PL247127B1 - Compounds, derivatives of 4-nitro-benzo-2,1,3-thiadiazole, method of obtaining them and their use - Google Patents

Abstract

The subject of the application are compounds, derivatives of 4-nitro-benzo-2,1,3-thiadiazole, of general formula 1, wherein R is an S atom or an SO group. The subject of the application is also a method of obtaining compounds of formula 1, consisting in the reaction of 4-nitro-benzo-2,1,3-thiadiazole with a morpholine derivative of formula 2, wherein R is an S atom or an SO group, used in an equimolar amount in relation to 4-nitro-benzo-2,1,3-thiadiazole, in a dichloromethane environment, at a temperature of 20°C, for 20 hours in an inert gas atmosphere and after completion of the reaction, removing the solvent under reduced pressure and purifying the final product by column chromatography. The application also concerns the use of the compound of formula 1.

Description

Description of the invention

The subject of the invention are compounds, derivatives of 4-nitro-benzo-2,1,3-thiadiazole, the method of their preparation and their use.

From the description of the patent application US2005282818, as well as from the journal ChemMedChem, 2015,10(5), 797-803, a compound, a benzoxadiazole derivative, of the formula a is known,

Pattern a,

The compound of formula a is obtained by reacting 4-chloro-7-nitro-2,1,3-benzoxadiazole with thiomorpholine in a dimethylformamide medium in an ice bath for 15 minutes and then at room temperature for 50 minutes.

From the journal Organic & Biomolecular Chemistry, 2016, 2016,14,11117-11124 a derivative of 4-nitro-benzo-2,1,3-thiadiazole, of formula b, is known,

Pattern b,

The compound of formula b is obtained by the reaction of 4-chloro-7-nitrobenzo-2,1,3-thiadiazole with morpholine in a dichloromethane medium, in the presence of N-ethyl-N,N-diisopropylamine, at a temperature of 20°C for 4 hours.

From the description of the patent application CN 106565694, as well as from the journal Organic & Biomolecular Chemistry, 2016, 2016,14,11117-11124, a compound is known, a benzothiadiazole derivative, of the formula c,

PL 247127 Β1

Pattern c,

The compound of formula c is obtained by the reaction of a mixture of mono-tBoc-piperazine and 4-chloro-7-nitrobenzofurazan in dichloromethane in the presence of N,N-di-isopropylethylamine at room temperature for 4 hours.

Known methods of obtaining 4-nitro-benzo-2,1,3-thiadiazole derivatives require the use of a basic environment using, among others, triethylamine or N,N-diisopropylethylamine, which requires the use of significantly larger amounts of organic solvents in order to wash out the above amines.

The aim of the invention is to produce new compounds - derivatives of 4-nitro-benzo-2,1,3-thiadiazole, which find application in the study of processes occurring in biological-chemical systems, for fluorescent detection of hypochlorous acid and as fluorescent dyes, as well as to develop a method for producing these compounds.

The subject of the invention are compounds, derivatives of 4-nitro-benzo-2,1,3-thiadiazole, of the general formula 1,

Formula 1 where R is an S atom or an SO group.

The compounds of formula 1 are solids (powders) of orange-red color, with a melting point of 194-195°C when R is an S atom and a melting point of 275°C when R is an SO group.

The method of obtaining compounds of formula 1, defined above, by reacting a 2,1,3-thiadiazole derivative with a morpholine derivative in a dichloromethane medium at a temperature of 20°C, according to the invention, consists in reacting a morpholine derivative of formula 2 with a 2,1,3-thiadiazole derivative in the form of 4-nitro-benzo-2,1,3-thiadiazole

PL 247127 Β1 .NH ^R^

Formula 2 wherein R is an S atom or an SO group, used in an equimolar amount relative to 4-nitro-benzo-2,1,3-thiadiazole, under stirring for 20 hours in an inert gas atmosphere and after completion of the reaction, the solvent is removed under reduced pressure. The final product is purified by column chromatography using a dichloromethane/methanol mixture in a volume ratio of 20:1 (v/v) as eluent.

The use according to the invention consists in that the compound of formula 1, in which R is an S atom, hereinafter referred to as the probe is used to determine chloric acid (I). Detection of chloric acid (I) consists in the reaction of the probe with chloric acid (I) used in an amount of 1 to 160% by moles in relation to the amount of the probe, in the medium of a mixture of acetonitrile and 0.1 M aqueous phosphate buffer solution at pH 7.4, used in a volume ratio of 1:9 v/v, for up to 1 minute, and as a result of this reaction, a compound of formula 1 is obtained, in which R is an SO group.

Compounds of formula 1, wherein R is an S or SO atom, are used in the procedure of detecting hypochlorous acid in biological systems, because they do not show reactivity towards glutathione, which is a compound present in the cellular environment in high concentrations, and do not show reactivity towards other biological oxidants, such as hydrogen peroxide and peroxynitrite. Since hydrogen sulphide (H2S) can interfere with the action of reagents and reaction products of the activated myeloperoxidase (MPO)/hydrogen peroxide (H2O2)/chloride anion (Cl) system, it has been shown that compounds of formula 1, wherein R is an S or SO atom, do not show reactivity towards H2S, which confirms their stability. Moreover, these compounds can potentially be used in cellular studies to monitor biological-chemical processes or as fluorescent dyes.

The new compound of formula 1, in which R is an S atom, used as a probe for detecting chlorous acid, facilitates the identification of the oxidation product without the need to identify undesirable reaction products, and indirectly also the qualitative and quantitative analysis of chlorous acid, the detection time is short and as a result of detection a product is selectively formed which constitutes an unambiguous confirmation of the presence of the detected chlorous acid.

The subject of the invention is illustrated by the following examples, with reference to the drawing, in which Fig. 1 shows chromatograms of the compound of formula 1, in which R denotes an S atom, used as a probe for detecting hypochlorous acid (HOCl) in an environment of an acetonitrile - 0.1 M phosphate buffer mixture at pH 7.4, with a ratio of 1:9 v/v, at a wavelength of 500 nm; Fig. 2A graphs of changes in fluorescence intensity spectra as a function of concentration for a system containing 4 μΜ of the compound of formula 1, in which R denotes an S atom, and 0-7 μΜ of HOCl acid, in an acetonitrile (MeCN) - 0.1 M phosphate buffer mixture at pH 7.4, with a ratio of 1:9 v/v; Fig. 2B is a graph of fluorescence changes as a function of concentration for a system containing 4 μM of the compound of formula 1, in which R is S, and 0-7 μM of HOCl acid, in a mixture of MeCN - 0.1 M phosphate buffer at pH 7.4, at a ratio of 1:9 v/v; Fig. 3 and Fig. 4 present graphs of the selectivity of the compound of formula 1, in which R is S (Fig. 3) and SO (Fig. 4), relative to HOCl acid, hydrogen peroxide (H2O2), peroxynitrite (ONOO) and glutathione (GSH); Figures 5A and 5B show chromatograms from hydrogen sulfide (H2S) stability studies: Figure 5A of the compound of formula 1, wherein R is S, Figure 5B of the compound of formula 1, wherein R is SO, in an aqueous solution containing phosphate buffer (50 mM, pH 7.4) and MeCN (10%) at room temperature.

Parts given in the examples are parts by weight.

Example 1

0.2 parts of 4-nitro-benzo-2,1,3-thiadiazole (NBD-S-Cl) was dissolved in 5 parts of dichloromethane, 0.2 parts of thiomorpholine was added, and the mixture was stirred at 20°C for 20 hours under argon atmosphere. The reaction mixture was then evaporated under reduced pressure. The crude product was purified by column chromatography using dichloromethane/methanol 20:1 (v/v) as eluent.

4-Thiomorpholino-7-nitro-2,1,3-benzenethiadiazole (NBD-S-TM) was obtained in 85% yield as an orange powder with a melting point of 194-195°C.

¹ H NMR spectrum and HRMS-ESI mass spectrum of the obtained compound:

^1H NMR (CDCl3, 400 MHz) d (ppm): 8.60-8.55 (m, 1H), 6.63-6.59 (m, 1H), 4.41-4.36 (m, 4H), 2.90-2.85 (m, 4H)

HRMS (ESI): m/z 283.0330 ([M+H]+)

HRMS (ESI-Na) m/z 305.0150 ([M+Na] ⁺ ) fully confirmed its structure.

The spectroscopic properties in MeCN - 0.1 M sodium phosphate buffer solution at pH 7.4, 1:4, (v/v) of the obtained compound were as follows:

λabs [nm] (ε [MW ¹ ]): 489'(263500), λabs [nm]: 550, λex [nm]: 489.

Example 2

0.2 part of 4-nitro-benzo-2,1,3-thiadiazole (NBD-S-Cl) was dissolved in 5 parts of dichloromethane, 0.2 part of thiomorpholine monoxide was added, and the mixture was stirred at 20°C for 20 hours under argon atmosphere. The reaction mixture was then evaporated under reduced pressure. The crude product was purified by column chromatography using dichloromethane/methanol 20:1 (v/v) as eluent.

Thiomorpholine-7-nitro-2,1,3-benzenethiadiazole-4-monoxide (NBD-S-TSO) was obtained in 50% yield as a dark orange powder with a melting point of 275°C.

1H NMR spectrum and HRMS-ESI mass spectrum of the obtained compound:

NBD-S-TSO: Spectrum: ¹ H NMR (CDCI3, 400 MHz) d (ppm): 8.62 (dd' J = 12.8, 8.3 Hz, 1H ) 6.75 (d, J = 8.9 Hz, 1H), 4.72 (d, J = 14.5 Hz, 2H), 4.43 (ddd, J = 14.1, 10.7, 3.1 Hz, 2H), 3.06-2.93 (m, 4H)

HRMS (ESI): m/z 299.0277 ([M+H] ⁺ )

HRMS (ESI-Na) m/z 321.0091 ([M+H] ⁺ ) fully confirmed its structure.

The spectroscopic properties in acetonitrile (MeCN) of the obtained compound were as follows: λabs [nm] (ε [M' ¹ cm- ¹ ]): 475 (22250), λem [nm]: 550, λθχ [nm]: 489.

Example 3

A sample solution was prepared, i.e. a solution of 0.00282 parts of NBD-S-TM compound in 9.997 parts of acetonitrile.

A solution of 0.017 parts HOCl acid in 9.983 parts water was also prepared.

Then, 0.154 parts of the prepared HOCl acid solution were diluted with 4.846 parts of water to obtain HOCl acid stock solution.

From the prepared solutions, compositions containing 75 μl of the NBD-S-TM sampler stock solution, 75 μl of MeCN and 0-120 μl of the HOCl acid stock solution were prepared.

The prepared compositions were diluted to a volume of 1.5 ml by adding 0.1 M phosphate buffer at pH 7.4. After 1 minute of sample incubation at room temperature, chromatographic analyses of the prepared samples were performed using a Shimadzu chromatograph with a DAD detector (model SPD-M20A); Kinetex Core-Shell (C18) column from Phenomenex (100 mm x 4.6 mm) with a grain diameter of 2.6 μm; mobile phase: water + 0.1% TFA - acetonitrile + 0.1% TFA with a gradient flow from 10 to 100% MeCN + 0.1% TFA for 15 minutes, v/v; flow rate 1.5 ml/minute; sample volume: 20 μl.

The conducted experiment confirmed the formation of the oxidation product of the NBD-S-TM probe with HOCl acid, i.e. the compound of formula 1, where R is the SO group (NBD-S-TSO), and thus confirmed the use of the NBD-S-TM probe for the detection of HOCl acid - Fig. 1 of the drawing shows chromatograms (DAD 450 nm detector) recorded after 1 minute of incubation of the NBD-S-TM probe (50 μM) with HOCl acid (0-80 μΜ) in an environment of pH 7.4 (mixture of MeCN and 0.1 M aqueous phosphate buffer solution, used in a volume ratio of 1:9).

Claims (4)

1. Compounds, derivatives of 4-nitro-benzo-2,1,3-thiadiazole, of the general formula 1, Formula 1 where R is an S atom or an SO group. 2. A method for obtaining compounds, 4-nitro-benzo-2,1,3-thiadiazole derivatives of the formula outlined in claim 1, by reacting a 2,1,3-thiadiazole derivative with a morpholine derivative in a dichloromethane environment at a temperature of 20°C, characterized in that a morpholine derivative of the formula 2 is reacted with a 2,1,3-thiadiazole derivative in the form of 4-nitro-benzo-2,1,3-thiadiazole Formula 2 wherein R is an S atom or an SO group, used in an equimolar amount relative to 4-nitrobenzo-2,1,3-thiadiazole, under stirring for 20 hours in an inert gas atmosphere and after completion of the reaction, the solvent is removed under reduced pressure and the final product is purified by column chromatography using a dichloromethane/methanol mixture in a volume ratio of 20:1 (v/v) as the eluent. 3. Use of the compound of formula 1 as defined in claim 1, wherein R is an S atom, for the detection of hypochlorous acid. 4. Use according to claim 3, characterized in that the detection of hypochlorous acid consists in the reaction of the compound of formula 1, in which R is an S atom, called the probe, with hypochlorous acid used in an amount of 1-160% by moles in relation to the amount of the probe, in the medium of a mixture of acetonitrile and 0.1 M aqueous phosphate buffer solution with pH 7.4, used in a volume ratio of 1:9 v/v, for up to 1 minute, and as a result of this reaction, a compound of formula 1 is obtained, in which R is an SO group.