WO2015137064A1

WO2015137064A1 - System for sequential controlled release of functional molecule

Info

Publication number: WO2015137064A1
Application number: PCT/JP2015/054423
Authority: WO
Inventors: 金井　求; 洋平相馬; 佳奈田辺
Original assignee: 国立大学法人東京大学
Priority date: 2014-03-10
Filing date: 2015-02-18
Publication date: 2015-09-17
Also published as: JP2015168669A

Abstract

[Problem] To provide a novel controlled-release carrier material capable of sequentially releasing a functional molecule such as a peptide or a fluorescent molecule. [Solution] A compound having repeating units represented by formula (I). (In formula (I), A is a group having a functional molecule; B is a spacer group; R¹ is a hydrogen atom, a carbonyl group, a C₁-C₅ alkyl which may have a substituent, alkoxy which may have a substituent, or aryl which may have a substituent; R² is a hydrogen atom or a C₁-C₁₀ alkyl which may have a substituent; and n is an integer of 1 or 2.)

Description

Sequential sustained release system of functional molecules

The present invention relates to a carrier characterized in that a specific functional molecule can be gradually and gradually released.

In recent years, a so-called drug delivery system (drug delivery system; DDS) that transports a necessary amount of a low molecular weight compound, peptide, nucleic acid, protein, or other drug to a necessary site in a living tissue or cell has been actively studied. . In particular, control of drug release in vivo (controlled release) is important as a technique for reducing side effects and obtaining a large therapeutic effect. A drug delivery material that can release a drug gradually or can be controlled and released makes it possible to maintain a medicinal effect for a long time, and an effective treatment is possible even with a small amount of drug administration. Moreover, since the concentration of the medicinal component is not excessively increased in the body or blood, side effects can be suppressed.

Until now, as a carrier for such drug delivery, liposomes (for example, Patent Document 1), polyethylene glycol (PEG), polymer micelles, emulsions, nanoparticles, water-soluble polymers, gels of hyaluronic acid and cellulose derivatives, etc. Various materials have been used. However, no technology has been reported so far that allows functional release of different types of functional molecules (drugs, etc.) to be sequentially and gradually released in a programmed release order.

JP 2003-226638 A

Therefore, an object of the present invention is to provide a novel sustained-release carrier material capable of sequentially releasing functional molecules such as peptides and fluorescent molecules.

As a result of intensive studies to solve the above problems, the present inventors use compounds having a serine-prolyl ester bond and a similar structure thereof (hereinafter collectively referred to as “serine-prolyl ester unit”). Thus, in a neutral aqueous solution, N-to-O acyl group transfer reaction with amide bond cleavage proceeds (diketopiperazine is formed), and subsequently functions by being hydrolyzed by the generated ester moiety. It has been found that functional molecules are released, and that different types of functional molecules can be sequentially released by designing the molecules so that such reactions occur continuously (cascade). Thus, the present invention has been completed.

That is, the present invention in one aspect,
(1) A compound having a repeating unit represented by the following formula (I):

[Wherein A is a group having a functional molecule; B is a spacer group; R ¹ is a hydrogen atom, carbonyl, C ₁ -C ₅ alkyl optionally having substituent, substituted An optionally substituted alkoxy, or an optionally substituted aryl; R ² is a hydrogen atom or an optionally substituted C ₁ -C ₁₀ alkyl; and , N is an integer of 1 or 2. ];
(2) The compound according to (1) above, wherein the functional molecule is selected from the group consisting of a fluorophore, a peptide, a nucleic acid, a fragrance compound, a taste-providing compound, and a low molecular weight pharmaceutical compound;
(3) The compound according to (1) above, wherein the functional molecule is a fluorophore serving as a donor of fluorescence resonance energy transfer (FRET);
(4) The compound according to (3) above, further having a quencher serving as an acceptor of fluorescence resonance energy transfer (FRET) in the terminal repeating unit;
(5) The compound according to (1) above, wherein in each repeating unit, the functional molecule is a different type of molecule;
(6) The compound according to (1) above, wherein B is a structure having an amide group;
(7) The compound according to (1) above, wherein R ¹ is carbonyl.
(8) The compound according to (1) above, wherein R ² is a hydrogen atom or methyl;
(9) The compound according to (1), wherein n is 1, and (10) the compound according to (1), wherein R ² is a hydrogen atom and n is 1. .

The present invention, in another preferred embodiment,
(11) A carrier for sequential and sustained release of functional molecules, comprising the compound according to any one of (1) to (10) above; and (12) any one of (1) to (10) above. A drug delivery system comprising the compound is provided.

According to the present invention, by connecting a plurality of repeating units having a serine-prolyl ester unit, an amide cleavage reaction occurs continuously, and different types of functions such as low molecular drug compounds, peptides, perfume compounds, fluorophores, etc. It becomes possible to sequentially release sex molecules in a desired order. Therefore, it can be expected to be applied to the creation of a novel sequential release type drug delivery system and a film preparation.

FIG. 1 shows the reaction mechanism of separation of repeating units and release of functional molecular sites in the compound of the present invention. FIG. 2 is a schematic diagram showing an embodiment of the present invention when the functional molecule is a peptide. FIG. 3 is a schematic diagram showing an embodiment of the present invention when the functional molecule is a fluorescence fluorophoretic energy transfer (FRET) donor fluorophore. FIG. 4 is an HPLC chart and Mass spectrum showing the results of monitoring the reaction of the compound of the present invention (Compound 5) over time. FIG. 5 is a diagram showing the results of the sequential release reaction of the compound of the present invention (compound 4) having a plurality of repeating units. FIG. 6 is a graph showing the change with time of the fluorescence spectrum of the compound of the present invention (compound 5). FIG. 7 is a schematic diagram showing a FRET mechanism by release of a functional molecule in the compound of the present invention (Compound 5).

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. Definition

In the present specification, “amino acid” may be any compound as long as it is a compound having both an amino group and a carboxy group, and includes natural and non-natural compounds. It may be any of neutral amino acids, basic amino acids, or acidic amino acids. In addition to amino acids that themselves function as transmitters such as neurotransmitters, bioactive peptides (in addition to dipeptides, tripeptides, tetrapeptides, An amino acid that is a constituent component of a polypeptide compound such as an oligopeptide or a protein can be used. As the amino acid, an optically active amino acid is preferably used. For example, as the α-amino acid, either D- or L-amino acid may be used, but it may be preferable to select an optically active amino acid that functions in a living body.

In the present specification, the “amino acid residue” means a residue having the same structure as the so-called N-terminal residue, which is the same as the remaining partial structure obtained by removing the hydroxyl group from the carboxy group of the amino acid. However, this does not exclude the case where A is constituted by linking a plurality of amino acid residues. In such a case, the C-terminal amino acid residue has a hydroxyl group from the carboxy group of the amino acid as described above. A partial structure in which a hydrogen atom is removed from the amino group is sufficient, and the intermediate and N-terminal amino acid residues can be linked in the same manner as a normal peptide chain.

In the present specification, “alkyl” may be any of an aliphatic hydrocarbon group composed of linear, branched, cyclic, or a combination thereof. The number of carbon atoms of the alkyl group is not particularly limited. For example, the number of carbon atoms is 1 to 20 (C _1-20 ), the number of carbons is 3 to 15 (C _{3 to 15} ), and the number of carbons is 5 to 10 (C _{5 to 10).} ). When the number of carbons is specified, it means “alkyl” having the number of carbons within the range. For example, C _1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like are included. In the present specification, the alkyl group may have one or more arbitrary substituents. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl part of other substituents containing an alkyl part (for example, an alkoxy group, an arylalkyl group, etc.).

In this specification, when a certain functional group is defined as “may have a substituent”, the type of substituent, the position of substitution, and the number of substituents are not particularly limited. When having the above substituents, they may be the same or different. Examples of the substituent group include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group, a dialkylamino group, and the like.

In the present specification, “alkenyl” refers to a linear or branched hydrocarbon group having at least one carbon-carbon double bond. For example, non-limiting examples include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl 4-pentenyl, 1,3-pentanedienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and 1,4-hexanedienyl). The double bond may be either cis or trans conformation.

In the present specification, “alkynyl” refers to a linear or branched hydrocarbon group having at least one carbon-carbon triple bond. For example, non-limiting examples include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl.

In the present specification, “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic ring system composed of the above alkyl. The cycloalkyl can be unsubstituted or substituted by one or more substituents, which can be the same or different, and non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl Non-limiting examples of polycyclic cycloalkyls include 1-decalinyl, 2-decalinyl, norbornyl, adamantyl and the like. In addition, the cycloalkyl may be a heterocycloalkyl containing one or more hetero atoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as ring-constituting atoms. Any —NH in the heterocycloalkyl ring may be protected, for example as a —N (Boc) group, —N (CBz) group and —N (Tos) group, a nitrogen atom in the ring or The sulfur atom may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. For example, non-limiting examples of monocyclic heterocycloalkyl include diazapanyl, piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, lactam and Examples include lactones.

As used herein, “cycloalkenyl” refers to a monocyclic or polycyclic non-aromatic ring system containing at least one carbon-carbon double bond. The cycloalkenyl may be unsubstituted or substituted by one or more substituents, which may be the same or different, and non-limiting examples of monocyclic cycloalkenyl include cyclopentenyl, cyclohexenyl And cyclohepta-1,3-dienyl, and non-limiting examples of polycyclic cycloalkenyl include norbornylenyl and the like. In addition, the cycloalkyl may be a heterocycloalkenyl which may be a heterocycloalkenyl containing one or more heteroatoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. Atoms may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide.

In the present specification, “aryl” may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring constituent atom Etc.) may be an aromatic heterocyclic ring. In this case, it may be referred to as “heteroaryl” or “heteroaromatic”. Whether aryl is a single ring or a fused ring, it can be attached at all possible positions. Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl Group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group or 1,2,4-oxadiazole-3 -Yl group and the like. Non-limiting examples of fused polycyclic aryl include 1-naphthyl group, 2-naphthyl group, 1-indenyl group, 2-indenyl group, 2,3-dihydroinden-1-yl group, 2,3 -Dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, Isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 , 3-dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazolyl group, fluorenyl group, thioxanthenyl group, etc. And the like. In the present specification, an aryl group may have one or more arbitrary substituents on the ring. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl. When the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing the aryl moiety (for example, an aryloxy group and an arylalkyl group).

In the present specification, “arylalkyl” represents alkyl substituted with the above aryl. The arylalkyl may have one or more arbitrary substituents. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. When the acyl group has two or more substituents, they may be the same or different. Non-limiting examples of arylalkyl include benzyl group, 2-thienylmethyl group, 3-thienylmethyl group, 2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group, 2-furylmethyl group, 3-furylmethyl group, 2-thiazolylmethyl group, 4-thiazolylmethyl group, 5-thiazolylmethyl group, 2-oxazolylmethyl group, 4-oxazolylmethyl group, 5-oxazolylmethyl group, 1-pyrazolylmethyl group 3-pyrazolylmethyl group, 4-pyrazolylmethyl group, 2-pyrazinylmethyl group, 2-pyrimidinylmethyl group, 4-pyrimidinylmethyl group, 5-pyrimidinylmethyl group, 1-pyrrolylmethyl group, 2-pyrrolylmethyl group, 3-pyrrolylmethyl group 1-imidazolylmethyl group, 2-imidazolylmethyl group, 4-imidazolylmethyl 3-pyridazinylmethyl group, 4-pyridazinylmethyl group, 3-isothiazolylmethyl group, 3-isoxazolylmethyl group, 1,2,4-oxadiazol-5-ylmethyl group Or a 1,2,4-oxadiazol-3-ylmethyl group etc. are mentioned.

Similarly, in the present specification, “arylalkenyl” represents alkenyl substituted with aryl.

In the present specification, the “alkoxy group” is a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include a saturated alkoxy group that is linear, branched, cyclic, or a combination thereof. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, cyclopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, cyclobutoxy group, cyclopropylmethoxy group, n- Pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, cyclopentylmethyloxy group, etc. are preferable Take as an example.

In the present specification, the “aryloxy group” is a group to which the aryl group is bonded through an oxygen atom. Examples of the aryloxy group include phenoxy group, 2-thienyloxy group, 3-thienyloxy group, 2-pyridyloxy group, 3-pyridyloxy group, 4-pyridyloxy group, 2-furyloxy group, and 3-furyl. Oxy group, 2-thiazolyloxy group, 4-thiazolyloxy group, 5-thiazolyloxy group, 2-oxazolyloxy group, 4-oxazolyloxy group, 5-oxazolyloxy group, 1-pyrazolyloxy group, 3-pyrazolyloxy group Group, 4-pyrazolyloxy group, 2-pyrazinyloxy group, 2-pyrimidinyloxy group, 4-pyrimidinyloxy group, 5-pyrimidinyloxy group, 1-pyrrolyloxy group, 2-pyrrolyloxy group, 3-pyrrolyloxy group, 1-imidazolyloxy group , 2-imidazolyloxy group, 4-imidazolyloxy group, -Pyridazinyloxy group, 4-pyridazinyloxy group, 3-isothiazolyloxy group, 3-isoxazolyloxy group, 1,2,4-oxadiazol-5-yloxy group, or Examples include a 1,2,4-oxadiazol-3-yloxy group.

In the present specification, “alkylamino” and “arylamino” mean an amino group in which a hydrogen atom of —NH ₂ group is substituted with 1 or 2 of the above alkyl or aryl. For example, methylamino, dimethylamino, ethylamino, diethylamino, ethylmethylamino, benzylamino and the like can be mentioned. Similarly, “alkylthio” and “arylthio” mean a group in which the hydrogen atom of the —SH group is substituted with the above alkyl or aryl. For example, methylthio, ethylthio, benzylthio and the like can be mentioned.

As used herein, “amide” includes both RNR′CO— (when R = alkyl, alkaminocarbonyl-) and RCONR′- (where R = alkyl, alkylcarbonylamino-).

As used herein, “ester” includes both ROCO— (in the case of R = alkyl, alkoxycarbonyl-) and RCOO— (in the case of R = alkyl, alkylcarbonyloxy-).

As used herein, “acyl” is a group obtained by removing a hydroxyl group from an organic acid such as carboxylic acid, and is represented by RCO— (in the case of R = methyl, acetyl).

As used herein, the term “ring structure”, when formed by a combination of two substituents, means a heterocyclic or carbocyclic group, such group being saturated, unsaturated, or aromatic. Can be. Accordingly, it includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperidinyl, imidazolyl, pyrrolidinyl, pyridyl and the like. In the present specification, a substituent can form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art will recognize a specific substitution, such as bonding to hydrogen. Can be understood. Therefore, when it is described that specific substituents together form a ring structure, those skilled in the art can understand that the ring structure can be formed by an ordinary chemical reaction and can be easily generated. Both such ring structures and their process of formation are within the purview of those skilled in the art.

2. Compound Having Serine-Prolyl Ester Unit In one aspect, the compound having sequential sustained release property of the present invention has a repeating unit represented by the following formula (I).

In the formula, A is a group having a functional molecule; B is a spacer group; R ¹ is a hydrogen atom, a carbonyl, an optionally substituted C ₁ -C ₅ alkyl, a substituent. R ² is a hydrogen atom or a C ₁ -C ₁₀ alkyl optionally having a substituent; and an optionally substituted alkoxy or an optionally substituted aryl; and n is an integer of 1 or 2.

As shown in the formula (I), the repeating unit is linked to each other at the terminal part of the acyl group on the nitrogen-containing cycloalkyl (5-membered ring or 6-membered ring) and the terminal part of the alkoxy group having R ² . The compound of the present invention has two or more repeating units linked at the terminal portion, for example, 2 to 50, preferably 3 to 20, more preferably 3 to 10 repeating units.

By having such a structure, the compound of the present invention is cleaved from the remainder of the compound by the diketopiperazine formation accompanying the N-to-O acyl transfer reaction in a neutral aqueous solution, and then The functional molecule can be sequentially released in a desired order by releasing a portion containing A having a functional molecule by hydrolysis of and repeating this. The reaction mechanism is shown in FIG.

Specifically, as shown in FIG. 1, separation of repeating units and release of functional molecular sites proceed by the following reaction (note that functional molecules are not specifically shown in the figure, A functional molecule is linked to the amino group at the left end of the “serine-prolyl ester unit”).
Step 1): The hydroxyl group at the site corresponding to serine in the structure of the repeating unit attacks the carbonyl at the adjacent amide site, resulting in an N-to-O acyl transfer reaction;
Step 2): The amino group produced by the transfer reaction and the ester group of nitrogen-containing cycloalkyl (5-membered ring or 6-membered ring) react to form a diketopiperazine ring, thereby linking with other repeating units. The resulting ester bond is separated from the compound of the invention;
Step 3): The separated repeating unit is hydrolyzed and the ester bond is cleaved, whereby the site having the functional molecule is released from the site having the diketopiperazine ring;
Step 4): The acyl group transfer reaction of Step 1) occurs again in the remainder of the compound of the present invention in which one repeating unit is cleaved by the above Step 2).

By designing the molecules so that these reactions occur continuously (cascade), a plurality of functional molecules of the same or different types can be sequentially released in a predetermined order. The compound of the present invention is characterized in that the pH is stable in the acidic region, and the above reaction proceeds under conditions near neutrality, thereby enabling sequential release. In that sense, it can be said to be a sequential sustained release carrier triggered by pH. The pH which becomes the said trigger is 5 or more, for example, Preferably it is 6.5 or more, More preferably, it is 7.4 or more. Therefore, it can function in an in vivo environment.

Examples of the functional molecule in A in formula (I) include, but are not limited to, compounds exhibiting taste such as fluorophores, peptides, nucleic acids, fragrance compounds, sweeteners, and low molecular weight pharmaceutical compounds. Any molecule that is useful to be sequentially released under neutral aqueous solution conditions can be used. The functional molecules can be the same or different types in each repeating unit, preferably different types.

As a preferred non-limiting example, the functional molecule in A is a peptide, and the embodiment of the present invention in that case is shown in FIG. Each repeat unit is linked to peptide 1 to 4. In accordance with the above steps 1) to 4) (in the figure, one cycle of these steps is shown as “first reaction” to “fourth reaction”), and separated from the compound of the present invention in order from peptide 1. Released. These peptides 1 to 4 can be the same or different types. In addition, here, only the release of peptide 4 by the fourth reaction is illustrated, but it is not necessarily limited to four, and it is designed to release more peptides by linking further repeating units. Those skilled in the art will understand that this is possible.

As another preferred example, the functional molecule in A is a fluorophore, and in particular, can be a fluorophore serving as a donor of fluorescence resonance energy transfer (FRET). This is suitable when the compound of the present invention is used for applications such as a fluorescent probe using the FRET mechanism. An embodiment of the present invention in that case is shown in FIG. By connecting a plurality of repeating units, it becomes possible to sequentially release the fluorophores, as in the case of FIG. In this case, in the repeating unit serving as the terminal of a plurality of repeating unit groups to be linked, for example, fluorescence resonance energy transfer (to the terminal on the acyl group side on the nitrogen-containing cycloalkyl (5-membered ring or 6-membered ring)) It is preferable to have a quencher that serves as an acceptor for (FRET). Thus, as shown in FIG. 3, the donor fluorophore is quenched by the quencher while it is linked to the compound of the present invention and does not emit fluorescence, but is released when separated from the compound of the present invention. Can fluoresce. By adjusting the substituent X or Y in the donor fluorophore in FIG. 3, fluorophores having fluorescence characteristics such as different emission wavelengths can be used.

For example, examples of such a fluorophore include those containing fluorene, coumarin, fluorescein, anthracene, and aminobenzyl, and examples of the quencher include groups containing azobenzene and dinitrophenol.

In the formula (I), B is a spacer group for linking A containing a functional molecule and a serine-prolyl ester unit. Accordingly, B is not particularly limited as long as it can be linked to the amide group on the serine-prolyl ester unit side, and examples thereof include a structure containing an alkyl group, an alkoxy group, an aryl group, and an acyl group. Preferably, B is a structure containing an amide group.

In formula (I), R ¹ has a hydrogen atom, a carbonyl, an optionally substituted C ₁ -C ₅ alkyl, an optionally substituted alkoxy, or a substituent. Can be any aryl, preferably carbonyl. R ² may be a hydrogen atom or C ₁ -C ₁₀ alkyl, preferably a hydrogen atom or methyl.

In formula (I), it can be an integer of 1 or 2, ie, when n is 1, the nitrogen-containing cycloalkyl in the serine-prolyl ester unit is a pyrrolidine ring, and when n is 2, piperidine It becomes a ring. Preferably n is 1. The nitrogen-containing cycloalkyl can be substituted with any substituent.

The compound represented by the above formula (I) may exist as a salt. Examples of such salts include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt. Examples thereof include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, paratoluenesulfonate, citrate, and oxalate. Examples of amino acid salts include glycine salts. However, the salt of the compound of the present invention is not limited to these.

The compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

The compound represented by the formula (I) may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.

The compound of the present invention may be used as a composition by blending additives usually used in the preparation of reagents as required. For example, additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for use in a physiological environment, and the amount of these can be appropriately selected by those skilled in the art. is there. These compositions can be provided as a composition in an appropriate form such as a powder-form mixture, a lyophilized product, a granule, a tablet, or a liquid.

In the examples of the present specification, production methods for representative compounds included in the compounds of the present invention represented by the formula (I) are specifically shown. Any compound included in formula (I) can be easily produced by referring to, and appropriately selecting starting materials and reagents as necessary. In addition, reaction conditions such as reaction temperature and reaction time in the production method will be described in detail as typical examples in the examples described later, but are not necessarily limited thereto, and those skilled in the art will not be limited thereto. If there are, they can be appropriately selected based on general knowledge in organic synthesis.

The compound represented by the formula (I) can be used as a carrier for sequential sustained release of functional molecules triggered by pH, for example, as a drug delivery system. Thereby, the same or different types of functional molecules such as drugs can be sequentially released in the desired order using pH as a trigger.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Example 1]
[Synthesis]
1. Synthesis of Z-Ser (H-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Pro) -NH ₂ (Compound 1)

NovaPEG Rink-amide resin (0.1 mmol) vs. Z-Ser-OH (59 mg, 0.25 mmol), N, N'-diisopropylcarbodiimide (DIC, 38 μL, 0.25 mmol), 1-hydroxybenzotriazole (HOBt, 38 mg, 0.25 mmol) and N, N-dimethylformamide (DMF, 2 mL) were added, and the mixture was shaken at room temperature for 2 hours. Subsequently, Fmoc-Pro-OH (505 mg, 1.5 mmol) was added, and the mixture was shaken in CH ₂ Cl _{2 in the} presence of DIC (231 μL, 1.5 mmol) and DMAP (1.2 mg, 0.01 mmol) for 24 hours. After removal of the N ^a -Fmoc group of Pro residues which was shaken by hand for 1 minute by adding 20% piperidine / DMF (v / v), the reaction solution was filtered, 10 minutes added freshly 20% piperidine / DMF This was done by shaking. Next, Fmoc-Gly-Ser (tBu) -OH (55 mg, 0.12 mmol) was added in DMF in the presence of DIC (18 μL, 0.12 mmol) and 1-hydroxybenzotriazole (HOBt, 18 mg, 0.12 mmol). Condensation was carried out by reacting for hours. Thereafter, removal of the Fmoc group and introduction of the Fmoc amino acid were sequentially repeated to construct a protected peptide resin. The tBu group was adopted as the side chain protecting group of Asp and Glu. The resin was washed with MeOH, dried with a vacuum pump (380 mg), stirred in triisopropylsilane-H ₂ O-trifluoroacetic acid (2.5: 2.5: 95) for 180 minutes, filtered, filtered and concentrated with Et ₂ O. A white solid was obtained via reprecipitation by (38 mg). A part of the crude peptide (8 mg) was purified by reverse phase HPLC (0.1% aqueous TFA-CH ₃ CN system), and lyophilized to obtain white amorphous form 1 (3.5 mg).
Yield: 13%; MALDI-MS (TOF): M _calc : 1287.3; M + Na _found : 1306.2; Retention time in reverse phase ODS column = 21.7 min (column: YMC-Pack ODS-AM (4.6 x 150 mm) , 0-100% CH ₃ CN in 0.1% aqueous TFA)

2. Synthesis of Z-Ser (H-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser- D-Pro) -NH ₂ (Compound 2)

Synthesis was performed in the same manner as for compound 1. Yield: 16%; ESI-MS (TOF): M _calc : 644.30; [M + 2H] ²⁺ _found : 644.29; Retention time in reversed-phase ODS column = 20.2 min (column: YMC-Pack ODS-AM (4.6 x 150 mm), 0-100% CH ₃ CN in 0.1% aqueous TFA)

3. Synthesis of Z-Thr (H-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Pro) -NH ₂ (compound 3)

Synthesis was performed in the same manner as for compound 1. Yield: 10%; ESI-MS (TOF): M _calc : 651.31; [M + 2H] ²⁺ _found : 651.29; Retention time in reversed-phase ODS column = 20.4 min (column: YMC-Pack ODS-AM (4.6 x 150 mm), 0-100% CH ₃ CN in 0.1% aqueous TFA)

4). Synthesis of Z-Ser (Z-Ala-Ser (Fmoc-Gly-Ser-Pro) -Pro)-(Arg) ₃ -NH ₂ (compound 4)

After constructing Z-Ser (H-Pro)-(Arg) ₃ -NH-resin by the same method as for Compound 1, Z-Ala-Ser is condensed and Z-Ser (Z-Ala-Ser-Pro)- (Arg) ₃ -NH-resin was synthesized. Using the same method as for compound 1, Fmoc-Pro-OH is condensed to the side chain hydroxy group of Ser (constructing the second ester bond), Fmoc group is removed, Fmoc-Gly-Ser (tBu) Compound 4 was synthesized by -OH condensation, deresinification / deprotection, and HPLC purification.
Yield: 2%; MALDI-MS (TOF): M _calc : 1559.7; M + H _found : 1560.9; Retention time in reverse phase ODS column = 23.3 min (column: YMC-Pack ODS-AM (4.6 x 150 mm) , 0-100% CH ₃ CN in 0.1% aqueous TFA)

5. Synthesis of peptide having fluorophore (compound 5)

For Dnp NovaTag resin (0.050 mmol), Fmoc-Arg (Pbf) -OH (82.9 mg, 0.125 mmol), 1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5- b] Add pyridinium 3-oxid hexafluorophosphate (HATU, 47.5 mg, 0.125 mmol), N, N-diisopropylethylamine (DIEA, 54.4 μL, 0.125 mmol) and N, N-dimethylformamide (DMF, 2 mL) for 1 hour at room temperature Shake. After removal of the N ^a -Fmoc group of Arg residues shaking manually for one minute by addition of 20% piperidine / DMF (v / v), the reaction solution was filtered, 10 minutes added freshly 20% piperidine / DMF This was done by shaking. After washing with DMF, add Fmoc-Arg (Pbf) -OH (82.9 mg, 0.125 mmol) and shake for 1 hour in DMF in the presence of HATU (47.5 mg, 0.125 mmol) and DIEA (54.4 μL, 0.125 mmol). did. Similarly, removal of Fmoc group and introduction of Fmoc-Arg (Pbf) -OH, removal of Fmoc group and introduction of Z-Ser-OH were performed. Subsequently, Fmoc-Pro-OH (253 mg, 0.75 mmol) was added, and the mixture was shaken in CH ₂ Cl ₂ for 20 hours in the presence of DIC (116 μL, 0.75 mmol) and DMAP (0.6 mg, 0.005 mmol). After removing the Fmoc group, react Fmoc-Gly-Ser-OH (48 mg, 0.125 mmol) in DMF for 1 hour in the presence of HATU (47.5 mg, 0.125 mmol) and DIEA (54.4 μL, 0.125 mmol). Condensed by Similarly, the Fmoc group was removed and 7-Methoxycoumarin-4-acetic acid (Mca-OH, 17.6 mg, 0.125 μmol) was introduced. The resin was washed with MeOH, dried with a vacuum pump (192 mg), then stirred in triisopropylsilane-H ₂ O-trifluoroacetic acid (2.5: 2.5: 95) for 60 minutes, filtered with resin, concentrated in filtrate, Et ₂ O A white solid was obtained through reprecipitation. A portion of the crude peptide was purified by reverse phase HPLC (0.1% aqueous TFA-CH ₃ CN system), and lyophilized to obtain white amorphous form 5 (0.5 mg).
Yield: 3%; ESI-MS (TOF): M _calc : 458.53; [M + 3H] ³⁺ _found : 458.53; Retention time on reverse phase ODS column = 19.9 min (column: YMC-Pack ODS-AM (4.6 x 150 mm), 0-100% CH ₃ CN in 0.1% aqueous TFA)

[Example 2]
[Amide cleavage reaction]
Compound 5 (0.17 mg, 0.1 μmol) obtained in Example 1 was dissolved in 0.1 M phosphate buffer (pH 7.4) / DMSO (95: 5, v / v, 100 μL) in a thermostatic chamber at 37 ° C. The amide cleavage reaction was monitored over time. The obtained HPLC chart and the result of Mass spectrum are shown in FIG. From this result, it was confirmed that the release reaction of the site having 7-methoxycoumarin progressed in almost all the compounds 5 after 24 hours.

Under similar solution conditions, amide cleavage was observed for compound 4 with fluorin in the first repeat unit and phenyl in the second repeat unit. As a result, as shown in FIG. 5, it was confirmed that the sequential release reaction in two stages proceeded.

[Example 3]
[Fluorescence spectrum measurement]
An aqueous solution obtained by diluting 3 μL of the reaction solution of Compound 5 to 3 mL with water was excited at 325 nm with a fluorometer to obtain a fluorescence spectrum. The results are shown in FIG. From FIG. 6, the fluorescence intensity peaking at 380 nm increased with time. This is because the fluorescence was suppressed by the quencher when the 7-methoxycoumarin moiety before the reaction was present in the molecule due to the FRET mechanism. Indicates that the recovery has occurred (FIG. 7).

Claims

A compound having a repeating unit represented by the following formula (I):

[Wherein A is a group having a functional molecule; B is a spacer group; R 1 is a hydrogen atom, carbonyl, C 1 -C 5 alkyl optionally having substituent, substituted An optionally substituted alkoxy, or an optionally substituted aryl; R 2 is a hydrogen atom or an optionally substituted C 1 -C 10 alkyl; and , N is an integer of 1 or 2. ]
The compound according to claim 1, wherein the functional molecule is selected from the group consisting of a fluorophore, a peptide, a nucleic acid, a fragrance, a compound exhibiting a taste, and a low molecular weight pharmaceutical compound.
The compound according to claim 1, wherein the functional molecule is a fluorophore serving as a donor of fluorescence resonance energy transfer (FRET).
The compound according to claim 3, further comprising a quencher that serves as an acceptor of fluorescence resonance energy transfer (FRET) in the terminal repeating unit.
The compound according to claim 1, wherein in each repeating unit, the functional molecule is a different type of molecule.
The compound according to claim 1, wherein B has a structure having an amide group.
The compound of claim 1, wherein R 1 is carbonyl.
The compound according to claim 1, wherein R 2 is a hydrogen atom or methyl.
The compound of claim 1, wherein n is 1.
The compound according to claim 1, wherein R 2 is a hydrogen atom and n is 1.
A carrier for sequential and sustained release of functional molecules, comprising the compound according to any one of claims 1 to 10.
A drug delivery system comprising the compound according to any one of claims 1 to 10.