CS242863B2 - Method of new compounds type acryloylaminodyes production - Google Patents

Abstract

The solution relates to a method of producing new ones in particular, acryloyl amine dye compounds of formulas I and II in which at least one of the radicals R 1 is an acryloyl radical. The method consists in having a dye that bears the ®NrI group and at least one of the residues R 1 is hydrogen, reacted with acrylic chloride.

Description

The invention relates to a process for the preparation of novel acryloylamine dyes.

The isolation of individual genes or a set of identical genes in a repeated arrangement from the genome of eukaryotic cells is not only of purely scientific importance but also of great practical importance in terms of gene technology. Until now, isolation has only been possible if the base composition of the gene or gene group is spacers, i. The spatial spacing moieties differed by at least 6 to 7% from the average base composition of the total genome.

Gradient bombardment based on cesium ion density with additives of base-specific substances such as deoxyribonucleic acids (DNA), such as those of silver, mercury or platinum or actinomycin or Hoechst dye, which is 2- (4-hydroxyphenyl) -5- [5- (N-methyl-N 1 -piperazinyl) -1,3,3-benzodiazol-2-yl] -1,3,3-benzodial. These methods have only limited capacity, are expensive and time consuming.

The development of materials for the affinity chromatography of biopolymers has greatly simplified the isolation of many biopolymers in recent years or made it possible for the first time to prepare them in pure form. These methods generally start from a carrier which, after chemical activation, is reacted with a substance which binds the bipolymer to be isolated as specifically as possible. Due to this specificity, the bipolymer of interest can ideally be selectively adsorbed from a mixture of similar compounds to the chromatographic material and then desorbed under suitable conditions. Said method is described by P. Cus-trecasase and C. B. Anfinsen, in Ann, Rev. Biochem. 40 (1971), 259-278.

Despite a number of examples of successful application of this method, so far no chromatographic material has been developed for many biopolymers that would allow such selective and programmable separation even for nucleic acid mixtures. However, high-resolution fractionation of gram-nucleic acid mixtures is a prerequisite for the desired isolation of individual genes or branches of the same genes.

In the prior art methods of separating low molecular weight nucleic acids, such as transfer ribonucleic acids, separation is achieved in different species on adsorption agents that combine the properties of ion exchangers with lipophilic interactions. see R. M. Kothari and V. Shankar, Journal of Chromatography 98 (1974) 449-475. This essentially utilizes the possibilities of carrier interactions with the rare bases of nucleic acids available in different transfer ribonucleic acids in varying amounts.

Since higher molecular weight ribonucleic acids and desoxyribonucleic acids generally do not contain any rare bases, the methods of their division can consist only of the following different features:

ratio of single strand to double strand difference in base composition difference in base sequence difference in molecular weights difference in tertiary structures

All of these features are indeed used today in conventional fractionation methods - see, for example, R. M. Kothari, Chromatograph. Roar. 12 (1970) 127-155.

In the most efficient method, salt gradient fractionation on an ultra centrifuge, differences in base composition, base sequence differences, and molecular weight differences lead to differences in buoyancy density for a particular type of deoxyribonucleic acid, which can be further increased by the addition of base-specific substances. The sharpness of division decreases with decreasing molecular weight, capacity with increasing molecular weight. Hydroxyapatite absorption chromatography yields fractionation essentially according to single-stranded to double-stranded ratios, and only to a minor extent due to differences in base composition, whereas quanine-rich and cytosine-rich nucleic acids are desorbed at slightly lower salt concentrations than adenine-rich components and thymine, see W. Pakroppa and W. Muller, Proc. Nat. Acad. Sci USA, 71 (3) (1974) 699-703.

By analogy, double-stranded nucleic acids can be fractionated on specific adsorptive reagents by protein-kieselguhr, for example methyl-serial albumin-kieselguhr, according to the difference in base composition; 1960) 66-77; N. Sueoka and Ts'i - Ying Chang, J. Mol. Biol. 4 (1963)

The intrinsic mechanism of action of these rather random adsorption agents is unknown. Therefore, the low sharpness of separation on these materials could not be substantially improved to this day.

Only in recent years, following the systematic study of numerous compounds that form complexes with nucleic acids, compounds have been found to be suitable for targeted synthesis of affinity chromatography materials, see W. Muller and D. M. Crothers,

Eur. J. Biochem. 54 (1975) 267-277; W. Muller, H. Bunemann and N. Dattagupta, Eur. a. Biochem. 54 (1975) 279-291 and W. Muller and F. Gautier, Eur. J. Biochem. 54 (1975) 385-394.

The benefits of using such well-studied compounds in the separation of nucleic acid mixtures may already have been demonstrated by the combined use of hydroxyapatite and ethidium bromide as base-specific addition substances for the separation of superhelical and helical desoxyribonucleic acids - see W. Pakroppa, W. Goebel and W. Muller Analytical Biochemistry 67 (1975) 372-383 and hydroxyapatite in combination with phenylneutral red derivatives as base-specific complexing agents in the separation of two-stranded desoxyribonucleic acid species, see W. Pakroppa and W. Muller,

Why. Nat. Acad. Sci. USA 71 (3) (1974) 699-703.

The resolution of these methods is comparable to the preparative cesium chloride density gradient, i. the fractions of desoxyribonucleic acid with different quanine and oytosine content can be separated from each other.

Despite the high capacity, the process is disadvantageous in that it is difficult to obtain mixtures of desoxyribonucleic acid with an average molecular weight of the components greater than 20 x 10 10 and that even a special hydroxyapatite to be used as an absorber must be prepared.

It has been known for some time that nucleic acid mixtures can be separated in the polyethylene-glycol-dextran system under certain conditions both in the single-stranded desoxyribonucleic acid and in the double-stranded desoxyribonucleic acid.

The double-stranded desoxyribonucleic acid is always enriched in the lighter polyethylene glycol phase, which means that it has a higher partition coefficient than single-stranded nucleic acids. The absolute values of the partition coefficients can be varied by about 3 to 4 orders of magnitude by adding potassium and lithium salts, but the fractionation according to the base composition fails in this system.

A method for the production of adsorbents is known by the same authors as in the present invention. These adsorbents can achieve a phase or structure-specific separation of biopolymers and in particular mixtures of single-stranded and / or double-stranded or so-called supercoiled and / or linear nucleic acids, in particular mixtures of deoxyribonucleic acids, at the same time high capacity.

Affinity specificity refers primarily to structural specificity and basic specificity. .

The aforesaid adsorbent comprises a polymeric carrier to which a residue with affinity for the biopolymer, optionally a basic and / or structurally specific group, is bonded directly or via other intermediate groups, in particular covalently.

Said adsorbent for affinity-specific separation of macromolecular substances, in particular biopolymers, such as nucleic acids, is produced by a process which is first produced in a manner known per se by polymerization or polycondensation of at least one monomer which additionally contains one functional group through which a moiety having a molecular weight of up to about 4,000, a spatial spacing, a nucleic acid affinity residue, a polymeric carrier, the polymeric carrier optionally pulverized to the desired particle size and then grafted either directly or by copolymerization in the presence of at least one additional monomer as copolymer, a nucleic acid affinity residue, or particles of a desired size are produced by peroxygen polymerization in a manner known per se.

Preferably, in the manufacture of said adsorbent, a copolymer of at least two mixed polymerizable monomers, one of which bears a nucleic acid affinity residue, is grafted onto a polymer having a low porosity and low compressibility.

As the residues with an affinity for biopolymers, the complexing agents basically and / or structurally specific as described in the cited literature can be used. Particularly suitable are dye residues which carry the group IR. ^It ° ^Color and ^VA of the formula I and II.

where they mean

X is CH- or N,

Y is O, S, NH- or a group of formula

r! independently of one another hydrogen atoms or methyl groups,

R is a hydrogen atom or a methyl group, a hydrogen atom or a methyl group,

R is hydrogen or methyl,

An anion, for example a chloride anion, a perchlorate anion or an oxalate anion.

Particularly preferred polymer-bound complexing agents and / or structurally specific residues are the following dyes:

1. Diaminophenylindole (DAPI)

NH

nh ₂

2. p-dimethylaminofuchsondimethylimine salt (Malachite green)

3. hexamethyl-p-rosaniline chlorohydrate (crystal violet)

4. Heptamethyl-p-rosaniline chloride (methyl green)

5, basic diphenylmethane dye (auramine)

NH

II

Cx / CH _3/2 nn / CH _3/2

6. 2- (4-Hydroxyphenyl) -5- [1-methylpiperazin-4-yl] benzimidazol-2-yl-benzimidazole (Hoechst dye 33 258)

7. Di-tert. butylproflavin / CH3 / H 3 C N ₂ ^C / CH _{3/3 NH2}

8. Di-tert-butylacriflavin

^CH 3

9. 3,7-Diamino-10-phenyl-5-oxanthracene salt

10. 3,7-Tetramethyldiamino-10-phenyl-5-oxantracene salt

11. 3,6-tetramethyldiamino-9-phenylacridine salt

12. 3- (10) -3,7-Tetramethyldiamino [5-oxaanthralene] -propionic acid ester salt

,. Xj / ^CH3 / 2N CH ₂ CH ₂ COOR ° ^ \ m / CH _3/2 Aktiflavin ... H ₂ hr ^ _NH2 ch ₃

14. 10- (propenyl) -proflavin salt

CH ₂ CH = CH ₂

16. 3,7-tetramethyldiamino-5-phenylphenazine salt

17. 2-methyl-3-amino-7-dimethylaminophenyl enazinová salt / CH _3/2 ^of n ≤ ^x ≤ n

NH,

18. 2-methyl-3-amino-6-dimetylaminoakridinová salt / CH _3/2 N ', CH ₃ NH ₂

19. 2-Methyl-3-amino-7-diethylaminophenothiazine salt

20. Proflavin

21. 3,7-Diamino-5-oxaanthracene salt

H, N

NH,

22. 3,7-Diamino-5-thiaanthracene salt

23. 3,7-diaminophenazine salt

24. 3,7-diaminophenoxazine salt

25. Thionine

H, N, nh ₂

26. 3,6-tetramethyldiaminoacridine (acridine orange)

27. Pyronine G

28. Thiopyronine

29. 3,7-tetramethyldiaminophenazine salt

30. 3,7-tetramethyldiaminophenoxazine salt

Me _From N $

NMe _z

31. Zinc chloride or disinium chloride 2,7-bis- (dimethylamino) -phenthiazonium hydrochloride (methylene blue)

32. N- (3-Methoxy-7-chloroquinolin-4-yl) -ethylenediamine nh-ch ₂ -ch ₂ -nh ₂ and ₃

Cl taking

Me in the above formulas is methyl

33. Ethidium bromide

Preferred basic and / or structurally specific complexing agents are in particular

and p-dimethylaminofuchsondimethylimine salt (malachite green)

These dyes are known per se and commercially available or can be prepared by methods known to those skilled in the art and described by W. Muller and DM Crothers Eur. J. Biochem. 54 (1975) 267-277, W. Muller, H. Bunemann and N. Dattagupta, Eur. J. Biochem. 54 (1975) 279 until 291 and W. Muller _F:. Gautier Eur.

J. Biochem. 54 (1975) 385-394.

These complexing agents, basic and / or structurally specific or dye residues, can be covalently bound to the support by methods known to the skilled person, for example by esterifying hydroxyl groups present on the support via a carboxyl group introduced into the dye molecule, amide formation, urethane formation and also copolymerization in the presence but also in the absence of other copolymerizable monomers via copolymerizable double bonds which are introduced into the dye molecule, for example via an acrylamido group, such as the preferred complexing agents in basic and / or structurally specific acrylic phenyl neutral and acrylmalachite green, of the following formulas . The names are trivial names, the exact name being acryloylaminophenylneutral red and acryloylmalaohite green.

CH = CH ₂

WHAT

CH-CH ₂

n / CH _3/2 · N ci / CH _3/2

These complexing agents, basic and / or structurally specific, can be prepared by methods known to those skilled in the art by introducing an acrylic residue into said dye molecules. This also applies to the molecules of the above dyes.

The designation acrylic residue is in this case equivalent to an acryloyl residue.

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of novel acryloylamine dyes of the general formula I and II.

where they mean

X is CH- or N,

Y is O, S, NH or a group of formula / N ^<Z ^ ^/> - ^N / ^R 1 + 2 (II)

R ¹ are independently hydrogen atoms or methyl groups,

R is hydrogen or methyl,

R ^J is hydrogen or methyl;

And an anion, for example a chloride anion, a perchlorate anion or an oxalate anion, wherein at least one of the radicals R1 is an acryloyl radical.

The essence of the process for the production of novel acryloylamine dyes, in which at least one of the radicals R @ ¹ is an acryloyl radical, consists in that the dye carrying a group Θ 1 '1

And at least one of R is hydrogen, reacted with acrylic chloride.

In particular, the invention relates to a process for the production of the following novel dyes: acryloylaminomalachite green, acryloylaminophenylneutral red, acryloylaminoacridine derivatives and acryloylaminophenanthridine derivatives. The preparation is carried out in a manner known per se by the usual acylation methods with acrylic chloride. The method is explained in more detail by the following examples.

Example 1

Production of acryloylaminomalachite green 1a) 15.1 g corresponding to 0.1 mol of 4-nitrobenzaldehyde and 36.3 g i.e. 38 ml corresponding to 0.3 mol / m, dim-dimethylaniline and 40.5 g of anhydrous zinc chloride The reaction mixture was heated to a bath temperature of 100 ° C. Initially, the easily moving mixture solidifies during the reaction to a solid green mass that can no longer be stirred. The reaction mixture was allowed to cool after 5 hours. The product is taken up in 150 ml of acetone, after which time the product dissolves with stirring and the zinc salt precipitates. The insoluble salt is filtered off with suction, washed with acetone and water is added to the filtrate until crystallization occurs. The crystals are filtered off with suction, washed with water, isopropyl alcohol and ether.

Yield: 28.7 g, corresponding to 76.6% of theory. Melting point: 168 DEG C., light sensitive.

ř;

1b) 3.75 g, corresponding to 0.01 mol, of the nitro compound obtained by the method of 1a), are dissolved in 200 ml of 99% glacial acetic acid and 20 ml of water. 4 g of zinc chloride are added. 20 g of zinc dust are then added in portions with stirring and cooling at 20-30 ° C. After stirring at room temperature for 30 minutes, the excess zinc is filtered off with suction and washed with glacial acetic acid. The filtrate was evaporated in vacuo at 50 ^Q C. The residue is partitioned between 150 ml of chloroform and 100 ml of water and separated, and the chloroform phase is shaken with 80 ml of 2N sodium carbonate solution and 100 ml water. The chloroform phase was separated and dried over anhydrous sodium sulfate and filtered, then the solution was concentrated in vacuo at 50 ° C to about 100 ml and used directly for the next reaction. The solution is faintly violet.

lc) Dilute 100 ml of the obtained chloroform solution of the amino compound with 50 ml of methanol. 10 g of anhydrous sodium carbonate and a spatula tip of 1,3-dinitrobenzene are added, and then 1.81 g (1.65 ml), equivalent to twice the amount of acryloyl chloride, is added dropwise with stirring and cooling to 0-5 ° C. The suspension was stirred overnight at room temperature, then the sodium carbonate was filtered off with suction and the filtrate was evaporated in vacuo at 35-40 ° C.

The residue is taken up in 100 ml of chloroform and 50 ml of water and shaken, the chloroform phase is separated, re-extracted with 50 ml of water, dried over sodium sulphate and evaporated in vacuo at 35-40 ° C. The oily greenish residue is triturated with 20 ml. isopropanol and cooled to crystallization. The crystals are filtered off with suction, washed with isopropyl alcohol and ether and dried in a desiccator, yield over steps 1b) and 1c): 2.9 g, corresponding to 72.7% of theory, melting point:

175 ° C.

Analysis

Calculated: C 78.2, H: 7.27, N: 10.52%,

Found: C 75.9, H: 7.21, N: 9.99%.

NMR Spectrum (hexadeuterodimethylsulphoxide): CH 2 -ZN-methyl (δ 2.83 PPM, = CH- (acrylic))

Q 5.7 PPM, CH = (acrylic) P 6.3 PPM, aromatic protons between 6.5 to 7.5 PPM.

1d) 250 mg (0.001 mol) of chloranil is dissolved in 15 ml of tetrahydrofuran and 0.4 g (0.001 mol) of acryloyl compound is added, as in 1c). The solution immediately turns blue and after standing for two hours at room temperature over The crystals are filtered off with suction, washed with tetrahydrofuran and ether and dried in a desiccator. For further purification, the substance is redissolved in water, neutralized with hydrochloric acid, and then the solution is shaken with ethyl acetate. the pure product is precipitated from the aqueous solution by the addition of sodium chloride.

Yield: 0.335 g, 84.2% of theory.

In the NMR spectrum of 1d), the shift of the N-methylproton signal to 3.3 PPM can be observed compared to 1c).

Example2

Preparation of acryloylaminophenylneutral red 1a) 6.9 g = 0.05 moles of 4-nitroaniline are dissolved in tetrahydrofuran / chloroform 1: 1, a spatula tip of 1,3-dinitrobenzene and 8 ml of triethylamine is added. 9 ml = 0.1 mol of acryoyl chloride is slowly added dropwise with stirring at 10 to 20 ° C. The solution was stirred at room temperature overnight, triethylamine hydrochloride crystallized. Thereafter, the tetrahydrofuran / chloroform mixture is evaporated at 50 ° C under vacuum, the residue is stirred in water, filtered off with suction and washed with water, isopropyl alcohol and ether. The product is dried under vacuum at 50 ° C.

1b) 2.5 g of the acryloyl compound 2a) are dissolved at 50 ° C with stirring in 60 ml of tetrahydrofuran and diluted with 60 ml of 99% glacial acetic acid and cooled to 30 ° C.

Then 10 g of zinc dust are added in portions, the temperature rising to 35-40 ° C under ice-cooling.

After stirring for a further 10 minutes at room temperature, the excess zinc is filtered off with suction and washed with concentrated acetic acid. The filtrate was evaporated in vacuo at 45 ° C. The oily residue is practically chromatographically pure and is used for the next step.

(1c) To a solution of 4.2 g = 0.02 mole of N, N-dimethyl-p-phenylenediammonium chloride and 2.88 g, i.e. 0.02 mole of o-toluidinium chloride in 400 ml of water, is slowly added at room temperature with stirring 12 g of sodium chromate, ie 0.04 mol in 100 ml of water. The precipitated green product is aspirated after 15 minutes, washed three times with water, leaving the wash solution green, and the precipitate is then immediately further processed by suspending it in a small amount of water and homogenizing.

Dilute the homogeneous suspension to 1.6 liters with water. After addition of 1.5 x 0.02 mole of N-acryloyl-β-phenylenediamone acetate in 100 ml of water, the pH of the reaction mixture was adjusted to 4.9 with 3M sodium acetate solution. The mixture is then heated to boiling with stirring. The solution is colored blue first, then dark violet while boiling. Allow the mixture to boil for 5 minutes to complete the reaction. The boiling hot solution is then suction filtered off, the filtrate, in an amount of about 2 liters, is treated with 240 g of sodium chloride to a 2 M solution and left to stand overnight in the refrigerator.

 and

4'3 242863

The precipitated crystals are aspirated and dried in a desiccator. It must not be washed with water as the substance is well soluble in water.

Yield: 2.4 g of crude product.

For further purification, 1.3 of the crude product was dissolved in 25 mL of methanol and 0.1 M sodium chloride 4: 1 and applied to a silica gel 60 (4 x 95 cm) column. It is then eluted with the same solvent. Fraction volume = 20 ml. Fractions 22 to 25 are combined and concentrated in vacuo to about 1.5 ml. The crystals are aspirated, washed with a small amount of 0.1 M sodium chloride and dried in a desiccator.

Yield; 0.665 g of chromatographically pure dye.

Example 3

Preparation of 9- (Acryolyolylaminoethylamino) -3-chloro-7-methoxyacridine

3,9-Dichloro-7-methoxyacridine (854 mg) was heated to 60 ° C with 1 ml of ethylenediamine and 7.7 g of phenol for 30 minutes in an oil bath. The melt is taken up in 200 ml of chloroform and 150 ml of water and the pH of the reaction mixture is adjusted and equilibrated to 4 by addition of acetic acid. The organic phase is reextracted 3 to 4 times with 0.1 M sodium acetate buffer and then discarded.

The aqueous extracts were adjusted to pH 9.5 with soda solution, extracted with chloroform: n-butanol (20: 1), the organic phase was filtered through silicone paper and evaporated in vacuo. The residue is dissolved in 0.1 M sodium acetate buffer, the dimeric product is filtered off and the solution is adjusted to pH 10. After standing for 1 hour at room temperature, the precipitate is filtered off, washed with a small amount of dilute ammonia solution at 4 ° C and dried. .

Yield: 50 to 80% of theory.

The amine compound (206 mg) was dissolved in chloroform (25 ml) and after cooling, acryloyl chloride (0.3 ml) was added. The solution darkens immediately. After a few minutes, the acrylic derivative precipitates. Evaporation and washing with a small amount of chloroform gave 122 mg = 50% of theory of (9-acryloylaminoethylamino) -3-chloro-7-methoxyacridine in chromatographically pure form.

Claims (5)

SUBJECT OF THE INVENTION 1. A process for the preparation of novel acryloylamine-type dyes, in particular of formulas I and II Ί4 where they stand X CH- or nitrogen atom, kyslí oxygen atom, sulfur atom, NH- group or general group, in formula n / Ri / ₂ R independently of one another is hydrogen or methyl R is hydrogen or methyl, R ^ is hydrogen or methyl, R is hydrogen or methyl; and Θ * An anion, for example a chloride anion, a perchlorate anion or an oxalate anion, ffl 1, characterized in that the colorant which carries the NR group and at least one of the residues 1 R is hydrogen, reacted with acrylic chloride. 2. A process for the preparation of acryloylaminomalachite green of the general formula characterized in that 4-nitrobenzaldehyde is reacted with reakce, Ν-dimethylaniline, the nitro group of the resulting triphenylmethane derivative is reduced and acylated with acrylohloride. A process for producing acryloylaminophenylneutral red of the formula CH = CH ₂ CO / CH _3/2 n, characterized in that N, N-dimethyl-p-phenylenediamine was reacted with o-toluidine, and the resulting reaction product is reacted with N-acryloyl-p-phenylenediamine. A process for the preparation of acridine derivatives of the formula characterized in that 3,9-diohloro-6-methoxyacridine is reacted with ethylenediamine and the 9-aminoethylamino compound obtained is acylated with acrylohloride. > _ř 5 5. A process for the preparation of an acryoylphenanthridine derivative of the general formula ## STR2 ## in which it denotes R represents a hydrogen atom or a methyl group, characterized in that the phenanthridine derivative of the formula