JPH01500189A - Photocleavage method of nucleic acids using 2,7-diazapyrenium dication and new diazapyrene derivatives - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 2. Photocleavage method of nucleic acids using 7-diazapyrenium dication and new diazapine Ren derivatives The present invention relates to 2,7-diazapyrenium dication, DAP", or its derivatives. The present invention relates to a method for photocleavage of nucleic acids using a dimeric species, "bis-DAP".

The present invention also relates to novel diazapyrene derivatives, which are capable of forming sication, D It is a salt that releases AP2+ or a tetracation, bis-DΔP4+.

Finally, the 2,7-diazapyrenium dication attached to the oligonucleotide is Photocleavage reagents can be made specific for nucleic acids.

In recent years, nucleic acids, more specifically deoxyribonuclei commonly referred to as DNA and RNA Much research has been done to discover synthetic reagents that can cleave acids and ribonucleic acids. It was.

Many studies have focused on D by oxygen-dependent redox processes or by photoactivation. It was carried out with metal complexes that cleave NA. Acridine-type dyes cause photodamage to DNA used.

Among the metal complexes used, in particular the EDTA-Fe (if) group and the 1.10 -phenanthroline-copper complex.

For example, F, E, B, S, Letters (1'E mouth S LCTTERS).

Volume 172, No. 1, 1984-6 Bootliff (BOIITORIN) Hokaha A reagent with EDTA-Fe(II) group suitable for direct cleavage of single-button nucleic acids is described. ing.

Similarly, SCIENCE, vol. 230, 1985.679-68 In No. 1, TULLIUS and others used EDTA-F ( II) is used.

HERTZBERG [Journal of the American ・Chemical Society (J, Am Chem, Soc,) +1982.1 04, pp. 313-315] has been shown to be effective in DNA cleavage for many antitumor antibiotics. The use of (methidium propyl-EDTA)-Fe(II) as a cofactor for Recommended.

The use of 1,10-furinandroline-copper complexes for DNA cleavage is particularly (REICH) et al. Journal of the American Chemical Society Yeti (J, Am Chem, Soc,).

103, No. 12, 1981, and Faziani (FAGG [ANI) et al. , Journal of the American Chemical Society (J, Ai+ , Che+s, Soc, ), Vol. 102, pp. 5419-5421, 1980. It is listed.

Furthermore, acridine-type dyes are known to damage DNA. for example, FREIFELDER (Biophysical Journal) (Biophysical Journal) + Volume 1.

19613, DNA irradiated with visible light in the presence of acridine orange depolymerized. , indicated that it would be disconnected.

Furthermore, 4,4'-bipyridinium ions, especially methylviologen, are present in various levels. It is recognized that it was used in dox and photoredox processes. Ya MOUYAL et al., Israeli Journal of Chemistry ( Israel Journal of Chemistry), 22. 1982 ．． [pages 117-123], these processes are carried out in the presence of photosensitizers or in the presence of ultraviolet light. Can only occur under irradiation.

Methyl viologen, i.e. 1,1'-dimethyl-4,4'-bipyridinium Chloride, commonly referred to as MV, can damage DNA under these conditions. can.

This time, we demonstrated that diazapyrene derivatives, especially 2,7-diazabirenium cations, are Among them, it was found that it can be used for photocleavage of nucleic acids without the need for photosensitizers.

Accordingly, the present invention relates to the process of photocleavage of nucleic acids, the process of catalyzing the process, and the resulting reaction. It consists of irradiating the mixture with visible light.

Diazapyrene derivatives suitable for the purposes of the invention correspond to the following formula (1): R and R' are the same or different, hydrogen, or optionally interrupted by one or more heteroatoms such as oxygen, sulfur or nitrogen. A straight or branched, saturated or unsaturated aliphatic hydrocarbon group that can be Therefore, hydrocarbon groups can also be conjugated with hydroxyl, amino or thio groups as appropriate. Aliphatic hydrocarbons which can be substituted by lyl groups or by heterocyclic groups or R and R' are the same and the formula R,Y- (wherein R1 is group (CHt) 3-, Y- is sulfonatoio7SO,-), or expression %formula% X- is an anion or at least one of the substituents R and R' has the formula R, If Y- is later, it does not exist, in which case the derivative is an inner salt] In this description, ``optionally interrupted by one or more heteroatoms'' is used. The term "aliphatic hydrocarbon radicals capable of" also includes the corresponding onium cations. aliphatic hydrocarbon radicals, especially aliphatic carbons containing one or more quaternary nitrogen atoms a hydrogen group (where Alc and Alcz contain, for example, 1 to IO carbon atoms) are the same or different alkylene groups; are R′ and R12 the same? or different, is hydrogen or a lower alkyl group containing 1 to 6 carbon atoms; R'3, R'a and R1 are the same or different, hydrogen or a saturated or unsaturated aliphatic hydrocarbon group, preferably containing 1 to 25 carbon atoms; rukyl group) Indicates the group of

Compounds of formula (3) in which R and R' are the same and represent hydrogen or a methyl group are Hunig (Angew, Chem), 1980. It is a well-known compound described by M19 (1968)).

In the method of the present invention, the general formula (I[)R, R' and X are as described above; , ■ can be optionally interrupted by one or more heteroatoms, and and/or optionally interrupted by one or more divalent aromatic or heterocyclic groups. saturated or unsaturated, straight or branched hydrocarbon chains that can be It is also possible to use bisdiazapyrene derivatives corresponding to the valent group ].

The hydrocarbon chain forming the group ■ contains two or more carbon atoms, oxygen, sulfur and one or more heteroatoms selected from nitrogen atoms be able to.

Preferred examples of the divalent group ■ are 2■～■: It is the basis of

In formulas (1) and (I[), the aliphatic hydrocarbon group preferably has 1 to 10 Contains carbon atoms. Straight or branched alkyl groups having 1 to 6 carbon atoms is particularly preferred.

In the method according to the present invention, general formula (III) (wherein p is an integer of 1 to 15) of the diazopyrene (sic) macrocycle, preferably equal to 8). The use is also possible.

The diazapyrene derivatives of formula (sic) (1) and (11) are all 2.7- Diazapyrene, or DAP, can be obtained from one of the following processes For convenience, the diazapyrene derivative of formula (1) and the derivative of formula (I[) are as follows: The terms rDAP” derivatives” and “bis-DAP” derivatives, respectively, More shown.

DAP + RX, ROAP" + RX, -DAP" -RX, -+R'X 2 + R-”DAP”-R’X,-X2-(X and X2 are the same or is different and has the same meaning as X) This process involves essentially two Nw substitutions of DAP with groups R and R', respectively. The resulting compound was then subjected to ion exchange to obtain xl and It can be suitably converted into the identical corresponding compound. R and R' are the same If so, it will be recognized that the two @conversion steps take place simultaneously. These steps are suitable solvents such as dimethylformamide (DMF), chloroform or acetate. Performed in nitrile. If it is desired to carry out two substitutions simultaneously (e.g. R= R' = CH5), DMF is suitable and the reaction is carried out at room temperature; otherwise In this case, chloroform is used in the first substitution stage and acetonitrile is used in the second stage (R6R'). in which case the first displacement stage is preferably carried out at room temperature and the second stage under reflux. be exposed.

This process is particularly suitable for DAP'' derivatives in which R and R' are aliphatic hydrocarbon radicals of the type described above. Production of conductors, especially N,N'-dimethyl-2,7-diazabirenium dichloride , hereinafter referred to as MDAP'''(CI-)z, which has the following scheme: %formula%() It is shown by.

Process B R′-″DAP　”-R,2CI- (In the formula, Ts is an anion, such as tosylate.) It will be appreciated that Ts- can be replaced by other anions.

This process is performed when R is an aliphatic hydrocarbon group substituted by a hydroxyl group, especially DAP” is a hydroxyalkyl group and R′ is an alkyl group such as methyl. Suitable for manufacturing conductors. The process begins with DAP and 2,4-dinitrophenyl salt such as tosylate in a suitable solvent such as a chloroform/methanol mixture. 2-dinitrophenyl-2,7-diazabilene by reacting under reflux A sic salt such as tosylate is prepared as a synthetic intermediate and then this intermediate is and an amine substituted by the group R, and the second step consists of reacting As in process A, N substitution of formula R'X with a halide is included.

to the process The process is performed when R' is an alkyl group, e.g. methyl, and R is replaced by an amino group. This process is suitable for producing DAP”° derivatives, which are aliphatic hydrocarbon groups The process is similar to Process B above, the only difference is that the amine used is one of the amino groups. is a protected diamino, and said group is then protected before the second substitution step. The group is removed. This process can be represented by the following diagram.

DAP"-Alk-NHz・Ts-+R'X=R'-"DAP" -A6 to -NHz ・Ts-X- [wherein, Rs -Aj! k　NHHz　(protected 2-dinitrophethyl-2,7-diazabirenium sic ) Tosylate is a novel compound useful as a synthetic intermediate, and is another main feature of the present invention. Compose the topic.

Process D This process uses DA Suitable for the preparation of P'' derivatives: this process involves a partial solution of DAP in anhydrous acetone. and propane sultone at reflux temperature.

This process applies to DAP in which R and R' are the same and is represented by a group of ``Suitable for producing derivatives.

Ts Therefore, this process involves collidine and collidinium chloride between DAP and aziridine. Consists of a reaction in the presence of lido.

Bis-DAP” The method for producing these bis=DAP''''' derivatives essentially consists of two molecules of DAP and formulas X, -■ -X+ (in the formula, XrXz and ■ are as described above) and one cybaride molecule The method consists of reacting the remaining nitrogen in the DAP molecule and then converting the remaining nitrogen in the DAP molecule to Ni.

Each step is preferably carried out in acetonitrile at reflux temperature.

This process can be represented by the following diagram: R’-“DAP”-〇- Synthesis of DAP" -R2X, -2X, -bis-DAP" macrocyclic compound Method for producing a macrocyclic compound of formula (also referred to as "bis-DAP" macrocycle) is naphthalene-1,4,5,8-tetracarboxylic anhydride of formula CH2), -NH2 with diamine in dimethylformamide in the presence of acetic acid. Consists of responses. The macrocycle formed by this reaction is then converted to aluminum hydride. Reduction in the presence of mulithium and aluminum chloride to form a compound of formula ■ , which is then converted into a compound of formula (1). The first step in this process is to refill ・Journal of the Royal Netherlands Chemical Society (R ecuel Journal or the Royal Netherlan ds Chemical 5oci-ety), 95/4. 1976-4.　 The protocol described by J.H. Verhoeven on page 89 It is a modified form of process.

The second and third stages of this process are described by Hünig et al. Ren der Hemi (8nn, CI+em,), 1973. Described by 339 This is an adaptation of the process.

The reaction scheme for this process is as follows:■ ■　■ The diazapyrene derivative according to the invention is a fluorescent compound.

Diazapyrene (DAP) used as a starting material in the above synthesis is a commercially available naphthalene of formula Obtained from ren-1,4,5,8-tetracarboxylic anhydride according to the following reaction scheme Can: CH.

meeting VI CH,X I Hunig (Hunig); Angewante Hemi (Angeii, Che+ w, ) +180 (1968), in the adaptation of the Na19 process, the formula ■ Naphthalene 1,4,6,8-carboxylic acid (sic) anhydride first with methylamine is converted to the corresponding bis(N-methylimide) of formula X by reaction at room temperature with Ru. Imide X was then refluxed in tetrahydrofuran to form LiA ICls mixture to form compound IIXI, which is then treated with selenium DAP is obtained by reacting for about 4 hours at a temperature of 265-300°C in the presence of DAP.

Compounds which are particularly suitable for the purposes of the present invention are those of the formula: 1) in which R and R' are methyl groups; Compound (I) or MDAP'' derivative (sic); 2) One of the substituents R or R' is lower alkyl, e.g. methyl, and the other an alkyl group substituted by a roxyl or thio group or an amino group, i.e. Formula -(CHz)ゎ-OH.

-(CHg　), -3H or -(CHz)-NHg (where n is preferably 1 ~10), and a compound of formula (1) which is 3) ■ corresponds to one of formulas ■ to V, and R and R' are lower alkyl groups, such as methyl is a compound of formula (II).

Formula (1) or (II) containing 1 to 2 2,7-diazapyrenium dications The diazapyrene derivative has a planar structure.

This planar structure makes these compounds suitable for intercalating drugs such as actinomycin and protein. Like reomycin, it interacts with nucleotides of DNA or RNA. Sara In addition, the effect of visible light irrigation (sic) Below, the derivatives according to the invention can cleave DNA or RNA into several strands.

The derivative of formula (n) or bis-DAP'' derivative has two 2,7-diazabilene Since the um cation is present in their molecules, the formula (derivatives of RI or DAP” It should be mentioned that it is more reactive than the derivative. They are also during photosection Has greater selectivity and greater stability.

Furthermore, one of the substituents R or R' is substituted by a hydroxyl or amino group. Derivatives of formula (I) which are aliphatic hydrocarbon groups can be modified or synthesized. An oligonucleotide or oligodeoxynucleotide containing a chain of naturally occurring nucleotides that can Can be covalently camped to leotide.

This bonding can be carried out by processes known to those skilled in the art, e.g. It is possible to use the synthesis process described by Lancer Patent No. 8301223. and that patent is incorporated herein by reference. These processes are inter alia DAP” derivative hydroxyl and oligonucleotide 3’-phosphodiester or oligonucleotides 3',5'-bis(phosphodiester) or with 5'-phosphodiester. with oligodeoxynucleotide 2'-phosphodiester) or oligodeoxynucleotide 2'-phosphodiester) oligonucleotide or oligodeoxynucleotide. Otide is properly protected. Therefore, oligonucleotides (or oligodeoxy nucleotide)/DAP” or DAP”/oligonucleotide (also oligodeoxynucleotide)/DAP” can be obtained. They can constitute highly selective photocleavage reagents.

The oligonucleotide or oligodeoxynucleotide chain of the coupling product can bind selectively to complementary sequences. The small amount in this coupling product (Due to the presence of one 2,7-diazapyrenium dication, DNA or R NA can be easily and selectively cleaved at a specific site, and that site is determined by the oligonucleotide that binds to.

The oligonucleotide/DAP” coupling product is then combined with other reagents and camprin. It can be labeled with any kind of indicator reagent, e.g. a label, especially an active It can be a lysine-type dye or an intercalating reagent, and one of the functionalities is photocleavage. A bifunctional camping product can be produced in which the other functionality is provided by the second reagent. can.

1 lower) Jυ艷1凱 The process of the invention is then limited for convenience and thereby to only these derivatives. Rather than simply mentioning MDAP “° or its dimeric species bis-MDAP”, It will be posted.

Tests conducted on DNA under the following conditions were conducted using the sication "MDAP" or its dimeric species. Bis-MDAP'' was shown to cleave DNA under irradiation with visible light.

The test uses double-stranded supercoiled DNA (2,6.10-”M) and MDAP” or pH 7 in Tris buffer containing chloride (2,6·10-'M) of ``S-MDAP'' , 8 and 3 ± 1°C through visible light (only visible light of λ > 395 nm). 1 by a 250W slit projector (equipped with a suitable filter) The 0 reaction performed by irradiation for a time is the presence of air (i2 element) or EDTA. Done under or without.

The double-stranded supercoiled circular DNA (hereinafter referred to as 5cDNA) is plasmid pBR3. Produced by known procedures using Escherichia coli (E. coli) infected with 22, and then was purified by electroelution to be virtually free of Cant 5 cDNA.

Plasmid pBR322 becomes linear when incubated with the restriction enzyme ECORI. A plasmid or double-stranded linear DNA was given, and it was subjected to the photocleavage test described above. It was used as a label in electrophoretic analysis.

For comparison, methyl viologen, MV"", was used instead of the derivative of the present invention. The same test was conducted. 5cDNA only solution, 5cDNA solution containing oxygen, or or a solution of 5cDNA containing oxygen and EDTA was also subjected to the same treatment (under the above conditions). irradiation with visible light) and served as a control.

The results obtained are shown in Figure 1 (sic), which shows the voltage of the solutions in the various tests. A photograph of the gel after pneumophoresis, annotated below: I indicates 5 cDNA, ■ indicates Cant 5 cDNA, ■ indicates linear DNA, The abbreviations used indicate: M=marker C = control ■＝Visible light 0 = oxygen A=EDTA The measurement of the optical density of each band gave the following form DI, the ratio of ■ and ■ in %.

(1) As shown by bands 9 to 13 in Figure 1, "MDAP" was used Under the conditions, 5 cDNA was effectively cleaved under visible light irradiation, and mainly single-stranded cleavage was achieved. 5cDNA cut by cleavage, but when the reaction was continued, both values were cut by cleavage. Give linear DNA.

At low concentrations of ``MDAP'', fewer cleavages occur (conversion to the Cant 5 cDNA form). is almost complete at a concentration of 2.10-'M and monotonous at 2.10-'M and below. only a small amount.

(2) It will be observed that there is no significant cleavage of the 5cDNA in the absence of light.

(3) When oxygen is removed by degassing through freezing and thawing, the efficiency of photocleavage is small. decreases to

(The addition of 41EDTA does not affect the reaction in the presence of oxygen, but It appears that running the reaction in the absence of oxygen reduces the reaction.

t5) It was observed that the movement of scDNA was slightly delayed depending on the photocleavage reagent used. , suggesting that the latter inserts into the 5 cDNA.

B) By “Bis-MDAP” (11 Under the same conditions, bis-MDAP'° is more effective than MDAP'. appears to give small DNA fragments due to multiple cleavage of both strands (band 1). 4-18).

(2) The cleavage reaction is very effective, making it difficult to exclude light. However, 5cDN It can be acknowledged that the movement of A is delayed considerably, and the Suggesting a strong and possibly multiple interaction with Sumid pBR322.

(3) Considering the efficiency of the reaction, the effect of any kind of oxygen or EDTA is accepted. I can't.

The effectiveness of bis-MDAP'° decreases at lower concentrations, and cleavage is still complete at 10-5 M. but only slightly at lower concentrations.

These tests were carried out under the same conditions with no or substantially no cutting at MV''. Furthermore, there is no reaction in the absence of a photocleavage reagent.

Other studies have shown that "MDAP" photooxidizes various substrates under irradiation with visible light. without being limited in any way to any particular theory, The cleavage reaction causes local photooxidation at the site of interaction by the excited state of the species MDAP. You can think of it as happening later. Ribose itself photoreduces MDAP. Since it was recognized that the electron donor can be ribose.

The retarded effect of EDTA in the absence of oxygen is due to the fact that the oxidation of the external donor is similar to the oxidation of the internal donor. It will show that it competes.

The slight but not negligible effect caused by the removal of oxygen is that the cleavage reaction is also photosensitive. This shows that reaction systems such as those based on sexually reduced metal complexes can be included. By photoreduction The DAP cation generated at the beginning of the reaction is then MV itself Biochim, Biophys, Acta, 1973 ．． 314°372] can react with oxygen to form superoxides. Good morning.

Another test was performed using the acridine-type dye proflavin under the same conditions as above. that proflavin is a DNA photocleavage reagent comparable to "bis-MDAP"; and a concentration of 2.10-'M to produce an extensive cleavage of DNA to give linear DNA. It was recognized that Efficiency decreases at low concentrations within the range of 2.lo-6 to 2.101M. Ru.

The photocleavage process by proflavin leads to the generation of reduced dye and the D It seems to involve photooxidation of NA. Acridine-type dye undergoes photoreduction along with oxidation of substrate Actually well known about being.

Because they are flat molecules, the derivatives of the present invention can be used with intercalating drugs such as actinomycin, preomers, etc. Like icine, it interacts with nucleotides in DNA or RNA. Sara In addition, it was observed that the fluorescence of the derivative disappeared when binding to DNA occurred. follow The process of the photocleavage reaction can be tracked using fluorescence.

- Other tests are performed using zero value DNA or 5sDNA and the following are reported.

MDAP” and bis-MDAP” at concentrations ranging up to 4.10-’M. A solution was prepared with double distilled water and adjusted to pH 7.6 with hydrochloric acid.

Circular 5cDNA and circular 5sDNA were transferred to plasmids pB, R322 and R322, respectively. and Ml 3mp 19 and purified by conventional electroelution methods.

next d Diazapyrene derivative MDAP" or bis-MDAP" 5sDNA or 5cD NA Tris buffer pH 7.6 Carefully mix appropriate amounts of 5-10-'M and 10-'M di- The concentrations of azapyrene derivatives were given. The container is kept under constant pressure of oxygen using known methods. Then, the tube was irradiated with visible light (250W projector; λ>395nm) for 2 hours. It was kept at 3°C in an ice water bath.

After irradiation, the tubes were kept in the dark and stored at 3°C. Mix the sample from the container with glycerin, Electrophoresis on agarose gel (Sigma type II EEO) (6 hours at 120V and 80mA), agarose (sic) was It was stained with umpromide and photographed. Attached Figure ■ (sic) is a photo of this gel. The bands above correspond to the various tests performed and the concentrations are shown below.

Bands 1 to 20 (sic) corresponding to the label and bands 2 and 3 are diazapine. 5cDNA and 5sDNA irradiated without the presence of ren derivatives; DNA is made publicly available by reaction with the enzymes EcoR1 and S1 nuclease, respectively. obtained by the method of knowledge.

This photo shows circular 5cDNA and 5sDNA in the presence of the diazapyrene of the present invention. Indicates that it will be disconnected.

The diazapyrene derivatives used in the process according to the invention are It has been found that it retains the ability to photocleave DNA even when incorporated into posomes. D It can be used as a vehicle to deliver to NA.

Diazapyrene derivatives containing long-chain hydrocarbon groups are It is particularly suitable for this type of use as it allows good adhesion to the cell.

The invention will now be described in more detail by the following examples.

Example 1: N-methyl=N'-benzyl-2,7- (sic) Lorido DAP 1 g (,0,0049-r-ole) was dissolved in chloroform 2o- Then excess methyl iodide (3m, 0.048 mmol) was added. obtained The reaction mixture was stirred at room temperature for 24 hours.

A yellow solid precipitated at that time. This yellow solid is separated from the reaction medium by filtration, Washing with chloroform and drying in vacuo yields N-methyl-2,7-cyazapyrenyo. 1.61 g (0,0047 mol) of MDAP''I- rate: 96%).

Since some protons have been added, this crude product can be mixed with slightly alkaline water (pHx 7.80; Deprotonation was performed by treatment with water + trace amounts of NH, OH).

The crude product was then recrystallized from boiling methanol to give yellow crystals.

The product had the following physicochemical characteristics: Elemental analysis: Cl5H+rNx I , 0.25H20MW-350,7 Calculated value: C51,37; H3,31 i N 7.99 Measured value: C51,31; H3,19; N 8.05' HNMR spectrum (Solvent DtO/CFs COOD; standard TMPS) 10.05 ppm (s) 10,03 ppm(s) δ. : 8.89ppm (J-9, 19Hz, AB, q) 5,00ppm (s) methyl The product obtained above (1 g; 0.0029 mol) was then dissolved in anhydrous acetonitrile. benzyl bromide ( 3d, 0.025d) was added. The resulting reaction mixture was refluxed for 12 hours with stirring. did.

The resulting yellow product was filtered, washed with chloroform and dried in vacuo.

Yield: 1.15 g (0,0022 mol), or 77%.

The obtained crude product was mixed with water (100 ml and 1i chloride (AgNOs3g) and (obtained from NaCj and properly washed with water) and stirred in the dark for 24 hours. did. The resulting solid was filtered and washed with hot water, then the filtrate was concentrated under reduced pressure and dissolved in acetate. A yellow precipitate formed when diluted with water (90d). Leave this mixture overnight to allow the precipitate to Filter and dry in vacuo.

Yield: 0.81 g (0,00213 mol), 73%.

The obtained product is N-methyl-N-benzyl-2,7-diazapylene dichloride. It has the following physicochemical characteristics: Elemental analysis: CzJ + sNz Cj t MW = 381.3 Calculated value: C6 9,29i H4,72; N 7.35 measurement value: C69,07: H4, 89,N 7.38IHNMR spectrum (solvent: D, Oi product standard TMPS) 9 ．． 9 8 ppm(s) 9.8 8 ppm(s) 8.675ppm (ABq) 7,41 ppm (m-benzyl) 6.2 4 ppm(s) 4,81 ppm (s, methyl) Example 2 Nidiazapyrene derivative, bis-〇AP'' (formula II: R=R'-C H3 ■=　CHz　Cb　Ha　CHz　Ch　Ha　CHz)－4.4-B (bromomethyl)diphenylmethane (0,174g) to 2,7-diazapyrene (0.2g) in anhydrous acetonitrile (15Ta1) under reflux while stirring. was allowed to react for 4 hours.

The resulting yellow precipitate was separated by filtration, washed with chloroform and dried in vacuo.

Yield: 0.34g; 85% The crude product was recrystallized from hot water and analyzed. The product has the formula: and it has the following physical Chemical characteristics: Elemental analysis: C45llioNaBrz H3Hto M W = 816.6 Calculated value: C63, 24H4, 44N 6.86 Measured value: C63,17H4,25N 6.65'HNMR spectrum (Solvent: D, O/CF, C00D standard TMPS) 10,09 ppm (s) 10, OOppm(s) δo = 8.8 1ppm (J-9,37Hz, AB-q) δo = 7 ．． 91ppm (J = 7.61Hz, AB -q) 6.29pp■ (s) 3.98pp■(s) The product (0.3 g) was dissolved in 50 ml of acetonitrile and 0.5 ml of methyl iodide. When suspended in acetonitrile and refluxed for 4 hours with stirring, an acetonitrile-insoluble brown-red solid appears. was gotten. The solid was filtered, washed with chloroform and dried in vacuo.

Yield: 0.37g; 90% The resulting di-salt (yosito/promide) is then suspended in water with silver chloride, Ion exchange was performed by stirring at room temperature for 24 hours. Filter the solid that forms. It was separated by filtration and washed with hot water. Crude product (yellow solid, very soluble in water) was recrystallized several times from methanol or methanol/ether mixtures.

According to another procedure, the product of the above formula and methyl bromide were mixed in a sealed flask at 40-50°C. The methylation step can be carried out by mixing for 2 to 3 days in a container. this is Gives a water soluble yellow product.

The products obtained in each case had the following physicochemical (sic) characteristics: Elemental analysis: CaJxJa Cl, ・9HzOMW=937 Calculated value: C5 7,70B 5.81N 5.98 Measured value: C57,80H5,04N 5.861HNMR spectrum (solvent: D, O; standard TMPS) 10, 15 p pm(s) 10,09 ppsi(s) 8.87ppm (AB - q; J = 9.47Hz) 7.55ppm + (AB −q; J=8.18Hz) 6, 3 9 ppsi(s) 5, 20 ppm(s) methyl 4.1 3 ppm(s) Example 3: Bis-DAP” derivative or -CHz-C4H6-0-ChHa-CHz-) as described in Production Example 2. 2. instead of 4.4'-bis(bromomethyl)diphenylmethane according to the procedure described above. 2'-(4,4'-bis(bromomethyl)diphenyl]propane (sic) and and 4.4'-bis(bromomethyl)diphenyl ether respectively: NMR spectrum of: 1 0.2 8ppm (s); 1 0.2 1ppm (s); 9.0 0 ppm (q-AB); 7.7 ppm (q-AB); 6.5 2 ppm (s ); 5.1 4 ppm (s); 2.3 9 ppm (s): compound, and the following NMR spectrum: 10.27 ppm (s); 10.1 8ppm (s): 8.97ppm (q-A[l); 7.82ppo+ (d) ;7.34ppm (d);6.51pp111(s);5,10ppm Formula with (S):% formula% The compound was obtained.

Example 4: 2. 4-dinitrophenyl-2,7-diazapyrenium silar (synthetic intermediate) 2,4-dinitrophenyl tosylate [tosyl chloride and 2,4-dinitrophe] 10.81 g (2,4·10-s mol) and and 0.49 g (4.10-' mol) of 2.7-diazapyrene were mixed with chloroform. Refluxed in a mixture with setonitrile (50150 v/v).

The yellow precipitate formed was filtered and redissolved in methanol, and the new precipitate formed was washed with excess alcohol. Washed in tel. The resulting yellow precipitate was then dried in vacuo.

Yield 10.96g, 73% Melting point: 252-254℃ (decomposition) The elemental analysis of this product was as follows: Calculated value: C59,78I+ 3.34 N 10.33 Measured value: C59, 82113, 10N 10.4 1' HN M Rx vector (solvent D2o; mark #; TMPS) 10.285 ppm(s) 9,975 ppm(s) 9.4 ppm(s) 9.0ppm (d, d J = 0.05 p) 8, 90ppm (d, J = 0.0 5 p) 8, 675 ppm < d J = 0.05 p) 8, 5 ppm (d J = 0.05 p) 7.575 flpm (d J = 0.04 i p) Example 5 : Diazapyrene derivative of formula I (R=H and R'--(CH,)2-NH,) (7) Operation of manufacturing process C According to the work, one amine group was replaced with a carbobenzoxy group, An amine of the formula H,N-(CH,),NH, protected by was used.

Dissolve 0.44 g of DAP+-DNP Ts-in 40 m of anhydrous methanol (!) to obtain The resulting solution was placed in a dropping funnel.

The amine was prepared separately from the corresponding ammonium salt and dissolved in 2M anhydrous methanol. (1.6·10-' mol).

A few drops of collidine were then added to the reaction mixture with continuous stirring.

DAP''-DNPTs- was then added dropwise over 1 hour. The solution turned from deep red to brown. It changed color. Stirring was continued for another hour, after which the solvent was removed with a Buchner funnel. Ta. Then, 300 d of anhydrous ether was added, resulting in some brown precipitate. Pour the solution The mixture was stirred for an additional 1 hour and 30 minutes and filtered. The obtained solid was placed in a desiccator overnight. Dry (sic).

Yield: 0.41g; 94%.

The obtained compound has the following physicochemical characteristics: Elemental analysis: MW = 537 Calculated value: C64, 53H5, 94N 7.51 Measured value: c 64.58 H5,00N 7.32 NMR (ppm) 9.87 (s); 8.53 (d) ;8.77(d);9.92(s);5,19(t);3.98(t);6 ．． 6 (s); 4.8 (s); 7.1 (s); 7.2 (d); 7, 7 ( d); 2.07 (s) The product obtained is then reacted with a mixture of acetic acid and hydrobromic acid at room temperature. The amine group was deprotected by

The obtained product is phased with 2′″DAP″″-(CHx)z　NH3″　・3Br- and has the following elemental analysis values: Calculated value: C39, 19H3, 28N 8.57 Measured value: 34.11H 3,227,43 The NMR spectrum (DiO, TMPS) of this product is 9. 93ppm, 8.80ppm, 8.86ppm, 10.09ppm , showed peaks at 5.56 ppm and 4.00 ppm. Expected product is salt obtained by reacting the group with the resulting product.

Example 6: N-methyl-N'-hydroxypentyl-2,7-diazabirenium Promid Preparation of (Formula 1: R=CH3, R(sic)--(CHz)s-OH) 5-amino Pentan-1-ol was dissolved in anhydrous methanol and added under argon under anhydrous conditions. and placed into a dry 100 d container.

The mixture was added dropwise to a solution of 5-aminopentan-1-ol at 20° C. while stirring.

A red and then dark red (sic) solution is obtained.

(Continued baking for another 2 hours, then removed methanol in vacuo until almost dry. Next, 250wd of ether was added to the residue to form a brown precipitate, which was filtered. It was separated, taken up in methanol and dried.

CHCj! */CH30H mixture (5%/95%) was used as eluent A single spot was observed on thin layer chromatography on silica gel.

Yield: 72% - melting point 250℃ or higher NMRDz O/TMPS: δ=9.65s δ=3.67t δ-2,32p δ=t, 5opp Example 7: Derivative of formula ■ Dissolve DAP 25e+g (1,22·10-' mol) in cold collidine 5yd. Ta. Add the next step to this solution. 48.2 g (2,44·1o-4 mol) of dosyl acylidine and collidinium 38.4++g (2,44·1o-4 mol) of chloride was added directly.

The entire mixture was heated from 20°C to 100°C for 2 hours with stirring. The solution is The color changes from dark brown to dark yellow and is evaporated by decanting and then heating to 50°C. It dried and solidified.

The obtained product was purified by thin layer chromatography on silica gel (methanol/CH, CI, eluting at 2%798%) gave a single spot.

Yield = 48% The NMR spectrum of the product is as follows: Example 8: Production of diazapyrene derivative (R=R'=R,Y-) of formula (■) 25 mg of DAP is anhydrous. Partially dissolved in acetone (1d), heated to reflux temperature and propane sultone (45m g) was added and the mixture was stirred under reflux overnight.

The obtained product was purified by thin layer chromatography on silica gel (eluent: methanol). /CHs Cj! s (sic) 10%/90%) was given as a single spot. a The setone was removed under reduced pressure.

The NMR spectrum in CF, Co2-D'' gives the following peaks Example 9: Diazapyrene derivatives This derivative is produced by process C above.

(a) IIJs NH+C+5HsJr-111>C+5Hit N (CH 3)! A solution of 1.6 bromide C+*HsJr in 10 d of ethanol was added to the amine (C Hs)z When reacted with a 33% solution of NH in methanol under reflux, the quaternary 42 g of grade amine C+aHsyN (CHs)zl was obtained.

After drying the resulting reaction mixture in vacuo, the amine was purified with CHCl.

and purified by extraction with basic water (containing LiOH). CHC1S 100% was filtered and dried.

1.4 g of the obtained amine was then dibromide Br-(CHz)5-Br4M1 and ethanol 10-, and the reaction mixture was refluxed for 20 hours. Then melt The medium was removed and the residue was dissolved in CHC1,5-. The resulting solution is processed without precipitation. It plunged into the ether until it reached its peak. The resulting pink precipitate is filtered and dried to yield the product. 1.8 g were obtained, which was recrystallized from 40 wl1 of ethyl acetate. this is expected 1.5 g (98% yield) of crystals of bromide was obtained, with the following 'HNMR spectrum. Do: δ (pp + *) = 0.875 (t); 1.25 (s): 1.4 5(t): 2.39(p); 3, OO(p); 3.39(p): 3 ．． 45(t); 3.58(t); 3.80(t), and the mixture is The mixture was heated at 110° C. for 4 hours and 30 minutes. The solvent was removed and the residue was separated using conventional methods. .

The 'IINMR spectrum of the obtained amine is as follows: δ (ppm): 0.78 (L); 1.15 (s); 1.25 (m); 1. G (t); 1.61 (L); 1, 8 (m); 2.55 (t) ; 2.72 (t); 3.00 (s); 3.15 (L); 3.40 (+ a) Production of the compound of the B0 invention According to the method of process C, DAP”-DNA Ts-43,5+og (&05 ・Dissolve amine (10-5 mol) in 15rn1 of methanol, and dissolve the resulting solution into amine (3 9.5 mg; 8.05·to-' mol) in methanol 6d was added dropwise to a solution of . 2 hours for slow addition, then 4 hours and 30 minutes under nitrogen. Solvent was removed.

Ether was then added and a precipitate formed, which was separated and purified by conventional methods.

18.6 mg (2,2·10-' mol) of the product obtained was then treated with methyl iodide ( 3 μf; 2.2·10 −4 mol) in DMF at 70° C. for 15 hours. profit When the resulting product is passed through an ion exchange column (Cj!-), the formula: was obtained, which has a molecular weight of 787 and whose NMR spectrum is as follows: Has characteristics: δ-0,78t δ3.00s δ-10,44gδ=1.15s δ=3. 24s δ-10, 52gδ=1.25 ■ δ=3.40 Ugly δ=1.6t 6=5.05s δ-1, Els δ-5, 45t δ=2.55t δ=9.09AB system δ=2.72t δ=10.26s Example 10: Diazapyrene derivative of formula (1) This derivative was also prepared by process B above. DAP”-DNATs- is produced with the formula: amine.

1.5g of amine in CIl, CN 20ml! Heat to 80℃ in a formal Dehyde 1.1mJ! and cool the solution. Then NaCNBH-0.19g and then gradually add 1 d of glacial acetic acid over 10 minutes. solution then 2 Stir for an hour and then add an additional 2 d of glacial acetic acid over 4 hours. obtained The solution was poured into ether, 3 times with 20 ml of IM-NaOH, and brine. Extract once at 20d. The ether phase was dried over CO3, filtered and the solvent removed. I left. 1.39 g of expected product was obtained.

The following was stirred under reflux for 14 days: CH2 (J! 200 mg of amine (obtained in (a)), dibromide [lr-(CH2)3- [1r 1+nj! and Ki (sic) 1 crystal.

The solvent was then removed to give 274 mg of the expected product (100% yield).

Following the procedure described in Example 9(C), confirmed by the following 'HNMR spectrum: δ (ppm): 0.83 (t); 1.20 (S ); 1.67 (t); 1.92 (p); 2.69 (U); 2.85 (t) ; 3.2Ht); 3.31(t); and 3.57(t) B6 compounds of the invention Manufacturing of The operation of Example 9B was repeated to successively obtain: (1) the following 'HNMR spectra; Spectrum: δ (ppm): 0.94 (t); 1.33 (s); 1.59 (p): 2.29 (s): 2.78 (p); 3. IHs): 3.20 (t); 5. 35(t); 7.14(d) and 7.61(d) (AB series); 8.63 d and 8.76d (AB system); 9, 84 (s) and 10.11 (s) The formula has: %formula%): The formula confirmed by: Example 11: Preparation of macrocyclic compound of formula m (p=8) 2 solutions were prepared.

Solution I: Naphthalene-1,4,5,8-tetracarboxylic anhydride2. '15 g (0,008 mol) in hot DMF<50-60t) 300rnI! dissolve in The liquid was filtered.

Solution n: 1.15 g (0,008 mol) of 1.8-diaminooctane was dissolved in hot DMF ( 50~60℃) 200mf, then add CH,C00H30rnl, Filter the solution.

Put 250 mf of DMF into a three-ring flask and heat it in an oil bath (110-120°C). Ta. Solutions I and ■ were added simultaneously to DMF over 3-4 hours. light brown precipitate occurred during the addition.

After addition of the 2 solutions, the reaction mixture was heated for an additional hour and the solvent was evaporated under reduced pressure.

The resulting solid was suspended in CHC#, 20 Omf and the suspension was refluxed for 15 minutes. and filtered. This operation was repeated three times.

The desired crude product was that which was insoluble in CHCl.

The collected filtrate was washed with 100 ml of 10% aqueous solution of NaC0− and brine IOM. and dried over Na25Oa.

The entire organic phase was passed onto a silica column and eluted with Cl-12cz. contaminated with by-products The expected product migrated in the form of yellowish pink spots.

The product obtained was suspended in 25 ml of CH2Cl2 and the suspension was heated to reflux temperature. , cooled and filtered. The expected product is only slightly soluble in CH,C12. case 0.3 g of virtually pure product was obtained as a CHC13/CH,OH mixture on silica gel ( Obtained by thin layer chromatography using 99/1) as eluent (yield: 10 %).

The resulting compound (imide) can be crystallized from hot nitrobenzene. So It has the following physicochemical characteristics: Elemental analysis: CaaHa. N40. 〚肚＝7 52.8 Calculated value: C70, 20H5, 36N 7.44 Measured value: C70 ,29H5,19N 7.40'HNMR spectrum (solvent S CD C1M> δ (ppm): 8.53 (s, Ar): 4.16 (tr; NCHz- ; J = 6.31 Hz); 1, 65 (m) and 1.56 (s) An CI! , , 0.26 g (0,002 mol) was dissolved in anhydrous THF 15 ad, L i Aj2 Ha 0.218 g (0,0058 mol) was added. mixture Stir and heat to reflux temperature, then add the previously obtained imide (0.3 g; ．． 0004 mol) was added in several portions. The resulting mixture was refluxed for 5 hours, then cooled. did. Decompose excess LiAZHn by adding THF/H,O (1:1), The solid was filtered and suspended in THF15ad, the suspension was refluxed for 10 minutes and filtered. . This extraction was repeated three times.

The collected organic filtrate was evaporated under reduced pressure and the crude product was dried.

The crude product was then dissolved in 120 ml of CHzClz, the solution was filtered and the solvent was evaporated. was allowed to evaporate, thereby removing inorganic compounds. The resulting product was dried in vacuo.

The compound (in the form of a yellowish-rimmed gel) was carried to the next step (NB (aromatization with S).

This compound was purified in the form of its hydrochloride salt by the following procedure: the crude product, i.e. An amine of formula ■ is dissolved in ethanol lO- and 37% aqueous hydrochloric acid to pH 1. , then the solvent was evaporated under reduced pressure. Pour the product (temperate red oily mixture) into a small volume of water. and precipitated by addition of 200 g of acetone. Filter the precipitate and add CHs OH/Hz　When recrystallized from O or CH30H/THF mixture, the amine is 0.15 g was obtained in the form of the hydrochloride (yield 48%).

Elemental analysis: CaaHshNa・4HCI MW”786.8 Calculated value: C64, 22H7, 84N 6.81 Measured value: C64, 50H7, 92N 6.27'HNMR spectrum (free base, CD C11s): δ (ppm ): 7.04 (s, 8H, Ar); 4.00 (s, 16H, NCHz) Ar); 2.47 (Tr, 8) 1. NCHz-(CHz)-(sic)); 1.6 and 1.2 (m, 24H, -aliphatic chain) IHNMR spectrum (hydrochloride, D, O): δ (ppm) near, 50 (s), A r); 3.1 (broad); 1.8 (broad); 1.2 (broad); approximately 4 ．． Solvent peak at 8 ppm.

Mass spectrum m/e = 640 (relative to free base).

Dissolve the previously obtained amine in hot CHZ COOH (approximately 50°C) 801R1, The liquid was filtered and then heated to reflux temperature.

0.93 g (0,0053 mol) of NBS was added. A reddish-brown solid settles out immediately. I was honored.

The mixture was refluxed for 30 minutes and the solvent was evaporated under reduced pressure. The crude product obtained (brown oil mixture) with a large amount of water (approx. Filtered. The filtrate (light orange solution) was mixed with Amberite D.C. It was passed through a 50H column (acid form) and the column was thoroughly washed with water.

The desired product was removed from the column with aqueous hydrochloric acid (10%, then 20% and finally 38%). 0, then dissolved in a small amount of water and precipitated by the addition of acetone. dried, filtered and dried in vacuo.

Yield: 0.11g - i.e. 26% based on imide Elemental analysis: Ca4HasNa CJ 4 MW = 774.7'HNMR7, vector (D, O): δ (ppm): 10.09 (s, Ar); 8.81 (s, Ar); 2 ．． 4 (broad, aliphatic chain); 1.5 (broad, aliphatic chain).

Procedural amendment (formality) Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1. Indication of case PCT/FR8710O1423, person making amendment Relationship to the case: Applicant Name: Company Auris Industrie 5, date of amendment order: 1986 September 27, 2015 6, Subject of amendment (1) Translation of description and scope of claims (2) Corporation citizenship certificate international search report international search report PJniEX To’hr:E INTERNAτl0NAL 5EARC! ! RE:’ORT 0NINTER) JAτ 0NAL APPLICATI ON No, PCτ/l 87100142 (SA 17004)

Claims (1)

[Claims] 1. A nucleic acid is contacted with a solution of a diazapyrene derivative, and the resulting reaction mixture is exposed to visible light. A method of photocleaving a nucleic acid comprising subjecting said diazapyrene derivative to irradiation. The conductor is (1) a compound of formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R and R' are the same or different, hydrogen, or optionally interrupted by one or more heteroatoms such as oxygen, sulfur or nitrogen. A straight or branched, saturated or unsaturated aliphatic hydrocarbon group that can be Therefore, hydrocarbon groups can also be conjugated with hydroxyl, amino or thio groups as appropriate. Aliphatic hydrocarbons which can be substituted by lyl groups or by heterocyclic groups or R and R' are the same and have the formula R1Y-, where R1 is group -(CH2)3- and Y- is a sulfonato ion SO3-, or or expression: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ represents the group of X- is an anion or at least one of the substituents R and R' has the formula R1 If it is Y-, it does not exist, in which case the derivative is an inner salt.] (2) Compound of formula II: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, R, R' and X are as described above, and ■ is one or more heteroatoms. and (or) one or more divalent Saturated or unsaturated, straight, optionally interrupted by aromatic or heterocyclic groups. is a divalent group representing a linear or branched hydrocarbon chain] and (3) Compound of formula III: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) (In the formula, p is an integer from 1 to 15. (preferably equal to 8) Method chosen from. 2. The method according to claim 1, wherein the nucleic acid is DNA or RNA. 3. New products of formula I or II: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R and R' are the same or different, hydrogen, or optionally interrupted by one or more heteroatoms such as oxygen, sulfur or nitrogen. A straight or branched, saturated or unsaturated aliphatic hydrocarbon group that can be Therefore, hydrocarbon groups can also be conjugated with hydroxyl, amino or thio groups as appropriate. Aliphatic hydrocarbons which can be substituted by lyl groups or by heterocyclic groups or R and R' are the same and the formula R1Y- (wherein R 1 is the group -(CH2)3-, Y- is the sulfonato ion SO3-) , or the expression: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ represents the group of X- is an anion or at least one of the substituents R and R' has the formula R1 Not present if Y-, in which case the derivative is an inner salt, provided that R and R' are not hydrogen or methyl groups at the same time] ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, R, R' and X are as described above, and ■ is one or more heteroatoms. and (or) one or more divalent Saturated or unsaturated, straight, optionally interrupted by aromatic or heterocyclic groups. a divalent radical representing a linear or branched hydrocarbon chain] Ren derivative. 4. Corresponds to formula I, where R is a lower alkyl group, such as a methyl group, and R' is thio, hydrogen. An alkyl group substituted by droxyl or an amino group, i.e. an alkyl group of the formula -(CH2 )n-SH, -(CH2)n-OH or -(CH2)n-NH2 (where n is 1 to 10). 5. R and R' are lower alkyl groups such as methyl, and ■ is of the formula: (III)▲There are mathematical formulas, chemical formulas, tables, etc.▼; (IV)▲Mathematical formulas, chemical formulas, tables, etc. There are ▼; or (V) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Select from divalent groups. The derivative according to claim 3, wherein the derivative is disclosed in claim 3. 6. N-methyl-N'-benzyl-2,7-diazabirenium dichloride , the derivative according to claim 3. 7. R=R'=CH3, Tetraque of formula II, where ■=-CH2-C4H4-CH2-C4H4-CH2- The derivative according to claim 3 or 5, which is a loride. 8. Novel intermediate, 2-dinitrophenyl-2,7-diazabirenium (sic) salt. 9. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 2-dinitrophenyl-2,7-diazabirenium (sic) tosylate. 9. The intermediate according to claim 8.