CN115991678A

CN115991678A - Diketopiperazine compound, preparation method and application thereof

Info

Publication number: CN115991678A
Application number: CN202111221371.9A
Authority: CN
Inventors: 刘娜; 赵晓东; 王浩
Original assignee: Shanghai Huiyong Pharmaceutical Research Co ltd
Current assignee: Shanghai Huiyong Pharmaceutical Research Co ltd
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2023-04-21

Abstract

The invention discloses a diketopiperazine compound, a preparation method and application thereof. The invention provides a diketopiperazine compound shown in a formula I or pharmaceutically acceptable salt thereof, which can be used as self-assembled drug-carrying microspheres to realize effective drug delivery.

Description

Diketopiperazine compound, preparation method and application thereof

Technical Field

The invention relates to a diketopiperazine compound, a preparation method and application thereof.

Background

Drug delivery systems have been an important direction of formulation research, traditional methods being affected by the digestive tract pH environment, various enzymes, etc., resulting in some bioactive substances, such as calcitonin, insulin, mucopolysaccharides, etc., being affected in the gastrointestinal tract, being rapidly destroyed or inactivated; meanwhile, due to the physicochemical properties of the medicine, partial sensitive medicines are easy to degrade, and the bioavailability of insoluble medicines is low. The development of a high-efficiency delivery system can enhance the efficacy of the medicine and reduce the toxic and side effects.

The diketopiperazine derivative is a novel material, and can be precipitated in an acidic solution and self-assembled into microspheres. The microsphere has large surface area, high drug loading, and simple preparation process, and is suitable for preparing various medicines, such as cationic medicine, anionic medicine, hydrophilic/lipophilic medicine, polypeptide, protein, and small molecule medicine with API molecular weight of 500-140000 Da.

Because of small diameter, the microsphere dry powder prepared by the technology can be used for pulmonary inhalation administration, and the in vivo absorption rate can reach the absorption rate of simulated arterial injection; or directly used for injection, and both approaches can avoid liver first pass effect and peripheral circulation degradation.

Preparation of microspheres with fumaryl diketopiperazine (FDKP, CAS: 176738-91-3) as a carrier

The technology is a novel drug delivery technology. Quick-acting insulin manufactured by Mannkind company with the trade name of Afrezza is approved by FDA to be marketed in 2014, and becomes the only insulin inhalation preparation on the current market.

The technical key support material FDKP patent (WO 2013/162764, CN 104797563) mentions the synthesis of this material. The mother nucleus is synthesized by dewatering and cyclizing amino acid ester derivative dimer, such as dipeptide ester, in 1946, and the synthesizing process of diketopiperazine organic compound in 1968 by Kopple is prepared from amino acid derivativeAnd thermally dehydrating in a high boiling organic solvent. Methods for the synthesis of 3, 6-bis (4-bis-fumaroylaminobutyl) -2, 5-diketopiperazine and its salt substituents are reported in CN 201010206311.5.

The diketopiperazine derivative can be self-assembled into drug-loaded microspheres by adjusting the pH value of a preparation system, and has certain advantages as a carrier, so that the novel diketopiperazine derivative replaces FDKP, and the development of a series of successfully and effectively-substituted substances and salts thereof has practical significance for realizing the effective delivery of drugs.

Disclosure of Invention

The invention aims to overcome the defect of single diketopiperazine derivative in the prior art, and provides a diketopiperazine compound with a novel structure, a preparation method and application thereof. The diketopiperazine compound provided by the invention can be used as self-assembled drug-carrying microspheres, so that the effective delivery of drugs is realized.

The invention provides a diketopiperazine compound shown in a formula I or pharmaceutically acceptable salt thereof,

wherein R is ^a And R is ^a ' is independently selected from unsubstituted or substituted with one or more R ⁴ Substituted as follows: -C ₁ -C ₆ Alkylene-,

-N(R ² )R ³ -5-10 membered heteroaryl-, -3-12 membered cycloaliphatic-and-3-7 membered heterocycloalkyl-; the heteroatom in the-5-10 membered heteroaryl-is selected from one or more of N, O and S, and the number of the heteroatom is 1-4; the hetero atom in the-3-7 membered heterocycloalkyl-is selected from N, O, S, S (=O) and S (=O) ₂ One or more of the above, the heteroatom number is 1-3; when the substituents are plural, the same or different;

R ⁴ selected from C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-O-, C substituted by one or more halogens ₁ -C ₆ Alkyl and-NHC (=o) -R ⁵ ；

R ¹ And R is ³ independently-C ₁ -C ₆ An alkylene group;

R ² and R is ⁵ Independently selected from H or C ₁ -C ₆ An alkyl group;

the carbon atoms with "×" represent, when chiral, S configuration, R configuration or mixtures thereof.

In the present invention, when R ^a And R is ^a ' independently is unsubstituted or substituted with one or more R ⁴ Take the place of-C ₁ -C ₆ Alkylene-at the time of said-C ₁ -C ₆ Alkylene is independently-C ₁ -C ₄ Alkylene-, said-C ₁ -C ₄ Alkylene-is preferably-methylene-, -CH ₂ CH ₂ -、-(CH(CH ₃ ))-、-CH ₂ CH ₂ CH ₂ -、-(C(CH ₃ ) ₂ )-、-CH ₂ CH ₂ CH ₂ CH ₂ -、-(CH(CH ₃ ))CH ₂ CH ₂ -or-CH ₂ CH(CH ₃ )CH ₂ -, e.g. -methylene-, -CH ₂ CH ₂ -or-CH ₂ CH ₂ CH ₂ -。

In the present invention, when R ^a And R is ^a ' independently is unsubstituted or substituted with one or more R ⁴ When substituted with a-5-10 membered heteroaryl-the-5-10 membered heteroaryl-is a-5-6 membered heteroaryl-in which the heteroatom is selected from one or more of N, O and S, the number of heteroatoms being 1-2, preferably-pyridinyl-, -thienyl-, e.g.

In the present invention, when R ^a And R is ^a ' independently is unsubstituted or substituted with one or more R ⁴ When substituted with a-3-12 membered cycloaliphatic radical, said-3-12 membered cycloaliphatic radical is preferably a-3-7 membered cycloalkyl radical or a-4-7 membered cycloalkenyl radical. Said-3-7 membered cycloalkyl-preferably-cyclopropyl-, -ringButyl-, -cyclopentyl-, -cyclohexyl-or-bicyclo [1.1.1]Pentanyl-, e.g

Said-4-7 membered cycloalkenyl-preferably-cyclopropenyl-, -cyclobutenyl-, -pentenyl-, -cyclohexenyl-, -cycloheptenyl-, e.g. +. >

In the present invention, when R ¹ And R is ³ independently-C ₁ -C ₆ Alkylene-at the time of said-C ₁ -C ₆ Alkylene is independently-C ₁ -C ₄ Alkylene-, said-C ₁ -C ₄ Alkylene-is preferably-methylene-, -CH ₂ CH ₂ -、-(CH(CH ₃ ))-、-CH ₂ CH ₂ CH ₂ -、-(C(CH ₃ ) ₂ )-、-CH ₂ CH ₂ CH ₂ CH ₂ -、-(CH(CH ₃ ))CH ₂ CH ₂ -or-CH ₂ CH(CH ₃ )CH ₂ -, e.g. -methylene-or-CH ₂ CH ₂ -。

In the present invention, when R ² And R is ⁵ Independently C ₁ -C ₆ In the case of alkyl, said C ₁ -C ₆ Alkyl of (a) is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl.

In the present invention, when R ⁴ Is C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-O-, C substituted by one or more halogens ₁ -C ₆ In the case of alkyl, said C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-O-, C substituted by one or more halogens ₁ -C ₆ C in alkyl ₁ -C ₆ Alkyl is independently C ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl groupPreferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl.

In the present invention, when R ⁴ For C substituted by one or more halogens ₁ -C ₆ In the case of alkyl, the number of halogens may be 1 to 3. The halogen may be fluorine, chlorine, bromine or iodine, for example fluorine or chlorine.

In certain preferred embodiments of the present invention, certain groups of the diketopiperazines of formula I are defined below (groups not mentioned are as described in any of the embodiments herein),

R ^a And R is ^a 'independently selected from one or more C' s ₁ -C ₆ alkyl-substituted-C ₁ -C ₆ Alkylene-, by one or more-NHCs (=o) -R ⁵ substituted-C ₁ -C ₆ Alkylene-,

-N(R ² )R ³ -5-to 10-membered heteroaryl-and-3-to 12-membered cycloaliphatic-groups-.

said-N (R) ² )R ³ The preferred manner of attachment is that the N atom is attached to the amide bond as in formula I, R ³ Is linked to the carboxyl group as described in formula I.

the said

Preferably alkenyl, is attached to said amide bond as in formula I, R ¹ Is linked to the carboxyl group as described in formula I.

R ² selected from H.

R ⁴ Selected from C ₁ -C ₆ Alkyl or-NHC (=o) -R ⁵ 。

R ⁵ selected from C ₁ -C ₆ An alkyl group.

R ^a and R is ^a ' same or different, e.g. the same.

in the diketopiperazine compound shown in the formula I,

the configuration of (2) is mainly->

(i.e.)>

)、/>

(i.e.)>

)、/>

(i.e.)>

)、/>

(i.e

) The configuration shown or mixtures thereof;

for example, greater than 80% ee for each chiral center; for example, for each chiral center, the configuration shown is about 85% -90% excess over the other configuration, more preferably about 95% -99% excess, still more preferably about 99% excess, or no other configuration is detected.

-N(R ² )R ³ -5-10 membered heteroaryl-and-3-12 membered cycloaliphatic-;

R ¹ and R is ³ independently-C ₁ -C ₆ An alkylene group;

R ² is H;

R ⁵ is C ₁ -C ₆ An alkyl group;

R ^a and R is ^a ' phaseAnd the same is true.

R ^a and R is ^a 'independently selected from one or more C' s ₁ -C ₆ alkyl-substituted-C ₁ -C ₆ Alkylene-,

-N(R ² )R ³ -5-10 membered heteroaryl-and-3-12 membered cycloaliphatic-;

R ¹ and R is ³ independently-C ₁ -C ₆ An alkylene group;

R ² is H;

R ⁵ is C ₁ -C ₆ An alkyl group;

R ^a and R is ^a 'same'.

R ^a and R is ^a’ Independently is

Wherein the bond at the b position is connected with the amide shown in the formula I, and the bond at the c position is connected with the carboxyl shown in the formula I.

In one embodiment of the present invention, the diketopiperazine compound represented by formula I is selected from any one of the following structures, isomers thereof, or mixtures thereof:

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In the present invention, the diketopiperazine compound shown in formula I or a pharmaceutically acceptable salt thereof has one or more chiral carbon atoms, so that an optically pure isomer, such as a pure enantiomer, or a racemate, or a mixed isomer, can be isolated. Pure single isomers may be obtained by separation methods in the art, such as chiral crystallization to form salts, or chiral preparative column separation.

In the invention, the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof can exist in a crystal form or an amorphous form. The term "crystalline form" refers to a form in which ions or molecules are strictly and periodically arranged in three dimensions in a defined manner and have a periodic recurrence pattern at certain intervals; due to the above-mentioned periodic arrangement, there may be various crystal forms, i.e., polymorphism. The term "amorphous" refers to a state in which ions or molecules are randomly distributed, i.e., the ions and molecules do not have a periodic arrangement rule.

In the present invention, the diketopiperazine compound represented by formula I or a pharmaceutically acceptable salt thereof may exist as a single stereoisomer or as a mixture thereof (e.g., a racemate) if a stereoisomer exists. The term "stereoisomer" refers to a cis, trans or optical isomer. These stereoisomers may be isolated, purified and enriched by asymmetric synthesis methods or chiral separation methods (including but not limited to thin layer chromatography, rotary chromatography, column chromatography, gas chromatography, high pressure liquid chromatography, etc.), and may be obtained by chiral resolution by bonding (chemical bonding, etc.) or salifying (physical bonding, etc.) other chiral compounds. The term "single stereoisomer" means that one stereoisomer of the compound of the present invention is present in an amount of not less than 95% by mass relative to all stereoisomers of the compound.

In the present invention, the diketopiperazine compound shown in formula I or a pharmaceutically acceptable salt thereof may exist in the form of a single tautomer or a mixture thereof, preferably in the form of a stable tautomer.

The invention also includes isotopically-labeled diketopiperazine compounds of formula I or pharmaceutically acceptable salts thereof, wherein one or more atoms are replaced by one or more atoms having a particular atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include, but are not limited to, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, sulfur, and chlorine (e.g. ² H， ³ H， ¹³ C， ¹⁴ C， ¹⁵ N， ¹⁸ O， ¹⁷ O， ¹⁸ F， ³⁵ S and ³⁶ cl). Isotopically-labeled compounds of the present invention are useful in the determination of the tissue distribution of a compound, prodrug thereof, and metabolite thereof; preferred isotopes for use in such assays include ³ H and ¹⁴ C. furthermore, in some cases, substitution with heavier isotopes, such as deuterium (2H or D), may afford increased metabolic stability, which may afford therapeutic advantages such as increased in vivo half-life or reduced dosage requirements.

Isotopically-labeled compounds of the present invention can generally be prepared according to the methods described herein by substituting an isotopically-labeled reagent for a non-isotopically-labeled reagent.

In the present invention, the diketopiperazine compound of the formula I or a pharmaceutically acceptable salt thereof can be synthesized by methods including methods similar to those known in the chemical arts, and the procedures and conditions can be referred to in the art for similar reactions, particularly according to the description herein. The starting materials are typically from commercial sources, such as Aldrich or can be readily prepared using methods well known to those skilled in the art (available via SciFinder, reaxys on-line databases).

In the invention, the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof can also be prepared by adopting a conventional method in the field to carry out peripheral modification on the prepared diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof so as to obtain other diketopiperazine compounds shown in the formula I or pharmaceutically acceptable salts thereof.

The necessary starting materials or reagents for preparing the diketopiperazine compounds of formula I or pharmaceutically acceptable salts thereof are commercially available or can be prepared by synthetic methods known in the art. The compounds of the invention may be prepared as free bases or as salts thereof with acids, as described in the experimental section below. The term pharmaceutically acceptable salt refers to a pharmaceutically acceptable salt as defined herein and has all the effects of the parent compound. Pharmaceutically acceptable salts can be prepared by adding the corresponding acid to a suitable organic solvent for the organic base, and processing according to conventional methods.

Examples of salification include: for base addition salts, it is possible to prepare salts of alkali metals (such as sodium, potassium or lithium) or alkaline earth metals (such as aluminum, magnesium, calcium, zinc or bismuth) by treating a compound of the invention having a suitably acidic proton with an alkali metal or alkaline earth metal hydroxide or alkoxide (such as ethoxide or methoxide) or a suitably basic organic amine (such as diethanolamine, choline or meglumine) in an aqueous medium.

Alternatively, for acid addition salts, salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; and salts formed with organic acids, such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, oxalic acid, pyruvic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, tartaric acid, citric acid, cinnamic acid, p-toluenesulfonic acid or trimethylacetic acid.

"the compound of the present invention" or "the compound of the present invention" includes any diketopiperazine compound of the formula I or a pharmaceutically acceptable salt thereof. The compounds of the present invention may also exist in the form of a hydrate or solvate.

The invention also provides a pharmaceutical composition, which comprises the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof, and one or more pharmaceutical active ingredients; one or more other pharmaceutical excipients can be also included; the pharmaceutically active ingredient may be in a therapeutically and/or prophylactically effective amount.

The pharmaceutical composition may be a microsphere drug delivery system. The diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof is self-assembled microsphere.

The weight percentage of the medicine active ingredient in the medicine composition is 10% -90%; for example 20% to 80%.

The active pharmaceutical ingredient (drug) can be cationic drug, anionic drug, hydrophilic/lipophilic drug, polypeptide, protein and small molecule drug with API molecular weight of 500-140000 Da; for example selected from insulin, dolutegravir (dolutegradvir), sildenafil, etc.

For example, the pharmaceutically active ingredient (drug) is a poorly soluble drug.

The methods of preparing the pharmaceutical compositions described above may be conventional in the art.

The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps:

Adding the solution containing the active pharmaceutical ingredient (drug) into an alkaline solution containing the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof, and then adjusting the pH value to precipitate solid to obtain the pharmaceutical composition. The mass ratio of the active pharmaceutical ingredient (drug) to the diketopiperazine compound shown in the formula I or the pharmaceutically acceptable salt thereof is 7:10; the alkaline solution can be obtained by adding 1% ammonia water (v/v); the pH may be 5.0; the pH can be adjusted by adding 10% glacial acetic acid aqueous solution (v/v); the obtained pharmaceutical composition is preferably dried after filtration and washing.

The invention also provides application of the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof as preparation auxiliary materials; for example, it forms drug-loaded microspheres. Specifically, diketopiperazine compounds shown in formula I or pharmaceutically acceptable salts thereof can be prepared into drug carrier particles with proper properties (such as particle size, shape, structural strength, solubility, low toxicity and the like), and drug microparticles formed by loading effective drug ingredients onto the drug carrier particles can be stable at low pH value and decompose at physiological pH value, so that the diketopiperazine compounds are suitable for oral administration, injection or inhalation and other administration modes to realize effective drug delivery. ).

The invention also provides a preparation method of the diketopiperazine compound shown in the formula I, which comprises the following steps: in a solvent, in the presence of alkali, performing saponification reaction shown in the formula II to obtain the diketopiperazine compound shown in the formula I;

wherein R is ^a And R is ^a ' all have the definition as described above; r 'and R' are independently C ₁ -C ₈ Alkyl (e.g. C) ₁ -C ₆ Alkyl radicals of (2), e.g. C ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; also for example methyl, t-butyl).

In the preparation method of the diketopiperazine compound shown in the formula I, the conditions and the operation of the saponification reaction can be conventional conditions and operations in the field of the reaction; the following are preferred in the present invention:

wherein the solvent may be an alcoholic solvent (e.g., methanol and/or ethanol). The amount of the solvent may be such that the reaction is not affected, for example, the volume to mass ratio of the solvent to the compound represented by formula II is 1mL/g to 50mL/g, for example, 5mL/g.

The base may be an alkali metal hydroxide (e.g., sodium hydroxide and/or potassium hydroxide).

The volume to mass ratio of the solvent to the base is 5mL/g to 20mL/g (e.g., 10 mL/g).

The molar ratio of the base to the compound of formula II may be from 2:1 to 10:1 (e.g., 4:1).

The temperature of the saponification reaction shown may be from room temperature to 80 ℃ (e.g. 60-70 ℃).

The progress of the saponification reaction shown can be monitored by conventional monitoring methods in the art (e.g., TLC, HPLC or NMR), typically with the compound of formula II shown as described as the end point of the reaction when it is lost or no longer reacted.

The preparation method can also comprise post-treatment and crystallization; the post-treatment may comprise the steps of: after the saponification reaction is finished, acid is added for neutralization (such as glacial acetic acid) to separate out solid, and the diketopiperazine compound shown in the formula I is obtained.

The crystallization may include the steps of: filtering the mixture of the diketopiperazine compound shown in the formula I and trifluoroacetic acid obtained after the post-treatment is finished, mixing filtrate with glacial acetic acid, cooling to separate out solid, filtering and washing to obtain the crystals of the diketopiperazine compound shown in the formula I. The mixture of the diketopiperazine compound shown in the formula I and the trifluoroacetic acid can be obtained by 50 ℃ -100 ℃ (for example, 80 ℃ -90 ℃) of the diketopiperazine compound shown in the formula I and the trifluoroacetic acid. The solvent used for the washing may be an alcoholic solvent (e.g., ethanol and/or ethanol).

The preparation method can further comprise the following steps of carrying out condensation reaction on the compound shown in the formula III in the presence of alkali and a condensing agent in a solvent to obtain the compound shown in the formula II;

wherein R is ^a 、R ^a The definitions of', R ' and R ' are as described above; and R is ^a And R is ^a ' same, R ' and R ' are the same.

The conditions and operations of the condensation reaction shown may be those conventional in the art; the following are preferred in the present invention:

wherein the solvent can be one or more of amide solvents (such as N, N-dimethylformamide DMF and/or N, N-dimethylacetamide DMA), sulfoxide solvents (such as dimethyl sulfoxide DMSO), halogenated hydrocarbon solvents (such as dichloromethane DCM) and cyclic ether solvents (such as tetrahydrofuran THF); such as amide solvents (e.g., N-dimethylformamide DMF and/or N, N-dimethylacetamide DMA), sulfoxide solvents (e.g., dimethylsulfoxide DMSO), halogenated hydrocarbon solvents (e.g., dichloromethane DCM), and cyclic ether solvents (e.g., tetrahydrofuran THF). The solvent is used in an amount that does not affect the reaction.

In the condensation reaction, the base may be an organic base (e.g., triethylamine). The molar ratio of the base to the compound of formula III is 1.5:1 to 3:1 (e.g., 2:1 to 2.5:1).

In the condensation reaction, the condensing agent may be one or more of HATU (2- (7-azabenzotriazol) -N, N '-tetramethylurea hexafluorophosphate), EDTA (ethylenediamine tetraacetic acid) and HBTU (benzotriazol-N, N' -tetramethylurea hexafluorophosphate), preferably HATU and/or EDTA. The molar ratio of the condensing agent to the compound of formula III is 1.0:1-2:1 (e.g., 1:1-1.5:1).

In the condensation reaction, the molar ratio of the compound shown in the formula IV to the compound shown in the formula III is 2:1-1:1.

The temperature of the condensation reaction may be from room temperature to 80 ℃ (e.g., 10-30 ℃).

The progress of the condensation reaction shown can be monitored by methods conventional in the art (e.g., TLC, HPLC or NMR), typically with the compound of formula III as shown as the end point of the reaction when it disappears or no longer reacts.

The preparation method can also comprise post-treatment and crystallization; the post-treatment and crystallization may comprise the steps of: after the condensation reaction is finished, crystallizing the filter cake obtained by filtering in glacial acetic acid and water, and separating out solid to obtain the compound shown in the formula II.

The invention also provides a preparation method of the compound shown in the formula II, which comprises the following steps of carrying out condensation reaction on the compound shown in the formula III in the presence of alkali and a condensing agent in a solvent to obtain the compound shown in the formula II;

The conditions and operation of the preparation process may be as described in any of the schemes above.

The invention also provides a compound shown as a formula II,

wherein R is ^a 、R ^a The definitions of', R ' and R ' are as described above.

In one embodiment of the present invention, the compound of formula II is any one of the following structures, isomers thereof, or mixtures thereof:

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in the present invention, the following definitions are used:

the following definitions as used herein should be applied unless otherwise indicated. For the purposes of the present invention, chemical elements are in accordance with CAS version of the periodic Table of the elements, and handbook of chemistry and physics, 75 th edition, 1994. In addition, general principles of organic chemistry may be referenced to the descriptions in "Organic Chemistry", thomas Sorrell, university Science Books, sausalato:1999, and "March's Advanced Organic Chemistry" by Michael b.smith and Jerry March, john Wiley & Sons, new york:2007, the entire contents of which are incorporated herein by reference.

In this specification, groups and substituents thereof can be selected by one skilled in the art to provide stable moieties and compounds. When substituents are described by conventional formulas written from left to right, the substituents also include chemically equivalent substituents obtained when writing formulas from right to left.

Certain chemical groups defined herein are preceded by a simplified symbol to indicate the total number of carbon atoms present in the group. For example, C ₁ -C ₆ Alkyl refers to an alkyl group as defined below having a total of 1, 2, 3, 4, 5 or 6 carbon atoms. The total number of carbon atoms in the reduced notation does not include carbon that may be present in a substituent of the group.

In this context, a numerical range as defined in substituents, such as 0 to 4, 1-4, 1 to 3, etc., indicates an integer within the range, such as 1-6 is 1, 2, 3, 4, 5, 6.

As is conventional in the art, are used herein in the structural formula

For depicting the bond at the point of attachment of the moiety or substituent to the core or backbone structure.

As is conventional in the art, "-" at the end of a group means that the group is attached to other fragments in the molecule through that site. For example, CH ₃ -C (=o) -means acetyl.

The term "one(s)" or "one(s) or two or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

The term "comprising" is an open-ended expression, i.e. including what is indicated by the invention, but not excluding other aspects.

The term "substituted" refers to any one or more hydrogen atoms on a particular atom being substituted with a substituent, including heavy hydrogen and variants of hydrogen, so long as the valence of the particular atom is normal and the substituted compound is stable.

In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with a specific substituent. Further, when the group is substituted with 1 or more of the substituents, the substituents are independent of each other, that is, the 1 or more substituents may be different from each other or the same. Unless otherwise indicated, a substituent group may be substituted at each substitutable position of the substituted group. When more than one position in a given formula can be substituted with one or more substituents selected from a particular group, then the substituents may be the same or different at each position.

In the various parts of the present specification, substituents of the presently disclosed compounds are disclosed in terms of the type or scope of groups. It is specifically noted that the present invention includes each individual subcombination of the individual members of these group classes and ranges. For example, the term "C ₁ -C ₆ Alkyl "or" C ₁ -C ₆ Alkyl "means in particular methyl, ethyl, C independently disclosed ₃ Alkyl, C ₄ Alkyl, C ₅ Alkyl and C ₆ An alkyl group; "C _1-4 Alkyl "refers specifically to independently disclosed methyl, ethyl, C ₃ Alkyl (i.e. propyl, including n-propyl and isopropyl), C ₄ Alkyl (i.e., butyl, including n-butyl, isobutyl, sec-butyl, and tert-butyl).

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "alkoxy" refers to the group-O-R ^X Wherein R is ^X Are alkyl groups as defined above.

The term "alkyl" as used herein refers to a straight or branched saturated hydrocarbon chain, such as a straight or branched saturated hydrocarbon chain containing from 1 to 20 carbon atoms. The term "C _x -C _y Alkyl "refers to a straight or branched chain saturated hydrocarbon containing from x to y carbon atoms. For example "C ₁ -C ₈ Alkyl "refers to straight or branched chain saturated hydrocarbons containing 1 to 8 carbon atoms. Representative examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl and n-decyl.

The term "cycloaliphatic radical" as used herein refers to a carbocyclic ring system containing from 3 to 10 carbon atoms, zero heteroatoms, which may be monocyclic or bridged, saturated or unsaturated (non-aromatic rings). Examples of saturated monocyclic ring systems ("cycloalkyl") include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The carbocyclic ring system of the saturated bridged ring system may contain one or two alkylene bridges, each alkylene bridge comprising one, two or three carbon atoms, each bridge linking two non-adjacent carbon atoms of the ring system. Representative examples of such bridged cycloalkyl carbocyclic ring systems include, but are not limited to: bicyclo [1.1.1] pentane, bicyclo [3.1.1] heptane, bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane, bicyclo [3.2.2] nonane, bicyclo [3.3.1] nonane, bicyclo [4.2.1] nonane, tricyclo [3.3.1.0, 7] nonane (octahydro-2, 5-methylenepentalene or noradamantane) and tricyclo [3.3.1.13,7] decane (adamantane). An unsaturated monocyclic carbocyclic ring system (e.g., "cycloalkenyl") may contain olefinic bonds, having from four to ten carbon atoms and zero heteroatoms. The "cycloalkenyl" of the four-membered ring system has one double bond, the "cycloalkenyl" of the five-or six-membered ring system has one or two double bonds, the "cycloalkenyl" of the seven-or eight-membered ring system has one, two or three double bonds, and the "cycloalkenyl" of the nine-or ten-membered ring system has one, two, three or four double bonds. Representative examples of unsaturated monocyclic carbocyclic ring systems include, but are not limited to: cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. An unsaturated monocyclic "cycloalkenyl" may contain one or two alkylene bridges, each of which includes one, two or three carbon atoms, with each bridge connecting two non-adjacent carbon atoms of the ring system. Representative examples of unsaturated bridged ring carbocyclic ring systems containing olefinic bonds include, but are not limited to: bicyclo [2.2.1] hept-2-ene, 4,5,6, 7-tetrahydro-3 aH-indene, octahydronaphthyl and 1, 6-dihydro-pentalene. Monocyclic and bridged ring "cycloaliphatic radicals" (e.g., "cycloalkyl", "cycloalkenyl") may be joined to the parent molecular moiety through any substitutable atom contained within the ring system.

The term "heterocycloalkyl" as used herein refers to a cyclic group of a specified number of ring atoms (e.g., 5-10 membered), of a specified number of heteroatoms (e.g., 1, 2, or 3), of a specified heteroatom species (one or more of N, O and S), which is a single ring, bridged ring, or spiro ring, and each ring is saturated. Heterocycloalkyl groups include, but are not limited to, azetidinyl, tetrahydropyrrolyl, tetrahydrofuranyl, morpholinyl, piperidinyl, and the like.

The term "heteroaryl" as used herein refers to a cyclic group of a specified number of ring atoms (e.g., 5-10 membered), of a specified number of heteroatoms (e.g., 1, 2, or 3), of a specified class of heteroatoms (one or more of N, O and S), which is monocyclic or polycyclic, and at least one ring has aromaticity (meeting the shock rule). Heteroaryl groups are linked to other fragments in the molecule through aromatic or non-aromatic rings. Heteroaryl groups include, but are not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, indolyl, and the like.

The term "solvate" refers to a substance formed by combining a compound of the invention with a stoichiometric or non-stoichiometric solvent. The solvent molecules in the solvate may be present in an ordered or unordered arrangement. Such solvents include, but are not limited to: water, methanol, ethanol, and the like.

The term "pharmaceutically acceptable" refers to salts, solvents, excipients, and the like, which are generally non-toxic, safe, and suitable for patient use. The "patient" is preferably a mammal, more preferably a human.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared with relatively non-toxic, pharmaceutically acceptable acids. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in pure solution or in a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids including, but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phosphorous acid, sulfuric acid, hydrogen sulfate, and the like. The pharmaceutically acceptable acid includes organic acids including, but not limited to: acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4' -methylene-bis (3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), and the like. When the compounds of the present invention contain relatively basic functional groups, they can be converted into acid addition salts. See, for example, berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science 66:1-19 (1977), or Handbook of Pharmaceutical Salts: properties, selection, and Use (P.Heinrich Stahl and Camille G.Wermuth, ed., wiley-VCH, 2002).

The terms "pharmaceutically acceptable salts" and "solvates" in "solvates of pharmaceutically acceptable salts" refer, as described above, to the compounds of the invention formed by combining 1 with 2, prepared with a relatively non-toxic, pharmaceutically acceptable acid or base, with a stoichiometric or non-stoichiometric amount of a solvent.

The terms "moiety", "structural moiety", "chemical moiety", "group", "chemical group" as used herein refer to a particular fragment or functional group in a molecule. Chemical moieties are generally considered to be chemical entities that are embedded or attached to a molecule.

When none of the listed substituents indicates through which atom it is attached to a compound included in the chemical structural formula but not specifically mentioned, such substituents may be bonded through any of their atoms. Combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

Where no substituent is explicitly indicated in a recited group, such a group is merely unsubstituted. For example when "C ₁ -C ₄ Alkyl "not previously" substituted or unsubstituted "refers only to" C ₁ -C ₄ Alkyl "as such or" unsubstituted C ₁ -C ₄ An alkyl group.

In the various parts of the invention, linking substituents are described. When the structure clearly requires a linking group, the markush variables recited for that group are understood to be linking groups. For example, if the structure requires a linking group and the markush group definition for that variable enumerates an "alkyl" or "aryl" group, it will be understood that the "alkyl" or "aryl" represents a linked alkylene group or arylene group, respectively.

In some specific structures, when an alkyl group is explicitly represented as a linking group, then the alkyl group represents a linked alkylene group, e.g., the group "halo-C ₁ -C ₆ C in alkyl' ₁ -C ₆ Alkyl is understood to mean C ₁ -C ₆ An alkylene group.

Unless otherwise specified, all technical and scientific terms used herein have the standard meaning of the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.

As used herein, the singular forms "a", "an", and "the" are understood to include plural referents unless the context clearly dictates otherwise. Furthermore, the term "comprising" is an open-ended limitation and does not exclude other aspects, i.e. it includes the content indicated by the invention.

Unless otherwise indicated, the present invention employs conventional methods of mass spectrometry, elemental analysis, and the various steps and conditions are referred to in the art by conventional procedures and conditions.

The present invention employs, unless otherwise indicated, standard nomenclature for analytical chemistry, organic synthetic chemistry and optics, and standard laboratory procedures and techniques. In some cases, standard techniques are used for chemical synthesis, chemical analysis, and light emitting device performance detection.

In addition, unless explicitly indicated otherwise, the description of the invention as "…" independently is to be understood broadly as meaning that each individual described may be independent of the other, and may be the same or different. In more detail, the description "… is independently" may mean that specific options expressed between the same symbols in different groups do not affect each other; it may also be expressed that specific options expressed between the same symbols in the same group do not affect each other.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that: the diketopiperazine compound provided by the invention can be used as self-assembled drug-carrying microspheres, so that the effective delivery of drugs is realized.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

EXAMPLE 1 Synthesis of Compounds S1 to S18

The compounds S1 to S18 are synthesized by the following general formula:

step 1: dehydration cyclization reaction: 36 g of ε -benzyloxycarbonyl-L-lysine and 59 g of m-cresol, and 6 g of phosphorus pentoxide were added to a conical flask, heated to 200℃and the water was distilled off during the reaction, and the reaction was cooled in a mixed solution of water and sodium hydroxide (10:1) and a precipitate was formed. After separating the precipitate and washing with 36 ml of ethanol, 24.8 g of a crude product of intermediate (2 s,5 s) -2, 5-bis [4- (N-benzyloxycarbonyl) aminobutyl ] -3, 6-diketopiperazine was obtained after filtration. The resulting crude product was heated in 120 ml of glacial acetic acid solution (100 ℃ C.), then 36 ml of purified water was added thereto, and then the crystals were washed with 60 ml of glacial acetic acid solution to obtain 13.5 g of an intermediate (2S, 5S) -2, 5-bis [4- (N-benzyloxycarbonyl) aminobutyl ] -3, 6-diketopiperazine (compound 1) as a refined product.

Step 2: hydrogenation reaction: 13.5 g of the product from the above step was dissolved in 60 ml of glacial acetic acid solution, and a catalyst (10% palladium on carbon) was added to conduct hydrogenation in a reaction vessel filled with hydrogen. The resulting mixture was cooled and filtered. The filtrate was then distilled to remove glacial acetic acid components, thereby obtaining acetate (compound 2) of (2 s,5 s) -2, 5-bis (4-aminobutyl) -3, 6-diketopiperazine.

MS：257(M+H)。

Step 3:

condensation reaction: the product of the above step was dissolved in DMF, and two equivalents of monocarboxylic acid (monocarboxylic acid compound structure shown in Table 1), two equivalents of triethylamine and an equivalent of HATU were added to conduct a condensation reaction at room temperature. After the liquid phase detection raw material disappears, adding water, then separating out the obtained solid substance,

thus obtaining the condensation product solid crude product. The obtained solid crude product was added with glacial acetic acid and then with water cooling. The crystals were then washed with glacial acetic acid solution to give the condensation products (compounds I1-18).

Step 4: saponification reaction: the condensation product obtained in the above step was heated to 70℃by adding a mixed solution of methanol and 4 equivalents of sodium hydroxide (volume/mass ratio of methanol to sodium hydroxide: 10 mL/g), and then cooled to room temperature by adding glacial acetic acid after filtration. The solid matter was separated and washed with water to obtain a crude product of the compound (S1-18).

Step 5: and (5) recrystallizing: and adding trifluoroacetic acid into the crude target molecule product obtained in the previous step, heating to 90 ℃, filtering the mixture, cooling the filtrate, adding glacial acetic acid, and further cooling. Separating the solid material, washing with methanol, washing with purified water, and drying to obtain compound (S1-18).

The structural formula and mass spectrum data of monocarboxylic acid and the prepared compounds I1-18 are shown in Table 1.

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The structures of the prepared compounds S1 to S18 are shown in Table 2.

TABLE 2

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Structural identification data for Compounds S1-18:

compound S1: ¹ H NMR(400MHz,DMSO)δ1.24～1.54(m,8H),1.77～1.78(m,4H),3.02～3.06(m,4H),3.98～3.99(m,2H),8.25～8.26(s,2H),8.72～8.99(m,8H),10.01～12.17(br,2H).ESI,555(M+H)。

compound S2: ¹ H NMR(400MHz,DMSO)δ1.21～1.22(m,4H),1.47～1.79(m,16H),2.98～3.02(m,4H),3.99～4.00(m,2H),8.01～8.01(s,2H),8.25～8.26(s,2H),11.00～13.17(br,2H).ESI,503(M+Na)。

compound S3: ¹ H NMR(400MHz,DMSO)δ1.23～1.53(m,8H),1.76～1.77(m,4H),3.18～3.19(m,4H),3.54～3.56(m,4H),3.98～3.99(m,2H),5.86(s,2H),6.29(s,2H),8.26～8.41(s,4H),12.20～12.22(br,2H).ESI,481(M+H)。

compound S4: ¹ H NMR(400MHz,DMSO)δ1.23～1.25(m,4H),1.50～1.75(m,12H),2.70～2.80(m,4H),3.17～3.18(m,4H),3.33～3.34(m,4H),3.98～3.99(m,2H),6.06(s,2H),6.20(s,2H),8.01(s,2H),8.25(s,2H),12.01～12.14(br,2H).ESI,607(M+Na)。

compound S5: ¹ H NMR(400MHz,DMSO)δ0.97～0.98(m,6H),1.24～1.78(m,12H),1.99～2.23(m,10H),2.99～3.01(m,4H),3.98～3.99(m,2H),7.70(s,2H),8.27(s,2H),11.60～11.80(br,2H).ESI,513(M+H)。

compound S6: ¹ H NMR(400MHz,DMSO)δ1.20～1.25(m,4H),1.40～1.81(m,24H),2.61～2.69(m,4H),3.17～3.18(m,4H),3.99～4.00(m,2H),8.00(s,2H),8.25(s,2H),12.00～12.13(br,2H).ESI,565(M+H)。

compound S7: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.39～1.80(m,12H),1.99～2.26(m,8H),3.00～3.01(m,4H),3.99～4.00(m,2H),7.99(s,2H),8.02(s,2H),13.80～13.85(br,2H).ESI,509(M+H)。

compound S8: ¹ H NMR(400MHz,DMSO)δ1.25～1.26(m,4H),1.50～1.52(m,4H),1.76～2.21(m,16H),3.02～3.03(m,4H),3.99～4.00(m,2H),8.01(s,2H),8.04(s,2H),12.0～12.02(br,2H).ESI,533(M+H)。

compound S9: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.45(s,12H),1.52～1.53(m,4H),1.76～1.77(m,4H),3.02～3.03(m,4H),3.98～3.99(m,2H),8.00(s,2H),8.27(s,2H),13.81～13.85(br,2H).ESI,485(M+H)。

compound S10: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.53～1.54(m,4H),1.76～1.77(m,4H),2.08～2.35(m,8H),2.81～2.90(m,4H),3.17～3.18(m,4H),3.99～4.00(m,2H),5.64～5.68(m,4H),8.01(s,2H),8.25(s,2H),12.14～12.16(br,2H).ESI,561(M+H)。

compound S11: ¹ H NMR(400MHz,DMSO)δ1.23～1.24(m,4H),1.33(s,12H),1.52～1.53(m,4H),1.76～1.77(m,4H),2.38(s,4H),3.02～3.03(m,4H),3.96～3.99(m,2H),8.01(s,2H),8.26(s,2H),13.13～13.14(br,4H).ESI,535(M+Na)。

compound S12: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.49～1.84(m,12H),2.18～2.23(m,4H),3.17～3.19(m,4H),3.99～4.00(m,2H),8.01(s,2H),8.25(s,2H),12.12～12.13(br,2H).ESI,503(M+Na)。

compound S13: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.52～1.53(m,4H),1.76～1.84(m,10H),2.06～2.08(m,4H),2.32～2.34(m,4H),3.18～3.19(m,4H),3.99～4.00(m,2H),4.44～4.46(m,2H),8.01(s,2H),8.32～8.34(m,4H),12.00～12.01(br,2H).ESI,599(M+H)。

compound S14: ¹ H NMR(400MHz,DMSO)δ1.25～1.26(m,4H),1.53～1.54(m,4H),1.76～1.73(m,10H),2.10～2.61(m,4H),3.18～3.19(m,4H),3.99～4.00(m,2H),4.08～4.09(m,2H),8.01(s,2H),8.30(s,4H),12.59～13.01(br,2H).ESI,571(M+H)。

compound S15: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.53～1.54(m,4H),1.76～1.77(m,4H),3.07～0.08(m,4H),3.99～4.00(m,4H),8.06～8.07(m,2H),8.24～8.26(d,2H),8.60～8.61(m,2H),9.13～9.14(m,2H),12.15～12.25(br,2H).ESI,555(M+H)。

compound S16: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.53～1.54(m,4H),1.76～1.77(m,4H),3.06～3.07(m,4H),3.98～3.99(m,2H),8.09～8.10(d,2H),8.25～8.50(m,6H),12.10～12.15(br,2H).ESI,565(M+H)。

compound S17: ¹ H NMR(400MHz,DMSO)δ1.25～1.26(m,4H),1.53～1.54(m,4H),1.77～1.78(m,4H),3.04～3.05(m,4H),3.99～4.06(m,6H),6.36(s,2H),8.27(s,2H),13.00～13.01(br,2H).ESI,459(M+H)。

compound S18: ¹ H NMR(400MHz,DMSO)δ1.24～1.25(m,4H),1.54～1.55(m,4H),1.77～1.78(m,4H),2.48～2.49(m,4H),3.05～3.06(m,4H),63～3.66(m,4H),3.99～4.00(m,2H),6.01～6.02(m,4H),8.26(s,2H),12.10～12.13(br,2H).ESI,487(M+H)。

EXAMPLE 2 evaluation of drug loading

The purpose of the experiment is as follows: preliminary drug loading studies of FDKP and derivatives thereof were performed using dolutegradvir (hereinafter referred to as Dol) as a model drug.

The experimental method comprises the following steps: 100mg of FDKP or a derivative thereof is precisely weighed, 15mL of 1% ammonia water (v/v) is added for dissolution, and insoluble substances are removed by a filtering membrane; accurately weighing 70mg of the rutevir, adding 1mL of aqueous solution for dissolution, dropwise adding the solution into the FDKP solution under the condition of magnetic stirring, continuously adjusting the pH to 5.0 by using 10% glacial acetic acid aqueous solution (v/v), precipitating solid, filtering, washing and drying to obtain the Dol-FDKP-Der.

Dissolving the obtained solution in ammonium acetate solution, adjusting pH to dissolve completely, and filtering to remove insoluble substances. The content of lurtevir was then checked by HPLC by area comparison.

The HPLC test method is as follows:

chromatographic column: ultimate XB-C18 (4.6X150 mm,3 μm)

Flow rate: 1.0ml/min

Wavelength: column temperature at 230 nm: 25 DEG C

Mobile phase a:0.1% phosphoric acid

Mobile phase B: acetonitrile

Experimental results: drug loading data results

TABLE 3 Table 3

Numbering of compounds	％
		FDKP	44.1
S1	77.5
		S2	52.4
S3	49.4
		S4	38.1
S5	50.0
		S6	67.1
S7	56.2
		S8	55.4
S9	55.0
		S10	65.2
S11	48.8
		S12	56.2
S13	24.7
		S14	33.7
S15	75.5
		S16	62.9
S17	49.2
		S18	47.7

EXAMPLE 3 cytotoxicity

1. Purpose of experiment

Toxicity of FDKP and derivatives to mouse fibroblasts (NIH/3T 3) was examined.

2. Test materials:

reagent: FDKP and derivatives, MTT, DMSO, DEME culture medium, gibico serum, etc.

And (3) equipment: 96-well plates, ep tubes, centrifuge tubes, and the like.

3. Experimental procedure

Preparation of FDKP and derivative solutions

15mg FDKP or derivative is precisely weighed and dissolved in 3ml of 5% basal medium to prepare 5mg/ml initial solution, which is further diluted to 2.5mg/ml,1.25mg/ml,0.625mg/ml,0.313mg/ml,0.156mg/ml,0.078mg/ml,0.039mg/ml with 5% basal medium.

MTT solution configuration

10mg of MTT was weighed and dissolved in 2ml of the pbs solution to obtain a MTT solution having a concentration of 5 mg/ml.

3. Specific test procedure

3T3 cells were diluted to the appropriate concentration, seeded in 96-well plates at a density of 5000 cells per well (n=6), and incubated at 37 ℃ under 5% co2 for 12 hours.

Subsequently, the medium was removed, 100ul of medium containing different concentrations of FDKP or derivative was added to each well and the incubation was continued for 24 hours, respectively.

The medium was removed from each well and 100ul of fresh medium and 10ul of MTT solution were added at 37℃with 5% CO ₂ The culture was continued for 4 hours.

Subsequently, the solution in the wells was removed and 150ul DMSO was added and shaken at room temperature to allow the crystals to dissolve well. The microplate reader records the OD of each group at 490 nm.

Cell viability was calculated according to the following formula:

percent (%) cell viability = [ OD experimental group-OD blank group)/(OD control group-OD blank group) ] × 100%.

TABLE 4 cytotoxicity test results

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Claims

1. A diketopiperazine compound shown in a formula I or pharmaceutically acceptable salt thereof,

wherein R is ^a And R is ^a' Independently selected from unsubstituted or substituted with one or more R ⁴ Substituted as follows: -C ₁ -C ₆ Alkylene-,

R ¹ And R is ³ independently-C ₁ -C ₆ An alkylene group;

R ² and R is ⁵ Independently selected from H or C ₁ -C ₆ An alkyl group;

2. The diketopiperazine compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the diketopiperazine compound of formula I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions:

(1) When R is ^a And R is ^a' Independently is unsubstituted or substituted with one or more R ⁴ Take the place of-C ₁ -C ₆ Alkylene-at the time of said-C ₁ -C ₆ Alkylene is independently-C ₁ -C ₄ Alkylene-, said-C ₁ -C ₄ Alkylene-preferably-alkyleneMethyl radical-, -CH ₂ CH ₂ -、-(CH(CH ₃ ))-、-CH ₂ CH ₂ CH ₂ -、-(C(CH ₃ ) ₂ )-、-CH ₂ CH ₂ CH ₂ CH ₂ -、-(CH(CH ₃ ))CH ₂ CH ₂ -or-CH ₂ CH(CH ₃ )CH ₂ -, e.g. -methylene-, -CH ₂ CH ₂ -or-CH ₂ CH ₂ CH ₂ -；

(2) When R is ^a And R is ^a' Independently is unsubstituted or substituted with one or more R ⁴ When substituted with a-5-10 membered heteroaryl-the-5-10 membered heteroaryl-is a-5-6 membered heteroaryl-in which the heteroatom is selected from one or more of N, O and S, the number of heteroatoms being 1-2, preferably-pyridinyl-, -thienyl-, e.g.

(3) When R is ^a And R is ^a' Independently is unsubstituted or substituted with one or more R ⁴ When the-3-12 membered cycloaliphatic radical is substituted, the-3-12 membered cycloaliphatic radical is a-3-7 membered cycloalkyl radical or a-4-7 membered cycloalkenyl radical; said-3-7 membered cycloalkyl-preferably-cyclopropyl-, -cyclobutyl-, -cyclopentyl-, -cyclohexyl-or-bicyclo [1.1.1]Pentanyl-, e.g

Said-4-7 membered cycloalkenyl-preferably-cyclopropenyl-, -cyclobutenyl-, -pentenyl-, -cyclohexenyl-, -cycloheptenyl-, e.g. +.>

(4) When R is ¹ And R is ³ independently-C ₁ -C ₆ Alkylene-at the time of said-C ₁ -C ₆ Alkylene is independently-C ₁ -C ₄ Alkylene-, said-C ₁ -C ₄ Alkylene-is preferably-methylene-, -CH ₂ CH ₂ -、-(CH(CH ₃ ))-、-CH ₂ CH ₂ CH ₂ -、-(C(CH ₃ ) ₂ )-、-CH ₂ CH ₂ CH ₂ CH ₂ -、-(CH(CH ₃ ))CH ₂ CH ₂ -or-CH ₂ CH(CH ₃ )CH ₂ -, e.g. -methylene-or-CH ₂ CH ₂ -；

(5) When R is ² And R is ⁵ Independently C ₁ -C ₆ In the case of alkyl, said C ₁ -C ₆ Alkyl of (2) is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl;

(6) When R is ⁴ Is C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-O-, C substituted by one or more halogens ₁ -C ₆ In the case of alkyl, said C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-O-, C substituted by one or more halogens ₁ -C ₆ C in alkyl ₁ -C ₆ Alkyl is independently C ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl;

(7) When R is ⁴ For C substituted by one or more halogens ₁ -C ₆ When alkyl is used, the number of the halogen is 1-3;

(8) When R is ⁴ For C substituted by one or more halogens ₁ -C ₆ In the case of alkyl, the halogen is fluorine, chlorine, bromine or iodine, for example fluorine or chlorine;

(9)R ^a and R is ^a 'independently selected from one or more C' s ₁ -C ₆ alkyl-substituted-C ₁ -C ₆ Alkylene-, by one or more-NHCs (=o) -R ⁵ substituted-C ₁ -C ₆ Alkylene-,

-N(R ² )R ³ -5-10 membered heteroaryl-and-3-12 membered cycloaliphatic-;

(10)-N(R ² )R ³ the connection being such that the N atom is bound to said amide bond as in formula I, R ³ Is connected with the carboxyl in the formula I;

(11)

in such a way that an alkenyl group is bonded to said amide bond as in formula I, R ¹ Is connected with the carboxyl in the formula I;

(12)R ² selected from H;

(13)R ⁴ selected from C ₁ -C ₆ Alkyl or-NHC (=o) -R ⁵ ；

(14)R ⁵ Selected from C ₁ -C ₆ An alkyl group;

(15)R ^a and R is ^a' The same or different, e.g., the same;

(16)

is of the configuration +.>

For example

3. The diketopiperazine compound according to claim 1, wherein,

R ^a and R is ^a' Independently selected from one or more C ₁ -C ₆ alkyl-substituted-C ₁ -C ₆ Alkylene-, by one or more-NHCs (=o) -R ⁵ substituted-C ₁ -C ₆ Alkylene-,

-N(R ² )R ³ -5-10 membered heteroaryl-and-3-12 membered cycloaliphatic-;

R ¹ And R is ³ independently-C ₁ -C ₆ An alkylene group;

R ² is H;

R ⁵ is C ₁ -C ₆ An alkyl group;

R ^a and R is ^a' The same applies.

4. The diketopiperazine compound according to claim 1, wherein,

R ^a and R is ^a’ Independently is

5. The diketopiperazine compound of any of claims 1-4, or a pharmaceutically acceptable salt thereof, of formula I, wherein the diketopiperazine compound of formula I is selected from any of the following structures, isomers thereof, or mixtures thereof:

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6. a pharmaceutical composition comprising a diketopiperazine compound of formula I according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, one or more pharmaceutically active ingredients.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition satisfies one or more of the following conditions:

(1) The medicine composition is a microsphere medicine carrying system; for example, the diketopiperazine compound as defined in any one of claims 1 to 5, as shown in formula I, or a pharmaceutically acceptable salt thereof, is an autonomously drug-loaded microsphere;

(2) The weight percentage of the pharmaceutically active ingredient in the pharmaceutical composition is 10% to 90%, for example 20% to 80%;

(3) The active pharmaceutical ingredient is a cationic drug, an anionic drug, a hydrophilic/lipophilic drug, a polypeptide, a protein or a small-molecule drug with the API molecular weight of 500-140000Da, such as insulin, dolutegravir (dolutegradvir) or sildenafil;

(4) The active ingredients of the medicine are insoluble medicines;

(5) The pharmaceutical composition also comprises pharmaceutically acceptable pharmaceutical excipients.

8. A method of preparing a pharmaceutical composition according to claim 6 or 7, comprising the steps of: adding the solution containing the active pharmaceutical ingredient into an alkaline solution containing the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof, and adjusting the pH to precipitate solid to obtain the pharmaceutical composition;

the mass ratio of the active pharmaceutical ingredient to the diketopiperazine compound shown in the formula I or the pharmaceutically acceptable salt thereof is 7:10; the alkaline solution can be obtained by adding 1% ammonia water (v/v); the pH may be 5.0; the pH can be adjusted by adding 10% glacial acetic acid aqueous solution (v/v); the obtained pharmaceutical composition is preferably dried after filtration and washing.

9. Use of a diketopiperazine compound of formula I as defined in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof as an adjuvant for formulations; for example, the diketopiperazine compound shown in the formula I or pharmaceutically acceptable salt thereof forms drug-loaded microspheres.

10. A process for the preparation of diketopiperazines of formula I as claimed in any one of claims 1 to 5 comprising the steps of: in a solvent, in the presence of alkali, performing saponification reaction shown in the formula II to obtain the diketopiperazine compound shown in the formula I;

wherein R is ^a And R is ^a' Is as defined in any one of claims 1 to 5; r 'and R' are independently C ₁ -C ₈ Alkyl group of said C ₁ -C ₈ The alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl, tert-butyl.

11. The method for producing diketopiperazine compound of claim 10, wherein the saponification reaction satisfies one or more of the following conditions:

(1) The solvent is an alcohol solvent, such as methanol and/or ethanol;

(2) The volume-mass ratio of the solvent to the compound shown in the formula II is 1mL/g-50mL/g, such as 5mL/g;

(3) The base is an alkali metal hydroxide, such as sodium hydroxide and/or potassium hydroxide;

(4) The volume to mass ratio of the solvent to the base is 5mL/g to 20mL/g, for example 10mL/g;

(5) The molar ratio of the base to the compound of formula II is from 2:1 to 10:1, for example 4:1;

(6) The saponification reaction is shown at a temperature of from room temperature to 80 ℃, for example 60-70 ℃;

(7) The preparation method also comprises post-treatment and crystallization; the post-treatment may comprise the steps of: after the saponification reaction is finished, adding acid for neutralization, and separating out solids to obtain the diketopiperazine compound shown in the formula I; the crystallization may include the steps of: filtering the mixture of the diketopiperazine compound shown in the formula I and trifluoroacetic acid obtained after the post-treatment is finished, mixing filtrate with glacial acetic acid, cooling to separate out solid, filtering and washing to obtain the crystals of the diketopiperazine compound shown in the formula I.

12. The method for preparing diketopiperazine compounds of formula I as recited in claim 10, characterized in that,

The preparation method further comprises the following steps of carrying out condensation reaction on the compound shown in the formula III in the presence of alkali and a condensing agent in a solvent to obtain the compound shown in the formula II;

wherein R is ^a And R is ^a' The definitions of R ' and R ' are as defined in any one of claims 1 to 5 and R ' is as defined in claim 10 ^a And R is ^a' Identical, R 'and R' are identical.

13. The method for preparing diketopiperazine compound of formula I according to claim 12, wherein the condensation reaction satisfies one or more of the following conditions:

(1) The solvent is one or more of amide solvents, sulfoxide solvents, halogenated hydrocarbon solvents and cyclic ether solvents; the amide solvent can be N, N-dimethylformamide and/or N, N-dimethylacetamide; the sulfoxide solvent can be dimethyl sulfoxide; the halogenated hydrocarbon solvent can be dichloromethane; the cyclic ether solvent can be tetrahydrofuran;

(2) The base is an organic base such as triethylamine;

(3) The molar ratio of the base to the compound of formula III is 1.5:1 to 3:1, e.g., 2:1 to 2.5:1;

(4) The condensing agent is one or more of HATU, EDTA and HBTU, such as HATU and/or EDTA;

(5) The molar ratio of the condensing agent to the compound shown in the formula III is 1.0:1-2:1, such as 1:1-1.5:1;

(6) The molar ratio of the compound shown in the formula IV to the compound shown in the formula III is 2:1-1:1;

(7) The temperature of the condensation reaction is from room temperature to 80 ℃, for example 10-30 ℃;

(8) The condensation reaction also comprises post-treatment and crystallization; the post-treatment and crystallization may comprise the steps of: after the condensation reaction is finished, crystallizing the filter cake obtained by filtering in glacial acetic acid and water, and separating out solid to obtain the compound shown in the formula II.

14. The preparation method of the compound shown in the formula II is characterized by comprising the following steps of carrying out condensation reaction on the compound shown in the formula III in the presence of alkali and a condensing agent in a solvent to obtain the compound shown in the formula II;

wherein R is ^a 、R ^a” R ' and R ' are as defined in any one of claims 1 to 5 and R ' is as defined in claim 10 ^a And R is ^a' Identical, R 'and R' are identical;

the conditions and operation of the preparation process are as defined in claim 12 or 13.

15. A compound of the formula (II),

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the compound shown in the formula II can be any one of the following structures, isomers or mixtures thereof:

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