JP3023773B2 - Nitrogen-containing heterocyclic compounds - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a nitrogen-containing heterocyclic compound.

[0002]

2. Description of the Related Art Naturally occurring nucleic acid bases having complementary molecular structures are selectively bonded to each other via several hydrogen bonding pairs. Adenine, which is a nucleobase, selectively binds to thymidine via hydrogen bonds. Bonding occurs between certain atoms on the nucleobase, in which case two hydrogen bonding pairs are formed. Similarly, cytidine selectively binds to guanine through a hydrogen bond,
Unlike the previous case, three hydrogen bond pairs are generated. The formation of a binding pair between specific nucleobases is a property essential for the expression of a biochemical function of a gene (nucleic acid) that is the basis of a life phenomenon, and has been frequently observed as a biochemical phenomenon.
Further, the formation of the selective binding of nucleic acid bases has been elucidated as a physicochemical phenomenon by various spectroscopic techniques, X-folding, and the like. Such complementary bonds between nucleobases based on hydrogen bonds have been used for recovery and purification of nucleic acid-related substances or useful substances having a partial structure of nucleobases. For example, an insoluble carrier in which a nucleobase having a structure complementary to the nucleobase to be recovered and purified is chemically bonded by an appropriate method is prepared in advance, and the nucleobase to be targeted to this carrier is prepared. The target nucleic acid base is selectively captured on the carrier by contacting the raw material containing it. Compared to the methods for recovering nucleic acid-related substances by reprecipitation, extraction, and ion exchange methods, the recovery method that uses the complementary binding force between nucleic acid bases has an excellent recovery rate of the target substance, and purification per step This is an important method that has the characteristic of high efficiency. In particular, the mutual separation between nucleic acid-related substances having very similar chemical behaviors, particularly the separation by liquid chromatography, requires a high number of theoretical plates, and thus the use of specific interaction of nucleobases is an essential method. Conventionally, for the purpose of the above-mentioned recovery / purification or analysis, natural nucleic acid-related compounds and partially modified compounds thereof have been exclusively used as materials for selectively capturing target nucleic acid-related substances. However, natural nucleic acid-related compounds as capture materials are small molecular species, so there are few functional groups available for immobilization on carriers,
The joining method is very limited. If the immobilization position on the carrier is improper, the formation of hydrogen bonds between the nucleobases is inhibited, the selective binding ability between the nucleobases having the original complementary structure is not exhibited, and the effectiveness of the carrier is reduced. Lost.

[0003]

The present invention relates to a novel compound
The challenge is to provide This compound is a heterocyclic compound having a binding strength, selectivity, and number of bonds equal to or higher than that of a natural nucleobase, and which is advantageously used as a nucleobase scavenger which does not inhibit the binding ability even by chemical modification.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above problems, and as a result, have completed the invention. According to the present invention, a nitrogen-containing heterocyclic compound represented by the following general formula (1) is provided.

Embedded image (Wherein the substituents R1 and R3 are hydroxyl groups,
R2 and R4 are NHR groups, and substituents R1 and R4 are
A xyl group, and R2 and R3 are an NHR group, a substituent R
1 is a hydroxyl group and R2, R3 and R4 are NH
R group and substituent R4 are hydroxyl groups;
2, and R3 are NHR groups, and substituents R1 and R3 are fluorine atoms
And R2 and R4 are NHR groups, and R is hydrogen
It is an atom or an alkyl group having 1 to 25 carbon atoms. This al
The kill group may be straight-chain or branched, and may be an aryl group or
Or an arylalkyl group.
The kill group consists of an alkoxy group, an ester group, and an acyl group
Including those substituted with a substituent selected from the group
No. )

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention has the following general formula:
It is a nitrogen-containing heterocyclic compound represented by (1).

Embedded image (Wherein the substituents R1 and R3 are hydroxyl groups,
R2 and R4 are NHR groups, and substituents R1 and R4 are
A xyl group, and R2 and R3 are an NHR group, a substituent R
1 is a hydroxyl group and R2, R3 and R4 are NH
R group and substituent R4 are hydroxyl groups;
2, and R3 are NHR groups, and substituents R1 and R3 are fluorine atoms.
And R ₂ and R 4 are NH ₂ or NHR groups
R is a hydrogen atom or an alkyl group. This alkyl
The group may be straight-chain or branched, and may be an aryl group or
Optionally substituted with an arylalkyl group,
Group is a group consisting of an alkoxy group, an ester group, and an acyl group
And the case where it is substituted by a substituent selected from the above. )
In order to impart appropriate solubility to the synthetic intermediate, it is preferable to introduce one or more substituted amino groups into the substituents. At this time, the substituent of the substituted amino group may be not only a simple linear alkyl group, but also a branched one or a ring one. Further, the substituted amino group is not limited to an alkyl group, and may have a substituent such as an aryl group or an arylalkyl group. Furthermore, these alkyl groups, aryl groups, and arylalkyl groups may have a substituent that does not hinder capture of the nucleobase, for example, a substituent such as an alkoxy group, an ester group, or an acyl group. . In the substituted amino group having an alkyl group, the alkyl group has 1 to 25, preferably 8 to 20 carbon atoms.

Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (2)

Embedded image (Wherein R represents hydrogen or an alkyl group) is a compound represented by the following general formula (3)

Embedded image (Wherein R has the same meaning as described above). In this case, as the hydrolyzing agent, KOH and 18-
Use of an aqueous solution containing a click round -6 are preferred. The compound represented by the general formula (3) is represented by the following general formula (1)
It can be obtained by reacting the compound represented by 6) with an amine.

Embedded image

[0007] Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (4)

Embedded image (Wherein, R represents hydrogen or an alkyl group) is a compound represented by the following general formula (5)

Embedded image Wherein R has the same meaning as described above, and an amine. The compound represented by the general formula (5) can be obtained by hydrolyzing one of the chlorine atoms contained in the compound represented by the following general formula (17).

Embedded image The hydrolysis is preferably performed in an aqueous solution containing hydrochloric acid and acetic acid.

Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (6)

Embedded image (Wherein R represents hydrogen or an alkyl group) is represented by the following general formula (7)

Embedded image (Wherein R has the same meaning as described above) by reacting with a amine. The compound represented by the general formula (7) is represented by the following general formula (18)

Embedded image After reacting a compound represented by the formula with an amine, the following general formula (19) is obtained.

Embedded image (Wherein, R represents hydrogen or an alkyl group) by hydrolyzing one chlorine contained in the compound represented by the formula:

Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (8)

Embedded image (Wherein, R represents hydrogen or an alkyl group) is a compound represented by the following general formula (9)

Embedded image (Wherein, R has the same meaning as described above) by hydrolyzing the chlorine at the 8- position contained in the compound represented by the formula: In this case, it is preferable to use an aqueous solution containing hydrochloric acid and NaOH as the hydrolyzing agent. The compound represented by the general formula (9) is a compound represented by the general formula (7)
Can be obtained by reacting the 6-position chlorine contained in the compound represented by the formula with an amine.

Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (10)

Embedded image (Wherein R represents hydrogen or an alkyl group) is a compound represented by the following general formula (11)

Embedded image (Wherein, R has the same meaning as described above), and can be obtained by reacting chlorine at the 2- position contained in the compound with an amine. The compound represented by the general formula (11) can be obtained by hydrolyzing chlorine at the 6-position contained in the compound represented by the general formula ( 7 ). In this case, the hydrolysis is preferably performed in an aqueous solution containing chlorine and acetic acid.

Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (12)

Embedded image (Wherein, R represents hydrogen or an alkyl group) is represented by the following general formula (13)

Embedded image (Wherein, R has the same meaning as described above), by reacting an amine with 2-position chlorine contained in the compound represented by the formula:

Among the nitrogen-containing heterocyclic compounds of the general formula (1) according to the present invention, the following general formula (14)

Embedded image (Wherein, R represents hydrogen or an alkyl group) is represented by the following general formula (15)

Embedded image (Wherein, R has the same meaning as described above), by converting chlorines at the 2- and 6-positions contained in the compound represented by the formula (II) into a fluorine atom. In this case, as the fluorinating agent, it is preferable to use a KF-18-single crown 6.

The general formulas (2), (4), (6),
The hydroxyl group contained in the compounds represented by (8) and (10) actually exists as a keto tautomer. For example, a keto-form tautomer of the compound of the general formula (2) is represented by the following general formula ( 20 ).

Embedded image The structure of the compound of the general formula (2) corresponds to a structure in which two molecules of cytidine, one of natural nucleobases, are condensed.
The structure of the compound of the general formula (4) corresponds to a structure in which a pyrimidine ring portion of a natural nucleobase adenine and cytidine are condensed. The structure of the compound of the general formula (6) corresponds to a structure in which a pyrimidine ring portion of adenine and a pyrimidine ring portion of guanine are condensed. The structure of the compound of the general formula (8) corresponds to a structure in which a pyrimidine ring portion of cytidine and guanine is condensed. The structure of the compound of the general formula (10) corresponds to a structure in which a pyrimidine ring portion of adenine is condensed with thymidine.

[0014]

The nitrogen-containing compound of the present invention represented by the general formula (1) is used as a nucleobase scavenger, and forms a hydrogen bond with a naturally occurring nucleic acid-related substance or a heterocyclic compound having a partial structure of a nucleobase. Selective binding allows efficient separation, recovery and purification of these substances. Further, because of its high selectivity, it can be used as a material for performing qualitative and quantitative analysis of nucleobase-related substances having similar properties.

[0015]

EXAMPLES The present invention will be described in detail with reference to examples.

Example 1 2,6-Dichloro-4,8-dioctadecyl-pyrimido [5,4-d] pyrimidine (in the general formula (3),
= Octadecyl) (compound 3) 2,4,6,8-tetrachloropyrimido [5,4-d]
2.50 g of pyrimidine (I) are dried in 25 m of chloroform.
of octadecylamine 5.49.
g and 2.64 g of ethyldiisopropylamine in 50 ml of a dry chloroform solution were added dropwise over 30 minutes. The reaction vessel was cooled to maintain the reaction temperature at 5-10 ° C. After the addition, the mixture was left at room temperature for 2 hours. The chloroform phase was washed with water and dried over magnesium sulfate to obtain the desired product. It was recrystallized from ethyl acetate and purified (4.84 g). Melting point 1
12 ° C. Similarly, 2,6-dichloro-4,8-didodecyl-pyrimido [5,4-d] pyrimidine. Melting point 102
° C. Synthesis of 2,6-dihydroxy-4,8-dioctadecyl-pyrimido [5,4-d] pyrimidine (in the general formula (2), R = octadecyl) (compound 2) 2,6-dichloro-4,8- Dioctadecyl-pyrimido [5,4-d] pyrimidine (Compound III) 300 mg,
To 1815-crown-6215 mg and potassium hydroxide 120 mg was added 2 l of triethylene glycol dimethyl ether, and the mixture was heated to 185 ° C. while stirring under a nitrogen atmosphere.
Heated for 2 hours. After the solvent was removed from the reaction solution by heating under vacuum, chloroform was added to the reaction product, and the mixture was washed with a dilute aqueous acetic acid solution and water. After drying the solution with magnesium sulfate, the target product was isolated by column chromatography (silica gel / acetone-chloroform). Yield 50%. Thin layer chromatography (silica gel / acetone: chloroform 1: 9) R
f value 0.3.

Example 2 2-chloro-4,8-dioctadecyl-6-hydroxypyrimido [5,4-d] pyrimidine (in the general formula (5), R = octadecyl) (compound 5) 8 ml of acetic acid and 2 ml of concentrated sulfuric acid were added to 300 mg of dichloro-4,8-dioctadecyl-pyrimido [5,4-d] pyrimidine (compound 3), and the mixture was heated and stirred at 110 ° C. Chloroform was added to the reaction solution, washed with water, 5% aqueous sodium hydrogen carbonate solution and water in that order, and dried over magnesium sulfate to obtain the desired product. Recrystallized from ethyl acetate and purified (light yellow solid, yield 235 mg), thin layer chromatography (silica gel / acetone: chloroform 1: 9), Rf value 0.4. 6-hydroxy-2,4,8-trioctadecylpyrimido [5,4-d] pyrimidine (in the general formula (4),
R = octadecyl) (compound 4) 2-chloro-4,8-dioctadecyl-6-hydroxypyrimido [5,4-d] pyrimidine (in the general formula (5), R = octadecyl) (compound 5) (80 mg) A mixture of 72 mg of octadecylamine and 2 ml of triethylene glycol dimethyl ether was heated to 180 ° C. for 6 hours. After cooling, chloroform was added to the reaction mixture, and
After washing with sodium carbonate and water, dried over magnesium sulfate. After removing the solvent and unreacted octadecylamine under vacuum heating, the residue was purified by recrystallization from ethyl acetate (yield 83 mg). Thin layer chromatography (silica gel / acetone: chloroform 1: 9) Rf value 0.5.

Example 3 4-octadecyl-2,6,8-trichloropyrimido [5,4-d] pyrimidine (in the general formula (19),
R = octadecyl) (compound 19) 2,4,6,8-tetrachloropyrimido [5,4-d]
2.88 g of pyrimidine (formula (16)) were suspended in 30 ml of dry chloroform by stirring, and 2.87 g of octadecylamine and 1.38 of ethyldiisopropylamine were suspended.
g of 30 ml of a dry chloroform solution was slowly added dropwise over 1 hour. The reaction vessel was cooled on ice and the temperature of the solution was kept at 0 ° C. The reaction solution was washed with water to obtain the desired product. Thin layer chromatography (silica gel / chloroform) Rf value 0.5. 4-octadecyl-8-hydroxy-2,6-dichloropyrimido [5,4-d] pyrimidine (in the general formula (7), R = octadecyl) (compound 7) 4-octadecyl-2,6,8-trichloro Pyrimido [5,4-d] pyrimidine (in the general formula (19),
5.0 g of R = octadecyl) (compound 19) was dissolved in 50 ml of tetrahydrofuran, 20 ml of a 1 mol / l aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 3 hours.
The reaction solution was poured into 300 ml of water, and the precipitate was collected. This was recrystallized from ethyl acetate and purified (yield 2.5 g).
Thin layer chromatography (silica gel / acetone: chloroform 1: 2) Rf value 0.05. Infrared absorption spectrum (carbonyl group) 1703cm-1. 165 ° C. 8-hydroxy-2,4,6-trioctadecylaminopyrimido [5,4-d] pyrimidine (in the general formula (6), R = octadecyl) (compound 6) 4-octadecyl-8-hydroxy-2,6 -Dichloropyrimido [5,4-d] pyrimidine (compound 7) 100
mg, octadecylamine 228 mg, and solvent dimethylimidazolidinone 2 ml were heated to 150 ° C. for 12 hours.
The reaction product was dissolved in chloroform, and the solution was washed with an aqueous solution of sodium carbonate and water, and dried over magnesium sulfate to obtain the desired product. It was purified by recrystallization from ethyl acetate (yield: 225 mg). Thin layer chromatography (silica gel / methanol: chloroform 1: 9) Rf value 0.3.

Example 4 2-Chloro-4,6-dioctadecylamino-8-hydroxypyrimido [5,4-d] pyrimidine (in the general formula (9), R = octadecyl) (compound 9) 4-octadecyl -8-Hydroxy-2,6-dichloropyrimido [5,4-d] pyrimidine (compound 7) 150
mg, 89 mg of octadecylamine, 77 mg of N, N-dibutylaniline, and 2 ml of N, N-dimethylformamide were heated to 90 ° C. for 5 hours. The reaction product was dissolved in chloroform, and the solution was washed with water and dried over magnesium sulfate. Recrystallization from ethyl acetate gave the desired product (yield 1).
31 mg). Thin layer chromatography (silica gel / methanol: chloroform 1: 9) Rf value 0.5. 4,6-dioctadecylamino-2,8-dihydroxypyrimido [5,4-d] pyrimidine (in the general formula (8), R = octadecyl) (compound 8) 2-chloro-4,6-dioctadecylamino -8-Hydroxypyrimido [5,4-d] pyrimidine (compound 9)
Acetic acid 1.5ml and 2mol / l hydrochloric acid 0.2 in 42mg
was added and heated to 80 ° C. for 12 hours. The reaction product was poured into water, and the resulting solid was washed with dilute aqueous ammonia and water to obtain the desired product (yield 33 mg). It was recrystallized from ethyl acetate and purified.

Example 5 2-Chloro-6,8-dihydroxy-4-octadecylaminopyrimido [5,4-d] pyrimidine (general formula (1)
In 1), R = octadecyl) (compound 11) 4-octadecylamino-8-hydroxy-2,6-dichloropyrimido [5,4-d] pyrimidine (compound 7)
10 ml of acetic acid and 2 ml of water were added to 357 mg, and the mixture was
Heated to 0 ° C. The reaction product was poured into water, and the precipitated solid was separated by filtration and washed with dilute aqueous ammonia and water (yield 346 m).
g). Thin layer chromatography (silica gel / methanol:
Chloroform 1: 2) Rf value 0.8. 2,4-dihydroxy-6,8-dioctadecylpyrimido [5,4-d] pyrimidine (in the general formula (10), R = octadecyl) (compound 10) 2-chloro-6,8-dihydroxy-4- Octadecylaminopyrimido [5,4-d] pyrimidine (general formula (1
In 1), R = octadecyl) (compound 11) 60
mg of ethanol and 0.13 ml of a 1 mol / l aqueous sodium hydroxide solution were added, and the mixture was stirred for a while.
The solvent was removed by evaporation. To the obtained solid, 37 mg of octadecylamine and 2 ml of dimethylpyrrolidinone were added, and the mixture was heated to 150 ° C. for 2 days. The reaction product was poured into water, and the precipitated solid was collected by filtration and purified by column chromatography (silica gel / methanol: chloroform 1: 4) (yield 75 m).
g). Thin layer chromatography (silica gel / methanol:
Chloroform 1: 4) Rf value 0.3. Melting point 78 ° C

Example 6 Synthesis of 6-chloro-2,4,8-trioctadecylamino-pyrimido [5,4-d] pyrimidine (R = octadecyl in the general formula (12)) (Compound 12) -Dichloro-4,8-dioctadecylamino-pyrimido [5,4-d] pyrimidine (in the general formula (13), R = octadecyl) (compound 13) 100 mg,
2 ml of N, N-dimethylacetamide was added to 73 mg of octadecylamine, and the mixture was heated at 100 ° C. for 12 hours. After the solvent was removed from the reaction solution under reduced pressure, chloroform was added. The organic phase was washed with 5% sodium carbonate and water, and then dried over magnesium sulfate. The composition was purified by column chromatography (silica gel / hexane-benzene 1: 1) to isolate the desired product (yield 112 mg). Thin layer chromatography (silica gel / cyclohexane-benzene 1: 1) Rf value 0.6.

Example 7 Synthesis of 2,6-difluoro-4,8-dioctadecylaminopyrimido [5,4-d] pyrimidine (in the general formula (14), R = octadecyl) (compound 14). 2,6-Dichloro-4,8-dioctadecylamino compound (in the general formula (15), R = octadecyl) (compound 15) (100 mg), 18-crown-611 mg, and 81 mg of potassium fluoride in dry acetonitrile 10
The mixture was heated at 140 ° C. for 2 days. After adding 100 ml of chloroform to the reaction solution and washing the organic phase with water, the chloroform phase was dried over magnesium sulfate. The solid obtained by removing the solvent under reduced pressure was purified by column chromatography (silica gel / hexane-benzene 2: 3) to isolate the desired product. Yield 39%, thin layer chromatography (silica gel / cyclohexane: benzene 1: 1), Rf value 0.4. 19-F NMR: δ (CDCl 3, internal standard substance hexafluorobenzene) 115.15 (s) 1H NMR: δ (CDCl 3): 6.86 (2H, t,
NH), 3.57 (4H, d, NCH2), 1.69.
(4H, t, NCCH2) 1.25 (60H, bs, CH2), 0.88 (6H,
t, CH3)

Example 8 2,6-Dichloro-4,8-bis (4-methoxybenzylamino) pyrimido [5,4-d] pyrimidine (in the general formula (3), R = octadecyl) (compound 3
Synthesis of (2)) 2,4,6,8-tetrachloropyrimido [5,4-d]
Pyrimidine formula (16) (1.33 g) in dry chloroform 3
The mixture was suspended in 0 ml by stirring, and 1.39 g of 4-methoxybenzylamine and 1.39 g of ethyldiisopropylamine were added.
25 ml of a dry chloroform solution of 31 g was added dropwise over 15 minutes. The reaction vessel was cooled to keep the reaction temperature at 5 ° C. After the addition, the mixture was left at room temperature for 2 hours. The solid obtained by evaporating the solvent under reduced pressure was washed with water to obtain the desired product (yield 2.2).
7g). It was purified by recrystallization from methoxyethanol. Thin layer chromatography (silica gel / chloroform) Rf value 0.6.

Example 9 Scavenging heterocyclic compound based on hydrogen bond of heterocyclic compound The heterocyclic compound was dissolved in chloroform and air-dried.
The stretching vibration of the carbonyl group of the heterocyclic compound was measured by the Br tablet method using an infrared spectrophotometer. Heterocyclic compound A-heterocyclic compound B (mixing molar ratio) Wave number of stretching vibration of carbonyl group (cm @ -1) Compound 2 (single) 1679 Compound 5 (single) 1694 Compound 9 (single) 1694 Compound 11 (single) 1702 Compound 5-Compound 9 (1: 1) 1686 Compound 11-Compound 12 (1: 1) 1652 Compound 14-Compound 9 (1: 2) 1702

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yuko Uchimaru 1-1-1, Higashi, Tsukuba, Ibaraki Pref. Institute of Industrial Science and Technology (72) Inventor Kazuhisa Hiratani 1-1-1, Higashi, Tsukuba, Ibaraki Pref. (56) References JP-A-50-101391 (JP, A) JP-A-50-106997 (JP, A) JP-A-51-136696 (JP, A) (58) Fields investigated ( Int.Cl. ⁷ , DB name) C07D 487/04

Claims (1)

(57) [Claims] 1. A nitrogen-containing heterocyclic compound represented by the following general formula (1). Embedded image (Wherein the substituents R1 and R3 are hydroxyl groups,
R2 and R4 are NHR groups, and substituents R1 and R4 are
A xyl group, and R2 and R3 are an NHR group, a substituent R
1 is a hydroxyl group and R2, R3 and R4 are NH
R group and substituent R4 are hydroxyl groups;
2, and R3 are NHR groups, and substituents R1 and R3 are fluorine atoms
And R2 and R4 are NHR groups, and R is hydrogen
It is an atom or an alkyl group having 1 to 25 carbon atoms. This al
The kill group may be straight-chain or branched, and may be an aryl group or
Or an arylalkyl group.
The kill group consists of an alkoxy group, an ester group, and an acyl group
Including those substituted with a substituent selected from the group
No. )