molecular encapsulation of 5-nitroindazole derivatives in 2-6-dimethyl beta ciclodestrina estudos electroquimicos e espectroscopicos

molecular encapsulation of 5-nitroindazole derivatives in 2-6-dimethyl beta...

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Molecular encapsulation of 5-nitroindazole derivatives in 2,6-dimethyl-b-cyclodextrin: Electrochemical and spectroscopic studies

Fernanda Pérez-Cruz a, Carolina Jullian a,*, Jorge Rodriguez b,c, Vicente J. Arán d, Claudio Olea-Azar b,*Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, ChileDepartamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, ChileDepartamento de Química, Facultad de Ciencias Básicas, Universidad Metropolitana de Ciencias de la Educación, Santiago, ChileInstituto de Química Médica, CSISC, Juan de la Cierva 3, 28006 Madrid, Spain article info

Article history: Received 30 January 2009 Revised 28 April 2009 Accepted 29 April 2009 Available online 5 May 2009

Keywords: 5-Nitroindazole Cyclodextrin Molecular modeling NMR ESR abstract

Four different 5-nitroindazole derivatives (1–4) and its inclusion with Heptakis(2,6-di-O-methyl)-bcyclodextrin (DMbCD) were investigated. The stoichiometric ratios and stability constants describing the extent of formation of the complexes were determined by phase-solubility measurements obtaining in all cases a type-AL diagram. Also electrochemical studies were carried out, where the observed change in the EPC value indicated a lower feasibility of the nitro group reduction. The same behavior was observed in the ESR studies. The detailed spatial configuration is proposed based on 2D NMR methods.

These results are further interpreted using molecular modeling studies. The latter results are in good agreement with the experimental data. 2009 Elsevier Ltd. All rights reserved.

The American trypanozomiasis is a disease that according to data from WHO, over 24 million people are infected or at least serologically positive for Trypanosoma cruzi.1 The importance of the investigations of nitroheterocycles derivatives lie in the existence of the group electroacceptor capable of generate radicals species (ROS) that act against the parasite producing oxidative stress. Since 2005 there began the study of a family of 5-nitroindazole (NI) derivatives in order to verify their anti-trypanocidal activity. A recent study has shown the in vitro and in vivo anti-trypanosomatide activity,2 besides, the mode of action of the NI against T. cruzi has being reported by the first time.3 Recently, the electrochemical study and the generation of the nitro anion radical of 5-nitroindazole derivative was done.4 Unfortunately many of these compounds showed low water solubility, which is a problem for its possible use as antichagasic drugs.

Cyclodextrins (cyclic oligosaccharides composed of D-glucose units) (Scheme 1) are known for their ability to bind non-covalently and forming inclusion complexes with wide variety organic compounds.5 In these complexes, a guest molecule is held within the lipophilic cavity of a cyclodextrin host molecule. The cyclodextrin cavity has an apolar character similar to an 80% dioxane/water solution and provides a slightly alkaline environment, because it is surrounded by glycosidic ethers.6 The main driving force of the complexation is the release of enthalpy-rich water molecules from the cavity.7–1 Replacement of water molecules by more hydrophobic guest molecules present in the solution results in formation of an inclusion complex between the host and the guest.12

In a previous work13 we verified that the incorporation of NI derivatives in cyclodextrins improved their solubility. In this sense, encapsulating the drug in the hydrophobic cavity of cyclodextrin seems to be a good approximation in order to increase the bioavailability and biological activity.

In this work, inclusion complex of four 5-nitroindazole derivatives in Heptakis(2,6-di-O-methyl)-b-cyclodextrin molecule were prepared in order to improve the aqueous solubility of the drugs. The inclusion complexes were analyzed by UV–vis, NMR spectroscopy, Differential pulse polarography, ESR and molecular modeling techniques. The structural information obtained and the geometry of the complexes, are necessary to clarify the complexation mechanism and the importance of the substituent groups on the indazole ring.

2. Results and discussion

The stoichiometric ratios and stability constants were derived from the changes in the solubility of the substrates 1–4 (Scheme 1) in the presence of increasing amounts of DMbCD, measured by

0968-0896/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.04.067

* Corresponding authors. E-mail address: (C. Jullian).

Bioorganic & Medicinal Chemistry 17 (2009) 4604–4611 Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage: w.else


ON 4’ 5’

N 4’



ON 4’ 5’

N 4’

DMβCDn = 7 R , R = CH and R = H

CH 5-nitroindazole

Scheme 1.



4.50x10 6.00x10

[ 4 ] / M

[ 3 ] / M




2.0x10 2.5x10

[ 1] / M

[ 2 ] / M

Figure 1. Phase solubility diagrams of compounds 1–4 with DMbCD in water at 30 C.

F. Pérez-Cruz et al./Bioorg. Med. Chem. 17 (2009) 4604–4611 4605

UV–vis spectroscopy. For all the NI/DMbCD systems, cyclodextrin enhance the aqueous solubility of NI derivatives, as shown in

Figure 1. The binding constant Ka of the complexes was calculated from the slopes of the linear phase-solubility plots, Table 1, accord- ing to the methodology described in the experimental part. These diagrams showed a linear relationship between the amount of NI solubilized and the concentration of CD in solution.

The binding constant for the nitroindazole derivatives with

DMbCD followed the rank order 3 > 4 > 1 > 2, reflecting an enhancement of binding on the different substituent’s of the nitroindazole derivative. We observed previously the same trend employing bCD instead of DMbCD14 (data not shown), but with less association constants, indicating that the complexation ability of the b-cyclodextrin is significantly enhanced by its methylation. Apparently, the presence of the methylated groups in the cyclodextrin, seems to be important for the binding of these compounds in the cyclodextrin cavity.13,15 These groups enlarge the cyclodextrin cavity, making its environment more hydrophobic and favor the adaptability of the cyclodextrin towards a guest, through an enhanced flexibility.

Also, the enhancement of the Ka values can be related to the different substituent groups at the positions R1 and R2 of the indazole ring. In this regards, morpholine and benzyloxy groups have a positive influence on the association constant and isopropyl and methoxyl groups have a negative influence on the Ka value. On the other hand, the ratio R was plotted against the difference of the absorbance of NI in presence and absence of DMbCD. The results of Job’s plot are shown in Figure 2. According to the continuous variation method, the maximum concentration of the complex will be present in the sample where the molar ratio R corresponds to thecomplexationstoichiometry. The maximumabsorbancevariation for DMbCD with the nitroindazole 1–4 derivatives was observed for R = 0.5, which might indicate that the main stoichiometry is 1:1, in agreement with the stoichiometry suggested from the phase-solubility study.

2.1. Differential pulse polarography (DPP) studies

Electrochemical behavior in protic media for this kind of 5-nitroindazole derivatives has been previously studied,3,13 indicating that NI derivatives are able to be reduced on the mercury electrode due to the four-electron and four-proton irreversible reduction of the nitroaromatic moiety to yield the hydroxylamine derivative according to the following overall reaction:

Consequently to evaluate changes in the polarograms, due to addition of DMbCD into a solution of NI, we used different NI/DMbCD mixtures in aqueous media at pH 7.4. The addition of DMbCD to a solution of NI causes two main changes in the polarograms. Firstly, the cathodic peak potential (EPC) shifted in a negative direction and secondly, the cathodic peak current IPC increased. Figures 3 and 4, show the displacement of cathodic peak potential (EPC) for compounds 3 and 4, when different concentrations of DMbCD are used.

The shift of signals 4-DMbCD and 3-DMbCD to more negative potentials indicates that the nitro group is reduced less favorably when the indazole ring is included in the hydrophobic cavity hindering the interaction with the electrode. Complexes 3-DMbCD and 4-DMbCD show very close reduction potentials, 0.57 V and

Table 1 Association constants for NI–DMbCD complexes

Inclusion complex K (M ) r

0.0 3.0x10

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