 Theoretical Kd vs. experimental ID50 values for CP–ligand

complexes analyzed. Notes: ID50 for derivative 23 was inferred by

extrapolation as ca. 400 mM; the line shows tendency.

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

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 thio-

semicarbazone (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 bio-

logical (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

Table 6 Principal CP residues in contact with compounds analyzed

Cys25 (S) Gly66 (N) Gly66 (O) Leu67

4 HBa (N6-Bfx)b HB (Y ¼ –O–) HB (N5-Bfx) Hphc (16 — — — —17 — — HB/N4, HB (N2) —18 — HB (O1) — —19 — HB/N1 HB/N4 Hph (a21 — — — —23 — — — —29 — — — —34 — — HB/N4 –mbthg — — HB/N2 Hph

a HB: hydrogen bond. b According to labelled structure. c Hph: hydrophobicg The same atomic labels at the thiosemicarbazone moiety as those of the hy

220 | Med. Chem. Commun., 2010, 1, 216–228

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 iden-

tified (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.

Leu157 Asp158 (O) His159 (Nd1) Glu205 (O32)

Bfxd) —e HB/N2 — HB/N3

Hph (Bfx) — — —— HB/N3 — —— — — HB/N4

llf) Hph (Bfx) — — —— HB/N2 HB/N4

— — — —Hph — — —— HB/N2 — —— — — —

interactions. d Bfx: benzofuroxan ring. e no interaction. f all: allyl moiety.brid derivatives analyzed.



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.

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

In the CP–19 complex (Fig. 2a) Gly66 formed stable

hydrogen bonds with the N1 (3.5 �A) and N4 (2.8 �A) thio-

semicarbazone 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 mM, 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 proton-

ated forms), CP–17 and CP–4, respectively, it could be seen that

when the molecule occupies the active-cleft with the benzofur-

oxan 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.


Experimental section

Chemistry

General methods. Compounds 4–6, 8 and 5-bromome-

thylbenzofuroxan 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 d relative to tetrame-

thylsilane 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 ioni-

zation (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 : 3)

until the disappearance of the reactants. The solvent was

Med. Chem. Commun., 2010, 1, 216–228 | 221


Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

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) d (ppm): 5.16 (s, 2H),

7.31 (m, 1H), 7.36–7.69 (bs, 3H), 7.49 (s, 1H), 7.53 (d, 1H, J ¼7.6 Hz), 7.55 (t, 1H, J1 ¼ 2.0 Hz, J2 ¼ 1.2 Hz), 10.00 (s, 1H).13C NMR (CDCl3, 100 MHz) d (ppm): 68.7 (CH2), 112.4 (Ar),

122.2 (Ar), 124.9 (Ar), 130.5 (Ar), 137.9 (C–C ¼ 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 concen-

trated in vacuo. The residue was purified by flash chromatog-

raphy (petroleum ether–EtOAc, 7 : 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) d (ppm): 5.37 (s, 2H), 7.15 (t, 1H, J ¼ 7.5 Hz),

7.33 (d, 1H, J ¼ 8.4 Hz), 7.69 (t, 1H, J1 ¼ 8.6 Hz, J2 ¼ 7.4 Hz),

7.57–7.77 (bs, 3H), 7.76 (d, 1H, J ¼ 7.5 Hz), 10.50 (s, 1H). 13C

NMR (CDCl3, 100 MHz) d (ppm): 69.5 (CH2), 114.8 (Ar), 122.2

(Ar), 125.5 (C–C ¼ 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) d (ppm): 2.59 (s, 3H), 5.41 (s, 2H), 7.09

(dt, 1H, J1 ¼ 7.2 Hz, J2 ¼ 7.8 Hz, J1 ¼ 0.8 Hz, J2 ¼ 0.4 Hz), 7.29

(d, 1H, J ¼ 8.4 Hz), 7.54 (m, 1H), 7.58–7.68 (bs, 3H), 7.71 (dd,

1H, J ¼ 7.6 Hz, J ¼ 1.6 Hz). 13C NMR (acetone-d6, 100 MHz)

d (ppm): 31.1 (CH3), 69.2 (CH2), 113.4 (Ar), 121.1 (Ar), 128.9 (C–

C ¼ N–NH–), 130.0 (Ar), 133.5 (Ar), 157.3 (C-O-CH2), 198.1

(C]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 : 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

222 | Med. Chem. Commun., 2010, 1, 216–228

(d). 1H NMR (DMSO-d6, 400 MHz) d (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) d (ppm): 68.44

(CH2), 113.1 (Ar), 117.9 (Ar), 121.9 (Ar), 130.4 (Ar), 147.0

(CH), 135.3 (C–C ¼ N–NH–), 155.7 (C ¼ 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) d (ppm): 5.23 (s, 2H), 5.31 (s, 2H),

5.89 (s, 2H), 6.97 (t, 1H, J1 ¼ 7.6 Hz, J2 ¼ 7.2 Hz), 7.12 (t, 1H,

J1 ¼ 8.4 Hz, J2 ¼ 9.2 Hz), 7.27 (t, 1H, J1 ¼ 7.2 Hz, J2 ¼ 8.0 Hz),

7.49–7.78 (bs, 3H), 8.00 (d, 1H, J ¼ 7.6 Hz), 8.39 (s, 1H). 13C

NMR (CDCl3, 100 MHz) d (ppm): 69.2 (CH2), 113.7 (Ar),

122.0 (Ar), 126.3 (C–C ¼ N–NH–), 126.5 (Ar), 129.8 (Ar),

139.0 (Ar), 156.0 (C-O-CH2), 161.3 (C ¼ 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) d (ppm): 2.56 (s, 3H),

5.32 (s, 2H), 7.08 (t, 3H, J1 ¼ 7.2 Hz, J2 ¼ 7.6 Hz), 7.27 (d, 1H,

J¼ 8.4 Hz), 7.52–7.89 (bs, 3H), 7.57 (dt, 1H, J1¼ 7.2 Hz, J2¼ 8.4

Hz, J¼ 2.0 Hz), 7.65 (dd, 1H, J ¼ 7.6 Hz, J ¼ 1.6 Hz). 13C NMR

(DMSO-d6, 100 MHz) d (ppm): 40.2 (CH3), 69.2 (CH2), 114.1

(Ar), 121.5 (Ar), 128.6 (C–C ¼ N–NH–), 130.2 (Ar), 134.3 (Ar),

155.8 (HNC¼NH), 157.2 (C–C ¼ N-NH-), 167.2 (CH3C¼N).

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,

n/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. N4-Phenylthiosemicarbazide or N4-allylth-

iosemicarbazide (1 eq.) was added to a solution of the corre-

sponding carbonyl reactant (8–11) (50 mg, 0.185 mmol) in dry

ethanol (10.0 mL) acetic acid (0.1%) and semicarbazide, thio-

semicarbazide. 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 : 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) d (ppm): 4.21 (t,

2H, J1 ¼ 5.2 Hz, J2 ¼ 5.6 Hz), 5.10 (dd, 1H, Jgem ¼ 1.6 Hz, Jcis ¼10.2 Hz), 5.15 (dd, 1H, Jgem ¼ 1.6 Hz, Jtrans ¼ 17.2 Hz), 5.25 (s,

2H), 5.91 (m, 1H), 7.11 (d, 1H, J ¼ 8.8 Hz), 7.42–7.68 (bs, 3H),

7.79 (d, 1H, J ¼ 8.8 Hz), 8.02 (s, 1H), 8.65 (t, 1H, J ¼6.0 Hz), 11.40 (s, 1H). 13C NMR (DMSO-d6, 100 MHz)

d (ppm): 46.2 (CH2), 69.6 (CH2), 115.5 (Ar), 115.9 (C]C), 127.9



Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

(C–C ¼ N–NH–), 129.4 (Ar), 135.6 (C ¼ C), 142.2, (C ¼ NH),

159.6 (C-O-CH2), 177.4 (C ¼ S). MS (ESI), m/z: 429 (M + 2Na+).

IR, n/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) d (ppm): 5.26 (s, 2H), 7.13 (d,

2H, J ¼ 8.8 Hz), 7.21 (t, 1H, J1 ¼ 7.6 Hz, J2 ¼ 7.2 Hz), 7.37 (t, 2H,

J1 ¼ 7.6 Hz, J2 ¼ 8.4 Hz), 7.56 (d, 2H, J ¼ 7.6 Hz), 7.60–7.84 (bs,

3H), 7.89 (d, 2H, J ¼ 8.8 Hz), 8.11 (s, 1H), 10.10 (s, 1H). 13C NMR

(DMSO-d6, 100 MHz) d (ppm): 68.5 (CH2), 115.5 (Ar), 125.7 (Ar),

126.3 (Ar), 127.7 (C–C ¼ N–NH–), 128.5 (Ar), 129.8 (Ar), 139.6

(Ar), 143.1 (C¼NH), 159.8 (C-O-CH2), 176.1 (C¼ 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, n/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) d (ppm): 5.24 (s, 2H), 7.10

(d, 1H, J ¼ 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) d (ppm): 68.6 (CH2), 112.5 (Ar),

117.3 (Ar), 121.7 (Ar), 130.4 (Ar), 136.2 (C–C¼N–NH–), 142.3

(C ¼ NH), 158.6 (C-O-CH2), 162.8 (Bfx), 178.5 (C ¼ 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 n:

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%).


c]1,2,5-oxadiazole N-oxide (19). Pale brown solid (80%), mp

149.6 �C (d). 1H NMR (DMSO-d6), 400 MHz) d (ppm): 4.23 (t,

2H, J1 ¼ 4.8 Hz, J2 ¼ 5.2 Hz), 5.10 (dd, 1H, Jgem ¼ 1.2 Hz, Jcis ¼12 Hz), 5.16 (dd, 1H, Jgem ¼ 1.2 Hz, Jtrans ¼ 18 Hz), 5.24 (s, 2H),

5.93 (m, 1H), 7.12 (d, 1H, J ¼ 6.8 Hz), 7.38 (d, 2H, J ¼ 6.8 Hz),

7.56 (s, 1H), 7.64–7.88 (bs, 3H), 8.04 (s, 1H), 8.74 (t, 1H, J ¼ 5.2

Hz), 11.60 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) d (ppm): 46.2

(CH2), 68.6 (CH2), 113.5 (Ar), 116.0 (C]C), 116.6 (Ar), 121.3

(Ar), 130.4 (Ar), 135.5 (C]C), 136.2 (C–C ¼ N–NH–), 142.2,

(C ¼ NH), 158.6 (C-O-CH2), 177.7 (C ¼ 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) d (ppm): 5.27 (s, 2H), 7.03 (dd,

1H, J1 ¼ 7.6 Hz, J2 ¼ 7.4 Hz), 7.20 (d, 1H, J1 ¼ 8.4 Hz), 7.42

(dd, 1H, J1 ¼ 7.4 Hz, J2 ¼ 8.2 Hz), 7.49–7.79 (bs, 3H), 7.96 (s,

1H), 8.15 (s, 1H), 8.16 (d, 1H, 3J¼ 7.6 Hz), 8.62 (s, 1H), 11.50 (s,

1H). 13C NMR (DMSO-d6, 100 MHz) d (ppm): 69.3 (CH2),

114.0 (Ar), 122.2 (Ar), 123.6 (C–C ¼N-NH-), 127.1 (Ar), 132.2

(Ar), 138.9 (CH), 157.1 (C-O-CH2), 163.2 (Bfx), 178.7 (C]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%).



191.4 �C (d). 1H NMR (MeOH-d4, 400 MHz) d (ppm): 4.36 (m,

2H), 5.11 (dd, 1H, Jgem ¼ 1.6 Hz, Jcis ¼ 10.0 Hz), 5.23 (dd, 1H,

Jgem¼ 1.6 Hz, Jtrans¼ 17.2 Hz), 5.36 (s, 2H), 6.00 (m, 1H), 7.05 (t,

1H, J ¼ 7.6 Hz), 7.24 (d, 1H, J ¼ 8.0 Hz), 7.43 (dt, 1H, J1 ¼ 7.2

Hz, J2¼ 8.4 Hz, J¼ 1.6 Hz), 7.56–7.78 (bs, 3H), 8.06 (dd, 1H, J¼7.6 Hz, J ¼ 1.6 Hz), 8.38 (s, 1H), 8.77 (s, 1H), 10.50 (s, 1H). 13C

NMR (MeOH-d4, 100 MHz) d (ppm): 45.9 (CH2), 69.3 (CH2),

113.5 (Ar), 115.6 (C]C), 121.9 (Ar), 123.6 (C–C ¼ N–NH–),

126.5 (Ar), 131.7 (Ar), 135.2 (C]C), 138.0, (CH), 157.2 (C-O-

CH2), 176.7 (C]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-


164.3 �C (d). 1H NMR (MeOH-d4, 400 MHz) d (ppm): 5.38 (s, 2H),

7.08 (t, 1H, J¼ 7.6 Hz), 7.20 (t, 1H, J1¼ 7.6 Hz, J2¼ 7.2 Hz), 7.27

(d, 1H, J ¼ 8.4 Hz), 7.37 (t, 2H, J ¼ 8.0 Hz), 7.46 (dt, 1H, J ¼ 7.6

Hz, J ¼ 1.6 Hz), 7.52–7.72 (bs, 3H), 7.77 (t, 1H, J ¼ 7.6 Hz), 8.20

(dd, 1H, J¼ 7.6 Hz, J¼ 1.6 Hz), 8.86 (s, 1H), 9.88 (s, 1H), 10.70 (s,

1H). 13C NMR (MeOH-d4, 100 MHz) d (ppm): 69.4 (CH2), 113.5

(Ar), 122.0 (Ar), 123.4 (C–C ¼ 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]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-oxa-

diazole N-oxide (24). Pale brown solid (56%), mp 237.6 �C (d).1H NMR (DMSO-d6, 400 MHz) d (ppm): 5.26 (s, 2H), 6.44 (s,

2H), 7.02 (t, 1H, J1 ¼ 7.2 Hz, J2 ¼ 7.6 Hz), 7.17 (d, 1H, J ¼ 8.8

Hz), 7.36 (t, 1H, J1 ¼ 8.6 Hz, J2 ¼ 7.6 Hz), 7.48–7.83 (bs, 3H),

7.49 (s, 1H), 8.04 (d, 1H, J1 ¼ 7.2 Hz), 8.36 (s, 1H), 10.32 (s, 1H).13C NMR (DMSO-d6, 100 MHz) d (ppm): 69.3 (CH2), 114.0 (Ar),

122.2 (Ar), 124.3 (C–C ¼ N–NH–), 126.6 (Ar), 131.2 (Ar), 135.6

(C ¼ NH), 156.5 (C-O-CH2), 157.6 (C]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 deriv-

ative (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 : 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) d (ppm): 1.62 (s, 6H), 5.19

Med. Chem. Commun., 2010, 1, 216–228 | 223


Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

(s, 2H), 7.10 (d, 1H, J ¼ 9.6 Hz), 7.28 (m, 4H), 7.93 (d, 2H, J ¼8.8 Hz), 9.94 (s, 1H). 13C NMR (acetone-d6, 100 MHz) d (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 ¼ 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.


5-(3-Formylphenyloxymethyl)-2,2-dimethyl-2H-benzimi-

dazole 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-benzimi-

dazole 1,3-di-N-oxide (14). Bordeaux solid (8%), mp 112.5–

113.4 �C. 1H NMR (acetone-d6, 400 MHz) d (ppm): 1.62 (s, 6H),

1.57 (s, 3H), 5.64 (s, 2H), 6.73 (d, 1H, J ¼ 9.6 Hz), 7.30 (d, 1H, J

¼ 9.6 Hz), 7.43 (t, 1H, J1 ¼ 6.2, J2 ¼ 7.4), 7.55 (dd, 2H, J ¼ 8.4

Hz, J1 ¼ 6.0 Hz, J2 ¼ 4.4 Hz), 7.87 (s, 1H), 7.93 (d, 1H, J ¼ 9.2

Hz). 13C NMR (acetone-d6, 100 MHz) d (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 ¼ N-NH-), 130.7 (Ar), 136.2 (Ar),

140.6 (Bfx), 157.4 (C-O-CH2), 198.0 (C]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) d (ppm): 1.57 (s,

6H), 5.06 (s, 2H), 7.07 (m, 3H), 7.27 (d, 2H, J ¼ 8.8 Hz), 7.77

(d, 2H, J¼ 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) d (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–C¼N-NH-), 141.5

(Bfx), 142.9 (CH), 160.0 (C-O-CH2), 178.61(C]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) d (ppm):

1.62 (s, 6H), 4.35 (t, 2H, J1 ¼ 4.6 Hz, J2 ¼ 5.0 Hz), 5.10 (s, 2H),

5.11 (dd, 1H, Jgem ¼ 1.6 Hz, Jcis ¼ 10.0 Hz), 5.22 (dd, 1H, Jgem ¼1.6 Hz, Jtrans ¼ 17.2 Hz), 5.98 (m, 1H), 6.90 (d, 1H, J ¼ 8.8 Hz),

7.11 (dd, 2H, J ¼ 6.8 Hz, J ¼ 2.0 Hz), 7.23 (d, 1H, J ¼ 9.6 Hz),

7.63 (d, 1H, J¼ 8.8 Hz), 7.76 (d, 2H, J¼ 8.8 Hz), 8.15 (s, 1H). 13C

NMR (acetone-d6, 100 MHz) d (ppm): 23.5 (CH3), 46.0 (CH2),

68.3 (CH2), 96.9 (C(CH3)2), 112.7 (Ar), 115.1 (Ar), 115.6 (C]C),

115.7 (Ar), 125.9 (Ar), 127.7 (C–C ¼ N-NH-), 128.8 (Ar), 128.9

(Ar), 130.2 (Ar), 134.9 (C]C), 140.3 (Bfx), 142.4 (CH), 159.8 (C-

O-CH2), 176.7 (C]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%).

224 | Med. Chem. Commun., 2010, 1, 216–228

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)

d (ppm): 1.62 (s, 6H), 5.12 (s, 2H), 7.08 (d, 1H, J ¼ 8.0 Hz), 7.14

(dd, 2H, J ¼ 8.4 Hz, J1 ¼ 2.0 Hz, J2 ¼ 1.6 Hz), 7.22 (t, 2H, J1 ¼9.0 Hz, J2¼ 11 Hz), 7.29 (s, 1H), 7.37 (t, 2H, J1¼ 7.6 Hz, J2¼ 8.0

Hz), 7.76 (d, 2H, J ¼ 7.6 Hz), 7.86 (d, 2H, J ¼ 8.8 Hz), 8.23 (s,

1H). 13C NMR (acetone-d6, 100 MHz) d (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–C¼N-NH-), 140.4 (Bfx), 142.4 (CH), 159.9 (C-O-CH2), 176.6

(C]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-benz-

imidazole 1,3-di-N-oxide (30). Bordeaux solid (25%), mp 123.3-

124.1 �C. 1H NMR (DMSO-d6, 400 MHz) d (ppm): 1.57 (s, 6H),

5.05 (s, 2H), 6.39 (s, 2H), 7.04 (m, 3H), 7.26 (d, 2H, J ¼ 8.8 Hz),

7.68 (d, 2H, J ¼ 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%).


benzimidazole 1,3-di-N-oxide (32). Bordeaux solid (20%),


1.62 (s, 6H), 5.09 (s, 2H), 7.08 (d, 1H, J ¼ 9.6 Hz), 7.13 (m,

1H), 7.24 (2, 1H, J ¼ 9.2), 7.55 (t, 1H, J ¼ 1.2 Hz), 7.39 (m,

2H), 7.59 (d, 1H, J ¼ 2.0 Hz), 8.17 (s, 1H). 13C NMR (acetone-

d6, 100 MHz) d (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 ¼ N-NH-),

140.5 (Bfx), 142.1 (C]NH), 158.7 (C-O-CH2), 178.7 (C]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) d (ppm): 1.62

(s, 6H), 5.24 (s, 2H), 7.16 (m, 2H), 7.25 (d, 1H, J ¼ 9.6 Hz),

7.34 (m, 2H), 7.67 (dt, 1H, J1 ¼ 7.6 Hz, J2 ¼ 8.2 Hz, J1 ¼ 1.6

Hz, J2¼ 2.0 Hz), 7.81(dd, 1H, J¼ 7.6 Hz, J¼ 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.



benzimidazole 1,3-di-N-oxide (34). Bordeaux solid (30%),

124.5–125.8 �C. 1H NMR (CDCl3, 400 MHz) d (ppm): 1.63 (s,

6H), 5.15 (s, 2H), 7.04 (s, 2H), 7.12 (d, 1H, J ¼ 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%).



Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

5-[2-(N4-Allylthiosemicarbazono)phenyloxymethyl]-2,2-dimethyl-

2H-benzimidazole 1,3-di-N-oxide (35). Bordeaux solid (20%),


1.62 (s, 6H), 4.37 (t, 2H, J¼ 5.6 Hz), 5.11 (dd, 1H, Jgem¼ 1.6 Hz,

Jcis ¼ 10.0 Hz), 5.14 (s, 2H), 5.24 (dd, 1H, Jgem ¼ 1.6 Hz, Jtrans ¼17.2 Hz), 6.00 (m, 1H), 7.03 (t, 1H, J1¼ 7.2 Hz, J2¼ 8.0 Hz), 7.12

(d, 1H, J¼ 9.6 Hz), 7.21(dd, 2H, J¼ 11.8 Hz, J¼ 3.6 Hz), 7.31(s,

1H), 7.41(dt, 1H, J1 ¼ 7.6 Hz, J2 ¼ 8.0 Hz, J ¼ 1.6 Hz), 8.04 (dd,

1H, J ¼ 8.0 Hz, J ¼ 1.6 Hz), 8.37 (s, 1H), 8.72 (s, 1H), 10.60 (s,

1H). 13C NMR (acetone-d6, 100 MHz) d (ppm): 23.9 (CH3), 45.1

(CH2), 69.4 (CH2), 97.3 (C(CH3)2), 113.1 (Ar), 113.5 (Ar), 115.6

(C]C), 116.1 (Ar), 121.8 (Ar), 123.7 (Ar), 126.5 (Ar), 130.7 (Ar),

131.6 (Ar), 135.2 (C]C), 138.0 (CH), 140.7 (Bfx), 157.30 (C-O-

CH2), 176.6 (C]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)

d (ppm): 1.62 (s, 6H), 5.17 (s, 2H), 7.19 (m, 1H), 7.24 (m, 5H),

7.36 (t, 2H, J1 ¼ 7.6 Hz, J2 ¼ 8.0 Hz), 7.44 (m, 1H), 7.78 (d, 2H,

J1 ¼ 8.0 Hz), 8.20 (dt, 1H, J1 ¼ 7.2 Hz, J2 ¼ 8.8 Hz, J ¼ 1.6 Hz),

8.88 (s, 1H), 9.89 (s, 1H), 10.90 (s, 1H). 13C NMR (acetone-d6,

100 MHz) d (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 ¼ 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 ¼ NH), 139.4 (Ar), 140.2 (Bfx),

157.2 (C-O-CH2), 176.8 (C]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-benz-

imidazole 1,3-di-N-oxide (37). Bordeaux solid (10%), mp 125.5–

126.3 �C. 1H NMR (acetone-d6, 400 MHz) d (ppm): 1.63 (s, 6H),

5.13 (s, 2H), 6.91 (d, 2H, J ¼ 8.4 Hz), 7.24 (d, 2H, J1 ¼ 8.0 Hz),

7.32 (s, 1H), 7.45 (d, 1H, J ¼ 8.0 Hz), 8.04 (dd, 1H, J ¼ 7.6 Hz,

J ¼ 1.6 Hz), 8.50 (s, 1H). 13C NMR (acetone-d6, 100 MHz)

d (ppm): 23.0 (CH3), 68.9 (CH2), 96.6 (C(CH3)2), 112.3 (Ar),

116.0 (Ar), 121.1 (Ar), 124.1 (C–C¼N–NH–), 125.3 (Ar), 129.3

(Ar), 130.0 (Ar), 136.0 (C]NH), 140.6 (C-O-CH2), 141.8 (Ar),

156.2 (Bfx), 158.0 (C]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 �[(Ap � A0p)/(Ac � A0c)]} � 100, where Ap ¼ A590 of the culture

containing the compound to be analyzed at day 5; A0p ¼ A590 of

the culture containing the compound to be analyzed just after

addition of the inocula (day 0); Ac ¼ A590 of the culture in the

absence of any compound (control) at day 5; A0c ¼ A590 in the

absence of the compound at day 0. To determine ID50 values,

parasite growth was followed in the absence (control) and pres-

ence 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 macro-

phage-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 mL 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 mM) for 48

h. Afterwards, cell viability was assessed by measuring the

mitochondrial-dependent reduction of MTT (Sigma) to for-

mazan. 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 mL), 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 sus-

pended in PBS to 2% w/v red blood cell solution. A volume of 400

mL of compound to be analyzed, in PBS (final doses 50, 100 and

200 mM), negative control (solution of PBS), or AmpB (final dose

1.5 mM) were added in 400 mL to the 2% w/v red blood cell solu-

tion. 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 MICRO-

WELL STRIP READER. Results are expressed as percentage of

Med. Chem. Commun., 2010, 1, 216–228 | 225


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

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

total haemoglobin released in the presence of the compounds.

This percentage was calculated using the equation percentage

haemolysis (%) ¼ [(A1 � A0)/A1 water] � 100, where A1 is the

absorbance at 405 nm of the test sample at t ¼ 24 h, A0 is the

absorbance at 405 nm of the test sample at t¼ 0 h, and A1 water is

the absorbance at 405 nm of the positive control (water) at t¼ 24

h. The experiments were done by quintuplicate.

Cruzipain inhibitory activity.30 CP (6 mM) 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 mM compound for 5

min at room temperature. Fluorogenic substrate Z-Phe-Arg-

AMC (KM ¼ 1.8 mM) was added to a concentration of 10 mM,

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 spec-

trophotometer. Compounds were added as solutions in DMSO

and positive controls contained only buffered solvent. The final

assay volume was 100 mL and the final DMSO concentration

never exceeded 10%. ID50 values were independently determined

from the three inhibitor concentrations. The CP-inhibitor refer-

ence 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 mL of this solution

was then added to 50 mL 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 ami-

dinohydrazones and as neutral form for the thio- and semi-

carbazones, were built with standard bond lengths and angles

using the molecular modeling package SYBYL 8.131 and their

226 | Med. Chem. Commun., 2010, 1, 216–228

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 minimi-

zation 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 �A 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 ¼ [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 confor-

mation 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 DG

binding and dissociation constants obtained from the difference

accessible surface area method using the STC (Structural Ther-

modynamics Calculations) program.35

Discussion and conclusions

We identified new benzofuroxan and benzimidazole 1,3-dioxide

derivatives with interesting trypanosomicidal activities.



Table 7 Estimated properties of compounds investigated.a,37

Compd miLogP nON nOHNH MW nviolations TPSA

8 3.28 6 0 270.2 0 77.810 3.23 6 0 270.2 0 77.811 3.33 6 0 284.3 0 77.816 4.86 8 2 419.5 0 97.118 3.40 8 3 343.4 0 111.119 4.43 8 2 383.4 0 97.123 4.82 8 2 419.5 0 97.132 2.94 8 3 385.4 0 117.135 3.93 8 2 425.5 0 103.236 4.35 8 2 461.5 0 103.237 2.37 9 3 369.4 0 134.2Nfx 0.71 8 0 287.30 0 108.71

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 (�A2).

Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

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).

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, benzo-

furoxan 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 thio-

semicarbazones (Fig. 3b).20,36 Theoretical results clearly indi-

cated that 3-substituted derivatives were able to interact via

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).


hydrogen bonding and hydrophobic interactions with CP

consistent with the more negative DG 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 substit-

uents (Fig. 4). In addition, no relationship between miLogPs and

CP inhibitory activity was found.

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 acknowl-

edged. 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.

Med. Chem. Commun., 2010, 1, 216–228 | 227


Publ

ishe

d on

11

Aug

ust 2

010.

Dow

nloa

ded

on 1

6/10

/201

3 12

:13:

01.

View Article Online

Notes and references

1 www.who.int/tdr.2 J. Rodrigues Coura and S. L. Castro, Mem. Inst. Oswaldo Cruz, 2002,

97, 3–24.3 C. J. Schofield, J. Jannin and R. Salvatella, Trends Parasitol., 2006,

22, 583–588.4 H. Cerecetto and M. Gonz�alez, Pharmaceuticals, 2010, 3, 810–838.5 J. A. Castro, M. M. de Meca and L. C. Bartel, Hum. Exp. Toxicol.,

2006, 25, 471–479.6 Chagas Disease; Special Programme for Research and Training in

Tropical Diseases (TDR), World Health Organization, Geneva,http://www.who.int/tdr/diseases/chagas/direction.htm.

7 S. Nwaka and R. G. Ridley, Nat. Rev. Drug Discovery, 2003, 2, 919–928.8 A. Cavalli and M. L. Bolognesi, J. Med. Chem., 2009, 52, 7339–7359.9 L. Otero, M. Vieites, L. Boiani, A. Denicola, C. Rigol, L. Opazo,

C. Olea-Azar, J. D. Maya, A. Morello, R. L. Krauth-Siegel,O. E. Piro, E. Castellano, M. Gonz�alez, D. Gambino andH. Cerecetto, J. Med. Chem., 2006, 49, 3322–3331; M. Vieites,L. Otero, D. Santos, J. Toloza, R. Figueroa, E. Norambuena,C. Olea-Azar, G. Aguirre, H. Cerecetto, M. Gonz�alez, A. Morello,J. D. Maya, B. Garat and D. Gambino, J. Inorg. Biochem., 2008,102, 1033–1043; M. Vieites, L. Otero, D. Santos, C. Olea-Azar,E. Norambuena, G. Aguirre, H. Cerecetto, M. Gonz�alez,U. Kemmerling, A. Morello, J. D. Maya and D. Gambino, J.Inorg. Biochem., 2009, 103, 411–418; A. Gerpe, I. Odreman-Nunez,P. Draper, L. Boiani, J. A. Urbina, M. Gonz�alez and H. Cerecetto,Bioorg. Med. Chem., 2008, 16, 569–577; A. Gerpe, G. �Alvarez,D. Ben�ıtez, L. Boiani, M. Quiroga, P. Hern�andez, M. Sortino,S. Zacchino, M. Gonz�alez and H. Cerecetto, Bioorg. Med. Chem.,2009, 17, 7500–7509; A. Gerpe, L. Boiani, P. Hern�andez,M. Sortino, S. Zacchino, M. Gonz�alez and H. Cerecetto, Eur. J.Med. Chem., 2010, 45, 2154–2164; W. Porcal, P. Hern�andez,M. Boiani, G. Aguirre, L. Boiani, A. Chidichimo, J. J. Cazzulo,N. E. Campillo, J. A. Paez, A. Castro, R. L. Krauth-Siegel,C. Davies, M. A. Basombr�ıo, M. Gonz�alez and H. Cerecetto, J.Med. Chem., 2007, 50, 6004–6015.

10 W. Porcal, P. Hernandez, L. Boiani, M. Boiani, A. Ferreira,A. Chidichimo, J. J. Cazzulo, C. Olea-Azar, M. Gonz�alez andH. Cerecetto, Bioorg. Med. Chem., 2008, 16, 6995–7004.

11 M. Boiani, L. Piacenza, P. Hern�andez, L. Boiani, H. Cerecetto,M. Gonz�alez and A. Denicola, Biochem. Pharmacol., 2010, 79,1736–1745.

12 G. B. Henderson, P. Ulrich, A. H. Fairlamb, I. Rosenmerg,M. Pereira, M. Sela and A. Cerami, Proc. Natl. Acad. Sci. U. S. A.,1988, 85, 5374–5378; N. Fujii, J. P. Mallari, E. J. Hansell,Z. Mackey, P. Doyle, Y. M. Zhou, J. Gut, P. J. Rosenthal,J. H. McKerrow and R. Kiplin Guy, Bioorg. Med. Chem. Lett.,2005, 15, 121–123; R. Siles, S.-E. Chen, M. Zhou, K. G. Pinney andM. Lynn Trawick, Bioorg. Med. Chem. Lett., 2006, 16, 4405–4409.

13 C. Olea-Azar, C. Rigol, F. Mendiz�abal, H. Cerecetto, R. Di Maio,M. Gonz�alez, W. Porcal, A. Morello, Y. Repetto and J. D. Maya,Lett. Drug Des. Discovery, 2005, 2, 294–301.

14 J. F. Faucher, T. Baltz and K. G. Petry, Parasitol. Res., 1995, 81, 441–443; M. Almeida-de-Faria, E. Freymuller, W. Colli and M. J. Alves,Exp. Parasitol., 1999, 92, 263–274.

15 M. Boiani, L. Boiani, A. Denicola, S. Torres de Ortiz, E. Serna,N. Vera de Bilbao, L. Sanabria, G. Yaluff, H. Nakayama, A. Rojasde Arias, C. Vega, M. Rol�on, A. G�omez-Barrio, H. Cerecetto andM. Gonzalez, J. Med. Chem., 2006, 49, 3215–3224; L. Boiani,C. Davies, C. Arredondo, W. Porcal, A. Merlino, A. Gerpe,M. Boiani, J. P. Pacheco, M. A. Basombr�ıo, H. Cerecetto andM. Gonz�alez, Eur. J. Med. Chem., 2008, 43, 2229–2237; L. Boiani,A. Gerpe, V. J. Ar�an, S. Torres de Ortiz, E. Serna, N. Vera deBilbao, L. Sanabria, G. Yaluff, H. Nakayama, A. Rojas de Arias,J. D. Maya, A. Morello, H. Cerecetto and M. Gonz�alez, Eur. J.Med. Chem., 2009, 44, 1034–1040.

228 | Med. Chem. Commun., 2010, 1, 216–228

16 S. Choi, A. Isaacs, D. Clements, D. Liu, H. Kim, R. W. Scott,J. D. Winkler and W. F. DeGrado, Proc. Natl. Acad. Sci. U. S. A.,2009, 106, 6968–6973.

17 V. Yardley and S. L. Croft, Am. J. Trop. Med. Hyg., 1999, 61, 193–197.

18 B. Sahu, V. Chenna, K. L. Lathrop, S. M. Thomas, G. Zon,K. J. Livak and D. H. Ly, J. Org. Chem., 2009, 74, 1509–1516.

19 J. M. Pereira, R. P. Severino, P. C. Vieira, J. B. Fernandes,M. F. G. F. da Silva, A. Zottis, A. D. Andricopulo, G. Oliva andA. G. Correa, Bioorg. Med. Chem., 2008, 16, 8889–8895;R. F. Freitas, I. M. Prokopczyk, A. Zottis, G. Oliva,A. D. Andricopulo, M. T. S. Trevisan, W. Vilegas, M. G. V. Silvaand C. A. Montanari, Bioorg. Med. Chem., 2009, 17, 2476–2482.

20 N. Fujii, J. P. Mallari, E. J. Hansell, Z. Mackey, P. Doyle,Y. M. Zhou, J. Gut, P. J. Rosenthal, J. H. McKerrow andR. Kiplin Guy, Bioorg. Med. Chem. Lett., 2005, 15, 121–123;A. C. Lima Leite, R. Souza de Lima, D. R. de M. Moreira,M. V. de O. Cardoso, A. C. Gouveia de Brito, L. M. Farias dosSantos, M. Zaldini Hernandes, A. Costa Kiperstok, R. Santana deLima and M. B. P. Soares, Bioorg. Med. Chem., 2006, 14, 3749–3757.

21 G. Veglia, M. Delfini, M. R. del Giudice, E. Gaggelli and G. Valensin,J. Magn. Reson., 1998, 130, 281–286.

22 K. Brak, P. S. Doyle, J. H. McKerrow and J. Ellman, J. Am. Chem.Soc., 2008, 130, 6404–6410.

23 L. S. Brinen, E. Hansell, J. Cheng, W. R. Roush, J. H. McKerrow andR. J. Fletterick, Structure, 2000, 8, 831–840.

24 L. Huang, L. S. Brinen and J. A. Ellman, Bioorg. Med. Chem., 2003,11, 21–29.

25 M. E. McGrath, A. E. Eakin, J. C. Engel, J. H. McKerrow,C. S. Craik and R. J. Fletterick, J. Mol. Biol., 1995, 247, 251–259.

26 S. A. Gillmor, C. S. Craik and R. J. Fletterick, Protein Sci., 1997, 6,1603–1611.

27 D. Bogdal, J. Pielichowsky and A. Boron, Synth. Commun., 1998, 28,3029–3039; R. Kamakshi and B. S. R. Reddy, Aust. J. Chem., 2005,58, 603–606.

28 N. Aggarwal and P. Mishra, J. Zhejiang Univ., Sci., 2005, 6b, 617–621.29 L. Fernandez, M. Calder�on, M. Martinelli, M. Strumia, H. Cerecetto,

M. Gonz�alez, J. J. Silber and M. Santo, J. Phys. Org. Chem., 2008, 21,1079–1085; R. Hinojosa Valdez, L. T. D€usman Tonin, T. Ueda-Nakamura, B. P. Dias Filho, J. A. Morgado-Diaz, M. Sarragiottoand C. Vataru Nakamura, Acta Trop., 2009, 110, 7–14.

30 R. S. Ferreira, C. Bryant, K. K. H. Ang, J. H. McKerrow,B. K. Shoichet and A. R. Renslo, J. Med. Chem., 2009, 52, 5005–5008.

31 SYBYL 8.1, Tripos Inc., 1699 South Hanley Rd., St. Louis, MO63144, USA, 2008.

32 M. Clark, R. D. Cramer III and N. Van Opdenbosch, J. Comput.Chem., 1989, 10, 982–1012.

33 R. Judson, Genetic algorithms and their use in chemistry, in Reviewsin Computational Chemistry, ed. K. B. Lipkowitz and D. B. Boyd, vol.10, VCH Publishers, New York, 1997, pp. 1–73.

34 V. Sobolev, A. Sorokine, J. E. Prilusky, E. Abola and M. Edelman,Bioinformatics, 1999, 15, 327–332.

35 P. Lavigne, J. R. Bagu, R. Boyko, L. Willard, C. F. B. Holmes andB. D. Sykes, Protein Sci., 2000, 9, 252–264.

36 D. C. Greenbaum, Z. Mackey, E. Hansell, P. Doyle, J. Gut,C. R. Caffrey, J. Lehrman, P. J. Rosenthal, J. H. McKerrow andK. Chibale, J. Med. Chem., 2004, 47, 3212–3219.

37 Compounds properties calculation.Polar surface area (TPSA), miLogP, and violations of Lipinski’s rule of five, were calculated usingMolinspiration online property calculation toolkit: http://www.molinspiration.com/cgi-bin/properties.

38 P. Gaillard, P. A. Carrupt, B. Testa and A. Boudon, J. Comput.-AidedMol. Des., 1994, 8, 83–96; A. J. M. Carpy and N. Marchand-Geneste,SAR QSAR Environ. Res., 2003, 14, 329–337.

39 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Adv.Drug Delivery Rev., 1997, 23, 3–25.

40 P. Ertl, B. Rohde and P. Selzer, J. Med. Chem., 2000, 43, 3714–3717.



Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma...

Documents

Transcript of Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma...