PL243397B1 - Reactive derivatives of Safirinium dyes and their use - Google Patents

Abstract

The subject of the invention are active compounds derived from Safirinium and their use for labeling chemical molecules in the cycloaddition reaction of azides and alkynes.

Description

Description of the invention

The subject of the invention are reactive triazolopyridinium and triazoloquinolinium derivatives of Safirinium dyes and their use for fluorescent labeling of chemical molecules in the cycloaddition reaction of azides and alkynes. The copper-catalyzed cycloaddition reaction of azides and alkynes, the products of which are 1,4-disubstituted triazoles (Scheme 1), is a "flagship" example of the "click chemistry" approach, proposed in 2001 by K. B. Sharpless. The basic assumption of this approach is the use of simple and efficient reactions taking place under mild conditions [1]. From the beginning, one of the main applications of this synthetic strategy was fluorescent labeling of biomolecules or biologically active substances. Numerous examples of such use of cycloaddition are presented in a number of review papers [2-5], of which the paper by Thirumurugan et al. [6] is particularly extensive and up-to-date.

R ¹ ® ... Cum mN

r ^z

Scheme 1. General scheme of the copper catalyzed cycloaddition reaction of azides and alkynes.

Since organic azides, terminal alkynes, and the 1,2,3-triazoles formed from them do not occur in the basic biological processes of living organisms, and the copper ion-catalysed cycloaddition reaction can proceed without much difficulty in the aqueous environment in the presence of ions and other molecules, it is considered as a "bioorthogonal" reaction, i.e. one that does not interfere with biological processes, and at the same time is not hampered by them. This property of the cycloaddition reaction of azides and alkynes is extremely valuable because, thanks to a properly designed labeling procedure, it allows for selective tracking of selected biological processes or biomolecules in vitro or even in vivo using the phenomenon of fluorescence. An example of such an application can be the method of tracking the incorporation of azidothymidine (an antiviral drug used in the treatment of HIV) into DNA described in 2011 by Koha et al. [7].

Fluorescent dyes combined with reactive fragments that can react with amino and other groups are known from the state of the art - a number of such compounds have been disclosed in the international patent application PCT WO 2012/129128A1, or in European patents EP2223086B1 and EP2686680 B1. Among the reactive functional groups that enable the dye to be combined with other molecules, we can distinguish A/-hydroxysuccinimide esters, which allow easy formation of amide bonds. The fluorescent system may also be provided with fragments such as an isothiocyanate group or others. Dyes containing a carboxyl group in their structure can be converted into reactive esters in situ using activating reagents such as N,Λ/'-dicyclohexylcarbodiimide (DCC) or carbonyldiimidazole (CDI). Examples of reactive fluorescent dyes containing an N-hydroxysuccinimide fragment (EP2686680B1 or EP2223086B1) or an isothiocyanate group (FITC) are shown in Scheme 2 below:

EP2686680B1

EP2223086B1

FITC

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From the scientific literature there are also known alkyne derivatives of fluorescent dyes that can undergo cycloaddition reactions with organic azides.

Scheme 3. Alkyne labels used in click chemistry fluorescence labeling: A - DIB-ET, B - prop-fmoc, C - Cy3, D - fluorescein, E - dansyl, F - cinchona alkaloid, G - BODIPY

Most alkyne tags have been designed by chemical modification of dyes known in the literature, such as DIB-ET (A) [8], prop-fmoc (B) [9], cyanine derivatives (Cy3, Cy5) (C) [7], xanthene derivatives (fluorescein) (D) [10], dansyl (E) [11], cinchona alkaloids (F) [12], BODIPY (G) [13] (Scheme 3). For most alkyne dyes, the acetylene moiety was introduced by amide or ester bond formation (prop-fmoc (B), cyanins (C), fluorescein (D), dansyl (E), BODIPY (G)). Reactions of forming such chemical bonds are fast and do not require special conditions. However, due to the presence of reactive sites in the structure of the dye molecules, which after modification do not affect the fluorescent properties, it is possible to introduce the alkyne by other synthetic methods. For example, in the case of the synthesis of DIB-ET (A), the carbon-carbon bond formation reaction was carried out in the reaction of dibenzoyl, ammonium acetate and 4-ethynyl bezaldehyde. In the case of cinchona alkaloids (F), in the first step, a dibromo derivative of the olefin is obtained, and then, after the action of a strong base, an alkyne fragment is formed as a result of elimination. Alkyne fluorescent tags are used to label mainly structures of biological importance [14], such as the drug azidothymidine [8,9,12], sugars [15], proteins [11], nucleotides [16], viruses [10]. The biological environment in different systems can vary enough that it is often necessary to adapt the reagents to the prevailing conditions. The structural complexity of fluorescent markers and the resulting difficulties in their synthesis constitute a serious limitation in scientific research [17]. The search for new dyes containing an alkyne fragment is an important element of the dynamic development of bioconjugation methods.

As in the case of alkyne derivatives of fluorescent dyes, a number of azide derivatives are known, obtained by modifying known fluorescent systems containing a carboxyl or amino group. Examples include derivatives of fluorescein (H [18]), rhodamine (I [19]), cyanine (M [20]). The azide fragment is also introduced directly into the structure of dyes, most often as a substituent of aromatic rings or alkyl fragments. Examples of known azide derivatives of fluorescent dyes are shown in Scheme 4.

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Scheme 4: Structures of fluorescent dyes containing an azide fragment: H - fluorescein derivative, I - rhodamine, J - coumarin, K and L - dansyl, M - cyanine, N - BODIPY [21,22].

The fluorescent dyes Safirinium-P (P1 and P2) and Safirinium-Q (P4 and P5) (Scheme 6) and the method of their preparation are disclosed in the Polish patent description PL 223740 B1. The common core of the above compounds is the PO fragment (Scheme 6) containing a 1,2,4-triazolopyridinium system substituted with a carbonyl group providing an attachment point for reactive groups. The disclosed structures were used for fluorescent labeling of bacterial spores [30] and in proteomics as ionization markers in the study of protein hydrolysates. [31]

AFTER

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Scheme 6. Fluorescent dyes Safirinium-P (P1 and P2) and Safirinium-Q (P4 and P5) disclosed in the patent description PL 223740 B1.

Physical and chemical properties of Safirinium dyes and fluorescent markers obtained on their basis in the form of active esters with N-hydroxysuccinimide (NHS), such as high fluorescence quantum yield, high Stokes shift, water solubility, chemical stability, resistance to photo-bleaching and the lack of dependence of optical properties on pH predispose this group of compounds to the role of fluorescent dyes with potential for use. In addition to the dyes described above, their derivatives containing substituents in the quinoline system, as well as analogs with a more developed skeleton, are also known. The synthesis, structure and physicochemical properties of these derivatives were revealed in the work of Fedorowicz et al. [32].

Despite the description of derivatives that can be used as fluorescent labels in the state of the art, there is still a lack of compounds that selectively bind to labeled molecules in a fast and efficient manner using techniques other than the amide bond formed as a result of the action of amines on N-hydroxysuccinimide esters. The state of the art does not describe compounds with a structure that would allow them to be used in the copper-catalysed bioorthogonal cycloaddition reaction of azides and alkynes. The above problems have been solved by the present invention.

1. The first object of the invention is a compound of formula (S1):

Where:

X is a moiety selected from the group consisting of: NH, O, where n is from 1 to 10 carbon atoms in the methylene moieties,

Ri is a substituent selected from the group consisting of the formulas (W1) to (W2):

W1 W2

R ² , R ³ - means an alkyl substituent with the number of carbons from 2 to 8,

R ⁴ , R ⁵ , R ⁶ - means a methyl substituent or a hydrogen atom, or R ⁴ means a methyl substituent or a hydrogen atom, while the substituents R ⁵ and R ⁶ are combined and form an aromatic system of the general formula S2, where the substituents R ⁷ - R ¹⁰ represent a hydrogen atom:

wherein the anion A ^- is an ion selected from the group consisting of: F ^- , Ch, Br, h.

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In a preferred embodiment of the invention, the compound of formula (S1) is described by the structure:

Where:

X is a moiety selected from the group consisting of: NH or O, where n denotes from 1 to 7 carbon atoms in the methylene moieties,

^R1 is a substituent selected from the group consisting of the formulas (W1) to (W2):

W1 W2

R ² , R ³ - denote an alkyl substituent with the number of carbon atoms from 2 to 8,

R ⁴ , R ⁵ , R ⁶ - means a methyl substituent or a hydrogen atom, where the anion A- is a bromide ion Br

In another preferred embodiment of the invention, the compound (S1) for the purposes of the present invention has been selected from the group consisting of derivatives of the formulas P6-P11:

2,2-Diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H-/,3H-/-[1,2,4]triazolo[4] bromide ,3-a]pyridine-2-yl (P6),

5,7-Dimethyl-2,2-dioctyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H-/,3H-/-[1,2,4]triazolo[4] bromide ,3-a]-pyridine-2-yl (P7),

2,2-Diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yloxy)carbonyl]-2H-/,3H-/-[1,2,4]triazolo[4] bromide ,3-a]-pyridine-2-yl (P8),

8-[(5-Azidopentyl)carbamoyl]-5,7-dimethyl-2,2-dioctyl-2H-/,3H-/-[1,2,4]triazolo[4,3-a]pyridine bromide -2-lead (P9),

8-[(5-Azidopentyl)carbamoyl]-5,7-dimethyl-2,2-diethyl-2H-/,3/-/-[1,2,4]triazolo[4,3-a]pyridine bromide -2-lead (P10),

- 8-[(7-Azidoheptyl)carbamoyl]-5,7-dimethyl-2,2-diethyl-2H-/,3/-/-[1,2,4]triazolo[4,3-a] bromide pyridine-2-ium (P11), shown below:

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Me O

me

P7 C ₈ H ₁₇

Preferably, the invention relates to a compound of formula (S2):

Where:

the X moiety is selected from the group consisting of: NH, O, wherein n is from 1 to 10 carbon atoms in the methylene moieties,

Ri - is a substituent selected from the group consisting of substituents with the formulas (W1) to (W2):

R ² , R ³ is an alkyl substituent with the number of carbons from 2 to 8,

R ⁴ - represents a substituent selected from the group consisting of: methylene or hydrogen, R ⁷ , R ⁸ , R ⁹ , R ¹⁰ - represents a hydrogen atom, the anion A ^- is an ion selected from the group consisting of: F ^- , Ch, Br, h.

PL 243397 B.I

In a preferred embodiment of the invention, the compound of formula (S2) is described by the structure:

Where:

the X moiety is an NH group, where n is from 1 to 7 carbon atoms in the methylene moieties, Ri - is a substituent (W1) or (W2):

W1 W2

R ² , R ³ is an alkyl substituent with the number of carbons from 2 to 8,

^R4 - represents a substituent selected from the group consisting of: methylene or hydrogen,

R ⁷ , R ⁸ , R ⁹ , R ¹⁰ - represents a hydrogen atom, the anion A- being the ion Br.

In another preferred embodiment of the invention, the compound (S2) is selected from the group consisting of derivatives of formulas Ρ12-P13:

2,2-Diethyl-4-[(prop-2-yn-1-yl)carbamoyl]-7H-/,2H-/-[1,2,4]triazolo[4,3-a]quinoline bromide -2-lead (P12),

2,2-Dioctyl-4-[(prop-2-yn-1-yl)carbamoyl]-7H-/,2H-/-[7,2,4]triazolo[4,3-a]quinoline bromide -2-lead (P13), shown below

A further object of the invention is the use of the compound of the invention as defined above for the labeling of chemical molecules in the cycloaddition reaction of azides and alkynes.

For the purposes of this specification, an "alkyl substituent" is considered to be a group having the general formula CnH2n+i with an unbranched carbon chain, eg methyl, ethyl, n-propyl, n-octyl.

For the purposes of this specification, an "alkenyl substituent" is considered to be a group having the general formula _CnH2n -i with an unbranched carbon chain, eg ethenyl, propenyl.

For the purposes of this specification, an "alkyne substituent" is considered to be a group containing at least one triple bond in a branched or unbranched carbon chain.

The advantage of the solution according to the invention is the increase in the selectivity of the process of combining the fluorescent dye with the target molecule by using the bioorthogonal cycloaddition reaction of azides and alkynes, which has found wide application in the labeling of biomolecules.

The presence of azide and alkyne fragments in the molecules of the presented dyes allows them to be used for labeling with the use of cycloaddition reactions of azides and alkynes in a copper(I) or non-copper catalyzed version. In addition to the 3+2 cycloaddition, both azide [35] and alkyne [33,34] derivatives can undergo a number of other chemical reactions.

The reactive dyes of the present invention can be used to label substances containing suitable compatible functional groups such as azide or alkyne. A particular example of such a reaction is the reaction of a dye containing an alkyne moiety linked by an amide bond with organic azides of various structures. In addition to the given examples, other azides may also undergo analogous reactions, including derivatives of known drugs and even more complex systems.

Implementation examples are shown below. Figure 1 shows the ¹ H-NMR spectrum of 2,2-diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H,3H-[1,2 ,4]triazolo[4,3-a]pyridin-2-ium (P6), Fig. 2 1H-NMR spectrum of 2,2-diethyl-5,7-dimethyl-8-{[(1-tetradecyl-1-bromide) H-1,2,3-triazol-4-yl)methyl]carbamoyl}-2H,3H-[1,2,4]triazolo[4,3-a]pyridin-2-ium, obtained by the procedure from Example 3, from compound P6 and tetradecyl azide, Fig. 3 Absorption and emission spectra in the UV-Vis range for pyridinium derivatives (S1), with emission spectra recorded at the excitation wavelength of 350-370 nm, Fig. 4 Absorption and emission spectra in the UV-Vis range for quinoline derivatives (S2), the emission spectra were recorded at the excitation wavelength of 350-370 nm.

Example 1. Synthesis of the active N-hydroxysuccinimide ester of Safirinium P according to publication [30]

4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one, diethylamine (1.1 equiv.) and formaldehyde (1.1 equiv.) in the form of a 37% aqueous solution were dissolved in methanol and heated in in a closed vessel at 50 degrees Celsius for 1 hour. After heating was complete, the reaction mixture was evaporated under reduced pressure.

The product of the reaction described above (2,2-diethyl-5,7-dimethyl-2,3-dihydro-[1,2,4]triazolo[4,3-a]pyridine-2-inium-8-carboxylate) was dissolved in methanol and acidified with methanolic HCl. The solvent was evaporated under reduced pressure, and the hydrochloride salt thus obtained was used for the next reaction.

The hydrochloride, N,N'-diisopropylcarbodiimide (DIC, 1.5 equiv.) and N-hydroxysuccinimide (NHS, 2 equiv.) obtained earlier were dissolved in DMF and stirred at room temperature for two hours. The reaction mixture was then evaporated under reduced pressure and the resulting solid was washed twice with small amounts of acetone.

Example 2. General procedure for the synthesis of Safirinium esters and propargylamides P (S1 where X = NH or O; R ¹ = W1) and Q (S2 where X = NH or O; R ¹ = W1)

To the N-hydroxysuccinimide ester of Safirinium P (Example 1) or Q was added 1.5 eq. propargylamine (for X = NH) or propargyl alcohol (for X = O), dissolved in dichloromethane, followed by the addition of 1.1 equivalents of anhydrous potassium carbonate. Then it was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was filtered through a pleated filter to remove potassium carbonate. The obtained filtrate was evaporated under reduced pressure.

Example 3. General procedure for the cycloaddition reaction of alkyne derivatives of dyes (S1 and S2, where ^R1 = W1) with organic azides.

To 100 mg of Safirinium propargylamide dissolved in 3 mL of dichloromethane was added 1.05 molar equivalents of organic azide, followed by 10 mol% 0.05 M aqueous CuSO4, and 10 mol% AMTC (2-{4-[(dimethylamino)methyl]-1 ,2,3-triazolyl}cyclohexan-1-ol). The reaction was started by adding 10 mol% sodium ascorbate as a 10 mg/mL aqueous solution. The mixture was supplemented with water so that the volume of water was 1.5 mL after addition of the sodium ascorbate solution. The reaction was carried out overnight at 30 degrees Celsius with vigorous stirring. After completion of the reaction, the solvents were evaporated under reduced pressure.

Example 4. General procedure for chromatographic purification of reactive dyes and their triazole derivatives (S1 and S2)

The chromatography column was filled with a degassed suspension of silica gel in methanol under reduced pressure. After the gel set up properly, the column was saturated with an inorganic salt by washing with a saturated methanolic salt solution (e.g. potassium iodide, sodium chloride, sodium bromide, potassium bromide, potassium chloride). After the column was saturated, the chromatographic separation was carried out according to the standard procedure of preparative liquid chromatography. Upon completion of the separation, the excess inorganic salt in the collected eluent fractions containing the substance to be purified was removed by crystallization.

Example 5. Synthesis of azidoalkylamide derivatives of Safirinium (S1 and S2 where X = NH; ^R1 = W2)

The azidoalkylamide derivatives of Safirinium dyes were obtained by reacting the active N-hydroxysuccinimide esters of Safirinium with the appropriate azidoalkylamine in the presence of anhydrous potassium carbonate.

Safirinium P N-hydroxysuxinimide ester (Example 1) was dissolved in dichloromethane, 2 molar equivalents of azidoalkylamine and 1.5 equivalents of anhydrous potassium carbonate were added. Then it was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was filtered through a pleated filter to remove potassium carbonate. The obtained filtrate was evaporated under reduced pressure. The resulting product was chromatographically purified according to the procedure described in Example 4.

Example 6 2,2-Diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H,3H-[1,2,4]triazolo[4,3-bromide a]pyridine-2-yl (P6)

The derivative was prepared according to the procedure described in examples 1-2 and purified according to the procedure described in example 4

1 H-NMR (CHLOROFORM-d, 300 MHz): δ = 8.40 (t, J = 4.7 Hz, 1H), 6.55 (s, 2H), 5.99 (s, 1H), 4, 11 (dd, J=5.0, 2.6 Hz, 2H), 3.99-4.08 (m, 2H), 3.69-3.83 (m, 2H), 2.58 (d, J = 7.6 Hz, 6H), 2.22 (t, J = 2.3 Hz, 1H), 1.43 (t, J = 6.7 Hz, 6H) ppm, ¹³ C-NMR (CHLOROFORM- d, 75 MHz): δ = 163.0, 159.6, 156.5, 144.3, 113.9, 107.4, 79.6, 73.0, 71.4, 62.6, 29, 6, 29.2, 22.9, 19.7, 8.6 ppm, LC-MS: R t = 2.04 min; 100%; m/z [M] ⁺ = 287

Example 7 5,7-dimethyl-2,2-dioctyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H,3H-[1,2,4]triazolo[4,3 -a]pyridine-2-yl (P7)

The derivative was prepared according to the procedure described in examples 1-2 and purified according to the procedure described in example 4

1 H-NMR (METHANOL-d4, 300 MHz): δ = 6.16 (s, 1H), 5.95 (s, 2H), 4.11 (d, J = 2.9 Hz, 2H), 3.553, 76 (m, 4H), 2.64 (t, J = 2.6 Hz, 1H), 2.42 (s, 3H), 2.34 (s, 3H), 1.81 (br. s, 4H ), 1.29-1.39 (m, 20H), 0.89 (t, J = 7.0 Hz, 6H) ppm, LC-MS: Rt = 7.24 min; 95.31%; m/z [M] ⁺ = 455.

Example 8 2,2-Diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yloxy)carbonyl]-2H,3H-[1,2,4]triazolo[4,3-a ]pyridine-2-yl (P8)

The derivative was prepared according to the procedure described in examples 1-2 and purified according to the procedure described in example 4

1 H-NMR (CHLOROFORM-d, 300 MHz): δ = 6.42 (s, 2H), 5.91 (s, 1H), 4.88 (d, J = 2.9 Hz, 2H), 4, 02-4.12 (m, 2H), 3.64-3.76 (m, 2H), 2.58 (s, 3H), 2.51 (t, J = 2.3 Hz, 1H), 2 .36 (s, 3H), 1.46 (t, J = 7.0 Hz, 6H) ppm, LC-MS: R t = 2.96 min; 95.19%; m/z [M] ⁺ = 288

Example 9 8-[(5-Azidopentyl)carbamoyl]-5,7-dimethyl-2,2-dioctyl-2H,3H-[1,2,4]triazolo[4,3-a]pyridinium bromide 2nd (P9)

The derivative was prepared according to the procedure described in examples 1 and 5 and purified according to the procedure described in example 4

1H-NMR (METHANOL-d4, 500MHz): δ = 6.12 (s, 1H), 5.81 (s, 2H), 3.71-3.80 (m, 2H), 3.51-3 .66 (m, 4H), 3.30-3.33 (m, 2H), 2.35 (s, 3H), 2.28 (s, 3H), 1.72-1.87 (m, 4H) ), 1.57-1.66 (m, 4H), 1.45-1.51 (m, 2H), 1.27-1.38 (m, 20H), 0.86-0.90 (m 6H)ppm, ^13C -NMR (METHANOL-d4, 126 MHz): δ = 164.6, 155.9, 153.1, 142.9, 111.5, 111.1,73.5, 66, 4, 51.1, 41.2, 38.9, 31.5, 28.9, 28.5, 28.2, 26.0, 23.8, 22.4, 22.3, 22.2, 19.1, 17.3, 13.0 ppm LC-MS: R t = 7.90 min; 84.24%; m/z [M] ⁺ = 528.63; UV-VIS: λmax = 257 nm; 362nm

Example 10 8-[(5-Azidopentyl)carbamoyl]-5,7-dimethyl-2,2-diethyl-2H,3H-[1,2,4]triazolo[4,3-a]pyridin- 2-way (P10)

The derivative was prepared according to the procedure described in examples 1 and 5 and purified according to the procedure described in example 4

1 H-NMR (CHLOROFORM-d, 500 MHz): δ = 7.85 (t, J = 5.7 Hz, 1H), 6.42 (s, 2H), 6.05 (s, 1H), 4, 04 (dq, J=13.3, 6.8 Hz, 2H), 3.71-3.83 (m, 2H), 3.34-3.43 (m, 2H), 3.23-3, 33 (m, 2H), 2.60 (s, 3H), 2.56 (s, 3H), 1.55-1.71 (m, 4H), 1.38-1.54 (m, 8H) ppm, ¹³ C-NMR (CHLOROFORM-d, 126 MHz):

δ = 163.3,158.7,156.7,143.3,114.1, 109.0, 100.0, 73.5, 62.8, 51.4, 51.1, 39.9, 39.4, 29.0, 28.6, 28.3,

26.9, 24.3, 23.8, 22.7, 20.2, 8.9 ppm, LC-MS: R t = 3.61 min; 100%; m/z [M] ⁺ = 360.34; UV-VIS: Xmax = 256 nm; 355nm

Example 11 8-[(7-Azidoheptyl)carbamoyl]-5,7-dimethyl-2,2-diethyl-2H,3H-[1,2,4]triazolo[4,3-a][4,3- a]-triazolo[4,3-o]pyridin-2-ium (P11)

The derivative was prepared according to the procedure described in examples 1 and 5 and purified according to the procedure described in example 4 ¹ H-NMR (CHLOROFORM-d, 500 MHz): δ = 7.81 (t, J = 5.4 Hz, 1H), 6 .51 (s, 2H), 5.98 (s, 1H), 3.98 (m, 2H), 3.74 (m, 2H), 3.27-3.34 (m, 2H), 3, 18-3.22 (m, 2H), 2.56 (s, 3H), 2.50 (s, 3H), 1.50-1.54 (m, 4H), 1.42 (t, J = 7.2 Hz, 6H), 1.30-1.33 (m, 6H) ppm, ¹³ C-NMR (CHLOROFORM-d, 126 MHz): δ = 163.3, 158.3, 156.7, 143.3, 113.9, 109.1,73.7 , 62.4, 53.6, 51.4, 39.5, 29.3, 28.8, 28.7, 26.9, 26.7, 26.7, 22.6, 20.0, 8.9 ppm, LC-MS: Rt = 4.42 min; 100%; m/z [M] ⁺ = 388.38; UV-VIS: Xmax - 255nm; 354nm

Example 12 2,2-Diethyl-4-[(prop-2-yn-1-yl)carbamoyl]-1H,2H-[1,2,4]triazolo[4,3-a]quinolin-2 bromide -i (P12)

The derivative was prepared according to the procedure described in example 2 and purified according to the procedure described in example 4 ^1H -NMR (METHANOL-d4, 300 MHz): δ = 8.76 (s, 1H), 7.91-7.97 (m, 1H), 7.78-7.87 (m, 1H), 7.357.47 (m, 2H), 6.09 (s, 2H), 4.24 (d, J = 2.9 Hz, 2H), 3.87-4.00 (m, 4H), 3.06-3.11 (m, 1H), 1.48 (t, J = 7.0 Hz, 6H) ppm, ^13C -NMR (DMSO- ds, 75 MHz): δ = 173.2, 160.9, 154.1, 145.0, 135.3, 134.5, 131.5, 124.3, 120.6, 115.2, 114, 9, 81.0, 74.0, 73.1, 61, 1, 29.5, 25.7, 8.4 ppm, LC-MS: R t = 2.85 min; 99.45%; m/z [M] ⁺ = 309

Example 13 2,2-Dioctyl-4-[(prop-2-yn-1-yl)carbamoyl]-1H,2H-[1,2,4]triazolo[4,3-a]quinolin-2-bromide ionic (P13)

The derivative was prepared according to the procedure described in Example 2 and purified according to the procedure described in Example 4. ¹ H NMR (CHLOROFORM-d, 300 MHz): δ = 8.79 (s, 1H), 8.21 (t, J = 3 Hz, 1H), 7.72-7.81 (m, 2H), 7.53 (d, J = 8.8 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 6, 56 (s, 2H), 4.27-4.36 (m, 2H), 4.25 (dd, J = 5.3, 2.3 Hz, 2H), 3.73-3.86 (m, 2H), 2.26-2.31 (m, 1H), 1.71-1.86 (m, 4H), 1.19-1.45 (m, 20H), 0.75-0.93 ( m, 6H) ppm, LC-MS: R t = 7.40 min; 94.06%; m/z [M] ⁺ = 477

Example 14 2,2-Diethyl-5,7-dimethyl-8-{[(1-tetradecyl-1H-1,2,3-triazol-4-yl)methyl]carbamoyl}-2H,3H bromide -[1,2,4]triazolo[4,3-a]pyridin-2-yl

The derivative was prepared according to the procedure described in example 3 and purified according to the procedure described in example 4 ¹ H-NMR (CHLOROFORM-d, 300 MHz): δ = 8.77 (br. s, 1H), 7.60 (s, 1H) 6.59 (s, 2H), 6.01 (s, 1H), 4.64 (d, J = 5.3 Hz, 2H), 4.32 (t, J = 7.3 Hz, 2H) , 3.98-4.12 (m, 1H), 3.75-3.87 (m, J = 12.3, 7.0 Hz, 2H), 2.61 (br.s, 3H), 2 .59 (s, 3H), 1.84-1.93 (m, 2H), 1.45 (t, J = 6.7 Hz, 6H), 1.22-1.32 (m, 24H), 0.84-0.90 (m, 3H) ppm, ¹³ C-NMR (CHLOROFORM-d, 75 MHz): δ = 163.3, 159.4, 156.5, 143.7, 122.2, 114 .0, 108.0, 73.6, 62.5, 50.6, 35.0, 31.9, 30.2, 29.6, 29.6, 29.5, 29.4, 29.3 , 29.0, 26.5, 22.9, 22.7, 20.0, 14.1, 8.8 ppm, LC-MS: R t = 7.40 min; 94.06%; m/z [M] ⁺ = 477

Example 15. Use of azidoalkylamide derivatives of Safirinium in the cycloaddition reaction of azides and alkynes

To 0.79 mmol (48 mg) 8-[(5-azidopentyl)carbamoyl]-5,7-dimethyl-2,2-dioctyl-2H,3H-[1,2,4]triazolo[4] bromide 3-a]-pyridin-2-yl was added 1.5 eq. phenylacetylene (1.18 mmol, 33 mg, 36 μL) and 2 mL of methylene chloride. Then 0.05 M aqueous solution of CuSO4 (1.58 mL, 10 mol% Cu), AMTC (20 mol%, 35 mg) and sodium ascorbate (10 mol%, 16 mg), dissolved in 2.5 mL of water were added so so that the total amount of water is 4 mL and the volume ratio of DCM:water is 1:2. The reaction was carried out at room temperature overnight. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and purified by chromatography according to the procedure described in Example 4.

The obtained product was analyzed by NMR and LC-MS.

¹ H-NMR (CHLOROFORM-d, 500 MHz): δ = 7.78 (br. s, 1H), 7.73 (m, 3H), 7.36 (t, J = 7.4 Hz, 2H) , 7.26-7.30 (m, 1H), 6.43 (br.s, 2H), 5.86 (s, 1H), 4.38 (t, J = 6.6 Hz, 2H), 3.81 (dt, J = 13.3, 6.8 Hz, 2H), 3.64 (br.s, 2H), 3.35 (q, J = 5.9 Hz, 2H), 2.50 (br.s, 3H), 2.46 (s, 3H), 1.90-2.00 (m, 2H), 1.66-1.82 (m, 4H), 1.61 (quin, J = 7.0 Hz, 2H), 1.19-1.40 (m, 22H), 0.81 ppm (t, J = 6.9 Hz, 6H), ¹³ C-NMR (CHLOROFORM-d, 126 MHz ): δ = 163.4, 158.1, 156.4, 143.1, 130.6, 129.0, 128.2, 125.6, 113.8, 109.0, 74.3, 67, 3, 50, 1, 42, 1, 38.8, 31.7, 29.5, 29.3, 29.2, 28.5, 26.3, 23.6, 23.4, 23.2, 22.7, 22.5, 20.2, 14.1 ppm, LC-MS: R t = 7.68 min; 82.88%; m/z [M] ⁺ = 630.66; UV-VIS: Xmax = 250 nm; 361nm

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Claims (7)

1. Compound of formula (S1): Where: X is a moiety selected from the group consisting of: NH, O, where n is from 1 to 10 carbon atoms in the methylene moieties, Ri is a substituent selected from the group consisting of the formulas (W1) to (W2): W1 W2 R ² , R ³ - means an alkyl substituent with the number of carbons from 2 to 8, ^R4 , ^R5 , ^R6 - represents a methyl substituent or a hydrogen atom or ^R4 is a methyl substituent or a hydrogen atom, while the substituents ^R5 and ^R6 are combined and form an aromatic system of the general formula S2, where the substituents ^R7 - ^R10 are a hydrogen atom: wherein the anion A ^- is an ion selected from the group consisting of: F ^- , Ch, Br, h. 2. A compound according to claim 1 with the formula (S1): Where: X is a moiety selected from the group consisting of: NH or O, where n denotes from 1 to 7 carbon atoms in the methylene moieties, ^R1 is a substituent selected from the group consisting of the formulas (W1) to (W2): ' ^{ν -'νθ} W1 W2 PL 243397 B.I R ² , R ³ - denote an alkyl substituent with the number of carbon atoms from 2 to 8, R ⁴ , R ⁵ , R ⁶ - means a methyl substituent or a hydrogen atom, where the anion A ^- is a bromide ion Br 3. The compound of claim 2 selected from the group containing derivatives with the formulas P6-P11: - 2,2-Diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H-/,3H-/-[1,2,4]triazolo[ bromide 4,3-a]pyridine-2-yl (P6), - 5,7-Dimethyl-2,2-dioctyl-8-[(prop-2-yn-1-yl)carbamoyl]-2H-/,3H-/-[1,2,4]triazolo[ bromide 4,3-a]triazolo[4,3-a]pyridin-2-yl (P7), - 2,2-Diethyl-5,7-dimethyl-8-[(prop-2-yn-1-yloxy)carbonyl]-2H-/,3H-/-[1,2,4]triazo I bromide o-[4,3-a]-p and η-2-i (P8), - 8-[(5-Azidopentyl)carbamoyl]-5,7-dimethyl-2,2-dioctyl-2H-/,3/-/-[1,2,4]triazolo[4,3-a] bromide triazolo[4,3-o]pyridin-2-ium (P9), - 8-[(5-Azidopentyl)carbamoyl]-5,7-dimethyl-2,2-diethyl-2H-/,3/-/-[1,2,4]triazolo[4,3-a] bromide [4,3-o]pyridin-2-ium (P10), - 8-[(7-Azidoheptyl)carbamoyl]-5,7-dimethyl-2,2-diethyl-2H-/,3/-/-[1,2,4]triazolo[4,3-a] bromide pyridine-2-ium (P11), shown below: P11 C ₈ H ₁₇ 4. The compound of claim 1 of the formula (S2): Where: the X moiety is selected from the group consisting of: NH, O, wherein n is from 1 to 10 carbon atoms in the methylene moieties, Ri - is a substituent selected from the group consisting of substituents with the formulas (W1) to (W2): PL 243397 B1 W1 W2 R ² , R ³ is an alkyl substituent with the number of carbons from 2 to 8, R ⁴ - is a substituent selected from the group consisting of: methylene or hydrogen atom R ⁷ , R ⁸ , R ⁹ , R ¹⁰ - is a hydrogen atom, with the anion A ^- being an ion selected from the group consisting of: F ^- Cl, Br, h 5. The compound of claim 4 of the formula (S2): Where: the X moiety is an NH group, where n is from 1 to 7 carbon atoms in the methylene moieties, Ri - is a substituent (W1) or (W2): W1 W2 R ² , R ³ is an alkyl substituent with the number of carbons from 2 to 8, R ⁴ - represents a substituent selected from the group consisting of: methylene or hydrogen, R ⁷ , R ⁸ , R ⁹ , R ¹⁰ - represents a hydrogen atom, with the anion A- being the Br ion. 6. The compound of claim 4 or 5 selected from the group containing derivatives of the formulas P12-P13: - 2,2-Diethyl-4-[(prop-2-yn-1-yl)carbamoyl]-7H-/,2/-/-[1,2,4]triazolo[4,3-a] bromide quinolin-2-ium (P12), - 2,2-Dioctyl-4-[(prop-2-yn-1-yl)carbamoyl]-7H-/,2/-/-[1,2,4]triazolo[4,3-a] bromide ]quinolin-2- -i (P13), shown below: P12 et 7. The use of a compound as claimed in claim 1-6 for labeling chemical molecules in the cycloaddition reaction of azides and alkynes.