CN116903506A - bis-S-alkyl isothiourea derivative, preparation method and application thereof - Google Patents

bis-S-alkyl isothiourea derivative, preparation method and application thereof Download PDF

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CN116903506A
CN116903506A CN202310781286.0A CN202310781286A CN116903506A CN 116903506 A CN116903506 A CN 116903506A CN 202310781286 A CN202310781286 A CN 202310781286A CN 116903506 A CN116903506 A CN 116903506A
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bis
derivative
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胡宏岗
刘超
陈宝宝
汪楠
丛薇
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University of Shanghai for Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/30—Isothioureas
    • C07C335/38—Isothioureas containing any of the groups, X being a hetero atom, Y being any atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention discloses a bis-S-alkyl isothiourea derivative, a preparation method and application thereof, and belongs to the technical field of biochemistry. The bis-S-alkylisothiourea derivative is a compound shown in a general formula 1 or pharmaceutically acceptable salt thereof,wherein R is saturated alkane or unsaturated alkane; x is any one of i, ii and iii; i is branched C 1 ‑C 20 Saturated alkanes or straight-chain C 1 ‑C 20 Saturated alkanes; ii is an unsaturated alkane; iii is a heteroatom-substituted alkane. The invention also discloses the bis-S-alkanePreparation method and application of thiosemicarbazide derivative, and a staple peptide and preparation method thereof. The bis-S-alkyl isothiourea derivative can be used for synthesizing the staple peptide, and the obtained staple peptide containing the guanidyl structure not only can keep various advantages of the traditional full-hydrocarbon staple peptide in polypeptide structure transformation, but also has the advantages of good water solubility and strong membrane permeation capability.

Description

bis-S-alkyl isothiourea derivative, preparation method and application thereof
Technical Field
The invention relates to a bis-S-alkyl isothiourea derivative, a preparation method and application thereof, and belongs to the technical field of biochemistry.
Background
Currently, the all-hydrocarbon stapling peptide strategy has been successfully applied in a number of active polypeptide modification efforts. However, the chemical kits for constructing stapled peptides are very limited compared to small molecule synthesis methods. More importantly, the introduction of the full-hydrocarbon stapling belt brings about the problem of reduced water solubility of the polypeptide in many cases, and causes certain difficulty in absorption and utilization of the medicaments in organisms. Thus, developing a stapling peptide construction strategy that differs from all-hydrocarbon stapling, particularly hydrophilic stapling, can further expand the potential of stapling strategies in the field of active polypeptide molecule construction.
Guanidine, also known as semicarbazide, is the most basic organic base (pka=13.6), is in a fully protonated state in physiological environments, has good hydrophilicity, and can maintain electropositive properties over a large pH range. Therefore, the compound with guanidine group is easy to form special interaction with receptor and enzyme and substrate through hydrogen bond or electrostatic action, and has high affinity to phosphate, carboxylate and metal ion. Due to the above properties, guanidine groups are present in many drugs, such as guanethidine, clonidine hydrochloride, metformin, phenformin, etc. In addition, guanidine groups are also side chain groups of arginine, a key residue for many polypeptides to acceptor and an important hydrogen bond donor. In addition, because guanidine groups can be protonated, the octa-poly-arginine (R8) sequence is often used as a membrane penetrating peptide with strong membrane penetrating capability, can form hydrogen bonds with negative electric residues on the surface of cells, and enables drugs to penetrate through cell membranes under the action of cell membrane potential.
However, at present, there is no staple peptide containing a guanidine group structure. Accordingly, there is a need to provide a staple peptide containing a guanidino structure that overcomes the deficiencies of the prior art.
Disclosure of Invention
It is an object of the present invention to provide a bis-S-alkylisothiourea derivative.
The technical scheme for solving the technical problems is as follows: a bis-S-alkylisothiourea derivative is a compound shown in a general formula 1 or pharmaceutically acceptable salt thereof,
wherein R is saturated alkane or unsaturated alkane; x is any one of i, ii and iii; i is branched C 1 -C 20 Saturated alkanes or straight-chain C 1 -C 20 Saturated alkanes; ii is an unsaturated alkane; iii is a heteroatom-substituted alkane.
The bis-S-alkylisothiourea derivative has the beneficial effects that:
1. the bis-S-alkyl isothiourea derivative can be used for synthesizing the staple peptide, and the obtained staple peptide containing the guanidyl structure not only can keep various advantages of the traditional full-hydrocarbon staple peptide in polypeptide structure transformation, but also has the advantages of good water solubility and strong membrane permeation capability.
2. The bis-S-alkyl isothiourea derivative provides a synthetic building block for constructing a new stapling strategy, and opens up a new way for deeply researching and developing the new stapling strategy.
On the basis of the technical scheme, the invention can be improved as follows.
Further, R is any one of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, alkene, alkyne, phenyl, benzyl, substituted phenyl and substituted benzyl.
Still further, R is ethyl.
Further, the i is any one of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, and the carbon atoms in the above structures are mono-or polysubstituted with polyethylene glycol, biotin, steroid and fluorescein like functional groups.
Further, the ii is C 1 -C 20 Olefins, C 1 -C 20 Alkyne, C 1 -C 20 Phenyl, C 1 -C 20 Benzyl, C 1 -C 20 Substituted phenyl and C of (2) 1 -C 20 Any one of the substituted benzyl groups, and the substituent is positioned at the ortho position, the meta position or the para position, and is mono-substituted or multi-substituted.
Further, said iii is C 1 -C 20 Oxaalkanes, C 1 -C 20 Thiaalkane, C 1 -C 20 Phosphaalkane, C 1 -C 20 And any of the subsequent modified derivatives on heteroatoms of the aforementioned heteroalkanes.
Still further, the modified derivative is any one of acetylation, biotinylation, pegylation, luciferization, and higher fatty acidification.
Further, the pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, bisulfate, hydrobromide, oxalate, citrate, and methanesulfonate.
Further, the specific structure of the compound of the general formula 1 is any one of the following structures:
the adoption of the method has the further beneficial effects that: the above are preferred compound structures.
The second object of the present invention is to provide a process for producing the bis-S-alkylisothiourea derivative.
The technical scheme for solving the technical problems is as follows: the preparation method of the bis-S-alkyl isothiourea derivative comprises the following steps:
the reaction route is as follows:
wherein R and X have the meanings given above;
step 1: preparation of Compound III
A diamine compound of formula II, in anhydrous methanol, with CS 2 And TEA at room temperature to give a precipitate, which is then added (Boc) 2 O and 4-dimethylaminopyridine react for 15min at room temperature to obtain a compound shown in a formula III;
step 2: preparation of formula 1
The compound of formula III is reacted in methanolic ammonia at room temperature, followed by reaction with Etl and (Boc) under catalysis of 4-dimethylaminopyridine 2 O, reacting for 12 hours at room temperature to obtain the compound shown in the general formula 1.
In the above step 1, the diamine-based compound is commercially available from Shanghai Taitan technologies Co., ltd, and has a specification of 100g.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the compound shown in the general formula 1 reacts with any one of hydrochloric acid, sulfuric acid, hydrobromic acid, oxalic acid, citric acid and methanesulfonic acid to generate corresponding salts.
The preparation method of the bis-S-alkyl isothiourea derivative has the beneficial effects that:
the preparation method provided by the invention is simple, low in cost and wide in market prospect, and is suitable for large-scale popularization and application.
It is a further object of the present invention to provide the use of the bis-S-alkylisothiourea derivative.
The technical scheme for solving the technical problems is as follows: the application of the bis-S-alkyl isothiourea derivative in the synthesis of staple peptides.
The application of the bis-S-alkyl isothiourea derivative has the beneficial effects that:
the invention confirms that the bis-S-alkyl isothiourea derivative can be applied to the construction of staple peptides through liquid phase, mass spectrum, nuclear magnetic resonance technology and the like, and has wide application prospect.
The fourth object of the present invention is to provide a staple peptide.
The technical scheme for solving the technical problems is as follows: a staple peptide is a compound shown in a general formula 2,
wherein AA represents an amino acid; y represents any one of lysine, ornithine, 2, 4-aminobutyric acid and 2, 3-aminopropionic acid.
The staple peptide has the beneficial effects that:
1. the invention uses circular dichroism to test the secondary structure of the staple peptide, which proves that the stapling strategy can enhance the helicity of the polypeptide.
2. The stability experiment of alpha-chymotrypsin proves that the stapling peptide strategy can greatly improve the proteolytic resistance.
3. According to the invention, through coupling of the polypeptide and the fluorescein, the stapling strategy is obtained by utilizing a high content cell drug screening and analyzing system, so that the membrane permeability of the polypeptide can be greatly improved.
4. According to the invention, through a water solubility experiment, the stapling strategy can greatly improve the water solubility of the polypeptide.
5. The staple peptide disclosed by the invention can improve the inhibition effect on HCT116 of human colon cancer cells.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the specific structure of the compound of the general formula 2 is any one of the following structures: wherein Z represents 2, 3-diaminopropionic acid, B represents 2, 4-diaminobutyric acid, O represents ornithine, and K represents lysine;
the expression of the peptides, amino acids and chemical groups used in the present invention are all art-recognized expression methods, wherein the abbreviations for amino acids are as defined in table 1. Amino acids are generally referred to herein as L-form amino acids unless otherwise indicated.
TABLE 1 amino acid abbreviations
Amino acids Three letter abbreviation One letter abbreviation Amino acids Three letter abbreviation One letter abbreviation
Alanine (Ala) Ala A Leucine (leucine) Leu L
Phenylalanine (Phe) Phe F Lysine Lys K
Serine (serine) Ser S Cysteine (S) Cys C
Glycine (Gly) Gly G Isoleucine (Ile) lle l
Valine (valine) Val V Glutamic acid Glu E
Glutamine Gln Q Threonine (Thr) Thr T
Asparagine derivatives Asn N Tryptophan Trp W
Arginine (Arg) Arg R Tyrosine Tyr Y
Proline (proline) Pro P Histidine His H
Methionine Met M Aspartic acid Asp D
The fifth object of the present invention is to provide a method for producing the above-mentioned staple peptide.
The technical scheme for solving the technical problems is as follows: the preparation method of the staple peptide comprises the following steps:
and coupling the S-alkyl isothiourea derivative shown in the general formula 1 with polypeptide to obtain the staple peptide shown in the general formula 2.
Drawings
FIG. 1 is a circular dichroism spectrum and helicity of the stapled peptide of example 3 of the present invention.
FIG. 2 is a graph showing the chymotrypsin degradation profile and half-life in trypsin for the staple peptides of example 4 of the invention.
FIG. 3 shows fluorescence intensity after permeation of the staple peptide in example 5 of the present invention.
FIG. 4 shows the anti-tumor activity and IC of the stapler peptide of example 6 of the present invention 50 Values.
FIG. 5 shows the solubility of the staple peptide in an aqueous solution in example 7 of the present invention.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
The structural formula and nuclear magnetic data of the preferred compounds of the present invention are shown in Table 2.
TABLE 2
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The chemical structure, yield and mass spectral data of the synthesized portion of the preferred stapled peptides of the present invention are shown in Table 3.
TABLE 3 Table 3
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Example 1: preparation of bis-S-alkylisothiourea derivatives (exemplified by M1)
Step 1: preparation of (3- ((thiomethylene) amino) propyl) azone
Absolute ethanol (40 mL)Propylene diamine (1 g,13.5 mmol) was added and CS was slowly added with stirring 2 (10.2 g,135 mmol) and TEA (1.36 g,13.5 mmol) and precipitation (dithiocarbamate) immediately occurred. The reaction mixture was stirred at room temperature for 30 min and then added (Boc) 2 O (2.95 g,13.5 mmol) and a catalytic amount of DMAP were reacted under ice bath conditions for 10 minutes and then left to react at room temperature for 30 minutes. After the reaction was completed, the solvent was removed, ethyl acetate (300 mL) was added, the organic layer was washed with water (3×100 mL), the organic layer was dried over saturated brine, and filtered, and the solution was concentrated in vacuo to give a yellow oily liquid which was purified by silica gel column chromatography (eluent, petroleum ether: ethyl acetate=30:1-1:1, v/v) to give 1.8g of a pale yellow liquid, yield 89%. 1 H NMR(600MHz，DMSO)：δ3.78(t，J＝13.2Hz，4H)，2.02(m，2H). 13 C NMR(150MHz，DMSO)：δ129.30，42.78，29.72。
Step 2: preparation of ((5Z, 12Z) -6- (ethylsulfanyl) -2, 2-dimethyl-4-oxo-3-oxa-13-thia-5, 7, 11-triaza-penta-5-en-12-ylidene) amino) methanoic acid-2-methylpropan-2-yl ester
The product of the previous step (1.5 g,9.55 mmol) was dissolved in methanol (50 mL) of ammonia and reacted at room temperature for 2h. The solvent was removed in vacuo to give a pale yellow oil. Dry methanol (100 mL) was added as a reaction solvent, and TEA (1.16 g,11.46 mmol) and Etl (1.78 g,11.46 mmol) were added to the reaction system to reflux for 6h, after which the solvent was removed in vacuo to give a pale yellow oily liquid. Dichloromethane (100 mL), TEA (1.16 g,11.46 mmol) and (Boc) were then added 2 O (3.6 g,16.68 mmol), stirred overnight at room temperature, water (500 mL) was added, extracted three times with dichloromethane, the organic phases were combined and washed with saturated brine, anhydrous Na 2 SO 4 Drying and concentration in vacuo gave a yellow oily liquid which was purified by silica gel column chromatography (eluent, petroleum ether: ethyl acetate=10:1-5:1, v/v) to give 2.82g of a colourless oily liquid in 66% yield. 1 H NMR(600MHz，DMSO)：δ3.28(d，J＝5.4Hz，4H)，2.95(d，J＝7.2Hz，4H)，1.76(s，2H)，1.40(s，18H)，1.21(t，J＝14.4Hz，6H). 13 C NMR(150MHz，DMSO)：δ160.86，78.26，28.40，24.88，14.59.ESI MS：HRMS m/z calcd for C 19 H 36 N 4 O 4 S 2 448.2178，found[M+H] + 449.50，[M+Na] + 471.50。
The practice of the present invention is not limited to the above examples and other compounds can be synthesized by repeating the steps of the above examples to the desired bis-S-alkylisothiourea derivatives. The reagents used in the examples were all commercially available analytical.
Example 2: solid phase synthesis of staple peptides (exemplified by staple peptide 4 d)
100mg of RINK AMIDE AM resin (substitution degree 0.33 mmol/g) was added to the polypeptide synthesis tube, and 5mL of DCM solution was added thereto for swelling for 20min. The Fmoc protecting group at the amino end of the resin was removed by using 5mL of DMF solution containing 20% piperidine, and after the reaction was performed for 10min, the reaction solution was drained and 5mL of DMF solution containing 20% piperidine was added again to react for 10min. The resin was washed sequentially 5 times with 5mL DMF, 5 times with 5mL DCM, 5 times with 5mL DMF. Fmoc-Met-OH (37 mg,0.1 mmol) and HCTU (41 mg,0.1 mmol) were placed in a 10mL centrifuge tube, 6mL DMF was added for dissolution, DIPEA (38 mg,0.3 mmol) was added, after shaking for 5min, it was poured into a polypeptide synthesis tube and shaking at room temperature for 40min. After the reaction was completed, the solution was removed by suction filtration, and the resin was washed successively with 5mL of DMF 5 times, 5mL of DCM 5 times, and 5mL of DMF 5 times. Fmoc-Val-OH (34 mg,0.1 mmol), fmoc-Arg (Pbf) -OH (65 mg,0.1 mmol), fmoc-Gln (Trt) -OH (61 mg,0.1 mmol), fmoc-Val-OH (34 mg,0.1 mmol) and Fmoc-His (Trt) -OH (62 mg,0.1 mmol) were condensed on the resin in this order according to the method for condensing Fmoc-Met-OH described above. After removal of the amino protecting group Fmoc of Fmoc-Gly-OH using 20% piperidine in DMF, the resin was washed 5 times with 5mL DMF, 5 times with 5mL DCM, 5 times with 5mL DMF. Fmoc-Dap (Alloc) -OH (27 mg,0.066 mmol) and HCTU (27 mg,0.066 mmol) were placed in a 10mL centrifuge tube, 6mL DMF was added to dissolve, DIPEA (26 mg,0.2 mmol) was added, after shaking for 5min it was poured into the polypeptide synthesis tube and shaking at ambient temperature for 2h. After the condensation reaction was completed, the resin was washed 5 times with 5mL DMF, 5 times with 5mL DCM, 5 times with 5mL DMF. Fmoc-Asp (tBu) -OH (41 mg,0.1 mmol), fmoc-Leu-OH (35 mg,0.1 mmol) and Fmoc-Ile-OH (35 mg,0.1 mmol) were then condensed on the resin. After removal of the amino protecting group Fmoc of Fmoc-lle-OH using 20% piperidine in DMF, the resin was washed 5 times with 5mL DMF, 5 times with 5mL DCM, 5 times with 5mL DMF. Fmoc-Dap (alloc) -OH (27 mg,0.066 mmol) and HCTU (27 mg,0.066 mmol) were placed in a 10mL centrifuge tube, 6mL DMF was added to dissolve, DIPEA (26 mg,0.2 mmol) was added, after shaking for 5min it was poured into the polypeptide synthesis tube and shaking at ambient temperature for 2h. After the condensation reaction was completed, the resin was washed 5 times with 5mL DMF, 5 times with 5mL DCM, 5 times with 5mL DMF. Fmoc-Glu (tBu) -OH (43 mg,0.1 mmol), fmoc-Pro-OH (34 mg,0.1 mmol), fmoc-Asn (tBu) -OH (60 mg,0.1 mmol), fmoc-Glu (tBu) -OH (43 mg,0.1 mmol) until all amino acids are joined. After Fmoc protecting group of Fmoc-Glu (tBu) -OH was removed using 20% piperidine in DMF, 5mL pyridine and 5mL acetic anhydride were added to the polypeptide synthesis tube and the procedure was repeated once by shaking at room temperature for 20min. After the reaction was completed, the solution was removed by suction filtration, and the resin was washed 5 times with 5mL of DMF, 5 times with 5mL of DCM, 5 times with 5mL of DMF, 5 times with 5mL of DCE. After further addition of tetrakis (triphenylphosphine) palladium (11 mg,0.01 mmol) and phenylsilane (36 mg,0.33 mmol) to react overnight in the absence of light, the resin was washed 5 times with 5mL DMF, 5 times with 5mL DCM, 5 times with 5mL DMF. To the polypeptide synthesis tube was added 5mL of a mixed solution of M4 (48 mg,0.1 mmol), silver acetate (17 mg,0.1 mmol) and triethylamine (13 mg,0.1 mmol) in DMF, and after reaction for 6h, the mixture was washed 5 times with 5mL of DMF, 5 times with 5mL of DCM, 5 times with 5mL of DMF, and 10 times with 5mL of DCE.
After 20min of the resin was drained using a water pump, 10mL of a solution containing TFA/TIPS/EDT/H was added to the polypeptide synthesis tube 2 O=95: 2:2:1, and cutting the resin for 2 hours. After the reaction was completed, the cut solution was collected and concentrated with argon gas to 2mL, and then, glacial ethyl ether was added to the concentrated solution to precipitate the polypeptide, followed by separation of the precipitate from ethyl ether by a centrifuge. The resulting polypeptide solid was washed 2 times again with glacial diethyl ether. After the obtained crude peptide was naturally air-dried, it was analyzed, identified and purified using HPLC and ESI-MS.
The practice of the invention is not limited to the above examples, and the synthesis of other staple peptides repeats the steps in the above examples. The reagents used in the examples were all commercially available analytical.
Example 3: round two chromatography for testing the secondary structure of staple peptides
PMIs (including staples 14, 15, 16a,16b and 16 c) were combined to form a series of staples, respectively dissolving in trifluoroethanol and water in the same proportion to obtain a sample to be tested with the final concentration of 50 mu M. PMI series polypeptide samples were tested separately using a Jasco-820 round dichroscope at 20 ℃. Ovality data of the samples were collected using quartz cuvettes with a thickness of 1 mm. The observation parameters of the circular dichroscope were set as follows: the wavelength is 190-250nm, the step length is 0.5nm, the bandwidth is 1nm, and the test is carried out for 3 times. The degree of helix of the polypeptide can be obtained from the following formula:
in the formula [ theta ]] 222 In deg.cm, the molar ellipticity at an absorption wavelength of 222nm 2 ·dmol -1 ；[θ] max = (-44000+250t) (1-k/n); k=4; n is the number of amino acids; t is the observed temperature of 20 ℃.
Experimental results: as shown in fig. 1, the linear peptide 14 shows a weak helix structure, the degree of helix is 14.25%, the degree of helix is increased to 24.35% after the conventional stapling modification, the novel stapling peptide has a helix range of 21.75% -27.09%, the change of the degree of helix is not obvious, and the novel stapling mode has no obvious advantage compared with the conventional stapling mode, in other words, the biguanide stapling mode does not change the original secondary structure of the polypeptide.
Example 4: alpha-chymotrypsin stability assay for staple peptides
1-2mg of the peptides 14, 15, 16a,16b and 16c were weighed and dissolved in a predetermined amount of DMSO to prepare a 1mg/mL stock solution. An amount of alpha-chymotrypsin enzyme was weighed out in phosphate buffer saline (1 x, ph=7.4) to a concentration of 100ng/μl of alpha-chymotrypsin. To a 2mL centrifuge tube, 900. Mu.L of a phosphate buffer solution containing chymotrypsin and 100. Mu.L of a polypeptide stock solution were added, respectively, to perform an enzymatic digestion reaction. Incubation was carried out at 37℃and 20. Mu.L of 1M hydrochloric acid was added to 100. Mu.L of each of the reaction solutions at the same time points of 0.5h, 1h, 2h, 4h and 6h to quench chymotrypsin activity. Following centrifugation, supernatants were taken and analyzed for residual amounts of polypeptide at various time points using HPLC.
Experimental results: as shown in fig. 2, the linear peptide degraded about 90% after 2h exposure, was almost completely degraded at 4h, and had a half-life of about 0.4h; compared with linear peptide, the full hydrocarbon stapling modification only degrades about 40% in 2h, the 4h polypeptide also retains 40% content, and typical full hydrocarbon stapling peptide truly enhances the protease hydrolysis resistance, but the novel stapling peptide designed and synthesized only degrades 20% in 2h, and the 4h polypeptide also retains 70% content, so that the protease hydrolysis resistance of the novel stapling peptide is greatly improved compared with full hydrocarbon stapling peptide.
Example 5: evaluation of the Tachypeptide Membrane permeation Capacity
MCF-7 cells were inoculated in 10% fetal bovine serum, 1% diabody in H-DMEM complete medium at 37℃with 5% CO 2 Is cultured in a constant temperature incubator; the MCF-7 cells were digested and then were 5X 10 per well 3 The concentrations of the individuals were plated on average into 96-well plates; the original medium in the 96-well plate was pipetted off, 100. Mu.L of diluted (25. Mu.M, 50. Mu.M and 100. Mu.M) sample solutions (FITC-14, FITC-15, FITC-16a, FITC-16b and FITC-16 a) were added to each well in this order, three wells were set, and at 37℃5% CO 2 Acting for 6 hours in an incubator; nuclei were stained with Hoechst33342 live cell stain for 15min according to the instructions, then washed three times with PBS, fixed with 4% neutral buffered formalin (ph=7.4) for 10min, then washed three times with PBS, and F-actin stained with TRITC Phalidin rhodamine-labeled Phalloidin to index cell range according to the instructions, then washed three times with PBS.
Experimental results: as shown in fig. 3, linear peptides hardly penetrated the cell membrane, and perhydro-and biguanide staplers significantly observed FITC fluorescence signals in a concentration-dependent manner in the nuclei and cytoplasm of MCF-7 cells. Among them, the novel staple peptides showed the strongest fluorescence intensity, indicating excellent membrane permeability, 3.66-4.3 fold enhancement compared to perhydro staple peptides at 100 μm. These results indicate that the novel stapling modification can enhance the permeance to a greater extent.
Example 6: determination of the anti-tumor Activity of Tachypeptide by CCK8 method
Wild p53 tumor strain HCT116 (human colon cancer cells) was inoculated into H-DMEM complete medium containing 10% fetal bovine serum and 1% diabody at 37℃with 5% CO 2 Is in the cell incubator; after the HCT116 cells had attached, they were digested at 1×10 per well 4 The concentration of each was spread evenly in 96-well plates, 100. Mu.L of cell suspension was added to each well, PBS was added only to the surrounding wells, and the wells were placed at 37℃with 5% CO 2 Is in the cell incubator; after 24h, 0. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M and 200. Mu.M polypeptides were added, respectively, and incubated with the cells for 72h. Sucking the complete culture medium containing the medicines in the plate, adding 10 mu L of mixed culture medium of CCK-8 and 90 mu L of culture solution into each hole, placing the mixed culture medium into a cell culture box for acting for 30 minutes, and finally measuring and recording an OD value at 570nm by using a full-wave multifunctional enzyme-labeled instrument; statistics and calculation of IC 50 The obtained OD value is counted by using GraphPad Prism 8.0 and fitted to obtain corresponding IC 50 Values.
Experimental results: as shown in fig. 4, at 100 μm, the inhibition of cells by all four stapled modified polypeptides was improved to a different extent than that of linear peptide 14; wherein 16a and 16c showed better inhibitory activity than perhydro-staple peptides, inhibiting 98% and 93% of tumor cells, respectively.
Example 7: evaluation of Water solubility of staple peptides
1-2mg 14, 15, 16a,16b and 16c are respectively weighed and dissolved in a certain amount of DMSO to prepare standard solution with the concentration of 1mg/mL, the polypeptide is continuously diluted to 200ng/mL,100ng/mL,50ng/mL,25ng/mL,12.5ng/mL and 6.25ng/mL, and six standard curves are drawn by utilizing HPLC to analyze the content of the polypeptides with different concentrations; a small amount of water was added to a 200 μl centrifuge tube, and a certain amount of polypeptides 14, 15, 16a,16b and 16c were added to the centrifuge tube, respectively, until they were no longer dissolved, followed by shaking the centrifuge tube overnight, centrifuging, collecting the supernatant, and analyzing the content of polypeptides using HPLC.
Experimental results: as shown in FIG. 5, the water solubility of the linear peptide 14 was 6.7. Mu.g/mL, whereas the water solubility of the all-hydrocarbon staple peptide 15 was 1.2. Mu.g/mL, which was nearly 6-fold reduced compared to the linear peptide. Surprisingly, biguanide stapling peptides 16a,16b and 16c have water solubility of 50.8mg/mL, 72.6mg/mL and 57.8mg/mL, respectively, which are 42333-fold, 60500-fold and 48166-fold enhanced, and this experimental result shows that biguanide stapling modification has a very great development potential.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (10)

1. A bis-S-alkylisothiourea derivative is characterized by being a compound shown in a general formula 1 or a pharmaceutically acceptable salt thereof,
wherein R is saturated alkane or unsaturated alkane; x is any one of i, ii and iii; i is branched C 1 -C 20 Saturated alkanes or straight-chain C 1 -C 20 Saturated alkanes; ii is an unsaturated alkane; iii is a heteroatom-substituted alkane.
2. The bis-S-alkylisothiourea derivative according to claim 1, characterized in that R is any one of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, alkene, alkyne, phenyl, benzyl, substituted phenyl and substituted benzyl; the i is any one of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, n-pentyl, cyclopentyl, n-hexyl and cyclohexyl, and carbon atoms in the structure are mono-substituted or polysubstituted by polyethylene glycol, biotin, steroid and fluorescein functional groups; by a means ofIi is C 1 -C 20 Olefins, C 1 -C 20 Alkyne, C 1 -C 20 Phenyl, C 1 -C 20 Benzyl, C 1 -C 20 Substituted phenyl and C of (2) 1 -C 20 Any one of the substituted benzyl groups, wherein the substituent is positioned at the ortho position, the meta position or the para position, and is mono-substituted or multi-substituted; iii is C 1 -C 20 Oxaalkanes, C 1 -C 20 Thiaalkane, C 1 -C 20 Phosphaalkane, C 1 -C 20 And any of the subsequent modified derivatives on heteroatoms of the aforementioned heteroalkanes.
3. The bis-S-alkylisothiourea derivative according to claim 2, characterized in that the modified derivative is any one of acetylation, biotinylation, pegylation, luciferization and higher fatty acidification.
4. The bis-S-alkylisothiourea derivative according to claim 1, characterized in that the pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, bisulfate, hydrobromide, oxalate, citrate and mesylate.
5. The bis-S-alkylisothiourea derivative according to claim 1, characterized in that the specific structure of the compound of formula 1 is any one of the following structures:
6. a process for the preparation of bis-S-alkylisothiourea derivatives as claimed in any one of claims 1 to 5, characterized in that it comprises the steps of:
the reaction route is as follows:
wherein R and X have the meanings given in claim 1;
step 1: preparation of Compound III
Diamine compound shown in formula II, and CS in anhydrous methanol 2 And TEA at room temperature to give a precipitate, which is then added (Boc) 2 O and 4-dimethylaminopyridine react for 15min at room temperature to obtain a compound shown as a formula III;
step 2: preparation of formula 1
The compound of formula III is reacted in methanolic ammonia at room temperature, followed by reaction with EtI and (Boc) under catalysis of 4-dimethylaminopyridine 2 O, reacting for 12 hours at room temperature to obtain the compound shown in the general formula 1.
7. Use of a bis-S-alkylisothiourea derivative as claimed in any one of claims 1 to 5 in the synthesis of a staple peptide.
8. A staple peptide is characterized by being a compound represented by the general formula 2,
wherein AA represents an amino acid; y represents any one of lysine, ornithine, 2, 4-diaminobutyric acid and 2, 3-diaminopropionic acid.
9. The stapling peptide of claim 8, wherein the specific structure of the compound of formula 2 is any one of the following structures: wherein Z represents 2, 3-diaminopropionic acid, B represents 2, 4-diaminobutyric acid, O represents ornithine, and K represents lysine;
10. the method for producing a staple peptide according to claim 8 or 9, comprising the steps of: and coupling the S-alkyl isothiourea derivative shown in the general formula 1 with polypeptide to obtain the staple peptide shown in the general formula 2.
CN202310781286.0A 2023-06-28 2023-06-28 bis-S-alkyl isothiourea derivative, preparation method and application thereof Pending CN116903506A (en)

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