Investigación

 

Cytotoxic Evaluation of a Series of Bisalkanoic Anilides and Bisbenzoyl Diamines

 

Luis Chacón-García, M. Elena Rodríguez, and Roberto Martínez*

 

Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, México, D.F. E-mail: robmar@servidor.unam.mx

 

Dedicated to Profesor Alfonso Romo de Vivar.

 

Recibido el 10 de marzo del 2003.
Aceptado el 26 de junio del 2003.

 

Abstract

A series of bisalkanoic anilides and bisbenzoyl diamines were synthesized with the aim of elucidating the relationship between molecular structure and cytotoxic activity. Twenty-one derivatives were synthesized and tested on three tumoral cell lines. No apparent relationship was observed between electronic effects and cytotoxic activity, but it was found that compounds in which the 4'-phenyl substituent is fluoride or bromide gave the best inhibition of tumoral cell growth.

Keywords: Diamides, alkanediamides, cytotoxic activity.

 

Resumen

El objetivo del presente trabajo fue encontrar la relación entre la estructura molecular y la actividad citotóxica de una serie de anilidas de diácidos y diamidas bisbenzoiladas, para lo cual se sintetizaron veintiuno de los compuestos mencionados. Los resultados de la evaluación citotóxica de estos derivados, en tres líneas celulares, no indicaron ninguna relación con respecto a efectos electrónicos de los substituyentes, si bien los derivados 4-bromofenil y 4-fluorofenil son los más activos.

Palabras clave: Diamidas, alcano diamidas, actividad citotóxica.

 

Introduction

DNA recognizing molecules such as DNA-intercalators and groove binders have been the subject of increasing interest due to the ongoing search for more active antitumoral compounds. DNA-groove binders have been widely studied as anticancer compounds. In addition, they have been studied as anti-HIV agents and have been incorporated as a linker in DNA bis-intercalators [1-4]. The most typical DNA-groove binders are the antibiotics Distamycine A (1) and Netropsin (2), which are characterized by polyamide and polyaromatic functional groups along the DNA recognizing chain [5]. The aromatic portion of these compounds is the pyrrolo system; however, recent studies have investigated compounds incorporating thiazolyl (3) or phenyl (4) (Fig. 1) instead of pyrrolyl, and groove binders that contain the benzimidazolyl moiety have been described in earlier reports [6-8]. Recently, we reported a series of N,N'-(diaminophenyl)alkanediamides 5 which differ in the length of the aliphatic portion. These compounds were shown to inhibit the growth of tumoral cell lines, indicating that this topographical factor has an important influence on DNA recognition [9]. However, the cytotoxic activity of the N,N'-(diaminophenyl)alkanediamides was low. The present investigation was undertaken to study the influence of aryl substituents in these compounds and to find compounds of this type with improved cytotoxic activity. To achieve this, we synthesized a series of bisalkanoic anilides and bisbenzoyl diammines (6-27) and their activities as cytotoxic agents were evaluated.

 

Results and discussion

The N,N'-diarylalkanediamides (6-20) (Fig. 2) were synthesized by condensation of the respective 4-substitued aniline (2 equiv.) with succinyl, glutaryl or adipoyl chloride (1 equiv.) in acetone while being stirred and cooled in an iced bath. The products were precipitated, filtered, and washed with acetone. Yields varied from 65 to 96 %.

Compounds 21-23 and 25-27 were obtained as described for 6-20 but from condensation of the respective benzoyl chloride and ethylenediamine, 1,2-propanediamine, or piperazine as shown in Figure 2. The compounds were obtained in yields of 75 to 95 %. Compound 24 was obtained by reduction of the nitro derivative 23, using Pd/C and hydrazine in ethanol at reflux for 1 h. Recrystallization from methanol afforded the amine derivative. The yields and spectroscopic data of compounds 6-27 are summarized in Table 1.

The percentage of inhibition of the growth of the three tumoral cell lines after treatment with each compound at a concentration of 31 µM is given in Table 1. The groups bonded at the 4' position were selected on the basis of their electron withdrawing or donating properties, and their hydrogen bonding capabilities.

The first series of compounds comprises N,N'-diarylalkanediamides with different numbers of methylenes in the aliphatic chain. The first compounds synthesized and probed were 6 to 10 (n = 2). These compounds displayed little activity in the three cell lines. The compound which inhibits cell growth to the greatest extent (57 % in K562) is 6 (R = F), followed by 7 (R = Br) in the same cell line.

To study the influence of the length of the aliphatic chain on cytotoxic activity, we prepared compounds with a four methylene chain (12, 14, 16-18). It should be pointed out that compounds 13 (R = Cl) and 15 (R = I) were included due to the apparent tendency of halogens to present activity. In addition, compounds 19 (R = NHCOCH₃) and 20 (R = CN) were included in the study to investigate the effects of the NHCOCH₃ and CN functional groups. In contrast to the almost complete lack of activity shown by the first series (n = 2), compound 12 (R = F) induced almost 100 % inhibition of growth in K562 cell line and the functional groups OMe (16) and OH (17) were found to enhance cytotoxicity. The rest of the compounds showed no activity.

Compounds 21-25 were examined to analyze the importance of the relative position of the amide group and the presence of branching in the aliphatic chain. Surprisingly, compound 21 was the most active in the bromide series, displaying relatively good inhibition in the three cell lines. Given the activity of 21, it is surprising that 22 was inactive. To complete the series of bromide compounds, 11 (n = 3, R = Br) was obtained; it showed greater activity than 7 (R = Br, n = 2) but less than 14 (R = Br, n = 4) in K562 cell line.

The inhibition resulted by 21, lead to the resentment that conformation could be implicated in the cytotoxic activity. To test this idea, compounds 26 and 27 were synthesized; however, both of these compounds were inactive. Although these molecules are structurally similar to 21-25, the formers (26 and 27) are not very capable of interacting by hydrogen bonding. This is a very important factor affecting cytotoxicity in DNA groove binders due to the stability of the DNA-ligand complex.

 

Conclusions

The data presented here are inconclusive regarding the relationship between electronic factors or hydrogen bonding capability and inhibition of the growth in tumor cell lines. The present results also show no clear link between the presence of halogens or the length of the aliphatic chain and the cytotoxicity of a compound. However, this study did reveal the interesting finding that the compounds which presented cytotoxic activities were primarily those containing fluoride or bromide.

 

Experimental

Chemistry

General procedure for the preparation of 6-20. Diacyl chloride (0.72 mmol) was added to a solution of 4-R-aniline (1.44 mmol) in 15 mL of acetone at 5 °C. After 2 h stirring, the mixture was filtered and washed with acetone to afford 6-20. 12: ¹H NMR (δ, J(Hz)): 1.60 (s, 4H), 2.30 (s, 4H), 7.10 (m, 4H), 7.57 (m, 4H), 9.94 (s, 2H); IR ν (cm⁻¹) 1652, 3305. 15: ¹H NMR (δ, J(Hz)): 1.59 (s, 4H), 2.31 (s, 4H), 7.41 (d, J = 8.8, 4H), 7.60 (d, J 8.7, 4H), 9.97 (s, 2H); IR ν (cm⁻¹) 1657, 3292. 19: ¹H NMR (δ, J(Hz)) 1.59 (m, 4H), 1.99 (s, 6H), 2.28 (s, 4H), 7.46 (s, 8H), 9.80 (s, 2H), 9.83 (s, 2H); IR ν (cm⁻¹) 1659, 3298.

General procedure for the preparation of 21-23 and 25-27. 4-bromobenzoyl chloride (1 mmol) was added to a solution of diamine (0.7 mmol) in 15 mL of acetone at 5 °C. After 2 h stirring, water was added and the precipitated filtered and washed with water and acetone to afford 21-23 or 25. 21: ¹H NMR (δ, J(Hz)) 1.15 (d, J 6.64, 3H), 3.35 (t, J 9, 2H), 4.23 (m, 1H), 7.64 (d, J 8.6, 4H), 7.76 (d, J 8.5, 2H), 8.34 (d, J 8.2, 1H) 8.63 (t, J 5.6, 1H); IR ν (cm⁻¹) 1637, 3301.

22: ¹H NMR (δ, J(Hz)): 1.33 (s, 4H), 7.65 (d, J 8.85, 4H), 7.77 (d, J 8.5, 4H), 8.67 (s, 2H); IR ν (cm⁻¹) 1633, 3287. 23: ¹H NMR (δ, J(Hz) 3.47 (d, J=2.7, 4H), 8.07 (d, J = 8.8, 4H), 8.30 (d, J 8.9, 4H), 9.00 (s, 2H); IR ν (cm⁻¹) 1640-3319. 25: ¹H NMR (δ, J(Hz)): 1.2 (d, J 6.7, 3H), 3.45 (t, J 6.3, 2H), 4.3 (m, 1H), 8.03 (d, J 8.96, 2H), 8.05 (d, J 9, 2H), 8.63 (d, J 8.14, 1H), 8.92 (t, J 5.6, 1H); IR ν (cm⁻¹) 1661, 3318. 26: ¹H NMR (δ, J (Hz)) 3.54 (m, 8H), 7.37 (d, J 8.4, 4H), 7.64 (d, J 8, 4H); IR ν (cm⁻¹). 1635.

Preparation of 24.

Ethanol (10 ml), Pd/C 5% (0.046 g), Hidrazine (0.818 ml, 25.9 mmol), water (0.93 ml) and 23 (756 mg, 2.59 mmol) were mixed in a bottom flask. The mixture was refluxed for 2h. The resulting solid was dissolved in methanol with heat and filtered at vacuum. Methanol was eliminated up precipitation of a solid that was filtered and crystallized from methanol to afford 24. ¹H NMR (δ, J(Hz)): 3.33 (d, J 7.2, 4H), 5.58 (d, J 3.18, 4H), 6.51 (d, J 8.5, 4H), 7.54 (d, J 8.5, 4H), 8.12 (s, 2H); IR ν (cm⁻¹) 1600, 3333, 3437.

Cytotoxic Activity

Tumoral cell lines were supplied by the National Cancer Institute. The cytotoxicity assays were carried out at 5000 to 7500 cells / mL as reported by Skehan et al. and Monks et al using the sulforhodamine B (SRB) protein assay to estimate cell growth [17, 18]. Compounds were dissolved in DMSO which has not effect on the inhibition has shown by the control. The percentage of inhibition of the growth described for all compounds were obtained from three different experiments. The percentage growth was evaluated spectrophotometrically in a Bio kinetics reader spectrophotometer. Daunomicyne and 5-fluorouracyl were used as references. These compounds under the described conditions gave 100 % of inhibition. Each experiment was made two times by gave triplicate.

 

Acknowledgment

We thank CONACyT (32633-E) and DGAPA-UNAM (IN-211601) for financial support. We also thank M.T. Ramírez Apan for obtaining the biological data, R. Patiño, H. Rios, A. Peña, L. Velasco and J. Pérez for technical assistance. Contribution No. 1765 from Instituto de Química, UNAM.

 

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