Synthesis of tetrazole compounds as a novel type of potential antimicrobial agents and their synergistic effects with clinical drugs and interactions with calf thymus DNA

Ling-Ling Dai; Hui-Zhen Zhang; Sangaraiah Nagarajan; Syed Rasheed; Cheng-He Zhou

doi:10.1039/C4MD00266K

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DOI: 10.1039/C4MD00266K (Concise Article) Med. Chem. Commun., 2015, 6, 147-154

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Synthesis of tetrazole compounds as a novel type of potential antimicrobial agents and their synergistic effects with clinical drugs and interactions with calf thymus DNA†

Ling-Ling Dai , Hui-Zhen Zhang , Sangaraiah Nagarajan‡ , Syed Rasheed§ and Cheng-He Zhou *
Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China. E-mail: zhouch@swu.edu.cn; Fax: +86-23-68254967; Tel: +86-23-68254967

Received 21st June 2014 , Accepted 22nd September 2014

First published on 22nd September 2014

Abstract

A series of tetrazole derivatives were synthesized and characterized by NMR, IR, MS and HRMS spectroscopy. The bioactive assay manifested that most of the target compounds exhibited good antifungal activity, especially compound 6g displayed comparable or even stronger antifungal efficiency in comparison with the reference drug Fluconazole. The combination of tetrazole derivative 6g with antibacterials Chloromycin and Norfloxacin, or antifungal Fluconazole respectively was more sensitive to methicillin-resistant MRSA and Fluconazole-insensitive Aspergillus flavus. Further research revealed that compound 6g could effectively intercalate into Calf Thymus DNA to form a 6g–DNA complex which might block DNA replication to exert its good antimicrobial activities.

1. Introduction

Microbial infections have become alarming recently due to the increasing emergence of multidrug-resistant strains, intractable pathogenic microorganisms and newly arising pathogens.^1–4 Various synthetic and semi-synthetic antimicrobial agents have been discovered and extensively used in the clinic to treat various community and hospital acquired microbial infections. Especially, the FDA approved drug auranofin exerts an incredible effect on Gram-positive and multidrug-resistant bacteria and has attracted much attention recently.^5,6 However, there are still some unresolved problems such as narrow antimicrobial spectrum, adverse effects, high toxicity and so on for clinical drugs. The combination therapy with two or more agents, which could usually overcome multi-drug resistance, is one of the important strategies to improve the efficiency and bioavailability as well as treat mixed diseases in the clinic.^7,8 However, the discovery and development of structurally novel antimicrobial agents with good pharmacological profiles and excellent activity towards resistant strains are highly desirable.^9–11

Tetrazole is an important five-membered aromatic heterocyclic compound with poly-nitrogen electron-rich planar structural features. This unique structure allows tetrazole derivatives to readily bind with various enzymes or receptors in organisms via weak interactions such as coordination bonds, hydrogen bonds, cation–π, π–π stacking, hydrophobic effect, van der Waals force and so on, thus displaying a broad spectrum of biological activities and a considerable role in the pharmaceutical field.^12,13 The tetrazole ring can be used as an attractive linker to combine or stabilize different pharmacophore fragments to generate special functionalized molecules.^14,15 The tetrazole ring is also an isostere of carboxyl,^16,17 amide¹⁸ and some heterocycles (triazole,¹⁹ benzotriazole,²⁰ imidazole,²¹ benzimidazole,²² carbazole,²³etc.) in designing various new types of drug molecules. So far many tetrazole-based derivatives have been successfully developed and prevalently used as clinical drugs such as antihypertensives Lorsartan²⁴ and Valsartan,²⁵ antibiotics Flomoxef²⁶ and Cefonicid,²⁷ and antinociceptive Alfentanil²⁸ to treat various diseases. Some literature has revealed that tetrazole derivatives could effectively prevent the biosynthesis of microbial proteins^29,30 to inhibit the growth of various microorganisms in recent years, which suggests the large potentiality of tetrazole compounds as a new type of antimicrobial agents.

Fluconazole is one of the most important triazole-based compounds recommended as the first-line antifungal drug by the World Health Organization (WHO). It has been prevalently employed to treat fungal infection by Candida albicans, Cryptococcus neoformans, Dermatitis blastomycosis, etc. due to its potent activity, excellent safety profile, and favorable pharmacokinetic characteristics.^31,32 The triazole ring of Fluconazole could efficiently coordinate with the iron(II) ion of heme to inhibit the biosynthesis of ergosterol and thus inhibits the growth of fungi. However, the emergence of fluconazole-resistant Candida albicans isolates, increasingly serious drug resistance, narrow antifungal spectrum, and low activity against invasive mycoses have attracted great efforts towards modifying the side chain of Fluconazole or exploiting its new analogues.³³ In our previous work,^34–36 it has been demonstrated that the tertiary amine type of Fluconazole analogues displayed large potentiality as a new type of antimicrobial agents in which the tertiary alcohol moiety in Fluconazole was replaced by a tertiary amine fragment that may exert the same function with the active site residue H310 as the tertiary alcohol moiety in Fluconazole. However, to the best of our knowledge, so far the combination of a tetrazole ring with a tertiary amine fragment has not been reported.

In view of the above considerations and as an extension of our continuous work, we have great interest in investigating tetrazole tertiary amines as a novel type of potential antimicrobial agents. Herein, we would like to report the synthesis of tetrazole compounds 4a–d and 6a–l with different lengths of alkyl chains or halogen-substituted aralkyl moieties. All the new compounds were screened for their antibacterial and antifungal activities in vitro, and the synergistic effects of the most active tetrazole compound with clinical drugs were also evaluated in vitro. The ionization constants (pK_a) and lg P of title compounds were determined by the UV-vis absorption spectroscopic method to evaluate the antimicrobial activity. In this work, with the consideration to explore the preliminary mechanism of action, the interaction of the most active compound with calf thymus DNA was also investigated.

2. Chemistry

The target tetrazole compounds 4a–d and 6a–l were prepared via multi-step reactions and the synthetic process was outlined in Scheme 1. Commercially available substituted anilines were treated by chloroacetonitrile in acetonitrile to give N-phenyl glycinonitriles 2a–c in 50–65% yields. The N-alkylation of compounds 2a–c with a series of alkyl bromides in refluxing ethanol using potassium carbonate as the base respectively afforded the intermediates 3a–d in yields of 50–55%, which showed that the length of the aliphatic chain in alkyl bromides exerted a slight effect on the formation of tertiary amines. Halobenzyl halides and intermediates 2a–c underwent N-alkylation to produce compounds 5a–l in yields of 65–70%. Compounds 4a–d and 6a–l were easily synthesized in high yields ranging from 75% to 80% by the cycloaddition of compounds 3a–d and 5a–l respectively with sodium azide using ammonium chloride as the catalyst in DMF at 120 °C. The total yields of target compounds were ranging from 18% to 36%. Moreover, in the procedure for the preparation of N-phenyl glycinonitriles, it was found that no anticipated products were observed when the aniline ring was substituted by electron-withdrawing groups such as fluoro, chloro, nitro and so on. This phenomenon might be attributed to the electron-withdrawing properties of these groups which made the electron density of the nitrogen atom in the aniline ring low and made it difficult to perform the nucleophilic substitution. All the new compounds were confirmed by IR,¹H NMR, ¹³C NMR, MS and HRMS spectroscopy (See the ESI 1†).


	Scheme 1 Reagents and conditions: (i) chloroacetonitrile, potassium carbonate, CH₃CN, 80 °C; (ii) substituted halobenzyl halide, potassium carbonate, CH₃CH₂OH, reflux; (iii) sodium azide, ammonium chloride, DMF, 120 °C; (iv) alkyl bromide, potassium carbonate, CH₃CH₂OH, reflux.

3. Results and discussion

3.1 Antimicrobial activities

The results of antibacterial activity (ESI 2†) in Table 1 showed that intermediates 3a–d and 5a–l exhibited weak or no antibacterial efficacy against all the tested bacterial strains. In addition, some of the halobenzyl tertiary amines among 5a–l exerted relatively better activities in inhibiting the growth of tested strains in comparison with alkyl tertiary amines 3a–d. Moreover, the bioactivity of target compounds 4a–d and 6a–l were improved by the introduction of a tetrazole ring into substituted benzyl tertiary amines, which indicated that the tetrazole ring was helpful for enhancing antibacterial activity. The types of substituents on the aniline ring exhibited no significant effects on biological activity. Moreover, substituents on benzyl moieties displayed notable effects on antibacterial activity, especially 6g (MIC = 16–32 μg mL⁻¹) with the chlorine atom on the 2-position of the benzyl ring gave better bioactivity against all the tested bacterial strains than others.

Table 1 Antimicrobial activities in vitro for the prepared compounds expressed as MIC (μg mL⁻¹)^a^,^b^,^c

Compds	Gram-positive bacteria				Gram-negative bacteria				Fungi
Compds	S. A	MRSA	B. S	M. L	B. P	E. C	P. A	S. D	C. A	C. M	C. U	A. F
a Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. b MRSA, Methicillinresistant Staphylococcus aureus N315; S. A, Staphylococcus aureus ATCC25923; B. S, Bacillus subtilis ATCC6633; M. L, Micrococcus luteus ATCC 4698; E. C, Escherichia coli DH52; S. D, Shigella dysenteriae ATCC51252; P. A, Pseudomonas aeruginosa ATCC27853; B. P, Bacillus proteus ATCC13315; C. A, Candida albicans ATCC90029; C. M, Candida mycoderma; C. U, Candida utilis ATCC9950; A. F, Aspergillus flavus. c A = Chloromycin; B = Norfloxacin; C = Fluconazole.
3a	256	512	256	256	256	128	512	512	256	256	256	128
3b	256	256	256	256	256	128	512	512	256	256	64	256
3c	128	128	64	128	128	64	256	512	128	256	128	128
3d	256	512	256	128	512	256	512	512	256	256	256	128
4a	128	256	128	128	128	128	128	128	64	64	64	64
4b	128	128	128	64	128	64	256	128	64	128	32	128
4c	64	32	32	32	64	32	128	64	32	16	32	32
4d	128	128	64	128	256	128	256	256	128	64	64	32
5a	256	256	128	256	256	128	256	256	128	128	64	256
5b	256	256	128	256	256	256	256	128	64	256	128	256
5c	128	128	256	256	512	128	128	512	128	128	128	128
5d	64	128	128	256	128	256	256	128	64	128	128	256
5e	128	256	256	256	256	64	128	256	128	512	256	256
5f	256	128	128	512	256	128	256	128	64	128	128	128
5g	128	128	256	256	128	256	128	128	128	64	64	128
5h	128	256	128	128	128	128	64	128	128	128	128	256
5i	128	128	512	512	512	256	256	256	64	64	512	128
5j	128	256	256	128	128	128	256	128	64	128	128	64
5k	256	256	128	128	256	256	128	256	128	128	256	128
5l	128	128	256	256	128	256	256	128	64	128	64	128
6a	32	64	32	64	32	16	32	64	16	8	8	16
6b	128	128	64	128	128	64	64	64	64	32	16	32
6c	64	64	32	64	64	64	64	128	32	32	16	64
6d	64	64	32	64	64	32	64	32	32	16	32	32
6e	64	32	64	64	64	64	32	64	64	16	16	32
6f	64	64	64	256	128	64	64	64	32	16	16	32
6g	32	32	16	32	16	16	16	32	8	4	4	8
6h	64	128	32	64	32	32	64	64	16	32	16	32
6i	32	16	64	32	32	16	32	32	16	16	8	16
6j	64	64	64	64	64	64	32	64	32	32	16	32
6k	64	64	64	64	64	64	64	64	32	16	16	32
6l	32	64	32	32	32	32	32	32	16	8	8	16
A	8	16	32	8	32	16	16	32	—	—	—	—
B	4	1	2	1	4	4	1	2	—	—	—	—
C	—	—	—	—	—	—	—	—	1	4	8	256

The antifungal evaluation in vitro revealed that the activities of most of the compounds were relatively better in comparison to their antibacterial activities. The halobenzyl substituted target compounds 6a–l and alkyl substituted target compounds 4a–d exhibited moderate to excellent antifungal activities against the tested strains. Especially the target 2-chlorobenzyl compound 6g displayed comparable or even a stronger antifungal efficiency (MIC = 4–8 μg mL⁻¹) against the tested fungi in comparison with the reference drug Fluconazole (MIC = 1–256 mg mL⁻¹). The length of the aliphatic chain also exhibited obvious effects on antifungal activity. The suitable length of the alkyl chain to exert the best antifungal efficacy was observed to be (CH₂)₉, and decyl tetrazole 4c gave generally better activity in comparison with other alkyl tetrazole compounds with shorter or longer chain length.

In addition, the combinations of tetrazole compound 6g with clinical antibacterials Chloromycin and Norfloxacin, or antifungal Fluconazole respectively against the tested bacteria and fungi were investigated. To our excitement, the tested results showed excellent antimicrobial efficacies with less dosage and a broad antimicrobial spectrum as described in Tables 2 and 3. The FIC index was less than 0.8 which suggested that the combinations of compound 6g with clinical drugs have good synergistic effects. As shown in Table 2, the combinations of compound 6g with Chloromycin (4 μg mL⁻¹) or Norfloxacin (0.5 μg mL⁻¹) were four- or two-fold more potent than themselves alone against MRSA. Interestingly, the combination of compound 6g with Fluconazole also displayed good activity against all the tested fungi with low MIC values of 2–4 μg mL⁻¹ as described in Table 3. Especially, this combination showed excellent activity against Fluconazole-insensitive Aspergillus flavus in comparison with Fluconazole alone (MIC = 256 μg mL⁻¹). These results manifested that the combinations of tetrazoles with clinical drugs could efficiently enhance antimicrobial activity, overcome drug resistance and broaden the antimicrobial spectrum. The advantages of combinations might be attributed to the different binding sites of these compounds towards the tested microorganism.

Table 2 Synergistic effects of compound 6g with antibacterial Chloromycin and Norfloxacin^a^,^b^,^c

Bacteria	Compds	MIC	FIC index	Effect	Compds	MIC	FIC index	Effect
a Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. b MRSA, Methicillinresistant Staphylococcus aureus N315; S. A, Staphylococcus aureus ATCC25923; B. S, Bacillus subtilis ATCC6633; M. L, Micrococcus luteus ATCC 4698; E. C, Escherichia coli DH52; S. D, Shigella dysenteriae ATCC51252; P. A, Pseudomonas aeruginosa ATCC27853; B. P, Bacillus proteus ATCC13315; C. A, Candida albicans ATCC90029; C. M, Candida mycoderma; C. U, Candida utilis ATCC9950; A. F, Aspergillus flavus. c A = Chloromycin; B = Norfloxacin; C = Fluconazole.
S. A	A	4	0.750	Synergistic	B	2	0.500	Synergistic
S. A	6g	16	0.750	Synergistic	6g	16	0.500	Synergistic
MRSA	A	4	0.500	Synergistic	B	0.5	0.750	Synergistic
MRSA	6g	8	0.500	Synergistic	6g	8	0.750	Synergistic
B. S	A	4	0.375	Synergistic	B	1	0.625	Synergistic
B. S	6g	16	0.375	Synergistic	6g	8	0.625	Synergistic
M. L	A	2	0.500	Synergistic	B	0.5	0.750	Synergistic
M. L	6g	8	0.500	Synergistic	6g	8	0.750	Synergistic
B. P	A	8	0.750	Synergistic	B	1	0.500	Synergistic
B. P	6g	32	0.750	Synergistic	6g	16	0.500	Synergistic
E. C	A	8	0.750	Synergistic	B	0.5	0.625	Synergistic
E. C	6g	16	0.750	Synergistic	6g	8	0.625	Synergistic
P. A	A	4	0.500	Synergistic	B	0.5	0.750	Synergistic
P. A	6g	16	0.500	Synergistic	6g	16	0.750	Synergistic
S. D	A	8	0.500	Synergistic	B	0.5	0.625	Synergistic
S. D	6g	8	0.500	Synergistic	6g	4	0.625	Synergistic

Table 3 Synergistic effects of compound 6g with antifungal Fluconazole^a^,^b^,^c

Fungi	Compds	MIC (μg mL⁻¹)	FIC index	Effect
a Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. b MRSA, Methicillinresistant Staphylococcus aureus N315; S. A, Staphylococcus aureus ATCC25923; B. S, Bacillus subtilis ATCC6633; M. L, Micrococcus luteus ATCC 4698; E. C, Escherichia coli DH52; S. D, Shigella dysenteriae ATCC51252; P. A, Pseudomonas aeruginosa ATCC27853; B. P, Bacillus proteus ATCC13315; C. A, Candida albicans ATCC90029; C. M, Candida mycoderma; C. U, Candida utilis ATCC9950; A. F, Aspergillus flavus. c A = Chloromycin; B = Norfloxacin; C = Fluconazole.
C. A	C	0.5	0.750	Synergistic
C. A	6g	2	0.750	Synergistic
C. M	C	1	0.500	Synergistic
C. M	6g	2	0.500	Synergistic
C. U	C	2	0.500	Synergistic
C. U	6g	4	0.500	Synergistic
A. F	C	64	0.500	Synergistic
A. F	6g	4	0.500	Synergistic

3.2 Values of ionization constant (pK_a) and lg P

The ionization constant (pK_a) of a molecule is an important physicochemical parameter which has significant influence on its lipophilicity, solubility, protein binding as well as permeability. Therefore the pK_a values are usually used to evaluate the extent of ionization of bioactive molecules at different pH values, which is of fundamental importance in the consideration of their pharmacokinetic behavior such as absorption, distribution, metabolism, and excretion in organisms.^37,38 A compound with a pK_a < 4 or > 10 will be charged at physiological pH and display slower diffusion rate across biological membranes such as the blood–brain barrier. UV spectroscopy is an efficient method for the determination of ionization constants which employs readily available equipment with a fast determination. Structural transposition of title tetrazole compounds would be generated in buffer solutions at different pH values, thus resulting in the changes in UV absorbance. The changes observed in the UV spectrum of compound 6g in different buffer solutions are clearly displayed in Fig. 1. With the pH values ranging from 2.0 to 8.5, the structural formula of compound 6g was changed from I to II (Scheme 2). As shown in Fig. 2, the spectral differences between 283 and 309 nm were chosen to analyze the pK_a of target tetrazole compounds. Data analysis included normalization of the raw scans (Abs₃₃₀ nm = 0), and then the spectral difference between the acid spectra and the spectra obtained at every other pH was calculated. The wavelengths of maximum positive and negative deviations were determined graphically, and the absolute values of the absorbance difference at the chosen wavelengths were summed. The total absorbance difference was then plotted versus pH, and the data were fit to eqn (1)


	(1)

where ε_HA and ε_A− are the extinction coefficients of the acid and base forms of the compound, respectively, and [S_t] is the total compound concentration. When using absorbance differences, ε_HA and ε_A− are simply the minima and maxima of the curve.³⁹ As shown in Table 4, the pK_a values of compounds 4a–d and 6a–l displayed an appropriate range 4.13–4.83, which suggested their good pharmacokinetic behavior as bioactive agents.


	Fig. 1 UV spectra (λ = 260–330 nm) of compound 6g in different aqueous buffer solutions ranging from pH 2.0 to 8.5. The absorbances are normalized to zero for λ = 330 nm.


	Scheme 2 The structural formula of compound 6g was changed from I to II in buffer solutions with the increase of pH values.


	Fig. 2 Plot of the spectral difference between different solutions of compound 6g. The maximum positive deviation occurred at 309 nm, while the maximum negative deviation occurred at 283 nm.

Table 4 pK_a, lg P and c lg P of target compounds 4a–d and 6a–l^a

Compds	pK_a	lg P	c lg P
a NE = No experimental data. It is difficult to obtain the lg P data of compounds 4d due to their quite low water solubility.
4a	4.78	2.18 ± 0.16	4.11
4b	4.83	2.24 ± 0.24	5.17
4c	4.87	4.92 ± 0.18	6.23
4d	4.92	NE	7.28
6a	4.54	1.67 ± 0.19	4.56
6b	4.49	1.63 ± 0.14	4.44
6c	4.22	1.28 ± 0.11	3.28
6d	4.19	1.29 ± 0.13	3.28
6e	4.13	1.36 ± 0.14	3.42
6f	4.20	1.26 ± 0.16	3.28
6g	4.34	1.09 ± 0.11	3.85
6h	4.38	1.39 ± 0.21	3.85
6i	4.26	1.99 ± 0.12	5.06
6j	4.23	1.42 ± 0.13	3.92
6k	4.31	1.37 ± 0.11	3.45
6l	4.29	1.58 ± 0.10	4.59

Lipophilicity/hydrophilicity plays a significant role in determining where drugs are distributed and how rapidly they are metabolized and excreted in the body, for example, hydrophobic drugs are preferentially distributed to hydrophobic compartments such as lipid bilayers of cells while hydrophilic drugs are preferentially found in hydrophilic compartments like blood serum.⁴⁰ Therefore, the lipophilicity/hydrophilicity expressed as lg P was calculated theoretically using ChemDraw Ultra 10.0 software and experimentally by a traditional saturation shake flask approach combined with a UV-vis spectrophotometric method. The obtained results are given in Table 4, the c lg P of compounds 4a–d increased with the increase in the length of the alkyl chain, and an enhancement of the antimicrobial activities was observed in compounds 4a–c, but decreased in that of compounds 4c–d, these might be explained by the possibility that higher lipophilic compounds were unfavourable for being delivered to the binding sites. This phenomenon also indicted that suitable lipophilicity is necessary for good activities in drug design (Fig. 3).


	Fig. 3 Plot of the total absorbance difference vs. pH to determine the pK_a of compound 6g. The total absorbance difference is the sum of the absolute absorbance difference values at the chosen wavelengths from 283 to 309 nm.

3.3 Interaction with calf thymus DNA

3.3.1 UV-vis absorption spectral study. The application of UV-vis absorption measurement is one of the most important techniques in DNA-binding studies. Generally, hypochromism and hyperchromism are important spectral features to distinguish the change of DNA double-helical structure. The hyperchromism originates from the breakage of the DNA duplex secondary structure; while the hypochromism generates from the stabilization of the DNA duplex by either the intercalation binding mode or the electrostatic effect of small molecules.⁴¹ The hypochromism demonstrated a close proximity of the aromatic chromophore to the DNA bases, which might be attributed to the strong interaction between the electronic states of the intercalating chromophore and that of the DNA base. With a fixed concentration of DNA, UV-vis absorption spectra were recorded with the increasing amount of compound 6g. As shown in Fig. 4, UV-vis spectra showed that the maximum absorption of DNA at 260 nm displayed a proportional increase with the increasing concentration of compound 6g. Simultaneously, the absorption value of the sum of free DNA and free compound 6g was a little greater than the measured value of the 6g–DNA complex, which was observed in the inset of Fig. 4. These indicated that a weak hypochromic effect existed between DNA and compound 6g. Moreover, the intercalation of the aromatic chromophore of compound 6g into the helix and the strong overlap of π–π* states in the large π-conjugated system with the electronic states of DNA bases were consistent with the observed spectral changes.^42,43


	Fig. 4 UV absorption spectra of DNA with different concentrations of compound 6g (pH = 7.4, T = 303 K). Inset: comparison of absorption at 260 nm between the 6g–DNA complex and the sum values of free DNA and free compound 6g. c(DNA) = 4.52 × 10⁻⁵ mol L⁻¹, and c(compound 6g) = 0–1.6 × 10⁻⁵ mol L⁻¹ for curves a–i respectively at increment 0.2 × 10⁻⁵.

On the basis of the variations in the absorption spectra of DNA upon binding to tetrazole derivative 6g, eqn (2) can be utilized to calculate the binding constant (K).


	(2)

A ⁰ and A represent the absorbance of DNA in the absence and presence of compound 6g at 260 nm, ξ_C and ξ_D−C are the absorption coefficients of compound 6g and 6g–DNA complex respectively. The plot of A⁰ (A⁻¹−A⁰) versus 1/[compound 6g] is constructed by using the absorption titration data and linear fitting (Fig. 5), yielding the binding constant, K = 4.16 × 10⁴ L mol⁻¹, R = 0.999, and SD = 0.17 (R is the correlation coefficient. SD is standard deviation).


	Fig. 5 The plot of A⁰ (A⁻¹−A⁰) versus 1/[compound 6g].

3.3.2 Absorption spectra of NR interaction with DNA. Neutral Red (NR) is a planar phenazine dye and is structurally similar to other planar dyes like acridine, thiazine and xanthene kind. Recently, it has been demonstrated that the binding of NR with DNA is an intercalation binding.⁴⁴ Therefore, NR was employed as a spectral probe to investigate the binding mode of 6g with DNA in the present work.

The absorption spectra of the NR dye upon the addition of DNA are shown in Fig. 6. It is apparent that the absorption peak of the NR at around 460 nm showed a gradual decrease with the increasing concentration of DNA, and a new band at around 530 nm developed. This was attributed to the formation of the DNA–NR complex. An isosbestic point at 504 nm provided evidence of DNA–NR complex formation.


	Fig. 6 UV absorption spectra of NR in the presence of DNA at pH 7.4 under room temperature. c(NR) = 2 × 10⁻⁵ mol L⁻¹, and c(DNA) = 0–3.61 × 10⁻⁵ mol L⁻¹ for curves a–i respectively at increment 0.45 × 10⁻⁵.

3.3.3 Absorption spectra of competitive interaction of compound 6g and NR with DNA. As shown in Fig. 7, the competitive binding between NR and 6g with DNA was observed in the absorption spectra. With the increasing concentration of compound 6g, an apparent intensity increase was observed around 276 nm. In comparison with the absorption around 276 nm of the NR–DNA complex, the absorbance at the same wavelength exhibited the reverse process (inset of Fig. 5). These various spectral changes were consistent with the intercalation of compound 6g into DNA by substituting for NR in the DNA–NR complex.


	Fig. 7 UV Absorption spectra of the competitive reaction between 6g and neutral red with DNA.

4. Conclusions

In conclusion, a novel type of tetrazole was successfully synthesized for the first time via an easy, convenient and efficient synthetic procedure starting from commercially available substituted anilines. The antimicrobial tests demonstrated that some prepared compounds exhibited comparable or even superior antifungal activity against the tested strains relative to the reference drug Fluconazole. Especially compound 6g was found to be four-fold more potent than Fluconazole against Aspergillus flavus (MIC = 256 μg mL⁻¹). Meanwhile, the combinations of tetrazoles with antibacterials Chloromycin and Norfloxacin or antifungal Fluconazole gave striking enhanced antimicrobial efficiency with less dosage as well as a broader antimicrobial spectrum than their separate use. Notably, the combined system was more sensitive to Fluconazole-insensitive Aspergillus flavus and methicillin-resistant MRSA. The specific interaction of compound 6g with calf thymus DNA, which was studied by UV-vis absorption spectroscopy, manifested that compound 6g could intercalate into DNA to form the 6g–DNA complex which might further block DNA replication to exert its good antibacterial and antifungal activities. All these results should be a promising starting point to optimize the structures of tetrazole-derived tertiary amines to access potent antimicrobial agents.

Acknowledgements

This work was partially supported by the National Natural Science Foundation of China [no. 21172181, 21372186, 81350110523, 81450110095 (The Research Fellowship for International Young Scientists from International (Regional) Cooperation and Exchange Program)], the key program from the Natural Science Foundation of Chongqing (CSTC2012jjB10026), the Specialized Research Fund for the Doctoral Program of Higher Education of China (SRFDP 20110182110007) and Chongqing Special Fund for Postdoctoral Research Proposal (Xm2014127).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4md00266k

‡ Postdoctoral fellow from School of Chemistry, Madurai Kamaraj University, India.

§ Postdoctoral fellow from Department of Chemistry, University of Hyderabad, India.