3-(Hydroxyimino)imidazo[1,2- a ]pyridin-2(3 H )-ylidene)-1-arylethanones as new red heterocyclic dyes: Synthesis,spectral studies,quantum-chemical investigations,and antibacterial activities

Abstract

The synthesis, optical properties, theoretical calculations, and antibacterial activities of a series of new red heterocyclic dyes derived from imidazo[1,2-a]pyridine are presented. 3-(Hydroxyimino)imidazo[1,2-a]pyridin-2(3H)-ylidene)-1-arylethanones are obtained from the reaction of 3-nitroimidazo[1,2-a]pyridine with substituted acetophenone derivatives in good yields (65%–72%). The structures are confirmed by spectral and analytical data, and the optical properties of the dyes are characterized by spectrophotometry. Density functional theory calculations are performed to provide the optimized geometries and relevant frontier orbitals. Calculated electronic absorption spectra are also obtained by the time-dependent density functional theory method. Moreover, the antibacterial activities (minimum inhibitory concentration) of the new dyes against Gram-positive and Gram-negative bacterial species are determined (minimum inhibitory concentration: 5–200 μg mL⁻¹).

Introduction

Heterocyclic dyes have wide applications in the dyestuff industries.^1,2 The use of these dyes in the electronic industry, for example, as colorimetric sensors, nonlinear optical (NLO) devices, and liquid crystalline displays (LCDs), has been investigated, while their use as potential sensitizers for photodynamic therapy (PDT) has attracted much attention. ³ In addition, they have been evaluated and employed as optoelectronic devices, ⁴ photoconductors, ⁵ sensitizers, ⁶ biomedical probes, ⁷ photo/catalysts, ⁸ for solar-energy utilizations, ⁹ and so on. Furthermore, heterocyclic dyes have been widely used in the preparation of disperse dyes with outstanding dischargeability on cellulose acetate. ¹⁰

Imidazo[1,2-a]pyridines, as an important type of bicyclic nitrogen heterocycle, have been used for the production of dyes and some valuable drugs. They exhibit interesting biological properties such as antiviral, ¹¹ anticancer, ¹² anxiolytic, ¹³ antimalarial, ¹⁴ hypnotic, ¹⁵ antiprotozoal, ¹⁶ and anti-inflammatory ¹⁷ activities, which has rendered this ring system an attractive target. Recently, imidazo[1,2-a]pyridine scaffolds became of interest as dye and fluorescent compounds.^18–20 In some case, imidazo[1,2-a]pyridines with suitable functionalization can use as acid–base indicators. ²¹

Based on these aspects and in continuation of our previous studies on the synthesis of new heterocyclic dyes,^18–23 we have synthesized three new heterocyclic dyes in good yields from the reaction of 3-nitroimidazo[1,2-a]pyridine with substituted acetophenone derivatives in a basic medium. The optical properties, density functional theory (DFT)/time-dependent density functional theory (TD-DFT) calculations, and antibacterial activity of the dyes have also been examined.

Results and discussion

Synthesis and structures of the dyes 3a–c

In order to the synthesis new dyes, the precursor 3-nitroimidazo[1,2-a]pyridine (1) was prepared in two steps. Initial reaction of pyridine-2-amine with 2-bromo-1,1-dimethoxyethane led to the formation of imidazo[1,2-a] pyridine. ²⁴ Subsequent nitration of imidazo[1,2-a] pyridine in H₂SO₄ and HNO₃ produced 3-nitroimidazo[1,2-a]pyridine (1) according to a literature method. ²⁵ The new 3-(hydroxyimino)imidazo[1,2-a]pyridin-2(3H)-ylidene)-1-arylethanones (3a–c) dyes were obtained in good yields from the condensation of 3-nitroimidazo[1,2-a]pyridine (1) and different acetophenone derivatives 2a–c in basic MeOH solution via nucleophilic substitution ²⁶ (Scheme 1).

Scheme 1.

Synthesis of new dyes 3a–c.

The structural assignments of compounds 3a–c were based on analytical and spectral data. In the ¹H NMR spectrum of 3a the four signals at 8.87, 9.29, 10.04, and 10.08 ppm assignable to OH protons were attributed to four non-isolable isomers of 3a (Figure 1). As depicted in Scheme 2, the most plausible reaction mechanism to explain the formation of the four isomers of 3a–c involves attack of the anion derived from 2a–c on compound 1 followed by dehydration of A and the formation of intermediate B. Finally, tautomerization of B in basic medium led to the formation of four isomers each of compounds 3a–c. These isomeric relationships were not reported in similar reactions.^18–23

Figure 1.

Structures of the isomers of 3a.

Scheme 2.

A plausible reaction mechanism for the formation of 3a–c.

We made a several attempts to isolate the isomers by classical methods, but they could not be isolated from each other. This implies that the isomers can convert into each other. Recently, we performed DFT calculations by using the B3LYP hybrid functional and the 6-311++G(d,p) basis set to determine the most reasonable mechanism for the formation of 3H-imidazo[4,5-a]acridines. ²⁷ According to these results, conversion of the isomers into each other can be facilitated by their tautomerization into intermediate B. In order to clarify the optimized geometry and energy of each isomer, DFT calculations were carried out at the B3LYP/6-311++G(d,p) level. The optimized geometries of the isomers 3a can be found in Figure 2. Also, the sum of the electronic and zero-point energy and the relative energies (kJ mol⁻¹) of all the isomers in MeOH solution are reported in Table 1. As can be seen in Table 1, the energy difference between the four isomers is low and the isomer 3a ³ is the most stable isomer in MeOH solution.

Figure 2.

Optimized geometries of the isomers of 3a.

Table 1.

Energies (kJ mol⁻¹) of the isomers of 3a.

Isomer	E (pcm) a	ΔE (PCM) b
3a 1	2344720.247	1.016
3a 2	2344707.889	13.274
3a 3	2344721.263	0
3a 4	2344705.942	15.321

PCM: Polarized Continuum Model.

a

Sum of the electronic and zero-point energies.

b

Relative energy to 3a³.

The data from the NOESY experiment on compound 3a did not show a cross-peak between the OH protons and other protons in the isomers, and we are thus unable to assign the OH groups of the four isomers in DMSO-d6 (not shown). However, the DFT-calculated chemical shifts of compounds 3a–c can help identify the structures of the isomers. For example, the DFT-calculated chemical shifts of the isomers of 3a show that the shift OH protons in 3a¹–a⁴ are 9.05, 9.33, 9.91, and 10.01 ppm, respectively, and therefore, in the ¹H NMR spectrum of 3a, the signals at 8.87, 9.29, 10.04, and 10.08 ppm are assigned to the OH protons of 3a¹, 3a², 3a³, and 3a⁴, respectively.

The percentage of isomers can be easily obtained from integrations. Similarly, the percentage of each isomer in compounds 3a–c can be determined from the ¹H NMR spectra. The isomer ratios of each isomer in compounds 3a–c can be obtained from the relative integrations of the OH group in the ¹H NMR spectra of compounds 3a–c. Table 2 shows the detailed results of each reaction, including the yields and isomer ratios in compounds 3a–c.

Table 2.

The detailed results of each reaction including the yields and percentage of isomers in compounds 3a–c.

Entry	Compound	R	Yield %	Isomer 1 (Z, Z) (%)	Isomer 2 (E, Z) (%)	Isomer 3 (Z, E) %	Isomer 4 (E, E) (%)
1	3a	H	72	27.9	23.5	24	24.6
2	3b	Br	69	27.5	23.9	24.2	24.4
3	3c	NO2	65	28.2	23	23.4	25.4

Optical properties

The new dyes 3a–c were deep red in color. The compounds were characterized by their UV-Vis spectra. Figure 3 shows the visible absorption spectra of dyes 3a–c in methanol solution (3 × 10⁻⁵ M). The characteristics of the absorption spectra of 3a–c in MeOH are presented in Table 3. The values of the extinction coefficient (ε) were calculated as the slope of the plot of absorbance versus concentration. The absorbance intensity and extinction coefficient (ε) of red dye 3c were the highest.

Figure 3.

Visible absorption spectra of dyes 3a–c in methanol solution (3 × 10⁻⁵ mol L⁻¹).

Table 3.

Spectroscopic data for 3a–c at 298 K showing dependence on the solvent.

Solvent	λabs (nm) a 3a	ε × 10−3 (M−1 cm−1) b 3a	λabs (nm) a 3b	ε × 10−3 (M−1 cm−1) b 3b	λabs (nm) a 3c	ε × 10−3 (M−1 cm−1) b 3c
n-hexane	405	5.8	430	3.9	425	11.0
EtOAc	430	9.5	460	7.0	460	15.6
MeCN	440	14.5	478	10.8	480	22.0
DMF	445	15.0	485	11.0	490	22.5
DMSO	445	15.4	490	11.5	495	23.3
MeOH	449	17	498	13	494	25.7

a

Wavelengths of maximum absorbance (λ_max).

b

Extinction coefficients.

The absorption spectra of dyes 3a–c were measured in different solvents. As shown in Table 4, the absorption spectra of 3a–c in polar solvents undergo a red shift. Increasing the solvent polarity stabilizes the excited state molecule comparative to the ground-state molecule, with the observed red shift of the absorption maximum as the experimentally observed result (Table 3). For example, in the absorption spectra of dye 3c, λ_abs shifts from 425 to 495 nm as the solvent changes from n-hexane to DMSO (Table 4).

Table 4.

Antibacterial activity (MIC, μg mL⁻¹) of references and compounds 3a–c.

Compound	S.a. (MRSA) a	B.s. (ATCC 6633) b	P.a. (ATCC 27853) c	E.c. (ATCC 25922) d
3a	>200	>200	25	20
3b	>200	>200	15	15
3c	>200	>200	10	5
Ampicillin	62	0.50	125	8
Penicillin G	60	8	–	–
Sulfamethoxazole	16	16	62	16

a

Staphylococcus aureus (methicillin-resistant S. aureus).

b

Bacillus subtilis.

c

Pseudomonas aeruginosa.

d

Escherichia coli.

The color intensity of the dyes 3a–c indicates efficient intramolecular charge transfer (ICT) states^18–23 from the donor site (endocyclic N-4 and OH group) to the acceptor moiety (C=O group) (Scheme 3). To gain a deeper insight into the optical properties and the UV-Vis absorption spectra of the dyes, we performed DFT and TD-DFT calculations at the B3LYP/6-311++G(d,p) level and obtained HOMO and LUMO frontier orbitals and electronic spectra of isomers 3a¹ –a⁴.

Scheme 3.

Some mesomeric structures of 3a³.

The energy difference between the HOMO and LUMO frontier orbitals is one of the important characteristics of molecules, which has a determining role in such cases as electric properties, electronic spectra, and photochemical reactions. The HOMO and LUMO maps of 3a¹ –a⁴ are shown in Figure 4. As can be seen, the HOMOs and LUMOs of 3a¹ –a⁴ are totally localized on the OH (donor site) and C=O (acceptor site) groups. The separation energies between the HOMO and LUMO (Δε = ε_LUMO − ε_HOMO) in isomers 3a¹ –a⁴ are 2.77, 2.81, 2.79, and 2.88 eV, respectively.

Figure 4.

The HOMO (bottom) and LUMO (up) frontier orbitals of the dyes 3a¹ –a⁴.

The calculated electronic absorption spectra were also obtained by the time-dependent density functional theory (TD-DFT) method. The TD-DFT electronic spectra calculations on of 3a¹ –a⁴ show two electronic transition bands. There is a relatively sharp peak at 347–353 nm (oscillator strength: 0.1041–0.2682), which can be attributed to π − π* transitions (donor endocyclic N-4 to the acceptor C=O group) (experimental value: 389 nm) and a relatively broad band at 505–520 nm with an oscillator strength of 0.2674–0.3492, which can be linked to n − π* transitions from the donor OH group to the acceptor C=O group, compared with the experimental values of 450–550 nm.

The calculated electronic absorption spectra of non-isolable isomers 3a¹ –a⁴ are shown in Figure 5.

Figure 5.

The calculated electronic absorption spectra of isomers 3a¹ –a⁴.

Antibacterial studies

The antibacterial activity of new dyes 3a–c was tested against a panel of strains of Gram positive (Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) clinical isolated and Bacillus subtilis (ATCC 6633)) and Gram negative (Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli, (ATCC 25922)) bacterial species (Table 4) using the broth microdilution method as previously described. ²⁸ Ampicillin, penicillin G, and sulfamethoxazole were used as references. The lowest concentration of the antibacterial agent that prevents growth of the test organism, as detected by lack of visual turbidity (matching the negative growth control), is designated the minimum inhibitory concentration (MIC). Experimental details of the tests can be found in our earlier study.^29,30

As shown in Table 4, compounds 3a–c are effective against Gram-negative bacteria. Also, the results revealed that new dye 3c, with an Ar = C₆H₄NO₂ group, displayed greater antibacterial activity against Gram-negative bacteria compared with the well-known antibacterial agents ampicillin and sulfamethoxazole (Table 4).

Conclusion

In conclusion, we have synthesized three new red dyes in good yields via nucleophilic substitution of the hydrogen of 3-nitroimidazo[1,2-a]pyridine with different acetophenone derivatives in basic methanol solution. The ¹H NMR spectra of the products revealed that the dyes exist as four non-isolable isomers. Although data from NOESY experiments did not allow assignment of the OH groups of the four isomers, the DFT-calculated chemical shifts of compounds 3a–c helped to identify the structures of the isomers. The optical properties of the dyes were examined and the solvent effects on the absorption spectra of the dyes were studied. DFT and TD-DFT calculations of the dyes were performed to obtain HOMO and LUMO frontier orbitals and electronic spectra by using the B3LYP hybrid functional and the 6-311++G(d,p) basis set. Furthermore, the electronic spectra of the dyes were in relatively good agreement with visible absorption spectra. Moreover, results from the antimicrobial screening tests showed that these new compounds were effective against standard strains of Gram-negative growth inhibitors. This property, together with optical properties, offers an excellent opportunity for the study of the physiological functions of bacteria such as at single-cell level. ³¹

Further investigations into the scope and application of these new dyes are in progress and will be reported soon.

Experimental

Materials

All reagents and solvents used in this work were purchased from Merck. The microorganisms Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 were purchased from the Pasteur Institute of Iran and MRSA was isolated from different specimens which were referred to the Microbiological Laboratory of the Ghaem Hospital of the Medical University of Mashhad, Iran, and its methicillin resistance was tested according to the national committee for clinical laboratory standards (NCCLS) guidelines. ³² All solvents were dried according to standard procedures. Compound 1 was synthesized according to a literature method.^24,25

Equipment

Absorption spectra were recorded on a Varian Cary 50-bio UV-Vis spectrophotometer. UV-Vis scans were recorded from 200 to 1000 nm. Melting points were measured on an Electro thermal type-9100 melting-point apparatus. The IR (as KBr disks) spectra were obtained on a Tensor 27 spectrometer and only noteworthy absorptions are listed. The ¹³C NMR (100 MHz), ¹H NMR (400 MHz) and NOESY spectra were recorded on a Bruker Avance DRX-400 Fouriertransform spectrometer in DMSO-d₆. Chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS) as the internal standard; coupling constants (J) are given in hertz. The mass spectra were recorded on a Varian Mat, CH-7 at 70 eV. Elemental analysis was performed on a Thermo Finnigan Flash EA microanalyzer. All measurements were carried out at room temperature.

Computational methods

The DFT calculations were performed with the Gaussian 98 software package ³³ by using the B3LYP hybrid functional ³⁴ and the 6-311++G (d,p) basis set. The geometries of the compounds were fully optimized in MeOH solution.

Here, one of self-consistent reaction field methods, the sophisticated Polarized Continuum Model (PCM) ³⁵ was used for investigation of the solvent effects. The PCM calculations were performed in MeOH, and zero-point corrections were considered to obtain the energies. Based on the optimized geometries and using time-dependent density functional theory (TD-DFT)^36–38 methods, the electronic spectra of the compounds were predicted.

The ¹H NMR chemical shifts of the compounds were predicted with respect to TMS. Here, the gauge-independent atomic orbital method (GIAO) was used for prediction of DFT nuclear shielding. ³⁹

Synthesis of 3a–c from 1 and 2a–c

3-Nitroimidazo[1,2-a]pyridine (1) (3.26 g, 20 mmol) and substituted acetophenones 2a–c (30 mmol) were added with stirring to a solution of KOH (20 g, 357 mmol) in methanol (50 mL). The mixture was stirred at reflux for 4 h and then poured into water. After neutralization with dilute HCl solution, the precipitate was collected by filtration, washed with water, and then air on silica gel dried to give crude 3a–c. Further purification was achieved by column chromatography on silica gel.

3-(Hydroxyimino)imidazo[1,2-a]pyridin-2(3H)-ylidene-1-phenylethanone (3a): Purified by silica gel column chromatography (CHCl₃-MeOH, 15:3) to give a dark red powder, yield (72%), mp > 300 °C. IR (KBr): 1569 (m) (C=C), 1631 (s) (C=O), 3374 (m) (OH) cm⁻¹. ¹H NMR (DMSO-d₆): δ = 6.75 (1H, d, J = 6.7 Hz, pyridine H), 6.97 (1H, t, J = 7.1 Hz, pyridine H), 7.35–7.65 (6H, m, ArH), 7.71–7.84 (2H, m, ArH); 8.87 (br s, 27.9% of 1H, OH), 9.29 (br s, 23.5% of 1H, OH), 10.04 (br s, 24.0% of 1H, OH), 10.08 (br s, 24.6% of 1H, OH). ¹³C NMR (DMSO-d₆): δ = 102.9, 112.5, 122.5, 132.0, 133.3, 134.2, 136.7, 141.9, 147.3, 152.8, 159.8, 167.7, 192.5. MS (m/z): 265 (M⁺). Anal. Calcd for C₁₅H₁₁N₃O₂ (265.3): C, 67.92; H, 4.18; N, 15.84, Found: C, 68.12; H, 4.21; N, 15.65.

1-(4-Bromophenyl)-2-(3-(hydroxyimino)imidazo[1,2-a]pyridin-2(3H)-ylidene)ethanone (3b): Purified by a silica gel column chromatography (CHCl₃-MeOH, 18:4) to give a dark red powder, yield (69%), mp > 300 °C. IR (KBr): 1569 (m) (C=C), 1631 (s) (C=O), 3374 (m) (OH) cm⁻¹. ¹H NMR (DMSO-d₆): δ = 6.77 (1H, d, J = 6.7 Hz, pyridine H), 7.01 (1H, t, J = 7.1 Hz, pyridine H), 7.39 (1H, s, Alkene H), 7.55 (2H, d, J = 8.6 Hz, 4-bromophenyl H), 7.69–7.87 (4H, m, ArH); 8.96 (br s, 27.5% of 1H, OH), 9.33 (br s, 23.9% of 1H, OH), 10.09 (br s, 24.2% of 1H, OH), 10.12 (br s, 24.4% of 1H, OH). ¹³C NMR (DMSO-d₆): δ = 103.4, 113.4, 123.4, 128.3, 134.8, 135.1, 135.7, 141.6, 147.2, 152.8, 158.5, 166.8, 191.9. MS (m/z): 345 [M(⁸¹Br)]⁺, 343 [M(⁷⁹Br)]⁺. Anal. Calcd for C₁₅H₁₀N₃O₂Br (344.2): C, 52.35; H, 2.93; N, 12.21, Found: C, 52.17; H, 2.90; N, 12.55.

3-(Hydroxyimino)imidazo[1,2-a]pyridin-2(3H)-ylidene)-1-(4-nitrophenyl)ethanone (3c): Purified by silica gel column chromatography (CHCl₃-MeOH, 4:1) to give a dark red powder, yield (65%), mp > 300 °C. IR (KBr): 1335, 1575 (s) (NO₂), 1569 (m) (C=C), 1631 (s) (C=O), 3374 (m) (OH) cm⁻¹. ¹H NMR (DMSO-d₆): δ = 6.81 (1H, d, J = 6.7 Hz, pyridine H), 7.02 (1H, t, J = 7.1 Hz, pyridine H), 7.41 (1H, s, Alkene H), 7.73–7.85 (2H, m, ArH), 8.17 (2H, d, J = 8.7 Hz, 4-nitrophenyl H), 8.49 (2H, d, J = 8.7 Hz, 4-nitrophenyl H), 8.99 (br s, 28.2% of 1H, OH), 9.36 (br s, 23.0% of 1H, OH), 10.05 (br s, 23.4% of 1H, OH), 10.09 (br s, 25.4% of 1H, OH). ¹³C NMR (DMSO-d₆): δ = 103.6, 113.9, 122.2, 124.8, 134.3, 135.1, 135.9, 147.8, 152.3, 152.8, 158.5, 166.8, 191.7. MS (m/z): 310 (M⁺). Anal. Calcd for C₁₅H₁₀N₄O₄ (310.3): C, 58.07; H, 3.25; N, 18.06, Found: C, 57.85; H, 3.21; N, 17.90.