Synthesis and Study of Antitumor Activity of Substituted Imidazolecarboxamides

Document Type: Research Paper


1 Biotechnology and Pharmaceutical Research Center, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Biotechnology and Pharmaceutical Research Center, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Biotechnology and Pharmaceutical Research Cente

4 Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


      In view of potential biological activities of small molecule polyamides, we synthesized some novel N-(2-aminoethyl)-1-benzyl-2- (alkylthio)-1H-imidazole-5-carboxamide (7a,b), and N-(2-(1-benzyl-2-(alkylthio) -1H-imidazole- 5-carboxamido) ethyl)-1-benzyl-2- (alkylthio)-1H-imidazole-5-carboxamides (8a,b) as antitumor agents. The antitumor activity of compounds 7a,b and 8a,b was studied at concentrations of 0.01, 0.1 and 1 mg/ml, using the potato disk bioassay technique. Vincristine at 0.25 mg/ml employed as positive control and had -67.24% tumor inhibition. Maximum % inhibition for potato tumors was found to be -75.52 for 7b at 1 mg/ml.


1. Introduction

        In every human cell, genetic information is stored on a string-like DNA polymer which is approximately 1 meter in length and contains 3×109 units of information in the form of base pairs, within which is encoded approximately 80,000 to 100,000 genes or sets of instructions [1]. The specific interaction of proteins such as transcription factors with DNA controls the regulation of genes and hence cellular processes [2]. A wide variety of human conditions ranging from cancer to viral infection arise from malfunctions in the biochemical machinery that regulates gene-expression [3]. Designed small molecules which target specific DNA sequences offer a potentially general approach for gene-specific regulation [4]. Such molecules could be powerful therapeutics for combating life threatening diseases which result from misregulation in transcription.

       The structures of two small molecules isolated from natural sources are shown in Figure 1. Among these DNA-binding molecules, distamycin is distinguished by its structural simplicity, having no chiral centers and an oligopyrrolecarboxamide core structure [5]. Structural studies of distamycin-DNA complexes reveal modular complexes in which adjacent pyrrolecarboxamides makes similar contacts with adjacent DNA base pairs. The relative simplicity of distamycin, with respect both to its chemical structure and its complexes with DNA, guided the initial decision to use distamycin as a basis for designed polyamides having novel DNA-binding sequences specificity [6]. The structure of the complex between the minor groove binder netropsin and d(GGCC AATTGG) was recently determined via single-crystal X-ray techniques [7].

      Polyamides containing N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provide a model for the design of artificial molecules for recognition of double helical DNA [8]. In this work, N-(2-aminoethyl)-1-benzyl-2-(alkylthio)-1H-imidazole-5-carbox-amide (7a,b), and N-(2-(1-benzyl-2-(alkylthio)-1H-imidazole-5-carboxamido)ethyl)-1-benzyl-2-(alkylthio)-1H-imidazole-5-carboxamide (8a,b) were synthesized as netropsin analogues. A test that has proven useful in monitoring the inhibition of crown-gall tumour is the potato disc assay [9-11]. It has been shown that inhibition of crown gall tumour initiation on potato discs has good agreement with compounds and plant extracts known to be active in the 3PS (in vivo, mouse leukaemia) antitumor assay [12]. In the present work, antitumor results are presented from such a screening of synthesized carboxamides (7a,b, 8a,b).


2. Experimental procedures

2.1. Chemistry

      Melting points were determined on Capillary Electrothermal apparatus and are uncorrected. The IR spectra were measured on a Unicam SP-1100 spectrophotometer, using samples prepared as KBr disks. 1H-NMR spectra were obtained on Bruker Ac-80 spec-trophotometer and chemical shifts (d) are in ppm relative to internal tetramethylsilane. Elemental microanalyses were carried out with a Perkin-Elmer 240 °C apparatus and were within ±0.4% of the theoretical values for C, H, and N. All solvents and reagents were purchased from the Fluka, Aldrich or Merck Chemical Company. Compounds 1-6 were synthesized as it has been reported previously.


2.2. Synthesis of N-(2-aminoethyl)-1-benzyl-2-(methylthio)-1H-imidazole-5-carboxamide (7a)

      To ethylenediamine (0.24 g, 4.0 mmol) in dry tetrahydrofuran (15 ml) was added dropwise at ice bath, crude acid halide 6a (1.07 g, 4.0 mmol) in dry tetrahydrofuran (5 ml) and then stirred overnight at room temperature. The resulting mixture was basified by adding saturated solution of sodium bicarbonate and extracted with chloroform (3×100 ml). Chloroform was evaporated to give 0.87 g (3 mmol) of 7a (75%); IR(KBr): ν 1680 cm-1(C=O); 1H-NMR (CDCl3): δ7.80 (s, 1H, H4-imidazole), 7.30-7.27(m, 5H, arom), 5.50 (s, 2H, CH2N), 3.70(m, 4H, NCH2CH2N), 2.67ppm(s, 3H,CH3).


Figure 1. Structure of two natural antitumor minor groove binding agents.

       Anal. Calcd. for C14H18N4OS: C, 57.91; H,6.25; N, 19.29. Found: C, 57.71; H, 6.35; N, 19.09%.


2.3. Synthesis of N-(2-aminoethyl)-1-benzyl-2-(ethylthio)-1H-imidazole-5-carboxamide hydrochloride (7b)

       It was prepared as described for 7a, yield 65%; IR (KBr): ν 1680cm-1 (C=O); 1H-NMR (CDCl3): δ7.8 (s, 1H, H4-imidazole), 7.5-7.0 (m, 6H, arom, NH), 5.47(s, 2H, CH2N), 3.70 (m, 4H, NCH2CH2N), 3.14 (q, 2H, CH2S), 1.28ppm (t, 3H, CH3).

       Anal. Calcd. for C15H20N4OS: C, 59.18; H,6.62; N, 18.41. Found: C, 59.39; H, 6.74; N, 18.30%.


2.4. Synthesis of N-(2-(1-benzyl-2-(methylthio)-1H-imidazole-5-carboxamido) ethyl)-1-benzyl-2-(methyl thio)-1H-imidazole-5-carboxamides (8a)

       To crude viscous liquid acid halide 6a (1.07 g, 4.0 mmol) in dry tetrahydrofuran (15 ml) was added dropwise at ice bath, eth-ylenediamine (0.12 g, 2.0 mmol) in dry tetrahydrofuran (5 ml) and then stirred overnight at room temperature. The resulting mixture was basified by adding saturated solution of sodium bicarbonate and extracted with chloroform (3×100 ml). The combined organic layers were dried (Na2SO4) and evaporated to dryness. The crude was purified by silica gel column chromatography (CHCl3/EtOH = 90:10) to afford 0.84 g (1.61 mmol) of 7a, yield 40%; IR (KBr): ν 1713 cm-1 (C=O); 1H-NMR (DMSO-d6): δ 7.80 (s, 2H, H4-imidazole), 7.30-7.27 (m, 12H, arom, NH), 5.5 (s, 4H, CH2N), 3.7 (s, 4H, NCH2CH2N), 2.7ppm (s, 3H, CH3).

      Anal. Calcd. for C26H28N6O2S2: C, 59.98; H, 5.42; N, 16.14. Found: C, 59.77; H, 5.53; N, 16.14%.


2.5. Synthesis of N-(2-(1-benzyl-2-(ethylthio)-1H-imidazole-5-carboxamido)ethyl)-1-benzyl-2-(ethylthio)-1H-imidazole-5-carboxamides (8b)

      It was prepared as described from 6b for 7a, yield 35%; IR(KBr):ν1700 cm-1(C=O); 1H-NMR (DMSO-d6): δ 7.8-7.0 (m, 14H, arom, H4-imidazole, NH), 5.47 (s, 4H, CH2N), 3.5-3.0 (m, 8H, NCH2CH2N, CH2S), 1.2ppm (t, 3H, CH3).

      Anal. Calcd. for C28H32N6O2S2: C, 61.29; H, 5.88; N, 15.32. found: C, 61.42; H, 5.65; N, 15.13%.


 Scheme 1. Synthesis of compounds 2 to 5.


2.6. Screening with the potato disc assay

     Fresh, disease-free potatoes were obtained from a local market. Tubers of moderate size were surface sterilized by immersion in sodium hypochlorite 0.1% for 20 min. Ends were removed and the potatoes were soaked for an additional 10 min. in sodium hypochlorite solution. A core of the tissue was extracted from each tuber with a surface-sterilized 1.0 cm cork borer. Pieces of 2 cm were removed from each end and discarded. The remainder of the cylinder was cut into 0.5 cm discs with a surface-sterilized scalpel. The discs were then transferred to agar plates (1.5 g of agar dissolved in 100 ml double distilled water (DDW), autoclaved for 20 min. at 121 °C, 20 ml poured into each Petri dish). Each plate contained 5 discs and 3-5 plates, were used for each sample dilution.


Scheme 2. Synthesis of compounds 7 and 8.

     Agrobacterium tumefaciens (ATCC 23341)was cultivated in Soybean Casein Digest Agar. For inoculation of the potato discs, 48 h broth culture containing 5×109 cells/ml were used. Samples were dissolved in 5% DMSO, filter sterilized, diluted and mixed with the bacterial culture for inoculation.

      The potato discs were incubated for 20 days at 25 °C incubator, after which Lugol's solution (I2/KI) was added, the tumor counts were made and compared with negative controls (bacterial suspension containing 5% DMSO). The results were expressed as E percentage versus the number of tumors on the control discs. Significant activity was indicated by consistent negative values of ca. 20% or greater inhibition. Vincristine was used as positive control.

%inhibition = [(average number of tumors in sample - average number of tumors in negative control) average number of tumors in negative control]×100

      Minimum inhibitory concentration (MIC) of samples on A. tumefaciens was determined by microplate MTT-based method in nutrient broth. Gentamicin was used as positive control.


3. Results and discussion

3.1. Chemistry

       Compounds 2-6 were synthesized as it has been previously reported [11]. Benzylamine hydrochloride (1) was stirred with 1,3-dihy-droxyacetone dimmer and potassium thiocyanate to give 5-hydroxymethyl-2-mercapto-1-benzylimidazole (2). Subsequent alkylation of compound 2 with alkyl halides afforded 2-alkylsulfanyl-1-benzyl-5-hydroxy-methylimidazole (3). Oxidation of 3 with manganese dioxide gave 4, which wasfurther oxidized by boiling in alkaline solution of silver nitrate to give 2-alkylsulfanyl-1-benzylimidazole-5-carboxylic acid (5) (Scheme 1).

      Compound 5 was converted to its acid halide (6), which was then added dropwise to ethylenediamine to give N-(2-aminoethyl)-1-benzyl-2-(alkylthio)-1H-imidazole-5-carboxa mide (7a,b) (Scheme 2). Reverse addition of ethylenediamine to acid halide (6) afforded N-(2-(1-benzyl-2-(alkylthio)-1H-imidazole-5-carboxamido)ethyl)-1-benzyl-2-(alkylthio)-1H-imidazole-5-carboxamides (8a,b) (Scheme 2).

       The proposed structure of compounds was confirmed by IR and 1H NMR spectroscopy. Interpretation of the 1H NMR spectra was base on the chemical shifts, multiplicities, and integral intensities of the signals.


3.2. Biological assay

       Minimum inhibitory concentration (MIC) of samples on A. tumefaciens was found to be greater than 1 mg/ml, and at lower concentrations formazan pink color developed after MTT addition that did not differ with that of negative control, indicating no inhibition for A. tumefaciens growth. The concentrations above the MIC would produce false positive results in potato disc assay due to the antibacterial activity of the compounds. Therefore, dilutions below MIC including 0.01, 0.1 and 1 mg/ml were used for potato disc assay. The results are shown in Table 1. Vincristine at 0.25 mg/ml was employed as positive control and caused -67.24% inhibition. Compounds 7a and 8a had stimulatory effect on tumors at 1 mg/ml with positive values as +37.5 and +15.75, respectively. All compounds at other concentrations had negative %inhibition, indicating tumor inhibition activity. Compound 7b at 1mg/ml caused -75.52% tumor inhibition with highest activity among compounds tested.


Table 1. Antitumor activity of compounds 7a-d, 8a-d on potato disc model.



       This work was supported by a grant from Research Council of Mashhad University of Medical Sciences.


[1]      Watson JD. Double helix: Recombination DNA: Gene therapy; predictive genetics. Gene 1993; 135: 309-15.


[2]      Roeder RG. The role of general initiation factors in transcription by RNA polymerase II. TIBS 1996; 9: 327-35.


[3]      Tijan R. Molecular machines that control genes.Scientific America 1995; 2: 54-61.


[4]      Gottesfeld JM. Regulation of gene expression by small molecules. Nature 1997; 387: 202-5.


[5]      Zimmer C, Wahnert U. Nonintercalating DNA-binding ligands: Specificity of the interaction and their use as tools in biophysical, biochemical and biological investigations of the genetic material. Prog Biophys Mol Biol 1986; 47: 31-112.

[6]      Dervan PB. Design of sequence-specific DNA-binding molecules. Science 1986; 232: 464-71.


[7]      van Hecke K, Nam PC, Nguyen MT, van Meervelt L. Netropsin interactions in the minor groove of d(GGCCAATTGG) studied by a combination of resolution enhancement and ab initio calculations. FEBS 2005; 272: 3531-41.


[8]      Dervan PB. Preparation and use of bifunctional molecules having DNA sequence binding specificity. US Patent 6506906. California Institute of Technology, 2003.


[9]      Ferrigni NR, Putnam JE, Anderson B, Jacobsen LB, Nichols DE, Moore DS, Mclanghlin JL, Powell RG, Smith Jr CR. Modification and evaluation of the potato disc assay and antitumor screening of euphorbiaceae seeds. J Nat Prod 1982; 45: 679-86.


[10]   Lellau F, Liebezeit TF. Cytotoxic and antitumor activities of ethanolic extracts of salt Marsh plants from the lower saxonian Wadden sea, Southern North sea. Pharm Biol 2003; 41: 293-300.

[11]  Hadizadeh F, Ghodsi R. Synthesis of novel N-substituted imidazolecarboxylic acid hydrazides as monoamine oxidase inhibitors. IL Farmaco 2005; 60: 237-240.


[12]  Galsky AG, Kozimor R, Piotrowski D, Powell RG. The crown-gall potato disk bioassay as a primary screen for compounds with antitumor activity. J Natl Cancer Inst 1981; 67: 689-92.