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Boletín de la Sociedad Chilena de Química

versão impressa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.47 n.1 Concepción mar. 2002

http://dx.doi.org/10.4067/S0366-16442002000100009 

NEW SYNTHESIS AND CHARACTERIZATION OF THE SODIUM 3-
N-ACETYLAMINO-2,4,6-TRIIODOBENZOATE. ANTIBACTERIAL
ACTIVITY EVALUATION

VICTOR KESTERNICH 1*, PEDRO GARCÍA1 and ROLANDO MARTÍNEZ2

1Departamento de Química, Universidad Católica del Norte. Avda. Angamos 0610, Casilla 1280, Fax (56-55) 355621, Chile, e-mail: vkestern@ucn.cl.

2Instituto de Química, Universidad Austral de Chile, Casilla 567, Valdivia, Chile

(Received: September 10, 2001 - Accepted: December 27, 2001)

ABSTRACT

A new synthesis and chemical characterization by mass and infrared spectroscopy of sodium 3-N-acetylamine-2,4,6-triiodobenzoate (sodium acetrizoate), a compound used as contrast agent, has been developed. For its preparation, a new method consisting of three stages has been worked out, using 3-aminobenzoic acid as a substrate. Both the intermediate 4 and the product 5 showed antibacterial activity over Pseudomona aeruginosa ATCC and. Shigella flexneri.

Keywords: organoiodine, bactericidal effect, contrast agent.

RESUMEN

Se ha desarrollado una nueva síntesis y caracterización química por espectroscopía de masas e infrarroja, de 3-N-acetilamino-2,4,6-triyodo benzoato de sodio, un compuesto empleado como agente de contraste. Para su preparación se ha desarrollado un método nuevo que consta de tres etapas, empleando como sustrato el ácido 3-aminobenzoico. Tanto el intermedio 4 como el producto 5, mostraron actividad antibacteriana sobre Pseudomonas seruginosa ATCC Shigella flexneri.

Palabras claves: organoyodado, efecto bactericida, agente de contraste.

INTRODUCTION

Organic iodine compounds are used in medicine as X-ray contrast agent due to its unusual high opacity in front of this type of radiation1. Because they have iodine atoms in their chemical structure, they present a greater X-ray absorbance with respects to human body organs.

Among the most used compounds are those derived from 3,5-diaminobenzoic acid, for example, gastrografin2 1 and those derived from 3-aminobenzoic acid like iodipamida3 2 (Figure 1). Although these compounds have been prepared before, most of the synthesis methods are dated several years ago, therefore their performance is low and the procedures are generally long and complex. This makes information on most of the spectroscopic data insufficient.

 

Fig. 1. Structure of gastrografin 1 and iodipamide 2

The main drawback of the synthesis is the iodination stage since, in most cases, mixtures of reagents such as iodides and silver salts4 or iodine salts and ammonium and cerium nitrate5 Ce(NH3)2(NO3)6 are used, thus making the synthesis process more expensive. Besides, reagents highly toxic and difficult to handle are used, this being the case of monochloride iodine6 or iodine mixtures in presence of strong oxidant agents.

The sodium acetrizoate synthesis method used at present was worked out by V.H. Wallingford et. al7 and, to date, more efficient alternatives have not been published. In this respect, this study proposes, on the one hand, a route with fewer stages and better overall performance. On the other hand, it provides spectroscopic data (IR and EM) of both the intermediate and the product. As to our knowledge, this is the first time the bacterial activity of intermediates and products is evaluated from the biological viewpoint.

RESULTS AND DISCUSSION

Acetrizoate synthesis was carried out in three stages, starting from the 2-aminobenzoic acid (Scheme 1).

 

Scheme 1. Synthesis of sodium acetrizoate 6

The iodination of 3 lasted three hours since, after this period, a di-iodine compound was observed. Substitution between two goal-arranged groups is difficult, particularly if they have a high electronic density. Undoubtedly, this was the key stage of the synthesis where iodination effectiveness was proven by using hydrogen peroxide as oxidizing agent, aimed at keeping a high iodine concentration from the HI generated in the process. Structural determination was based mainly on mass (MS) and infrared (IR) spectra. The presence of the molecular ion M+ 515.9 and also the fragments m/z 338.9 (100%), 262.1 and 134.9 corresponding to the losses of one, two or three iodine atoms, respectively, were clearly observed. In IR, two bands of primary aromatic amine at 3430 and 3320 cm-1, the acid broad band between 3100 and 2500 cm-1 and the acid carbonyl at 1665 cm-1 can be seen.

The obtainment of 5 as a crystalline product was possible by making 4 react with an excess acetic dehydrate in pyridine for 3 hours. The molecular ion M+ 556.87 as well as, a fragment m/z 495.20 corresponding to the loss of acetate as acetic acid (M+-HOAc) are observed in its mass spectrum. Besides, the fragments corresponding to the losses from the molecular ions of the three iodine atoms m/z 431.00, 303.91 and 107.12, respectively, can bee seen. The infrared spectrum IR clearly shows the band of the bonding N-H at 3100 cm-1 and of the amide carbonyl at 1690 cm-1.

Finally, the treatment of 5 with an NaOH aqueous solution at 20% until its complete dissolution allowed access to the sodic salt, sodium acetrizoate 6.

As literature states that the organoiodine compounds show various citotoxicity levels depending on their structures, and, that they can show anthelmintic and antimicrobial effects8, we decided to do a preliminary study of the biological activity on five bacterial strains, summarized in Table 1, shows that all the compounds evaluated, particularly 4 and 5 showed an important inhibitory effect of the growth of two bacterial strains, Pseudomona aeruginosa and Shigella flexneri, respectively. These results encourage us to continue working on these molecules in other bacterial strains and later carry out tests in vivo with laboratory specimens.

Table 1. Effect of intermediate and product on the bacterial growth.

Compounds

E. coli

HB 101

E. coli

enteropatógena

Shigella

flexneri

Pseudomona

aeruginosa ATCC

Baccillus

subtilus ATCC

4

+

++

+

+++

++

5

++

+

+++

++

+

6

++

+

+

+

+

Oxytetracycline

+++

nt

nt

+++

+++

- No activity
+ Inhibition hale <5 mm
++ Inhibition hale from 5 to 10 mm
+++ Inhibition hale >10 mm
nt no tested

Finally, as opposed to the Walllingfor et al’s method, in using this synthesis the product is obtained in fewer stages resulting in the simplification of the process. Thus this synthesis opens a way to access other aromatic organic iodine compounds with a good performance.

EXPERIMENTAL

Melting points were determined on a Kofler-type apparatus and are uncorrected. The IR was taken on a Perkin-Elmer 1310 spectrophotometer nujol, as solvent. The MS spectra were measured on a VG-Micromass ZAB-2F.

Synthesis of 3-amino-2,4,6-triiodinebenzoic acid, 4. In a rounded-bottom three-necked balloon (250 ml), 56 ml of ethanol and 12.8 g of iodine (0.05 mol) were poured into a glycerin bath with a condenser and agitation system. Once the iodine dissolved completely, 5.0 g (0.0365 mol) of 3-aminobenzoic acid 3 were added. The mixture was heated at reflux (80ºC) and 10 ml of H2O2 at 30% were slowly added in 30 minutes. After a three-hour reaction, it was cooled and 250 ml of sodium metabisulfite at 10% were added. Then, it was vacuum filtered with a Büchner funnel and the filtrate was dried at 90ºC, obtaining 16 g (88%) of 4. Mp 198-200°C. IR (cm-1): 3430 y 3320 (NH2); 3100-2500 (CO2-H); 1665 (carbonyl). MS (m/z): 515.9 (M+); 338.9 (M+-I); 262.1 (M+-2I); 134.9 (M+-3I).

Synthesis of 3-N-acetylamino-2,4,6-triiodobenzoic acid, 5. In a reaction balloon with an agitation system and refrigerants, 4.0 g (7.8 mol) of 3-amino-2,4,6-triiodobenzoic acid 4 and 45 ml of acetic anhydride were added and left at reflux for 3 hours at 80ºC over a thermally regulated glycerin bath. Once the reaction was over, it cooled and then 50 ml of distilled water were added. Later, it was vacuum cooled and the product was dried at 90ºC, weighing 4.0 g (92 %). Mp. 270-272 ºC. IR (cm-1): 3100-2500 (CO2-H); 3220 (NH); 1680 (carbonyl). MS (m/z): 556.87(M+), 495.20 (M+-HOAc). 431.00 (M+-I); 303.91 (M+-2I) and 107.12 (M+-3I).

Synthesis of sodium 3-N-acetylamino-2,4,6-triiodobenzoate, 6. A solution of NaOH at 20% (0.7) was slowly dropped into a 100 ml beaker placed on an agitator, with 0.423 g of 5 in 20 ml of water, until it dissolved completely. The solvent was evaporated by distillation, obtaining 0.28 g of 6 (82%). Mp 295-297°C. IR; 3215 (N-H), MS (m/z). 451.85 (M+); 324.92 (M+-I); 198.01 (M+-2I) and 71.16 (M+-3I).

Measurements of Antibacterial Activity

The method used for the determination of antibacterial activity was that of Chabert which, consists on spreading the different assay bacteria over Müeller-Hinton agar plates. After incubation the zones of growth inhibition around for Whatman Nº1 paper disk (6.5 mm) were observed. The paper disk was impregnated with different concentrations of the compounds to be tested: 0.25, 0.5, 1.0 and 5.0 m g/ml, respectively. Results were measured after 24 h of incubation at 37º.

ACKNOWLEDGEMENTS

This study has been partially financed by DGICT, Universidad Católica del Norte, Antofagasta, and DID (project S-200156), Universidad Austral de Chile, Valdivia. Also, we thanks Prof. Ana Tejeda, Facultad de Humanidades, Universidad Católica del Norte.

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