CS242863B2 - Process for the production of novel compounds of the acryloyl amine type - Google Patents

Abstract

The solution relates to a method for producing new compounds of the acryloylamino dye type, in particular of the general formulas I and II, in which at least one of the R1 residues is an acryloyl residue. The method consists in reacting a dye carrying the ®Nr1 group and at least one of the R1 residues is hydrogen with acrylic chloride.

Description

The invention relates to a method for producing new compounds of the acryloylamino dye type.

The isolation of individual genes or sets 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 from the point of view of gene technology. Until now, isolations were possible only if the base composition of the gene or gene group with its spacers, i.e. acupins with a molecular weight of up to about 4,000, which create a spatial distance, differed by at least 6 to 7% from the average base composition of the entire genome.

The separation method used was usually cesium ion density gradient spin-coating with additives specific for deoxyribonucleic acid (DNA) bases, such as silver, mercury or platinum ions, or actinomycin or the Hoechst dye, which is 2-(4-hydroxyphenyl)-5-[5-(N-methyl-N-piperazinyl)-L,3-benzodiazol-2-yl]-[1,3-benzodial]. These methods have only limited capacity, are expensive and time-consuming.

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

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

In the present methods of separating low molecular weight nucleic acids, for example transfer ribonucleic acids, separation into different species is achieved 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 to 475. In this case, the possibilities of carrier interactions with rare nucleic acid bases, which are available in different amounts in different transfer ribonucleic acids, are essentially used.

Since higher molecular weight ribonucleic acids and deoxyribonucleic acids generally do not contain any rare bases, their division can only be based on the following different characteristics:

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 actually used today in common fractionation methods - see e.g. R. M. Kothari, Chromatograph. Rev. 12 (1970) 127 to 155.

In the most efficient method, fractionation in a salt gradient in an ultracentrifuge, differences in base composition, differences in base sequence, and differences in molecular weights lead to differences in the buoyancy density for each type of deoxyribonucleic acid, which can be further increased by the addition of base-specific substances. The sharpness of the separation decreases with decreasing molecular weight, the capacity with increasing molecular weight. In absorption chromatography on hydroxyapatite, fractionation occurs essentially according to the ratios of single strand to double strand of nucleic acids and only to a small extent according to the difference in base composition, while nucleic acids richer in quanine and cytosine are desorbed at a slightly lower salt concentration than components richer in adenine and thymine, see W. Pakroppa and W. Muller, Proc. Nat. Acad. Sci USA, 71 (3) (1974) 699 to 703.

In a completely analogous manner, double-stranded nucleic acids can be fractionated on specific adsorption agents protein-diatomaceous earth, for example methyl-serium albumin-diatomaceous earth, according to the difference in base composition, whereby again the species of deoxyribonucleic acids richer in guanine and cytosine are eluted first, see J. D. Mandeli and A. D. Hershey, Analytical Biochemistry 1 (1960) 66 to 77; N. Sueoka and Ts 'ai - Ying Chang, J. Mol. Biol. 4 (1962)

161 to 172. The actual mechanism of action of these rather accidentally discovered adsorption agents is unknown. Therefore, the low resolution of these materials has not been significantly improved to date.

Only in recent years, after a systematic study of numerous substances that form complexes with nucleic acids, compounds were found that seemed suitable for the targeted synthesis of materials for affinity chromatography, 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 to 394.

The advantages of using such well-studied substances in the separation of nucleic acid mixtures could already be shown by the examples of the combined use of hydroxyapatite and ethidium bromide as base-specific additions for the separation of superhelical and helical deoxyribonucleic acids - see W. Pakroppa, W. Goebel and W. Muller, Analytical Biochemistry 67 (1975) 372 to 383 and hydroxyapatite in combination with phenyl neutral red derivatives as base-specific complexing agents for the separation of double-stranded species of deoxyribonucleic acids - see - W. Pakroppa and W. Muller,

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

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

Despite the high capacity, the method has the disadvantage that it is difficult to obtain mixtures of deoxyribonucleic acid with an average molecular weight of the components higher than 20 x 10® and that special hydroxyapatite used as an absorbent must even be prepared.

It has been known for a long time that mixtures of nucleic acids can be separated in the polyethylene-glycol-dextran system under certain conditions into single-stranded deoxyribonucleic acid and double-stranded deoxyribonucleic acid.

In the lighter polyethylene glycol phase, double-stranded deoxyribonucleic acid is always enriched, which means that it has a higher partition coefficient than single-stranded nucleic acids. The absolute values of the partition coefficients can be changed by adding potassium and lithium salts by about 3 to 4 orders of magnitude, but fractionation according to base composition is not possible in this system.

A method for producing adsorption agents is known, by the same authors as in the case of the present invention. These adsorption agents can achieve 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, while simultaneously providing high capacity.

Affinity specificity refers primarily to structural specificity and base specificity.

The above-mentioned adsorbent comprises a polymeric carrier to which a residue with affinity for a biopolymer, or a basic and/or structurally specific group, is directly or via further intermediate groups, in particular covalently bound.

The said adsorbent for the affinity-specific separation of macromolecular substances, in particular biopolymers, such as nucleic acids, is produced by a process in which at least one monomer is first formed in a known manner by polymerization or polycondensation, which additionally contains one functional group through which it is possible to bind directly or through a group with a molecular weight of up to about 4,000, acting as a spatial distance, a residue with affinity for nucleic acids, a polymer carrier, this polymer carrier is optionally ground to the desired particle size and then grafted either directly or by copolymerization in the presence of at least one further monomer as a copolymer, a residue with affinity for nucleic acids, or particles of the desired size are produced in a known manner by bead polymerization.

Preferably, in the production of the said adsorbent, a copolymer of at least two mixture-polymerizable monomers, one of which carries a residue with affinity for nucleic acids, is grafted onto a polymer carrier with small pores and low compressibility.

As residues with affinity for biopolymers, basic and/or structurally specific complexing agents can be used, which are described in the cited literature. Particularly suitable are residues of dyes which carry the group ^IR^. ^{These are dyes of} the general formula I and II.

where they mean

X is a CH- group or a nitrogen atom,

Y is an oxygen atom, a sulfur atom, an NH- group or a group of the general formula

r! independently of each other hydrogen atoms or methyl groups,

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

R is a hydrogen atom or a methyl group,

A® anion, for example, chloride anion, perchlorate anion or oxalate anion.

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

1. Diaminophenylindole (DAPI)

NH

nh ₂

2. p-dimethylaminofuchsonedimethylimine salt (Malachite green)

3. hexamethyl-p-rosaniline chlorhydrate (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 /CH3/3C h ₂ n ^c /ch ₃ / ₃ nh ₂

8. Di-tert-butyl acriflavine

^CH 3

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

10. 3,7-tetramethyldiamino-10-phenyl-5-oxanthracene salt

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

12. 3-(10)-3,7-tetramethyldiamino-5-oxaanthrylene-propionic acid ester salt

, . Xj / ^CH 3/2N ch ₂ ch ₂ color °^\m/ch ₃ / ₂ Aktiflavin .. . H ₂ hrs ^NH ₂ ch ₃

14. 10-/propenyl/-proflavine salt

_CH2CH = _CH2

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

17. 2-methyl-3-amino-7-dimethylaminophenazine salt /ch ₃ / ₂ n ^z ^ ^x ^n

NH,

18. 2-methyl-3-amino-6-dimethylaminoacridine salt /ch ₃ / ₂ n' ,CH ₃ nh ₂

19. 2-methyl-3-amino-7-dimethylaminophenothiazine 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. Pyronin G

28. Thiopyronine

29. 3,7-tetramethyldiaminophenazine salt

30. 3,7-tetramethyldiaminophenoxazine salt

Me _Z N $

NMe _from

31. zinc chloride, or disintegrating 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 och ₃

Cl while

Me in the above formulas means methyl group

33. Ethidium bromide

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

and p-dimethylaminofuchsonedimethylimine salt (malachite green)

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

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

These basic and/or structurally specific complexing agents, or dye residues, can be covalently bound to the support by methods known to the person skilled in the art, for example by esterification of hydroxyl groups present on the support via a carboxyl group introduced into the dye molecule, by amide formation, by urethane formation and also by copolymerization, preferably in the presence or absence of other copolymerizable monomers via copolymerizable double bonds introduced into the dye molecule, for example via an acrylamido group, as is the case with the preferred complexing agents in basic and/or structurally specific, namely acrylphenyl neutral red and acrylmalachite green, of the following formulas. The designations given are trivial names, the exact name is acryloylaminophenyl neutral red and acryloylmalachite green.

CH = CH ₂

WHAT

CH-CH ₂

,n/ch ₃ / ₂ ci· n/ch ₃ / ₂

These basic and/or structure-specific complexing agents can be produced in a manner known to the skilled person by introducing an acrylic residue into the dye molecules mentioned. This also applies to the molecules of the above-mentioned dyes.

The designation acrylic residue is equivalent to an acryloyl residue in the cases indicated.

The subject of the invention is a method for producing new compounds of the acryloylamino dye type, especially of general formulas I and II.

where they mean

X is a CH- group or a nitrogen atom,

Y is an oxygen atom, a sulfur atom, an NH- group or a group of the general formula /n— ^< ^Z/ ^> — ^N / ^R 1^2 (II),

R ¹ independently of each other hydrogen atoms or methyl groups,

R is a hydrogen atom or a methyl group,

R ^J hydrogen atom or methyl group, θ

A anion, for example a chloride anion, a perchlorate anion or an oxalate anion, wherein at least one of the radicals R 1 represents an acryloyl radical.

The essence of the method for producing new compounds of the acryloylamino dye type, in which at least one of the R ¹ residues is an acryloyl residue, consists in the fact that the dye, which carries the group Θ 1 „ 1

NR^ and at least one of the R radicals is hydrogen, is allowed to react with acrylic chloride.

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

Example 1

Production of acryloylaminomalachite green la) 15.1 g, which corresponds to 0.1 mol of 4-nitrobenzaldehyde and 36.3 g, i.e. 38 ml, which corresponds to 0.3 mol / N,N-dimethylaniline and 40.5 g of anhydrous zinc chloride are mixed and the reaction mixture is heated to a bath temperature of 100 ° C. The initially easily mobile mixture solidifies during the reaction to a solid green mass that can no longer be stirred. The reaction mixture is allowed to cool after 5 hours. The product is taken up in 150 ml of acetone, and after a certain time the product dissolves with stirring and the zinc salt is precipitated. The insoluble salt is filtered off with suction, washed with acetone and enough 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, which corresponds to 76.6% of theory, melting point: 168 °C, the substance is sensitive to light.

ř ;

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

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

The residue is taken up in 100 ml of chloroform and 50 ml of water and shaken, the chloroform phase is separated, extracted again with 50 ml of water, dried over sodium sulfate and evaporated in vacuo at a temperature of 35 to 40 ° C. The oily greenish residue is triturated with 20 ml of isopropyl alcohol and cooled to crystallize. The crystals are filtered off with suction, washed with isopropyl alcohol and ether and dried in a desiccator, yield over stages lb) and lc): 2.9 g, which corresponds 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 (hexadeuterodimethylsulfoxide): CH^-ZN-methyl/ S 2.83 PPM, =CH-/acrylic/

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

ld) 250 mg, corresponding to 0.001 mol of chloranil, are dissolved in 15 ml of tetrahydrofuran and 0.4 g, corresponding to 0.001 mol of the acryloyl compound are added, analogously to the case of 1c). The solution immediately turns blue and after about two hours of standing at room temperature overnight, the crystals are then 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 adding sodium chloride.

Yield: 0.335 g, i.e. 84.2% of theory.

In the NMR spectrum ld) a shift of the N-methyl proton signal to 3.3 PPM can be observed compared to 1c).

Example2

Preparation of acryloylaminophenyl neutral red la) 6.9 g = 0.05 mol of 4-nitroaniline are dissolved in a 1:1 tetrahydrofuran/chloroform mixture, 1,3-dinitrobenzene and 8 ml of triethylamine are added on the tip of a spatula. 9 ml = 0.1 mol of acryloyl chloride are slowly added dropwise while stirring at a temperature of 10 to 20 °C. The solution is stirred at room temperature overnight, during which triethylamine hydrochloride crystallizes. The tetrahydrofuran/chloroform mixture is then evaporated at a temperature of 50 °C in vacuo, the residue is stirred in water, filtered off with suction and washed with water, isopropyl alcohol and ether. The product is dried in vacuo at a temperature of 50 °C.

lb) 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 is added in portions, the temperature rising to 35 to 40 °C under ice cooling.

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

lc) A solution of 12 g of sodium chromate, i.e. 0.04 mol in 100 ml of water, is added slowly at room temperature and with stirring to a solution of 4.2 g = 0.02 mol of N,N-dimethyl-p-phenylenediammonium chloride and 2.88 g, i.e. 0.02 mol of o-toluidinium chloride in 400 ml of water. The green product that precipitates is filtered off with suction after 15 minutes, washed three times with water, the washing solution remaining green, and the precipitate is then immediately processed further by suspending it in a small amount of water and homogenizing.

The homogeneous suspension is diluted with water to a volume of 1.6 liters. After adding 1.5 x 0.02 mol of N-acryloyl-P-phenylenediamonium acetate in 100 ml of water, the pH of the reaction mixture is adjusted to 4.9 with 3 M sodium acetate solution. The mixture is then heated to boiling with stirring. The solution first turns blue, then turns dark purple on boiling. To complete the reaction, the mixture is left to boil for 5 minutes. The boiling hot solution is then filtered off with suction, the filtrate, in an amount of about 2 liters, is adjusted to a 2 M solution by adding 240 g of sodium chloride and left to stand overnight in the refrigerator.

and

4'3 242863

The precipitated crystals are filtered off with suction and dried in a desiccator. They must not be washed with water because the substance is highly soluble in water.

Yield: 2.4 g of crude product.

For further purification, 1.3 g of the crude product is dissolved in 25 ml of methanol and 0.1 M sodium chloride in a ratio of 4:1 and applied to a silica gel 60 column (4 x 95 cm). 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 filtered off with suction, 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-(acrylolylaminoethylamino)-3-chloro-7-methoxyacridine

854 mg of 3,9-dichloro-7-methoxyacridine are heated with 1 ml of ethylenediamine and 7.7 g of phenol for 30 minutes in an oil bath at 60°C. 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 adding acetic acid. The organic phase is re-extracted 3 to 4 times with 0.1 M sodium acetate buffer and then discarded.

The aqueous extracts are adjusted to pH 9.5 with sodium hydroxide solution, extracted with a mixture of chloroform and n-butanol in a ratio of 20:1, the organic phase is filtered through silicon paper and evaporated in vacuo. The residue is dissolved in 0.1 M sodium acetate buffer, the remains of the dimer product are filtered off and the solution is adjusted to pH 10. The precipitate is filtered off after standing for 1 hour at room temperature, washed with a small amount of dilute ammonia solution at 4 °C and dried.

Yield: 50 to 80% of theory.

206 mg of the obtained amino compound are dissolved in 25 ml of chloroform under heat and after cooling 0.3 ml of acryloyl chloride is added. The solution immediately darkens. After a few minutes the acryloyl derivative is separated. After evaporation and washing with a small amount of chloroform 122 mg = 50% of theory of /9-acryloylaminoethylamino/-3-chloro-7-methoxyacridine is obtained 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