SCIENCE CHINA Chemistry • ARTICLES •
March 2015 Vol.58 No.3: 483–494 doi: 10.1007/s11426-014-5296-3
Novel benzimidazole derived naphthalimide triazoles: synthesis, antimicrobial activity and interactions with calf thymus DNA Yun-Lei Luo, Kishore Baathulaa†, Vijaya Kumar Kannekanti‡, Cheng-He Zhou* & Gui-Xin Cai* Key Laboratory of Applied Chemistry of Chongqing Municipality; Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China Received July 30, 2014; accepted October 16, 2014; published online January 19, 2015
A novel series of benzimidazole derived naphthalimide triazoles and some corresponding triazoliums have been successfully synthesized and characterized by 1H NMR, 13C NMR, 1H-1H COSY, IR and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro by two-fold serial dilution. 2-Chlorobenzyl triazolium 8g and compound 9b with octyl group exhibited the best antibacterial activities among all the tested compounds, especially against S. aureus with inhibitory concentration of 2 μg/mL which was equipotent potency to Norfloxacin (MIC=2 μg/mL) and more active than Chloromycin (MIC=7 μg/mL). Triazoliums 8g and 8f bearing 3-fluorobenzyl moiety displayed the best antifungal activities (MIC=219 μg/mL) against all the tested fungal strains without being toxic to PC12 cell line within concentration of 128 µg/mL. Further investigations by fluorescence and UV-Vis spectroscopic methods revealed that the compound 8g could effectively intercalate into calf thymus DNA to form the 8g-DNA complex which could block DNA replication, exerting powerful antimicrobial activities. naphthalimide, benzimidazole, triazole, antibacterial, antifungal, calf thymus DNA
1 Introduction Naphthalimides with a naphthalene framework and cyclic double imides moiety have been widely investigated as anticancer agents, and some naphthalimide compounds such as Amonafide and Elinafide have been found to possess large clinical potentiality in the treatment of cancers acting by intercalating deoxyribonucleic acid (DNA) [1–3]. This special mechanism has attracted much interest in exploiting other medicinal potentialities of naphthalimides especially their antibacterial and antifungal behaviors [4]. Recently, some researches have revealed that the introduction of some nitrogen-containing heterocycles into naphthalimide back*Corresponding authors (email:
[email protected];
[email protected]) †Postdoctoral fellow from Department of Chemistry, Kakatiya University, Warangal 506009, India ‡Postdoctoral fellow from Indian Institute of Chemical Technology, Hyderabad 500607, India
© Science China Press and Springer-Verlag Berlin Heidelberg 2015
bone is beneficial for the pharmaceutical properties [5,6]. This has inspired increasing effort to investigate naphthalimide-based azoles as a novel type of potential antimicrobial agents. Our previous work indicated that the introduction of five-membered triazole heterocycle into the N atom of imides could effectively inhibit the growth of the tested bacteria and fungi. Most of derivatives or analogues of ZYY (Figure 1) with a (CH2)4 linker between naphthalimide backbone and triazole ring showed better antimicrobial activity as compared to the standard drugs like Norfloxacin, Chloromycin and Fluconazole [7,8]. Thus, the hybrids of naphthalimide with triazole could not only enhance the antimicrobial activities, but had also broadened the antimicrobial spectrum; and this molecular motif is worth further investigation as possible potential antimicrobial agents. Reasonably, this attracts our special interest to modify the structure of ZYY. The design is shown in Figure 2. chem.scichina.com
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Figure 1 potency.
Figure 2
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Naphthalimide-derived triazole with strong antimicrobial
Design of benzimidazole derived naphthalimide trizoles.
It is well known that the special benzimidazole ring could readily interact with various active targets in biological system via non-covalent interactions, like hydrogen bonds, coordination, ion-dipole, cation-, - stacking and hydrophobic effect as well as Van der Waals force, and thereby exhibited potential applications in medicinal field [9–17]. So far many benzimidazole-based compounds have been successfully developed as clinical drugs such as antiparasitic Albendazole, anti-anabrotic Omeprazole, antihistaminic Astemizole and antihypertensive Candesartan to prevalently treat various diseases [18,19]. Benzimidazole is structurally similar to purine, and its derivatives could compete with them, inhibiting the nucleic acids and protein synthesis thereby killing the microorganisms or disrupting their growth [20]. Recently, the benzimidazole-modified derivatives have been discovered to effectively intercalate into calf thymus DNA and block DNA replication and cleavage, thus exerting powerful antimicrobial activities [21,22]. Clearly, benzimidazole-based derivatives possess large potential for developing into new antibacterial and antifungal agents by blocking DNA replication. Reasonably, the hybrid of benzimidazole moiety and naphthalimide backbone has attracted our special interest. The thioether bridge between azole ring and target molecule has been found to enhance antimicrobial potency [23]. The presence of sulfur moiety as an electron-rich center is able to improve lipophilicity and modulate electron density of the azole ring while changing their interaction modes with biomolecules and thus exhibiting excellent bioactivity. Therefore, the thioether bond is incorporated between benzimidazole ring and naphthalimide skeleton assuming it to contribute positively to the antimicrobial activities. The
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introduction of sulphur atom is expected to bring in a bulky polarisable atom for potent pharmacological activities, and thus alter its geometry, polarisability, stability, lipophilicity, steric and electronic characteristics of the molecule. Researches revealed that the aliphatic and aromatic groups might greatly improve the physiochemical properties which could beneficially enhance the absorption rate and transport of drugs in vivo [24–26]. Thus a variety of alkyl and aryl substituents were introduced into target naphthalimide system in order to study their effect on antimicrobial activities. A lot of works have shown that the transformation of triazolyl ring into triazolium resulted in improving antibacterial and antifungal efficacy and broading antimicrobial spectrum [27–34]. This happens because the electropositive triazolium moiety was useful for increasing water solubility and membrane permeability. Reasonably, the naphthalimide triazoles were transformed into the corresponding triazoliums by diverse halobenzyl or alkyl halides. Noticeably, coumarin-based compounds as medicinal drugs have been increasingly attracting special interest due to their outstanding contributions in the prevention and treatment of diseases [35–38]. Thereby, it is reasonable that coumarin backbone was incorporated to naphthalimide triazole with the aim of surveying its effect on antimicrobial activities. In view of the above consideration, a novel series of benzimidazole derived naphthalimide triazoles were designed and prepared from commercially available 6-bromobenzo [de]isochromene-1,3-dione according to the synthetic routes as shown in Schemes 1 and 2. All the new compounds were screened for antibacterial and antifungal activities in vitro. As is known, DNA, a helical polyanion built by the union of two linear polymeric strands that are composed of sugars (deoxyribose) linked by phosphates and is viewed as the informational molecule used in the development and function of almost all the known living organisms because it plays a central role in replication, transcription, and regulation of genes. Drugs or bioactive small molecules could conjugate with DNA in biological tissues, enzymes and receptors, thus exhibiting potent bioactivities in vivo [39–41]. Actively ongoing researches focus on the mechanism of action of drugs, their binding with DNA and explore their functions with enzymes and receptors for the rational design and construction of efficiently novel drugs [42–44]. Therefore, it is important to investigate the pharmacokinetics of the newly synthesized highly active compound by evaluating the interactions with calf thymus DNA.
2 Experimental 2.1
Materials and measurements
Melting points were recorded on X–6 melting point apparatus and uncorrected. TLC analysis was done using pre-
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coated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer (Bio-Rad, Cambridge, USA) using KBr pellets in the 400–4000 cm1 range. 1H NMR and 13C NMR spectra were recorded on a AVANCE III 600 spectrometer using TMS as an internal standard. The following abbreviations were used to designate aryl groups: NAPH=naphthalimide, Bim=benzimidazolyl, Tri=triazole.
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The chemical shifts were reported in parts per million (ppm), the coupling constants (J) were expressed in hertz (Hz) and signals were described as singlet (s), doublet (d), triplet (t) as well as multiplet (m). The mass spectra were recorded on LCMS-2010A and HRMS. All chemicals and solvents were commercially available and were used without further purification.
Scheme 1 Synthetic routes of naphthalimide triazoles. Conditions and reagents: (i) aqueous ammonia, 45 °C, 8 h; (ii) 1,4-dibromobutane, K2CO3, DMF, 40 °C, 12 h; (iii) 1,2,4-triazole, K2CO3, CH3CN, 70 °C; (iv) 1H-benzimidazole-2-thiol, K2CO3, DMF, 100 °C; (v) alkyl bromides, K2CO3, CH3CN, 70 °C ; (vi) halobenzyl chlorides, K2CO3, CH3CN, 70 °C; (vii) halobenzyl halides, CH3CN, reflux; (viii) alkyl bromides, CH3CN, reflux.
Scheme 2 Synthetic routes of naphthalimide-coumarin triazole 13. Conditions and reagents: (ix) ethyl acetoacetate, oxalic acid, 100 °C; (x) 1,6-dibromohexane, K2CO3, acetone, reflux; (xi) compound 6g, CH3CN, reflux.
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Synthesis
All new compounds were synthesized via multi-step reactions starting with 6-bromobenzo[de]isochromene-1,3-dione and confirmed by IR, 1H NMR, 13C NMR and HRMS spectra (see the Supplementary Information online). 2.3
Biological assays
Minimal inhibitory concentration (MIC, μg/mL) is defined as the lowest concentration of analyte that completely inhibits the growth of bacteria, and is determined by standard two-fold serial dilution method in 96-well microtest plates following the National Committee for Clinical Laboratory Standards (NCCLS) [45,46]. The tested microorganisms were provided by the School of Pharmaceutical Sciences, Southwest University and the College of Pharmacy, Third Military Medical University. Chloromycin, Norfloxacin and Fluconazole, were used as control drugs. DMSO was used as positive control to ensure that the solvent had no effect on bacteria growth. All the bacteria and fungi growth was monitored visually and spectrophotometrically, and the experiments were repeated at least five times. 2.3.1 Antibacterial assays The prepared compounds 4–9 and 13 were evaluated for their antibacterial activities against Gram-positive bacteria like S. aureus (ATCC25923), MRSA (N315), B. subtilis (ATCC6633) and M. luteus and Gram-negative bacteria like B. proteus (ATCC13315), E. coli (JM109), P. aeruginosa and B. typhi. The bacterial suspension was adjusted with sterile saline to a concentration of 1×105 CFU. The tested compounds were dissolved in DMSO to prepare the stock solutions. The tested compounds and reference drugs were prepared in Mueller-Hinton broth (Guangdong huaikai microbial sci. & tech co., Ltd, China) by two-fold serial dilution to obtain the required concentrations. These dilutions were inoculated and incubated at 37 °C for 24 h. To ensure that the solvent had no effect on bacterial growth, a control was set using test medium supplemented with DMSO at the same dilutions as used in the experiment. 2.3.2 Antifungal assays The synthesized compounds were evaluated for their antifungal activity against C. albicans ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cerevisia and A. flavus. A spore suspension in sterile distilled water was prepared from 1-day old culture of the fungi growing on Sabouraud agar (SA) media. The final spore concentration was 1×103–5×103 spore/mL. From the stock solutions of the tested compounds and reference antifungal drug, Fluconazole, dilutions in sterile RPMI 1640 medium (Neuronbc Laboraton Technology CO., Ltd, Beijing, China) were made to obtain eleven desired concentrations of each tested compound. These dilutions were inoculated and incubated at
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35 °C for 24 h. 2.3.3 Cytotoxicity assays CF12 cells were plated in 96-well plates having 100 L of medium and allowed to attach for 24 h, to which 100 L of medium containing an appropriate dilution of the tested compounds was added. Cells and compounds were incubated at 37 °C in 5% CO2 humidified incubator for 2 d. Cell viability was determined by using CCK-8 kit. Absorbance of converted dye was measured in an ELISA plate reader at the wavelength of 450 nm. The cytotoxicity of the compounds was tested thrice and calculated as the reduction percentage of viable cells observed in the drug-free control cell culture.
3 3.1
Results and discussion Chemistry
The benzimidazole derived naphthalimide triazole derivatives 5–9 and 13 were prepared via multi-step reactions starting with 6-bromobenzo[de]isochromene-1,3-dione 1 and the synthetic procedures were outlined in Schemes 1 and 2. The commercially available compound 1 was treated with aqueous ammonia to give intermediate naphthalimide 2 of 92% yield, and then the later was further reacted with 1,4-dibromobutane in DMF at 40 °C to produce the naphthalimide bromide 3 with a yield of 72%. The N-alkylation of 1,2,4-triazole with compound 3 in acetonitrile using potassium carbonate as base produced naphthalimide triazole 4 with a yield of 74%. The further thio-etherification of naphthalimide triazole 4 with benzimidazole-thiol in DMF afforded the benzimidazole derived naphthalimide triazole 5 in a yield of 73%. Subsequently, the N-alkylation with benzyl or alkyl halides in acetonitrile using potassium carbonate as base produced compounds 6a–6i and 7a–7d with excellent yields, and then the quaternization of compound 6 with excessive benzyl chlorides or alkyl bromides gave the corresponding triazoliums 8a–8h and 9a–9c with high yields ranging from 49% to 75%. Besides, the quaternization of compound 6g with excessive coumarin bromide could afford the corresponding compound 13 in the moderate yield. 3.2
Spectral analysis
All new compounds were confirmed by 1H NMR, 13C NMR, 1 H-1H COSY, IR and HRMS spectra. The analytical data were in accordance with the assigned structures, and the spectral data have been given in the protocol section. 3.2.1 IR spectra In IR spectra, the carbonyl group of cyclic imides in naphthalimide derivatives 5–9 and 13 possessed characteristic stretching frequencies ranging from 1715 to 1654 cm–1,
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while the aromatic frame exhibited absorption between 1616 and 1500 cm–1. In addition, the moderate absorption band at 3210–3009 cm–1 was attributed to the stretching vibration of aromatic CH, while the aliphatic ones showed absorption peaks in the range of 2985–2847 cm–1 were ascribed to the vibration of CH bond of CH2 and CH3 in aliphatic ones. All the other absorption bands were also observed at expected regions. 3.2.2 1H NMR spectra In 1H NMR spectra, compound 6 gave singlets at 5.55–5.38 ppm assigned to the CH2 protons linked with benzimidazole ring, while the N–CH2 protons of alkyl chain in compound 7 gave lower shift signals at 4.30–4.23 ppm. Triazole ring in compounds 4–6 led to downfield shifts of N–CH2 protons up to 4.29–4.28 ppm, which were higher than those of N–CH2 protons attached naphthalimide moiety because of the stronger electron-withdrawing ability of triazole moiety in comparison with naphthalimide moiety. It was also observed that the peaks of protons 4-H, 5,6-H and 7-H in benzimidazole ring in target compounds 59 and 13 appeared at 7.89−7.73, 7.40−7.30 and 7.31−7.28 ppm. It was particularly noticed that naphthalimide triazoles 67 gave two singlets with values of 8.14–8.13 and 7.92–7.90 ppm assigning to the two protons Ha and Hb on triazole ring as shown in Table 1. The further conversion of compound 6 to its corresponding triazoliums 89 and 13 resulted in dramatically downfield shifts for triazole protons Ha (=9.41–9.27 ppm) and Hb (=10.42–10.18 ppm) along with the formation of permanent positive charges with strong electrophilic triazole ring. Additionally, all the other aromatic and aliphatic protons also appeared at the appropriate chemical shifts and Table 1 Some 1H NMR data ( (ppm)) of naphthalimide triazoles and their triazoliums
Compounds 6a 6b 6c 6d 6e 6f 6g 6h 6i 7a 7b 7c 7d
Ha 8.13 8.13 8.13 8.14 8.13 8.14 8.13 8.13 8.14 8.14 8.13 8.12 8.12
Hb 7.92 7.91 7.92 7.92 7.92 7.92 7.92 7.92 7.91 7.90 7.91 7.91 7.91
Compounds 8a 8b 8c 8d 8e 8f 8g 8h 9a 9b 9c 9d 13
Ha 10.34 10.35 10.36 10.41 10.31 10.36 10.32 10.39 10.20 10.18 10.23 10.20 10.42
Hb 9.39 9.38 9.40 9.41 9.36 9.39 9.38 9.41 9.27 9.26 9.29 9.27 9.36
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integral values. 3.2.3 13C NMR spectra The 13C NMR spectra were in accordance with the assigned structures. The signals at 51.9–48.9 ppm in compounds 6, 89 and 13 were assigned to the methylene carbon in halobenzyl groups. Similarly, the carbon signals of N–CH2 group of alkyl chain in compound 7 were resonated at 49.0 ppm. The signals at 163.9–163.1 ppm in compounds 6, 79 and 13 were assigned to the carbons of triazole ring. Furthermore, all the other carbons gave 13C peaks in the expected regions. 3.3
Effect of ClogP values on antimicrobial activity
The lip/water partition of drugs played an important role in their bioactivities by influencing the absorption and transportation of the compounds in biological organisms [47,48]. The estimated lip/water partition coefficients (ClogP) of all prepared compounds by ChemDraw Ultra 10.0 were shown in Table 2. The ClogP values have been widely employed to predict the bioactivity of target molecules which showed that compounds with lower absolute values of ClogP exhibited more efficient antimicrobial activities. Triazoliums 8a–8h showed low ClogP values in contrast to their precurosors 6a–6i which means that these triazoliums had more reasonable lip/water partition, thus possessing more potent antimicrobial efficacy. The high lipophilic compounds containing various alkyl chains had higher absolute values of ClogP showed poor antimicrobial activities. In conclusion, as revealed from the above discussion, the conversion of naphthalimide triazoles into triazoliums remarkably modulated their lip/water partition, thus improving antimicrobial potency of these naphthalimide triazoliums. 3.4
Biological activity
The in vitro antimicrobial screening for all the synthesized compounds was evaluated for 4Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Methicillin-resistant Staphylococcus aureus N315 (MRSA), Micrococcus luteus (ATCC4698) and Bacillus subtilis ATCC 21216), 4 Gramnegative bacteria (Escherichia coli ATCC 8099, Pseudomonas aeruginosa ATCC 27853, Bacillus typhi and Bacillus proteus ATCC 13315) and 5 fungi (Candida albicans ATCC 76615, Candida mycoderma, Candida utilis, Saccharomyces cerevisia and Aspergillus flavus) using two fold serial dilution technique as recommended by National Committee for Clinical Laboratory Standards (NCCLS) with the positive control of clinically antimicrobial drugs Chloromycin, Norfloxacin and Fluconazole [45,46]. The tested microbial strains were provided by the School of Pharmaceutical Sciences, Southwest University and the College of Pharmacy, Third Military Medical University. Minimal inhibitory concentration (MIC, g/mL) is defined
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as the lowest concentration of the analyte that completely inhibited the growth of microbial strains, and determined by the means of standard two-fold serial dilution method in 96-well microtest plates taking Chloromycin, Norfloxacin and Fluconazole as reference drugs. To ensure that the solvent had no effect on bacterial growth, a control test was performed by testing medium supplemented with DMSO at the same dilutions as used in the experiment. The antibacterial and antifungal data were depicted in Tables 2 and 3. 3.4.1 Antibacterial activity The antibacterial results in Table 2 indicated that all newly prepared compounds were effective growth inhibitors for both Gram-positive and Gram-negative bacteria with the exception of intermidates 4 and 5 that were insensitive to most of the tested bacterial stains. The N-alkylation of compound 5 with halobenzyl halides, Table 2
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which yielded compounds 6a–6i, resulted in relatively improved activities in inhibiting the growth of the tested strains. Particularly, naphthalimide 6e containing 4-chloro gave good anti-B. subtilis activity with an MIC value of 4 g/mL, which was about 8-fold more active than the reference drug Chloromycin (MIC=29 g/mL), and it also showed equivalent potency against E. coli as Chloromycin at the concentration of 14 g/mL. Additionally, compound 6g bearing 2,4-dichlorobenzyl moiety could effectively inhibit the growth of B. proteus and E. coli with MIC values of 4 and 14 g/mL, respectively. The substitution of halobenzyl groups by alkyl chain was performed to explore their effects on antimicrobial activities. However, alkyl compounds 7a–7d exhibited relatively weaker antibacterial activity than halobenzyl ones in compounds 6a–6i. This phenomenon might manifest that the aliphatic chains were un-
ClogP values and antibacterial data as MIC (g/mL) for compounds 49, 13 a)
Compounds
ClogP b)
4 5 6a 6b 6c 6d 6e 6f 6g 6h 6i 7a 7b 7c 7d 8a 8b 8c 8d 8e 8f 8g 8h 9a 9b 9c 9d 13 Chloromycin Norfloxacin
3.23 4.68 6.61 6.61 6.61 7.17 7.17 7.17 7.89 7.77 6.21 7.34 8.40 10.51 NE d) 5.93 3.61 6.05 5.46 4.89 4.89 5.46 6.17 5.92 7.69 NE d) NE d) NE d) –1.09 0.58
S. aureus 512±0 230±57.2 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 230±57.2 230±57.2 512±0 14±3.5 512±0 512±0 57±14.3 57±14.3 2±0.9 19±7.1 57±14.3 2±0.9 57±14.3 230±57.2 57±14.3 7±1.8 2±0.9
Gram-positive bacteria c) MRSA B. subtilis 230±57.2 512±0 512±0 115±28.6 512±0 230±57.2 512±0 115±28.6 512±0 512±0 512±0 512±0 512±0 4±0.9 512±0 512±0 115±28.6 512±0 512±0 115±28.6 512±0 512±0 512±0 230±57.2 512±0 512±0 512±0 230±57.2 512±0 230±57.2 512±0 512±0 14±3.5 14±3.5 230±57.2 512±0 115±28.6 512±0 230±57.2 57±14.3 115±28.6 205±70.1 14±3.5 4±0.9 230±57.2 7±1.8 57±14.3 115±28.6 29±7.1 4±0.9 57±14.3 19±7.1 512±0 512±0 115±28.6 115±28.6 57±14.3 29±7.1 1±0.4 1±0.4
M. luteus 230±57.2 512±0 512±0 512±0 512±0 512±0 512±0 115±28.6 512±0 512±0 512±0 512±0 205±70.1 230±57.2 205±70.1 512±0 29±7.1 512±0 512±0 115±28.6 115±28.6 14±3.5 57±14.3 115±28.6 29±7.1 115±28.6 512±0 57±14.3 57±14.3 2±0.9
B. proteus 512±0 205±70.1 512±0 230±57.2 115±28.6 512±0 512±0 512±0 4±0.9 512±0 14±3.5 512±0 512±0 230±57.2 230±57.2 14±3.5 19±7.1 512±0 512±0 512±0 512±0 7±1.8 230±57.2 205±70.1 19±7.1 57±14.3 512±0 29±7.1 29±7.1 1±0.4
Gram-negative bacteria c) E. coli P. aeruginosa 205±70.1 512±0 512±0 230±57.2 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 14±3.5 512±0 512±0 512±0 14±3.5 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 29±7.1 14±3.5 512±0 512±0 512±0 512±0 512±0 230±57.2 512±0 512±0 7±1.8 14±3.5 115±28.6 205±70.1 115±28.6 57±14.3 29±7.1 19±7.1 57±14.3 230±57.2 230±57.2 512±0 115±28.6 115±28.6 14±3.5 19±7.1 4±0.9 1±0.4
B. typhi 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 230±57.2 205±70.1 512±0 512±0 512±0 512±0 205±70.1 205±70.1 9±3.5 230±57.2 205±70.1 19±7.1 115±28.6 512±0 115±28.6 29±7.1 1±0.4
a) Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates; b) ClogP values were calculated by ChemDraw Ultra 10.0; c) S. aureus, Staphylococcus aureus (ATCC25923); MRSA, Methicillin-Resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus (ATCC4698); B. proteus, Bacillus proteus (ATCC13315); E. coli, Escherichia coli (JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; d) NE=no experimental data. It is difficult to obtain the ClogP data of compounds 7d, 9c9d and 13 due to their quite low water solubility.
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favorable for the antibacterial activity. As observed in Table 2, the transformation of benzimidazole derived naphalimide triazoles into their triazoliums 8a–8h increased biological activities. As was expected, triazolium 8b with 4-fluorobenzyl moiety exhibited potent activities and broad antibacterial spectrum against the tested strains with MIC values ranging from 14 to 29 g/mL except for B. typhi. The replacement of 4-fluorobenzyl group by 2,4-dichlorobenzyl fragment to afford compound 8h that gave effectively inhibitory activity against B. subtilis at 7 g/mL. Noticeably, 2-chlorobenzyl triazolium 8g exhibited the best activity (MIC=214 g/mL) among all the prepared compounds, especially against S. aureus with inhibitory concentration of 2 g/mL which was equipotent to Norfloxacin (MIC=2 g/mL) and more efficient than Chloromycin (MIC=7 g/mL). Moreover, the quaternization of naphthalimide triazole 6g by alkyl bromides, which yielded their corresponding triazoliums 9a–9d, also displayed considerable activity against all the tested bacterial strains. Notably, compound 9b with octyl group exhibited broad spectrum and good antibacterial efficacies with MICs between 2 and 29 g/mL, and its anti-S. aureus and anti-B. subtilis Table 3
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abilities (MIC=2 and 4 g/mL, respectively) were better than Chloromycin (MIC=7 and 29 g/mL, respectively). Introduction of coumarin ring has been found to improve antimicrobial potency [23,35]. In our work, it was also observed that the coumarin compound 13 remarkbly improved antimicrobial activities against some of the tested strains with MIC values ranging from 29 to 115 g/mL in comparison with its precursor 5. Consequently, this series of benzimidazole derived naphthalimide triazoliums needed further investigation as potential antibacterial drugs. Further study is necessary to elucidate their mechanisms and structure-activity relationships. 3.4.2 Antifungal activity The antifungal results in Table 3 showed the unsubstituted benzimidazole intermidate 5 gave weak activity towards all the tested fungi strains. However, further transformation of compound 5 with 3-chlorobenzyl chloride, which yielded compound 6d, resulted in good activity (7 g/mL) in inhibiting S. cerevisiae. Moreover, the 3,4-dichlorobenzyl compound 6g, the analogue of compound 6d, also gave excellent anti-C. albicans activity with MIC value of 7 μg/mL,
Antifungal data as MIC (μg/mL) for compounds 49, 13 a)
Compounds 4 5 6a 6b 6c 6d 6e 6f 6g 6h 6i 7a 7b 7c 7d 8a 8b 8c 8d 8e 8f 8g 8h 9a 9b 9c 9d 13 Fluconazole
C. albicans 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 7±1.8 512±0 29±7.1 115±28.6 115±28.6 512±0 230±57.2 512±0 19±7.1 512±0 512±0 115±28.6 4±0.9 14±3.5 29±7.1 115±28.6 512±0 115±28.6 205±70.1 205±70.1 29±7.1
C. mycoderma 230±57.2 512±0 230±57.2 512±0 230±57.2 512±0 14±3.5 512±0 512±0 512±0 512±0 230±57.2 230±57.2 512±0 205±70.1 512±0 19±7.1 230±57.2 115±28.6 29±7.1 4±0.9 9±3.5 7±1.8 57±14.3 512±0 57±14.3 512±0 29±7.1 29±7.1
C. utilis 230±57.2 512±0 512±0 115±28.6 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 205±70.1 512±0 512±0 512±0 3.6±0.9 512±0 512±0 115±28.6 14±3.5 19±7.1 29±7.1 205±70.1 512±0 57±14.3 512±0 230±57.2 7±1.8
S. cerevisiae 512±0 512±0 512±0 512±0 512±0 7±1.8 512±0 512±0 512±0 57±14.3 14±3.5 512±0 512±0 205±70.1 230±57.2 512±0 512±0 512±0 512±0 115±28.6 4±0.9 2±0.9 19±7.1 230±57.2 512±0 29±7.1 512±0 115±28.6 14±3.5
A. flavus 205±70.1 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 512±0 19±7.1 57±14.3 230±57.2 115±28.6 4±0.9 4±0.9 9±3.5 512±0 512±0 57±14.3 512±0 115±28.6 230±57.2
a) C. albicans, Candida albicans (ATCC76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisia, Saccharomyces cerevisia; A. flavus, Aspergillus flavus.
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which was 4-fold more active than the reference drug Fluconazole (MIC=29 g/mL). Additionally, compound 6i containing 4-nitrobenzyl group showed equivalent potency against S. cerevisiae and C. albicans as Fluconazole at the concentration of 14 and 29 g/mL, respectively. In comparison with halobenzyl benzimidazole compounds 6a–6i, the length of aliphatic chain in compounds 7a–7d exhibited little effects on the improvement of antifungal activity. The target triazoliums 8a–8h exerted higher activity than their precursors, which might be attributed to the improved water solubility. 2,4-Dichlorobenzyl triazolium 8h (MIC=7–29 μg/mL) showed more efficient activity than Fluconazole (MIC=7–230 μg/mL). Moreover, bis(4-fluorobenzyl) triazolium 8b (MIC=419 μg/mL) also exhibited good activities against the tested strains except for S. cerevisiae. Noticeably, target triazoliums 8f and 8g bearing 3-fluorobenzyl and 2-chlorobenzyl groups displayed the best activities (MIC=219 g/mL) against all the tested fungal strains. In addition, the target triazoliums 9a–9d exhibited obvious effects on antifungal activity and the suitable length of alkyl chain with (CH2)11 substituent exerted the best antifungal efficacy. Additionally, the target compound 13 with coumarin moiety was investigated that showed moderate antifungal activities with MIC value of 29230 μg/mL. Thus, these naphthalimide triazoliums could be employed for further researches as potential antifungal agents. 3.5
Cell toxicity
The highly bioactive benzimidazoles 8f and 8g were further examined for their cytotoxic properties on FC12 cell line by means of CKK-8 Kit, where the absorbance of viable cells (450 nm) increased linearly as cells proliferate. Compounds 8f and 8g were dissolved in DMSO to prepare the stock solutions, and then the tested compounds were prepared in medium to obtain the required concentrations of 256, 128, 64, 32, 16 and 8 g/mL. Cells were cultured for 24 h in these solutions. The cell viability was examined by microplate reader. Cytotoxicity results (Figure 3) showed that the cell viability of the tested compounds was at least more than 81% within concentration of 128 g/mL, which indicated the tested compounds did not affect the viability of FC12 cell line within the concentration of 128 g/mL. Moreover, with the increasing concentrations of the tested compounds, there was absence of obvious reducing trends in the cell viability. 3.6
Interaction with calf thymus DNA
DNA is an informational molecule encoding the genetic information used in the development and function of almost all the known living organisms, which make it one of the targets for the studies of biologically important small mole-
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Figure 3 Cytotoxic assay of target compounds 8f and 8g on FC12 tested by CCK-8 Kit. Each data bar represents an average of three parallels, and error bars indicate one standard deviation from the mean.
cules such as antimicrobial drugs. Recently, DNA as a therapeutic target is increasingly investigated for interaction with small molecules in the rational design and construction of new and efficient drugs. To explore the possible antimicrobial action mechanism, calf thymus DNA was selected as DNA model for its medical importance, low cost and ready availability properties to study the binding behavior of compound 8g with DNA on molecular level in vitro using neutral red (NR) dye as a spectral probe by UV-Vis spectroscopic methods. 3.6.1 Absorption spectra of DNA in the presence of compound 8g Absorption spectroscopy is one of the most useful techniques used in DNA-binding studies. Hypochromism and hyperchromism are very important spectral features to distinguish the change of DNA double-helical structure in absorption spectroscopy. Due to the strong interaction between the electronic states of intercalating chromophore and that of the DNA base, the observed large hypochromism strongly displayed a close proximity of the aromatic chromophore to the DNA bases. With a fixed concentration of DNA, UV-Vis absorption spectra were recorded with the increasing amount of compound 8g. As shown in Figure 4, UV-Vis spectra suggested that the maximum absorption peak of DNA at 260 nm exhibited proportional increase and red shift with the increasing concentration of compound 8g. Meanwhile the absorption value of simply sum of free DNA and free compound 8g was a little greater than the measured value of 8g-DNA complex. This meant that a strong hypochromic effect existed between DNA and compound 8g. Furthermore, the intercalation of the aromatic chromophore of compound 8g into the helix and the strong overlap of -* states in the large -conjugated system with the electronic states of DNA bases were consistent with the observed spectral changes.
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Figure 5
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The plot of A0/(AA0) versus 1/[compound 8g].
Figure 4 UV absorption spectra of DNA with different concentrations of compound 8g (pH 7.4, T=290 K). Inset: comparison of absorption at 260 nm between the 8g-DNA complex and the sum values of free DNA and free compound 8g. c(DNA)=5.95×105 mol/L, and c(compound 8g)= 0–2.00×105 mol/L for curves (a–i) respectively at increment 0.25×105.
On the basis of the variations in the absorption spectra of DNA upon binding to 8g, Eq. (1) can be utilized to calculate the binding constant (K):
C C A0 1 0 D C C D C C K [Q] A A
(1)
where A0 and A represent the absorbance of DNA in the absence and presence of compound 8g at 260 nm, C and
D C are the absorption coefficients of compound 8g and 8g-DNA complex respectively. The plot of A0/(AA0) versus 1/[compound 8g] is constructed by using the absorption titration data and linear fitting (Figure 5), yielding the binding constant, K=2.16×104 L/mol, R=0.9993, SD=0.0047 (R is the correlation coefficient; SD is standard deviation).
3.6.2 Absorption spectra of NR interaction with DNA To further understand the interaction between compound 8g and DNA, the absorption spectra of competitive interaction of compound 8g were investigated. Neutral Red (NR) is a planar phenazine dye which is structurally similar to other planar dyes with lower toxicity, higher stability and more convenient application in comparison with other common probes. Furthermore, the binding of NR with DNA is an intercalative mode by spectrophotometric and electrochemical techniques has been sufficiently demonstrated in recent years [49]. Therefore, NR was employed as a spectral probe to investigate the binding mode of 8g with DNA in the present work. The absorption spectra of the NR dye upon the addition of DNA are showed in Figure 6. Apparently, the absorption peak of the NR at around 460 nm showed gradual decrease with the increasing concentration of DNA, and a
Figure 6 UV absorption spectra of NR in the presence of DNA at pH 7.4 and room temperature. c(NR)=2×105 mol/L, and c(DNA)=0–3.81×105 mol/L for curves (a–i) respectively at increment 0.48×105.
new band at around 530 nm developed. This was attributed to the formation of the new DNA-NR complex. An isosbestic point at 504 nm provided evidence of DNA-NR complex formation. 3.6.3 Absorption spectra of competitive interaction of compound 8g and NR with DNA Figure 7 displayed the absorption spectra of the competitive binding between NR and 8g with DNA. As shown, with the gradually increasing concentration of 8g, an apparent maximum absorption around 530 nm of the DNA-NR complex decreased, but a slight intensity increase was observed in the developing band around 460 nm. Compared with the absorption band at around 460 nm of NR-DNA complex (Figure 6), the absorbance at the same wavelength in the inset of Figure 7 exhibited the reverse process. The results suggested that compound 8g intercalated into the double helix of DNA by substituting for NR in the DNA-NR complex.
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Figure 7 UV absorption spectra of the competitive reaction between 8g and neutral red with DNA. c(DNA)=4.17×105 mol/L, c(NR)=2×105 mol/L, and c(compound 8g)=0–2.5×105 mol/L for curves (a–i) respectively at increment 0.25×105. Inset: absorption spectra of the system with the increasing concentration of 8g in the wavelength range of 350–600 nm absorption spectra of competitive reaction between compound 8g and NR with DNA.
3.6.4 Fluorescence quenching spectra of compound 8g with DNA Investigation of the binding characteristics of compound 8g with DNA, was done by fluorescence measurement. With a fixed concentration of compound 8g, fluorescence spectra were recorded with increasing amount of DNA. The fluorescence intensity of the peak around 504 nm decreased gradually with increasing concentrations of DNA. Figure 8 meant that the binding of compound 8g with DNA helix was found to strongly quench the fluorescence of compound 8g. From the experimental results, it was concluded that compound 8g could intercalate into the DNA helix to form 8g-DNA complex, and thereby decreased its fluorescence intensity to come true fluorescence quench. Furthermore, gel electrophoresis was also employed to investigate the interactions between the prepared highly active compound 8g and calf thymus DNA following standard protocol [50]. The results (Figure 9) suggested that it was difficult to cleave DNA with compound 8g, which indicated that DNA cleavage was not the main reason to exert antimicrobial activity. This further revealed the binding of 8g and DNA with an intercalative mode.
4 Conclusions In summary, a series of benzimidazole derived naphthalimide triazoles and the corresponding triazoliums have been successfully prepared by convenient and efficient procedures starting from commercial 6-bromobenzo[de] isochromene-1,3-dione. All the new compounds were confirmed by NMR, IR and HRMS spectra. The in vitro antimicrobial evaluation manifested that some naphthalimide
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Figure 8 Fluorescence spectra of compound 8g in the presence and absence of DNA at different concentrations (pH 7.4, T=310 K). c(compound 8g)=4×105 mol/L, and c(DNA)=0–7.0×105 mol/L for curves (a–h) at increment 1×105. Red line shows the emission spectrum of compound 8g only.
Figure 9 Gel electrophoresis of DNA in the presence of increasing amounts of 8g. ri=[8g]/[DNA]=0.0, 0.1, 0.2, 0.3, 0.4, 0.5.
triazoliums exhibited better antimicrobial efficiency than their precursor triazoles, which might be attributed to the improved water solubility, especially in case of triazolium 8g bearing 2-chlorobenzyl fragment that exhibited superior bioactivities to other analogs with MIC values ranging from 2 to 19 μg/mL and no obvious toxicity against PC12 cell line within concentration of 128 µg/mL. The interactive investigations by fluorescence and UV-Vis spectroscopic methods revealed that compound 8g could effectively intercalate into calf thymus DNA to form 8g-DNA complex which might further block DNA replication, and thus exerted their powerfully antimicrobial activities. These results manifested that the prepared benzimidazole modified naphthalimide triazole derivatives had broad antimicrobial spectrum, and they not only exerted good antibacterial activity but also gave antifungal activity. Conclusively, the new type of benzimidazole derived naphthalimide triazoles could be exploited further as potential intriguing antimicrobial agents. Supporting information The supporting information is available online at chem.scichina.com and link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.
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This work was partially supported by the National Natural Science Foundation of China (21172181, 21372186, 81450110094), the key program from Natural Science Foundation of Chongqing (CSTC2012jjB10026), the Specialized Research Fund for the Doctoral Program of Higher Education of China (SRFDP 20110182110007), the Doctoral Fund of Southwest University (SWU111075), the Research Funds for the Central Universities (XDJK2013C112) and Chongqing Special Foundation for Postdoctoral Research Proposal (Xm201450). 1 2
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