Optical properties and fluorescence quenching of biologically active ethyl 4-(4-N,N-dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) dye as a probe to determine CMC of surfactants

Abstract

4-(4-N,N-Dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) was prepared via the multi-component reaction of indane-1,3-dione with 4-(dimethylamino)benzaldehyde, ethyl acetoacetate and ammonium acetate. Data obtained from FT-IR, ¹H-NMR, ¹³C-NMR, EI-MS and elemental analysis were consistent with the chemical structure of the newly prepared DDPC. Electronic absorption and emission spectra of DDPC were measured in different solvents. The DDPC dye exhibits a red shift in its emission spectrum as the solvent polarity increases, indicating a large change in the dipole moment upon excitation due to intramolecular charge transfer within the excited DDPC molecules. Excited state intermolecular hydrogen bonding has an effect upon the energy of the emission spectrum and the fluorescence quantum yield of the DDPC molecules. The DDPC dye undergoes solubilization into different micelles and can be used as a probe to determine the critical micelle concentration (CMC) of SDS and CTAB. The DDPC dye can also be used to probe of the polarity and hydrogen bonding properties of its local micro-environment. The antibacterial activity of DDPC was tested in vitro for the first time by using a disk diffusion assay against two Gram-positive and two Gram-negative bacteria, and the minimum inhibitory concentration (MIC) was also determined with reference to the standard drug tetracycline.

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Introduction

Nitrogen containing heterocyclic compounds especially pyridine, occupy a special place in industry and have attracted considerable attention because of their broad pharmacological activity, including antibacterial, anti-tumor, anticancer,^1–3 antiviral,⁴ anti-inflammatory,⁵ antimicrobial,⁶ anti-diabetic,⁷ anti-hypertensive⁸ and osteogenic properties,⁹ in addition to their use in the treatment of CNS disorders.¹⁰ Various synthetic methods have been reported for the synthesis of pyridine derivatives and bi-cyclic heterocyclic compounds such as pyrazolo-pyridine, thiazolo-pyridine and triazolo-pyridine have synthesized by the cyclization of pyridine.¹¹ Such compounds have also been used as ligands for metal complexes by coordinating with transition metals.¹² Due to the presence of the long range π bond conjugation systems in pyridine derivatives they are also used within fields of materials science such as non-linear optical devices,¹³ photonic materials,¹⁴, optical limiting devices,¹⁵ electrochemical sensors,¹⁶ light-emitting devices,¹⁷ Langmuir films,¹⁸ and solar cells.¹⁹ Moreover, pyridine has been used extensively as the photo-alignment and photo-crosslinking unit in polymers. The study of physicochemical characteristics, such as solvatochromism, piezochromism, oscillator strength, dipole moment, fluorescence quantum yield and photostability, are also important for determining the physical behavior of compounds.¹⁹ Donor (D)–π–acceptor (A) π-bond conjugated systems impart good physicochemical behavior due to intramolecular charge transfer through their π-bonds from the donor group to the acceptor group.²⁰ Based on literature survey we find that lot of work have been done on chromophores,²¹ but to the best of our knowledge, there has not been a deep investigation into the present topic, which we are reporting for the first time. Therefore, due to the possible importance of D–π–A chromophores we have great interest in the development of heterocycle-based D–π–A chromophores. In the present study, we report the synthesis of a new heterocycle-based chromophore. One-pot multi-component reactions (MCR) have attracted significant attention in synthetic chemistry as they can produce target products from readily available starting materials in one reaction step without isolating the intermediates, thus reducing reaction times, labor costs and waste production.²² Therefore, in the present paper, we report the synthesis of 4-(4-N,N-dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) by MCR and physicochemical studies on its properties such as oscillator strength, dipole moment, fluorescence quantum yield and fluorescence quenching in organized media. The DDPC dye undergoes solubilization into different micelles and can be used as a probe to determine the critical micelle concentration (CMC) of SDS and CTAB.

Experimental

Apparatus

FT-IR spectra were recorded on a Nicolet Magna 520 FT-IR spectrometer. ¹H-NMR and ¹³C-NMR experiments were performed in CDCl₃ on a Bruker DPX 600 MHz spectrometer using tetramethyl silane (TMS) as an internal standard at room temperature. Melting points were recorded on a Thomas Hoover capillary melting apparatus without correction. UV-vis electronic absorption spectra were acquired on a Shimadzu UV-1650 PC spectrophotometer. Absorption spectra were collected using a 1 cm quartz cell. Steady state fluorescence spectra were measured using a Shimadzu RF 5301 PC spectrofluorophotometer with a rectangular quartz cell. Emission spectra were monitored at a right angle. All of the fluorescence spectra were blank subtracted before proceeding with data analysis.

Chemicals and reagents

The required chemicals; indane-1,3-dione, 4-(dimethylamino)benzaldehyde, ethyl acetoacetate and ammonium acetate were purchased from Acros Organics. Other reagents and solvents (A.R.) were obtained commercially and used without further purification, except dimethylformamide (DMF), ethanol and methanol.

4-(4-N,N-Dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC)

To an alcoholic solution (50 mL) of indane-1,3-dione (0.01 mol), 4-(dimethylamino)benzaldehyde (0.01 mol), ethyl acetoacetate (0.01 mol), ammonium acetate (0.02 mol) and a drop of piperidine were added and the mixture was refluxed for 2 h. The reaction mixture was concentrated to half of its original volume and allowed to cool in an ice-chest. The resulting separated solid was filtered, washed with ice cold aqueous ethanol and crystallized from petroleum ether (60–80 °C)–chloroform (1 : 1) (Scheme 1).

Scheme 1 The synthesis of DDPC.

Dark brown solid: mp 122 °C; EIMS m/z (rel. int. %): 390 (72) [M + 1]⁺; IR (KBr) ν_max cm⁻¹: 3254 (N–H), 2964 (C–H), 1656 (C=O), 1585 (C=C); 1246 (C–N); ¹H-NMR (600 MHz CDCl₃) (δ/ppm): 9.16 (s, NH), 8.51–6.47 (m, 9H, CH aromatic), 4.07 (s, 3H, –CH₃), 4.00 (s, 3H, –CH₃), 3.96 (s, 3H, –CH₃), 3.85–3.75 (q, CH₂–CH₃), 1.61 (t, –CH₂–CH₃); ¹³C-NMR (CDCl₃) δ: 168.22 (C=O), 156.25 (pyridine C), 139.91, 134.76, 134.61, 124.81, 122.86, 122.78 (Ar-C). Element. anal. calc. for C₂₄H₂₄N₂O₃: C, 74.21; H, 6.23; N, 7.21. Found: C, 74.18; H, 5.97; N, 6.16.

The fluorescence quantum yield (ϕ_f) of DDPC (1 × 10⁻⁵ M) was evaluated in different solvents. Rhodamine 6G (1 × 10⁻⁵ M) in ethanol (ϕ_f = 0.94) was selected as a standard sample since rhodamine 6G absorbs at the same excitation wavelength (λ_ex = 385 nm) as DDPC in ethanol and the same concentration of solution for DPPC and rhodamine 6G was used to obtain the absorption spectra.²³ It was, therefore, expected that the same number of photons should be absorbed by both samples (rhodamine 6G and DDPC). The fluorescence quantum yield of DDPC can be related to that of the standard via the following relationship:²⁴

where ϕ is the quantum yield, I is the integrated emission intensity, A is the absorbance at the excitation wavelength, and n is the refractive index of the solvent. The subscript r refers to the reference fluorophore with a known quantum yield.

Organism culture and in vitro screening

The antibacterial activity of 4-(4-N,N-dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) was measured using a disk diffusion method with minor modifications. S. aureus, S. pyogenes, S. typhimurium and E. coli were sub-cultured in BHI medium and incubated for 18 h at 37 °C, and then the bacterial cells were suspended, according to the McFarland protocol in saline solution to produce a suspension of about 10⁻⁵ CFU mL⁻¹. 10 μL of this suspension was mixed with 10 mL of sterile antibiotic agar at 40 °C and poured onto an agar plate in a laminar flow cabinet. Five sterile paper discs (6.0 mm diameter) were fixed onto nutrient agar plates, 1 mg of DDPC was dissolved in 100 μL DMSO to prepare a stock solution to form different concentrations of DDPC (10, 20, 25, 50, and 100 μg μL⁻¹). Each different concentration was then poured over a disk plate. Tetracycline (30 μg per disk) was used as a standard drug (positive control). A DMSO poured disk was used as a negative control. The susceptibility of the bacteria to DDPC was determined by the formation of an inhibitory zone after 18 h of incubation at 36 °C. Table 2 reports the inhibition zones (mm) of DDPC and the controls. The minimum inhibitory concentration (MIC) was evaluated by using a macro dilution test with standard inoculums of 10⁻⁵ CFL mL⁻¹. A series of dilutions of the DDPC solution, previously dissolved in dimethyl sulfoxide (DMSO), was prepared to give final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg mL⁻¹ and 100 μL of 24 h old inoculum was added to each tube. The MIC was defined as the lowest concentration of DDPC that inhibited visible growth, determined visually after incubation for 18 h at 37 °C and the results are presented in Table 2. Tests using DMSO and tetracycline as negative and positive controls were also performed.

Results and discussion

Chemistry

4-(4-N,N-Dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) was prepared via the multi-component reaction of indane-1,3-dione with 4-(dimethylamino)benzaldehyde, ethyl acetoacetate, ammonium acetate.²⁵ The purified dye was characterized by FT-IR, ¹H-NMR, ¹³C-NMR, EI-MS m/z (rel. int. %), and elemental analysis. The FT-IR spectrum of DDPC shows the characteristic band at 1676 cm⁻¹ for the ν (C=O) peak of indane-1,3-dione has been shifted to a lower frequency of 1656 cm⁻¹ for DDPC. This is due to one C=O bond being utilized for the formation of pyridine. The IR spectrum of DDPC shows a characteristic band at 3254 cm⁻¹ due to the presence of a –NH group. The IR spectrum shows sharp peak at 1246 cm⁻¹ due to the presence of the C–N–C stretching mode, which confirms the formation of a pyridine ring. The ¹H-NMR spectrum of DDPC measured at room temperature shows a singlet at 9.16 ppm for the NH group. The appearance of multiplets between δ 8.51–6.47 is due to aromatic protons and the three singlets at δ 4.07, 4.00 and 3.96 correspond to the three methyl groups present in DDPC. Moreover, the ¹³C-NMR (CDCl₃) spectrum of DDPC was recorded in CDCl₃ and the spectral signals are in good agreement with the predicted structure. The carbonyl carbon of DDPC usually appears at δ 168.22 in the ¹³C-NMR spectrum. The ¹³C-NMR spectrum shows signals in the range of δ 139.91–122.78 ppm due to the presence aryl carbons. Details of the ¹³C-NMR spectrum of DDPC is given in the Experimental section. Finally, characteristic peaks were observed in the mass spectrum of DDPC. The mass spectrum of DDPC shows a molecular ion peak of (M⁺) m/z 390.

Spectral behavior of DDPC in different media

UV-vis absorption spectra of DDPC (1 × 10⁻⁵ M) were measured in various non-polar, polar aprotic and polar protic solvents such as ethanol, methanol, dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane, carbon tetrachloride, acetonitrile, dioxan and tetrahydrofuran. Fig. 1 shows the absorption spectra of 1 × 10⁻⁵ mol dm⁻³ solutions of DDPC in these solvents as examples. As it can be seen from Fig. 1, in all of the solvents tested the main band of DDPC is located in the spectral range of 371–400 nm from CCl₄ to DMSO. These wavelength band in the UV region was observed for the studies of DDPC in different solvent systems, which can be assigned to a π to π* transition from the benzenoid system toward the other ring, which is characteristic of the high electron donating and electron accepting character present in the structure. Upon excitation at 385 nm, within the λ range of emission between 395 nm and 700 nm, the emission spectrum of DDPC (1 × 10⁻⁵ M) shows a smooth correlation with increasing polarity of the solvent, which broadens and becomes red shifted (Fig. 2 and Table 1) as the solvent polarity increases. The red shift from 475 nm in CCl₄ to 548 in DMSO indicates that photoinduced intramolecular charge transfer (ICT) occurs in the singlet excited state and, therefore, the polarity of DDPC increases on excitation.²⁶ The change in the fluorescence peak in alcoholic solvents can be assigned to solute–solvent hydrogen bonding interactions in the singlet excited state, which causes a red shift in the observed spectra (Table 1).²⁷

Fig. 1 Electronic absorption spectra of 1 × 10⁻⁵ mol dm⁻³ solutions of DDPC in different solvents.

Fig. 2 Emission spectra of 1 × 10⁻⁵ mol dm⁻³ solutions of DDPC in different solvents (λ_ex = 385 nm).

Table 1 Spectral data for DDPC in different solvents

Solvent	Δf	ET^N	ET (30) kcal mol^−1	λab (nm)	λem (nm)	ε (M^−1 cm^−1)	f	μ12 Debye	Δ[small nu, Greek, macron] (cm^−1)	ϕf
EtOH	0.305	1.28	72.38	395	559	8410	0.24	4.48	7427	0.092
DMSO	0.266	1.25	71.47	400	548	11 480	0.31	5.12	6752	0.29
MeOH	0.308	1.32	73.49	389	561	8073	0.25	4.53	7881	0.035
DMF	0.263	1.35	74.26	385	540	9590	0.28	4.77	7396	0.33
CHCl3	0.217	1.35	74.65	383	532	9158	0.26	4.59	7313	0.44
CH2Cl2	0.255	1.33	73.87	387	530	7810	0.21	4.14	6972	0.40
Acetonitrile	0.274	1.38	75.43	379	542	9183	0.29	4.82	7935	0.30
dioxan	0.148	1.41	76.65	373	490	11 840	0.30	4.86	6401	0.43
THF	0.263	1.41	76.44	374	497	8614	0.22	4.17	6617	0.34
CCl4	0.024	1.43	77.06	371	475	9752	0.23	4.25	5902	0.16

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A simplified description of the hydrogen bonding in DDPC is shown in Scheme 2. Type (a) hydrogen bonding is strengthened in the excited state, since the charge density at the carbonyl oxygen is enhanced in the ICT excited state. On the other hand, type (b) hydrogen bonding is weakened by photoexcitation, because the charge density at N–(CH₃)₂ decreases in the excited state.

Scheme 2 Types of hydrogen bonding in DDPC.

The energy of absorption (E_a) and emission (E_f) of DDPC in different solvents correlate with the empirical Dimroth polarity parameter E_T (30) of the solvent (Fig. 3).²⁸ A linear correlation between the energy of absorption and emission versus the polarity of the solvent was obtained (eqn (2) and (3)), implying the potential application of these parameters to probe the micro-environment of DDPC.

E_a = 75.17 − 0.1032 × E_T(30) (2)

E_f = 68.28 − 0.257 × E_T(30) (3)

Fig. 3 Plots of the energy of absorption (E_a) and emission (E_f) versus E_T (30) for the different solvents.

Analysis of the solvatochromic behavior allows us to estimate the difference in the dipole moment between the excited and ground states (Δμ_e − Δμ_g). This was achieved by applying simplified Lippert–Mataga equations, eqn (4) and (5).²⁹

where Δν is the Stokes shift, which increases with increasing solvent polarity, pointing to a stronger stabilization of the singlet excited state in polar solvents, h is Planck constant, c is the speed of light, a is the Onsager cavity radius, while k and n are the dielectric constant and refractive index of the solvent, respectively. The constant (const.) represents higher order terms which are usually neglected. The Onsager cavity radius was taken as 5.7 Å.³⁰ Fig. 4 shows a plot of the Stokes shift versus the orientation polarizability (Δf). Changes in the dipole moment (Δμ) upon excitation was calculated from slope of the plot and the cavity radius is Δμ = 5.58 Debye. This change in dipole moment is caused by redistribution of atomic charges in the excited state as a result of charge transfer from the electron rich –N–(CH₃)₂ group to the electron accepting keto-group fragment.

Fig. 4 Plot of the Stokes shift (Δν) versus polarity (Δf) of the solvent for DDPC.

The oscillator strength (f) and transition dipole moment (μ₁₂) of the electronic transition for DDPC from ground to excited singlet state (S₀ → S₁) was calculated in different solvents using the following equations, eqn (6) and (7).³¹

where ε the numerical value for the molar decadic extinction coefficient, which is measured in dm³ mol⁻¹ cm⁻¹, ν is the value of the wavenumber, measured in cm⁻¹, and E_max is the energy maximum of the absorption band in cm⁻¹. The values of f and μ₁₂ are listed in Table 1 and they indicate that the S₀ → S₁ is a strongly allowed transition.

The empirical Dimroth polarity parameter, E_T (30), and E_T^N of DDPC were also calculated according to the following equation:^32,33

where λ_max corresponds to the peak wavelength (nm) in the red region of the intramolecular charge transfer absorption of DDPC. The red (bathochromic) shift from CCl₄ to DMSO indicates that photoinduced intramolecular charge transfer (ICT) occurs in the singlet excited state, and the polarity of DDPC, therefore, increases on excitation.

Fluorescence quantum yield

The fluorescence quantum yield (ϕ_f) of DDPC depends strongly on the solvent properties (Table 1). The fluorescence quantum yield can be correlated with E_T (30) of the solvent, where E_T (30) is the solvent polarity parameter introduced by Reichardt.³⁴ The fluorescence quantum yield of DDPC increases with increasing solvent polarity from 0.16 in a non-polar solvent (CCl₄) to 0.43 in a moderately polar solvent (dioxan). With a further increase in solvent polarity the fluorescence quantum yield seems to decrease, i.e., it is 0.29 in a strongly polar solvent (DMSO) (Fig. 5). This indicates the occurrence of both a negative solvatokinetic effect and a positive solvatokinetic effect during the course of an increase in the solvent polarity.³⁵ One of the reasons for the negative solvatokinetic effect (an increase in ϕ_f with a suitable enhancement of ICT) could be due to biradicaloid charge transfer involving the un-bridged double bonds and another cause could be related to the proximity effect in compounds with n–π and π–π* electron configurations. In other words, in non-polar solvents, these effects will result in an effective non-radiative decay of the excited states. In strongly polar solvents, the fluorescence quantum yield decreases, due to a large degree of intramolecular charge transfer, which causes an increase in the rate of radiationless relaxation of the excited state, giving rise to a positive solvatokinetic effect (a reduction in ϕ_f via strong ICT). Moreover, the much lower fluorescence quantum yields observed in protonated solvents can be attributed to hydrogen bond interactions between the molecules and the surrounding solvent, which results in an additional non-radiative decay process as observed in other dipolar molecules.

Fig. 5 Plots of ϕ_f versus E_T (30) for DDPC in different solvents.

The effect of surfactants on the emission spectrum of DDPC

The emission spectrum of 1 × 10⁻⁵ mol dm⁻³ of DDPC was also measured with cationic micelles of cetyltrimethyl ammonium bromide (CTAB) and anionic micelles of sodium dodecyl sulphate (SDS). As shown in Fig. 6 and 7, the emission intensity of DDPC increases with increasing concentration of the surfactant and an abrupt change in fluorescence intensity is observed at surfactant concentrations of 7.30 × 10⁻⁴ and 7.45 × 10⁻³ mol dm⁻³. We used Carpena’s method³⁶ to obtain the value of CMC from the emission intensity data and found the same CMC values shown in Fig. 6 and 7, which are very close to the CMCs of CTAB and SDS,³⁷, thus DDPC can be employed as a probe to determine the CMC of a surfactant. It was well known that aromatic molecules are generally solubilized in the palisade layer of a micelle.^38,39 Therefore, the enhancement in the emission intensity can be attributed to the passage of the dye molecule from the aqueous bulk solution to the palisade layers of the micelles. The decrease in polarity of the micro-environment around the dye molecule results in a reduction of the non-radiative decay rate from the ICT state to a low-lying singlet or triplet state due to an enlargement the energy gap between them, which also leads to an increase in the emission intensity. The CMC of SDS and CTAB with DDPC were further confirmed using a conductometric method.

Fig. 6 A plot of I_f versus the concentration of CTAB.

Fig. 7 A plot of I_f versus the concentration of SDS.

Fluorescence quenching of DDPC with alcoholic solvents

The fluorescence quenching of DDPC in dioxan (λ_ex = 385 nm) was studied using different concentrations of various polar protic solvents of different acidity (such as methanol, ethanol, 2-propanol and n-butanol) as quenchers (Fig. 8(a)–11(a)). As shown in the figures, the fluorescence spectrum undergoes very complex changes upon the addition of different concentrations of each alcoholic solvent, e.g., the spectrum is shifted to a longer wavelength, possesses changed half widths and different band profiles for the emission spectrum. This behavior indicates that, in such solutions, an extra factor contributes to the well known dipole–dipole interactions, i.e., hydrogen-bonding interactions between the DDPC and the alcohol.⁴⁰ Stern–Volmer constants (K_SV) were calculated from the Stern–Volmer plots shown in Fig. 8(b)–11(b). The K_SV constant was determined to be 0.206, 0.169, 0.99 and 0.094 M⁻¹ for methanol, ethanol, 2-propanol and butanol, respectively, which increases according to the acidity (α) of the alcohol. It seems that the K_SV value in the case of methanol is higher than that for the other solvents, which indicates that there is a possibility that hydrogen bonding with the solute increases with decreasing number of carbon atoms in the solvent molecule. The dependence of the fluorescence characteristics on the solvent properties implies that there is potential for DDPC to be used probe of the polarity and hydrogen bonding properties of its local micro-environment. The Stern–Volmer, K_SV constant can be calculated by the eqn (10):

I₀/I_f = 1 + K_SV[Q] (10)

where I₀ and I_f are the relative integrated fluorescence intensities without and with the quencher of concentration [Q] and K_SV is the Stern–Volmer constant.

Fig. 8 (a) Fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ DDPC in dioxan (λ_ex = 385 nm) by MeOH. The concentrations of MeOH with decreasing emission intensity are 0, 0.49, 1.47, 2.46, 3.45, 4.43, 5.43, 5.42, 6.41, 7.31 and 8.38 mol dm⁻³. (b) A Stern–Volmer plot of the fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ of DDPC in dioxan by MeOH.

Fig. 9 (a) Fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ DDPC in dioxan (λ_ex = 385 nm) by EtOH. The concentrations of EtOH with decreasing emission intensity are 0, 0.34, 1.02, 1.71, 2.39, 3.07, 3.76 and 4.44 mol dm⁻³. (b) A Stern–Volmer plot of the fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ of DDPC in dioxan by EtOH.

Fig. 10 Fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ DDPC in dioxan (λ_ex = 385 nm) by ⁱPrOH. The concentrations of ⁱPrOH with decreasing emission intensity are 0, 0.23, 0.71, 1.18, 1.66, 2.13, 2.61, 3.08 and 3.56 mol dm⁻³. (b) A Stern–Volmer plot of the fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ of DDPC in dioxan by ⁱPrOH.

Fig. 11 (a). Fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ DDPC in dioxan (λ_ex = 385 nm) by BuOH. The concentrations of BuOH with decreasing emission intensity are 0, 0.21, 0.65, 1.08, 1.52, 1.95, 2.39, 2.82 and 3.25 mol dm⁻³. (b) A Stern–Volmer plot of the fluorescence quenching of 1 × 10⁻⁵ mol dm⁻³ of DDPC in dioxan by BuOH.

Antibacterial activity

Disk diffusion assay. 4-(4-N,N-Dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) was tested for its antibacterial activity using a disk diffusion method with nutrient broth medium containing (g L⁻¹) beef extract (3 g) and peptone (5 g) at pH 7.0.⁴¹ The Gram-positive bacteria and Gram-negative bacteria utilized in this study were S. aureus, S. pyogenes, S. typhimurium and E. coli. In the disk diffusion method, sterile paper disks (0.5 mm) impregnated with DDPC and dissolved in dimethylsulfoxide (DMSO) at a concentration 100 μg mL⁻¹ were used. The paper disks were impregnated with a solution of the test DDPC and were then placed on the surface of the media inoculated with the microorganism. The plates were incubated at 35 °C for 24 h. After incubation, the growth inhibition zones are shown in Table 2.

Table 2 Antibacterial activity of DDPC, including a positive control (tetracycline) and a negative control (DMSO), measured by the Halo Zone test (unit, mm) and MIC (μg mL⁻¹)

	Corresponding effect on microorganisms
	S. aureus	S. pyogenes	S. typhimurium	E. coli
DDPC (disk)	11.4 ± 0.3	12.5 ± 0.5	14.3 ± 0.2	15.2 ± 0.2
DDPC (MIC)	64	64	32	32
Tetracycline (disk)	13.0 ± 0.5	20.0 ± 0.5	12.0 ± 0.5	14.0 ± 0.5
Tetracycline (MIC)	32	32	32	32
DMSO	—	—	—	—

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Assessment of the minimum inhibitory concentration (MIC). The MIC of the synthesized DDPC and a standard drug were investigated against two Gram-positive and two Gram-negative bacteria using a broth dilution method (BDM). The data is reported as MIC, which is defined as the lowest concentration required to inhibit 90% growth in comparison to a nagative control (absence of DDPC) for each isolate. Table 2 summarizes the in vitro susceptibilities of both types of isolates (Gram-positive and Gram-negative) against DDPC. Evaluation of MIC showed that DDPC is active in vitro against all of the tested microorganisms with varying degrees of inhibition, within the reference range.

Conclusion

A novel donor–accepter heterocyclic chromophore 4-(4-N,N-Dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) was prepared via the multi-component reaction of indane-1,3-dione with 4-(dimethylamino)benzaldehyde, ethyl acetoacetate, ammonium acetate and characterized by various spectral techniques. The optical properties of the DDPC dye including singlet absorption, extinction coefficient, Stokes shift, oscillator strength, dipole moment and fluorescence quantum yield were investigated on the basis of the polarity of the solvent used. The absorption and emission spectra of DDPC exhibit an intramolecular charge transfer band, which exhibited positive solvatochromism in different solvents. Emission spectra of DDPC also revealed the character of the intramolecular charge transfer band. These findings confirm that there is significant electron transfer between the donating moiety and the accepting fragment through π conjugation. The DDPC dye undergoes solubilization into different micelles and may be used in the determination of the CMC of surfactants (SDS and CTAB). The DDPC dye can also be used to probe the polarity and hydrogen bonding properties of its local micro-environment. The antibacterial activity of DDPC was examined using cultures of different bacteria and the results showed that DDPC has better antibacterial activity for both types of bacteria (Gram-positive and Gram-negative) when compared to the reference drug tetracycline.

Acknowledgements

The authors are thankful to the Chemistry Department at King Abdulaziz University for providing research facilities.

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