PL236882B1 - New derivative of styrylquinoline and its application - Google Patents

Abstract

The subject of the application is a new styrylquinoline derivative, which is characterized by having a chemical structure according to formula (1). The subject of the application is also the use of a new styrylquinoline derivative of formula (1) as a fluorescent dye for coloring biological structures and for detecting metal ions, in particular zinc ions, in biological structures, preferably in human or animal cells.

Description

Description of the invention

The subject of the invention is a novel styrylquinoline derivative and its use. Compounds based on the quinoline structure are known in particular for their broad spectrum of biological activity. Among the wide group of therapeutics with this group, we can find antibacterial, antifungal, anticancer and antimalarial drugs. In addition to its therapeutic properties, quinoline also has a number of attractive features used in the design of new fluorophores and fluorescent sensors. It is a low-molecular chromophore with a nitrogen atom that allows complexing metal ions or creating hydrogen bonds. Imaging of cell structures with fluorescent dyes is of particular importance in the diagnosis of neoplastic diseases. A compound based on the structure of styrylquinoline has also been reported, allowing the imaging of β-amyloid plaques characteristic of Alzheimer's disease (M. Staderini, ACS Med. Chem. Lett, 2012). Thanks to the tool which is fluorescence microscopy, we can study the mechanisms taking place in the cell or monitor the concentration of ions important for our body. The selectivity of imaging is possible thanks to appropriate probes that connect to the target of our interest, activating the fluorescence process. Both the quinoline molecule and the imine moiety are often used as fragments in the design of such compounds. Dyes based on a vinylbenzene structure (styryl) are also known, such as those described, inter alia, in the patent documents: US5486616, US6794509 and US7781187. However, they do not include quinoline as the main nucleus of the molecule. There are also reports of styrylquinoline fluorophores used for multi-color staining. Such compounds arouse particular interest as they allow for multi-colored, simultaneous imaging of selected cell structures. The fluorescence wavelength, and thus the color of the emitted light, is modulated in this case by introducing various cations into the solution, influencing the intramolecular charge transfer process (Shiraishi Y, Chem - AEur J. 2011).

Properties desirable for fluorescent dyes are, above all, high fluorescence intensity and the Stokes shift understood as the distance between the absorption band and the emission band of a given substance. A larger shift allows a more accurate measurement due to the efficient separation of the sample luminescence from the radiation source of the excitation. The presence of an imine moiety in the fluorophore structure typically provides this feature. To be used in the staining of cellular systems, the compounds must also have a number of other properties. The first is the appropriate excitation and emission wavelength. It is believed that suitable characterization should include excitation above 350 nm and fluorescence in the range 500-900 nm. Moreover, such compounds should bind appropriately well to target structures and be easily removed from the measurement environment when unbound. Considering the site of use, it is also required that the compound does not have biological activity in order to avoid additional influences on the measurement process.

According to the research results, the presented structure based on the styrylquinoline skeleton may have favorable fluorescence and physicochemical parameters enabling its use in cell staining.

The essence of the invention is a novel styrylquinoline derivative characterized in that it has the chemical structure according to formula 1.

The novel styrylquinoline derivative of the formula I is suitable for use as a fluorescent dye for staining biological structures.

The invention also relates to the use of the novel styrylquinoline derivative of the formula I for the detection of metal ions, in particular zinc ions, in biological structures, in particular in human or animal cells.

The invention is illustrated by the following examples.

P r z k ł a d 1

Synthesis of 6-amino-1,3-dimethyl-5 - [(E) -N- {4 - [(E) -2- (quinolin-2-yl) ethenyl] phenyl} carboximidoyl] -1,2,3, 4-tetrahydropyrimidine-2,4-dione with the designation BCl15.

Step 1: 2 - [(E) -2- (4-nitrophenyl) ethylene] quinoline 2-methylquinoline (10 mmol) was dissolved in acetic anhydride (40 cm) and p-nitrobenzaldehyde (10 mmol) was added. The mixture was heated on a magnetic stirrer for 17 h at 130 ° C.

PL 236 882 Β1

Then acetic anhydride was evaporated under reduced pressure. The crude product was crystallized from ethanol. A light yellow solid with a melting point of 167 ° C is obtained in a yield of 73%.

¹ H NMR (400 MHz, DMSO) δ 8.41 (d, J = 8.6 Hz, 1H), 8.27 (d, J = 8.6 Hz, 2H), 8.02 (dd, J = 8.2, 4.2 Hz, 3H), 8.00 - 7.90 (m, 3H), 7.78 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 16.4 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H). ¹³ C NMR (126 MHz, DMSO) δ 155.27, 148.11, 147.36, 143.49, 137.24, 133.63, 132.22, 130.52, 129.30, 128.67, 128.35, 127.77, 127.17, 124.53, 120.82.

Stage 2: 2 - [(E) -2- (4-aminophenyl) ethylene] quinoline

[(E) -2- (4-nitrophenyl) -ethylene] -quinoline and anhydrous tin (II) chloride SnCl2 in ethanol in a molar ratio of 1: 5 were placed in a round bottom flask. inert gas atmosphere. After the reaction was complete, the mixture was cooled, transferred to a beaker with ice and adjusted to pH 7-8 with 5% NaHCO 3 solution. Extraction with ethyl acetate was performed, the organic layer was washed with brine then dried with anhydrous Na2SO4 and evaporated under reduced pressure. A red solid with a melting point of 174 ° C is obtained in a yield of 61%. ¹ H NMR (400 MHz, DMSO) δ 8.26 (d, J = 8.6 Hz, 1H), 7.91 (dd, J = 14.2, 8.3 Hz, 2H), 7.77 (d, J = 8.6 Hz, 1H), 7.69 ( dd, J = 17.8, 11.8 Hz, 2H), 7.50 (t, J = 7.4 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 16.2 Hz, 1H), 6.61 ( d, J = 8.3 Hz, 2H), 5.54 (s, 2H). ¹³ C NMR (126 MHz, DMSO) δ 157.03, 150.39, 148.22, 136.54, 135.54, 130.07, 129.21, 128.81, 128.18, 127.10, 125.95, 124.22, 123.28, 120.06, 114.33.

Step 3: BCl15: 6-amino-1,3-dimethyl-5 - [(E) -N- {4 - [(E) -2- (quinolin-2-yl) ethenyl] phenyl} carboximidoyl] -1, 2,3,4-tetrahydropyrimidine-2,4-dione - according to the formula 1.

In a 15 ml tube, 2 - [(E) -2- (4-aminophenyl) -ethylene] -quinoline was placed together with 6-amino-1,3-dimethyl-5-formyluracil in a 1: 1 molar ratio. Then 5 ml of ethanol and 3 drops of acetic acid were added. The mixture thus prepared was subjected to a 50 W microwave field at 83 ° C for 20 minutes. A yellow precipitate is obtained, m.p. 276 ° C in a yield of 60%. ¹ H NMR (400 MHz, DMSO) δ 8.81 (s, 1H), 8.38 (d, J = 13.6 Hz, 1H), 8.35 (d, J = 8.6 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.86 (dd, J = 12.1, 10.1 Hz, 1H), 7.75 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.4 Hz, 1H), 7.44 (d, J = 16.3 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 3.38 (s, 1H), 3.21 (s, 1H). ¹³ C NMR (126 MHz, DMSO) δ 162.02, 157.27, 156.25, 155.79, 151.96, 150.73, 148.16, 136.88, 134.21, 133.37, 130.27, 129.07, 128.91, 128.26, 128.04, 127.43, 126.53, 121.77, 120.39, 86.75, 29.76, 27.98.

Example 2

Spectroscopic properties of the described quinoline derivative.

The most important spectroscopic parameters confirming that the presented derivative is suitable for imaging biological structures with the method according to the invention are presented in the table below.

· ^: · Range and absorption / nm Location of the absorption band ¹¹ λ / nm j (ε / Af ¹ cm ' ¹ )' Maximum n --b 1 luorescence / nm The intensity of Π uorescence ^L Shift Slokes / nm BCI15 250 - 450 386 (45.4 * 10 ^λ 3) 266 (19.3 * 1Ο ^Λ 3) 504 1358 118

Table 1. Spectroscopic properties of a quinoline derivative in DMSO.

^a for a concentration of 2.5 * 10-5 mol / L ^b for a concentration of 6.25 * 10-6 mol / L

PL 236 882 Β1

Exemplary absorption spectra are presented in Fig. 1 for concentrations in the range of 50 μΜ, where the absorption spectra in DMSO are shown, concentrations in the graph (from the highest): 5e-5, 2.5e-5, 1.25e-5, 6.25e6, 3.125e- 6 [mol / L],

The emission spectrum of the compound described by the invention is shown in Fig. 2: emission spectrum (excitation 386 nm), concentration in the diagram: 6.25e-6 [mol / L],

Example 3

Solvatochromic properties of BCI15 derivative.

Table 2 shows the dependence of the spectroscopic properties of the compound on the type (polarity) of the solvent, the so-called solvatochromic properties. Solvents sorted by polarity index (Burdick & Jackson).

solvent DMSO Location of the bands: absorption λ / nm .. Fluorescence maximum λ, _1Μ / ηιή. 504 Fluorescence intensity Shift. Stokcsa / nin acetonitrile 378 488 390 110 methanol 376 494 440 116 chloroform 376 470 585 94 toluene 382 464 100 82

Table 2. Solvatochromic properties of the described bisquinoline derivative - positive solvatochromism.

^a for a concentration of 2.5 * 10 -5 mol / L ^b for a concentration of 5.25 * 10-5 mol / L

Example 4

Spectroscopic properties in the presence of zinc ions.

Table 3 shows the changes in spectroscopic properties after the addition of Zn ²⁺ ions to the dye solution, which proves the complexing properties of the described compound and the possibility of its application to detect this metal ions in cells. There is a noticeable change in the color of the staining from yellow to green and an 18-fold increase in the fluorescence intensity.

relationship Absorption range / other The position of the bands; tbn> rp ^J λ / nni Maximum how many. lrescence ¹ λ / nm lnlens \ nce ł liHwwrncji Shift Stokes / nm BCI15 250 - 500 386 504 220 118 BCI15 + ZnCh (1: 1) 250 - 500 390 483 4016 93

Table 3. Spectroscopic properties of the compound before and after the complexation of Zn ²⁺ ions in DMSO (concentration: 2.5 * 10-5 mol / L)

PL 236 882 B1

The emission spectra of the compound showing changes in fluorescence properties depending on the concentration of Zn ²⁺ ions present in the solution are shown in Fig. 3: the fluorescence spectra of the compound depending on the concentration of Zn ²⁺ ions in the DMSO solution, molar ratio BCI15: Zn ²⁺ in the graph from the highest): 1: 1; 2: 1; 3: 1; 4: 1; 5: 1; 1: 0.

P r z k ł a d 5

Exemplary staining procedure for HCT116 + / + human colorectal cancer cells. Cells were seeded in the amount of 120 thousand. (along with 0.8 mL of DMEM) onto pre-prepared glass slides coated with poly-L-lysine and outlined with a hydrophobic marker, followed by incubation for 24 h at 37 ° C, 5% CO2. After this time, a 25 µM solution of BCl15 (DMSO solvent) in DMEM culture medium was prepared, the final volume on the slide was 0.8 mL. The solution was then infused into the cells and incubated for 2 h at 37 ° C to allow compounds to penetrate the cell membrane. After incubation, cells were washed twice with DMEM (without FBS and phenol red) and then observed by fluorescence microscopy after excitation with a DAPI filter.

P r z k ł a d 6

An exemplary procedure for staining human colorectal cancer cells of the HCT116 + / + line in the presence of zinc ions.

Cells were seeded in the amount of 120 thousand. (along with 0.8 mL of DMEM) onto pre-prepared glass slides coated with poly-L-lysine and outlined with a hydrophobic marker, followed by incubation for 24 h at 37 ° C, 5% CO2. After this time, solutions of BCl15 derivative (DMSO solvent) were prepared at a concentration of 25 µM with the addition of ZnCl2 at a concentration of 25 µM and 50 µM. The solutions were prepared in DMEM culture medium, the final volume on the slide was 0.8 mL. The solutions were then administered to the cells and incubated for 2 h at 37 ° C to allow the compounds to penetrate the cell membrane. After incubation, cells were washed twice with DMEM (without FBS and phenol red) and then observed by fluorescence microscopy after excitation with a DAPI filter.

Claims (2)

Patent claims 1. A novel styrylquinoline derivative, characterized in that it has the chemical structure according to formula 1. 2. The use of a new styrylquinoline derivative of the formula I for the detection of metal ions, in particular zinc ions, in biological structures, especially in human or animal cells.