Preliminary Communication Special Focus: Trypanosomatid Diseases For reprint orders, please contact [email protected]
Identification of novel benzimidazole derivatives as anti-Trypanosoma cruzi agents: solid-phase synthesis, structure–activity relationships and molecular docking studies Background: In this paper, we report the solid-phase synthesis of 33 novel 1,2,5-tri-substituted benzimidazole derivatives and their in vitro activity on cruzipain and Trypanosoma cruzi epimastigotes. Results: Seven compounds were potent inhibitors of T. cruzi growth with IC50 values in the range 6–16 µM. Applying structure–activity relationships and principal component analysis strategies we were able to determine ring substituent effects and physicochemical properties that are important for the antichagasic activity of these novel derivatives, as well as get an insight into their possible mechanisms of action. Molecular docking studies revealed the binding orientation of the ligands in the active site of cruzipain providing new guidelines for the further design of better inhibitors. Conclusion: Compound 2a constitute a promising hit compound for novel anti-T. cruzi agents showing that the benzimidazole scaffold may represent an interesting therapeutic alternative for the treatment of Chagas disease. Neglected tropical diseases are still a major public health concern for most developing countries [1–3]. Emerging and reemerging parasitic diseases caused by protozoa, such as malaria, leishmaniasis and Chagas disease (CD), result in serious situations for national health systems in Latin America [4–6]. CD is a histidic and hematic parasitic infection produced by the flagellate protozoan Trypanosoma cruzi [7,8]. The search for solutions to prevent and treat such diseases should be a crucial point for governments, pharmaceutical companies and private agencies. Due to the lack of interest from the pharmaceutical industry in developing new drugs for these neglected diseases, current efforts in this direction rely almost exclusively on academic research [9–11]. In the absence of effective vaccines, chemotherapy plays a critical role in the control of CD. Nifurtimox (Nfx; Lampit ®; Bayer, Leverkusen, Germany) and benznidazole (Lafepe Benznidazol®; Lafepe Laboratory, Pernambuco, Brazil) are the only currently approved drugs. These compounds originally registered to treat acute T. cruzi infections, remain, to this date, the only available drugs for the specific treatment of CD [12]. The major limitation of currently available drugs is their lower antiparasitic activity in the chronic form of the disease. On the other hand, both drugs have unwanted side effects that can lead to treatment discontinuation. Therefore, improved therapeutic agents against CD with higher efficacy and
lower toxicity are needed, especially since efforts to target the disease have been limited [13]. The identification of valid drug targets, coupled with rational, structure-based design of specific smallmolecule inhibitors, can be an effective strategy for discovering new drugs [14–16]. In this context, in the 1990s the authors’ group worked in the R&D of drugs designed to act selectively on the parasite metabolism, as a main objective for the development of novel compounds able to act both as inhibitors of key enzymes and on the redox metabolism of the parasite [17–19]. These biochemical processes are considered to be excellent therapeutic targets in the chemotherapy of CD. Cysteine proteases (CP) from parasites, as well as from mammals, are promising drug targets for parasitic infections and systemic human diseases, respectively [20,21]. The major characterized CP in T. cruzi is cruzipain (TcCP), which is expressed as a mixture of isoforms [22]. In adition, there is clear evidence that TcCP is critical for intracellular parasite survival. The C-terminal truncated equivalent of the major TcCP isoform was successfully expressed in Escherichia coli and the recombinant enzyme, termed cruzain, has been crystallized, becoming the family archetype for structure–function-based studies. In this context, the authors’ group is interested in the development of rapid synthesis of azaheterocyclic molecules with anti-T. cruzi activity by exploring the possibility to obtain benzimidazole libraries. Benzimidazole is a
Natalia Ríos1, Javier Varela1, Estefania Birriel1, Mercedes González1, Hugo Cerecetto1, Alicia Merlino2 & Williams Porcal*1
10.4155/FMC.13.160 © 2013 Future Science Ltd
Future Med. Chem. (2013) 5(15), 1719–1732
ISSN 1756-8919
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay 2 Laboratorio de Química Teórica y Computacional, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay *Author for correspondence: Tel.: +598 25258618/7216 Fax: +598 25250749 E-mail: [email protected] 1
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Preliminary Communication | Ríos, Varela, Birriel et al. Key Terms Nifurtimox and benznidazole: Two
nitro-substituted heterocyclic drugs are, to date, still the only clinically available drugs for the chemotherapy of Chagas’ disease (American trypanosomiasis).
Microwave-assisted solid-phase synthesis:
Combination of microwave with solid support. In recent years, microwave-assisted solid-phase synthesis has emerged as a powerful synthetic tool because not only does it ensure greater purity of the product but it also results in improved yields, mainly due to a decrease in the formation of side products.
Molecular docking: Key tool in structural molecular biology and computer-assisted drug design. The goal of ligand– protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known 3D structure. Docking can be used to perform virtual screening on a large library of compounds, rank the results and propose structural hypotheses of how the ligands inhibit the target, which is invaluable in lead optimization.
very important pharmacophore in drug discovery, and its derivatives are an important class of bioactive molecules in the field of drugs and pharmaceuticals [23,24]. Benzimidazoles are also veterinary drugs widely used for prevention and treatment of parasitic infections. The authors’ research group has synthesized a large number of benzimidazole N-oxide derivatives with antiparasitic activity, resulting in 2H-benzimidazole 1,3-dioxide, the most active compound in vitro against T. cruzi and Leishmania spp. [18,25,26]. On the other hand, the authors’ group has been investigating different chemical scaffolds as anti-TcCP pharmacophores finding different behaviors against this enzyme [18,25,27,28]. Here, the microwave-assisted solid-phase synthesis of benzimidazole derivatives and their in vitro activity on TcCP and T. cruzi epimastigotes is described (Figure 1). Structure–activity relationship analysis and molecular docking studies were performed for all compounds in order to get insight into structural and physicochemical properties responsible for the observed activity profile. Experimental NMR studies See supplementary material for the general procedure and NMR results for 1q, 2a–c, 3a–f, 4a–e and 5a & b. Trypanocidal
activity T. cruzi epimastigotes (Tulahuen 2 strain) were grown at 28°C in BHI–tryptose medium supplemented with 5% fetal calf serum. Cells were harvested in the exponentially growing phase, resuspended in fresh culture medium, counted
X Y
NO2
in a Neubauer chamber, and placed in 24-well plates (3 × 106 cells/ml). The parasites were incubated with 25 µM of each compound diluted in DMSO during 5 days. The final concentration of DMSO in the experiments never exceeded 0.8%. Cultures containing nontreated epimastigote forms and 0.8% DMSO were included as negative controls, while Nifurtimox was used as positive control. Cell growth was measured as the absorbance of the culture at 610 nm, which was proved to be proportional to the number of cells present. In order to determine the antiproliferative IC50, parasite growth was followed in the absence (control) and presence of increasing concentrations of the corresponding compound. At day 5, the absorbance of the culture was measured and related to that of the control. The IC50 value was taken as the concentration of compound needed to reduce the absorbance ratio to 50%. T.
cruzi CP inhibition assays CP was purified to homogeneity from epimastigotes of the Tulahuen 2 strain by ConA– Sepharose affinity chromatography, as previously described [29]. Cruzipain (0.25 µM; e = 58,285 M-1cm-1) was incubated in 50 mM Tris-Saline buffer pH 7.6 containing 150 mM NaCl, 5 mM EDTA, 5 mM DTT and 100 µM inhibitor was added, shaking the solution for 5 min at 37°C. Then the fluorogenic substrate Z-Phe-Arg-AMC (10 µM) was added and the fluorescence was measured (excitation at 380 nm and emission at 460 nm) using a Varioskan Flash Spectrophotometer. No differences were observed with or without Triton® 0.01%
o-fluronitrobenzene polymer-supported
F
Triphenylphosphine or Wang resin
o-nitroaniline polymer-supported NO2
Z
Z
NO2
F
Linker
NHR1
i) Reduction ii) Condensation ii) Cleavage R5
H N
i) Reduction ii) Oxidative cyclization ii) Cleavage R5
X
N
N
R2
R1
R1
Benzimidazolone derivatives
N
Benzimidazole derivatives
Figure 1. General scheme for the synthesis of benzimidazole derivatives.
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Identification of novel benzimidazole derivatives in any case. The derivatives were added as solutions in DMSO, and the controls contained the same solvent concentration. E-64 was used as a positive control of inhibition. Cytotoxicity
assay In vitro cytotoxicity assays were performed on J774 murine macrophages. 1 × 105 cells/well were seeded in 200 µl of Complete Medium (RPMI1640 supplemented with 20% heat-inactivated fetal bovine serum) in 96-well plates (Nunclon). The plates were incubated at 37°C under a 5% CO2 atmosphere for 48 h to allow cell adhesion prior to drug testing. The compounds were dissolved in DMSO and cells were exposed to the compounds at various concentrations (25– 400 µM) for 48 h. Cells were washed with PBS and incubated at 37°C with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 0.4 mg/ml) for 3 h. Cell respiration was followed as an indicator of cell viability and was detected by reduction of MTT to formazan, a colored product. Formazan crystals were dissolved in DMSO and absorbance measurements at 595 nm were performed. The IC50 value was defined as the drug concentration at which 50% of the cells are viable relative to the control (no drug added). Theoretical
methods
Molecular modeling
The structure of the ligands was fully optimized in vacuo at the density functional theory (DFT) level using the B3LYP hybrid functional [30] with the 6–31+G(d,p) basis set [31] with the Spartan 08 v1.2.0 package (Wavefunction, Inc., OK, USA). The nature of each optimized structure as a stable species was carefully inspected checking the eigenvalues of the diagonalized Hessian. Solvent effects have been included at the same level of theory by single point calculations using the SM8 continuum model [32]. Physicochemical descriptors
Nine structural and reactivity 1D descriptors were initially selected for describing the ligands at the DFT level in aqueous solution including Vol and PSA, natural atomic charges derived from Weinhold’s natural population analysis [33], Kohn-Sham’s frontier orbitals energies, electronegativity, hardness, and electrophilicity calculated as reactivity descriptors in a conceptual DFT framework, according to the following expressions [34]: \=
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1 2^E HOMO + E LUMOh
h=
| Preliminary Communication
1 2^E LUMO - E HOMOh 2 ~= \ 2h
All the descriptors were generated and graphically analyzed using Spartan software (Wavefunction, Inc.). 3D MEPs mapped on an isosurface of electron density of 0.002 au have also been inspected to analyze differences in the charge distribution and molecular shape between active and inactive compounds. PCA
Previous to conduct the PCA the data showed in Table 1 was converted in a matrix of 33 compounds (objects) and nine variables (descriptors). Descriptors were scaled to obtained data of the same order of magnitude for comparison with each other. The chemometric analysis was conducted using Statistica software version 6.0 (StatSoft Inc., CA, USA). Molecular docking studies
Molecular docking of the compounds into the 3D x-ray structure of cruzain (PDB ID: 1ME3) was carried out with Autodock 4.2 using the implemented empirical free energy function and the Lamarckian Genetic Algorithm. The AutoDockTools package was employed to generate the docking input files and to analyze the docking results [35]. Gasteiger-Marsilli charges were used for the protein and ligands. In all docking experiments a grid map with 60 × 60 × 66 points and a gridpoint spacing of 0.375 Å was applied. The maps were centered on the S atom of the catalytic Cys25 residue. Each docking consisted of 50 independent runs, with an initial population of 150 individuals, a maximum number of 2.5 × 105 energy evaluations and a maximum number of 2.7 × 104 generations. Mutation and crossover were applied to the population at rates of 0.02 and 0.80, respectively. For the remaining parameters, default values were used. Results differing by less than 2.0 Å in root square-deviation were grouped into the same cluster. Results & discussion Chemistry Several advanced techniques, such as microwave synthesis, ionic liquid synthesis, sonication, polymer-support synthesis, and multicomponent condensation, are broadly used to accelerate synthetic organic chemistry [36–40]. These modern techniques in organic synthesis integrating a variety of advanced technologies for the rapid generation of www.future-science.com
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Preliminary Communication | Ríos, Varela, Birriel et al. Table 1. Density functional theory theoretical descriptors for benzimidazole derivatives†. Compound
1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 2a 2b 2c 3a 3b 3c 3d 3e 3f 4a 4b 4c 4d 4e 5a 5b
NPA atomic charges (au)
Reactivity descriptors (eV)
Molecular size
N1
N3
EHOMO
ELUMO h
c
w
Vol (Å 3 )
PSA (Å 2)
-0.402 -0.403 -0.389 -0.397 -0.395 -0.401 -0.384 -0.398 -0.399 -0.401 -0.385 -0.392 -0.389 -0.396 -0.385 -0.392 -0.392 -0.387 -0.398 -0.395 -0.394 -0.389 -0.448 -0.450 -0.520 -0.535 -0.457 -0.393 -0.399 -0.387 -0.392 -0.456 -0.386
-0.538 -0.542 -0.509 -0.524 -0.522 -0.535 -0.515 -0.526 -0.540 -0.544 -0.529 -0.531 -0.527 -0.542 -0.532 -0.535 -0.525 -0.527 -0.526 -0.564 -0.523 -0.527 -0.624 -0.623 -0.520 -0.535 -0.628 -0.512 -0.526 -0.519 -0.629 -0.628 -0.519
-6.05 5.96 6.27 -5.90 -5.98 -5.15 -6.22 -5.62 -6.06 -5.96 -5.60 -5.87 -5.94 -5.16 -5.94 -5.61 -5.24 -5.61 -5.76 -5.53 -5.25 -5.75 -5.89 -5.90 -6.12 -5.19 -5.55 -6.15 -5.22 -6.12 -5.20 -5.55 -5.69
-0.53 -0.49 -1.53 -1.07 -1.29 -0.88 -1.56 -1.05 -0.51 -0.46 -1.20 -1.10 -1.34 -0.91 -0.91 -1.08 -1.28 -1.22 -1.16 -0.67 -1.28 -1.33 -1.11 -1.11 -1.22 -0.89 -0.14 -1.43 -1.08 -1.45 -1.08 -0.15 -1.44
-3.29 -3.23 -3.90 -3.48 -3.64 -3.02 -3.89 -3.34 -3.29 -3.21 -3.40 -3.49 -3.64 -3.04 -3.43 -3.35 -3.26 -3.42 -3.46 -3.10 -3.27 -3.54 -3.50 -3.51 -3.67 -3.04 -2.85 -3.79 -3.15 -3.79 -3.14 -2.85 -3.57
1.96 1.90 3.21 2.51 2.82 2.13 3.25 2.43 1.94 1.87 2.63 2.55 2.88 2.17 2.33 2.47 2.68 2.66 2.60 1.98 2.69 2.84 2.56 2.56 2.75 2.15 1.50 3.04 2.40 3.07 2.39 1.50 2.99
266 358 377 354 368 399 329 374 219 311 281 307 320 351 352 327 361 328 401 283 408 389 236 283 362 394 273 462 493 415 446 226 484
8.98 8.93 14.50 9.03 8.99 9.86 34.30 25.30 8.99 8.93 14.50 8.64 8.61 9.65 33.60 24.90 10.90 12.50 27.40 27.30 41.70 42.60 41.30 42.60 45.20 46.50 25.60 23.70 24.70 23.50 24.40 60.20 60.20
2.76 2.74 2.37 2.41 2.35 2.14 2.33 2.29 2.78 2.75 2.20 2.39 2.30 2.13 2.52 2.27 1.98 2.20 2.30 2.43 1.99 2.21 2.39 2.40 2.45 2.15 2.71 2.36 2.07 2.34 2.06 2.70 2.13
N1 and N3 are the benzimidazole nucleus nitrogens according to the standard numbering. h: Chemical hardness; c: Electronic chemical potential; w: Electrophilicity; EHOMO /ELUMO: Energies of the frontier KS orbitals in aqueous solution; PSA: Polar surface area; Vol: Molecular volume. †
numerous multifunctionalized molecule libraries can provide the high speed path for drug discovery. In this context, the synthesis of benzimidazoles 1a–q (Figure 2), recently described by the authors’ group, was performed by a combination of solid-phase organic synthesis and microwaveassisted organic synthesis, which are powerful techniques for rapid generation of structurally diverse molecules [41]. Triphenylphosphine resin was used to obtain benzimidazole derivatives with a methyl group in position 5, as it is shown in Figures 2 & 3. In recent years, the application of 1722
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a traceless phosphonium linker during the synthesis of heterocycles has been reported [42]. The phosphonium linker (I), obtained by reaction between polymer-supported triphenylphosphine and 4-fluoro-3-nitrobenzyl iodide, underwent aromatic substitution with primary amines followed by one-pot reaction with aldehydes in the presence of SnCl2·2H2O, yielding the benzimidazole system under microwave irradiation (Figure 2). The final products were released from the resin with NaOH under microwave irradiation and were obtained in good overall yields. An future science group
Identification of novel benzimidazole derivatives
| Preliminary Communication
NO2
I
F
Ph
PPh2 µW
Ph
NO2
P + – I
NH2
R1
Ph
+
µW
F
Ph
I
Ph R2
NHR1
Ph N
P + – I
R2
NaOH / MeOH
N
SnCl2.2H2O µW
N R2
µW
N
R1
III
1a R1: CH2CH2Ph 1b R1: CH2CH2Ph 1c R1: CH2CH2Ph 1d R1: CH2CH2Ph 1e R1: CH2CH2Ph 1f R1: CH2CH2Ph 1g R1: CH2CH2Ph 1h R1: CH2CH2Ph 1i R1: (CH2)3CH3
–
II
I
OHC
NO2
P
1
R1
1j R1: (CH2)3CH3 R2: (CH2)4CH3 1k R1: (CH2)3CH3 R2: 2-Furyl 1l R1: (CH2)3CH3 R2: 4-F-Ph 1m R1: (CH2)3CH3 R2: 4-Br-Ph 1n R1: (CH2)3CH3 R2: 4-N(Me)2-Ph 1o R1: (CH2)3CH3 R2: 4-NHCOMe-Ph 1p R1: (CH2)3CH3 R2: 3,4-O,O(CH2)-Ph 1q R1: (CH2)3N(CH2CH3)2 R2: 4-Br-Ph [28%]
R2: H R2: (CH2)4CH3 R2: 2-Furyl R2: 4-F-Ph R2: 4-Br-Ph R2: 4-N(Me)2-Ph R2: 4-NHCOMe-Ph R2: 3,4-O,O(CH2)-Ph R2: H
Figure 2. Synthesis of 5-methylbenzimidazoles 1,2-disubstituted employing triphenylphosphine resin. Global yield is shown in square brackets.
alternative route was performed to obtain 5-methylbenzimidazolones 2a–c (Figure 3). Reduction of the nitro-functionality in II was accomplished by treatment with SnCl2 in N-methylpyrrolidone (NMP), producing the desired o-phenylenediamines IV. Progress of reduction was followed by on-bead fourier transform infrared spectroscopy monitoring by the disappearance of characteristic nitro group absorbance and presence of characteristic aromatic NH vibrations. For the next step, the authors supported on a previous study in which the authors showed that 5,6-diamines can
be transformed to 8-thionexanthines in one-step reaction using potassium ethylxanthate under microwave irradiation [43]. Thus, we carry out condensation of potassium ethylxanthate with o-phenylenediamines IVa to produce immobilized benzimidazol-2-thione Va under microwave irradiation at 120°C for 10 min (Figure 3). Treatment of o-phenylenediamines, IVb–c, with excess carbonyldiimidazole gave conversion to their corresponding cyclic analogues, benzimidazol-2-one 2b–c (Figure 3). Cleavage from the solid support was performed by the use of a 10% NaOH X=S
II
SnCl2.2H2O NMP, r.t.
Ph
i) EtOC = SSK/DMF
Ph
Ph
NH2 µW 120°C,10 min
P + – I
NHR1 IV
X=O
H N
r.t 4 h
N 2
X N
V
X = O; S
R1
2a R1: CH2CH2Ph X: S [7%] 2b R1: CH2CH2Ph X: O [28%]
X R1
H N
P + – I
ii) CO(Im)2/THF µW 150°C, 15 min
NaOH 10%/MeOH
Ph
X = O; S
2c R1: (CH2)3CH3 X: O [39%]
Figure 3. Synthesis of 5-methylbenzimidazole derivatives (2a–c) employing triphenylphosphine resin. Global yields are shown in square brackets.
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Preliminary Communication | Ríos, Varela, Birriel et al. O NO2
HO
O O
Cl
OH
O NO2
DCC / DMAP
H2N—R1
µW 70°C 20 min VII O
O
R3
NH2
N
O
CH2CI2/TFA
R2
DDQ r.t. 24 h
NHR1
DMF r.t. 24 h
NHR1
VI OHC
O
SnCl2·2H2O
DMF
Cl
CH2CI2:THF/100W, 1h
NO2
O
r.t. 2 h
N R1
IX
VIII
O
O i) HBTU/ OHBt/ THF r.t. 1 h
N
HO
R2 N 3
N
N
R2 N
ii)
R1
R1
N r.t. 24 h H
4
3a R1: CH2CH2Ph R2: Br [56%] 3b R1: CH2CH2Ph R2: N(Me)2 [49%] 3c R1: (CH2)3CH3R2: Br [77%] 3d R1: (CH2)3CH3 R2: N(Me)2 [73%] 3e R1: (CH2)3N(CH2CH3)2R2: Br [79%] 3f R1: (CH2)3N(CH2CH3)2R2: N(Me) [70%]
4a R1: (CH2)3N(CH2CH3)2R2: Br [52%] 4b R1: CH2CH2Ph R2: Br [41%] 4c R1: CH2CH2Ph R2: N(Me)2 [46%] 4d R1: (CH2)3CH3R2: Br [27%] 4e R1: (CH2)3CH3 R2: N(Me)2 [21%]
Figure 4. Synthesis of 5-carboxybenzimidazoles employing Wang resin. Global yields are shown in square brackets.
solution in MeOH, providing benzimidazolones 2a–c in 7–39% overall average yields. Wang resin was used to obtain benzimidazole derivatives with carboxylic group at position 5, as it is shown in Figures 4 & 5. Initially, 4-chloro-3-nitrobenzoic acid was anchored on polymer by standard esterification conditions under microwave heating. 4-chloro-3-nitrobenzoic acid was treated with Wang resin in the presence of DCC and DMAP in DCM/THF mixture (9:1) under microwave irradiation (100 W, Tmax = 100°C) for 1 h to afford polymer-bound 4-chloro-3-nitrobenzoate VI in quantitative yield (Figure 4). Loading of O VIII
CO(Im)2
the resin was verified after cleavage with 50% TFA/CH2Cl2 at room temperature for 1 h. The authors have also performed this transformation under conventional conditions (room temperature, 48 h) leading to identical results. As mentioned above, the first step in the synthesis of a benzimidazole scaffold is the aromatic nucleophilic substitution reaction of a halogen atom, preferably a fluorine or chlorine atom, with an amine (Figure 2). Three amines (benzylamine, n-butylamine and N 1,N 1-diethylpropane1,3-diamine) were selected for aromatic nucleophilic substitution with VI. For the synthesis of O
H N
O
TFA/CH 2Cl2 O
N
NMP r.t. 48 h X
R1
r.t. 1 h
HO
H N O N
R1 5 5a R1: CH 2CH2Ph [30%] 5b R1: (CH 2)3CH3 [38%]
Figure 5. Synthesis of 5-carboxybenzimidazolones using Wang resin. Global yields are shown in square brackets.
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Identification of novel benzimidazole derivatives benzimidazol-5-carboxylic acid derivatives the reactions were driven to completion by employing an excess of the corresponding amine with polymer-bound 4-chloro-3-nitrobenzoate VI under microwave heating at 70°C for 20 min in NMP (Figure 4). Subsequently, reduction of nitro group in polymer-supported VII was achieved at room temperature by using SnCl2 in NMP. The formation of the benzimidazole nucleus IX was accomplished by the treatment of o-phenylenediamines VIII with aromatic aldehydes in the presence of DDQ. Cleavage from the solid support was performed by the use of a 50% TFA solution in CH2Cl2, providing benzimidazoles 3a–f in 49–79% overall average yields, after filtration through a short column. The obtained carboxylic acids were used to synthesize the corresponding amides 4a–e by conventional synthesis, using HBTU as the activating agent and piperidine as nucleophile (Figure 4). An alternative route was performed to obtain benzimidazol-2-ones 5a & b (Figure 5). The remaining step completing the synthetic sequence involved cyclization of immobilized diamine VIII. Benzimidazole formation was attempted with carbonyldiimidazole in NMP at room temperature for 48 h. Considering that the reactions needed extensive time, the authors expected microwave irradiation to facilitate the transformation. Thus, when the reaction was conducted under microwave heating at 150°C for 15 min, similar results were obtained. Biology
Trypanocidal activity
The benzimidazole derivatives were initially tested in vitro against the epimastigote form of T. cruzi, Tulahuen 2 strain. Epimastigotes and intracellular amastigotes are both proliferative forms; therefore, a drug inhibiting epimastigote proliferation should exhibit antiproliferative activity in the intracellular parasite. In this context, it is clear that testing against epimastigotes is relevant for the development of new anti-T. cruzi agents [14]. Furthermore, it was confirmed that the antiproliferative epimastigote activity is related to the in vivo anti-T. cruzi activity with compounds from the authors’ chemical library [44,45]. The benzimidazoles were incorporated into the media at 25 µM and their ability to inhibit growth (percentage of inhibition [PI]; Table 2) of the parasite was evaluated in comparison with the control (no drug added to the media). Nifurtimox and benznidazole were used as the reference trypanosomicidal drugs. In addition, the IC50 was determined for the most active derivatives (Table 2). Some of future science group
| Preliminary Communication
the most active benzimidazole derivatives were selected to assess their unspecific mammalian cytotoxicity in vitro in the 50–400 µM range, using J774 murine macrophages as the cellular model (Table 3). The selectivity indexes (SI) were expressed as the ratio between IC50 in macrophages and IC50 in T. cruzi. This screening revealed some preliminary–activity relationships (SARs) among the series, and a few hit compounds showing Table 2. Trypanocidal activity and cruzipain inhibition of herein developed benzimidazole derivatives. Compound 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 2a 2b 2c 3a 3b 3c 3d 3e 3f 4a 4b 4c 4d 4e 5a 5b
Nifurtimox Benznidazole
Anti-Trypanosoma cruzi† PGI‡ (25 µM)
IC50
TcCP §# (PI)
0 4 30 35 82 95 2 41 51 90 49 80 83 9 68 57 18 92 4 0 2 0 0 15 8 4 18 0 14 11 87 6 0 95 90
– – – – 16.3 6.1 – 25.9 25.0 15.2 25.6 16.7 13.3 – 18.6 20.9 – 13.8 – – – – – – – – – – – – 12.0 – – 7.7 7.4
15 0 0 45 33 2 0 0 14 0 0 28 55 43 30 6 0 22 0 0 0 0 0 0 5 17 12 39 0 15 10 0 0 – –
The results are the means of three different experiments with a SD
Identification of novel benzimidazole derivatives as anti-Trypanosoma cruzi agents: solid-phase synthesis, structure–activity relationships and molecular docking studies Background: In this paper, we report the solid-phase synthesis of 33 novel 1,2,5-tri-substituted benzimidazole derivatives and their in vitro activity on cruzipain and Trypanosoma cruzi epimastigotes. Results: Seven compounds were potent inhibitors of T. cruzi growth with IC50 values in the range 6–16 µM. Applying structure–activity relationships and principal component analysis strategies we were able to determine ring substituent effects and physicochemical properties that are important for the antichagasic activity of these novel derivatives, as well as get an insight into their possible mechanisms of action. Molecular docking studies revealed the binding orientation of the ligands in the active site of cruzipain providing new guidelines for the further design of better inhibitors. Conclusion: Compound 2a constitute a promising hit compound for novel anti-T. cruzi agents showing that the benzimidazole scaffold may represent an interesting therapeutic alternative for the treatment of Chagas disease. Neglected tropical diseases are still a major public health concern for most developing countries [1–3]. Emerging and reemerging parasitic diseases caused by protozoa, such as malaria, leishmaniasis and Chagas disease (CD), result in serious situations for national health systems in Latin America [4–6]. CD is a histidic and hematic parasitic infection produced by the flagellate protozoan Trypanosoma cruzi [7,8]. The search for solutions to prevent and treat such diseases should be a crucial point for governments, pharmaceutical companies and private agencies. Due to the lack of interest from the pharmaceutical industry in developing new drugs for these neglected diseases, current efforts in this direction rely almost exclusively on academic research [9–11]. In the absence of effective vaccines, chemotherapy plays a critical role in the control of CD. Nifurtimox (Nfx; Lampit ®; Bayer, Leverkusen, Germany) and benznidazole (Lafepe Benznidazol®; Lafepe Laboratory, Pernambuco, Brazil) are the only currently approved drugs. These compounds originally registered to treat acute T. cruzi infections, remain, to this date, the only available drugs for the specific treatment of CD [12]. The major limitation of currently available drugs is their lower antiparasitic activity in the chronic form of the disease. On the other hand, both drugs have unwanted side effects that can lead to treatment discontinuation. Therefore, improved therapeutic agents against CD with higher efficacy and
lower toxicity are needed, especially since efforts to target the disease have been limited [13]. The identification of valid drug targets, coupled with rational, structure-based design of specific smallmolecule inhibitors, can be an effective strategy for discovering new drugs [14–16]. In this context, in the 1990s the authors’ group worked in the R&D of drugs designed to act selectively on the parasite metabolism, as a main objective for the development of novel compounds able to act both as inhibitors of key enzymes and on the redox metabolism of the parasite [17–19]. These biochemical processes are considered to be excellent therapeutic targets in the chemotherapy of CD. Cysteine proteases (CP) from parasites, as well as from mammals, are promising drug targets for parasitic infections and systemic human diseases, respectively [20,21]. The major characterized CP in T. cruzi is cruzipain (TcCP), which is expressed as a mixture of isoforms [22]. In adition, there is clear evidence that TcCP is critical for intracellular parasite survival. The C-terminal truncated equivalent of the major TcCP isoform was successfully expressed in Escherichia coli and the recombinant enzyme, termed cruzain, has been crystallized, becoming the family archetype for structure–function-based studies. In this context, the authors’ group is interested in the development of rapid synthesis of azaheterocyclic molecules with anti-T. cruzi activity by exploring the possibility to obtain benzimidazole libraries. Benzimidazole is a
Natalia Ríos1, Javier Varela1, Estefania Birriel1, Mercedes González1, Hugo Cerecetto1, Alicia Merlino2 & Williams Porcal*1
10.4155/FMC.13.160 © 2013 Future Science Ltd
Future Med. Chem. (2013) 5(15), 1719–1732
ISSN 1756-8919
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay 2 Laboratorio de Química Teórica y Computacional, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay *Author for correspondence: Tel.: +598 25258618/7216 Fax: +598 25250749 E-mail: [email protected] 1
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Preliminary Communication | Ríos, Varela, Birriel et al. Key Terms Nifurtimox and benznidazole: Two
nitro-substituted heterocyclic drugs are, to date, still the only clinically available drugs for the chemotherapy of Chagas’ disease (American trypanosomiasis).
Microwave-assisted solid-phase synthesis:
Combination of microwave with solid support. In recent years, microwave-assisted solid-phase synthesis has emerged as a powerful synthetic tool because not only does it ensure greater purity of the product but it also results in improved yields, mainly due to a decrease in the formation of side products.
Molecular docking: Key tool in structural molecular biology and computer-assisted drug design. The goal of ligand– protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known 3D structure. Docking can be used to perform virtual screening on a large library of compounds, rank the results and propose structural hypotheses of how the ligands inhibit the target, which is invaluable in lead optimization.
very important pharmacophore in drug discovery, and its derivatives are an important class of bioactive molecules in the field of drugs and pharmaceuticals [23,24]. Benzimidazoles are also veterinary drugs widely used for prevention and treatment of parasitic infections. The authors’ research group has synthesized a large number of benzimidazole N-oxide derivatives with antiparasitic activity, resulting in 2H-benzimidazole 1,3-dioxide, the most active compound in vitro against T. cruzi and Leishmania spp. [18,25,26]. On the other hand, the authors’ group has been investigating different chemical scaffolds as anti-TcCP pharmacophores finding different behaviors against this enzyme [18,25,27,28]. Here, the microwave-assisted solid-phase synthesis of benzimidazole derivatives and their in vitro activity on TcCP and T. cruzi epimastigotes is described (Figure 1). Structure–activity relationship analysis and molecular docking studies were performed for all compounds in order to get insight into structural and physicochemical properties responsible for the observed activity profile. Experimental NMR studies See supplementary material for the general procedure and NMR results for 1q, 2a–c, 3a–f, 4a–e and 5a & b. Trypanocidal
activity T. cruzi epimastigotes (Tulahuen 2 strain) were grown at 28°C in BHI–tryptose medium supplemented with 5% fetal calf serum. Cells were harvested in the exponentially growing phase, resuspended in fresh culture medium, counted
X Y
NO2
in a Neubauer chamber, and placed in 24-well plates (3 × 106 cells/ml). The parasites were incubated with 25 µM of each compound diluted in DMSO during 5 days. The final concentration of DMSO in the experiments never exceeded 0.8%. Cultures containing nontreated epimastigote forms and 0.8% DMSO were included as negative controls, while Nifurtimox was used as positive control. Cell growth was measured as the absorbance of the culture at 610 nm, which was proved to be proportional to the number of cells present. In order to determine the antiproliferative IC50, parasite growth was followed in the absence (control) and presence of increasing concentrations of the corresponding compound. At day 5, the absorbance of the culture was measured and related to that of the control. The IC50 value was taken as the concentration of compound needed to reduce the absorbance ratio to 50%. T.
cruzi CP inhibition assays CP was purified to homogeneity from epimastigotes of the Tulahuen 2 strain by ConA– Sepharose affinity chromatography, as previously described [29]. Cruzipain (0.25 µM; e = 58,285 M-1cm-1) was incubated in 50 mM Tris-Saline buffer pH 7.6 containing 150 mM NaCl, 5 mM EDTA, 5 mM DTT and 100 µM inhibitor was added, shaking the solution for 5 min at 37°C. Then the fluorogenic substrate Z-Phe-Arg-AMC (10 µM) was added and the fluorescence was measured (excitation at 380 nm and emission at 460 nm) using a Varioskan Flash Spectrophotometer. No differences were observed with or without Triton® 0.01%
o-fluronitrobenzene polymer-supported
F
Triphenylphosphine or Wang resin
o-nitroaniline polymer-supported NO2
Z
Z
NO2
F
Linker
NHR1
i) Reduction ii) Condensation ii) Cleavage R5
H N
i) Reduction ii) Oxidative cyclization ii) Cleavage R5
X
N
N
R2
R1
R1
Benzimidazolone derivatives
N
Benzimidazole derivatives
Figure 1. General scheme for the synthesis of benzimidazole derivatives.
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Identification of novel benzimidazole derivatives in any case. The derivatives were added as solutions in DMSO, and the controls contained the same solvent concentration. E-64 was used as a positive control of inhibition. Cytotoxicity
assay In vitro cytotoxicity assays were performed on J774 murine macrophages. 1 × 105 cells/well were seeded in 200 µl of Complete Medium (RPMI1640 supplemented with 20% heat-inactivated fetal bovine serum) in 96-well plates (Nunclon). The plates were incubated at 37°C under a 5% CO2 atmosphere for 48 h to allow cell adhesion prior to drug testing. The compounds were dissolved in DMSO and cells were exposed to the compounds at various concentrations (25– 400 µM) for 48 h. Cells were washed with PBS and incubated at 37°C with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 0.4 mg/ml) for 3 h. Cell respiration was followed as an indicator of cell viability and was detected by reduction of MTT to formazan, a colored product. Formazan crystals were dissolved in DMSO and absorbance measurements at 595 nm were performed. The IC50 value was defined as the drug concentration at which 50% of the cells are viable relative to the control (no drug added). Theoretical
methods
Molecular modeling
The structure of the ligands was fully optimized in vacuo at the density functional theory (DFT) level using the B3LYP hybrid functional [30] with the 6–31+G(d,p) basis set [31] with the Spartan 08 v1.2.0 package (Wavefunction, Inc., OK, USA). The nature of each optimized structure as a stable species was carefully inspected checking the eigenvalues of the diagonalized Hessian. Solvent effects have been included at the same level of theory by single point calculations using the SM8 continuum model [32]. Physicochemical descriptors
Nine structural and reactivity 1D descriptors were initially selected for describing the ligands at the DFT level in aqueous solution including Vol and PSA, natural atomic charges derived from Weinhold’s natural population analysis [33], Kohn-Sham’s frontier orbitals energies, electronegativity, hardness, and electrophilicity calculated as reactivity descriptors in a conceptual DFT framework, according to the following expressions [34]: \=
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1 2^E HOMO + E LUMOh
h=
| Preliminary Communication
1 2^E LUMO - E HOMOh 2 ~= \ 2h
All the descriptors were generated and graphically analyzed using Spartan software (Wavefunction, Inc.). 3D MEPs mapped on an isosurface of electron density of 0.002 au have also been inspected to analyze differences in the charge distribution and molecular shape between active and inactive compounds. PCA
Previous to conduct the PCA the data showed in Table 1 was converted in a matrix of 33 compounds (objects) and nine variables (descriptors). Descriptors were scaled to obtained data of the same order of magnitude for comparison with each other. The chemometric analysis was conducted using Statistica software version 6.0 (StatSoft Inc., CA, USA). Molecular docking studies
Molecular docking of the compounds into the 3D x-ray structure of cruzain (PDB ID: 1ME3) was carried out with Autodock 4.2 using the implemented empirical free energy function and the Lamarckian Genetic Algorithm. The AutoDockTools package was employed to generate the docking input files and to analyze the docking results [35]. Gasteiger-Marsilli charges were used for the protein and ligands. In all docking experiments a grid map with 60 × 60 × 66 points and a gridpoint spacing of 0.375 Å was applied. The maps were centered on the S atom of the catalytic Cys25 residue. Each docking consisted of 50 independent runs, with an initial population of 150 individuals, a maximum number of 2.5 × 105 energy evaluations and a maximum number of 2.7 × 104 generations. Mutation and crossover were applied to the population at rates of 0.02 and 0.80, respectively. For the remaining parameters, default values were used. Results differing by less than 2.0 Å in root square-deviation were grouped into the same cluster. Results & discussion Chemistry Several advanced techniques, such as microwave synthesis, ionic liquid synthesis, sonication, polymer-support synthesis, and multicomponent condensation, are broadly used to accelerate synthetic organic chemistry [36–40]. These modern techniques in organic synthesis integrating a variety of advanced technologies for the rapid generation of www.future-science.com
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Preliminary Communication | Ríos, Varela, Birriel et al. Table 1. Density functional theory theoretical descriptors for benzimidazole derivatives†. Compound
1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 2a 2b 2c 3a 3b 3c 3d 3e 3f 4a 4b 4c 4d 4e 5a 5b
NPA atomic charges (au)
Reactivity descriptors (eV)
Molecular size
N1
N3
EHOMO
ELUMO h
c
w
Vol (Å 3 )
PSA (Å 2)
-0.402 -0.403 -0.389 -0.397 -0.395 -0.401 -0.384 -0.398 -0.399 -0.401 -0.385 -0.392 -0.389 -0.396 -0.385 -0.392 -0.392 -0.387 -0.398 -0.395 -0.394 -0.389 -0.448 -0.450 -0.520 -0.535 -0.457 -0.393 -0.399 -0.387 -0.392 -0.456 -0.386
-0.538 -0.542 -0.509 -0.524 -0.522 -0.535 -0.515 -0.526 -0.540 -0.544 -0.529 -0.531 -0.527 -0.542 -0.532 -0.535 -0.525 -0.527 -0.526 -0.564 -0.523 -0.527 -0.624 -0.623 -0.520 -0.535 -0.628 -0.512 -0.526 -0.519 -0.629 -0.628 -0.519
-6.05 5.96 6.27 -5.90 -5.98 -5.15 -6.22 -5.62 -6.06 -5.96 -5.60 -5.87 -5.94 -5.16 -5.94 -5.61 -5.24 -5.61 -5.76 -5.53 -5.25 -5.75 -5.89 -5.90 -6.12 -5.19 -5.55 -6.15 -5.22 -6.12 -5.20 -5.55 -5.69
-0.53 -0.49 -1.53 -1.07 -1.29 -0.88 -1.56 -1.05 -0.51 -0.46 -1.20 -1.10 -1.34 -0.91 -0.91 -1.08 -1.28 -1.22 -1.16 -0.67 -1.28 -1.33 -1.11 -1.11 -1.22 -0.89 -0.14 -1.43 -1.08 -1.45 -1.08 -0.15 -1.44
-3.29 -3.23 -3.90 -3.48 -3.64 -3.02 -3.89 -3.34 -3.29 -3.21 -3.40 -3.49 -3.64 -3.04 -3.43 -3.35 -3.26 -3.42 -3.46 -3.10 -3.27 -3.54 -3.50 -3.51 -3.67 -3.04 -2.85 -3.79 -3.15 -3.79 -3.14 -2.85 -3.57
1.96 1.90 3.21 2.51 2.82 2.13 3.25 2.43 1.94 1.87 2.63 2.55 2.88 2.17 2.33 2.47 2.68 2.66 2.60 1.98 2.69 2.84 2.56 2.56 2.75 2.15 1.50 3.04 2.40 3.07 2.39 1.50 2.99
266 358 377 354 368 399 329 374 219 311 281 307 320 351 352 327 361 328 401 283 408 389 236 283 362 394 273 462 493 415 446 226 484
8.98 8.93 14.50 9.03 8.99 9.86 34.30 25.30 8.99 8.93 14.50 8.64 8.61 9.65 33.60 24.90 10.90 12.50 27.40 27.30 41.70 42.60 41.30 42.60 45.20 46.50 25.60 23.70 24.70 23.50 24.40 60.20 60.20
2.76 2.74 2.37 2.41 2.35 2.14 2.33 2.29 2.78 2.75 2.20 2.39 2.30 2.13 2.52 2.27 1.98 2.20 2.30 2.43 1.99 2.21 2.39 2.40 2.45 2.15 2.71 2.36 2.07 2.34 2.06 2.70 2.13
N1 and N3 are the benzimidazole nucleus nitrogens according to the standard numbering. h: Chemical hardness; c: Electronic chemical potential; w: Electrophilicity; EHOMO /ELUMO: Energies of the frontier KS orbitals in aqueous solution; PSA: Polar surface area; Vol: Molecular volume. †
numerous multifunctionalized molecule libraries can provide the high speed path for drug discovery. In this context, the synthesis of benzimidazoles 1a–q (Figure 2), recently described by the authors’ group, was performed by a combination of solid-phase organic synthesis and microwaveassisted organic synthesis, which are powerful techniques for rapid generation of structurally diverse molecules [41]. Triphenylphosphine resin was used to obtain benzimidazole derivatives with a methyl group in position 5, as it is shown in Figures 2 & 3. In recent years, the application of 1722
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a traceless phosphonium linker during the synthesis of heterocycles has been reported [42]. The phosphonium linker (I), obtained by reaction between polymer-supported triphenylphosphine and 4-fluoro-3-nitrobenzyl iodide, underwent aromatic substitution with primary amines followed by one-pot reaction with aldehydes in the presence of SnCl2·2H2O, yielding the benzimidazole system under microwave irradiation (Figure 2). The final products were released from the resin with NaOH under microwave irradiation and were obtained in good overall yields. An future science group
Identification of novel benzimidazole derivatives
| Preliminary Communication
NO2
I
F
Ph
PPh2 µW
Ph
NO2
P + – I
NH2
R1
Ph
+
µW
F
Ph
I
Ph R2
NHR1
Ph N
P + – I
R2
NaOH / MeOH
N
SnCl2.2H2O µW
N R2
µW
N
R1
III
1a R1: CH2CH2Ph 1b R1: CH2CH2Ph 1c R1: CH2CH2Ph 1d R1: CH2CH2Ph 1e R1: CH2CH2Ph 1f R1: CH2CH2Ph 1g R1: CH2CH2Ph 1h R1: CH2CH2Ph 1i R1: (CH2)3CH3
–
II
I
OHC
NO2
P
1
R1
1j R1: (CH2)3CH3 R2: (CH2)4CH3 1k R1: (CH2)3CH3 R2: 2-Furyl 1l R1: (CH2)3CH3 R2: 4-F-Ph 1m R1: (CH2)3CH3 R2: 4-Br-Ph 1n R1: (CH2)3CH3 R2: 4-N(Me)2-Ph 1o R1: (CH2)3CH3 R2: 4-NHCOMe-Ph 1p R1: (CH2)3CH3 R2: 3,4-O,O(CH2)-Ph 1q R1: (CH2)3N(CH2CH3)2 R2: 4-Br-Ph [28%]
R2: H R2: (CH2)4CH3 R2: 2-Furyl R2: 4-F-Ph R2: 4-Br-Ph R2: 4-N(Me)2-Ph R2: 4-NHCOMe-Ph R2: 3,4-O,O(CH2)-Ph R2: H
Figure 2. Synthesis of 5-methylbenzimidazoles 1,2-disubstituted employing triphenylphosphine resin. Global yield is shown in square brackets.
alternative route was performed to obtain 5-methylbenzimidazolones 2a–c (Figure 3). Reduction of the nitro-functionality in II was accomplished by treatment with SnCl2 in N-methylpyrrolidone (NMP), producing the desired o-phenylenediamines IV. Progress of reduction was followed by on-bead fourier transform infrared spectroscopy monitoring by the disappearance of characteristic nitro group absorbance and presence of characteristic aromatic NH vibrations. For the next step, the authors supported on a previous study in which the authors showed that 5,6-diamines can
be transformed to 8-thionexanthines in one-step reaction using potassium ethylxanthate under microwave irradiation [43]. Thus, we carry out condensation of potassium ethylxanthate with o-phenylenediamines IVa to produce immobilized benzimidazol-2-thione Va under microwave irradiation at 120°C for 10 min (Figure 3). Treatment of o-phenylenediamines, IVb–c, with excess carbonyldiimidazole gave conversion to their corresponding cyclic analogues, benzimidazol-2-one 2b–c (Figure 3). Cleavage from the solid support was performed by the use of a 10% NaOH X=S
II
SnCl2.2H2O NMP, r.t.
Ph
i) EtOC = SSK/DMF
Ph
Ph
NH2 µW 120°C,10 min
P + – I
NHR1 IV
X=O
H N
r.t 4 h
N 2
X N
V
X = O; S
R1
2a R1: CH2CH2Ph X: S [7%] 2b R1: CH2CH2Ph X: O [28%]
X R1
H N
P + – I
ii) CO(Im)2/THF µW 150°C, 15 min
NaOH 10%/MeOH
Ph
X = O; S
2c R1: (CH2)3CH3 X: O [39%]
Figure 3. Synthesis of 5-methylbenzimidazole derivatives (2a–c) employing triphenylphosphine resin. Global yields are shown in square brackets.
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Preliminary Communication | Ríos, Varela, Birriel et al. O NO2
HO
O O
Cl
OH
O NO2
DCC / DMAP
H2N—R1
µW 70°C 20 min VII O
O
R3
NH2
N
O
CH2CI2/TFA
R2
DDQ r.t. 24 h
NHR1
DMF r.t. 24 h
NHR1
VI OHC
O
SnCl2·2H2O
DMF
Cl
CH2CI2:THF/100W, 1h
NO2
O
r.t. 2 h
N R1
IX
VIII
O
O i) HBTU/ OHBt/ THF r.t. 1 h
N
HO
R2 N 3
N
N
R2 N
ii)
R1
R1
N r.t. 24 h H
4
3a R1: CH2CH2Ph R2: Br [56%] 3b R1: CH2CH2Ph R2: N(Me)2 [49%] 3c R1: (CH2)3CH3R2: Br [77%] 3d R1: (CH2)3CH3 R2: N(Me)2 [73%] 3e R1: (CH2)3N(CH2CH3)2R2: Br [79%] 3f R1: (CH2)3N(CH2CH3)2R2: N(Me) [70%]
4a R1: (CH2)3N(CH2CH3)2R2: Br [52%] 4b R1: CH2CH2Ph R2: Br [41%] 4c R1: CH2CH2Ph R2: N(Me)2 [46%] 4d R1: (CH2)3CH3R2: Br [27%] 4e R1: (CH2)3CH3 R2: N(Me)2 [21%]
Figure 4. Synthesis of 5-carboxybenzimidazoles employing Wang resin. Global yields are shown in square brackets.
solution in MeOH, providing benzimidazolones 2a–c in 7–39% overall average yields. Wang resin was used to obtain benzimidazole derivatives with carboxylic group at position 5, as it is shown in Figures 4 & 5. Initially, 4-chloro-3-nitrobenzoic acid was anchored on polymer by standard esterification conditions under microwave heating. 4-chloro-3-nitrobenzoic acid was treated with Wang resin in the presence of DCC and DMAP in DCM/THF mixture (9:1) under microwave irradiation (100 W, Tmax = 100°C) for 1 h to afford polymer-bound 4-chloro-3-nitrobenzoate VI in quantitative yield (Figure 4). Loading of O VIII
CO(Im)2
the resin was verified after cleavage with 50% TFA/CH2Cl2 at room temperature for 1 h. The authors have also performed this transformation under conventional conditions (room temperature, 48 h) leading to identical results. As mentioned above, the first step in the synthesis of a benzimidazole scaffold is the aromatic nucleophilic substitution reaction of a halogen atom, preferably a fluorine or chlorine atom, with an amine (Figure 2). Three amines (benzylamine, n-butylamine and N 1,N 1-diethylpropane1,3-diamine) were selected for aromatic nucleophilic substitution with VI. For the synthesis of O
H N
O
TFA/CH 2Cl2 O
N
NMP r.t. 48 h X
R1
r.t. 1 h
HO
H N O N
R1 5 5a R1: CH 2CH2Ph [30%] 5b R1: (CH 2)3CH3 [38%]
Figure 5. Synthesis of 5-carboxybenzimidazolones using Wang resin. Global yields are shown in square brackets.
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Identification of novel benzimidazole derivatives benzimidazol-5-carboxylic acid derivatives the reactions were driven to completion by employing an excess of the corresponding amine with polymer-bound 4-chloro-3-nitrobenzoate VI under microwave heating at 70°C for 20 min in NMP (Figure 4). Subsequently, reduction of nitro group in polymer-supported VII was achieved at room temperature by using SnCl2 in NMP. The formation of the benzimidazole nucleus IX was accomplished by the treatment of o-phenylenediamines VIII with aromatic aldehydes in the presence of DDQ. Cleavage from the solid support was performed by the use of a 50% TFA solution in CH2Cl2, providing benzimidazoles 3a–f in 49–79% overall average yields, after filtration through a short column. The obtained carboxylic acids were used to synthesize the corresponding amides 4a–e by conventional synthesis, using HBTU as the activating agent and piperidine as nucleophile (Figure 4). An alternative route was performed to obtain benzimidazol-2-ones 5a & b (Figure 5). The remaining step completing the synthetic sequence involved cyclization of immobilized diamine VIII. Benzimidazole formation was attempted with carbonyldiimidazole in NMP at room temperature for 48 h. Considering that the reactions needed extensive time, the authors expected microwave irradiation to facilitate the transformation. Thus, when the reaction was conducted under microwave heating at 150°C for 15 min, similar results were obtained. Biology
Trypanocidal activity
The benzimidazole derivatives were initially tested in vitro against the epimastigote form of T. cruzi, Tulahuen 2 strain. Epimastigotes and intracellular amastigotes are both proliferative forms; therefore, a drug inhibiting epimastigote proliferation should exhibit antiproliferative activity in the intracellular parasite. In this context, it is clear that testing against epimastigotes is relevant for the development of new anti-T. cruzi agents [14]. Furthermore, it was confirmed that the antiproliferative epimastigote activity is related to the in vivo anti-T. cruzi activity with compounds from the authors’ chemical library [44,45]. The benzimidazoles were incorporated into the media at 25 µM and their ability to inhibit growth (percentage of inhibition [PI]; Table 2) of the parasite was evaluated in comparison with the control (no drug added to the media). Nifurtimox and benznidazole were used as the reference trypanosomicidal drugs. In addition, the IC50 was determined for the most active derivatives (Table 2). Some of future science group
| Preliminary Communication
the most active benzimidazole derivatives were selected to assess their unspecific mammalian cytotoxicity in vitro in the 50–400 µM range, using J774 murine macrophages as the cellular model (Table 3). The selectivity indexes (SI) were expressed as the ratio between IC50 in macrophages and IC50 in T. cruzi. This screening revealed some preliminary–activity relationships (SARs) among the series, and a few hit compounds showing Table 2. Trypanocidal activity and cruzipain inhibition of herein developed benzimidazole derivatives. Compound 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 2a 2b 2c 3a 3b 3c 3d 3e 3f 4a 4b 4c 4d 4e 5a 5b
Nifurtimox Benznidazole
Anti-Trypanosoma cruzi† PGI‡ (25 µM)
IC50
TcCP §# (PI)
0 4 30 35 82 95 2 41 51 90 49 80 83 9 68 57 18 92 4 0 2 0 0 15 8 4 18 0 14 11 87 6 0 95 90
– – – – 16.3 6.1 – 25.9 25.0 15.2 25.6 16.7 13.3 – 18.6 20.9 – 13.8 – – – – – – – – – – – – 12.0 – – 7.7 7.4
15 0 0 45 33 2 0 0 14 0 0 28 55 43 30 6 0 22 0 0 0 0 0 0 5 17 12 39 0 15 10 0 0 – –
The results are the means of three different experiments with a SD
