The enzyme NRH:quinone oxidoreductase 2 (NQO2) plays an important role in the pathogenesis of various diseases such as neurodegenerative disorders, malaria, glaucoma, COVID-19 and cancer. NQO2 expression is known to be increased in some cancer cell lines. Since 3-arylidene-2-oxindoles are widely used in the design of new anticancer drugs, such as kinase inhibitors, it was interesting to study whether such structures have additional activity towards NQO2. Herein, we report the synthesis and study of 3-arylidene-2-oxindoles as novel NRH:quinone oxidoreductase inhibitors. It was demonstrated that oxindoles with 6-membered aryls in the arylidene moiety were obtained predominantly as E-isomers while for some 5-membered aryls, the Z-isomers prevailed. The most active compounds inhibited NQO2 with an IC50 of 0.368 µM. The presence of a double bond in the oxindoles was crucial for NQO2 inhibition activity. There was no correlation between NQO2 inhibition activity of the synthesized compounds and their cytotoxic effect on the A549 cell line.
Keywords: oxindoles; indolin-2-ones; NQO2; NRH:quinone oxidoreductase 2; 3-arylidene-2-oxindoles; anticancer
Human NRH:quinone oxidoreductase 2 (NQO2) is an enzyme that belongs to the quinone oxidoreductase gene family. The biological activity of NQO2 is quite multifaceted. NQO2 can be considered a detoxifying agent because it catalyzes two-electron reduction of quinones and quinoid compounds into hydroquinones [[
NQO2 was linked to the development of various diseases such as neurodegenerative disorders [[
Owing to the extensive research of Boutin and his team, it is generally assumed that NQO2 is the third melatonin binding site, MT3 [[
Previously, we demonstrated that 2-oxindole derivatives can act as NQO2 inhibitors and bind to NQO2 binding site, analogous to melatonin [[
To obtain 3-arylidene-2-oxindoles, the following synthetic approach was used (Scheme 1). The aromatic and heteroaromatic aldehydes were condensed with 2-oxindoles in presence of piperidine as a base with good yields. The synthesis of compounds 1–4, 7–15, 17–20, 24, 25, 27–29, 31, 32, 34–44, 46 and 48–52 was performed according to the our previously published procedure [[
The detailed structures of the obtained 3-arylidene-2-oxindoles are presented in Table 4.
The simultaneous reduction of the double bond and nitro group in compound 4 was performed using Zn/HCl (Scheme 2). Compound 47 was prepared analogously [[
3-arylidene-2-oxindoles were often obtained as mixtures of isomers in various ratios, so it was important to establish convenient criteria for determining the predominant isomer. For several arylidene derivatives, we have identified the characteristic signals in the NMR spectra.
For some 4′-substituted benzylidene-2-oxindoles, it was previously found that the chemical shift for the 2′(6′) protons of benzylidene moiety in the
In order to determine the main configuration of our products, we analyzed the NMR spectra of the obtained 4′-substituted benzylidene-2-oxindoles (Table 1). We can confirm that this method for establishing the configuration is very convenient since the characteristic signals in Z-isomers significantly shift downfield and can be easily distinguished from other aromatic signals.
We found the similar spectral trend for 3-(pyridin-2-ylmethylidene)-substituted oxindole derivatives. In E-isomers, the 1H NMR signal which belongs to the H
A pair of doublets (H
We found Z-isomer to be predominant in some pyrazole derivatives. The NOE experiment for 3-(1-methyl-1H-pyrazol-4-ylmethylidene)-2-oxindole 41, obtained as a single isomer, showed the interaction of the double bond proton with the proton H
The configurations of pyrazole derivative 45 was assigned by comparison of its 1H NMR spectrum with one of the reference compounds. The spectral characteristics of the aromatic region of the 1H NMR spectrum of 45 suggested the predominance of the Z-isomer in this compound as well.
As can be seen from the above studies, usually one isomer predominates in the synthesized oxindoles. Since E- and Z-isomers may have different biological activities, we searched for new reaction conditions that could increase the content of the minor isomer. We investigated the solvent effect and the influence of microwave activation (MW) on the yields and isomeric composition of the products. The reactions were carried out using standard thermal activation and a standard protic solvent (ethanol), an aprotic low-polar solvent (dioxane) and an aprotic polar solvent (ethyl acetate). As model experiments, both reactions with donor and acceptor aldehydes were carried out (Table 3).
It is interesting that in case of pyrazole derivatives 39 and 44, the ratio of isomers in all conditions indicated in Table 3 was not dependent on solvent or activation method while for both 6-membered aryls, the quantity of the minor Z-isomer increased with changing ethanol to an aprotic solvent (Table 3). When carrying out the reaction with an acceptor aldehyde in an aprotic solvent, intermediate 1a precipitated after 10 min and can be isolated. Interestingly, in the case of dioxane, the reaction went further until complete conversion to compound 1 as a mixture of isomers, while in ethyl acetate the reaction stopped at the stage of formation of product 1a (Scheme 3).
Thus, for non-pyrazole aldehydes, changing the solvent and MW can increase the yield of the minor Z-isomer, but does not lead to a complete inversion of the isomeric composition of the products. To obtain the E-isomer, standard reaction conditions remain preferable.
The inhibition activity of all synthesized compounds at a concentration of 10 µM was preliminarily tested in vitro using human recombinant NRH:quinone oxidoreductase (NQO2). For active compounds, IC
The mechanism of NQO2 inhibition by one of the lead compounds, 15, was elucidated using a kinetic experiment (Figure 4 and Figure S22). The reaction rate was monitored under a range of inhibitor (0–25 µM) and BNAH substrate concentrations (9.375–150 µM). We found that both maximum rate V
The cytotoxicity of the synthesized oxindoles was studied using the A549 cell line in which the levels of NQO2 are increased (Table 4). We found that some oxindoles had moderate cytotoxic effects in the micromolar range of concentration, but no correlation between NQO2 inhibition activity and influence on cell viability was observed. The cytotoxicity of the compounds may be due to the inhibition of other enzymes associated with the activation of apoptosis in cancer cells, for example GSK3b or tyrosine kinases, or with the general non-specific toxicity of these compounds. Thus, the influence of NQO2 on A549 cell viability has not been established.
To analyze the mode of binding of the obtained oxindole derivatives to the NQO2 active site, we performed ligand–protein (Induced Fit) calculations using the Schrodinger software package. The 3OWH PDB structure [[
The possibility of π–π stacking of both E- and Z-isomers of 15 with FAD are shown. For the E-isomer, the binding geometry (the first calculated positions according to the docking score) was extremely similar to MCA-NAT (Figure 5A) (PDB 3OVM [[
All solvents were used as received without further purification. The reactions were monitored by thin layer chromatography (TLC) carried out on Merck TLC silica gel plates (60 F254), using UV light for visualization. Flash column chromatography purifications were carried out using silica gel 60 (particle size 0.040–0.060 mm).
Isatin, 5-methoxyisatin and 5-bromoisatin were purchased from Merck. N-methyloxindole was obtained by N-alkylation using a previously described procedure [[
2-oxindole (1 eq.) and the corresponding carboxaldehyde (1 eq.) were dissolved in 2–5 mL of solvent and 60–120 µL of piperidine was added. The reaction mixture was refluxed until the parent oxindole disappeared (monitored by TLC). The reaction mixture was then cooled down to room temperature. If a precipitate formed, it was filtered off and washed with ethyl acetate and diethyl ether and dried. If no precipitate appeared, the solution was concentrated in a vacuum and the resulting product was washed with ethyl acetate and diethyl ether and dried. The following compounds were obtained according to this procedure, with the reactions carried out in ethanol:
- (_I_E_i_,_I_Z_i_)-3-(2-Pyridinylmethylidene)-5-acetamido-2-oxindole _B_5
- From 0.18 g (0.94 mmol) of 5-acetamido-2-oxindole, 87 µL (0.94 mmol) of 2-pyridinecarboxaldehyde and 75 µL (0.88 mmol) of piperidine with ethanol as solvent the reddish-brown powder (0.156 g, 59%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 7.15:1, respectively. -
E isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 2.02 (s, 3H, CH3 ), 6.76 (d, J = 8.4 Hz, 1H, H7 ), 7.42–7.48 (m, 1H), 7.49–7.55 (m, 2H), 7.82 (d, J= 7.7 Hz, 1H), 7.92 (td, J1 = 7.7 Hz, J2 = 1.8 Hz, 1H), 8.90 (d, J = 3.7 Hz, 1H, H3′ ), 9.30 (d, J = 2.0 Hz, 1H, H4 ), 9.89 (br.s, 1H, NH), 10.53 (br. s, 1H, NH). - Selected peaks of Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 1.96 (s, 3H, CH3 ), 6.85 (d, J = 7.9 Hz, 1H, H7 ), 7.66–7.71 (m, 1H), 8.00–8.05 (m, 1H), 8.80 (d, J = 4.8 Hz, 1H, H3′ ). -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 24.35, 109.61, 120.54, 121.91, 122.44, 124.45, 128.47, 130.18, 133.79, 134.19, 137.61, 139.66, 150.16, 153.59, 168.25, 169.77. - HRMS (ESI), m/z: (M+H) found 280.1073, C
16 H14 N3 O2 requires 280.1086, (M + Na) found 302.0891, C16 H13 N3 O2 Na requires 302.0905. - (_I_E_i_,_I_Z_i_)-3-(2-Pyridinylmethylidene)-5-benzoylamino-2-oxindole _B_6
- From 0.100 g (0.4 mmol) of 5-benzoylamino-oxindole, 43 µL (0.4 mmol) of 2-pyridinecarboxaldehyde and 30 µL (0.35 mmol) of piperidine with ethanol as solvent the light brown powder (0.084 g, 62%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 6:1, respectively. -
E isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 6.84 (d, J = 8.4 Hz, 1H), 7.41–7.58 (m, 5H), 7.61 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.88–7.97 (m, 4H), 8.88 (d, J = 3.9 Hz, 1H, H3′ ), 9.46 (d, J = 1.7 Hz, 1H, H4 ), 10.22 (br. s, 1H, NH), 10.67 (br.s, 1H, NH). - Selected peaks of Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 7.02 (d, J = 8.8 Hz, 1H, H7 ), 7.19–7.30 (m, 3H), 7.72 (d, J = 7.7 Hz, 1H). -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 109.27, 121.50, 121.68, 123.85, 124.12, 127.69, 128.14, 128.37, 129.74, 131.35, 132.98, 133.93, 135.30, 137.24, 139.97, 149.83, 153.20, 165.37, 169.49. - HRMS (ESI), m/z: (M + H) found 342.1233, C
21 H16 N3 O2 requires 342.1242, (M + Na) found 364.1051, C21 H15 N3 O2 Na requires 364.1062, (M+K) found 380.0790, C21 H15 N3 O2 K requires 380.0801. - (_I_E_i_,_I_Z_i_)-3-(4-Hydroxybenzylidene)-5-acetamido-2-oxindole _B_16
- From 0.192 g (1 mmol) of 5-acetamido-2-oxindole, 0.122 g (1 mmol) of 4-hydroxybenzaldehyde and 80 µL (0.94 mmol) of piperidine with ethanol as solvent the yellow powder (0.189 g, 64%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 2.6:1 respectively. - E isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 1.99 (s, 3H, CH3 ), 6.78 (d, J = 8.3 Hz, 1H, H7 ), 6.87 (d, J = 8.6 Hz, 2H, H3′ ,H5′ ), 7.40 (td, J1 = 8.3 Hz, J2 = 1.9 Hz, 1H, H6 ), 7.51 (s, 1H, CH=), 7.62 (d, J = 8.7 Hz, 2H, H2′ ,H6′ ), 8.12 (d, J = 1.9 Hz, 1H, H4 ), 9.80 (s, 1H, NH), 10.42 (br.s, 1H, NH). - Selected peaks of Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 2.02 (s, 3H, CH3 ), 6.70–6.83 (m, 3H), 7.22 (dd, J1 = 8.3 Hz, J2 = 1.9 Hz, 1H, H6 ), 7.48 (s, 1H, CH=), 7.82 (d, J = 1.9 Hz, 1H, H4 ), 8.39 (d, J = 8.8 Hz, 2H, H2′ ,H6′ ), 9.68 (s, 1H, NH), 10.46 (br.s, 1H, NH). -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 24.29, 24.49, 109.98, 114.47, 116.11, 116.46, 120.83, 121.84, 124.45, 124.58, 132.57, 133.56, 135.58, 137.40, 138.44, 161.08, 168.27, 169.79. - (_I_E_i_,_I_Z_i_)-3-(4-Methoxybenzylidene)-5-benzoylamino-2-oxindole _B_21
- From 0.100 g (0.4 mmol) of 5-(benzoylamino)oxindole, 0.055 g (0.4 mmol) of 4-methoxybenzaldehyde and 30 µL (0.35 mmol) of piperidine with ethanol as solvent the greenish grey powder (0.113 g, 77%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 2:1, respectively. - E isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.81 (s, 3H, CH3 ), 6.88 (d, J = 8.3 Hz, 1H, H7 ), 7.07 (d, J = 8.7 Hz, 2H), 7.43–7.66 (m, 5H), 7.76 (d, J = 8.3 Hz, 2H), 7.91 (d, J = 7.2 Hz, 2H), 8.24 (s, 1H, CH=), 10.18 (br.s, 1H, NH), 10.60 (br.s, 1H, NH). - Selected peaks of Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.82 (s, 3H, CH3 ), 6.82 (d, J = 8.4 Hz, 1H, H7 ), 7.03 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.6 Hz, 1H, Hind ), 8.00 (d, J = 7.1 Hz, 2H), 8.09 (s, 1H, CH=), 8.51 (d, J = 8.7 Hz, 2H, H2′ ,H6′ ), 9.85 (br.s, 1H, NH), 10.22 (br.s, 1H, NH). -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 55.40, 109.72, 113.80, 114.38, 115.63, 121.12, 121.85, 122.59, 124.28, 125.29, 125.54, 126.53, 126.92, 127.59, 127.64, 128.36, 128.42, 131.41, 131.47, 131.79, 131.85, 132.84, 132.88, 134.59, 135.12, 136.24, 136.85, 136.88, 138.94, 160.61, 161.29, 165.24, 165.36, 167.62, 169.24. - (_I_E_i_)-3-(4-Ethoxybenzylidene)-2-oxindole _B_22
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.115 g (0.8 mmol) of 4-ethoxybenzaldehyde and 60 µL (0.7 mmol) of piperidine with ethanol as solvent the yellow powder (0.146 g, 73%) was obtained as a single E isomer.
- m.p. = 170–171 °C (m.p.
lit. 169–171 [[35 ]]) -
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 1.35 (t, J = 7.0 Hz, 3H, CH3 ), 4.07–4.14 (m, 2H, CH2 ), 6.83–6.89 (m, 2H, H7 ,Hind ), 7.06 (d, J = 8.7 Hz, 2H, H3′ ,H5′ ), 7.21 (td, J1 = 7.0 Hz, J2 = 0.7 Hz, 1H, Hind ), 7.56 (s, 1H, CH=), 7.65 (d, J = 7.8 Hz, 1H, H4 ), 7.69 (d, J = 8.7 Hz, 2H, H2′ ,H6′ ), 10.55 (br.s, 1H, NH). - (E,Z)-3-(3,4,5-Trimethoxybenzylidene)-2-oxindole 23
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.143 g (0.8 mmol) of 3,4,5-trimethoxybenzaldehyde and 60 µL (0.7 mmol) of piperidine with ethanol as solvent the yellow powder (0.187 g, 84%) was obtained as a mixture of two isomers. According to
1 H NMR stereoisomer ratio is 1.66:1. [[36 ]] - Major isomer
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.89 (s, 6H, CH3 ), 3.95 (s, 3H, CH3 ), 6.86–6.95 (m, 2H, Hind ), 7.24 (t, J = 7.6 Hz, 1H, Hind ), 7.27 (s, 2H, H2′ ,H6′ ), 7.79–7.83 (m, 2H, Hind ,CH=), 8.13 (br.s, 1H, NH). - Selected peaks of minor isomer
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.98 (s, 9H, CH3 ), 6.88 (d, J = 7.7 Hz, 1H, H7 ) 7.06 (t, J = 7.4 Hz, 1H, Hind ), 7.49 (s, 1H, CH=), 7.54 (d, J = 7.5 Hz, 1H, H4 ), 8.02 (br.s, 1H, NH) - (_I_E_i_,_I_Z_i_)-3-(3,5-Dimethoxy-4-hydroxybenzylidene)-5-benzoylamino-2-oxindole _B_26
- From 0.100 g (0.4 mmol) of 5-benzoylamino-oxindole, 0.089 g (0.4 mmol) of 3,5-dimethoxy-4-hydroxybenzaldehyde and 30 µL (0.35 mmol) of piperidine with ethanol as solvent the black powder (0.133 g, 79%) was obtained as a mixture of two isomers. According to
1 H NMR stereoisomer ratio is 1.66:1. - Major isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.78 (s, 6H, CH3 ), 5.04 (br.s, 1H, OH), 6.83 (d, J = 8.3 Hz, 1H, H7 ), 7.05 (s, 2H, H2′ ,H6′ ), 7.37–7.59 (m, 4H), 7.87 (d, J = 7.2 Hz, 2H, HAr ), 8.06 (s, 1H, CH=), 8.52 (br.s, 1H, H4 ), 9.51 (br.s, 1H, NH), 10.15 (br.s, 1H, NH); - Selected signals of minor isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.72 (s, 6H, CH3 ), 6.78 (d, J = 8.3 Hz, 1H, H7 ), 7.01 (s, 2H, H2′ ,H6′ ), 7.91 (s, 1H, CH=), 7.96 (d, J = 7.0 Hz, 2H, HAr ), 8.02 (s, 1H, H4 ), 10.13 (br.s, 1H, NH); -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 56.08, 56.15, 108.11, 108.45, 109.93, 111.80, 114.39, 122.00, 122.08, 122.93, 123.81, 127.91, 128.76, 128.79, 131.77, 133.27, 135.39, 135.49, 138.22, 139.03, 148.57, 165.66, 169.89. - (_I_E_i_,_I_Z_i_)-3-(4-Dimethylaminobenzylidene)-2-oxindole _B_30
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.12 g (0.8 mmol) of 4-(dimethylamino)benzaldehyde and 60 µL (0.7 mmol) of piperidine with ethanol as solvent the reddish-brown powder (0.107 g, 54%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 1:1 respectively [[30 ]]. -
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.01 (s, 6H, CH3 ), 3.02 (s, 6H, CH3 ), 6.70–6.95 (m, 8H), 7.10 (t, J = 7.7 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 7.51 (s, 1H, CH=), 7.57–7.67 (m, 4H), 7.78 (d, J = 7.5 Hz, 1H, H4 ), 8.44 (d, J = 8.7 Hz, 2H, H2′ ,H6′ )*, 10.54 (br. s, 2H, NH). - *– Z isomer.
- (_I_E_i_,_I_Z_i_)-3-(4-Fluorobenzylidene)-2-oxindole _B_33
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.95 g (0.8 mmol) of 4-fluorobenzaldehyde and 60 µL (0.7 mmol) of piperidine with ethanol as solvent yellow powder (0.122 g, 68%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 2:1, respectively [[37 ]]. - E isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 6.78–6.89 (m, 2H, H7 ,Hind ), 7.17–7.25 (m, 1H, Hind ), 7.27–7.38 (m, 2H, H3′ ,H5′ ), 7.49 (d, J = 7.6 Hz, 1H, H4 ), 7.58 (s, 1H CH=), 7.73–7.75 (m, 2H, H2′ ,H6′ ), 10.62 (br. s 1H, NH). - Selected peaks of Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 6.98 (t, J = 7.5 Hz, 1H, Hind ), 7.69 (d, J = 7.5 Hz, 1H, H4 ), 7.8 (s, 1H, CH=), 8.45–8.5 (m, 2H, H2′ , H6′ ), 10.66 (br.s, 1H, NH). - (_I_E_i_,_I_Z_i_)-3-(1-[2-(Methoxycarbonyl)ethyl]-1H-pyrazol-4-ylmethylidene)-2-oxindole _B_45
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.142 g (0.8 mmol) methyl 3-(4-formyl-1H-pyrazol-1-yl)propanoate and 60 µL (0.7 mmol) of piperidine with ethanol as solvent the yellow powder (0.187 g, 84 %) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 1:3.5, respectively. -
Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 2.87–2.97 (m, 2H, CH2 ), 3.59 (s, 3H, CH3 ), 4.39–4.49 (m, 2H, CH2 ), 6.81 (d, J = 7.6 Hz, 1H, H7 ), 6.91–7.00 (m, 1H, Hind ), 7.14 (t, J = 7.4 Hz, 1H, Hind ), 7.57 (d, J = 7.3 Hz, 1H, H4 ), 7.67 (s, 1H, CH=), 8.24 (s, 1H, CHpyr ), 8.83 (s, 1H, NHpyr ), 10.54 (br. s, 1H, NHind ). - Selected peaks of E-isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 2.87–2.97 (m, 2H, CH2 ), 3.59 (s, 3H, CH3 ), 4.39–4.49 (m, 2H, CH2 ), 6.86 (d, J = 7.6 Hz, 1H, H7 ), 7.21 (t, J = 7.6 Hz, 1H, Hind ), 7.45 (s, 1H, CH=), 7.82 (d, J = 7.4 Hz, 1H), 8.00 (s, 1H), 8.42 (s, 1H), 10.5 (br. s, 1H, NHind ). -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 33.87, 47.31, 51.64, 109.28, 116.98, 118.89, 120.87, 121.77, 124.81, 126.30, 127.82, 134.70, 139.98, 143.53, 167.65, 169.30, 171.10. - HRMS (ESI), m/z: (M+H) found 298.1178, C
16 H16 N3 O3 requires 298.1192, (M+Na) 320.0095 C16 H15 N3 O3 Na requires 320.1011.
The general synthesis procedure was used for the synthesis of followed compounds with some variations: 1,4-dioxane or ethyl acetate was used instead of ethanol.
- (_I_E_i_)-3-(2-Pyridinylmethylidene)-2-oxindole _B_(_I_E_i_)-1
- From 0.200 g (1.5 mmol) of 2-oxindole, 0.16 mL (1.5 mmol) of 2-pyridinecarboxaldehyde and 120 µL (1.5 mmol) of piperidine with ethanol as solvent the brown powder (0.109 g, 61%) was obtained as a single E isomer [[
28 ]]. -
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 6.84 (d, J = 7.7 Hz, 1H, H7 ), 6.95 (td, J1 = 7.7 Hz, J2 = 0.9 Hz, 1H, H5 ), 7.25 (td, J1 = 7.5 Hz, J2 = 1.3 Hz, H6 ), 7.41 (ddd, J1 = 7.5 Hz, J2 = 4.8 Hz, J3 = 1.1 Hz, 1H, H4′ ), 7.54 (s, 1H, CH=), 7.82 (d, J = 7.5 Hz, 1H, H6′ ), 7.90 (td, J1 = 7.7 Hz, J2 = 1.8 Hz, 1H, H5′ ), 8.4 (d, J = 3.9 Hz, 1H, H3′ ), 8.98 (d, J = 7.3 Hz, 1H, H4 ), 10.61 (br.s, 1H, NH). - (_I_E_i_,_I_Z_i_)-3-(2-Pyridinylmethylidene)-2-oxindole _B_(_I_E_i_,_I_Z_i_)-1
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.08 mL (0.8 mmol) of 2-pyridinecarboxaldehyde and 60 µL (0.7 mmol) of piperidine with 1,4-dioxane as solvent the brown oil (0.145 g, 81%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomer ratio is 1.3:1 respectively.1 H NMR (DMSO-d6 ) for E isomer is identical to described above. - Selected signals of Z-isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 7.06 (d, J = 7.9 Hz, 1H, H4 ), 7.63 (s, 1H, CH=). - 3-(Hydroxy(pyridin-2-yl)methyl)-2-oxindole _B_1a
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.08 mL (0.8 mmol) of 2-pyridinecarboxaldehyde and 60 µL (0.7 mmol) of piperidine with ethyl acetate as solvent the beige powder (0.088 g, 49%) was obtained. According to
1 H NMR the ratio of two diastereoisomers of 1a and the final condensation product 1 is 6.92:4.17:1 respectively. - Major isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 4.08 (d, J = 2.4 Hz, 1H), 5.33 (dd, J1 = 4.7 Hz, J2 = 1.9 Hz, 1H), 6.62 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 7.02–7.10 (m, 1H), 7.34 (dd, J1 = 7.2 Hz, J2 = 2.2 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.85 (td, J1 = 7.7 Hz, J2 = 1.6 Hz, 1H), 8.62 (d, J = 4.2 Hz, 1H), 10.39 (br.s, 1H) - Selected peaks of minor isomer
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 3.96 (d, J = 3.0 Hz, 1H), 5.27 (t, J = 3.5 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 7.13 (dd, J1 = 7.1 Hz, J2 = 1.8 Hz, 1H), 7.25 (d, J = 7.3 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.66 (td, J1 = 7.8 Hz, J2 = 1.7 Hz, 1H), 8.35 (d, J = 4.1 Hz, 1H), 10.17 (br.s, 1H). - 1H spectrum also contains signals of 1.
-
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 57.07, 57.29, 77.74, 78.66, 113.84, 114.22, 125.66, 125.79, 125.91, 127.01, 127.45, 129.06, 129.43, 131.52, 132.66, 133.04, 141.01, 141.75, 148.43, 148.94, 152.96, 153.87, 166.51, 167.34, 181.07, 182.69. - HRMS (ESI), m/z: (M+H) found 241.0979, C
14 H13 N2 O2 requires 241.0977; (M+Na) found 263.0797, C14 H12 N2 O2 Na requires 263.0796. - (_I_E_i_,_I_Z_i_)-3-(4-Hydroxybenzylidene)-2-oxindole _B_(_I_E_i_)-14
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.092 g (0.8 mmol) of 4-hydroxybenzaldehyde and 60 µL (0.7 mmol) of piperidine with ethanol as solvent the yellow powder (0.136 g, 76%) was obtained as a mixture of two isomers. According to
1 H NMR E/Z stereoisomeric ratio is 19:1, respectively [[29 ]]. -
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 6.82–6.93 (m, 4H), 7.19 (t, J = 7.6 Hz, 1H, Hind ), 7.53 (s, 1H, CH=), 7.61 (d, J = 8.5 Hz, 2H, HAr ), 7.69 (d, J = 7.6 Hz, 1H, H4 ), 10.14 (br.s, 1H, OH), 10.52 (br. s, 1H, NH). - (_I_E_i_,_I_Z_i_)-3-(4-Hydroxybenzylidene)-2-oxindole _B_(_I_E_i_,_I_Z_i_)-14
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.092 g (0.8 mmol) of 4-hydroxybenzaldehyde and 60 µL (0.7 mmol) of piperidine in 1,4-dioxane or ethyl acetate as solvent the yellow powder was obtained as a mixture of two isomers. Yields: 0.150 g, 84% (1,4-dioxane); 0.109 g, 61% (ethyl acetate). According to _SP_1_sp_H NMR _I_E_i_/_I_Z_i_ stereoisomeric ratio is 1.75:1, respectively. _SP_1_sp_H NMR (400.13 MHz, DMSO-_I_d_SB_6_sb__i_, ppm) for _I_E_i_ isomer was identical to _B_14.
- Selected peaks of Z isomer,
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 6.79 (d, J = 7.6 Hz, 1H, H7 ), 6.94 (td, J1 = 7.5 Hz, J2 = 0.6 Hz, 1H, Hind ), 7.13 (td, J1 = 7.7 Hz, J2 = 0.6 Hz, 1H, Hind ), 7.64 (s, 1H, CH=), 8.39 (d, J = 8.7 Hz, 2H, H2′ , H6′ ) - (_I_E_i_,_I_Z_i_)-3-((5-Ethoxycarbonyl-1H-pyrazol-4-yl)methylidene)-2-oxindole _B_(_I_E_i_,_I_Z_i_)-39
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.134 g (0.8 mmol) of ethyl 4-formyl-1H-pyrazole-5-carboxylate and 60 µL (0.7 mmol) of piperidine with ethanol, 1,4-dioxane or ethyl acetate as solvent the yellow powder was obtained as a mixture of two isomers. Yields: 0.136 g, 76% (ethanol); 0.154 g, 86% (1,4-dioxane); 0.120 g, 67% (ethyl acetate). According to
1 H NMR major to minor stereoisomer ratio is 1.6:1 in all solvents [[29 ]]. - Major isomer
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 1.35 (t, J = 7.1 Hz, 3H, CH3 ), 4.33–4.40 (m, 2H, CH2 ), 6.5 (m, 1H, H7 ), 6.99 (td, J1 = 7.5 Hz, J2 = 0.7 Hz, 1H, Hind ), 7.17–7.24 (m, 1H, Hind ), 7.47 (d, J = 7.5 Hz, 1H, H4 ), 8.23 (s, 1H, CH=), 9,28 (br.s, 1H, Hpyrazol ), 10.64 (s., 1H, NHind ). - Selected peaks of minor isomer
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 1.27 (t, J = 7.1 Hz, 3H, CH3 ), 4.25–4.33 (m, 2H, CH2 ), 6.90 (td, J1 = 7.6 Hz, J2 = 0.9 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H, H4 ), 7.82 (s, 1H, CH=), 8.42 (br.s, 1H, Hpyrazol ), 10.54 (s, 1H, NHind ). - 3-((3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene)-2-oxindole _B_44
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.160 g (0.8 mmol) of 3,5-dimethyl-1-phenyl-1H-pyrazole-4-carbaldehyde and 60 µL (0.7 mmol) of piperidine with ethanol, 1,4-dioxane or ethyl acetate as solvent yellow powder was obtained as single isomer. Yields: 0.159 g, 63% (ethanol); 0.157 g, 62% (1,4-dioxane); incomplete conversion of started oxindole (76% conversion) in ethyl acetate. According to
1 H NMR a single isomer was obtained in all solvents. [[29 ]] -
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 2.15–2.22 (m, 6H, CH3 ), 6.87 (d, J = 7.6 Hz, 1H, H7 ), 6.92 (td. J1 = 7.6 Hz, J2 = 0.7 Hz, 1H, Hind ), 7.07 (d, J = 7.7 Hz, 1H, H4 ), 7.20 (td, J1 = 7.6 Hz, J2 = 0.8 Hz, 1H, Hind ), 7.4–7.48 (m, 2H, Har, CH=), 7.53 (t, J = 7.5 Hz, 2H, Har ), 7.57–7.62 (m, 2H, Har ), 10.60 (br.s, 1H).
2-Oxindole (1 eq.) and corresponding carboxaldehyde (1 eq.) were dissolved in 2–3 mL of 1,4-dioxane and 60 µL of piperidine was added. Reaction mixture was subjected to microwave irradiation (260W) for 11.5 min via a repeated process of 0.5–1 min of MW activation followed by a 1–2 min cooldown period. The reaction was carried out until the parent oxindole was no longer detected by TLC. The reaction mixture was cooled down to room temperature.
If the precipitate formed, it was filtered off, washed with ethyl acetate and diethyl ether and dried.
If no precipitate appeared, the solution was concentrated in vacuum and the resulting product was washed with ethyl acetate and diethyl ether and dried. The following compounds were obtained according to this procedure:
- (_I_E_i_,_I_Z_i_)-3-(2-Pyridinylmethylidene)-2-oxindole _B_(_I_E_i_,_I_Z_i_)-1
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.08 mL (0.8 mmol) of 2-pyridinecarboxaldehyde and 60 µL (0.67 mmol) of piperidine the brown oil (0.149 g, 83%) was obtained as a mixture of isomers. According to
1 H NMR E:Z stereoisomer ratio is 3.7:1, respectively.1 H NMR signals were identical to described in Section 3.1.2. for 1. - (_I_E_i_,_I_Z_i_)-3-(4-Hydroxybenzylidene)-2-oxindole _B_(_I_E_i_,_I_Z_i_)-14
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.092 g (0.8 mmol) of 4-hydroxybenzaldehyde and 60 µL (0.67 mmol) of piperidine the yellow powder (0.159 g, 89%) was obtained as a mixture of two isomers. According to _SP_1_sp_H NMR _I_E_i_/_I_Z_i_ stereoisomer ratio is 1.75:1, respectively. _SP_1_sp_H NMR signals were identical to described in Section 3.1.2. for _B_14.
- (_I_E_i_,_I_Z_i_)-3-((5-Ethoxycarbonyl-1H-pyrazol-4-yl)methylidene)-2-oxindole _B_(_I_E_i_,_I_Z_i_)-39
- From 0.100 g (0.8 mmol) of 2-oxindole, 0.134 g (0.8 mmol) of ethyl 4-formyl-_I_1H_i_-pyrazole-5-carboxylate and 60 µL (0.67 mmol) of piperidine the yellow powder (0.089 g, 50%) was obtained as a mixture of two isomers. According to NMR _SP_1_sp_H major to minor stereoisomer ratio is 1.6:1. _SP_1_sp_H NMR signals were identical to described in Section 3.1.2. for _B__I_(E,Z_i_)-39.
- 3-((3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene)-2-oxindole _B_44
-
From 0.100 g (0.8 mmol) of 2-oxindole, 0.160 g (0.8 mmol) of 3,5-dimethyl-1-phenyl-_I_1H_i_-pyrazole-4-carbaldehyde and 60 µL (0.67 mmol) of piperidine the yellow powder was obtained as single isomer _SP_1_sp_H NMR signals were identical to described in Section 3.1.2 for _B_44. According to
1 H NMR conversion is 68%.
- To the suspension of 0.146 g (0.6 mmol) 3-(2-pyridinylmethylidene)-5-nitro-2-oxindole and 0.350 g (5.6 mmol) zinc powder in 5 mL of MeOH the 0.6 mL of HCl conc. was rapidly added with vigorous stirring. After 30 min, the reaction was terminated by addition of NaHCO
3 and pH was adjusted to 8. The reaction mixture was extracted with EtOAc, organic fraction was dried with Na2 SO4 and the solvent was removed under reduced pressure. The compound (0.086 g, 65%) was obtained as brown powder. -
1 H NMR (400.13 MHz, DMSO-d6 , ppm): 2.88–3.0 (m, 1H, CH2 ), 3.28–3.36 (m, 1H, CH2 ), 3.83–3.90 (m, 1H, CH), 4.51 (br.s, 2H, NH2 ), 5.98 (s, 1H, H4 ), 6.33 (dd, J1 = 7.6 Hz, J2 = 1.8 Hz, 1H, H6 ), 6.49 (d, J = 8.1 Hz, 1H, H7 ), 7.2–7.26 (m, 2H, Hpy ), 7.65–7.72 (td, J1 = 7.7 Hz, J2 = 1.8 Hz, 1H, Hpy ), 8.495 (d, J = 4.1 Hz, 1H, H3′ ), 9.99 (br. s, 1H, NH). -
13 C NMR (100.6 MHz, DMSO-d6 , ppm): 38.15, 45.04, 109.46, 111.31, 112.63, 121.76, 123.60, 130.41, 132.34, 136.42, 143.20, 149.03, 158.41, 178.07. - HRMS (ESI), m/z: (M+H) found 240.1128, C
14 H14 N3 O requires 240.1137, (M+Na) found 262.0939, C14 H13 N3 ONa requires 262.0956.
The activity of recombinant human NQO2 (Sigma #Q0380, St. Louis, MA, USA) was evaluated kinetically using menadione and N-benzyl-dihydronicotinamide (BNAH) as the substrate and co-substrate, respectively. All reagents and test compounds were dissolved in 50 mM Hepes-KOH (pH 7.4) containing 1 mM of β-octyl-D-glucopyranoside, 0.1 mg/mL BSA and 1 μM FAD. In a 96-well black flat-bottom plate, 50 μL of the test compounds were introduced at a final concentration of 10 μM for primary screening, and a range of final concentrations from 10 nM to 100 μM was used to determine IC
A549 lung carcinoma cells were purchased from the ATCC (CCL-185). The A549 cells were maintained in F12-K (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, UK), penicillin (100 UI mL
The effects of the synthesized compounds on cell viability were determined using the MTT colorimetric test. All examined cells were diluted with the growth medium to 3.5 × 10
The in silico studies were carried out according to the standard algorithms of the Schrodinger software package and our previous work [[
The synthesis and study of 3-arylidene-2-oxindoles as novel NRH:quinone oxidoreductase 2 inhibitors was performed. It was shown that the E-isomer was the predominant product in the synthesis of 3-benzylidene and 3-(pyridylmethylidene) derivatives, while for pyrazole-4-carbaldehyde derivatives, the Z-isomer sometimes predominated. An NMR criterion for determining the (E/Z)-configuration of 3-(pyridin-2-ylmethylidene)-2-oxindoles was proposed. It was shown that for E-isomers, the signals of protons in the 4th position of the oxindole ring are characteristic in the 1H NMR spectra and lie in the region of 8.9–10 ppm. The molecular modeling of binding to the active site of NQO2 demonstrated that both the E- and Z-isomers are capable of π–π stacking with the FAD cofactor and are located in the active site of the enzyme similar to MCA-NAT (a selective inhibitor of NQO2). The presence of hydroxy groups in the arylidene moiety and the introduction of a methyl group to the oxindole nitrogen led to an increase in binding affinity. The most active compounds 15, 17, 18, 24, and 39 inhibited NQO2 with IC
Graph: Figure 1 Proposed design of novel oxindole-based NQO2 inhibitors [[
Graph: Scheme 1 Synthesis of 3-arylidene-2-oxindoles.
Graph: Scheme 2 The simultaneous reduction of nitro group and double bond in compound 4.
Graph: Figure 2 NOE correlations for E-isomer of compound 1.
Graph: Figure 3 NOE correlation for Z-isomer of compound 41.
Graph: Scheme 3 The isolation of intermediate product 1a using ethyl acetate as solvent.
Graph: Figure 4 Kinetic evaluation of NQO2 inhibition by compound 15.
MAP: Figure 5 Proposed binding models of MCA-NAT and 15 to active site of NQO2 (blue) with FAD (green). (A): binding pose of MCA-NAT (purple) and E-isomer (yellow) of 15. (B): E- (yellow) and Z- (red) isomers of 15. (C): interaction map for the E-isomer of 15. (D): interaction map for the Z-isomer of 15.
Table 1 Preferred configurations of 4′-substituted benzylidene-2-oxindoles and corresponding characteristic chemical shifts.
№ R R1 R2 Chemical Shift of E:Z E Z 14 -OH H H 7.47 8.24 19:1 15 -OH H Me 7.48 - 1:0 16 -OH NHC(O)CH3 H 7.60 8.38 2.6:1 17 -OH NHC(O)OCH3 H 7.63 8.41 25:1 18 -OH NH(2-furoyl) H 7.66 8.40 2:1 19 -OH NHBz H 7.67 8.42 2.3:1 20 -OMe H H 7.70 8.47 5:1 21 -OMe NHBz H 7.76 8.51 2:1 22 -OEt H H 7.69 - 1:0 28 -NO2 H H 7.94 8.27 10:1 29 -NO2 NHC(O)OCH3 H 7.93 8.26 3:1 30 -NO2 NHBz H 7.85 8.25 2:1 31 -N(Me)2 H H 7.57–7.67 1 8.44 1:1 32 -Br Br H 7.73 8.31 1.6:1 33 -Br NHC(O)OCH3 H 7.67 8.31 12.5:1 34 -F H H 7.73–7.75 1 8.45–8.50 1 2:1
Table 2 Preferred configuration of 3-(pyridin-2-ylmethylidene)-2-oxindoles and corresponding characteristic chemical shifts.
№ R1 R2 E:Z Ratio H H 8.98 8.40 1:0 H CH3 8.85–8.91 1 1:0 Br H 9.27 8.87 1:0 NO2 H 10.11 8.85 1:0 AcNH H 9.31 8.89 7.7:1 BzNH H 9.46 8.88 5:1
Table 3 Influence of the reaction conditions on the ratio of isomers.
Activation Method Thermal Activation (reflux) MW Solvent Ethanol 1,4-dioxane Ethyl Acetate 1,4-dioxane 1:0 3.7:1 120 90 10 11.5 63 81 49 83 19:1 2.3:1 2.3:1 2.3:1 60 60 30 11.5 76 84 61 89 1.6:1 1.6:1 1.6:1 1.6:1 120 120 20 11.5 61 86 67 50 1:0 1:0 1:0 1:0 150 120 120 11.5 63 62 Conversion 76% Conversion 68%
Table 4 Evaluation of enzyme inhibition activity and compound cytotoxicity against A549 cancer cell line.
NQO2 A549, CC50 (μM) № R R1 R2 Inhibition 1, % IC50 ± SEM (μM) 1 2-pyridyl H H 19.56 >>50 47.97 ± 4.42 2 2-pyridyl Me H 64.43 16.78 ± 0.09 - 2 3 2-pyridyl H Br 18.63 >>50 90.56 ± 1.50 4 2-pyridyl H NO2 1.85 >>50 n.a. 3 5 2-pyridyl H AcNH 46.35 41.13 ± 2.44 135.9 6 2-pyridyl H BzNH 43.13 n.d 2. 17.43 ± 1.80 7 3-pyridyl H H 59.17 n.d. 67.45 ± 7.58 8 3-pyridyl H Br 44.24 n.d. 22.30 ± 1.42 9 3-pyridyl H NO2 4.25 >>50 428.59 ± 23.97 10 4-pyridyl H H 27.29 >>50 80.27 ± 9.87 11 4-pyridyl H Br 41.92 n.d. 22.30 ± 1.42 12 4-pyridyl H NO2 19.23 >>50 n.a. 13 3-OH-C6H4 H AcNH 89.07 0.64 ± 0.04 175.83 ± 4.76 14 4-OH-C6H4 H H 31.68 >>50 80.85 ± 12.22 15 4-OH-C6H4 Me H 98.87 0.62 ± 0.04 n.a. 16 4-OH-C6H4 H AcNH 70.41 0.99 ± 0.03 n.a. 17 4-OH-C6H4 H MeOC(O)NH 93.63 0.44 ± 0.02 44.43 ± 0.29 18 4-OH-C6H4 H 2-furoylNH 96.4 0.37 ± 0.02 146.25 ± 11.31 19 4-OH-C6H4 H BzNH 62.89 n.d. 412.17 ± 3.98 20 4-OMe-C6H4 H H 40.82 n.d. 54.76 21 4-OMe-C6H4 H BzNH 30.89 >>50 42.33 ± 8.30 22 4-OEt-C6H4 H H 25.41 >>50 - 23 3,4,5-triOMe-C6H2 H H 85.71 1.6 ± 0.14 25.72 24 3,4,5-triOMe-C6H2 H AcNH 92.78 0.37 ± 0.01 148.99 ± 2.63 25 3,4,5-triOMe-C6H2 H 2-furoylNH 90.98 0.66 ± 0.02 200.90 ± 22.65 26 3,4-diOMe-4-OH-C6H2 H BzNH 40.47 n.d. n.a. 27 4-NO2-C6H4 H H 4.66 >>50 - 28 4-NO2-C6H4 H MeOC(O)NH 74.0 n.d. n.a. 29 4-NO2-C6H4 H BzNH 21.79 >>50 - 30 4-NMe2-C6H4 H H 27.30 >>50 n.a. 31 4-Br-C6H4 H Br 68.63 n.d. 241.71 ± 3.19 32 4-Br-C6H4 H MeOC(O)NH 95.46 0.61 ± 0.03 n.a. 33 4-F-C6H4 H H 19.46 >>50 38.56 ± 10.45 34 2-furyl H H 59.91 n.d. 594.74 ± 25.37 35 2-furyl H Br −15.2 >>50 449.07 ± 18.72 36 2-furyl H NO2 59.55 n.d. 328.55 ± 17.00 37 2-thienyl H H 38.1 n.d. 491.01 ± 19.85 38 2-thienyl H Br 29.9 >>50 426.53 ± 6.20 39 H H 47.32 0.34 ± 0.02 231.3 40 H NO2 92.3 1.88 ± 0.08 - 41 H H 59.76 9.40 ± 0.12 - 42 H H 54.35 n.d. - 43 H H 77.81 7.61 ± 0.19 218.9 44 H H 43.58 n.d. n.a. 45 H H 28.97 >>50 n.a. 46 4-NH2-C6H4 H H 19.05 >>50 - 47 2-pyridyl H NH2 26.93 >>50 n.a. 48 4-Me-C6H4 H OMe −50.28 >>50 n.a. 49 4-Me-C6H4 H Br −85.22 >>50 n.a. 50 4-MeO-C6H4 H Br 78.82 n.d. 473.55 ± 18.21 51 4-Me-C6H4 H 2-furoyl-NH 25.63 >>50 n.a. 52 4-Me-C6H4 H H −92.90 >>50 170.63 ± 13.41 Melatonin 63.5 ± 2.7 Quercetin 98 0.08 ± 0.02
Conceptualization, N.A.L. and S.E.S.; methodology, D.A.B. and N.A.L.; validation, D.R.B., E.N.B., M.D., D.D.M. and S.E.S.; investigation, E.N.B., M.D., D.D.M., V.G.K., O.B.G., E.V.S. and A.S.B.; resources, A.A.S.; data curation, D.A.B., N.A.L.; writing—original draft preparation, E.N.B. and N.A.L.; writing—review and editing, D.A.B., A.S.B., N.A.L. and S.E.S.; supervision, S.E.S.; project administration, N.A.L., S.E.S. and A.A.S.; funding acquisition, S.E.S. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The data presented in this study are available on request from the corresponding authors.
The authors declare no conflict of interest.
The following supporting information can be downloaded at:
By Natalia A. Lozinskaya; Elena N. Bezsonova; Meriam Dubar; Daria D. Melekhina; Daniil R. Bazanov; Alexander S. Bunev; Olga B. Grigor'eva; Vladlen G. Klochkov; Elena V. Sokolova; Denis A. Babkov; Alexander A. Spasov and Sergey E. Sosonyuk
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