In vitro photodynamic inactivation of B. subtilis and E. coli O157 by a new chalcone conjugate photosensitizer tethered with the NMe 2 group as fluorescence donor auxochrome

doi:10.21203/rs.3.rs-2961179/v1

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In vitro photodynamic inactivation of B. subtilis and E. coli O157 by a new chalcone conjugate photosensitizer tethered with the NMe 2 group as fluorescence donor auxochrome

https://doi.org/10.21203/rs.3.rs-2961179/v1

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4-Azido-2,6-dimethylquinolie 2 was prepared and coupled under the CuAAC conditions with a set of propargylated chalcones 3a-e & 4a,b, and a ferrocene-chalcone conjugate 5. These couplings afforded a new series of tripod and tetrapod 1,4-disubstituted-1,2,3-triazoles 6a-e, 7a,b, and 8 for potential divergent applications. In vitro antimicrobial screening disclosed their negative antifungal activity against A. flavus and C. albicans. Similarly, they were inactive against S. aureus and E. coli except the chalcone 6e which was 39% as active as the antibiotics that target these organisms. Compound 6d displayed fluorescence in DMSO under the influence of 365 nm UV irradiation, absorption at λ_max = 421 which falls within the blue range, and fluorescence emission at λ_max = 523 nm. As photosensitizer (PS) of ROS in the presence of blue LED irradiation at a dose of 12 J/cm², it displayed better in vitro photodynamic inactivation than antibiotics against B. subtilis and E. coli O157. The activity against the positive-type bacteria was better than the Gram-negative one due to penetration intolerance by the outer membrane of the negative type as displayed by the confocal microscope images. Therefore, the activity is attributed to intracellular bacterial poisoning by the photo-generated ROS rather than membrane disruption.

Quinoline. 1

3&minus

Triazole. Chalcone. Ferrocene. Photodynamic inactivation. Confocal microscope

The bacterial immune system [1] is renewable machinery that is anticipated to hinder antibiotic-mediated therapy of pathogenic bacterial infections by 2050 [2]. This system can detoxify antibiotics through hydrolytic enzymes, e.g. β-lactamases in case of β−lactam antibiotics [3], drug efflux proteins [4], and even by encapsulation within resistant biofilms made of polysaccharides, proteins, and lipid architectures [5, 6]. Therefore, the search for alternative therapeutic solutions has become an urgent necessity.

In the meantime, photodynamic inactivation (PDI) of bacteria emerged as a potential lifeline. Under aerobic conditions, an organic photosensitizer (PS) upon photoexcitation can undergo an electronic state transition from a singlet-to-triplet spin state (S₁→T₁) through the radiation-less intersystem crossing. The T₁-state is stable enough to affect bactericidal chemical and/or physical changes in the bacterial cell. Thus, the T₁-state can acquire an electron from a nucleophile to reduce ground-state molecular oxygen giving a superoxide radical anion (^•O−O⁻). Then, this anion is reduced into H₂O₂ and finally splits into hydroxyl radicals HO^•. These PS-mediated chemical reactions are classified as a Type I pathway. Fortunately, molecular oxygen exists in its ground state in a non-toxic triplet spin state, therefore, the PS in its T₁-state is physically allowed to transfer its energy to triplet molecular oxygen (³O₂) and flip it into the excited singlet oxygen (¹O₂), a process that is named Type II pathway. The oxygen species arising from both types are well known as reactive oxygen species (ROS) which are harmful oxidants to biological systems [7, 8]. According to the penetrability of the PS within the bacterial cell, these ROS can attack the bacterial cell wall/membrane disrupting its integrity, cytoplasmic biomolecules, and organelles to potentiate bactericidal effects. For efficient PDI of bacteria, the PS is recommended to have no dark toxicity, photo-stability, high absorption coefficient, elevated quantum yield of its T₁−state as well as the ¹O₂, sufficient solubility, and selectivity for the microorganism [9]. Conjunction, PDI is still effective in hypoxic tissues, i.e. anaerobic conditions, where the PS can produce inorganic radicals of bactericidal effects as strong as the ROS in the so-called Type−III photochemical pathway. For instance, inorganic salts such as KI were found to reduce the effective dose of the PS 1000−fold and raise its efficiency against bacterial biofilms that are obstacles in face of PDI [7, 10].

For optimal PDI, minimal PS concentration and irradiation time are basic targets in PDI research since the light source must not harm the tissues and match the maximum absorption wavelength of the PS. Light-emitting diodes (LED) fulfill these requirements [11]. Most reports claim that the prospective development of bacterial resistance against PDI is nearly excluded as the PS needs not to enter the cell as the ROS can disrupt the bacterial membrane. Moreover, the ¹O₂ is able not only to resist the action of antioxidant enzymes such as catalase but also able to deactivate these defensive enzymes [12].

Artificial cationic dyes and common natural pigments were investigated as PSs in FDI of bacteria. Thus, Methylene blue as a cationic dye has the affinity to adhere to the highly negative bacterial membranes and induces efficient PDI of Gram−positive as well as Gram−negative bacterial species. It is approved for disinfecting blood products such as plasma and platelets. However, it's poor penetrability and allergenic effects are disadvantageous [13−15]. Riboflavin and psoralen are similarly approved in the same regard. Other PSs employed in PDI research entail porphyrin, xanthenes [16], ferrocenes [17, 18], chalcones [19], and quinolone-based alkaloids [20]. To increase the efficiency of the PDI, the bioavailability of the PSs within the bacterial cells was improved by penetration enhancers like EDTA [11] and cationic polypeptides that inflict disorganization of the outer membrane in Gram-negative bacteria [21]. As well, the incorporation of the PSs within artificial liposomes improves their intracellular accumulation and controls their release [22]. The adhesion of porphyrins to bacterial cells and even their endocytosis was modified by the glycosylation of pigments such as porphyrins [23]. Formulation of the PSs as nano-particles and carbon nanodots (CNDs) improved their accumulation and ¹O₂ production [23−25]. Dispersion or covalent binding of the PS in polymeric materials was described for manufacturing of coating surfaces which achieves continuous use without environmental leaching [22, 26].

Despite, the current facts about the advantages of PDI, bacterial resistance should not be secured and it is obligatory to continually develop renewable PSs of soft ROS high yielding potency, biocompatibility, low immunogenicity, and host toxicity. For this endeavor, a series of chalcones were prepared on a quinoline-1,2,3-triazole scaffold through CuAAC [27]. Their UV absorption and emission spectra were recorded. The −NMe₂ crowned derivative 6d, as fluorescence donor auxochrome, displayed an intense fluorescence emission spectrum, and its PDI of S. aureus and E. coli O157 are discussed.

Materials and methods

Chemistry

All chemicals and solvents were purchased from a collection of companies comprise Sigma-Aldrich, Acros, Advent/India, Loba/India, Oxford chemicals/India and Al Nasr/Egypt. Melting points were determined on the Electrothermal apparatus. Flash chromatography was carried out on silica gel (Baker, 30–60 µm) (Type-I silica gel) and LiChroprep Si 60 (Merck; Ø (15–25 µm) (Type-II silica gel). If not noted that Type-II is used, then the purification was carried out on Type -I. TLC Monitoring tests were carried out using plastic sheets precoated with silica gel 60 F245 (layer thickness 0.2 mm) purchased from Merck. Spots were visualized by their fluorescence under UV−lamp (λ 254 and 365 nm) or staining with iodine vapor, 15% H₂SO₄, KMnO₄, Hanessian’s stain (cerium ammonium molybdate stain or Mostain). NMR spectra were recorded on Bruker 400 MHz spectrometer, NMR unit, Faculty of Pharmacy, El Mansoura University. IR spectra were recorded on Thermo Fisher FT−IR Spectrophotometer from 500 to 4000 cm^− 1 at the microanalytical unit, Faculty of Science, El Mansoura University. Mass spectra were measured in the microanalytical unit at Al Azhar University, Cairo, Egypt. The UV−visible spectra were recorded using the Jasco v630 UV−visible double beam spectrophotometer, Japan, Faculty of science at Damietta University.

Synthesis of 4-Azido-2,6-dimethylquinoline (2)

A mixture of compound (1) [28] (2.0 g, 10.4 mmol) and NaN₃ (2.5 g, 38.4 mmol) in DMF (4.0 ml) was heated in a sand bath at 95−100 °C overnight. The Mixture was evaporated in vacuo, and the residue was taken in acetone and then co-evaporated with silica gel in vacuo. Flash chromatography (petroleum ether/ethyl acetate, 4:1) afforded compound 2 (1.19 g, 57%) as creamy crystals. R_f 0.28 (petroleum ether/ethyl acetate, 4:1), Mp 70−72 ^°C. IR (ύ, cm⁻¹): 3043, 3015 (C−H_str.Ar), 2914 (C−H_asy.str.Me), 2856 (C−H_sym.str.Me), 2111 (N_3str.), 1383 (CH_3Rock.). ¹H NMR (400 MHz, CDCl₃): δ_H 7.86 (d, 1H, J_7,8 8.0 Hz, H−8), 7.73 (s, 1H, H−3), 7.52 (dd, 1H, J_5,7 4.0, J_7,8 8.0 Hz, H−7), 6.94 (d, 1H, J_5,7 4.0 Hz, H−5), 2.70 (s, 3H, CH₃−2), 2.50 (4, 3H, CH₃−6); ¹³C NMR (100 MHz, CDCl₃): δ_C 157.93 (C = N), 147.10, 147.60, 135.73, 132.79, 127.92, 120.62, 119.90, 109.17 (8 C−Ar), 25.18 (CH₃−2), 21.63 (CH₃−6) ppm. EI−MS (m/z, %) for C₁₁H₁₀N₄ (198.23): 198.34 (M+), 197.56 (M-1,100), 184.09 (46.27), 160.29 (71.70), 156.30 (80), 125.99 (51.41), 121.12 (66.66), 119.10 (82.33), 115.14 (49.88), 10.32 (67.79), 94.29 (59.63), 81.13 (73.83).

General procedure for the synthesis of propargyl derivatives (3b and 3e)

A mixture of the hydroxy chalcones (E)-1-(4-hydroxyphenyl)-3-(p-tolyl)prop-2-en-1-one [29] or (E)-3-(4-chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one [30] (1.0 mmol), propargyl bromide (4.98 mmol, 5.0 eq.), K₂CO₃ (1.19 mmol, 1.2 eq.) and KOH (0.98 mmol, 1.0 eq.) in Acetone (5.0 ml) was stirred overnight at ambient temperature then purified by flash chromatography.

Characterization details of the two newly prepared propargylated chalcones

(E)-3-(4-Methylphenyl)-1-(4-(prop-2-ynyloxy)phenyl)prop-2-en-1-one (3b). Creamy crystals; (0.828 g, 71%) by (petroleum ether/ethyl acetate, 7:3); R_f 0.59 (petroleum ether/ethyl acetate, 7:3); Mp 92−94 °C; IR (ύ, cm⁻¹): 3284 (≡ C−H_str), 3027 (= C−H_str.), 2916 (−C−H_str.), 2119 (C ≡ C_str.), 1654 (C = O_str.), 1598 (C = C_str.), 1225 (C_Ar−O_str), 1017 (C_Al.−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.05 (d, 2H, J_AB 12.0 Hz, Ar), 7.80 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.54 (d, 2H, J_AB 8.0 Hz, Ar), 7.51 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.22 (d, 2H, J_AB 8.0 Hz, Ar), 7.06 (d, 2H, J_AB 8.0 Hz, Ar), 4.76 (d, 2H, J_gem., J_1,3 < 1.0 Hz, OCH₂), 2.60 (dd, 1H, J_1,3, J_1,3` < 1.0 Hz, ≡C−H), 2.38 (s, 3H, CH₃−4) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.73 (C = O), 161.15, 144.13, 140.95, 132.25, 131.92, 130.73, 130.36, 129.73, 129.43, 128.69, 127.38, 125.91, 120.70, 114.69 (12 C_Ar, CH = CHCO), 77.92−76.33 (≡ C_ipso, H−C≡), 55.87 (OCH₂), 21.58 (CH₃) ppm. EI−MS: (m/z, %) for C₁₉H₁₆O₂ (276.34): 276.45 (M⁺, 22.00), 238.34 (32.24), 211.88 (29.36), 186.74 (86.18), 137.30 (77.44) 100.84 960.42), 77.96 (100), 65.00 (70.96), 44.70 (32.82).

(E)-3-(4-Chlorophenyl)-1-(4-(prop-2-ynyloxy)phenyl)prop-2-en-1-one (3e). Yellow crystals; (0.34 g, 74%) by (petroleum ether/ethyl acetate, 4:1); R_f 0.33 (petroleum ether/ethyl acetate, 7:3); Mp 98−100 °C; IR (ύ, cm⁻¹): 3298 (≡ C−H_str.), 3069 (= C−H_str.), 2924 (−C−H_str.), 2123 (C ≡ C_str.), 1657 (C = O_str.), 1600 (C = C_str.), 1224 (C_Ar−O_str.), 1009 (C_Al.−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.06 (d, 2H, J_AB 8.0 Hz, H−2`_Ar, H−6`_Ar), 7.76 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.58 (d, 2H, J_AB 8.0 Hz, H−2_Ar, H−6_Ar), 7.52 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.40 (d, 2H, J_AB 8.0 Hz, H−3_Ar, H−5_Ar), 7.08 (d, 2H, J_AB 8.0 Hz, H−3`_Ar,H−5`_Ar), 4.78 (d, 2H, J_gem., J_1,3 4.0 Hz, OCH₂), 2.59 (dd, 1H, J_1,3, J_1,3` 4.0 Hz, ≡C−H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.83 (C = O), 161.30 (CH = CHCO), 142.71, 136.26, 133.50, 131.01, 130.79, 130.35, 129.57, 129.24, 128.76, 128.30, 127.78, 122.18, 114.78 (12 C_Ar, CH = CHCO), 77.77−76.24 (≡ C_ipso, ≡C−H), 55.92 (OCH₂) ppm. EI−MS (m/z, %) for C₁₈H₁₃ClO₂ (296.75): 296.72 (M⁺, 40.00), 295.47 (M−1, 10.78), 294.60 (M−2, 17.13), 267.26 (80.81), 242.21 (80.73), 201.72 (73.15), 159.11 (36.49), 125.23 (100.00), 91.10 (32.91), 66.22 (91.79).

General procedure for the synthesis of the target triazoles

A mixture of the terminal alkynes 3a−e, 4a,b [31−33], 5 [34] (0.65 mmol), 4-azido-2,6-dimethylquinoline 2 (0.5 mmol), CuSO₄. 5H₂O (0.24 mmol) and L-ascorbic acid (1.4 mmol) in THF−H₂O (4:1, 5 ml) was gently refluxed with stirring for 4 h. The mixture was diluted with acetone then co-evaporated with silica gel in vacuo then purified by flash chromatography.

Characterization details of the newly synthesized of the target triazoles

(E)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-phenylprop-2-en-1-one (6a). yellow crystals; (0.11 g, 73%) on Type-II silica (petroleum ether/ethyl acetate, 2:1); R_f 0.32 (petroleum ether/acetone, 2:1); Mp 120−125 °C; IR (ύ, cm⁻¹): 3055 (= C−H_str.), 2919 (−C−H_Asy.str.), 1658 (C = O_str), 1603 (C = N_str., C = C_str.), 1338 (C_Ar−N_str.), 1223 (C_Ar−O_str), 1020 (C_Aliph−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.05−8.00 (m, 4H, J_AB 8.0 Hz, H−5_Triaz., H−8_Quin., 2Ar), 7.98−7.96 (d, 2H, J_AB 8.0 Hz, Ar), 7.93−7.90 (dd, 1H, J_AB 4.0, 4.0 Hz, H−7_Quin.), 7.76−7.72 (d, 1H, J_α,β 16.0, CH = CHCO), 7.58−7.56 (d, 2H, J_AB 8.0 Hz, Ar.), 7.54 (s, 1H, H−3_Quin.), 7.50−7.49 (d,1H, J_AB 4.0 Hz, H−5_Quin.), 7.49−7.46 (d, 1H, J_α,β 12.0, CH = CHCO), 7.09−7.07 (d, 2H, J_AB 8.0 Hz, Ar), 7.04−7.02 (d, 1H, J_AB 8.0 Hz, Ar), 5.40 (s, 2H, OCH₂), 2.73 (s, 3H, C₂−CH₃), 2.42 (s, 3H, C₆−CH₃) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.68 (C = O), 161.85, 161.77, 157.93, 144.33, 144.23, 140.99, 138.49, 134.96, 133.67, 131.84, 130.93, 130.73, 130.49, 129.35, 128.98, 128.42, 128.26, 124.93, 121.68, 121.27, 120.62, 117.38, 114.68, 114.56, 114.52 (23 C_Ar, CH = CHCO), 61.95 (OCH₂), 26.42 (C₂−CH₃), 21.94 (C₆−CH₃) ppm. EI-MS (m/z, %) for C₂₉H₂₄N₄O₂ (460.54): 459.99 (M⁺, 33.71), 457.72 (M−2, 21.87), 422.23 (64.13), 393.54 (62.15), 345.34 (55.32), 296.24 (68.79), 255.27 (70.18), 212.55 (100.00), 159.82 (70.29), 120.02 (80.55). UV−vis (DMSO): λ_max (nm); 269, 320.

(E)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(p-tolyl)prop-2-en-1-one (6b). Faint brown crystals; (0.22 g, 86%) by (Petroleum ether/Acetone, 2:1); R_f 0.26 (petroleum ether/acetone, 2:1); Mp 156−158 °C; IR (ύ, cm⁻¹): 3021 (= C−H_str.), 2918 (−C−H_Asy.str.), 2868 (−C−H_Sym.str.), 1657 (C = O_str),1601 (C = N_str., C = C_str.), 1337 (CH_3Rock.), 1229 (C_Ar−O_str), 1030 (C_Al−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.14 (s, 1H, H−5_Triaz.), 8.10−8.05 (2d, 3H, J_AB 8.0, 12.0 Hz, H−8_Quin., 2Ar), 7.83−7.79 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.64 (d, 1H, J_AB 8.0, H−7_Quin.), 7.57−7.51 (2d, 3H, J_AB 8.0, J_αβ 16.0, CH = CHCO, 2Ar), 7.41 (s, 1H, H−5_Quin.), 7.28 (s, 1H, H−3_Quin.), 7.25−7.23 (d, 2H, J_AB 8.0, Ar), 7.16 (d, 2H, J_AB 8.0, Ar), 5.49 (s, 2H, OCH₂), 2.82 (s, 3H, C₂−CH₃), 2.51 (s, 3H, C₆-CH₃), 2.41 (s, 3H, Tol-CH₃) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.79 (C = O), 161.69, 157.93, 146.81, 144.43, 144.25, 141.03, 138.49, 133.69, 132.21, 131.97, 131.46, 130.88, 129.72, 129.49, 128.98, 128.45, 128.01, 124.89, 121.29, 120.66, 120.62, 117.36, 114.64 (23 C−Ar, CH = CHCO), 61.95 (OCH₂), 24.54 (C₂−CH₃), 21.93(C₆−CH₃), 21.56 (Tol−CH₃) ppm; EI−MS (m/z,%) for C₃₀H₂₆N₄O₂ (474.56): 472.73 (M−2, 20.31), 461.40 (80.63), 368.40 (62.16), 327.95 (29.92), 255.33 (100), 159.35 (36.05), 79.08 (50.06). UV−vis (DMSO): λ_max (nm); 269, 327.

((E)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4methoxyphenyl)prop-2-en-1-one (6c). Yellow crystals; (0.3 g, 63%) by (petroleum ether/acetone, 2:1); Mp 124−126 °C; IR (ύ, cm⁻¹): 3139 (= C−H_str), 2921 (−C−H_str.Asy.), 1656 (C = O_str), 1601 (C = N_str., C = C_Str.), 1223 (C_Ar−O_str.), 1033 (C_Al−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.14 (s, 1H, H−5_Triaz.), 8.08 (d, 1H, J_AB 12.0 Hz, H−8_Quin.), 8.06 (d, 2H, J_AB 12.0 Hz, Ar), 7.82, 7.79 (d, 1H, J_α,β 12.0 Hz, CH = CHCO), 7.65−7.58 (2d, 4H, J_AB 8.0, 12.0 Hz, H−3_Quin., H−7_Quin., 2Ar), 7.47, 7.43 (d, 1H, J_α,β 16.0, CH = CHCO), 7.41 (s, 1H, H−5_Quin.), 7.16, 6.95 (2d, 4H, J_AB 8.0, 8.0 Hz, H−3, H−3`, H−5, H−5`), 5.49 (s, 2H, OCH₂), 3.86 (s, 3H, OCH₃), 2.82 (s, 3H, C₂−CH₃), 2.51 (s, 3H, C₆−CH₃) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.733 (C = O), 161.64, 161.61, 158.10, 147.70, 144.16, 144.12, 140.49, 138.05, 133.23, 132.07, 131.52, 131.36, 130.81, 130.18, 128.69, 127.68, 124.90, 121.09, 120.59, 119.33, 117.35, 114.62, 114.42, 113.61, 101.38 (23 C−Ar, CH = CHCO), 61.97 (OCH₂), 55.44 (OCH₃), 25.01 (C₂−CH₃), 21.80 (C₆−CH₃) ppm; EI−MS (m/z, %) for C₃₀H₂₆N₄O₃ (490.56): 490.08 (M⁺, 30), 479.58 (51.85), 338.83 (41.61), 283.47 (76.48), 255.47(63.57), 202.97(51.44), 185.28(100), 132.50(38.70), 97.84(44.68), 74.33(70.06), 41.29(84.01). UV−vis (DMSO): λ_max (nm); 269, 343.

(E)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4(dimethylamino)phenyl)prop-2-en-1-one (6d). Orange crystals; (0.11 g, 98%) by (petroleum ether/ethyl acetate, 1:1); R_f 0.17 (petroleum ether/ethyl acetate, 1:1); Mp 124−126 °C; IR (ύ, cm⁻¹): 3147 (≡ C−H_str), 2921 (−C−H_Asy.str.), 1651 (C = O_str), 1601 (C = N_str., C = C), 1228 (C_Ar−O_str.), 1170 (C_Al−N_str.), 1009 (C_Al−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.13 (4, 1H, H−5_Triaz.), 8.09−8.05 (m, 3H, J_AB 8.0 Hz, Ar), 7.83−7.80 (d, 1H, J_α,β 12.0 Hz, CH = CHCO), 7.65−7.63 (dd, 1H, H−7_Quin.), 7.57 (d, 3H, J 8.0 Hz, Ar), 7.41 (s, 1H, Ar), 7.37 (d, 1H, J_α,β 16.0 Hz CH = CHCO), 7.15 (d, 2H, J_AB 8.0 Hz, Ar), 6.71 (d, 2H, J_AB 8.0 Hz, Ar), 5.49 (s, 2H, OCH₂), 3.06 (s, 6H, NMe₂), 2.83 (s, 3H, C₂−CH₃), 2.51 (s, 3H, C₆−CH₃) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.83 (C = O), 161.27, 157.78, 151.91, 145.32, 144.50, 141.46, 138.85, 134.05, 132.64, 130.64, 130.37, 127.43, 124.85, 122.83, 121.47, 120.65, 117.37, 116.46, 114.50, 111.94 (23 C−Ar, CH = CHCO), 61.91 (OCH₂), 40.23 (NMe₂), 21.97 (CH₃) ppm; EI−MS (m/z, %) for C₃₁H₂₉N₅O₂ (503.61): 503.45 (M⁺), 477.10 (50.32), 458.59 (18.07), 442.02 (26.16), 406.15 (39.15), 375.00 (52.13), 333.75 (57.36), 294.85 (47.79), 255.35 (73.84), 238.30 (95.41), 195.16 (60.58), 182.23 (100). UV−vis (DMSO): λ_max (nm); 269, 290, 421.

(E)-3-(4-chlorophenyl)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)prop-2-en-1-one (6e). Yellow crystals; (0.06 g, 20%) by (petroleum ether/acetone, 2:1); R_f 0.25(petroleum ether/acetone, 2:1); Mp 152−154 °C; IR (ύ, cm⁻¹): 3144 (= C−H_str.), 2920 (−C−H_Asy.str.), 2852 (−C−H_Sym.str.), 1657 (C = O_str), 1604 (C = N_str., C = C_str.), 1227 (C_Ar−O_str.), 1014 (C_Al−O_str.); ¹H NMR (400 MHz, CDCl₃): δ_H 8.22− 8.20 (d, 2H, H−5_Triaz., H−8_Quin.), 8.09−8.07 (d, 2H, J_AB 8.4 Hz, Ar), 8.00−7.98 (d, 2H, J_AB 8.8 Hz, Ar), 7.78−7.74 (d, 1H, J_αβ 15.6 Hz, CH = CHCO), 7.67 (s, 1H, H−3_Quin.), 7.59−7.57 (d, 1H, J_α,β 8.0 Hz, CH = CHCO), 7.48 (s, 1H, H−5_Quin.), 7.41− 7.39 (d, 1H, J_AB 8.0 Hz, H−7_Quin.), 7.17− 7.15 (d, 2H, J_AB 8.4 Hz, Ar), 7.12−7.10 (d, 2H, J_AB 8.4 Hz, Ar), 5.48− 5.46 (d, 2H, J_gem., J_1,3 9.2 Hz, OCH₂), 2.893 (s, 3H, C₂−CH₃), 2.523 (s, 3H, C₆-CH₃) ppm; ¹³C NMR (100 MHz, CDCl₃): δ_C 188.42 (C = O), 161.85, 157.87, 146.27, 144.24, 142.85, 141.31, 138.76, 136.36, 133.97, 133.44, 131.65, 130.96, 130.74, 129.57, 129.25, 128.75, 128.47, 127.55, 124.95, 124.92, 122.07, 121.40, 120.63, 117.40, 114.51 (23 C−Ar, CH = CHCO), 61.88 (OCH₂), 24.24 (C₂−CH₃), 21.94 (C₆−CH₃) ppm; EI−MS (m/z, %) for C₂₉H₂₃ClN₄O₂ (494.98): 495.60 (M⁺,18.1), 468.61 (59.90), 381.47 (85.42), 363.57 (100.00), 272.28 (45.94), 205.66 (70.17), 137.75 (36.99), 76.23 (52.20). UV−vis (DMSO): λ_max (nm); 269, 322.

( E)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(furan-2yl)prop-2-en-1-one (7a). Creamy crystals; (0.28 g, 11%) by (petroleum ether/acetone, 1.5:1); R_f 0.22 (Petroleum ether/Acetone, 1.5:1); Mp 158−160 °C; IR (ύ, cm⁻¹): 3921 (−C−H_Asy.str.), 1658 (C = O_str.), 1604 (C = N_str., C = C_str.), 1229 (C_Ar−O_str), 1015 (C_Al−O_str.); ¹H NMR (400 MHz,): δ_H 8.11 (s, 1H, H−5_Triaz.), 8.01−7.99 (d, 3H, J_AB 6.4 Hz, H−8_Quin., 2Ar), 7.58 (s, 1H, H−3_Quin.), 7.53−7.39 (3d, 4H, J_α,β 15.6, 15.2 Hz, CH = CHCO, 1H_Fur., H−7_Quin.), 7.20 (s, 1H, H−5_Quin.), 7.07−7.05 (d, 2H, J_AB 6.4 Hz, Ar), 6.64 (1H_Fur.), 6.45 (1H_Fur.), 5.39 (s, 2H, OCH₂), 2.79 (s, 3H, C₂−CH₃), 2.453 (s, 3H, C₆−CH₃) ppm; ¹³C NMR (100 MHz, DMSO): δ_C 188.10 (C = O), 161.74, 157.88, 151.72, 146.44, 144.86, 144.28, 141.21, 138.67, 133.87, 131.81, 130.85, 130.30, 129.58, 127.68, 124.91, 121.36, 120.62, 118.95, 117.38, 116.15, 114.64, 112.70 (21 C−Ar, CH = CHCO), 61.91 (OCH₂), 24.36 (C₂−CH₃), 21.94 (C₆−CH₃) ppm; EI−MS (m/z, %) for C₂₇H₂₂N₄O₃ (450.50): 450.93 (M⁺, 22.51), 375.29 (40.72), 255.44 (100), 209.42 (35.26), 144.44 (54.19), 82.36 (49.61), 44.29 (22.93). UV−vis (DMSO): λ_max (nm); 269, 344.

(E)-1-(4-((1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3(thiophen-2-yl)prop-2-en-1-one (7b). Faint brown crystals; (0.46 g, 98%) by (petroleum ether/acetone, 2:1); R_f 0.27 (petroleum ether/acetone, 2:1); Mp 200−202 °C; IR (ύ, cm⁻¹): 3063 (= C−H_str.), 2922 (−C−H_Asy.str.), 1650 (C = O_str), 1598 (C = N_str., C = C_str.), 1221 (C_Ar−O_str), 1019 (C_Al−O_str.); ¹H NMR (400 MHz, DMSO): δ_H 8.98 (s, 1H, H−5_Triaz.), 8.17 (d, 2H, J_AB 8.0 Hz, Ar), 8.02 (d, 1H, J_AB 8.0 Hz, H−8_Quin.), 7.91 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.80 (d, 1H, J_AB 8.0 Hz, H−7_Quin.), 7.73 (s, 1H, H−3_Quin.), 7.71−7.70 (d, 1H, J_AB 4.0 Hz, Ar), 7.64−7.60 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.55 (s, 1H, H−5_Quin.), 7.31−7.28 (d, 2H, J_AB 12.0 Hz, Ar), 7.22−7.20 (t, 1H, J_AB 4.0 Hz, Ar), 5.49 (s, 2H, OCH₂), 2.75 (s, 3H, C₂−CH₃), 2.47 (s, 3H, C₆−CH₃) ppm; ¹³C NMR (100 MHz, DMSO): δ_C 187.34 (C = O), 162.33, 158.86, 147.73, 143.39, 140.32, 140.26, 138.49, 137.68, 136.50, 133.22, 133.07, 131.30, 131.21, 131.08, 130.70, 129.18, 129.08, 127.58, 121.55, 120.77, 120.69, 118.31, 115.37 (21 C−Ar, CH = CHCO), 61.66 (OCH₂), 25.09 (C₂−CH₃), 21.82 (C₆−CH₃) ppm; EI-MS (m/z, %) for C₂₇H₂₂N₄O₂S (466.56): 466.05 (M+, 11.71), 429.70 (39.95), 378.07 (39.78), 375.29 (40.72), 331.58 (37.88), 255.44 (100), 221.00 (13.11). UV−vis (DMSO): λ_max (nm); 269, 344.

(2E)-3-[4-[[1-(2,6-dimethylquinolin-4-yl)-1H-1,2,3triazol-4-yl]methoxy]phenyl]-1-(ferrocen-3-yl)prop-2-en1-one (8). Red crystals; (0.33 g, 97%) by (petroleum ether/acetone, 2:1); R_f 0.26 (petroleum ether/acetone, 2:1); Mp 172−174 °C; IR (ύ, cm⁻¹): 3130 (= C−H), 2923 (−C−H_Asy.str.), 1645 (C = O_str.), 1588 (C = N_str.), 1239 (C_Ar-O_str.), 1021 (C_Ar−O_str.); ¹H NMR (400 MHz, DMSO): δ_H 8.97 (s, 1H, H−5_Triaz.), 8.03−8.01 (d, 2H, J_AB 8.0 Hz, Ar), 7.90−7.88 (d, 1H, J_AB 8.0 Hz, Ar), 7.73−7.71 (m, 2H, J_AB 8.0 Hz, Ar), 7.65−7.62 (d, 1H, J_α,β 12.0 Hz, CH = CHCO), 7.55 (s, 1H, H−5_Quin.), 7.38−7.34 (d, 1H, J_α,β 16.0 Hz, CH = CHCO), 7.24−7.22 (d, 2H, J_AB 8.0 Hz, Ar), 5.43, 5.06, 4.67 (s, 7H−Fc), 4.23 (s, 5H, OCH₂, 3H−Fc), 2.74 (s, 3H, CH₃−2_Quin.), 2.48 (s, 3H, CH₃−6_Quin.) ppm; ¹³C NMR (100 MHz, DMSO): δ_C 133.84 (C = O), 132.68 (1C), 70.0 (1C), 41.88, 40.64, 40.44, 40.23, 40.02, 39.82, 39.61, 39.41 (8 C), 24.33 (CH₃) ppm; EI−MS (m/z, %) for C₃₃H₂₉FeN₄O₂ (568.16): 566.30 (M−2, 32.50), 539.85 (61.49), 488.46 (84.09), 390.61 (83.15), 315.54 (45.00), 249.25 (100), 217.55 (55.89), 173.32 (57.09), 137.74 (37.83), 94.42 (46.94), 48.63 (67.45). UV−vis (DMSO): λ_max (nm); 269, 334, 488.

Biological evaluations

In vitro antimicrobial activity

The antimicrobial activity of the new triazoles was determined using a modified Kirby-Bauer disc diffusion method (Bauer, et al., 1966) [35]. Briefly, 100 µl of the test bacteria/fungi were grown in 10 ml of fresh media until they reached a count of approximately 10⁸ cells/ml for bacteria or 10⁵ cells/ml for fungi (Pfaller, et al., 1988). 100 µl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained.

Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method (NCCLS, 1993, NCCLS, 1997).

Plates inoculated with filamentous fungi as Aspergillus flavus at 25^oC for 48 hours; Gram (+) bacteria as Staphylococcus aureus (ATTC 12600), and Gram (−) bacteria as Escherichia coli (ATCC 11775). They were incubated at 35−37^oC for 24−48 hours and yeast as Candida albicans were incubated at 30^oC for 24−48 hours and, then the diameters of the inhibition zones were measured in millimeters (Bauer et al., 1966).

Standard discs of Ampicillin and Kanamycin (Antibacterial agent), and Amphotericin B (Antifungal agent) served as positive controls for antimicrobial activity but filter discs impregnated with 10 µl of DMSO were used as a negative control. Blank paper disks (Schleicher & Schuell, Spain) with a diameter of 8.0 mm were impregnated with 10 µl of stock solutions of the new triazoles. When a filter paper disc impregnated with a tested chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a “Zone of inhibition” or " Clear zone". For the disc diffusion, the zone diameters were measured with slipping calipers of the National Committee for Clinical Laboratory Standards (NCCLS, 1993).

The antibacterial activity of the new triazoles in vitro was evaluated against E.coli (ATCC 11775) and S. aureus (ATTC 12600) according to the Kirby–Bauer disc diffusion method (Table 1). DMSO was used as a solvent and negative control, while Kanamycin was used as a positive control in the case of S. aureus and ampicillin in the case of E. coli.

Photodynamic Inactivation of Bacteria

Two standard suspensions of bacteria were used. The first suspension represents Escherichia coli O157 (E. coli O157), delegated as a Gram-negative (−ve) bacteria. The second suspension is Bacillus subtilis (B. subtilis), representative as a Gram-positive (+ ve) bacteria. Bacteria were grown overnight aerobically on nutrient agar at 37°C. The standard suspension of E.coliO157 strain and B. subtilis strain containing 10⁹/mL viable cells were prepared as follows:- (1) A single colony of each type of bacteria was isolated separately from an agar plate and inoculated in Luria Bertani (LB) broth medium. (2) Each inoculated broth was incubated for 48 h at 37^◦C. (3) The number of viable cells in each bacterial suspension was measured by using a Uv–visible power wave microplate spectrophotometer (BioTech, Vermont, USA) to an optical density (OD) of 0.5 at 600 nm (corresponding to 1 × 10⁹ cells/mL). (4) The bacterial suspension was diluted with LB broth media to start the inoculum of approximately 10⁶ colony-forming units/ml. These colonies were indicated by agar plate counts.

Light source

A light-emitting-diode (LED) with a blue wavelength was used to excite the PS 6d as a source of irradiation with homogeneous and stable irradiation. LED (LED Edixeon, Edison Opto Corporation, New Taipei City, Taiwan) produces a light emission at the blue wavelength of 460 nm with a power density of 100 mW. The irradiances (energy fluencies) of the blue LED array tested in this study were 6, 12, 24, and 48 J/cm², using a PM100D power/energy meter (Thorlabs, Inc., Newton, NJ). This was adapted by regulating the distance between the bacterial cells and the LED array aperture [36].

Growth profiles

Ideally, 1% of bacterial cells were placed in Luria Bertani (LB) medium broth in a flask, in which the nutrients and environmental conditions are controlled. Sterile 96-well tissue culture plates were used. The bacterial kinetic growth can be measured as a function of time in the 96-well microplates (as turbidity) with a microplate reader (Tecan Infinite 200). The optical density at 600 nm (OD₆₀₀) of the cultures was measured by a spectrophotometer.

Photodynamic inactivation effect

The bacterial cells were treated with compound 6d at five different concentrations 1, 2, 4, 8, and 10 m and each concentration was prepared in three replicates. 150 µL of each bacterial suspension was seeded in a 96-well microtiter plate with a flat bottom. Each bacterial strain was divided into five groups. These groups are categorized as (1) negative control group. (2) positive control group and (3) dark toxicity group. The bacteria were incubated with five different concentrations of the photo probe. (4) Irradiated group only, in which both bacterial strains were displayed to four different doses of the blue LED (6, 12, 24, and 48 J/cm²), without the three compounds. (5) PDT group, both bacterial strains were kept in the dark condition at 37°C for 30 min (pre-irradiation time) with different concentrations of the probe. The PDT experiment group was irradiated by a blue LED at a dose of 12 J/cm². The constant temperature conditions in 96-well plates were provided. Samples were analyzed under a temperature of 37°C, shaking between measurements and data recording after 48h. Survival was calculated from the last point in the growth curves, related to the control value.

Live cell imaging by confocal laser scanning Microscopy (CLSM)

The cellular internalization efficiency of the probe by the bacteria was estimated by live cell imaging. About 30 µM from each bacterial strain was exposed to the probe at a concentration of 10 mM placed on a glass slide. The sample was covered with a glass slide and then examined by CLSM. The experiments on live bacteria were immediately started after adding the samples. Images were taken by using 0.2% of the 488 nm laser line.

Statistical analysis

The experimental five groups were performed in triplicates. The significant difference between the control group and the other treated groups for each bacterial species was assessed by a One-way ANOVA test. Statistical significant values set at p < 0.05. The data were analyzed using statistical analysis software.

Chemistry

New eight chalcone conjugates 6a−e, 7a,b, and 8 were prepared to consider their latent antibacterial activity and then their UV−visible absorption spectra to select the dark non-toxic fluorescent conjugate(s) for PDI investigations (Scheme 1). Structurally, these conjugates are chalcones supported on a quinoline scaffold through a 1,2,3-triazole ring. The chalcone moiety is tethered with different tags entailing simple aryls as ring B. The N,N-dimethyl tag was amongst as it was observed to endow fluorescence, subsequently, it is described as fluorescence donor auxochrome. Exceptionally, compound 8 is crowned with a bulky ferrocene moiety as ring A for its biologically and optically significant redox potential.

Generally, the synthetic protocol was quite simple and based on the CuAAC of 4-azido-6-chloro-2-methylquinoline 2 with the propargylated chalcones 3a−e, 4a,b, and 5.

The quinoline scaffold 4-azido-2,6-dimethylquinoline 2 was prepared by azidolysis of 4-chloro-2,6-dimethylquinoline 2 [28] with NaN₃ in warm DMF (Scheme 1). Compound 2 displayed a strong N_3str. band at 2111 cm⁻¹. The chemical shift (¹H NMR) of the two CH₃ groups was assigned based on a comparison with the known 4-azido-6-chloro-2-methylquinoline [29]. The ¹³C NMR spectrum accounted for the theoretical number of carbon atoms and the 135º−DEPT spectrum displayed only positive signals.

All terminal alkyne substrates are known from previous investigations [31−33] in our group except compounds 3b,e. This pair was prepared by propargylation of the relevant hydroxychalcone with propargyl bromide in acetone containing K₂CO₃. They showed vibrational frequencies at about 3290, 2120, and 1655 cm⁻¹ corresponding to the ≡C−H_str., C≡C_str. and C = O_str., respectively.

Their ¹H NMR spectra, for instance, chalcone 3b, showed the signals of the terminal propargyl group at δ 4.7 ppm (CH₂), 2.6 ppm (H−C≡C) as well as the CH₃ at 2.4 ppm. In the ¹³C NMR spectrum, the characteristic signals of this group were observed at δ 76 ppm (C≡C), and 56 ppm (CH₂).

Coupling of the azido and propargyl substrates under CuAAC conditions afforded the target 1,4-disubstituted-1,2,3-triazoles 6a−e, 7a,b and 8 in yields ranging from weak to nearly quantitative. The mass values agreed with the expected formulas. The IR spectra hold the bathochromic shifted C = O_str band at ύ ≈1658 cm⁻¹ with the concurrent demise of the N₃ and the H−C≡C− stretching bands. ¹H NMR spectra showed a sum of the substrate signals free of the alkyne proton signal. Both the ¹H NMR and the ¹³C NMR spectra displayed the key signals of the reactants. At least, the 2CH₃ groups of quinolone as well as the C = O of the chalcones.

UV-Visible absorption and fluorescence spectroscopy

The fluorescence of the dilute solution of the chalcones 6a‒e, 7a,b, and 8 under UV irradiation at 365 nm is viewed in (Fig. 1). DMSO was chosen as a solvent since it is biocompatible for prospective biological investigations. Fortunately, it does not quench fluorescence. The figure displays the strong green fluorescence of the −NMe₂-tagged chalcone derivative 6d as expected.

The UV-visible absorption spectrum of the previous solutions (Fig. 2), obviously shows two principal λ_max for all chalcones and an exceptional third one in the case of compounds 6d and 8. The first absorption band at a fixed value of 269 nm in all chalcones is attributed to the allowed π−π* transition of the olefinic bond in the β−enone moiety. The second absorption maximum was observed within the range 320−344 nm except in the case of compound 6d which is hypthochromic shifted to 290 nm. This band has lower intensity compared to the first one since it is attributed to the forbidden n−π* transition of an electron from a lone pair of electrons of the carbonyl oxygen atom in the β−enone unit. The blue shift of the last transition in compound 6d might be attributed to the perpendicular orientation of the −NMe₂ group on the benzene ring. This situation prohibits the delocalization of the nitrogen atom lone-pair of electrons with the benzene ring and consequently the enone system. Then the electronegativity of the nitrogen atom in this case inhibits polarization of the carbonyl group and decreases the energy of the HOMO then increase the E_g with the LUMO.

Compound 6d displayed a third absorption band at λ_max 421 nm corresponding to the n−π* of one electron of the lone-pair of electrons of the nitrogen atom in the −NMe₂ group. This absorption requires less energy compared to that of the oxygen atom which is logical in terms of the electronegativity difference between both atoms. The ferrocene derivative 8 displayed a third band of quite low intensity at 488 nm (Table 1).

Table 1

Absorption maxima by compounds **6a−e, 7a,b**, and 8 in the UV-vis. Spectrum.
Compd.	λ_max (π−π*)_nm/A	λ_max [n−π* (O)] _nm/A	λ_max [n−π* (N)] _nm/A	λ_max (Emission)_nm
6a	269/2.0805	3201/0.4436	−	−
6b	2690/0.7743	327/0.9490	−	−
6c	269/0.7301	343/0.7915	−	−
6d	269/2.2118	2901.1684	421	539
6e	269/2.3104	322/0.8425	−	−
7a	269/0.8320	344/0.8269	−	−
7b	269/0.6909	344/0.4997	−	−
8	269/2.0949	334/1.3772	−	−

Finally, the fluorescence emission spectrum of compound 6d in DMSO showed a unique strong band at 539 nm (Fig. 3). From these comparative spectroscopic features, compound 6d seemed to be qualified for PDI investigation on two bacterial strains representing Gram-positive (B. subtilis) and Gram-negative species (E. coli O157). Luckily, this probe was inactive in the in vitro antimicrobial screening assay, therefore it was passed into PDI investigations as well be discussed next.

Biology

Antimicrobial activity

The in vitro antibacterial activity of all chalcones was evaluated against E.coli (ATCC 11775) and S. aureus (ATTC 12600) according to the Kirby–Bauer disc diffusion method. DMSO was used as a solvent and negative control. Kanamycin was used as a positive control in the case of S. aureus and Ampicillin for E. coli.

The whole series was inactive against the Gram−positive bacteria S. aureus and the Gram−negative strain E. coli except the chalcone 6e (Table 2). This chalcone was 39% as active as Kanamycin against S. aureus but 56% less active than ampicillin in the case of E. coli. SAR analysis reveals a unique feature in this chalcone compared to other chalcones is the p-chloro residue that is responsible for emerging this weak activity. It might be speculated that only this chalcone can mainly penetrate the murein wall which is common in both Gram−positive and Gram-negative bacteria. Further, it can penetrate the outer membrane of the Gram-negative type.

Table 2

Inhibition zone diameter (mm) of the new chalcone series.
Entry	Compound	S. aureus	E. coli	A. flavus	C. albicans
1	6a	0.0	0.0	0.0	0.0
2	6b	0.0	0.0	0.0	0.0
3	6c	0.0	0.0	0.0	0.0
4	6d	0.0	0.0	0.0	0.0
5	6e	11.0^b ± 1.0	11.33^b ± 0.577	0.0	0.0
6	7a	0.0	0.0	0.0	0.0
7	7b	0.0	0.0	0.0	0.0
8	8	0.0	0.0	0.0	0.0
11	Kanamycin	28	−	−	−
12	Ampicillin	−	25	−	−
13	Amphotericin B	−	−	17	21

The antifungal activity of the whole series was investigated against A. flavus and C. albicans following the same procedure. The whole series was inactive against these fungal strains. Presumably, the fungal chitin shell is resistant to endocytosis of these chalcones.

Photodynamic inactivation investigations

From the previous physicochemical investigations, we concluded that compound 6d, as theoretically hypothesized, is qualified to investigate its PDI of model bacterial strains representing both Gram−positive and Gram−negative bacteria. However, to go on with these investigations, we reinvestigated its dark toxicity against (B. subtilis) as Gram−positive bacteria and (E. coli O157) which represents Gram−negative bacteria. Another interesting preliminary prerequisite before PDI investigations is to select the tolerant light dose for these species. These precautions will be addressed for investigating the real potential of compound 6d as PS in PDI of bacteria.

Thus, the antimicrobial effect of compound 6d on these species was carried out by the turbidimetric method using a microplate assay to evaluate its dark toxicity. Dimethyl sulfoxide (DMSO) was used as a solvent and its 5% aqueous solution was the negative control. Pre-investigations showed that it has no significant effect on the growth of both strains.

(Fig. 4A,a) concludes the dark toxicity results of the conjugate 6d at five different concentrations on B. subtilis and E. coli O157. For accurate measurement, we used two controls; negative control and positive control for each strain. Negative control represents the bacterial strain without any addition, but the positive control refers to the bacterial strains in the presence of a definite dose of Amoxicillin as an effective antibiotic against both Gram-positive and Gram-negative bacteria. The bar graphs show that compound 6d has a negative effect on the survival of B. subtilis and E. coli O157 when compared to the two controls. The proliferation effect did not affect by increasing the concentration of the probe, thus the probe met a requirement as potential PS.

Then we moved into the selection of the light source and its tolerant dose to the considered bacterial species. The UV−visible absorption spectrum of compound 6d (Fig. 2) displayed its efficient absorption within the blue range (400−500 nm), precisely at λ_max value of 421 nm. Then, a 460 nm blue light-emitting diode (LED) was used as a source of irradiation and its effect on the growth of both bacterial species was investigated in absence of compound 6d at different energy densities (Fig. 4B,b). The results displayed that the survival of both bacterial strains is inversely proportional to the elevation in the energy irradiation. Interestingly, the survival of the bacteria was consolidated with a dose of 12 J/cm². Consequently, this dose was chosen as a working irradiation in the next PDI experiments.

Then, the PDI effect of compound 6d on the survival of both bacterial strains was done at concentrations of 1.0, 2.0, 4.0, 8.0, and 10.0 mM and a standard blue LED irradiation density of 12 J/cm² (Fig. 4C,c). Interestingly, the results of the PDI indicated a significant reduction in bacterial survival of E. coli O157 and B. subtilis relative to the negative and positive controls in which neither the blue light nor the probe was used. The bactericidal effect of the probe was dose-responsive until reaching an optimum concentration of 4.0 mM where further increments become ineffective.

These results stand out as the influence of compound 6d as PS under the working blue LED light irradiation on the survival of both bacterial strains and its discriminatory bactericidal effect on the Gram−positive type over the negative one. In principle, the outer membrane of the Gram-negative type seems to be a crucial factor in this contrast.

To address the relation between the endocytosis capability of the probe and the strength of its bactericidal effect, the treated bacterial cells with the compound 6d at 10 mM were 3D-optically imaged by the confocal laser scanning microscope (CLSM) (Fig. 5a,b).

Comparing the fluorescence emission of the PS from E. coli O157 (Fig. 5a) and that of B. subtilis (Fig. 5b) discloses stronger emission from the cells of B. subtilis than those of E. coli O157. Thus, compound 6d has high penetration affinity through the bacterial murein-made cell wall. It accumulated inside Gram-positive bacteria more than in the negative type, despite the cell−wall of the later type of bacteria being thinner than the Gram−positive strains. Therefore, the outer membrane of the Gram−negative bacteria resists the translocation of the probe inside their cells. More than that, this PS has better penetration affinity through the cell wall than adhesion with it and the strength of the ROS generated by this PS is strong enough to resist both the bacterial antioxidant enzymes and drug efflux proteins if any. Also, they were able to harm intracellular macromolecules and structures, but they were weak to attack the tougher outer membrane. In other words, the PS works by intracellular disruption pathways rather than damaging the outer membrane.

In conclusion, a set of new chalcones of potential fluorescence were prepared to discover new probes for PDI of bacteria. Only compound 6d displayed intense absorption at λ_max 421 nm which falls within the blue range and fluorescence emission at λ_max 524 nm. Irradiation of E.coli O157 and B.subtilis bacterial strains with a blue light at 460 nm and power of 12 Jcm⁻² in the presence of this probe was investigated. The probe properly worked as PS and displayed bactericidal activity against both bacterial strains. The activity against the Gram−positive strain B. subtlis was higher than the Gram−negative one E. coli O157. Confocal laser scan imaging revealed better intracellular uptake by the Gram−positive strain than the negative one, which accounts for this deviation in activity. These results declared that the photo-generated ROS by this new PS was able to stimulate bactericidal intracellular reactions rather than membrane damage. In vivo studies using this probe are planned to be considered.

Acknowledgements The authors are grateful to Port Said University, Ain Shams University and Cairo University for the facilities and support during performing the work. The support of Dr. Maryhan, Damietta University in spectroscopic measurements is highly acknowledged.

Ethical Approval

Not applicable

Competing interests

The authors declare no competing interests.

Authors' contributions

The chemistry in this work is part of the M.Sc. thesis of Ms. Alaa K. El-Sayed Abd-Allah, supervised by Professors Aly, M. R. E., and Rizk S. A., while the PDI study is a contribution of Dr. Alexeree, S. M., and Prof. Attia Y. A. The idea of this work is based on a talk between Professors Aly, Rizk, and Attia. Ms. Alaa did the synthetic work and spectroscopic elucidation of the chemical compounds under the supervision of Professors Aly and Rizk. Prof. Aly did the spectrophotometric studies on the target compounds. Dr. Alexeree did the photodynamic studies and reported the results under the supervision of Prof. Attia. Prof. Aly wrote the manuscript then it was reviewed by all authors, each in his discipline.

Funding

Not applicable

Availability of data and materials

Data and materials are available by contacting the corresponding author.

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Scheme 1 is available in the Supplementary Files section

No competing interests reported.

floatimage1.png
Scheme 1. Reagents and conditions: (a) NaN₃, DMF, 80-100 °C (57%); (b) CuSO₄.5H₂O, L-ascorbic acid, THF–H₂O 4:1, rfx [6a, R = H (73%); 6b, R= CH₃ (86%); 6c, R= p-OMe (63%); 6d, R = p-NMe₂ (98%); 6e, R = Cl, (20%); 7a, X = O (11%); 7b, X = S (97%); 8 (97%)].
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In vitro photodynamic inactivation of B. subtilis and E. coli O157 by a new chalcone conjugate photosensitizer tethered with the NMe 2 group as fluorescence donor auxochrome

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Abstract

Figures

Introduction

Experimental section

Materials and methods

Chemistry

Synthesis of 4-Azido-2,6-dimethylquinoline (2)

General procedure for the synthesis of propargyl derivatives (3b and 3e)

Characterization details of the two newly prepared propargylated chalcones

General procedure for the synthesis of the target triazoles

Characterization details of the newly synthesized of the target triazoles

Biological evaluations

In vitro antimicrobial activity

Photodynamic Inactivation of Bacteria

Light source

Growth profiles

Photodynamic inactivation effect

Live cell imaging by confocal laser scanning Microscopy (CLSM)

Statistical analysis

Results and discussion

Chemistry

UV-Visible absorption and fluorescence spectroscopy

Biology

Antimicrobial activity

Photodynamic inactivation investigations

Conclusion

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

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