JPS6281371A - Novel acridine derivative - Google Patents

Abstract

NEW MATERIAL:A terephthalaldehyde bis(9'-acridinyl)hydrazone derivative expressed by formula I (R<1> and R<2> are H, lower alkyl, lower alkoxy or substituted amino group except when both R<1> and R<2> are H at the same time). USE:A staining agent for differential staining technique of human chromosome having a high molecular extinction coefficient and fluorescent intensity. PREPARATION:For example, o-chlorobenzoic acid expressed by formula II is reacted with an aniline derivative expressed by formula III to give an N- phenylanthranilic acid derivative expressed by formula IV, which is then subjected to cyclocondensation and chlorine substitution reaction with phosphorus oxychloride to afford a compound expressed by formula V. The resultant compound expressed by formula V is then reacted with hydrazine to give a 9- hydrazinoacridine derivative expressed by formula VI, which is then reacted with terephthalaldehyde to afford the aimed compound expressed by formula I.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a novel acridine derivative useful as a staining agent for dividing human chromosomes, etc. [Background of the Invention] In recent years, it has been widely adopted in human chromosome research. The emerging chromosome segmentation method dyes stripes (bands) on chromosomes using special dyes and fluorescent dyes after various pretreatments, and the fact that the relationship between the distribution and density is constant for each chromosome. This is used to identify chromosomes and detect chromosomal abnormalities, including congenital abnormalities.

He has greatly contributed to the development of both basic and applied research on genetic layers, malignant tumors, etc.

Chromosome partitioning methods include the C-band method in which chromosome specimens are stained with quinacrine mustard, the C-band method in which chromosomes are treated with trypsin and stained with Giemsa, and the C-band method in which the chromosomes are dyed with Giemsa after alkali treatment. Although the mechanism by which bands are formed is not well understood,
Regardless of which method is used, each method produces a unique striped pattern on the chromosome, so it is also effective for detailed analysis of partial chromosome deletions and translocations. The C-band method does not require any pretreatment and is a technology that reliably produces distinct horizontal stripes in specimens prepared using ordinary methods, and it has brought progress in precise analysis of chromosomes along with the G-band method. Ta. Subsequently, research has progressed further, and recently, in addition to the general separation methods such as the Q-band method and G-band method, newer high-precision separation methods are becoming popular, and new and more effective methods with increased specificity are becoming popular. At present, there is a need for a coloring agent with a unique color.

[Purpose of the invention]

An object of the present invention is to provide a novel acridine derivative that can be expected to be used as a staining agent for human chromosome sorting as described above.

[Structure of the invention]

(In the formula, R' * R2 each independently represents a hydrogen atom, a lower alkyl group, a lower alkoxy group, or a substituted amine group. However, this excludes cases where R' and R2 are both hydrogen atoms.) , is an invention of terephthalaldehyde bis(9'-acridinyl)hydrazone derivatives.

In general formula CI), the lower alkyl group represented by R1 and R2 includes, for example, a methyl group, an ethyl group, a propyl group, a butyl group, an amyl group, and a lower alkyl group having 1 to 5 ring carbon atoms. The lower alkoxy group may be either a chain or 9-branched type, and examples of the lower alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, an amyloxy group, and a lower alkoxy group having 1 to 5 ring carbon atoms. Examples of substituted amine groups include lower alkyl groups such as methyl, ethyl, propyl, and butyl groups (which may be linear or bibranched). Or, for example, -C2H40H group, -C,H60H group, -CI(2NH
Examples include amino groups substituted with substituted lower alkyl groups such as 8O2CH, groups, and groups. These lower alkyl groups, lower alkoxy groups, and substituted amino groups are all electron donating groups, so they have the effect of increasing the electron density of the acridine ring.
Therefore, the acridine derivatives of the present invention having these substituents all have a large molecular extinction coefficient and a high fluorescence intensity, and when used as a stain for sorting human chromosomes, for example, is extremely effective.

The acridine derivative of the present invention can be easily synthesized, for example, as described below.

That is, for example, using 0-chlorobenzoic acid (or a derivative thereof) as a test substance, the Ulmann reaction is performed by the formula (wherein, R
', R2 is the same as above. ) is synthesized, and then this is subjected to condensation ring closure and chlorine substitution reaction with phosphorus oxychloride to form 9-chloro represented by t (wherein R1 and R2 are the same as above). This is converted into an acridine derivative, which is further reacted with total hydrazine to form a 9-hydrazinoacridine derivative represented by N~(J5-tube (Vj, where R' and R2 are the same as above). By reacting with terephthalaldehyde, the acridine derivative of the present invention represented by the desired general formula (wherein Hl and R2 are the same as above) can be obtained.

The Ulmann reaction in the first step may be carried out by a conventional method of reacting in the presence of Cu. The reaction is preferably carried out using a suitable neutral reaction solvent such as amyl alcohol, and is usually carried out by heating. The condensation ring closure and chlorine substitution reaction in the second step can be carried out using, for example, Org, Syn, Co11°Vol.
, II p, 53 (1955), the N-phenylanthranilic acid derivative and phosphorus oxychloride are heated to react. The reaction with hydrazine in the third step is described, for example, in J. Chem, Sac, 1965.
．． Heating a 9-chloroacridine derivative and hydrazine hydrate in an alcohol aqueous medium according to the method described in 4656,
Make a substitution reaction. The reaction with terephthalaldehyde in the fourth step is, for example, a reaction with a 9-hydrazinoacridine derivative,
In contrast, 0.5 to 0.75 times the mole of terephthalaldehyde is added in a hydrophilic organic medium such as methanol, ethanol, acetone, dioxane, dimethylformamide (DMF), etc., usually at 50 to 60°C. If the reaction is stirred for minutes to ν hours, crystals of the target substance will precipitate and precipitate.
The desired novel acridine derivative of the present invention can be easily obtained in the form of red-orange to red crystals by taking it in a T-furnace, washing, drying, and if necessary recrystallizing with an appropriate solvent according to a conventional method. can. In addition, as the above-mentioned recrystallization solvent, organic solvents such as methanol, ethanol, dioxane, DMF, etc., mixed solvents of these in arbitrary proportions, or mixed solvents of these with water further added, etc. are used. Accordingly, glacial acetic acid and the like can also be used as a reconsolidation solvent.

The novel acridine derivatives of the present invention are extremely stable in the atmosphere, and most of them are insoluble in water, but they are not soluble in +A solvents such as methanol, ethanol, dioxane, DMF, chloroform, carbon tetrachloride, etc. It is soluble in acetic acid and turns yellow in acetic acid and red-orange to deep red in other organic solvents.

The novel compound of the present invention has two chemical species depending on the difference in -. That is, at pH 3.5 or lower, it exists as a compound having a structure represented by formula (A) in which a proton is added to the nitrogen of the acridine ring, and it also exists as a compound with a structure represented by formula (A) in which a proton is added to the nitrogen of the acridine ring.
Or, if R2 is a substituted amino group (9,0 or more), it exists as a compound having a structure represented by the formula CB'), each having its own maximum absorption wavelength.

For example, when the novel compound of the present invention is used as a staining agent for sorting human chromosomes (by the R-staining method), it can be applied to specific regions, such as the center of chromosome C group 9, which is difficult to identify using conventional staining methods. It has an affinity for the body and emits strong fluorescence near it. Therefore, the use of the compounds of the present invention makes it possible to identify, ie, confirm and determine the location of a specific chromosome, greatly contributing to the discovery and elucidation of various diseases and abnormalities.

The present invention will be described in more detail below with reference to i/C examples, but the present invention is not limited in any way by these examples.

〔Example〕

The novel acridine derivatives of the present invention were all synthesized by the following synthetic route.

R' Anthranilic acid derivative j 9-Chloracridine derivative (1'a) 9-hydrazinoacridine derivative (to ■) (1) Synthesis of N-(fW-substituted phenyl)anthranilic acid Method A) 0-Chlorobenzoic [8
& (0,051 mol) was mixed with 10 g of potassium carbonate and 67 μl of amyl alcohol, then 12 m of toluene
After gt-injection, it was concentrated under normal pressure to completely remove fraction 20. Next, aniline derivative (0,051 mol) and copper powder 0.2.
After all of the F was added, the reaction was carried out at 120° C. for 3 hours. After cooling the reaction solution, 150 tR1 of water and 1 g of activated carbon were added for treatment, insoluble materials were removed, fc was carried out, and the lachrymal fluid was concentrated. Residue whole water 150m
After dissolving 1, dilute hydrochloric acid was added to adjust the pH to 4, and crystals were precipitated.
P was removed, washed with water, and dried to obtain an anthranilic acid derivative.

The results are shown in Table 1.

Method B) 8 g (0.051 mol) of 0-chlorobenzoic acid, an aniline derivative (0.24 mol) and 0.29 mol of copper powder were mixed, and a reflux reaction was carried out for 2.5 hours. After cooling the reaction solution, dilute hydrochloric acid was added to adjust the pH to 1.0, and the precipitated crystals were washed with water.
i. Dry to obtain an anthranilic acid derivative. The results are shown in Table 1.

The following margin (2) Synthesis of 9-chloroacridine derivative (1'a) Anthranilic acid derivative (0.05 mol) was added to phosphorus oxychloride 9
The mixture was mixed with 0- and refluxed for 3 hours. After the reaction, concentrate
An aqueous solution of sodium carbonate was poured into the residue to give a concentration of -(8).Then, the methylene chloride layer was separated, washed with water, and dried over anhydrous MgSO4.After removing the desiccant, the solvent was distilled off, and the residue was diluted with n-hexane. The product was crystallized, separated, and dried to obtain 9-chloroacridine derivative (1'a)'. The results are shown in Table 2.

Margin below (3) Synthesis of 9-hydrasonoacridine derivative (r'b) Hydrazine hydrate 15- is mixed with ethanol 300- and 9-chloroacridine derivative (0,03-
A solution prepared by dissolving 30 moles of ethanol in ethanol was added dropwise. After refluxing for 3 hours, the medium was distilled off under reduced pressure.

The concentrated residue was separated by column chromatography (Wako Glu C-200
, Eluent: methylene chloride/n-hexane = 571 (
v/v)) The king spot portion of L, Rfo, 15 to 0.35 was fractionated and concentrated to obtain a 9-hydrazinoacridine derivative (1'b). The results are shown in Table 3.

Space below (4) Synthesis of terephthalaldehyde 9 bis(9'-acridinyl)hydrazone derivative (■') 9'-hydrazinoacridine derivative (1'b) (0,005 mol) was dissolved in methanol 50-75 TrLt. To this, 30 ml of terephthalaldehyde (0,0025 mol) in a methanol bath Q was added dropwise at 50 to 60°C, and the mixture was stirred and reacted at the same temperature for 2 hours. The reaction solution was cooled, and the precipitated crystals were collected, washed with a small amount of methanol, and then recrystallized from a suitable medium to obtain terephthalaldehyde V bis(9'-acrizonyl)hydrazone derivative (1'). The results are shown in Table 4.

Margins below [Effects of the Invention] As described above, the present invention provides a novel acridine derivative useful as a staining agent for sorting human chromosomes, for example, in a sorting method using the acridine derivative of the present invention. In this case, it has a remarkable effect in that it becomes possible to identify and locate specific parts that are difficult to identify with conventional staining methods, that is, chromosomes of group C & 9, and contributes to this industry. This is a truly great invention.

Claims (1)

[Claims] General formula [I] ▲ Numerical formulas, chemical formulas, tables, etc.▼ [I] (In the formula, R^1 and R^2 are each independently a hydrogen atom, a lower alkyl group, a lower alkoxy group) terephthalaldehyde bis(9'
-acridinyl)hydrazone derivatives.