WO2023149487A1

WO2023149487A1 - Method for producing pyrrole-imidazole polyamide

Info

Publication number: WO2023149487A1
Application number: PCT/JP2023/003274
Authority: WO
Inventors: 哲宏根本; 誠也中島; 篤志金田
Original assignee: 国立大学法人千葉大学
Priority date: 2022-02-02
Filing date: 2023-02-01
Publication date: 2023-08-10

Abstract

The present invention pertains to a method for producing a pyrrole-imidazole polyamide, the method comprising the following steps A1 and A2. Step A1: A reduction step for reducing compound 1 to obtain compound 2; Step A2: An extension step of reacting compound 3 with compound 2 to obtain compound 4 [Compounds 1-4 are as defined in the specification.]

Description

Method for producing pyrrole-imidazole polyamide

The present invention relates to a method for producing pyrrole-imidazole polyamide.

Pyrrole-imidazole polyamide (hereinafter also referred to as “PIP”) is a direct structure in which N-methylpyrrole units (hereinafter also referred to as “Py”) and N-methylimidazole units (hereinafter also referred to as “Im”) are linked via amide bonds. It is a chain-like low molecule. The molecular design of PIP is derived from the DNA-binding ability of Distamycin A, an anticancer natural product having a pyrroleamide trimer structure.

PIP enters the minor grooves of DNA, as expected from its molecular design, with Im-Py for GC base pairs, Py-Im for CG base pairs, and AT base pairs. Or Py-Py can bind to the TA base pair. Since PIP can specifically recognize and bind to the base sequence of DNA and strongly suppress the transcriptional activity of target genes, it is expected to be applied to epigenetic drug discovery to suppress the expression of target genes.

PIP synthesis methods include a solid phase synthesis method using a condensing agent developed by Dervan et al. have proposed a cross-coupling method using a metal catalyst (Patent Document 1).
In the solid-phase synthesis method, a compound supported on a polymer is used to extend the peptide chain, so the atomic efficiency is very poor, and large-scale synthesis is difficult. In addition, since many impurities are synthesized by side reactions, the purity of the crude purified product is low, and column chromatography is required for purification, which is time-consuming.
Liquid-phase synthesis methods using trichloroacetyl derivatives can only synthesize dimers, and trichloroacetyl derivatives having imidazole are unstable and difficult to handle. Furthermore, when the reaction is actually carried out, the solubility of the substrate is low, and large-scale synthesis is difficult.
The cross-coupling method does not require expensive condensing agents or polymers, but requires multistep processes for substrate synthesis.

Patent Literature 2 discloses sequence-selective pyrrole and imidazole polyamide metal complexes and methods for preparing the same.
Non-Patent Document 5 discloses monocyclic and multicyclic pyrrole containing N-formamides and imidazole-2-carboxylic acids for use in the preparation of polyamide molecules.

WO2017/169503 U.S. Patent Application Publication No. 2007/0265240

The present invention relates to the provision of a method for producing a pyrrole-imidazole polyamide that can produce a pyrrole-imidazole polyamide in a simple manner.

The present invention includes aspects [1] to [11] below.
[1] A method for producing a pyrrole-imidazole polyamide, comprising the following steps A1 and A2.
Step A1: Reduction step of reducing compound 1 represented by the following formula (1) to obtain compound 2 represented by the following formula (2) Step A2: compound 3 represented by the following formula (3); Elongation step of reacting with compound 2 to obtain compound 4 represented by the following formula (4)

[In formula (1), X ⁰ each independently represents N or CH, R ^a is a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms, a aryl group, an aralkyl group having 7 to 25 carbon atoms, a nitrile group, —COOR ^p (R ^p represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 20 carbon atoms.), or —CONR ^q R ^r (R ^q and R ^r independently represent a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 20 carbon atoms), and n is 0 or 1 Represents an integer greater than or equal to . ]

[In formula (2), X ⁰ , R ^a and n are the same as above. ]

[In the formula (3), each X ¹ independently represents N or CH, and Y represents a halogen atom. m represents an integer of 0 or 1 or more. ]

[In formula (4), X ⁰ , X ¹ , R ^a , n and m are the same as above. ]
[2] The production method according to [1] above, wherein the step A2 is a flow reaction.
[3] The production method according to [1] or [2] above, wherein the step A1 is a flow reaction.
[4] The production method according to any one of [1] to [3] above, wherein the step A2 is performed in a microreactor.
[5] The production method according to any one of [1] to [4] above, wherein the step A1 is performed in the presence of a palladium carbon catalyst.
[6] The production method according to any one of [1] to [5] above, wherein the step A1 is performed in an aprotic polar solvent.
[7] The production method according to [6] above, wherein the aprotic polar solvent is N,N-dimethylformamide.
[8] The production method according to any one of [1] to [7] above, wherein m is 0.
[9] The production method according to any one of [1] to [8] above, wherein the compound 4 is continuously extended by using it as the compound 1 in the next step A1.
[10] A method for producing a pyrrole-imidazole polyamide according to any one of [1] to [9] above, further comprising the following steps B1 and B2.
Step B1: A reduction step to obtain a compound 5 represented by the following formula (5) by reducing the compound 4 obtained by the production method according to any one of [1] to [9] above Step B2: The following formula Elongation step of reacting compound 6 represented by (6) with compound 5 to obtain compound 7 represented by the following formula (7)

[In Formula (5), X ⁰ , X ¹ , R ^a , n and m are the same as above. ]

[In formula (6), X ² each independently represents N or CH, Y represents a halogen atom, R ^b represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 12 carbon atoms, a It represents an alkoxy group of 1 to 12 or an aryl of 6 to 10 carbon atoms. p represents an integer of 0 or 1 or more; ]

[In formula (7), X ⁰ , X ¹ , X ² , R ^a , R ^b , n, m, and p are the same as above. ]
[11] The production method according to [10] above, wherein each of m and p is 0.

According to the present invention, a method for producing a pyrrole-imidazole polyamide is provided by which a pyrrole-imidazole polyamide can be produced in a simple manner.

Schematic diagram of synthesis of PIP using a flow microreactor.

The method for producing a pyrrole-imidazole polyamide of the present invention includes the following steps A1 and A2.
Step A1: Reduction step of reducing compound 1 represented by the following formula (1) to obtain compound 2 represented by the following formula (2) Step A2: compound 3 represented by the following formula (3); Elongation step of reacting with compound 2 to obtain compound 4 represented by the following formula (4)

[In formula (2), X ⁰ , R ^a and n are the same as above. ]

[In formula (4), X ⁰ , X ¹ , R ^a , n and m are the same as above. ]

<Step A1>
In step A1, compound 1 represented by the above formula (1) is reduced to obtain compound 2 represented by the above formula (2).
[Compound 1]
Compound 1, which is the starting compound of Step A1, is a monomer of N-methylpyrrole or N-methylimidazole having a nitro group, or a pyrrole-imidazole polyamide having a nitro group, and is represented by the above formula (1).
Each X ⁰ in the above formula (1) independently represents N or CH. When X ⁰ represents N, the nitrogen-containing 5-membered ring containing X ⁰ is a pyrrole ring, and when X ⁰ represents CH, the nitrogen-containing 5-membered ring containing X ⁰ is an imidazole ring.
n represents an integer of 0 or 1 or more. n is preferably an integer of 0 to 3, more preferably 0 or 1, still more preferably 0, from the viewpoint of reactivity.
R ^a is a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms, an aryl group having 6 to 20 carbon atoms, an aralkyl group having 7 to 25 carbon atoms, a nitrile group, —COOR ^p (R ^p represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 20 carbon atoms.), or —CONR ^q R ^r (R ^q and R ^r are independently a hydrogen atom, a carbon represents an alkyl group having 1 to 12 carbon atoms or an aryl group having 6 to 20 carbon atoms.).
The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, n-hexyl group and isohexyl group. The alkyl group may have 1 or more and 3 or less substituents.
The alkoxy group is preferably a linear or branched alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy. group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group, n-hexyloxy group, isohexyloxy group and the like. Among them, an alkoxy group having 1 or more and 3 or less carbon atoms is preferable.
The aryl group is preferably an aryl group having 6 or more and 10 or less carbon atoms. Specific examples include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl group may have 1 or more and 3 or less substituents.
A halogen atom, an alkyl group, an alkoxy group, etc. are mentioned as said substituent.
In the aralkyl group (arylalkyl group), the alkyl group is the above alkyl group, and the aryl group is the above aryl group. A benzyl group is mentioned as an aralkyl group.
Compound 1 can be obtained as a commercial product or from a commercial product by a known organic synthesis reaction.
R ^a is, among others, preferably a hydrogen atom or —COOR ^p , more preferably —COOR ^p .

[Compound 2]
The compound 2 obtained in step A1 is an N-methylpyrrole or N-methylimidazole monomer having an amino group, or a pyrrole-imidazole polyamide having an amino group, and is represented by the above formula (2).
X ⁰ , R ^a and n in formula (2) are the same as defined in formula (1).

[Reduction reaction]
In step A1, the nitro group of compound 1 is reduced to lead to an amino group to obtain compound 2.
A reduction reaction of a nitro group can be performed by a known method.
For example, catalytic reduction in which hydrogen gas is reacted in the presence of a palladium carbon catalyst, nickel catalyst, etc.; Bechamp reduction in which an acid such as ammonium chloride, hydrochloric acid, acetic acid, etc. hydride reduction, etc., in which metal oxides are reacted.
Among them, the reduction reaction is preferably carried out in the presence of a palladium carbon catalyst.
From the viewpoint of reaction progress, the reduction reaction is preferably carried out in a solvent. Examples of solvents include aqueous solvents such as water; alcohol solvents such as methanol and ethanol; ether solvents such as diethyl ether and tetrahydrofuran; Aprotic polar solvents such as dimethylsulfoxide (DMSO), hexamethylphosphoric triamide (HMPA), N-methylpyrrolidone (NMP), acetonitrile, acetone, and the like. A solvent can be used individually by 1 type or in mixture of 2 or more types.
The alcoholic solvent is preferably methanol. The aprotic polar solvent is preferably N,N-dimethylformamide (DMF), tetrahydrofuran (THF), or a mixed solvent of DMF and THF.
In one preferred embodiment of the present invention, the reduction reaction is carried out in an alcoholic solvent, for example, from the viewpoint of being widely used in reduction reactions carried out in the presence of a palladium carbon catalyst.
In another preferred embodiment, the pyrrole-imidazole polyamide is highly soluble, and the reduction reaction is performed in an aprotic polar solvent from the viewpoint of further improving the reaction rate, reaction progress, reproducibility of the reaction, and the like. . The solubility of pyrrole-imidazole polyamide (polymer) is lower than that of N-methylpyrrole or N-methylimidazole monomers, which may reduce the reactivity and reaction reproducibility of step A1. , the reduction reaction of the pyrrole-imidazole polyamide (polymer) having a nitro group is preferably carried out in an aprotic polar solvent. Thus, in one aspect, when n is an integer greater than or equal to 1 (eg, 1, 2 or 3), the reduction reaction is performed in a polar aprotic solvent.
The solvent may be either a single solvent or a mixture of two or more solvents.

The amount of the catalyst to be used is preferably 0.1 equivalent to excess amount, more preferably 0.2 equivalent to 5 equivalents, still more preferably 0.5 to 2 equivalents, relative to compound 1. The reaction temperature is preferably 0° C. to 100° C., and the reaction time is preferably 0.5 to 24 hours.
Step A1 may be either a batch method using a flask or the like or a flow reaction using a flow microreactor or the like. In one preferred embodiment, it is a flow reaction.

<Step A2>
Step A2 is an elongation step of reacting compound 3 represented by the above formula (3) with compound 2 to obtain compound 4 represented by the above formula (4).
[Compound 3]
The compound 3 to be reacted with the compound 2 in step A2 is an acid halide compound represented by the above formula (3).
Each X ¹ in the above formula (3) independently represents N or CH.
Y represents a halogen atom. A halogen atom is a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, or the like, and preferably a chlorine atom from the viewpoint of availability.
m represents an integer of 0 or 1 or more. From the viewpoint of reactivity, m is preferably an integer of 0 to 3, more preferably 0 or 1, still more preferably 0.

When m is 0, compound 3 is represented by the following formula (3').

[In Formula (3′), X ¹ and Y are the same as above. ]

Compound 3 can be obtained as a commercial product or from a commercial product by a known organic synthesis reaction.
A specific example is a method of reacting a carboxylic acid compound with a halogenating agent such as thionyl chloride or oxalyl chloride.
From the viewpoint of reaction progress, the reaction with the halogenating agent is preferably carried out in a solvent. The solvent is preferably an ether solvent such as diethyl ether or tetrahydrofuran. The solvent may be either a single solvent or a mixture of two or more solvents.
The amount of the halogenating agent to be used is preferably 0.1 equivalent to excess, more preferably 0.2 to 5 equivalents, still more preferably 0.5 to 2 equivalents, relative to the carboxylic acid compound.
The reaction temperature is preferably 0° C. to 100° C., and the reaction time is preferably 0.5 to 24 hours.

[Compound 4]
Compound 4 obtained in step A2 is a pyrrole-imidazole polyamide and is represented by the above formula (4).
X ⁰ , X ¹ , R ^a , n and m in formula (4) are the same as defined in formulas (1), (2) and (3) above.

When m is 0, compound 4 is represented by the following formula (4').

[In Formula (4′), X ⁰ , X ¹ , R ^a and n are the same as above. ]

[Elongation reaction]
In step A2, compound 4 is obtained by reacting the acid halide group of compound 3 with the amino group of compound 2 to form an amide bond.
Preferably 0.1 to 10 equivalents, more preferably 0.5 to 2 equivalents of compound 2 can be reacted with respect to compound 3.
The elongation reaction can be carried out in the presence of preferably 0.1 to excess mol, more preferably 0.5 to 10 mol of base relative to 1 mol of compound 3, if necessary. Examples of the base include organic bases such as pyridine, triethylamine and N,N-diisopropylethylamine (DIPEA).
From the viewpoint of reaction progress, the reaction is preferably carried out in a solvent. Examples of solvents include organic solvents such as ethyl acetate, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,4-dioxane, tetrahydrofuran (THF), acetonitrile and dichloromethane. The solvent may be either a single solvent or a mixture of two or more solvents.

Step A2 may be either a batch method using a flask or the like or a flow reaction using a flow microreactor or the like. In one preferred embodiment step A2 is a flow reaction, more preferably the reaction is carried out in a microreactor.
In particular, it is more preferable that both steps A1 and A2 are flow reactions from the viewpoint of easy mass synthesis.

<Flow reaction>
A flow reaction, which is a preferred embodiment of the present invention, is a reaction in which multiple components are mixed in a channel.
The inner diameter of the channel in the flow reaction is not particularly limited. The inner diameter of the channel is preferably 0.05-2 mm, more preferably 0.1-1 mm. The cross-sectional shape of the channel is, for example, circular or rectangular.
The channel length in the flow reaction is preferably 0.5 m or more and 10 m or less, more preferably 0.9 m or more and 8 m or less.
The mode of mixing in the flow reaction may be either laminar mixing or turbulent mixing.
Flow reactions are preferably carried out in microreactors. The microreactor is not particularly limited as long as it has a diameter on the order of micrometers, that is, 1000 μm or less, and examples of the microreactor include T-shaped or Y-shaped micromixers.
It is preferable to use a pump such as a plunger pump, a gear pump, a rotary pump, a diaphragm pump, a syringe pump, or the like to transfer the liquid in the flow reaction.
Preferably, the reaction temperature in the microreactor is controlled.
The flow rate of each component in the flow reaction is controlled so that the reaction time (residence time) is the above reaction time, and each component is independently preferably 0.01 mL/min to 100 mL/min, more preferably is 0.05 mL/min to 50 mL/min.
The concentration of each component in the flow reaction is preferably 0.005M to 0.5M, more preferably 0.01M to 0.1M for each component independently.

<Continuous reaction>
In the method for producing a pyrrole-imidazole polyamide of the present invention, the compound 4 is used as the compound 1 in the next step A1 to continuously extend the pyrrole-imidazole polyamide to obtain a desired sequence and desired length of pyrrole • It is possible to produce imidazole polyamides.

<Step B1 and Step B2>
The method for producing the pyrrole-imidazole polyamide of the present invention can further include the following steps B1 and B2.
Step B1: Reduction step of reducing compound 4 obtained by the above production method to obtain compound 5 represented by the following formula (5) Step B2: Compound 6 represented by the following formula (6) and compound 5 above Elongation step of obtaining compound 7 represented by the following formula (7) by reacting

[In Formula (5), X ⁰ , X ¹ , R ^a , n and m are the same as above. ]

[In formula (7), X ⁰ , X ¹ , X ² , R ^a , R ^b , n, m, and p are the same as above. ]

By including the step B1 and the step B2 in the method for producing a pyrrole-imidazole polyamide of the present invention, a pyrrole-imidazole polyamide having no terminal nitro group or amino group can be produced.

[Step B1]
Step B1 can be carried out in the same manner as Step A1 above, except that Compound 4 obtained by the above production method is used in place of Compound 1.

When m is 0, compound 5 is represented by the following formula (5').

[In Formula (5′), X ⁰ , X ¹ , R ^a and n are the same as above. ]

[Step B2]
Step B2 can be performed in the same manner as Step A2 above, except that Compound 5 obtained in Step B1 above and Compound 6 above are used instead of Compound 3.

[Compound 6]
The compound 6 to be reacted with the compound 5 in step B2 is an acid halide compound represented by the above formula (6). Compound 6, unlike compound 3, does not have a nitro group.
Each X ² in the above formula (6) independently represents N or CH.
Y represents a halogen atom. A halogen atom is a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, or the like, and preferably a chlorine atom from the viewpoint of availability.
R ^b represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms, or an aryl group having 6 to 20 carbon atoms.
Halogen atoms include fluorine, chlorine, bromine and iodine atoms.
Examples of the alkyl group, the alkoxy group, and the aryl group include the same modes as those for ^Ra described above.
p represents an integer of 0 or 1 or more; p is preferably an integer of 0 to 3, more preferably 0 or 1, still more preferably 0, from the viewpoint of reactivity.

When p is 0, compound 6 is represented by the following formula (6').

[In Formula (6'), X ¹ and Y are the same as above. ]

Similar to compound 3, compound 6 can be obtained as a commercial product or from a commercial product by a known organic synthesis reaction.

In each step of the production method of the present invention, the reaction can be stopped by means such as changing the temperature or pH, adding water, a solvent, a saturated aqueous solution of sodium hydrogen carbonate, or the like, if necessary. In addition, the target compound can be obtained through isolation steps such as filtration, concentration, and extraction, if necessary. By obtaining the target compound, the solvent used in the next step can be exchanged. Obtainment of the desired compound in each step can be confirmed by known means such as ¹ H-NMR measurement, ¹³ C-NMR measurement and mass spectrometry.

[Compound 7]
Compound 7 obtained in step B2 is a pyrrole-imidazole polyamide and is represented by the above formula (7).
X ⁰ , X ¹ , X ² , R ^a , n, m, and p in formula (7) are the same as defined in formulas (5) and (6) above.

When m is 0 and p is 0, compound 7 is represented by the following formula (7').

[In Formula (7′), X ⁰ , X ¹ , X ² , R ^a , R ^b and n are the same as above. ]

Thus, a pyrrole-imidazole polyamide is produced.
According to the production method of the present invention, a pyrrole-imidazole polyamide in which pyrrole rings and imidazole rings are arranged in a desired order can be produced freely and simply.
The compounds used in the present invention can be easily synthesized from commercially available compounds, and are economical because they do not use condensing agents that tend to be expensive.
In addition, the production method of the present invention does not require a condensing agent or a support (polymer used for solid-phase synthesis), and can be carried out without purification such as column chromatography, so that waste is generated during production. very little quantity.
Furthermore, when a flow microreactor is used, the reaction efficiency is high and the formation of by-products such as branched structures can be prevented. The production method using a flow microreactor has a very short reaction time compared to the batch production method, and can easily produce a large amount of pyrrole-imidazole polyamide.

In the present examples, various measurements were performed by the following methods.
・Infrared absorption spectrum (IR)
Measurements were made with a Fourier transform infrared spectrometer equipped with ATR (total reflection measurement).
・Nuclear Magnetic Resonance (NMR)
A 400 MHz measuring device was used. For chemical shifts in DMSO-d ₆ , the solvent signal (DMSO) (=2.50 ppm) was used as an internal standard in ¹ H-NMR. In ¹³ C-NMR, the solvent signal (DMSO (=39.52 ppm)) was used as an internal standard for chemical shifts.
- Mass spectrometry Measurement was performed by an electrospray ionization method (ESI-TOF).

Synthesis Example 1: Dimer Synthesis by Batch Method

Methanol (MeOH) was added to each of commercially available methyl 4-nitro-1-methylpyrrole-2-carboxylate (Py) and methyl 4-nitro-1-methylimidazole-2-carboxylate (Im) monomers. A suspension solution of 0.1 M was prepared and 10% palladium on carbon catalyst (Pd/C) was added under an argon atmosphere. After decompressing the inside of the flask using an aspirator, the inside of the flask was replaced with hydrogen gas filled in a rubber balloon. After reacting at room temperature (RT) for one day, hydrogen gas was distilled off using an aspirator, and the inside of the flask was replaced with argon. The reaction solution was filtered through celite using methanol to remove Pd/C. The resulting solution was concentrated under reduced pressure using a rotary evaporator to obtain monomer (iii). Yield was 100% (quant).

Pyrrole or imidazole carboxylic acid derivative 1 was dissolved in tetrahydrofuran (THF) to make a 0.035 M solution, and under an argon atmosphere, in the presence of 1.1 equivalents of N,N-dimethylformamide (DMF) with respect to the carboxylic acid derivative (i). , and 1.1 equivalents of oxalyl chloride were acted to lead to the monomer (ii).
The pyrrole or imidazole derivative monomer (iii) was dissolved in tetrahydrofuran (THF) to make a 0.058 M solution, and under an argon atmosphere, 3.0 equivalents of N,N-diisopropylethylamine (DIPEA) was added to the monomer (iii). ) was added at room temperature.
A condensation reaction in which the solution (solution B) of the monomer (iii) is added dropwise to the solution (solution A) of the monomer (ii) is carried out by: For (iii), the pyrrole derivative and the imidazole derivative were used in four combinations to synthesize the dimer (iv). A saturated aqueous sodium hydrogencarbonate solution was added to stop the reaction, and the solution was subjected to liquid separation and extraction treatment with ethyl acetate, and the organic solvent was distilled off from the organic layer under reduced pressure using a rotary evaporator. The residue was suspended in methanol and filtered to obtain an insoluble matter containing dimer (iv) as a yellow solid. Yields are shown in Table 1.

In all four combinations, the target dimers (iv-a) to (iv-d) were obtained with a yield of 53-75% by performing the reaction for 24 hours. This reaction was very fast, and dimers 4a to 4d could be produced even in 30 seconds. The content of the PyPy dimer (iv-a) in the insoluble matter was 99% by mass or more.

(iv-a): PyPy dimer

yellow solid;
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 3.74 (s, 3H), 3.85 (s, 3H), 3.95 (s, 3H), 6.8 (s, 1H), 7 .46 (s, 1H), 7.55 (s, 1H), 8.19 (s, 1H), 10.28 (s, 1H);
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 36.2, 37.4, 51.0, 107.6, 108.3, 118.8, 120.8, 122.1, 126.1, 128 .3, 133.8, 156.9, 160.7;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₃ H ₁₄ N ₄ NaO ₅ ⁺ 329.0862; Found 329.0856.

(iv-b): PyIm dimer

yellow solid;
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 3.83 (s, 3H), 3.95 (s, 3H), 3.97 (s, 3H), 7.66 (s, 1H), 7 .79 (s, 1H), 7.79 (s, 1H), 8.15 (s, 1H);
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 35.2, 37.4, 48.4, 51.6, 108.6, 115.5, 125.2, 128.4, 133.9, 137 .0, 157.2, 158.7;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₂ H ₁₃ N ₅ NaO ₅ ⁺ 330.0814; Found 330.0804.

(iv-c): ImPy dimer

yellow solid;
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 3.74 (s, 3H), 3.85 (s, 3H), 4.05 (s, 3H), 7.08 (d, J=2. 4 Hz, 1 H), 7.50 (d, J = 2.4 Hz, 1 H), 8.54 (s, 1 H), 10.7 (s, 1 H);
¹³ C-NMR (150 MHz, DMSO-d ₆ ) δ 36.0, 36.2, 50.7, 109.1, 118.9, 121.2, 121.5, 126.3, 137.5, 144 .2, 154.6, 160.5;
IR(ATR) v 2360, 2338, 1695, 1542, 1452, 1384, 1309, 1255, 1203, 1125 ^{cm -1} ;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₂ H ₁₃ N ₅ NaO ₅ ⁺ 330.0814; Found 330.0812.

(iv-d): ImIm dimer

yellow solid;
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 3.83 (s, 3H), 3.96 (s, 3H), 4.04 (s, 3H), 7.68 (s, 1H), 8 .57 (s, 1H), 10.62 (s, 1H);
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 35.4, 36.1, 51.6, 116.1, 116.2, 126.4, 131.5, 135.7, 137.0, 155 .1,158.6;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₁ H ₁₂ N ₆ NaO ₅ ⁺ 331.0767; Found 331.0760.

Synthesis Example 2: Trimer Synthesis by Batch Method

The dimer (iv) obtained in Synthesis Example 1 was reduced using 10% Pd/C in a hydrogen atmosphere in the same manner as in Synthesis Example 1 to obtain dimer (v). Yields are shown in Table 2. In the table, "quant" indicates that the reaction proceeded quantitatively.
The reduction of dimers (iv-a)-(iv-c) to dimers (va)-(vc) proceeded in high yields.

Trimer (vii) was obtained in the same manner as in Synthesis Example 1 above, except that monomer (iii) was replaced with dimer (v). Yields are shown in Table 3.
Compounds (vii-a) to (vii-d) were obtained in yields of 12 to 71% in a reaction time as short as 30 seconds.

(vii-a): PyPyPy trimer

yellow solid;
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 3.73 (s, 3H), 3.83 (s, 3H), 3.85 (s, 3H), 3.90 (s, 3H), 6 .93 (s, 1H), 7.11 (s, 1H), 7.29 (s, 1H), 7.48 (s, 1H), 7.68 (s, 1H), 8.20 (s, 1H), 10.08(s, 1H), 10.48(s, 1H);
^13C -NMR (150MHz, DMSO- _d6 ) .71, 121.43, 122.76, 122.85, 126.24, 127.97, 133.76, 156.89, 158.35, 160.70;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₉ H ₂₀ N ₆ NaO ₆ ⁺ 451.1342; Found 451.1337.

(vii-a): PyPyIm trimer

yellow solid;
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 3.83 (s, 3H), 3.88 (s, 3H), 3.94 (s, 3H), 3.97 (s, 3H), 7 .12(s, 1H), 7.35(s, 1H), 7.60(s, 1H), 7.65(s, 1H), 8.13(s, 1H), 10.20(s, 1H), 10.61 (s, 1H);
¹³ C-NMR (150 MHz, DMSO-d ₆ ) δ 35.13, 36.06, 37.14, 51.42, 105.53, 107.47, 115.33, 119.62, 121.28, 121 .98, 126.17, 127.82, 130.67, 133.71, 137.63, 156.88, 158.49, 158.72;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₈ H ₁₉ N ₇ NaO ₆ ⁺ 452.1295; Found 452.1305.

(vii-a): PyImPy trimer

yellow solid;
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 3.76 (s, 3H), 3.86 (s, 3H), 3.98 (s, 3H), 3.99 (s, 3H), 7 .01 (s, 1H), 7.49 (s, 1H), 7.55 (s, 1H), 7.75 (s, 1H), 8.15 (s, 1H), 9.93 (s, 1H), 10.65 (s, 1H);
¹³ C-NMR (150 MHz, DMSO-d ₆ ) δ 34.7, 36.0, 37.3, 50.7, 108.4, 108.6, 115.2, 118.8, 120.7, 121 .7, 125.3, 128.3, 133.9, 134.2, 135.3, 155.7, 157.4, 160.4;
IR(ATR) v 2361, 1665, 1542, 1441, 1409, 1312, 1216, 1118, 899, 784 ^{cm -1} ;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₁₈ H ₁₉ N ₇ NaO ₆ ⁺ 452.1295; Found 452.1288.

(vii-a): ImPyPy trimer

yellow solid;
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 3.75 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 4.07 (s, 3H), 6 .93 (d, J = 2.4Hz, 1H), 7.24 (s, 1H), 7.30 (s, 1H), 7.44 (s, 1H), 8.57 (s, 1H), 9.89 (s, 1H), 10.8 (s, 1H);
¹³ C-NMR (150 MHz, DMSO-d ₆ ) δ 35.9, 35.9, 36.3, 50.7, 105.1, 108.5, 118.5, 119.1, 120.7, 120 .9, 122.7, 122.9, 126.3, 137.7, 144.2, 154.6, 158.3, 160.6;
IR(ATR) v 1688, 1548, 1441, 1365, 1308, 1205, 1112, 1060, 1005, 828 ^{cm -1} ;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcdfor C ₁₈ H ₁₉ N ₇ NaO ₆ ⁺ 452.1295; Found 452.1307.

Synthesis Example 3: Dimer Synthesis Using a Flow Microreactor FIG. 1 shows a schematic diagram of synthesis using a flow microreactor.
A T-shaped micromixer having an inner diameter of 0.25 mm in the mixing section was used, and a tube made of PTFE (polytetrafluoroethylene) having an inner diameter of 0.8 mm was used.
The concentration of solution A containing monomer (ii) obtained in the same manner as in Synthesis Example 1 was 0.035 M, the flow rate was 2.0 mL/min, and the monomer (iii) obtained in the same manner as in Synthesis Example 1. The concentration of solution B containing was set to 0.058 M, and the flow rate was set to 1.2 mL/min. After passing through a tube of a predetermined length, saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture to stop the reaction, followed by separation and extraction with ethyl acetate, and the organic solvent was distilled off from the organic layer under reduced pressure using a rotary evaporator. left. The residue was suspended in methanol and filtered to obtain an insoluble matter containing dimer 4 as a yellow solid.

3-1: Synthesis of PyPy dimer (iv-a) As monomer (ii) and monomer (iii), compounds that are pyrrole derivatives are used, respectively, and tubes after solution A and solution B are merged PyPy dimer (iv-a) was synthesized by setting the length of and the reaction temperature at various values. Moreover, the content of the PyPy dimer (iv-a) in the insoluble matter obtained by synthesis under the conditions of a tube length of 410 cm and a reaction temperature of 0° C. was 99% by mass or more. Table 4 shows the yield under each condition.

3-2: Synthesis of PyIm dimer (iv-b) Using a pyrrole derivative as monomer (ii) and an imidazole derivative as monomer (iii), PyIm dimer is prepared in the same manner as in 3-1 above. (iv-b) was synthesized. Table 5 shows the yield under each condition.

3-3: Synthesis of ImPy dimer (iv-c) Using imidazole derivative as monomer (ii) and pyrrole derivative as monomer (iii), ImPy dimer is prepared in the same manner as in 3-1 above. Synthesis of body (iv-c) was carried out. Table 6 shows the yield under each condition.

3-4: Synthesis of ImIm dimer (iv-d) Using imidazole derivative compounds as monomer (ii) and monomer (iii), respectively, in the same manner as in 3-1 above, ImIm dimer Synthesis of body (iv-d) was carried out. Table 7 shows the yield under each condition.

Synthesis Example 4: Trimer Synthesis Using Flow Microreactor 4-1: Synthesis 1 of PyPyPy Trimer (vii-a)
Using the flow microreactor used in Synthesis Example 3 above, using a pyrrole derivative as the monomer (ii) and a PyPy dimer (va) as the monomer (iii), after liquid A and liquid B are merged Synthesis of PyPyPy trimers (vii-a) was carried out by setting various tube lengths and reaction temperatures. The content of the PyPyPy trimer (vii-a) in the insoluble matter obtained by synthesis under the conditions of a tube length of 410 cm and a reaction temperature of 0° C. was 99% by mass or more. Table 8 shows the yield under each condition.

4-2: Synthesis 2 of PyPyPy trimer (vii-a)
The PyPy dimer (iv-a) was treated with oxalyl chloride in the presence of DMF, leading to a PyPy dimer (va).
PyPyPy trimer ( vii-a) was synthesized. Table 9 shows the yield under each condition.

4-3: Synthesis of PyPyIm(vii-b) trimer Using pyrrole derivative as monomer (ii) and PyIm dimer (vb) as monomer (iii), in the same manner as in 4-1 above. , the synthesis of the PyPyIm trimer (vii-b). Table 10 shows the yield under each condition.

4-4: Synthesis of PyImPy trimer (vii-c) Using pyrrole derivative and ImPy dimer (vc) as monomer (ii), PyImPy trimer (vii -c) was synthesized. Table 11 shows the yield under each condition.

4-5: Synthesis of ImPyPy trimer (vii-d) Using an imidazole derivative as monomer (ii) and a PyPy dimer (va) as monomer (iii), ImPyPy trimer (vii-d ) was synthesized. Table 12 shows the yield under each condition.

Synthesis Example 5: Tetramer synthesis using a flow microreactor 5-1: Synthesis of PyPyPyPy tetramer (ix-a) The PyPyPy trimer (vii-a) obtained in 4-1 above was added to 10% Reduction with Pd/C gave the PyPyPy trimer. Reduction proceeded quantitatively.
Using the flow microreactor used in Synthesis Example 3, using the pyrrole derivative as the monomer (ii) and the PyPyPy trimer obtained above as the monomer (iii), PyPyPyPy tetramer (ix-a) was synthesized. Table 13 shows the yield under each condition.

(ix-a): PyPyPyPy tetramer

yellow solid;
1H-NMR (600 MHz, DMSO-d ₆ ) δ 3.75 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 3. 98 (s, 3H), 6.93 (d, J = 1.8Hz, 1H), 7.05 (d, J = 2.4Hz, 1H), 7.07 (d, J = 1.8Hz, 1H) ), 7.22 (d, J = 1.8 Hz, 1 H), 7.25 (d, J = 1.2 Hz, 1 H), 7.44 (d, J = 1.2 Hz, 1 H), 7.58 (d, J = 2.4 Hz, 1H), 8.14 (d, J = 1.2 Hz, 1H), 9.83 (s, 1H), 9.88 (s, 1H), 10.19 (s , 1H);
IR(ATR) v 3468, 1649, 1438, 1310, 1110, 889, 815, 780, 751, 734 ^{cm -1} ;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₂₅ H ₂₆ N ₈ NaO ₇ ⁺ 573.1822; Found 573.1823.

5-2: Synthesis of PyPyImPy tetramer (ix-b) The PyImPy trimer (vii-c) obtained in 4-4 above was reduced using 10% Pd/C in a hydrogen atmosphere to obtain a PyImPy trimer. got a body Reduction proceeded quantitatively.
PyPyImPy tetramer (ix-b) was synthesized using a pyrrole derivative and the PyImPy trimer obtained above as monomer (ii). Synthesis was carried out in the same manner as in 5-1 above, except that the solvent of liquid B was changed from THF to DMF.
The target tetramer was successfully obtained with a yield of 39% by reaction at room temperature for 57.5 seconds.

(ix-b): PyPyImPy tetramer

yellow solid;
¹ H-NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 3.97 (s, 3H), 3. 99 (s, 3H), 7.01 (d, J = 2.0Hz, 1H), 7.10 (d, J = 2.0Hz, 1H), 7.34 (d, J = 1.6Hz, 1H ), 7.50 (d, J = 2.0 Hz, 1 H), 7.53 (s, 1 H), 7.60 (d, J = 2.0 Hz, 1 H), 8.14 (s, 1 H), 10.03 (s, 1H), 10.15 (s, 1H), 10.23 (s, 1H);
¹³ C-NMR (150 MHz, DMSO-d6) δ 34.7, 36.0, 36.0, 37.2, 50.7, 105.3, 107.5, 108.6, 114.9, 118. 8, 119.4, 120.8, 121.4, 121.9, 122.1, 126.2, 127.9, 133.7, 133.9, 135.9;
IR(ATR) v 3461, 2945, 2350, 2338, 1440, 1365, 900, 743, 664, 641 cm ^-1 ;
HRMS (ESI-TOF) m/z: [M+Na] ⁺ Calcd for C ₂₄ H ₂₅ N ₉ NaO ₇ ⁺ 574.1775; Found 574.1797.

Synthesis Example 6: Tetramer synthesis by flow method 6-1: Synthesis of PyPyPyPy tetramer (ix-a) The PyPy dimer (iv-a) obtained in 3-1 above was treated with 10% Pd /C to give the PyPy dimer (va). Reduction proceeded quantitatively.
The PyPy dimer (iv-a) obtained in 3-1 above was suspended in a 1:1 mixed solution of H ₂ O and EtOH, and then 5 equivalents of sodium hydroxide was added under ice-cooling. Stir in the bath for 1 hour. Thereafter, the mixture was ice-cooled again, and 10 equivalents of a 1 mol/l hydrochloric acid aqueous solution was added. The resulting yellow precipitate was filtered using a Kiriyama funnel to obtain PyPy-CO ₂ H(xa) with a yield of 50%.

Using the flow microreactor used in Synthesis Example 3 above, using PyPy—CO ₂ H (xa) as monomer (ii) and the PyPy dimer obtained above as monomer (iii), PyPyPyPy tetramer (ix-a) was synthesized. Synthesis was carried out in the same manner as in 5-1 above except that the concentration of solution A was changed to 0.018M and the concentration of solution B was changed to 0.03M. By reaction at room temperature for 57.5 seconds, PyPyPyPy tetramer (ix-a) was successfully obtained with a yield of 64%.

Test example: Investigation of solvent for hydrogenation reduction reaction

In Test Examples 1 to 4 below, differences in hydrogenation reduction reaction efficiency due to differences in solvents (DMF or methanol) were evaluated.

(Test Example 1) Hydrogenation reduction of PyPyPyPy tetramer in DMF

0.385 g (0.7 mmol) of the PyPyPyPy tetramer (ix-a) (hereinafter also referred to as NO ₂ Py ₄ OMe) obtained in Synthesis Example 5-1 above and 0.039 g of a palladium carbon catalyst were added to 7 mL of N, In N-dimethylformamide (DMF), under a slightly pressurized hydrogen atmosphere with a rubber balloon, a hydrogenation reduction reaction is carried out by stirring at room temperature for 41 hours to obtain NH ₂ Py ₄ OMe (xi-a). rice field.
As the palladium carbon catalyst (Pd/C), palladium-activated carbon was used, and the amount was 10 parts by mass with respect to 100 parts by mass of NO ₂ Py ₄ OMe (ix-a). The concentration of _NO2Py4OMe (ix-a) in DMF was 0.1M _. NO ₂ Py ₄ OMe was completely dissolved in DMF from the beginning of the reaction, and the reduction reaction proceeded well in the solution.
After the reaction, disappearance of the raw material was confirmed by thin layer chromatography (TLC). Ethyl acetate was used as the developing solvent for TLC.

The inside of the flask was replaced with argon to remove hydrogen, 7 mL of tetrahydrofuran (THF) was added to the reaction solution, and the mixture was stirred for 15 minutes. The reaction solution was passed through a column packed with 4.9 g of amino-modified silica gel to adsorb the product, and 49 mL of ethyl acetate was passed through to elute the product. After that, the eluate was concentrated under reduced pressure. The amount of amino-modified silica gel used was 0.7 g per 1 mL of DMF used in the reaction. The amount of ethyl acetate used was 7 times the amount of DMF used in the reaction.
0.115 g (0.68 mmol) of 1,1,2,2-tetrachloroethane was added to the concentrated residue, diluted with heavy chloroform to obtain a homogeneous solution, and ¹ H-NMR was measured. In the NMR chart, the yield of NH ₂ Py ₄ OMe (xi-a) is calculated from the integral ratio of an arbitrary peak of NH ₂ Py ₄ OMe (xi-a) and the peak of 1,1,2,2-tetrachloroethane. The calculated yield was 96%.

(Test Example 2) Hydrogenation reduction of PyPyPyPy tetramer in methanol A hydrogenation reduction reaction was performed in the same manner as in Test Example 1 except that the solvent was changed from DMF to methanol, and the reaction was performed for 41 hours in the same manner as in Test Example 1. reacted. NO ₂ Py ₄ OMe did not completely dissolve in methanol and the reaction proceeded in a suspended state. The suspended state was not resolved even at the end of the reaction.
Column purification and concentration were performed in the same manner as in Test Example 1.
0.115 g (0.68 mmol) of 1,1,2,2-tetrachloroethane was added to the concentrated residue, diluted with heavy chloroform to obtain a homogeneous solution, and ¹ H-NMR was measured. In the NMR chart, the yield of NH ₂ Py ₄ OMe (xi-a) is calculated from the integral ratio of an arbitrary peak of NH ₂ Py ₄ OMe (xi-a) and the peak of 1,1,2,2-tetrachloroethane. The calculated yield was 11%.

(Test Example 3) Hydrogenation reduction of PyPyPy trimer in DMF

A reduction reaction and quantification of the product were carried out in the same manner as in Test Example 1 except that the PyPyPyPy tetramer (ix-a) was replaced with the PyPyPy trimer (vii-a) obtained in Synthesis Example 4-1 above. gone. NO ₂ Py ₃ OMe was completely dissolved in DMF from the start of the reaction, and the reduction reaction proceeded well in the solution.
The reaction was completed in 15 hours _and the yield of _NH2Py3OMe (vii-b) was 100%.

(Test Example 4) Hydrogenation reduction of PyPyPy trimer in methanol A hydrogenation reduction reaction was performed in the same manner as in Test Example 3 except that the solvent was changed from DMF to methanol, and the reaction was performed for 15 hours in the same manner as in Test Example 3. reacted. NO ₂ Py ₃ OMe did not completely dissolve in methanol and the reaction proceeded in suspension. The suspended state was not resolved even at the end of the reaction.
The yield of _NH2Py3OMe (vii-b) was 13% _.

The results of Test Examples 1 to 4 are shown in Table 14 below.

When the reaction solvent is methanol, the PIP substrate containing the nitro group has low solubility in methanol. going to start. In this case, at the beginning of the reaction, only the substrate dissolved in the solvent is reduced, and as the reaction progresses, the undissolved substrate gradually dissolves, so that the reduction reaction proceeds. In the case of a monomer, the reaction proceeded without problems after stirring for one day (Synthesis Example 1), but the longer the PIP length, the lower the solubility in the MeOH solvent. Decreased.
On the other hand, when DMF is used as a solvent, the PIP substrate containing a nitro group is highly soluble, and the reduction reaction can be initiated with the entire amount of the substrate dissolved. Admitted. The effect was particularly remarkable when multimers, especially PIP containing a nitro group longer than a trimer, were used as substrates. Moreover, even when a mixed solvent of THF and DMF (1:1) is used as a solvent, the solubility of the PIP substrate (trimer) containing a nitro group is improved, and the reduction reaction efficiency is improved. is confirmed.
These results suggest that the use of an aprotic polar solvent such as DMF or THF in the hydrogenation-reduction reaction of PIP containing a nitro group improves the reactivity of the reduction reaction.

Claims

A method for producing a pyrrole-imidazole polyamide, comprising the following steps A1 and A2.
Step A1: Reduction step of reducing compound 1 represented by the following formula (1) to obtain compound 2 represented by the following formula (2) Step A2: compound 3 represented by the following formula (3); Elongation step of reacting with compound 2 to obtain compound 4 represented by the following formula (4)

[In formula (1), X 0 each independently represents N or CH, R a is a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms, a aryl group, an aralkyl group having 7 to 25 carbon atoms, a nitrile group, —COOR p (R p represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 20 carbon atoms.), or —CONR q R r (R q and R r independently represent a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 20 carbon atoms), and n is 0 or 1 Represents an integer greater than or equal to . ]

[In formula (2), X 0 , R a and n are the same as above. ]

[In the formula (3), each X 1 independently represents N or CH, and Y represents a halogen atom. m represents an integer of 0 or 1 or more. ]

[In formula (4), X 0 , X 1 , R a , n and m are the same as above. ]
The manufacturing method according to claim 1, wherein the step A2 is a flow reaction.
The manufacturing method according to claim 1, wherein the step A1 is a flow reaction.
The manufacturing method according to claim 1, wherein the step A2 is performed in a microreactor.
The production method according to claim 1, wherein the step A1 is performed in the presence of a palladium carbon catalyst.
The production method according to claim 1, wherein the step A1 is performed in an aprotic polar solvent.
The production method according to claim 6, wherein the aprotic polar solvent is N,N-dimethylformamide.
The manufacturing method according to claim 1, wherein said m is 0.
The production method according to claim 1, wherein the compound 4 is continuously extended by using it as the compound 1 in the next step A1.
2. The method for producing a pyrrole-imidazole polyamide according to claim 1, further comprising the following steps B1 and B2.
Step B1: Reduction step of reducing compound 4 obtained by the production method according to claim 1 to obtain compound 5 represented by the following formula (5) Step B2: Compound 6 represented by the following formula (6) , an elongation step of obtaining a compound 7 represented by the following formula (7) by reacting with the compound 5

[In Formula (5), X 0 , X 1 , R a , n and m are the same as above. ]

[In formula (6), X 2 each independently represents N or CH, Y represents a halogen atom, R b represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 12 carbon atoms, a It represents an alkoxy group of 1 to 12 or an aryl of 6 to 10 carbon atoms. p represents an integer of 0 or 1 or more; ]

[In formula (7), X 0 , X 1 , X 2 , R a , R b , n, m, and p are the same as above. ]
The manufacturing method according to claim 10, wherein said m and p are each 0.