WO2022184163A1 - Modified nonapeptide, and preparation method therefor and application thereof - Google Patents

Abstract

Disclosed in the present invention is a modified nonapeptide, which is an alkyl acylated and/or phosphorylated nonapeptide. The amino acid sequence of the alkyl acylated nonapeptide is sequence I: R-Co-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu, wherein R is a straight-chain or branched chain alkane containing 1-18 carbon atoms. The amino acid sequence of the phosphorylated nonapeptide is sequence II: Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO3H)-Gly-Glu. The amino acid sequence of the alkyl acylated and phosphorylated nonapeptide is sequence III: R-CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO3H)-Gly-Glu, wherein R is a straight-chain or branched chain alkane containing 1-18 carbon atoms. The sleep-inducing activity of the modified nonapeptide of the present invention is higher than the activity of the δ-sleep-inducing peptide (DSIP) which is the strongest in sleep-reducing activity in known peptides, and the duration of action is longer. Moreover, it can antagonize diseases such as anxiety and depression efficiently.

Description

Modified nonapeptide and its preparation method and application

The present invention claims the priority of the patent application number 2021102391122 submitted to the State Intellectual Property Office of China on March 4, 2021, and the invention title is "modified nonapeptide and its preparation method and application". The entire contents of this earlier application are hereby incorporated by reference.

technical field

The invention belongs to the field of medicines for the prevention and treatment of neurological diseases related to insomnia, and particularly relates to a modified nonapeptide and a preparation method and application thereof.

Background technique

In one's life, sleep accounts for about one-third of life, and good sleep is the main symbol of physical and mental health. However, with the development of modern society, people's life rhythm has generally accelerated, and the phenomenon of insomnia caused by various pressures is increasing. According to the research data of Professor Pan Jiyang in China, the current number of Chinese families suffering from insomnia is as high as 10% to 20%. Lack of sleep directly leads to slow thinking, nervousness, inability to concentrate, slow action, boredom, anxiety, burnout, lack of enthusiasm for work, and decreased work initiative and willingness to work, thereby reducing work efficiency. For a long time, people with insufficient sleep will feel particularly tired and hard, emotionally unstable, irritable, irritable, lack of self-control, causing serious psychological problems, and causing serious anxiety and depression. Therefore, the development of efficient and safe anti-insomnia drugs has become an urgent medical and social issue.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a modified nonapeptide, which has higher activity, stronger ability to penetrate the blood-brain barrier, and longer duration than the currently most insomniac peptide with the strongest hypnotic activity. The duration of action can effectively exert strong sleep-inducing activity, anti-anxiety and depression ability. Another aspect of the present invention provides a method for preparing the above-mentioned modified nonapeptide. Another aspect of the present invention provides the application of the above-mentioned modified nonapeptide for combating neurological diseases such as insomnia, anxiety and depression. The modified nonapeptide involved in the present invention has strong sleep-inducing activity and can be used for treating neurological diseases related to insomnia.

According to one aspect of the present invention, a modified nonapeptide is proposed, which is an alkylacylation-modified nonapeptide (sequence I) or a phosphorylation-modified nonapeptide (sequence II) or Alkylation-modified and phosphorylated-modified nonapeptides (sequence III).

In some more preferred embodiments of the present invention, the nonapeptide is further modified by alkylacylation and phosphorylation to obtain a modified nonapeptide.

In some preferred embodiments of the present invention, the modification site of the alkyl acylation modification is tryptophan (Trp) at position 1 of the N-terminal of the nonapeptide, and the sequence modification method is as follows:

The first Trp is modified by alkyl acylation to obtain a nonapeptide modified by alkyl acylation, and its amino acid sequence is sequence I: R-CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly- Glu, wherein R is a straight or branched chain alkyl group containing 1-18 carbon atoms.

In some preferred embodiments of the present invention, the modification site of the phosphorylation modification is serine (Ser) at position 7 of the N-terminal of the nonapeptide, and the sequence modification method is as follows:

The 7th Ser is phosphorylated to obtain a phosphorylation-modified nonapeptide, and its amino acid sequence is sequence II:

Trp-Ala-Gly-Gly-Phe-Ala-Ser( _PO3H )-Gly-Glu.

In some preferred embodiments of the present invention, the amino acid sequence of the alkylacylated and phosphorylated modified nonapeptide is sequence III: R-CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser (PO ₃ H)-Gly-Glu, wherein R is a straight or branched chain alkyl group containing 1-18 carbon atoms.

In some embodiments of the present invention, R is a straight or branched chain alkyl group containing 3-12 carbon atoms. For example, R is a straight or branched chain alkyl group containing 5 to 10 carbon atoms. For example R is hexyl, heptyl, octyl, nonyl, decyl. In some embodiments, R is n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecyl.

In some embodiments of the present invention, the amino acid sequence of the modified nonapeptide is: C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu, Trp-Ala-Gly- Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu, or C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu.

According to a preferred embodiment of the present invention, the modified nonapeptide has at least the following beneficial effects:

Compared with the current sleep-inducing peptide with the highest sleep-inducing activity, the modified nonapeptide has higher activity, stronger ability to penetrate the blood-brain barrier and longer duration of action, and can effectively exert strong sleep-inducing activity and anti-anxiety. and depression capacity.

According to another aspect of the present invention, a method for preparing the above-mentioned modified nonapeptide is provided. The method is a solid-phase synthesis method, which can obtain a high-yield, high-purity modified nonapeptide, and the process is simple and feasible.

According to the present invention, the preparation comprises the following steps:

S1. Synthesis of nonapeptide

S2. Modification of nonapeptides

Alkylation and/or phosphorylation of nonapeptides:

S3. Excision and precipitation to obtain a crude modified nonapeptide;

S4. Purification of the crude modified nonapeptide.

In some embodiments of the present invention, the modified nonapeptide is synthesized and purified by solid phase synthesis, the method comprising the steps of:

S1. Synthesis of nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu

① Activation of resin: Put Wang resin in a solid-phase reactor, add anhydrous dichloromethane (DCM) to soak and swell for 18 hours, and remove the solvent anhydrous dichloromethane under reduced pressure;

②Coupling the first amino acid to Wang resin: Fmoc-Glu(OtBu), HBTU((3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-yl)-1oxide, O-benzotriazol Azole-tetramethylurea hexafluorophosphate) and DMAP (4-dimethylaminopyridine, 4-dimethylaminopyridine) were dissolved in DMF (dimethylformamide, N,N-dimethylformamide), and DIEA (diisopropylethylamine) was added. diisopropylethylamine), the reaction was shaken at room temperature for 18 hours, the solvent was removed, and the resin was washed with DMF.

③ Block unreacted functional groups on the resin: add a mixed solution of acetic anhydride, pyridine and DMF, shake for 20-40 minutes, and block the unreacted functional groups on the resin.

④Deprotection of functional groups: Wash the resin with DMF, DCM and DMF in turn, and deprotect with piperidine/DMF; pick a resin with a capillary, and use Kaiser's reagent to check whether the deprotection is complete. If the reaction is negative, it means that the deprotection is not complete. , repeat steps 1-3 in Table 1, if the reaction is positive, it means that the deprotection is complete, and proceed to the next step.

⑤ Peptide receiving reaction: After washing the completely deprotected resin with DMF, the peptide receiving reaction was started according to the fourth step in Table 1, and each round was carried out according to the procedure in Table 1.

Further, the Kaiser's reagent detection method is:

Component A: 1.000 g of ninhydrin hydrate was dissolved in 20.0 mL of absolute ethanol.

Component B: 20.000 g of phenol was dissolved in 5.0 mL of ethanol.

Component C: 4.0*10-5mol/L potassium cyanide solution in pyridine.

Take two drops of each of components A, B, and C into a small amount of resin sample, react in an oil bath at 120°C for 5 minutes, and observe the color change of the resin and the solution. Resin and solution turned dark blue or reddish-brown (serine and threonine) for positive reaction, light yellow for negative reaction.

Table 1: Steps for Fmoc Solid Phase Synthesis of Peptides

step	operate	reagent	time (min)	frequency
1	deprotection	20% piperidine/DMF	5	1
2	deprotection	20% piperidine/DMF	20	1
3	Kaiser's test	Two drops each of A, B, and C, 120°C	5	1
4	washing		DMF	3	3
5	coupling	Activated Fmoc-AA-OH	120	1
6	washing		DMF	3	3
7	washing		DCM	3	3
8	Kaiser's test	Two drops of A, B, C each at 120°C	5	1
9	washing		DMF	3	3

The sequence of the peptide-attachment reaction is to access Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc- Ala-OH and Fmoc-Trp-OH are carried out according to the above-mentioned deprotection, detection, washing, coupling, washing and detection conditions, so that the polypeptide chain is extended until the last amino acid Fmoc-Trp-OH is connected, forming a continuous Resin with protective group of polypeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu.

S2. Modification of nonapeptides

①Alkyl acylation modification

Alkyl acid (R-COOH), Oxyma (ethyl 2-oxime cyanoacetate) and DIC (Diisopropylcarbodiimide, diisopropylcarbodiimide) are mixed in NMP (1-methyl-2-pyrrolidinone, N-methyl carbodiimide) pyrrolidone), added to the resin obtained in the above S1, shaken for 2-4 hours, developed color by ninhydrin-phenol, monitored the reaction progress, and formed a polypeptide R-CO-Trp with a protective group connected with the resin -Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu.

② phosphorylation modification

First, use diphenyl N,N'-diisopropylamine phosphite (amine phosphite reagent, 2.5 times the amount, based on the input amino acid) under the catalysis of tetrazolium (6 times the amount, based on the input amino acid) , the polypeptide containing serine Ser obtained in the above-mentioned S1 or obtained after the above-mentioned alkyl acylation generates phosphite triester, and the reaction time is 1-2 hours; The amino acid meter) is oxidized to the corresponding phosphotriester: a resin-linked polypeptide with a protective group Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu, or R- CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu, the reaction time was 30-60 minutes.

S3. Excision and Precipitation

Use TFA (trifluoroacetic acid, trifluoroacetic acid) solution to excise the resin and selectively remove the protecting group to obtain the target product of alkyl acylation and/or phosphorylation: nonapeptide modified by alkyl acylation (I) R- CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu, phosphorylated nonapeptide (II) Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly -Glu or alkylacylation-modified and phosphorylation-modified nonapeptide (III) R-CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu. The above-mentioned excision solution was blown off the solvent and precipitated with ether to obtain the modified nonapeptide crude product;

S4. Purification of crude modified nonapeptide

①Purification of nonapeptide modified by alkyl acylation

The crude product was dissolved in acetonitrile and water and purified by preparative RP-HPLC.

Preparative column: YMC-Pack ODS-AQ (250×20mml.D, S-5μm, 12nm); detection wavelength 214nm; mobile phase A is 0.1% TFA aqueous solution, mobile phase B is 0.1% TFA acetonitrile solution, gradient elution ( 0～60min, mobile phase B: 40%～90%); flow rate 15.0mL·min-1. The main peak was collected, and the pure product was obtained after freeze-drying, which was a white powdery solid.

②Purification of phosphorylation-modified nonapeptide or alkylacylation and phosphorylation-modified nonapeptide

Use filled with 20-50μm/200-500

The phosphorylated polypeptide purification device of titanium dioxide nanoparticles is used to purify the crude product of nonapeptide (II) modified by phosphorylation or nonapeptide (III) modified by alkyl acylation and phosphorylation.

S5. Identification of modified nonapeptide: The modified nonapeptide was analyzed and identified by chromatography and mass spectrometry.

The present invention also provides a pharmaceutical composition comprising the modified polypeptide of the present invention.

According to the present invention, the pharmaceutical composition may also optionally comprise at least one pharmaceutically acceptable auxiliary material;

According to the present invention, the pharmaceutical composition is used for preventing and/or treating neurological diseases such as insomnia, anxiety or depression.

According to the present invention, the pharmaceutical compositions of the present invention can be formulated into dosage forms suitable for administration by methods known in the art.

The present invention also provides the use of the modified polypeptide in the preparation of medicine. The drug is used, for example, for neurological diseases such as insomnia, anxiety or depression.

The present invention also provides a method for treating and/or preventing neurological diseases such as insomnia, anxiety or depression, the method comprising administering a therapeutically effective amount of the modified polypeptide of the present invention to a subject.

Beneficial effects of the present invention:

1. Compared with the hypnotic peptide with the highest hypnotic activity at present, the modified nonapeptide of the present invention has higher activity, stronger ability to penetrate the blood-brain barrier and longer duration of action, and can effectively play a strong role. Sleep-inducing activity, anti-anxiety and depressive abilities.

2. The nonapeptide modified by alkyl acylation significantly shortened the incubation period (P=0.041) and increased the deep sleep time (P=0.023), which could improve the content level of monoamine transmitters in insomnia animals to make it closer to normal animals. It can be seen that the alkyl acylation modified nonapeptide of the present invention has higher activity and longer duration of action than delta-hypnotic peptide (DSIP) which has the strongest sleep-inducing activity among the known peptides.

3. The nonapeptide modified by alkyl acylation can reduce the secretion of excitatory transmitter GABA caused by insomnia in serum; it can reduce the secretion of NE, which has excitatory effects such as vasoconstriction, in serum. Further experiments have shown that it can effectively Antagonize anxiety and depression and other diseases, can be used in neurological diseases related to insomnia.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, wherein:

FIG. 1 is an HPLC chromatogram of the sleep decoy peptide Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu of Comparative Example 1. FIG.

FIG. 2 is an ESI-MS image of the sleep decoy peptide Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu of Comparative Example 1. FIG.

3 is the HPLC chromatogram of the nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu of Example 1 of the present invention.

4 is an ESI-MS image of the nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu of Example 1 of the present invention.

5 is an HPLC chromatogram of the n-octanoylated nonapeptide C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu of Example 2 of the present invention.

6 is an ESI-MS image of the n-octanoylated nonapeptide C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu of Example 2 of the present invention.

7 is an HPLC chromatogram of the phosphorylated nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu of Example 3 of the present invention.

8 is an ESI-MS image of the phosphorylated nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu of Example 3 of the present invention.

Figure 9 is the n-octanoylation and phosphorylation modified nonapeptide C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu of Example 4 of the present invention HPLC chromatogram.

Figure 10 is a graph showing the relationship between sleep latency of the nonapeptides of Examples 2 and 3 of the present invention on the PCPA model.

Figure 11 is a graph showing the deep sleep time relationship of the nonapeptides of Examples 2 and 3 of the present invention on the PCPA model.

Figure 12 is a graph of the sleep latency dose-effect relationship of the n-octanoylated nonapeptide of Example 2 of the present invention on the PCPA model.

13 is a graph showing the dose-effect relationship of the n-octanoylated nonapeptide of Example 2 of the present invention on the PCPA model.

Figure 14 is a graph showing the body weight change of the n-octanoylated nonapeptide of Example 2 of the present invention on the PCPA model.

Fig. 15 is a graph showing the change of GABA of n-octanoylated nonapeptide in PCPA model of Example 2 of the present invention.

Fig. 16 is a graph showing the NE change relationship of the n-octanoylated nonapeptide of Example 2 of the present invention on the PCPA model.

Fig. 17 is a graph showing the DA change of the n-octanoylated nonapeptide of Example 2 of the present invention on the PCPA model.

Figure 18 is a graph of the dose-response relationship of the n-octanoylated nonapeptide of Example 2 of the present invention on the anxiety model of PTSD mice (open field test).

OT% refers to the percentage of time to enter the open arm; OE% refers to the percentage of times to enter the open arm.

Figure 19 is a graph showing the results of the open field test of the n-octanoylated nonapeptide (1 mg/kg) of Example 2 of the present invention on the PTSD anxiety model.

20 is a graph showing the results of the elevated plus maze on the PTSD anxiety model of n-octanoylated nonapeptide (1 mg/kg) of Example 2 of the present invention.

Figure 21 is a graph showing the results of the open field test of the rat CUMS depression model.

Figure 22 is a graph showing the results of the sugar water preference test of the rat CUMS depression model.

23 is a graph showing the results of an open field test of the n-octanoylated nonapeptide of Example 2 of the present invention in a rat CUMS depression model.

Detailed ways

The concept of the present invention and the technical effects produced will be clearly and completely described below with reference to the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts are all within the scope of The scope of protection of the present invention.

The Wang resin was purchased from Gill Biochemical (Shanghai) Co., Ltd.

The Fmoc-Glu (OtBu) was purchased from Gill Biochemical (Shanghai) Co., Ltd., and the HPLC purity was ≥98%.

Example 1

In this example, a nonapeptide was prepared, the sequence of which is: Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu.

The nonapeptide is prepared by solid-phase synthesis, and the method comprises the following steps:

S1. Synthesis of nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu

① Activation of resin: put 1 g of Wang resin in a solid-phase reactor, add 5.0 mL of anhydrous dichloromethane (DCM), soak and swell for 18 hours, and remove the solvent anhydrous dichloromethane under reduced pressure;

②Coupling the first amino acid to Wang resin: Dissolve 63.83mg (0.15mmol) Fmoc-Glu(OtBu), 56.85mg (0.15mmol) HBTU and 15mg DMAP in 4.0mL DMF, add 0.15ml DIEA, room temperature The reaction was shaken for 18 hours, the solvent was removed, and the resin was washed with DMF (4.0 mL*5).

③ Block unreacted functional groups on the resin: add a mixed solution of acetic anhydride, pyridine and DMF, wherein the volume ratio of acetic anhydride:pyridine:DMF is 2:1:3, shake for 30 minutes, and block the unreacted functional groups on the resin.

④ Deprotection of functional groups: Wash the resin 3 times with DMF, DCM and DMF in turn, and deprotect with 20% piperidine/DMF for 2 times for 5 minutes and 20 minutes; pick a resin with a capillary tube, and detect it with Kaiser's reagent. Whether the deprotection is complete, if the reaction is negative, it means that the deprotection is not complete, repeat steps 1-3 in Table 2, if the reaction is positive, it means that the deprotection is complete, and proceed to the next step.

Among them, the detection method with Kaiser's reagent is:

Component A: 1.000 g of ninhydrin hydrate was dissolved in 20.0 mL of absolute ethanol.

Component B: 20.000 g of phenol was dissolved in 5.0 mL of ethanol.

Component C: 4.0*10-5mol/L potassium cyanide solution in pyridine.

Two drops of each of components A, B and C were added to the above resin, reacted in an oil bath at 120°C for 5 minutes, and the color changes of the resin and the solution were observed. Resin and solution turned dark blue or reddish-brown (serine and threonine) for positive reaction, light yellow for negative reaction.

⑤ Peptide receiving reaction: After washing the completely deprotected resin with DMF for 3 times, the peptide receiving reaction was started according to step 4 in Table 2, and each round was carried out according to the procedure in the table. The method of amino acid activation is as follows: 0.3mmol Fmoc-AA-OH, 0.15mmol TBTU, 0.15mmol HOBt and 0.15ml DIEA are dissolved in 4.0mL DMF, and the reaction solution is added to the solid phase reactor and shaken at room temperature for 2 hours. After coupling, the ligation efficiency was checked again with Kaiser's reagent. If the reaction is positive, it means that the coupling is incomplete, repeat step 2 in Table 2 to start the re-peptide reaction until the coupling is complete.

Table 2: Steps for Fmoc Solid Phase Synthesis of Peptides

step	operate	reagent	time (min)	frequency
1	deprotection	20% piperidine/DMF	5	1
2	deprotection	20% piperidine/DMF	20	1
3	Kaiser's test	Two drops each of A, B, and C, 120°C	5	1
4	washing		DMF	3	3

5	coupling	Activated Fmoc-AA-OH	120	1
6	washing		DMF	3	3
7	washing		DCM	3	3
8	Kaiser's test	Two drops of A, B, C each at 120°C	5	1
9	washing		DMF	3	3

The sequence of the peptide-attachment reaction is to access Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc- Ala-OH and Fmoc-Trp-OH are carried out according to the above-mentioned deprotection, detection, washing, coupling, washing and detection conditions, so that the polypeptide chain is extended until the last amino acid Fmoc-Trp-OH is connected, forming a continuous Resin with protective group of polypeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu.

S2. Excision of the nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu:

The polypeptide-containing resin obtained in S1 was washed three times with DMF, DCM, and DMF, respectively, and then vacuum-dried. Pour the peptide-containing resin into a 10.0 mL round-bottom flask, and slowly add trifluoroacetic acid: pure water: anisole: EDT: phenol = 82.5: 5: 5: 2.5: 5 (v/v) solution 5.0 at room temperature mL, and reacted for 4 hours. Filter, dry the solvent with nitrogen until 1/10 of the volume of the solution remains, pour 5.0 mL of anhydrous ether into the residual liquid, and a white flocculent precipitate appears. 5.0 mL of anhydrous ether was added to the mixture, shaken, centrifuged for 10 minutes under the same conditions, and repeated once to remove most of the impurities, and the precipitate was vacuum-dried for 24 hours to obtain a crude polypeptide product.

S3. Separation and purification of nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu:

The crude product obtained in S2 was dissolved in an aqueous solution and separated by high performance liquid chromatography. The chromatographic conditions are as follows: a C ₁₈ semi-preparative column, the mobile phase is 0.1% trifluoroacetic acid (TFA)/acetonitrile, the flow rate is 3.000 ml/min, and the detection wavelength is 220 nm. The main peak product was collected, and the collected product was rotary-evaporated under reduced pressure to remove acetonitrile in the collected product (its boiling point is low, which is not conducive to freeze-drying). Then, the aqueous solution of the sample was frozen with liquid nitrogen and dried in a freeze dryer to obtain a white flocculent solid product, which was stored at 20°C. The HPLC analysis results are shown in Figure 3, and the mass spectrometry is shown in Figure 4.

Example 2

In this example, a n-octanoylated nonapeptide was prepared, the sequence of which is: C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu.

The n-octanoylation-modified nonapeptide is prepared by solid-phase synthesis, and the method comprises the following steps:

The raw materials needed to prepare the above nonapeptide are protected amino acids, which are Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Trp -OH. In addition, the raw material Fmoc-n-octanoic acid needs to be used in the preparation to condense it with the nonapeptide to obtain the modified target polypeptide.

The polypeptide synthesis method and separation and purification method are the same as in Example 1, wherein the reaction process of n-octanoic acid and the polypeptide is as follows: Fmoc-n-octanoic acid (1 mmol), Oxyme (142 mg) and DIC (155 μl) are mixed in 7 ml of NMP, and added to the mixture. In the polypeptide with a protective group of the resin, shake at room temperature for 2 hours, and monitor the progress of the reaction through ninhydrin-phenol color development. After the reaction is completed, the modified polypeptide is excised from the resin, and then separated and purified.

The HPLC analysis result is shown in Figure 5, and the mass spectrum is shown in Figure 6.

Example 3

In this example, a phosphorylation-modified nonapeptide was prepared, the sequence of which is: Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu.

The phosphorylation-modified nonapeptide is prepared by solid-phase synthesis, and the method includes the following steps:

S1. Synthesis of nonapeptide

① Activation of resin: Weigh 1 g of Wang resin, add it to 20 mL of dichloromethane (DCM) for 5 hours, filter off the solution, and then add 10 mL of dichloromethane (DCM) and 10 mL of N,N-dimethylformamide (DMF) Soak for 12h, filter out the solution;

②Coupling the first amino acid to Wang resin: Dissolve 638.3mg (1.5mmol) Fmoc-Glu(tBu), 568.5mg (1.5mmol) HBTU and 150mg DMAP in 10.0mL DMF, add 1.5mL DIEA, shake at room temperature The reaction was carried out for 18 hours, the solvent was removed, and the resin was washed with DMF (4.0 mL*5).

③ Block unreacted functional groups on the resin.

④Deprotection of functional groups: wash the resin with DMF (4.0mL*3), DCM (4.0mL*3) and DMF (4.0mL*3) in turn, deprotect 20% piperidine/DMF (4.0mL*2), time 5 minutes and 20 minutes. A resin was then picked with a capillary and checked for complete deprotection with Kaiser's reagent. If the reaction is positive, then the second amino acid Fmoc-Gly-OH is added, and the steps of coupling, detection, washing and deprotection of the first amino acid are repeated until the ninth amino acid Fmoc-Trp-OH is received. A crude polypeptide with a protective group attached to the resin was obtained.

The sequence of the peptide-attachment reaction is to sequentially access Fmoc-Glu-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Trp-OH to form a connection Resin-bearing polypeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu.

S2. Phosphorylation modification of nonapeptide

The above-mentioned crude polypeptide with protective groups of the connecting resin was dried under vacuum, and under nitrogen protection, 1.554g of diphenyl N,N'-diisopropylphosphoramidite, 794.3mg of tetrazolium, 9mL of DMF were added, The reaction was carried out for 1 h, rinsed three times with 20 mL of DCM, and then 811 mg of 70% aqueous tert-butanol peroxide solution and 3 mL of DMF were added, and the reaction was stirred for 30 min. The phosphorylated modified nonapeptide Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO3H)-Gly-Glu with a protective group attached to the resin was obtained.

S3. Excision of phosphorylated nonapeptide

Trifluoroacetic acid: phenol: water: EDT: phenylene sulfide = 82.5:5:5:2.5:5 (V/V) solution was added, and the reaction was stirred for 12 h. The phosphorylated nonapeptide was excised from Wang resin to obtain Crude phosphorylated nonapeptide.

S3. Isolation and purification of phosphorylated nonapeptides

Weigh 5g of titanium dioxide, put it into a glass tube with a sieve plate, add 100mL of a 70% acetonitrile/3% TFA solution, shake up and down and mix for 1 minute, filter the solution under reduced pressure at 0.05-0.09mbar, and wait for the Weigh 0.5 g of the phosphorylated nonapeptide crude product, dissolve it in 200 mL of a 70% acetonitrile/3% TFA solution, shake up and down for 10 minutes, and filter the solution under reduced pressure at 0.05 to 0.09 mbar. Remove; add 200 mL of 60% acetonitrile/0.1% TFA solution, shake up and down for 5 minutes, and filter the solution under reduced pressure at 0.05-0.09 mbar; add 200 mL of 50% acetonitrile/0.1% TFA solution, shake up and down to mix Homogenize for 5 minutes, filter the solution under reduced pressure at 0.05-0.09 mbar; add 200 mL of 50% acetonitrile solution, shake up and down for 1 minute, and filter off the solution under reduced pressure; finally add 0.1 mol/L ammonia solution to wash Three times, the volumes are 200 mL, 100 mL, and 100 mL, respectively, shake up and down for 5 minutes, and filter the solution under reduced pressure into a clean container under the condition of 0.05-0.09 mbar. Samples were taken for LC-ESI-MS analysis.

The HPLC analysis result is shown in Figure 7, and the mass spectrum is shown in Figure 8. It can be seen from Figure 7 that the obtained phosphorylated nonapeptide has a high purity (98.2417%).

Example 4

In this example, a nonapeptide modified by alkyl acylation and phosphorylation is prepared, and its sequence is: C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly -Glu.

The alkyl acylation and phosphorylation modified nonapeptide is prepared by solid-phase synthesis, and the method comprises the following steps:

The preparation of n-octanoylated nonapeptide is the same as that in Example 2. After the peptide is attached, phosphorylation modification is performed first and then deprotection is performed. The preparation of phosphorylation is the same as that in Example 3.

The method for removing, separating and purifying the nonapeptide after n-octanoylation and phosphorylation modification is the same as that in Example 3.

The HPLC analysis results are shown in Figure 9.

Comparative Example 1

In this comparative example, a hypnotic peptide Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu was prepared by solid-phase synthesis. The amino acids in the sequence need to be protected with amino acids, namely Fmoc-Glu(OtBu) -OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp-OH.

The polypeptide synthesis method and separation and purification method are the same as those in Example 1, except that the Phe at the 5th position in the N segment is changed to Asp.

The HPLC analysis result is shown in Figure 1, and the mass spectrum is shown in Figure 2.

Example 5

This example tests the pharmacodynamics of Example 2, Example 3 and Comparative Example 1 on the PCPA-induced insomnia model.

1. Method:

(1) Animal modeling and grouping.

First, 60 KM mice were randomly divided into 2 groups: 10 in the blank group and 50 in the modeling group; PCPA suspension was prepared with NaHCO ₃ , and the model was created by intraperitoneal injection.

The preparation method of PCPA suspension: Dissolve _NaHCO3 tablet in 30℃～40℃ warm water, prepare 5% _NaHCO3 aqueous solution, slowly drop _NaHCO3 aqueous solution in 0.9% NaCl solution, prepare pH7～pH8, stir Then add an appropriate amount of Tween20, add PCPA, and sonicate for 15 minutes after mixing.

PCPA modeling: the injection dose was 600 mg/kg, the injection volume was 10 μL/g b.w., at 17:00 p.m. every day, continuously for 3 days; the control group was intraperitoneally injected with the same volume of normal saline.

At 9:00 a.m. on the fourth day after the first intraperitoneal administration of PCPA, 50 animals in the modeling group were randomly divided into a model group and an administration group according to their body weight.

(2) Pentobarbital sodium correction test to verify the efficacy.

At 9:00 a.m. on the fourth day after the first intraperitoneal administration of PCPA, the grouped animals were injected with the corresponding drugs in the tail vein according to the dose, and the injection volume was 10 μL/g b.w., and the control group was injected with the same volume of normal saline through the tail vein.

The righting reflex was observed by intraperitoneal injection of 55 mg/kg sodium pentobarbital saline solution 0.5 h after the injection of the test product, and the injection volume was 10 μL/g b.w. The pentobarbital sodium injection time T1, the righting reflex disappearing time T2, and the righting reflex appearing time T3 were recorded (three times the righting reflex was determined).

Latency (s) = T2-T1 deep sleep time (s) = T3-T2

(3) The effects on animal body weight and physiological state before and after administration were compared.

40 female KM mice aged 6-8 weeks were selected, 8 were selected as blank control group, and the remaining 32 mice were intraperitoneally injected with pre-prepared PCPA solution (600 mg·kg ^-1 ) for 3 consecutive days. After the first intraperitoneal administration of PCPA At 9:00 a.m. on the fourth day, 32 animals in the modeling group were randomly divided into four groups according to their body weight: model group and administration group (different doses and different administration frequencies). Intravenous administration, the administration volume is 10mL/kg. The changes of body weight and physiological state were observed continuously for 3 days after treatment.

(4) ELISA method to explore the mechanism of drug efficacy.

At the end of the pentobarbital sodium correction test in the above (2), the eyeballs were removed after anesthesia and blood was collected. After the blood was overnight at 4°C, centrifuged at 3000 rpm for 10 min, and the supernatant was aspirated. The levels of serum monoamine transmitters γ-aminobutyric acid, norepinephrine and dopamine were determined using ELISA kits.

In this example, polypeptide 1 is the prototype of the sleep decoy peptide of Comparative Example 1, and its structural sequence is: Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu; polypeptide 2 is n-octanoyl of Example 2 Peptide ₃ is the nonapeptide modified by phosphorylation of Example ₃ , its structure The sequence is: Trp-Ala-Gly-Gly-Phe-Ala-Ser( _PO3H )-Gly-Glu.

2. Test:

By constructing an animal model of PCPA-induced insomnia, the following experiments were performed:

Test (1) carry out preliminary screening of sleep-promoting drug effect on polypeptide 1, polypeptide 2, and polypeptide 3;

Test (II) Select a compound with better efficacy to carry out a drug efficacy test to determine the effective dose;

Test (III) compares the effects on animal body weight and physiological state before and after administration;

Experiment (IV) The pharmacodynamic mechanism was preliminarily explored by ELISA method.

3. Results:

The results of the pharmacodynamic test on the PCPA-induced insomnia model showed that among the three peptides, peptide 2 and peptide 3 had stronger anti-insomnia or sleep-promoting effects than peptide 1; compared with the model group, peptide 2 significantly shortened the incubation period ( P=0.041), increased deep sleep time (P=0.023); the hypnotic effect of polypeptide 2 has a dose-dependent relationship, the effective dose is about 5μmol·kg ^-1 , and the administration method is tail vein injection; There was no significant difference between repeated administration (3 consecutive days) and single administration in weight loss caused by insomnia; Polypeptide 2 could improve the content of monoamine transmitters in insomnia animals and make them closer to normal animals.

In test (I), compared with the control group, the model group prolonged the latency period and decreased the deep sleep time; while compared with the model group, polypeptide 2 significantly shortened the latency period (P=0.041) and increased the deep sleep time (P=0.041). P=0.023), it is preliminarily determined that polypeptide 2 has better anti-insomnia efficacy, and the results are shown in Figures 10 and 11.

In test (II), polypeptide 2 was divided into group A (1μmol·kg ^-1 ), group B (2.5μmol·kg ^-1 ) and group C (5μmol·kg ^-1 ) according to the dose. Significant difference; in terms of deep sleep time, compared with the control group, the model group decreased the deep sleep time (no significant difference), and the polypeptide 2B group (2.5μmol·kg ^-1 ) increased the deep sleep time compared with the model group (P =0.043), the polypeptide 2C group (5 μmol·kg ⁻¹ ) increased the time of deep sleep compared with the model group (P=0.004). The anti-insomnia efficacy of polypeptide 2 has a dose-effect relationship, and the results are shown in Figures 12 and 13.

In test (III), in the process of modeling, except for the blank control group, the body weights of the other groups gradually decreased. In the process of drug administration after modeling, except the model group, the body weights of the other drug administration groups all recovered, and the body weight of polypeptide 2 (10 μmol·kg ^-1 , QD×1) recovered on the third day after administration There is a significant difference compared to the model group. It is preliminarily inferred that polypeptide 2 has the effect of adjusting the physiological state of animals by improving sleep and increasing body weight in insomniac animals. During the three-day treatment, there is no difference in weight gain between daily repeated administration and single administration. The results are shown in Figure 14.

In test (IV), after modeling, the excitatory transmitter GABA in the serum of the model group was significantly increased, while the GABA was significantly decreased after administration of 5 μmol·kg ^-1 of polypeptide 2, indicating that polypeptide 2 can reduce the insomnia caused by insomnia in the serum. The secretion of excitatory transmitter GABA, the results are shown in Figure 15; after modeling, the level of NE in the serum of the model group did not change, while the NE was significantly reduced after administration of 5 μmol·kg ^-1 of polypeptide 2, indicating that polypeptide 2 can reduce serum levels of NE. The secretion of NE, which has excitatory effects such as vasoconstriction, is shown in Figure 16; the level of dopamine in serum is related to the anxiety or depression-like state in behavior. After kg ^-1 of polypeptide 2, the level of dopamine in the serum of animals increased significantly, suggesting that their anxiety or depression-like behavior may be alleviated. The results are shown in Figure 17.

Example 6

Since Polypeptide 2 of Example 2 has the strongest sleep-inducing effect, it may improve anxiety symptoms caused by insomnia. Therefore, this test case tested the pharmacodynamics of n-octanoylated nonapeptide on the anxiety model of PTSD mice.

1. Method

(1) Animal modeling and grouping

ICR mice, SPF grade, male, 18g-20g, 16 mice. After being acclimated to feeding (4 mice/cage) for 3 days, 16 mice were randomly divided into model group and control group according to body weight, with 8 mice in each group (4 mice/cage). The first day of modeling after acclimatization to rearing for 3 days is called Day1, D1. On D1, the animals in the model group were placed in 50mL centrifuge tubes (with a 0.5cm diameter round opening at the bottom for the mice to breathe) for 2 hours of restraint stress, and then the animals (4 animals/group) were placed in acrylic glass buckets (water depth). 25cm, water temperature 23-25°C) for forced swimming for 15min, dry the animals, and put them back into the cage. The control group was fed normally without manipulation. On D2, after the model group and the control group were adapted to the behavioral laboratory environment for 5 minutes, the elevated plus maze test (EPM) and the open field test (OF) were used to test whether the modeling was successful.

(2) Behavioral test to evaluate drug efficacy

The day of the first administration was called D1, Day1; on D7, the behavioral test was performed 0.5h after administration. After the animals to be tested are acclimated to the behavioral laboratory environment for 5 minutes, the elevated plus maze test (EPM) is firstly performed, and the animals are placed in the EPM with their faces facing the open arm. Time, after each animal test, wipe the EPM with 75% alcohol to remove odor. After the EPM, the animals were put into the open field, and the open field experiment (OF) was performed, and the total movement distance of the animals in 5 minutes, the movement distance of the central area, and the movement distance of the surrounding area were recorded.

(3) Statistical methods

Data were entered and statistically analyzed by Excel 2016 and SPSS 20.0 software. The measurement indicators were first tested by LEVENE variance homogeneity test. When the variance was homogeneous (p>0.05), the results of the variance analysis could be directly used to judge whether the overall difference was statistically significant. When the overall difference was statistically significant (p≤0.05), use The LSD test was used to compare the differences between groups. When the overall difference was not statistically significant (p>0.05), the statistical analysis was over; -Wallis H test), when the Kruskal-Wallis H test showed that the overall difference was statistically significant (p≤0.05), the Mann-Whitney U test was used to compare the differences between groups, when the Kruskal-Wallis H test showed that the overall difference was not statistically significant When significant (p>0.05), the statistical analysis was terminated.

2. Results

First, different doses of n-octanoylated nonapeptide were administered, and the pharmacodynamic effects of single and multiple subcutaneous injections were mainly investigated. The results showed that the drug effect of low-dose administration is stronger than that of high-dose, which may be due to the strong sleep-inducing effect of the drug. Excessive dose will lead to sleep-like behavior and affect its anti-anxiety effect; repeated administration does not produce more sleep-like behavior. The best anxiolytic effect, the results are shown in Figure 18.

Secondly, the pharmacodynamic effect of n-octanoylated nonapeptide was investigated in single, low-dose, subcutaneous injection. The results of the open field experiment showed that subcutaneous administration of 1 mgl·kg ^-1 of n-octanoylated nonapeptide can significantly improve the total distance of exercise, the residence time in the central area, and the number of times of entering the central area. The results are shown in Figure 19; The elevated plus maze test showed that a single subcutaneous administration of n-octanoylated nonapeptide changed to 1 mgl·kg ^-1 and the EPM test 24 hours later could still significantly increase the time for the mice to enter the open arms. The results are shown in Figure 20. This shows that the polypeptide 2 of Example 2 has an obvious anti-anxiety effect.

Example 7

Since Polypeptide 2 of Example 2 has the strongest hypnotic effect, it may improve depressive mood. Therefore, this test case tested the pharmacodynamics of the n-octanoyl-modified nonapeptide on the CUMS rat depression model.

1. Method

(1) Animal modeling and grouping.

SD rats, SPF grade, male, 220-240g, 52 rats. After 5 days of acclimatization (1 rat/cage), 52 rats were randomly divided into 2 groups according to their body weight, 12 in the blank control group and 40 in the modeling group. The CUMS model was established according to different stimulation schedules, and the success of the CUMS model was judged by the sugar water preference test (SPT) and the open field test. The model was successfully established on about 37 days.

(2) Efficacy of sugar water preference and open field test.

On the 43rd day, 40 rats in the modeling group were grouped by body weight and divided into a model group and a drug-administered group. After 21 days of administration, efficacy evaluation was performed.

Sugar Preference Test (SPT): One bottle (100ml) of 1% sucrose water and one bottle (100ml) of purified water were freely ingested by animals for 1 hour and fed normally. After 1 hour, the consumption in the two bottles was counted, and the sugar water preference % was used as the degree of depression. The lower the value, the higher the degree of depression.

Sugar water preference % = [sugar water consumption/(sugar water consumption+purified water consumption)]×100%

Open field test: Record the total movement distance of the animal, the movement distance in the central area, and the movement distance in the surrounding area.

(3) Statistical methods.

Data were entered and statistically analyzed by Excel 2016 and SPSS 20.0 software. The measurement indicators were first tested by LEVENE variance homogeneity test. When the variance was homogeneous (p>0.05), the results of the variance analysis could be directly used to judge whether the overall difference was statistically significant. When the overall difference was statistically significant (p≤0.05), use The LSD test was used to compare the differences between groups. When the overall difference was not statistically significant (p>0.05), the statistical analysis was over; -Wallis H test), when the Kruskal-Wallis H test showed that the overall difference was statistically significant (p≤0.05), the Mann-Whitney U test was used to compare the differences between groups, when the Kruskal-Wallis H test showed that the overall difference was not statistically significant When significant (p>0.05), the statistical analysis was terminated.

2. Results

Construction of the CUMS rat depression model: SD rats, male, establish the CUMS model according to different stimulation schedules, and use the sugar water preference test (SPT) and the open field test to determine the success of the CUMS model. The model was successfully built around the day, and the results are shown in Figures 21 and 22.

On the 43rd day, 40 rats in the modeling group were grouped by body weight and divided into a model group and a drug-administered group. One week after administration, the drug efficacy was evaluated by the open field test, and it was found that polypeptide 2 could significantly improve the total distance of exercise in mice, with significant differences, indicating that polypeptide 2 has a better antidepressant effect. The results are shown in Figure 23.

The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments, and within the scope of knowledge possessed by those of ordinary skill in the art, various Variety. Furthermore, the embodiments of the present invention and features in the embodiments may be combined with each other without conflict.

Claims (9)

1. A modified nonapeptide, which is a nonapeptide modified by alkyl acylation and/or modified by phosphorylation, wherein the amino acid sequence of the nonapeptide is:

   Sequence I: R-CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu;

   Sequence II: Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu;

   Sequence III: R-CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu;

   wherein R is a straight or branched chain alkyl group containing 1 to 18 carbon atoms.
2. The modified nonapeptide according to claim 1, wherein R is a straight-chain or branched-chain alkyl group containing 3-12 carbon atoms.
3. The modified nonapeptide according to claim 1, wherein R is a straight-chain or branched-chain alkyl group containing 5-10 carbon atoms.
4. The modified nonapeptide according to claim 1, wherein the amino acid sequence of the modified nonapeptide is: C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu, Trp -Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu, or C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly- Glu.
5. The preparation method of the modified nonapeptide according to any one of claims 1-4, characterized in that, comprising the following steps:

   S1. Synthesis of nonapeptide

   S2. Modification of nonapeptides

   Alkylation and/or phosphorylation of nonapeptides:

   S3. Excision and precipitation to obtain a crude modified nonapeptide;

   S4. Purification of the crude modified nonapeptide.
6. The method for preparing the modified nonapeptide according to claim 5, wherein the synthesis of the nonapeptide described in S1 comprises the following steps: 1. activation of the resin; 2. coupling the first amino acid to the Wang resin; 3. blocking the resin Unreacted functional groups on it; ④ Deprotection of resin functional groups; ⑤ Reaction of peptides.
7. The method for preparing the modified nonapeptide according to claim 4, wherein the synthesis of the nonapeptide adopts a solid-phase synthesis method.
8. A pharmaceutical composition comprising the modified polypeptide of any one of claims 1-4.
9. Use of the modified nonapeptide according to any one of claims 1-4 in the preparation of anti-insomnia, anti-anxiety or anti-depressant drugs.

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