TWI535467B - Conformations of divergent peptides with mineral binding affinity (1) - Google Patents

Description

Polymorphic peptide configuration with mineral affinity (1)

The present invention relates to a configuration in which a polymoid compound containing aspartic acid is favorably coordinated with a calcium ion on a biomineral to enhance the potential of a target biomineral.

With the improvement of human quality of life, research on biomineralization has had certain influences on the progress of the medical field in recent years, such as the discussion of the mechanism of bone diseases and the development of treatment modes, the reconstruction of teeth or the treatment of oral diseases, and even other abnormal mineralization in the body. and many more. Bio-mineral is a mineral product produced by organisms. It is mainly divided into two major categories, carbonate and phosphate. Its composition includes not only inorganic components but also organic structures. The basic mineral composition of biominerals is hydroxylapatite (HAp), whose chemical composition is the crystal of Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ .

Molecules to be associated with biominerals should generally contain basic structures such as polycarboxylic acids or polyphosphoric acids that can be coordinated by calcium ions. In-depth observation of phenomena found in the biological world; in addition to collagen, organisms secrete acidic substances to provide polyanions to bind calcium in minerals, and then form nucleation to form biominerals contained in bone and tooth structures. The binding substances are all capable of providing calcium binding using a functional machine having a rich carboxylic acid in the structure or a post-translational modification such as phosphorylation or vulcanization. These acidic substances include polysaccharides or proteoglycans, and proteins.

The biomimetic reagents used to affinity bone in the literature mostly use polyaspartic acid to mimic the aspartic acid-rich protein in natural biominerals. Since polyaspartic acid is a known mineral affinity peptide, the required length is also clearly found to be composed of 6 to 12 aspartic acid, which has a mineral affinity effect. For example, Wang D. et al . published in Bioconjugate Chem. in 2003 and 2007 to compare the affinity of different substances for bone tissue. It was found that the 8 peptides composed of repeated aspartic acid have good affinity for bone. Together. They used the 8 peptides bound by a polymer containing a fluorescent substance for animal experiments, and it was observed that a large amount of fluorescent substances adhered to the skeletal system. In dentistry applications, it has also become more and more prosperous in recent years. For example, Liu, X.-M. and other scholars have proposed that a double phosphate drug (alendronate) is bonded to cyclodextrin for oral diseases.

According to Ugliengo, the P. team found that glycine can form a complex with hydroxyapatite via its carboxyl and amine groups, resulting in a more stable binding structure. In biological systems, the functional groups involved in calcium ion binding in the protein structure are the aspartic acid, glutamic acid and aspartate and the oxygen atom on the side chain of phosphorylated serine, the main protein backbone and water molecules. The number of acidic amino acids provides the electrostatic properties of the protein and also directly affects calcium ion binding. Studies have confirmed that the appropriate peptide sequence arrangement, the appropriate peptide sequence can not only provide the ability to selectively bind to calcium ions, and even provide a more specific performance for the stacking and binding of biominerals.

Since 1970, Vögtle, F. and other scholars have designed the first divergent synthetic spherical dendritic polymer, revealing the characteristics of dendritic polymeric molecules different from linear polymers. Dendrimer, as its name suggests, is a dendrimer-like molecule that is a branch of a polymer; its molecules are highly divergent polymolecules with a precise single molecular weight distribution. Different from the traditional linear polymer, it provides a three-dimensional radial arrangement, starting from the core molecules and extending outward evenly. A layer of divergence is defined as a generation; The defined generation is usually the calculation of the branching of the dendritic polymer from the center to the surrounding. The intersection between the same layer and the layer (also known as the branch point) is used as the basis for calculation, from the core center to The surrounding focal points are called an algebra, and five intersections can be represented as a fifth-generation dendrimer; as shown in the first figure, the dendrimer is the only one, so that only one core is called the zeroth generation. The structure has no intersection.

Apparent dendritic polymers are known, such as polyamindoden dendrimer (PAMAM), polyester dendrimer (polyester type) Dendrimer); polyglycerol dendrimer, triazine based dendrimer, poly(propyleneimine) dendrimer, Newkome dendrimer Newkome-type dendrimer, polylysine dendrimer, and other hybrids.

At present, the structural characteristics of the divergent molecules and the self-assembly behavior of the macromolecules are used to create many different performances from the small molecules. The interaction between many small molecules is not strong. When the distance between the molecules and the molecules is close to the unit area, it will help to improve their interaction, which means that the increase in concentration per unit area makes up for the lack of weak interaction between molecules. Therefore, the multi-valent and multi-phase bonding of divergent molecules promotes the ability to enhance binding. In recent years, the use of biomedical materials has gradually covered the fields of drug release, gene therapy, cell membrane penetration, cell structure control and medical imaging.

Therefore, the present invention utilizes the feasibility of combining an amine group of an amino acid with a carboxylic acid to form a hydroxyapatite, whereby the asparagine monomer or the polymer short peptide provides a positive charge to chelate the hydroxyapatite. The multivalent bond characteristics provided by the divergent skeleton, the invention of the multi-dissimilar peptide sequence, give the biomineral better binding ability. In order to improve the concept of combining bio-minerals, the use of polyaspartic acid as a binding target, the introduction of Percec, V. team's per unit area rich multi-functional base topology concept, the combination of aspartic acid or short peptide in divergent type The surface of the skeleton; the invented divergent peptide can exhibit more prominent mineral affinity due to the multivalent binding strategy.

The bone of the organism binds to the intra-bone calcium phosphate with the negatively charged end of collagen and acidic proteins. Proteins bound to biominerals are rich in aspartic acid, glutamic acid and aspartame and phosphorylated serine monomers. In 2003, Johnson, G.A. et al. and LeGeros, R. Z. et al., 2008, elaborated on the importance of osteopontin in organisms. Osteopontin has a polyaspartic acid sequence, so it can not only bind bone but also bone Cell.

The above studies also show that polyaspartic acid does have a binding ability for bones. For example, to develop a system for bone drug delivery, polyaspartic acid is also an important option. Observing Rimola, A. et al. proposed the surface crystal structure of hydroxyapatite; hydroxyapatite contains calcium ions, phosphate ions and oxygen atoms, and the ability to study hydroxyapatite with glycine. It is also believed that the amino acid possesses both a carboxyl group and an amine group, and can provide a binding site for the administration of calcium ions, phosphate ions and oxygen atoms; and it is speculated that the helical peptide is more efficiently bound to the hydroxyapatite, and with various sequences The difference affects its ability to combine. Therefore, in order to overcome the difference in binding produced by the secondary structure of the protein, the bifurcated peptide designed by the present invention will provide the stereoscopicity of the surface of the hydroxyapatite, and the difference in binding ability depends on the distribution in the stereo space. .

Among the compounds, carboxylic acid, phosphoric acid or polyethylene diamine dendrimer (G: 1.5), diethylene triamine pentaacetic acid (DTPA), ethylene diazide tetra Ethylene dinitrilotetra acetic acid (EDTA) and Propylene glycol methylether acetate (PMA) can provide chelating coordination of calcium ions. Among them, EDTA is a wide range of metal chelating coordination reagents, as well as Keum, D.-K. et al., 2003. Bull. Chem. Soc. Jpn . , as shown in the second figure, semi-integer generation (G: 1.5). The polyethylenediamine dendrimer polymer also utilizes structural dispersibility and metal group chelation coordination.

Zaupa, G. et al. developed a dendritic polymer using lysine as a monomer to provide a choice of stereoscopic space. Introducing cysteine into the sequence, forming a lyophilic acid dendrimer using a disulfide bond, and modifying 1,4,7-triazacyclononane on the periphery of the polymer (1,4,7 -triazacyclononane, TACN), TACN will chelate zinc ions. It has been tested that the fourth-generation lysine dendrimer can significantly cleave the effect of ribonucleic acid (RNA) because it exerts multivalent linkage. Therefore, it is considered that the stereoscopic space effect and the multi-directional active position promote the activity. The main reason for playing.

In the past, the combination of a single polyaspartic acid peptide and minerals produced a nearly one-dimensional relationship, but the surface of the hydroxyapatite has multiple calcium ions and phosphates, so The affinity that can be exhibited by a single peptide should be limited, especially if the peptide sequence is not long. If you can create more points, that is, provide a second-degree space coordination opportunity, you should be able to enhance the combination through the clustering effect; this concept has been echoed in the results of the Percec, V. team. They point out that the incorporation of reactive functional groups into the same backbone creates the ability to enrich a polyfunctional group per unit area so that a single molecule exhibits a polyfunctional group sufficient to perform better.

After careful experimentation and research, and the spirit of perseverance, the inventor finally conceived the "multi-disparity peptide configuration with mineral affinity" in this case, and designed a series of peptides to effectively achieve the same as hydroxyapatite. Biomineral molecular affinity provides future biomedical materials or applications for pharmaceutical development. Through the existence of binding elements such as polycarboxylic acid or polyphosphoric acid, the design of the divergent configuration and the biominerality to produce a proper two-dimensional combination relationship, the development of a minimum two-dimensional spatial arrangement of the combination mode, can complement the deficiencies of the prior art. The output of this case will include three types of polymorphic peptides. The potential mineral affinity potential of the invention will be assessed by observing the location of the primary binding unit and using the screening platform to analyze the biomineralization capacity. The following is a brief description of the case.

One of the ideas of the "multi-disparity peptide configuration with mineral affinity" is based on the divergent structure of polyethylenediamine dendrimer and polylysine. The surface is aspartic acid. Form different algebras. To provide a coordination of the second or third degree of space, the binding force can be enhanced by the clustering effect. The invention utilizes the feasibility of combining the amine group of the amino acid with the carboxyl group to form the hydroxyapatite, whereby the asparagine monomer or the polymer short peptide provides a positive charge to chelate the hydroxyapatite, and cooperates with the polyhedral skeleton. The multi-valent bonding property is provided to form a divergent peptide sequence, giving the biomineral better binding ability.

According to the above concept, the present invention provides a bifurcated peptide configuration compound of the formula (1A), the formula (1B), the formula (2) and the formula (3), wherein the U system is selected from the group consisting of U-: Lys-, -Lys-(Asp-NHAc) _J , -NHAc, -Lys-(Asp-) _K -NHAc, (Asp-NHAc) _J ; G and R2 are selected from the group consisting of poly Diamine dendrimer (PAMAM), polyester type dendrimer, polyglycerol dendrimer, triazine based dendrimer, Poly (propyleneimine) dendrimer, Newkome-type dendrimer and polylysine dendrimer; R1 is -( Asp-) _K ; and A, B, J, K, T and Y are both natural numbers, the value of the Y series algebra (generation), the sum of A and B (4 x 2 ^Y ), J = 1 or 2, 20≧K≧1, 32≧T≧1.

The amino acid system represents Aspartic acid with Asp, Lys stands for lysine, Ala stands for alanine, and Gly stands for glycine. NHAc stands for N-Acetyl and OH stands for hydroxyl.

Another aspect of the present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier; and an effective amount of a polymorphic peptide configuration; the pharmaceutical composition can be formulated by a formulation method It is administered to various dosage forms in mammals to exhibit the above-mentioned medical functions. The above-mentioned mammals refer to mammals referred to by humans or biology.

Another concept of the "polymorphic peptide configuration with mineral affinity" of the present invention is to obtain a controllable molecular size and a three-dimensional molecule having a plurality of functional groups on the periphery through a stepwise synthesis step, which facilitates subsequent application and derivatization. And the properties of conventional dendritic polymers or linear polymers The characteristic is that the polymer of the polymorphic peptide has the following characteristics: (1) structural stability and small particle size, (2) easy regulation of hydrophilicity and hydrophobicity, (3) configuration of approximately spherical shape, (4) Preparation according to different algebra to control size and configuration.

1‧‧‧ core

2‧‧‧Differential end

3‧‧‧End functional groups

The first picture is a tree-shaped polymer schematic

1: core 2: divergent end 3: terminal functional group

The second figure of the 1.5th generation of polyethylene diamine dendrimer polymer

Figure III Hydrogen nuclear magnetic resonance spectroscopy of compound B3

The magnetic field is 200 MHz and the solvent is yttrium oxide.

Figure 4 High performance liquid chromatography analysis of compound B1c

(The horizontal axis is the chromatographic time and the vertical axis is the relative intensity. The extract is 0.1% trifluoroacetic acid in methanol, the flow rate is 0.4 ml per minute, and the detection wavelength is 220 nm.)

Figure 5 High performance liquid chromatography analysis of compound B1d

(The horizontal axis is time and the vertical axis is absorption intensity. The extract is 0.1% trifluoroacetic acid in methanol, the flow rate is 0.4 ml per minute, and the detection wavelength is 220 nm.)

Figure 6 The residence time of compound L1c in the hydroxyapatite column

The horizontal axis is time and the vertical axis is absorption intensity.

Figure 7 The residence time of compound B1d in the hydroxyapatite column

The horizontal axis is time and the vertical axis is absorption intensity.

Figure 8 The residence time of compound B3 in the hydroxyapatite column

The horizontal axis is time and the vertical axis is absorption intensity.

In order to achieve the above concept, the present invention is controllable through a stepwise synthesis step. Molecular size, and three-dimensional molecules with numerous functional groups on the periphery. Although the traditional dendritic polymer has various biomedical applications, it does not have the application of mineral affinity; and the linear multi-aspartic acid peptide reference element L1b is known to have the ability to combine with hydroxyapatite, but most The nature of the appropriate number or spatial arrangement has not yet been revealed. The polydisperse peptide polymer developed by the invention can provide a three-dimensional radial arrangement configuration, enhance the binding ability through the multi-phase bonding of the divergent molecules, and create a combination of rich multi-functional groups per unit area.

According to Almora-Barrios, N. et al. and Capriotti, LA et al., the peptide has a secondary configuration that contributes to the production of mineral affinity, such as the control element L1a. Also, George, A. et al., published in Chem. Rev. in 2008, have 6-12 aspartic acid continuous short peptides such as the control element L1b which have significant bone affinity. It is known that the single polyaspartic acid peptide has almost a one-dimensional relationship with mineral production, but there are multiple calcium ions and phosphates on the surface of the hydroxyapatite. It is concluded that the affinity of a single peptide can be limited. Provides a coordination space for the second space, which enhances the combination through the clustering effect. Meanwhile, the functional group of the protein developed by Masica, DL et al . , published in J. Am. Chem. Soc. , 2010, which binds to HAp, should be appropriately arranged to enhance its surface affinity with HAp. Therefore, the present invention uses L1a and L1b as a control, and further uses L1b as a binding basic element, and is modified in a multi-aliased skeleton to evaluate the most appropriate distance between the bifurcated peptide configuration compound and the hydroxyapatite.

Based on A+B=4 x 2 ^Y , when the sum of A and B is 32, the formed multi-alias peptide configuration is 3 algebras, and its diameter is about 4 nm. The sum of A and B is 64, and the resulting multi-alias peptide configuration is 4 algebras with a diameter of about 5 nm. The sum of A and B is 128, and the resulting multi-alias peptide configuration is 5 algebras with a diameter of about 6 nm. The sum of A and B is 256, and the formed multi-alias peptide configuration is 6 algebras, and its diameter is about 7 nm. The R1 of the formula (3) represents -(Asp-) _K , wherein the K value can indicate the number of attached Asp. Although it has been reported that peptides composed of 8-12 aspartic acids have a good affinity for bone, only the hydroxylapatite column test (HAp column test) is used to evaluate mineral affinity. As shown in Figure 1, the residence time of the polydisperse peptide configuration prepared in accordance with an embodiment of the present invention on the hydroxyapatite column is generally divided into 12, 13 and 14 minutes.

Among the linear peptides, the reference element L1b having mineral affinity is known in the literature, and its basic structure is HO-Gly-(Asp) ₆ -NHAc retention time of 13.3 minutes. The L1c of the peptide prepared by the present invention belonging to the formula (1B) has a basic structure of ₂ HN-(Gly) ₃ -(Asp) ₃ -NHAc, and the residence time is 13.0 minutes. The amount of aspartic acid in L1c is half that of L1b alone, and the binding force is only slightly different between 13.0 minutes and 13.3 minutes.

Although the linear peptide control element L1a does not have a functional group (carboxyl group) capable of binding to calcium ions, its secondary helical structure can still provide excellent binding force, and the residence time can be up to 14.1 minutes, thus confirming that the configuration is arranged in a continuous arrangement. The number of aspartic acid is more contributing to the combination of hydroxyapatite.

By comparing the residence time of the divalent peptide configuration compound in the hydroxyapatite column, it is found that: (1) the same number of branches, such as compound B1b, which is separated from the amine acid branching chain by 2 aspartic acid Only a single aspartic acid bound to compound B1a did not show a significant increase in affinity for minerals (2) The total number of aspartic acid was the same, and the amino acid dendrimer backbone of formula (1A) peptide (HO) -Gly-Lys{NHAc}-Lys{Asp-NHAc}-Asp-NHAc), the second algebraic compound B1c has 4 branches and the third algebraic compound B1d has 8 branches, and the total number of Asp modified by the two includes the skeleton having 8 The carboxylic acid retention time was 13.6 and 14.5 minutes, respectively (Table 1). It is clear that the B1d combination with more branches has better performance.

In the compound of the formula (2), the third algebra B2a of the polyethyleneimine dendrimer (PAMAM) has 32 branches, and the sixth algebra B2b has 256 branches, and the residence time is 14.1 and 13.9 minutes, respectively. Large, but B2a has a longer stay. Due to the difficulty of synthesis, the branch of the modified aspartic acid of the B2a compound accounted for 46% of the surface functional groups, and the branch of the single aspartic acid of the B2b compound accounted for 21%, but both were more linear than the linear control element L1b (13.3). Minutes) have a strong combination, showing that the arrangement of multi-dissimilar space can improve the ability to combine with hydroxyapatite.

Coupling element L1b is coupled to the 3rd algebraic skeleton to construct a compound B3 of formula (3) modified with a 16-branched 6-day aspartate-glycine short peptide ((Asp) ₆ -Gly-OH), and 15 branches of Asparagus The amino acid (Asp) was modified to the compound B2a prepared by the same algebraic skeleton synthesis, and the results showed that the B3 retention time was 12.5 minutes and the B2a was 14.1 minutes, and the control element L1b was 13.3 minutes. Each branch of compound B2a modifies only a single aspartic acid, while compound B3 has 6 aspartic acid modified per branch of control element L1b, perhaps arranging an appropriate amount of aspartic acid from the polydisperse space. The compound, B2a, shows a better hydroxyapatite binding ability. Theoretically, it represents that the K value of the total amount of synthetic aspartic acid (Asp) can be modified up to 50 or 100 or even higher. However, it is considered that the aspartic acid has a suitable K value of 20 ≧K≧1 for the binding ability. More preferably 15≧K≧1. The value of T changes with algebra, but due to synthetic factors, the theoretical number of branches can be reached ≧T value.

The skeleton of formula (2) and formula (3) can be selected from the same dendritic polymer, such as polyethylenediamine dendrimer, the sum of A and B of formula (2) and the maximum value of formula (3) Both represent the number of surface functional machines for each algebra, where the B and T values represent the number of modifications and thus A+B≧T≧1.

According to the above concept, another concept of "multi-disparity peptide configuration with mineral affinity" is to promote the binding ability between molecules and molecules through the multi-valent and multi-phase bonding of the polymorphic peptide. . The application of polymorphic peptides in biomedical materials covers drug release, gene therapy, cell membrane penetration, cell structure control, and medical imaging. Therefore, the polymorphic peptide configuration can exhibit more prominent mineral affinity due to the combination of multivalent.

The "polymorphic peptide configuration with mineral affinity" of the present invention has the G value of the formula (2) and R2 of the formula (3) both represent a polyethyleneimine dendrimer polymer (PAMAM), according to the above concept. The polymorphic peptide configuration prepared according to the embodiment of the present invention can be applied to a known dendritic polymer skeleton, such as a polyester type dendrimer developed by Fréchet; a gathering of Frey and Haag Polyglycerol dendrimer, Simanek triazine based dendrimer, Meijer poly(propyleneimine) dendrimer, Newkome dendrimer Newkome-type dendrimer, polylysine dendrimer, and other types. Polyethylenediamine dendrimer (PAMAM), triazine based dendrimer, poly(propyleneimine) dendrimer and polylysine Skeletons such as polylysine dendrimer are more suitable for direct application; other types or mixed polymer skeletons, if the surface functional group is non-amino group (NH ₂ ), must be subjected to appropriate chemical treatment to carry out the reaction.

According to the above concept, the peptide preparation method of the "polymorphic peptide configuration with mineral affinity" of the present invention is a preparation method for modifying the standard solid phase peptide synthesis strategy, and (flow 1) is based on solid phase peptide synthesis. The Solid Phase Peptide Synthesis (SPPS) comprises the following steps: (1) selecting an appropriate resin as a starting material and immersing in an organic solvent; and (2) removing a methoxycarbonyl group by using a deprotection reagent (DEP). a nitrogen-terminal protecting group of (fluorenylmethyloxycarbonyl, Fmoc); (3) an amino acid and a coupling reagent such as benzotriazole-1-yl-oxytrispyrrolidinophosphonium hexafluoro -phosphate, PyBOP), adding an active solution (ACT) for coupling; (4) after the reaction is over, stopping the incomplete resin with a capping reagent (CAP); (5) repeating the above steps (2) ), (3) and (4).

As the raw material for synthesizing the above amino acid, a commercially available product such as N-9-Fluorenylmethoxycarbonyl-alanine (Fmoc-Ala-OH) or methoxycarbonyl hydrazine-lysine can be used. (tert-Butoxycarbonyl)-carboxyl (Fmoc-Lys(Boc)-OH), fluorenylmethoxycarbonyl-aspartic acid (tert-butyl ester)-carboxyl (Fmoc-Asp(tBu)-OH), methoxyl Carbonyl hydrazine-lysine [4,4-dimethyl-2,6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (Fmoc-Lys(ivDde)-OH), fluorenylmethoxycarbonyl醯-Lysine (芴methoxycarbonyl oxime)-carboxyl (Fmoc-Lys(Fmoc)-OH), can also be coupled with 4-carboxybenzaldehyde to obtain the desired multi-alias The peptide configuration.

The organic solvent for soaking the raw material is selected from N , N -dimethylformamide (DMF) or other fat-soluble solvent to promote the wetting and expansion of the resin to assist the subsequent reaction. The degree of the reaction was confirmed by an amine detecting reagent (Kaiser test); and after the reaction was completed, the unreacted resin was terminated with a terminating reagent (CAP). With the length of the sequence and the characteristics of the amino acid, the above coupling reaction can be repeated to obtain the desired multi-dimensional peptide configuration, in which the deprotection reagent (DEP) and the activation solution (ACT) are operated with the amino acid at the sequence length. Different types.

Considering the stereoscopic spatial effect and the multivalent effect, the present invention develops different algebraic dendrimer dendrimers; the third to sixth generation polyethylenediamine dendrimer polymers utilize commercially available raw materials. The aspartic acid is modified on the amino group (NH ₂ ) of the desired backbone to synthesize the compound B1a, B1b, B1c, B1d of the formula (1A) as shown in Table 2; the compound B1e of the formula (1B); the compound B2a of the formula (2) , B2b and formula (3) B3 and other polymorphic peptides.

Synthetic element L1a is synthesized in the same manner as in the first step of the procedure, using fluorenylmethoxy-alanine-carboxyl group (Fmoc-Ala-OH) as the amino acid raw material, and sequentially bonding 5 alanine to Alanine. In the sequence, as the number of reactions increases, the reaction time also increases, and product 2 can be obtained; the methoxycarbonyl hydrazine-decateuric acid (tert-butoxycarbonyl) carboxyl group (Fmoc-Lys(Boc)-OH) is coupled with PyBOP. The product 3 can be obtained; the product L1a (Scheme 2) can be obtained by removing from the resin with 95% trifluoro acetic acid (TFA).

The procedure for synthesizing L1b is compared to the step of product L1a, as set forth in Scheme 3. Using a pre-charged methoxycarbonyl-glycine king resin, deprotected with methoxycarbonyl hydrazine-aspartate The coupling reaction, continuous synthesis of 6 aspartic acid on the sequence, can obtain the product 5; finally, the peptide is cut off from the resin to obtain the product L1b.

According to the above procedure, after deprotecting the resin, three times of hydrazine-glycine-carboxyl (Fmoc-Gly-OH) coupling and three times of trimethyl methoxycarbonyl-aspartate-carboxyl (Fmoc-Asp) were introduced continuously. (tBu)-OH) Coupling, and then removing the peptide from the resin, the product L1c can be obtained smoothly (Scheme 4).

The "polymorphic peptide configuration with mineral affinity" of the present invention is prepared by adjusting the compound of formula (1A) or formula (1B) according to the basic strategy of synthesis of the peptide (Scheme 1).

As shown in Scheme 5-1, after removing the resin's fluorenylmethoxycarbonyl (Fmoc) protecting group, it is associated with methoxycarbonyl hydrazine-lysine ([4,4-dimethyl-2,6-dioxo) After the coupling of the cyclohex-1-ylideneethyl)-carboxyl group (Fmoc-Lys(ivDde)-OH) is completed, the fluorenylmethoxycarbonyl (Fmoc) protecting group of the product 6 is removed, and the methoxycarbonyl is used.醯-Lysine (芴methoxycarbonyl hydrazine)-carboxyl group (Fmoc-Lys(Fmoc)-OH) was subjected to the next coupling reaction to obtain a product 7 containing two protecting groups of fluorenylmethoxycarbonyl (Fmoc). The first divergence point of the dendrimer-like polymer appears; then the fluorenylmethoxycarbonyl (Fmoc) protecting group is removed, and the fluorenylmethoxycarbonyl-aspartate (tert-butyl ester)-carboxyl group (Fmoc-Asp (Fmoc-Asp) is added. tBu)-OH) is coupled to obtain product 8, which is removed to remove the protecting group, and the resin is removed to obtain B1a. If the number of aspartic acid is increased, the above-mentioned peptide synthesis step can be repeated after removing the methoxycarbonyl hydrazine (Fmoc) protecting group of the product 8, and the methoxycarbonyl hydrazine-aspartate is added. (tert-Butyl ester)-carboxyl group (Fmoc-Asp(tBu)-OH) can obtain B1b containing 2 aspartic acid continuous.

If the sequence of the polypyretic peptide is modified, as shown in Scheme 5-2, after removing the protective group of the methoxycarbonyl hydrazine (Fmoc) of the product 7, the methoxycarbonyl hydrazine-lysine is used. The methoxycarbonyl hydrazine-carboxyl group (Fmoc-Lys(Fmoc)-OH) is coupled to obtain a new skeleton branching point, and in this way, various algebraic polydisperse lysine polymer skeletons can be obtained by repeating the reaction. Depending on the target, at the appropriate branching point, each branch is modified with 1-2 methoxycarbonyl hydrazine-aspartate (tert-butyl ester)-carboxyl groups (Fmoc-Asp(tBu) , -OH), a polyaspartic acid polymer having a polydisperse lysine polymer as a skeleton, such as compounds B1c and B1d, can be obtained.

A single or a pair of addition products can be obtained in Scheme 5-1 or 5-2 using the same or different amine side chain and amino terminal protecting groups. For example, fluorenylmethoxycarbonyl- lysine (fluorenylmethoxycarbonyl)-carboxyl (Fmoc-Lys(Fmoc)-OH) can be removed under the treatment of a piperidine while removing the side. a chain and an amino terminal protecting group, followed by a pair of addition compounds of the formula (1B), or a single addition of a compound of the formula (1A) via an equivalent number; for example, fluorenylmethoxycarbonyl-lysine ([ 4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (Fmoc-Lys(ivDde)-OH) as a raw material, its methoxycarbonyl hydrazine and [4 , 4-Dimethyl-2,6-dioxocyclohex-1-ylidene] Ethyl can be removed by using piperidine and hydrazine, respectively, and can be used to obtain individual additions at various positions. Compound.

Process 5-2

The peptide compound of the formula (2) of the "polymorphic peptide configuration with mineral affinity" of the present invention can be prepared under liquid phase homogenization conditions.

To complete the reaction, a microwave system was introduced to assist in the synthesis of compounds B2a and B2b. Mixing different algebraic polyethylenediamine dendrimers (PAMAM) with methoxymethoxyindole-aspartate (tert-butyl ester)-carboxyl (Fmoc-Asp(tBu)-OH) in methylmorpholine (N-methyl morpholine) in a solution of dimethylformamide (DMF), a small amount of ethanol can be added to assist the smooth dissolution of the dendritic polymer, and the reaction is carried out in a microwave reaction tank for 5 hours. Subsequent removal of all protecting groups, preliminary purification using a dialysis membrane, and the resulting crude product was subjected to Sephadex G50 colloidal permeation chromatography. Purification, elution with water as the mobile phase, to obtain the compound B2a as a yellow oil. B2b was obtained according to the synthesis and purification mode of B2a, but the total microwave reaction time was 9 hours.

Another concept of the present invention is the preparation of a polyethylenediamine dendrimer containing a multi-day aspartic acid short peptide, which refers to the preparation step of the multi-alias peptide configuration of the formula (3), and the preparation is repeated six times. The peptide product of aspartic acid 5 . The product 5 is protected with a fluorenylmethoxycarbonyl (Fmoc) protecting group, 4-carboxylic benzylaldehyde (4-carboxylbenzaldehde) is attached to the peptide, and then reacted with a third-generation polyethylenediamine dendrimer. Compound B3 can be obtained by cutting off the resin.

The final product of the present invention was judged for its purity by high performance liquid chromatography, and its structure was identified by mass spectrometry and nuclear magnetic resonance, and the obtained compound was confirmed by the data. For example, compound B3, as shown in the third graph, shows that there is no aldehyde group signal between 10 and 8 ppm, and there are two sets of double-cleavage signals between 8 and 7 ppm, which are the signals of hydrogen on the disubstituted benzene ring, 3.2~ There are many groups of signals between 2.8ppm, which are thought to be signals of polyethylenediamine dendrimers, and between 2.8 and 2.6ppm there is a signal for the beta position of aspartic acid.

According to the above concept, the oligopeptide derivative of the present invention can be administered in combination with an acceptable pharmaceutical carrier, adjuvant, diluent, excipient or solvent as a pharmaceutical composition when administered to an individual to provide a therapeutic effect. . The aforementioned multi-peptides can be formulated into dosage forms for use in different routes of administration using conventional methods, for example, the pharmaceutical compositions can be directly delivered to the medicinal site. The dosage form is determined by the route of administration, the nature of the formulation, the condition of the disease, the size, weight, surface area, age and sex of the patient, and other compatible drugs. The efficacy of the pharmaceutical composition can be initially assessed in vitro and then administered to the animal to assess the efficacy of the composition on the medicinal site.

The above excipients, or "pharmaceutically acceptable carriers or excipients", "bioavailable carriers or excipients", include solvents, dispersing agents, coatings, antibacterial or antifungal agents, or Any suitable compound suitable for the preparation of a dosage form such as an absorbent is delayed. Usually such carriers or excipients do not themselves have the activity of treating diseases, and the derivatives disclosed in the present invention, together with pharmaceutically acceptable carriers or excipients, are prepared for administration to animals or humans. Causes adverse reactions, allergies or other inappropriate reactions. Thus, the derivatives disclosed herein, in combination with pharmaceutically acceptable carriers or excipients, are suitable for use in clinical and human applications. The therapeutic effect can be achieved by administering the dosage form of the compound of the present invention intravenously, orally, by inhalation or by local or sublingual administration such as nasal, rectal, vaginal or the like.

The carrier will vary with each dosage form, and the sterile injectable compositions may be solution or suspended in a non-toxic intravenous diluent or solvent such as 1,3-butanediol. A carrier acceptable therebetween may be Mannitol or water. In addition, a fixed oil or a suspension of a synthetic mono- or bis-glycoside oil ester is a commonly used solvent. Fatty acids, such as oleic acid, olive oil or castor oil, and their oleic acid oil ester derivatives, especially in the form of polyoxyethylation, can be used as an injection preparation and are natural pharmaceutically acceptable oils. These oil solutions or suspensions may contain long chain alcohol diluents or dispersants, carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tween, Spans or other similar emulsifiers or are used in the general pharmaceutical manufacturing industry for pharmaceutically acceptable solid, liquid or other bioavailable enhancers which are useful in the development of dosage forms.

The composition for oral administration is in any orally acceptable dosage form, and the form thereof includes a capsule, a tablet, a tablet, an emulsifier, a liquid suspension, a dispersing agent, and a solvent. Oral dosage forms are generally used as carriers, and in the case of tablets, lactose, corn starch, and a lubricant such as magnesium stearate are basic additives. The diluent used in the capsules includes lactose and dried corn starch. The liquid suspension or emulsifier dosage form is prepared by suspending or dissolving the active substance in an oily interface combined with an emulsifier or a suspending agent, and adding a moderate sweetener, flavor or coloring pigment as needed.

Nasal gasifying sprays or inhalant compositions can be prepared according to known formulation techniques. For example, dissolve the composition in physiological saline, add benzyl alcohol or other suitable defense A humic agent, or an absorbent, to enhance bioavailability. The compositions of the compounds of the invention may also be formulated as a suppository for rectal or vaginal administration.

The compounds of the present invention may also be administered "intravenous administration", including subcutaneous, intraperitoneal, intravenous, intramuscular, or intra-articular, intracranial, intra-articular, intraspinal injection, aortic injection, intrathoracic injection, intralesional injection. , or other suitable drug delivery technology.

Therefore, the present invention is a rare and innovative invention, which has profound industrial value and is submitted in accordance with the law.

The "polymorphic peptide configuration with mineral affinity" set forth in this application will be fully understood by the description of the following examples, so that those skilled in the art can accomplish this. However, the embodiments of the present invention are to be construed as being unrestricted by those skilled in the art, and those skilled in the art can practice other embodiments in accordance with the spirit of the disclosed embodiments. The scope of protection of the scope of application for patents.

I. Drugs and equipment:

General chemicals were purchased from TEDIA, ACROS and Merck. All chemicals were not further purified unless specifically described. All amino acids, benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP) and preloaded Glycine Wang resin were purchased from Novabiochem. Polyethylenediamine dendrimer (PAMAM) was purchased from Dendritech. A second type of calcium phosphate hydroxyapatite (Hydrohxylapaptite) having a particle pore size of 20 microns was purchased from Bio-Rad. Carbon and 18 powders were adsorbed from Merck Corporation by purchasing reverse-phase thin-layer chromatography (RP-TLC). LH20 and G75 colloids were purchased from GE Healthcare as a stationary phase material for Liquid Chromatography (LC). The product was analyzed using a Varian 400 MHz nuclear magnetic resonance spectrum (NMR). The molecular weight of the product was identified by assisted matrix laser mass spectrometry (MALDI-TOF/MS spectra) of Bruker's Autoflex III. The purity of the product was analyzed using Agilent's 1100 Series High-performance Liquid Chromatography (HPLC) with a carbon 5 trans-column.

II. Other reagents

1. methoxycarbonyl hydrazine-alanine-carboxyl group (Fmoc-Ala-OH)

2. 芴 methoxycarbonyl hydrazine - lysine (tert-butoxycarbonyl)-carboxyl (Fmoc-Lys (Boc)-OH)

3. 芴 methoxycarbonyl hydrazine - aspartate (tert-butyl ester) - carboxyl group (Fmoc-Asp (tBu)-OH)

4. 芴methoxycarbonyl hydrazine-lysine ([4,4-dimethyl-2,6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (Fmoc-Lys(ivDde)-OH )

5. 芴 methoxycarbonyl hydrazine - lysine (芴methoxycarbonyl hydrazine) - carboxyl group (Fmoc-Lys (Fmoc)-OH)

6. 4-carboxybenzaldehyde

7. Phenoxytriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP)

III. Reagent preparation

1. Deprotection reagent (DEP for short), which is 20% apiperidine dimethylformamide (DMF).

2. The activation solution (ACT for short) is formulated as 10% N-methyl-N-methylmorpholine dimethylformamide (DMF).

3. Stop reagent (abbreviated as CAP), prepared with 5 ml of dimethylformamide and 1 ml of acetic anhydride (Ac ₂ O).

4. Kaiser test (Kaiser test) is also known as amine detection or ninhydrin detection, with 0.28M ninhydrin ethanol solution, pyridine and 42.37M phenol ethanol solution and Three solutions such as pyridine were added to the resin in equal proportions, and the color change was observed after heating to 120 ° C; the blue-violet color showed an amine group, and the pale yellow color showed no amine residue. Unless otherwise specified, the deprotected methoxycarbonyl oxime protection step of the present invention is successful with blue-violet color; the amino acid coupling step is successful with light yellow color.

Hydroxyapatite column test (HAp column test)

Binding tests were carried out using hydroxyapatite particles packed in a column. The diameter and length of the hydroxyapatite column were 0.7 and 5 cm, respectively. The experiment was performed with a high performance liquid chromatography system. The extract was 0.5 mM PBS. (pH 7.4), the flow rate is set to 0.25 ml per minute, and the detection wavelength is 220nm. The residence time of each compound is summarized in Table 1; and the representative residence time, such as L1c, B1d, and B3, is shown in the sixth to eighth figures.

Example 1 Preparation of Synthetic Element L1a

0.2 mM (0.328 g) of rink amide resin was placed and reacted with deprotecting reagent for five minutes, followed by addition of 0.125 g of methoxymethoxy hydrazine-alanine-carboxyl (0.4 mM) and 0.208 g of hexafluorophosphate. And triazol-1-yl-oxytripyrrolidinylphosphine (0.4 mmol), and the activation solution was reacted for 30 minutes. After the coupling was determined to be successful, the termination reagent was added for 25 minutes. The subsequent five consecutive additions of alanine were carried out in the same dosages and steps with a reaction time of 40, 50, 60, 60 and 60 minutes. Thereafter, the terminal methoxycarbonyl protecting group was removed, followed by 0.187 g of methoxycarbonyl hydrazine-lysine (tert-butoxycarbonyl)-carboxyl (0.4 mM) and 0.208 g of benzotriazole hexafluorophosphate. Synthesis of -1-yl-oxytripyrrolidinylphosphine (0.4 mmol), after 90 minutes of reaction, the terminal methoxycarbonyl protecting group was removed, and then added to 3 ml with 1 ml of acetic anhydride. The dimethylformamide-soaked resin was reacted for 20 minutes, the resin was taken out, reacted with 95% trifluoroacetic acid for two hours, the solid was removed, and the filtrate was concentrated under reduced pressure to give the desired L1a peptide.

The purity was analyzed by high performance liquid chromatography, and a carbon 18 column was used, and the mobile phase was a methanol solution of trifluoroacetic acid having a weight percentage of 0.1. The detection wavelength was set to 220, the chromatographic ambient temperature was set to 25 ° C, and the flow rate was set to 0.4 ml per minute, by chromatographic analysis, it was found that there was a major single signal with a residence time of 6.5 minutes.

Example 2 Preparation of Synthetic Element L1b

Put 0.2 mmol of moir (0.328 g) of the king resin with glycine acid first, and use a deprotecting reagent for five minutes, then add 0.165 g of methoxymethoxyindole-asparagine (tert-butyl ester)-carboxyl group. (0.4 mmol) and 0.208 g of benzotriazol-1-yl-oxytripyrrolidinylphosphonate (0.4 mmol), and the activation solution was reacted for 30 minutes, and the termination reagent was added for 25 minutes. After the same dose and step, the addition of aspartic acid was continued for four consecutive days, and the reaction time was 45, 60, 60 and 60 minutes, respectively; the terminal methoxymethoxycarbonyl protecting group was removed, and then 1 ml of acetic acid was added. Add the anhydride to the resin soaked in 3 ml of dimethylformamide and react for 20 minutes. The resin was taken out, reacted with 95% by weight of trifluoroacetic acid for two hours, the solid was removed, and the filtrate was concentrated under reduced pressure to give the desired peptide, that is, L1b.

The crude product was dissolved in a mixed solution (MeOH:H ₂ O:MeCN=2:1:1), and purified using a medium pressure liquid chromatography system using a carbon 18 trans-polar column at a flow rate of one minute per minute. 0.5 ml was identified via a carbon 18 trans polar thin layer chromatography plate to obtain a main product having a main retention ratio (Rf) of 0.3. The purity was analyzed by high performance liquid chromatography, and a carbon 5 column was used, and the mobile phase was a methanol solution of trifluoroacetic acid having a weight percentage of 0.1. The detection wavelength was set to 220, and the chromatographic ambient temperature was set to 25 ° C. It was set to 0.4 ml per minute. Through chromatography analysis, it was found that there was a main single signal with a residence time of 6.1 minutes; matrix-assisted laser mass spectrometry analysis of L1b gave 788 (L1b theoretical molecular weight + sodium) signal.

Example 3 Preparation of Compound L1c

0.2 mM (0.328 g) of rink amide resin was placed and reacted with deprotecting reagent for five minutes, followed by addition of 0.119 g of methoxycarbonyl hydrazine-glycine (0.4 mM) and 0.208 g of hexafluorophosphate. Triazol-1-yl-oxytripyrrolidinylphosphine (0.4 mmol), and reacted with the activation solution for 30 minutes, after which the termination reagent was added for 25 minutes. The subsequent two glycine additions were carried out at the same dose and procedure for a reaction time of 45 minutes. Subsequently, the terminal fluorenylmethoxycarbonyl protecting group was removed, and then coupled with 0.165 g of methoxymethoxyindole-aspartate (tert-butyl ester)-carboxyl (0.4 mM) for 50 minutes, and the addition was terminated. The reagent, the addition of the fourth amino acid was completed; the subsequent aspartate addition was carried out in the same dose and procedure for 60 minutes. After the protective group of fluorenylmethoxycarbonyl hydrazine was removed, 1 ml of acetic anhydride was added to the resin soaked in 3 ml of dimethylformamide. After reacting for 20 minutes, the resin was taken out, and the weight was 95. The fluoroacetic acid was reacted for two hours, the solid was removed, and the filtrate was concentrated under reduced pressure to give the desired peptide, that is, L1c.

The crude product was dissolved in a mixed solution (MeOH:H ₂ O:MeCN=2:1:1), and purified using a medium pressure liquid chromatography system using a carbon 18 trans-polar column at a flow rate of one minute per minute. 0.5 ml was identified via a carbon 18 trans polar thin layer chromatography plate to obtain a main product having a main retention ratio (Rf) of 0.3. The purity was analyzed by high performance liquid chromatography, and a carbon 5 column was used, and the mobile phase was a methanol solution of trifluoroacetic acid having a weight percentage of 0.1. The detection wavelength was set to 220, and the chromatographic ambient temperature was set to 25 ° C. It was set to 0.4 ml per minute, and by chromatography analysis, it was found that there was a main single signal with a residence time of 6.5 minutes.

Example 4 Preparation of Compound B1a

Put 0.2 mmol of moir (0.328 g) into the glycine resin, and then protect for five minutes, then add 0.236 g of methoxycarbonyl hydrazine-lysine ([4,4-dimethyl-2, 6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (0.4 mM) and 0.208 g of benzotriazol-1-yl-oxytripyrrolidinylphosphonate (0.4 mmol) ()), reacted in the activation solution for 40 minutes, followed by the addition of the termination reagent for 25 minutes. The addition of the first amino acid. After removing the terminal methoxycarbonyl protecting group, it was coupled with 0.236 g of hydrazine methoxy hydrazine- lysine (fluorenylmethoxycarbonyl)-carboxyl (0.4 mM), and the reaction was added for 70 minutes. . After removing the two fluorenylmethoxycarbonyl protecting groups from the amine, 0.247 g of methoxymethoxyindole-aspartate (tert-butyl ester)-carboxyl (0.6 mM) and 0.312 g of hexafluorophosphoric acid were used. Benzotriazol-1-yl-oxytripyrrolidinylphosphine (0.6 mmol) was reacted for 60 minutes; then, after removal of the terminal fluorenylmethoxycarbonyl protecting group, ie, 95% by weight of trifluoro The acetic acid was reacted for two hours, the solid was removed, and the filtrate was concentrated under reduced pressure to give the desired B1a peptide.

The crude product is dissolved in a 1:9 mixture of methanol and water, separated by a carbon 18 trans-polar column, and detected and tested by a thin layer chromatography plate of trans polar carbon 18 to obtain a single main product. The purity was determined by high performance liquid chromatography, and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid of 0.1% by weight. The detection wavelength was set to 220 and the chromatographic ambient temperature was set to 25. °C, the flow rate was set to 0.4 ml per minute, and by chromatography analysis, it was found that there was a main single signal with a residence time of 8.0 minutes.

Example 5 Preparation of Compound B1b

Put 0.2 mmol of moir (0.328 g) into the glycine resin, and remove the protective reagent for five minutes, then add 0.236 g of methoxymethoxy hydrazine-lysine ([4,4-dimethyl -2,6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (0.4 mM) and 0.208 g of hexafluorophosphoric acid Benzotriazol-1-yl-oxytripyrrolidinylphosphine (0.4 mmol) was added to the activation solution for 40 minutes, and the terminating reagent was added for 25 minutes. After removing the terminal methoxymethoxy hydrazine protecting group, a coupling reaction was carried out with 0.236 g of hydrazine methoxy hydrazine- lysine (fluorenyl methoxycarbonyl)-carboxyl (0.4 mM) for 70 minutes, followed by addition of a reagent for termination. After 25 minutes; after removing the protective group of two fluorenylmethoxycarbonyl hydrazines, 0.247 g of hydrazine methoxy hydrazine-aspartate (tert-butyl ester)-carboxyl (0.6 mM) and 0.312 g of hexafluoride were used. After reacting benzotriazol-1-yl-oxytripyrrolidinylphosphoric acid phosphate (0.6 mmol) for 60 minutes, a stop reagent was added for 25 minutes. Subsequent aspartic acid addition was followed by the same procedure, reagents and doses for a reaction time of 80 minutes. All of the fluorenylmethoxycarbonyl protecting groups at the end were removed, i.e., reacted with 95% by weight of trifluoroacetic acid for two hours, the solids were removed, and the filtrate was concentrated under reduced pressure to give the desired B1b peptide. The crude product is dissolved in a 3:7 mixture of methanol and water, separated by a carbon 18 trans-polar column, and detected and tested by a carbon 18 trans-polar thin layer chromatography plate to obtain a single main The product was further subjected to an ethyleneation reaction with acetic anhydride for 30 minutes, and it was confirmed that ninhydrin was pale yellow, that is, B1b was obtained.

The purity was determined by high performance liquid chromatography, and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid of 0.1% by weight. The detection wavelength was set to 220 and the chromatographic ambient temperature was set to 25. °C, the flow rate was set to 0.4 ml per minute, and by chromatography analysis, it was found that there was a main single signal with a residence time of 8.1 minutes.

Example 6 Preparation of Compound B1c

0.2 mmol of moir (0.328 g) was placed in the pre-glycolic acid resin to deprotect the reagent for five minutes, followed by the addition of 0.236 g of methoxycarbonyl hydrazine-lysine ([4,4-dimethyl -2,6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (0.4 mM) and 0.208 g of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate 0.4 mmoles), the reaction was carried out for 40 minutes in the activation solution, followed by the addition of the terminating reagent for 25 minutes. After removing the terminal methoxymethoxy hydrazine protecting group, the coupling reaction was carried out with 0.236 g of hydrazine methoxy hydrazine- lysine (fluorenyl methoxycarbonyl)-carboxyl (0.4 mM) for 70 minutes, and then the reaction reagent was added. 25 minutes. The subsequent amino acid addition was carried out in the same step, and the dose and time were 0.354 g (0.6 mmol), respectively, and methoxycarbonyl hydrazine-lysine (芴methoxy) Carboxycarbonyl)-carboxyl and 90 minutes. After removing all the methoxycarbonyl protecting groups, 0.329 g of methoxymethoxyindole-aspartate (tert-butyl ester)-carboxyl (0.8 mmol) and 0.416 g of benzotriazole-1 hexafluorophosphate were added. -Base-oxytripyrrolidinylphosphine (0.8 mmol) was reacted for 90 minutes and reacted with a stop reagent for 25 minutes. Subsequent amino acid addition was carried out in accordance with the above synthetic procedure, and the amino acid dose and reaction time were respectively 0.329 g of methoxymethoxyindole-aspartate (tert-butyl ester)-carboxyl group (0.8 mmol) and 120 minutes. Finally, the terminal methoxy methoxy protecting group was removed, and then 1 ml of acetic anhydride was added to the resin soaked in 3 ml of dimethylformamide. After 20 minutes, the liquid was filtered off, followed by 4%. The hydrazine dimethyl decylamine was reacted for 10 minutes, and the resin was removed by filtration, and reacted with 95% by weight of trifluoroacetic acid for two hours to remove the solid, and the filtrate was concentrated under reduced pressure to give the desired B1c peptide.

Dissolve the crude product in water, with a carbon 18 trans-polar column and a high-performance liquid chromatograph UV detector, the wavelength is set to 320; multiple batches of products are separated, the first batch of column injection is 55 mg, the wavelength was set at 220, and the flow rate was 0.5 ml per minute to obtain the main isolate. The purity was analyzed by high performance liquid chromatography (Fig. 4), and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid having a weight percentage of 0.1, and the detection wavelength was set to 220. The ambient temperature was set to 25 ° C and the flow rate was set to 0.4 ml per minute. After chromatography analysis, it was found that there was a main single signal with a residence time of 9.6 minutes. Matrix-assisted laser mass spectrometry analysis of L1b yielded a 1740 (B1c theoretical molecular weight + sodium) signal. ^</ RTI><RTIgt; 2.84 (m, 16H), 2.34 (s, 3H), 1.97 (s, 9H), 1.90 (s, 3H), 1.7 (m, 8H), 1.43 (m, 8H), 1.30 (m, 8H).

Example 7 Preparation of Compound B1d

Put 0.2 mmol of moir (0.328 g) into the glycine resin, and then use a deprotecting reagent for five minutes, then add 0.236 g of methoxymethoxy hydrazine-lysine ([4,4-dimethyl Benzyl-2,6-dioxocyclohex-1-ylidene]ethyl)-carboxyl (0.4 mM) and 0.208 g of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (0.4 mil), and activation solution 40 minutes Clock, then add the termination reagent to complete the synthesis of this step; remove the terminal methoxymethoxy hydrazine protecting group, add 0.236 g of methoxymethoxy hydrazine-lysine (芴methoxycarbonyl hydrazine)-carboxyl (0.4 mmol) The coupling reaction was carried out for 70 minutes, followed by the addition of a terminating reagent to complete the synthesis of this step; the amino acid as in the above step was coupled twice with the step, only twice the equivalent amount in one more step. After removing the terminal methoxymethoxy hydrazine protecting group, 0.412 g of hydrazine methoxy hydrazine-aspartate (tert-butyl ester)-carboxyl (1.0 mM) and 0.521 gram of benzotriazole hexafluorophosphate were added. --Methoxy-p-pyrrolidinophosphoryl (1.0 mM) was reacted for 120 minutes, and a stop reagent was added for a further 25 minutes. After removing the terminal methoxymethoxy hydrazine protecting group, add 3 ml of dimethylformamide soaked resin, remove the resin liquid after 25 minutes of reaction, and add 4% by weight of hydrazine dimethyl The guanamine was reacted for 10 minutes, and the resin was taken out and reacted with trifluoroacetic acid of 95% by weight for two hours, the solid was removed, and the filtrate was concentrated under reduced pressure to give the desired B1d peptide.

The crude product B1d was dissolved in water and separated and purified using a medium pressure liquid chromatography system, and equipped with a carbon 18 trans-column and a high-performance liquid chromatograph ultraviolet light detector, wherein the wavelength was set to 320; The product was separated. The injection was 55 mg per batch, the wavelength was set to 320, the flow rate was 0.5 ml per minute, and the isolate was collected for 41 to 58 minutes to obtain the main isolate. The purity was analyzed by high performance liquid chromatography (figure 5), and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid having a weight percentage of 0.1, and the detection wavelength was set to 220. The ambient temperature was set to 25 ° C and the flow rate was set to 0.4 ml per minute. After chromatography analysis, it was found that there was a main single signal with a residence time of 9.4 minutes. ¹ H-NMR (solvent: D ₂ O; 400 MHz): δ 4.60 (m, 6H), 4.16 (m, 8H), 3.92 (m, 2H), 3.62 (s, 2H), 3.11 (br, 16H), 2.77 (m, 16H), 1.96 (s, 24H), 1.88 (s, 3H), 1.68 (s, 16H), 1.42 (m, 14H), 1.28 (m, 18H).

Example 8 Preparation of Compound B1e

0.2 mM (0.328 g) of rink amide resin was placed and reacted with deprotecting reagent for five minutes, followed by addition of 0.481 g of methoxymethoxy hydrazine- lysine (芴methoxycarbonyl hydrazine)-carboxyl (0.8 mM) And 0.416 g of benzotriazol-1-yl-oxytripyrrolidinylphosphonium phosphate (0.4 mmol), and the activation solution was reacted for 60 minutes, and the termination reagent was added to complete the combination of the steps. Repeatedly, the coupling of the lysine was repeated twice as described above, and the reaction time was 90 and 120 minutes respectively; the terminal methoxycarbonyl hydrazine protecting group was removed, and then 1 ml of acetic anhydride was added to the existing 3 ml of the second Methylformamide-soaked resin was reacted for 25 minutes, the resin was taken out, reacted with trifluoroacetic acid of 95% by weight for two hours, the solid was removed, and the filtrate was concentrated under reduced pressure to give the desired B1e peptide.

Example 9 Preparation of Compound B2a

In a 10 ml tube, 0.261 g of benzotriazol-1-yl-oxytripyrrolidinylphosphonate (0.5 mmol) and 0.206 g of methoxymethoxyindole-aspartate (tert-butyl ester) were placed. After the mixture of the carboxyl group (0.5 mM) and the activation solution for 30 minutes, 0.053 g of a third-generation polyethylenediamine dendrimer (7.6 mM) and 0.1 ml of ethanol were added; and then transferred to a microwave reactor. The wattage was set to 200 W, the temperature was 80 ° C, and the reaction time was five hours. The crude product was added to a solution of 20% piperidine in dimethylformamide, and the reaction was taken overnight. After removing the solvent, an aqueous solution of 95% trifluoroacetic acid was added thereto, and the reaction was carried out for two hours, and the solvent was removed to obtain a crude yellow product. After the crude product was dissolved in 10% aqueous solution of dimethyl sulfoxide, it was transferred into a dialysis membrane with a molecular weight of 3,500, and the external liquid was a 5% methanol aqueous solution. After changing water three times a day for four days, the solvent was removed to obtain an oily crude product. Sephadex G75 colloid was used as the static phase, the crude product was dissolved in water, placed in the column, and washed with water as the mobile phase. The collected liquid was identified by carbon 18 trans thin-layer chromatography plate. The compound of the same main spot ratio retention factor (Rf) was collected and concentrated to obtain a compound B2a as a yellow oil.

The purity was determined by high performance liquid chromatography, and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid of 0.1% by weight. The detection wavelength was set to 220 and the chromatographic ambient temperature was set to 25. °C, the flow rate was set to 0.4 ml per minute, and by chromatography analysis, it was found that there was a main single signal with a residence time of 4.7 minutes. (8th) ¹ H-NMR (solvent: D ₂ O; 200 MHz): 4.10 (t, J = 7 Hz, 17H), 3.55 (m, 74H), 3.26 (m, 64H), 3.05 (m, 80) , 2.80 (m, 78H), 2.43 (m, 43H), 2.25 (m, 44H).

Example 10 Preparation of Compound B2b

Synthesis of aspartic acid on the surface of the sixth generation of digital polymers, as The preparation method of B2a is only nine hours. The purity was determined by high performance liquid chromatography, and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid of 0.1% by weight. The detection wavelength was set to 220 and the chromatographic ambient temperature was set to 25. °C, the flow rate was set to 0.4 ml per minute, and by chromatography analysis, it was found that there was a main single signal with a residence time of 2.4 minutes.

Example 11 Preparation of Compound B3

The L1b element was first synthesized as described in Example 2, followed by removal of the terminal fluorenylmethoxycarbonyl protecting group, and addition of 4-carboxylic acid benzaldehyde (0.061 mg, 0.4 mmol) for 100 minutes. After adding the terminating reagent for 25 minutes, a third-generation polyethylenediamine dendrimer (0.021 g, 0.1 mmol) was added to the resin which had been immersed in dimethylformamide, and the reaction was further carried out for 30 minutes. The sodium triethoxy borohydride (3 mM) was stirred for two days and the end of the synthesis was determined. The reaction solution was filtered off, and then reacted with a 95% aqueous solution of trifluoroacetic acid for two hours. After removing the solid, the filtrate was washed with toluene to assist the removal of trifluoroacetic acid to obtain a crude yellow product. Sephadex G75 was used. As a static phase, the crude product is dissolved in water and placed in the column. The water was used as the mobile phase for the separation and separation. The collected liquid was identified by carbon 18 trans-thin chromatography plate. The same main spot ratio retention factor (Rf) was collected and concentrated to obtain yellow oil. Compound B3.

The purity was determined by high performance liquid chromatography, and a carbon 5 trans-polar column was used. The mobile phase was a methanol solution of trifluoroacetic acid of 0.1% by weight. The detection wavelength was set to 220 and the chromatographic ambient temperature was set to 25. °C, the flow rate was set to 0.4 ml per minute, and by chromatography analysis, it was found that there was a main single signal with a residence time of 7.8 minutes. ¹ H-NMR (solvent: D ₂ O; 400 MHz): 7.72 (d, J = 6 Hz, 32H), 7.43 (d, J = 8 Hz, 32H), 4.11 (m, 96H), 3.89 (s, 48H), 3.28 (m, 276H), 2.83 (m, 192H), 1.14 (m, 208H).

Other similar embodiments

A compound having a structure represented by formula (1A) or formula (1B),

Wherein U is selected from the group consisting of U-Lys-, -Lys-(Asp-NHAc) _J , -NHAc, -Lys-(Asp-) _K -NHAc, (Asp-NHAc) _J ; And K are natural numbers, J = 1 or 2, 20 ≧ K ≧ 1, Asp stands for aspartic acid, Lys stands for Lysine, and Gly stands for glycine.

2. A compound having a structure as shown in formula (2),

Wherein G is selected from the group consisting of polyethylenediamine dendrimer (PAMAM), polyester type dendrimer, polyglycerol dendrimer, Triazine based dendrimer, poly(propyleneimine) dendrimer, Newkome-type dendrimer; and A, B and Y is a natural number, the value of the Y-generation generation, the sum of A and B (4 x 2 ^Y ), and Asp stands for aspartic acid.

3. A compound having a structure as shown in formula (3),

Wherein R1 is -(Asp-) _K ; R2 is selected from the group consisting of polyethylenediamine dendrimer (PAMAM), polyester type dendrimer, polyglycerol tree Polyglycerol dendrimer, triazine based dendrimer, poly(propyleneimine) dendrimer, Newkome dendrimer (Newkome- Type dendrimer) and polylysine dendrimer; and T and K are natural numbers, 64≧T≧1, 20≧K≧1, and Asp stands for aspartic acid, Gly stands for glycine.

5. A compound as described above having a structure as shown in formula (2),

Among them, R is a polyethylene diamine dendrimer polymer (PAMAM).

6. A compound as described above having a structure as shown in formula (3),

Among them, R2 is a polyethylene diamine dendrimer polymer (PAMAM).

7. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and an effective amount of a principal component of formula (1A) or formula (1B),

Wherein U is selected from the group consisting of U-Lys-, -Lys-(Asp-NHAc) _J , -NHAc, -Lys-(Asp-) _K -NHAc, (Asp-NHAc) _J ; And K are natural numbers, J = 1 or 2, 20 ≧ K ≧ 1, Asp stands for aspartic acid, Lys stands for Lysine, and Gly stands for glycine.

8. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and an effective amount of the main component of the formula (2), wherein the G group is selected from the group consisting of polyethylenediamine dendrimer (PAMAM), polyester dendrimer Polyester type dendrimer, polyglycerol dendrimer, triazine based dendrimer, poly(propyleneimine) dendrimer Newkome-type dendrimer and polylysine dendrimer; and A, B and Y are both natural numbers and values of Y-generation algebras. The sum of A and B (4 x 2 ^Y ), Asp stands for aspartic acid.

9. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and an effective amount of a principal component of formula (3),

Wherein R1 is -(Asp-) _K ; R2 is selected from the group consisting of polyethylenediamine dendrimer (PAMAM), polyester type dendrimer, polyglycerol tree Polyglycerol dendrimer, triazine based dendrimer, poly(propyleneimine) dendrimer, Newkome dendrimer (Newkome- Type dendrimer) and polylysine dendrimer; and T and K are natural numbers, 64≧T≧1, 20≧K≧1, and Asp stands for aspartic acid, Gly stands for glycine.

10. The pharmaceutical composition according to the above embodiment, having a main component as shown in formula (2), Among them, R is a polyethylene diamine dendrimer polymer (PAMAM).

11. The pharmaceutical composition according to the above embodiment, having a main component as shown in formula (3), Among them, R2 is a polyethylene diamine dendrimer polymer (PAMAM).

12. A method of preparing a compound comprising the steps of:

(1) Rink amide or a resin with an amino acid (resin) is used as a synthetic raw material, and immersed in an organic solvent.

(2) removing the protecting group with a deprotecting reagent (DEP),

(3) taking amino acid and coupling reagent, adding activation solution (referred to as ACT)

(4) After the reaction is completed, the unreacted resin is terminated with a terminating reagent (abbreviated as CAP)

(5) Repeat steps (2), (3), and (4) above.

Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the scope of the present invention, and various modifications and refinements may be made without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims.

references

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Claims (4)

a compound having a structure as shown in formula (3), Wherein R1 is -(Asp-) _K ; R2 is selected from the group consisting of polyethylenediamine dendrimer (PAMAM), polyester type dendrimer, polyglycerol tree Polyglycerol dendrimer, triazine based dendrimer, poly(propyleneimine) dendrimer, Newkome dendrimer (Newkome- Type dendrimer) and polylysine dendrimer; and T and K are natural numbers, 64≧T≧1, 20≧K≧1, and Asp stands for aspartic acid, Gly stands for glycine. The compound of claim 1 has a structure as shown in formula (3), Among them, R2 is a polyethylene diamine dendrimer polymer (PAMAM). A pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and an effective amount of a main component of the formula (3), Wherein R1 Department - (Asp-) _K; R2 is selected from the group consisting of the following: polyethylenimine polymer dendrimer (PAMAM), polyester-type dendritic polymer (polyester type dendrimer), polyglycerol tree Polyglycerol dendrimer, triazine based dendrimer, poly(propyleneimine) dendrimer, Newkome dendrimer (Newkome- Type dendrimer) and polylysine dendrimer; and T and K are natural numbers, 64≧T≧1, 20≧K≧1, and Asp stands for aspartic acid, Gly stands for glycine. The pharmaceutical composition according to claim 3, which has the structure shown in formula (3), Among them, R2 is a polyethylene diamine dendrimer polymer (PAMAM).