SS-31 novel derivative as well as preparation method and application thereof
Novel SS-31 derivatives with tailored substituents address resistance issues by enhancing anti-inflammatory and mitochondrial protective activities, offering improved therapeutic outcomes in neurological disease treatment.
Patent Information
- Application Number
- CN202411245039.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-06
- Publication Date
- 2024-12-13
AI Technical Summary
Existing SS-31 derivatives face resistance issues, limiting their therapeutic efficacy in treating neurological diseases characterized by mitochondrial dysfunction and neuroinflammation, necessitating the development of alternative compounds to bypass resistance mechanisms and ensure sustained therapeutic effects.
Synthesis of novel SS-31 derivatives with specific substituents R1, R2, R3, and R4, utilizing a solid phase peptide synthesis method, followed by purification steps to obtain targeted peptides, which are then evaluated for anti-inflammatory and mitochondrial protective activities.
The new SS-31 derivatives demonstrate enhanced anti-inflammatory and mitochondrial protective effects, outperforming SS-31 in LPS-induced BV2 inflammatory cell models and rotenone-induced HT22 cell injury models, indicating potential therapeutic benefits for neurological diseases.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel SS-31 derivative and a preparation method and application thereof. Background Art
[0002] Neuroinflammation is a defense mechanism of the brain and is closely related to a variety of neurological diseases. Microglia are the main immune cells of the nervous system and play a major role in the initiation and continuation of inflammatory responses. Excessive activation of microglia leads to the release of various inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), which promote the occurrence and development of a variety of neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and stroke. Mitochondrial dysfunction is an important pathological feature of related neurological diseases, and it has a complex intrinsic relationship with neuroinflammation: on the one hand, the immune receptors of microglia recognize mitochondrial-derived damage-associated molecular patterns (DAMPs) and aggravate neuroinflammation; at the same time, the inflammatory factors released by activated microglia trigger intracellular cascade reactions, aggravate mitochondrial dysfunction, and the two form a vicious cycle to promote the development of the disease. Studies have shown that in the development of the disease, mitochondrial dysfunction occurs before neuroinflammation and can promote neuroinflammation. Therefore, developing mitochondrial-based molecular drugs to inhibit neuritis is crucial for the treatment of neurological diseases.
[0003] SS-31 (D-Arg-Dmt-Lys-Phe-NH2), also known as elamipretide, is a mitochondrial-targeted tetrapeptide that has attracted great attention in biomedical research for its potential therapeutic effects on mitochondrial dysfunction. Cardiolipin in the inner mitochondrial membrane is essential for mitochondrial function. SS-31 selectively targets cardiolipin in the inner mitochondrial membrane, improves mitochondrial biological function by stabilizing cardiolipin and maintaining mitochondrial membrane integrity, and reduces the generation of ROS. SS-31 has been mainly studied for its protective effects on mitochondria and has potential applications in various diseases involving mitochondrial dysfunction, such as cardiovascular disease, neurodegenerative diseases, and muscular dystrophy. Studies have shown that SS-31 can inhibit mitochondrial fission and has anti-inflammatory properties, thereby exerting neuronal protective effects. Recent clinical studies on SS-31 suggest that its clinical effect is poor. In order to improve the therapeutic effect of SS-31, we designed and synthesized a variety of new SS-31 derivatives and studied their anti-inflammatory and mitochondrial protective activities. In cases where SS-31 resistance may develop, SS-31 derivatives may provide alternative options to circumvent the resistance mechanism and ensure sustained therapeutic efficacy. Summary of the invention
[0004] In order to solve the problems existing in the prior art, the present invention provides novel SS-31 derivatives and preparation methods and applications thereof. In the case where SS-31 resistance may occur, the SS-31 derivatives can provide an alternative option to bypass the resistance mechanism and ensure a sustained therapeutic effect.
[0005] A new derivative of SS-31, the structural formula of which is as follows:
[0006]
[0007] Wherein, R1, R2, R3, and R4 represent substituents,
[0008] R1 is or R2 is H or R3 is or R4 is or And it does not include the SS-31 itself.
[0009] Preferably, R1, R2, R3, and R4 are different substituents.
[0010] Preferably, R1 is R4 is
[0011] Preferably, R3 is or
[0012] The preparation method of the novel SS-31 derivative comprises the following steps:
[0013] S1. Add RinkAmide MBHA resin into the peptide synthesis container, perform swelling treatment, and wash for later use.
[0014] S2, using a solid phase peptide synthesis method, adding the corresponding Fmoc-amino acid-OH to the peptide synthesis container in the order of R3, R4, R1 and R2 to perform a coupling reaction to obtain a product E1 with an Fmoc group, and after removing the Fmoc protecting group, obtaining a product E2,
[0015] S3, pour the product E2 into a cleavage bottle for peptide resin cleavage and deprotection, filter out the resin to obtain a crude product cleavage solution,
[0016] S4. The crude product lysate is concentrated, centrifuged and purified to finally obtain the target solid peptide.
[0017] Preferably, in step S1, DCM and DMF are used for washing alternately, and DCM or a mixture of DMF and DCM is used for swelling.
[0018] Preferably, in step S2, an HBTU / DIEA condensation system is used, and the ratio of Fmoc-amino acid-OH: HBTU: DIEA is 3:3:6.
[0019] Preferably, in step S3, the lysis solution is TFA:Tips:water=90:5:5.
[0020] Preferably, in step S4, the crude product lysate is first purged and concentrated with pure nitrogen, then precipitated with pre-cooled anhydrous ether, and the mixture is centrifuged to obtain a precipitated crude peptide; the crude peptide is then washed with ether and vacuum dried to obtain a powdered crude peptide; the crude peptide is then dissolved in a CH3CN / H2O solvent containing 0.1% TFA; finally, the target solid peptide is obtained by semi-preparative RP-HPLC purification and freeze-drying for 24-48 hours.
[0021] Application of novel SS-31 derivatives in the preparation of drugs for treating nervous system diseases.
[0022] Compared with the prior art, the present invention has the following beneficial effects: a variety of new SS-31 derivatives and their preparation routes are designed, and their anti-inflammatory activity and mitochondrial protection activity are studied. In the case where SS-31 resistance may occur, SS-31 derivatives can provide alternative options to bypass the resistance mechanism and ensure sustained therapeutic effects; in the LPS-induced BV2 inflammatory cell model, the anti-inflammatory effect of some new SS-31 derivatives is better than that of SS-31; in the rotenone-induced HT22 cell injury model, the ATP recovery effect of some new SS-31 derivatives is better than that of SS-31; the new SS-31 derivatives in this application have therapeutic potential for neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 It is the structural formula diagram of SS-31 in the prior art,
[0024] Figure 2 is the structural formula of the novel SS-31 derivative prepared in Examples 1 to 4,
[0025] Figure 3 is the structural formula of the novel SS-31 derivative prepared in Examples 5 to 8,
[0026] Figure 4 is the structural formula of the novel SS-31 derivative prepared in Examples 9 to 12,
[0027] Figure 5is the structural formula of the novel SS-31 derivative prepared in Examples 13 to 16,
[0028] Figure 6 is the structural formula of the novel SS-31 derivative prepared in Examples 17 to 19,
[0029] Figure 7 This is a schematic diagram of the preparation method of Example 1.
[0030] Figure 8 is the HPLC spectrum of the new SS-31 derivative of structural formula 5a,
[0031] Fig. 9 The HPLC spectrum of the new SS-31 derivative of structural formula 5f is shown in FIG.
[0032] Fig.10 The HPLC spectrum of the new SS-31 derivative with structural formula 5g is shown in FIG.
[0033] Fig.11 The ESI-MS spectrum of the new SS-31 derivative of structural formula 5a is shown in FIG.
[0034] Fig.12 This is the ESI-MS spectrum of the new SS-31 derivative with structural formula 5f.
[0035] Fig.13 This is the ESI-MS spectrum of the new SS-31 derivative with structural formula 5g.
[0036] Fig.14 The mass spectrometry results of the SS-31 derivatives shown in structural formulas 5a to 5s are summarized in the table.
[0037] Fig.15 The results of the effects of SS-31 and its derivatives on the LPS-induced inflammatory response of BV2 are shown in the figure.
[0038] Fig.16 The results of the test of SS-31 and its derivatives 5f and 5g inhibiting LPS-induced mitochondrial damage in BV2 cells.
[0039] Fig.17 This is the test result of SS-31 and its derivatives 5f and 5g inhibiting the reduction of ATP synthesis in HT22 cells induced by rotenone.
[0040] Fig.18 The results of the test of SS-31 and its derivatives 5f and 5g inhibiting rotenone-induced mitochondrial damage in HT22 cells.
[0041] Fig.19This is a test result of SS-31 and its derivatives 5f and 5g inhibiting rotenone-induced mitochondrial oxidative stress in HT22 cells. DETAILED DESCRIPTION
[0042] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0043] A new derivative of SS-31, the structural formula of which is as follows:
[0044]
[0045] Wherein, R1, R2, R3, and R4 represent substituents,
[0046] R1 is or R2 is H or R3 is or
[0047] R4 is or And it does not include the SS-31 itself.
[0048] In one embodiment, R1, R2, R3, and R4 are different substituents.
[0049] In one embodiment, R1 is R4 is
[0050] In one embodiment, R3 is or
[0051] The preparation method of the novel SS-31 derivative comprises the following steps:
[0052] S1. Add RinkAmide MBHA resin into the peptide synthesis container, perform swelling treatment, and wash for later use.
[0053] S2, using a solid phase peptide synthesis method, adding the corresponding Fmoc-amino acid-OH to the peptide synthesis container in the order of R3, R4, R1 and R2 to perform a coupling reaction to obtain a product E1 with an Fmoc group, and after removing the Fmoc protecting group, obtaining a product E2,
[0054] S3, pour the product E2 into a cleavage bottle for peptide resin cleavage and deprotection, filter out the resin to obtain a crude product cleavage solution,
[0055] S4. The crude product lysate is concentrated, centrifuged and purified to finally obtain the target solid peptide.
[0056] In one embodiment, in step S1, DCM and DMF are used for washing alternately, and DCM or a mixture of DMF and DCM is used for swelling.
[0057] In one embodiment, in step S2, an HBTU / DIEA condensation system is used, and the ratio of Fmoc-amino acid-OH: HBTU: DIEA is 3:3:6.
[0058] In one embodiment, in step S3, the lysis solution is TFA:Tips:water=90:5:5.
[0059] In one embodiment, in step S4, the crude product lysate is first purged and concentrated with pure nitrogen, then precipitated with pre-cooled anhydrous ether, and the mixture is centrifuged to obtain a precipitated crude peptide; the crude peptide is then washed with ether and vacuum dried to obtain a powdered crude peptide; the crude peptide is then dissolved in a CH3CN / H2O solvent containing 0.1% TFA; finally, the target solid peptide is obtained by semi-preparative RP-HPLC purification and freeze-drying for 24-48 hours.
[0060] Application of novel SS-31 derivatives in the preparation of drugs for treating nervous system diseases.
[0061] Example 1: A structural formula is Figure 2 The novel SS-31 derivative shown in 5a is prepared by combining Figure 7 To understand, the steps are as follows:
[0062] S1. Add 0.2 mmol RinkAmide MBHA resin (1 equiv, loading = 0.6 mmol / g) into a peptide synthesis container, add 10-20 ml of DCM to swell the resin for 60 min, then wash the resin with appropriate amounts of DCM and DMF for later use.
[0063] S2. Add 20 ml of 20% piperidine / DMF solution to the resin, shake on a shaker for 20 min to remove the Fmoc protecting group, then wash the resin with appropriate amounts of DCM and DMF, 20 ml*5 times, remove the solvent from the resin and set aside.
[0064] S3, Fmoc-Phe-OH: HBTU: DIEA in a ratio of 3:3:6 was dissolved in DMF and clarified, and then added to the resin in step S2. After shaking on a shaker for 30 minutes, the resin was washed with appropriate amounts of DCM and DMF, 20 ml * 3 times, to obtain amino acid resin compound 1a. The resin compound 1a was extracted with the solvent and set aside.
[0065] S4, add 20 ml of 20% piperidine / DMF solution to the resin compound 1a, shake on a shaker to remove the Fmoc protecting group for 20 min, then wash the resin with appropriate amounts of DCM and DMF, 20 ml*5 times, to obtain the resin compound 1b, remove the solvent from the resin compound 1b and set aside,
[0066] S5, Fmoc-Lys(Boc)-OH:HBTU:DIEA was dissolved in DMF in a ratio of 3:3:6 and then added to the resin compound 1b in step S4. After shaking on a shaker for 30 minutes, the resin was washed with appropriate amounts of DCM and DMF, 20 ml*3 times, to obtain an amino acid resin compound 1c. The resin compound 1c was used for standby after the solvent was removed.
[0067] S6. Add 20 ml of 20% piperidine / DMF solution to the resin compound 1c, shake on a shaker for 20 min to remove the Fmoc protecting group, then wash the resin with appropriate amounts of DCM and DMF, 20 ml*5 times, to obtain the resin compound 1d. Drain the solvent from the resin compound 1d and set aside.
[0068] S7, Fmoc-Tyr(otbu)-OH:HBTU:DIEA was dissolved in DMF in a ratio of 3:3:6 and then added to the resin compound 1d in step S6. After shaking on a shaker for 30 minutes, the resin was washed with appropriate amounts of DCM and DMF, 20 ml*3 times, to obtain an amino acid resin compound 1e. The resin compound 1e was used for standby after the solvent was removed.
[0069] S8, add 20 ml of 20% piperidine / DMF solution to resin compound 5, shake on a shaker to remove the Fmoc protecting group for 20 min, then wash the resin with appropriate amount of DCM and DMF, 20 ml*5 times, to obtain resin compound 2, remove the solvent from resin compound 2 and set aside,
[0070] S9, Fmoc-D-Arg(Pbf)-OH:HBTU:DIEA in a ratio of 3:3:6 was dissolved in DMF and clarified, and then added to the resin compound 2 in step 8. After shaking on a shaker for 30 minutes, the resin was washed with appropriate amounts of DCM and DMF, 20 ml*3 times, to obtain amino acid resin compound 3. The resin compound 3 was extracted with the solvent and set aside.
[0071] S10, add 20 ml of 20% piperidine / DMF solution to resin compound 3, shake on a shaker to remove the Fmoc protecting group for 20 min, then wash the resin with appropriate amount of DCM and DMF, 20 ml*5 times, to obtain resin compound 4, remove the solvent from resin compound 4 and set aside,
[0072] S11, wash the resin compound 4 with an appropriate amount of methanol, 20 ml*5 times, and dry the resin in a vacuum until constant weight is set aside.
[0073] S12, pour the resin peptide 4 obtained in step 11 into a cleavage bottle, add 15 ml of cleavage solution, the cleavage solution TFA: Tips: water = 90:5:5, and perform peptide resin cleavage and deprotection at 5°C for 2 hours.
[0074] S13, after filtering off the resin with a Buchner funnel, a crude product lysate was obtained, the crude product lysate was purged and concentrated with pure nitrogen, and then precipitated with pre-cooled anhydrous ether, and the mixture was centrifuged to obtain a precipitated crude peptide; the crude peptide was washed three times with ether and then vacuum dried to obtain a crude peptide in the form of an off-white powder; the crude peptide was then dissolved in a specific ratio of CH3CN / H2O solvent (containing 0.1% TFA, v / v), and finally, the target solid peptide 5 was obtained by semi-preparative RP-HPLC purification and lyophilization for 24-48 hours. The purity of the obtained peptide was analyzed by analytical RP-HPLC and ESI-MS.
[0075] Example 2-19: The structural formulas are Figure 2-6 The preparation process of the novel SS-31 derivatives shown in 5b to 5s is carried out according to Example 1, except that the Fmoc-amino acid-OH added in steps S3, S5, S7 and S9 is different, and the ratio of Fmoc-amino acid-OH:HBTU:DIEA remains unchanged.
[0076] for example:
[0077] When preparing the novel SS-31 derivative shown in 5b, in step S7, add
[0078] Fmoc-Tyr(PO(OBZL)OH)-OH, CAS:191348-16-0;
[0079] When preparing the novel SS-31 derivative shown in 5c, in step S7, add
[0080] Fmoc-DOPA(acetonide)-OH, CAS:852288-18-7;
[0081] When preparing the novel SS-31 derivative shown in 5d, in step S7, Fmoc-Tyr(3-Cl)-OH, CAS: 478183-58-3, is added;
[0082] When preparing the novel SS-31 derivative shown in 5e, in step S7, Fmoc-Tyr(3-NO2)-OH, Cas: 136590-09-5 was added;
[0083] When preparing the novel SS-31 derivative shown in 5f, in step S7, add
[0084] Fmoc-Tyr(2,6-Me2,4-tBu)-OH, CAS: 1043043-79-3; in step S9, a substituted arginine is added, wherein the substituted arginine is firstly introduced with a succinic acid linker arm on the amino group of arginine, and then reacted with taurine to form a substituted arginine;
[0085] When preparing the novel SS-31 derivative shown in 5g, in step S3, Fmoc-2-Nal-OH, CAS: 112883-43-9, is added;
[0086] When preparing the novel SS-31 derivative shown in 5h, in step S3, Fmoc-Phe(4-NO2)-OH, CAS: 95753-55-2, is added;
[0087] When preparing the novel SS-31 derivative shown in 5i, in step S3, Fmoc-HomoPhe-OH, CAS: 132684-59-4, is added;
[0088] When preparing the novel SS-31 derivative shown in 5j, in step S3, Fmoc-1-Nal-OH, CAS: 96402-49-2, is added;
[0089] When preparing the novel SS-31 derivative shown in 5k, in step S3, Fmoc-Trp(Boc)-OH, CAS: 143824-78-6, is added;
[0090] When preparing the novel SS-31 derivative shown in 5l, in step S3, Fmoc-L-Phe(4-ME)-OH, CAS: 199006-54-7, is added;
[0091] When preparing the novel SS-31 derivative shown in 5m, in step S3, Fmoc-Tyr(tbu)-OH, Cas: 71989-38-3, is added;
[0092] When preparing the novel derivative of SS-31 shown in 5n, in step S3, Fmoc-Tyr(Bzl)-OH, Cas: 71989-40-7 is added;
[0093] When preparing the novel SS-31 derivative shown in 5o, in step S3, Fmoc-Tyr(4-Et)-OH, CAS: 119894-20-1, is added;
[0094] When preparing the novel SS-31 derivative shown in 5p, in step S3, Fmoc-Tyr(Me)-OH, CAS: 77128-72-4, is added;
[0095] When preparing the novel SS-31 derivative shown in 5q, in step S5, Fmoc-Arg(Pbf)-OH, CAS: 154445-77-9, is added;
[0096] When preparing the novel SS-31 derivative shown in 5r, in step S5, Fmoc-Cit-OH, CAS: 133174-15-9, is added;
[0097] When preparing the novel SS-31 derivative shown in 5s, Fmoc-His(Trt)-OH, CAS: 109425-51-6, is added in step S5.
[0098] In the preparation of 5b to 5e in Example 2-5, in step S1, a DMF / DCM volume ratio of 3:1 was used for swelling for 30 minutes.
[0099] The purity of the obtained peptide was analyzed by analytical RP-HPLC and ESI-MS. Figure 8-14 As shown, Figure 8-13 The HPLC and ESI-MS spectra of the novel SS-31 derivatives with structural formula 5a, 5f and 5g are listed. Fig.14 This is a summary table of mass spectrometry detection results.
[0100] To further study the anti-inflammatory and mitochondrial protective activities of the novel SS-31 derivatives, relevant experiments were conducted as follows:
[0101] Experimental steps:
[0102] Drug preparation: Weigh the drug powder and use phosphate buffered saline (PBS) or dimethyl sulfoxide (DMSO) to prepare the stock solution. The drug solvents and concentrations are as follows: SS-31 (PBS, 10 mM), SS-31 derivatives (PBS, 10 mM), ginsenoside Rb1 (PBS, 20 mM), and idebenone (DMSO, 10 mM).
[0103] Cell culture and treatment: Mouse microglia (BV2) and mouse hippocampal neuron cells (HT22) were cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin. They were routinely cultured in an incubator at 37°C, 5% CO2 and saturated humidity, and cells in the logarithmic growth phase were used for the experiment. BV2 cells were cultured at 5×10 5 / well inoculated in a 12-well plate, 0.8×10 5 / well were seeded in 8-wellLab-Tekchamberslides, and inflammation was induced with LPS-5ng / mL, and then co-incubated with Rb1-100μΜ / SS-31-10μM / SS-31 derivative-10μM for 24h. HT22 cells were inoculated at 1×10 4 / well inoculated in 96-well plates or 0.5×10 5 The cells were seeded in 8-wellLab-Tek chamber slides, stimulated with rotenone 1 μM, and then co-incubated with idebenone-5 μM / SS-31 / 5f / 5g for 24 h.
[0104] Real-time quantitative PCR experiment: BV2 cells were seeded in 12-well plates as described above. After specific treatment, total RNA was isolated from BV2 cells using 5× All-In-One RT Master Mix, and then reverse transcribed into cDNA using ChamQ Universal SYBR qPCR Master Mix. The mRNA expression of IL-6, IL-1β, and TNF-α was analyzed on the LightCycler 480 SYBR Green I Master on the LightCycler 480 II system. GAPDH was used as an internal reference gene. According to 2-
[0105] [Ct(candidate)-Ct(GAPDH)] was used to calculate the fold change of gene expression.
[0106] ATP Assay: The CellTiter-Glo Cell Viability Assay Kit was used to assess changes in cellular adenosine triphosphate (ATP) levels in HT22 cells. HT22 cells were seeded in opaque white 96-well plates and treated as described above. The assay was performed according to the kit instructions.
[0107] SpectraMaxi3xMulti-ModeMicroplateReader records the fluorescence values.
[0108] Mitochondrial staining and imaging: BV2 and HT22 cells were plated in 8-wellLab-Tek chamber slides as described above. After specific treatments, MitoTracker TM
[0109] Dyeformitochondria Labeling was used to stain mitochondria, and the morphological changes of mitochondria in BV2 and HT22 cells were observed. Hoechst 33342 dye was used to label the cell nucleus. Confocal fluorescence microscopy was used to collect images.
[0110] Mitochondrial reactive oxygen species detection: using the mitochondrial superoxide indicator MitoSOX TM Red was used to evaluate mitochondrial superoxide production in HT22 cells. HT22 cells were plated in 8-wellLab-Tek chamber slides as described above. After the indicated treatments, cells were incubated with 5 μM MitoSOX TM The eggs were incubated at 37°C for 10 min in a working solution of 10 μg / mL Hoechst 33342 for staining. Images were acquired using a confocal fluorescence microscope.
[0111] Experimental results analysis:
[0112] For the convenience of description, the novel SS-31 derivatives with structural formula 5f and 5g in this application are abbreviated as 5f and 5g. Fig.15 There are three graphs A, B and C in the equation, of which graph A is denoted as Fig.15 A.
[0113] To evaluate the activity of SS-31 derivatives, we evaluated the effects of SS-31 derivatives on LPS-induced inflammatory responses in BV2 microglial cell line. Fig.15 Ginsenoside Rb1 was selected as the positive drug. Fig.15 As shown in A, LPS induced an increase in the expression of inflammatory factor IL-6 mRNA. SS-31 and its derivatives significantly inhibited the upregulation of IL-6 mRNA expression induced by LPS, among which 5f and 5g showed more significant inhibitory effects than SS-31. In order to further verify the anti-inflammatory activity of 5f and 5g, we detected the expression levels of inflammatory factors IL-1β and TNF-α, as shown in Fig.15 As shown in B and C, SS-31, 5f and 5g can inhibit the upregulation of IL-1β expression induced by LPS, but 5f and 5g have better anti-inflammatory activity than SS-31. More importantly, SS-31 has no significant inhibitory effect on the increase of TNF-α expression induced by LPS, while 5f and 5g can simultaneously inhibit the upregulation of TNF-α expression induced by LPS. These results together indicate that 5f and 5g have more significant anti-inflammatory activity than SS-31.
[0114] The protective effect of SS-31 and its derivatives 5f and 5g on mitochondria was analyzed. Mitochondria were labeled with MitoTrackerTM. Fig.16As shown in A, BV2 cells treated with LPS showed a decrease in mitochondrial tubular structures and mitochondrial fragmentation. SS-31 and its derivatives 5f and 5g could significantly inhibit LPS-induced mitochondrial fragmentation. Fig.16 As shown in B, we counted at least 60 mitochondria in each group and found that the length of mitochondria in BV2 cells treated with LPS decreased, and SS-31, 5f, and 5g could inhibit the reduction of mitochondrial length induced by LPS. At the same time, compared with SS-31, 5f and 5g could better maintain the mitochondrial morphology, and their protective effect on mitochondria was basically the same as that of the positive drug Rb1. This result suggests that the better anti-inflammatory activity of 5f and 5g is based on their better protective effect on mitochondria.
[0115] To evaluate the protective effect of 5f and 5g on mitochondria, we used the rotenone injury model in the mouse hippocampal neuron cell line HT22 cells to verify the mitochondrial protective activity of 5f and 5g, and used idebenone as a positive drug control. First, we used an ATP kit to detect the ATP level in HT22 cells. Fig.17 As shown in the results, compared with the control group, the intracellular ATP level decreased by about 50% after 12 hours of rotenone treatment, idebenone could significantly restore the intracellular ATP level, SS-31 had little effect on the recovery of ATP level, and SS-31 derivatives 5f and 5g could significantly inhibit the reduction of ATP production induced by rotenone in a dose-dependent manner, and they played a good role at 50nM. This result further verified that compared with SS-31, 5f and 5g have stronger mitochondrial protective activity, thereby playing a neuronal protective role.
[0116] MitoTrackerTM was used to label mitochondria to evaluate the direct protective effects of 5f and 5g on mitochondria in HT22 cells. Fig.18 As shown in A, HT22 cells treated with rotenone showed a decrease in mitochondrial tubular structures and mitochondrial fragmentation. Idebenone and SS-31 derivatives 5f and 5g could significantly inhibit rotenone-induced mitochondrial fragmentation. Fig.18 As shown in B, we counted at least 60 mitochondria in each group and found that the mitochondrial length of HT22 cells treated with rotenone was reduced, and idebenone, 5f, and 5g could inhibit the reduction of mitochondrial length induced by rotenone. The mitochondrial morphology and length of SS-31 were the same as those of the modeling group. This result further verified that compared with SS-31, 5f and 5g had a direct protective effect on neuronal inhibition of mitochondria, and their activity was significantly higher than that of SS-31, which could directly partially inhibit the neuronal toxicity of the mitochondrial damaging agent rotenone.
[0117] MitoSOX, a mitochondrial-specific superoxide indicator TM Red further evaluated the intervention effects of SS-31 derivatives on mitochondrial oxidative stress. Fig.19As shown, treatment of HT22 cells with 1 μM rotenone for 12 h significantly increased mitochondrial ROS levels, with visible perinuclear mitochondrial MitoSOX TM The intensity of immunofluorescence was significantly enhanced, and the dye entered the nucleus, indicating that the nuclear membrane was damaged. Idebenone and SS-31 derivatives 5f and 5g could significantly inhibit the generation of mitochondrial ROS, while SS-31 had no significant inhibitory effect on rotenone-induced mitochondrial ROS generation. This result further verified the direct protective effect of 5f and 5g on neuronal mitochondria, and its effect was significantly better than SS-31.
[0118] In the LPS-induced BV2 inflammatory cell model, some new SS-31 derivatives have better anti-inflammatory effects than SS-31; in the rotenone-induced HT22 cell injury model, some new SS-31 derivatives have better ATP recovery effects than SS-31, such as 5f and 5g, which have therapeutic potential for neurological diseases. The next step is to try to use them in the preparation of drugs for the treatment of neurological diseases.
[0119] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
Claims
1. A novel derivative of SS-31, characterized in that: Its structural formula is as follows: Wherein, R1, R2, R3, and R4 represent substituents, R1 is or R2 is H or R3 is or R4 is or And it does not include the SS-31 itself.
2. The novel SS-31 derivative according to claim 1, characterized in that: R1, R2, R3, and R4 are different substituents.
3. The novel SS-31 derivative according to claim 2, characterized in that: R1 is R4 is 4. The novel SS-31 derivative according to claim 3, characterized in that: R3 is or 5. The method for preparing the novel SS-31 derivative according to any one of claims 1 to 4, characterized in that: The steps include: S1. Add RinkAmide MBHA resin into the peptide synthesis container, perform swelling treatment, and wash for later use. S2, using a solid phase peptide synthesis method, adding the corresponding Fmoc-amino acid-OH to the peptide synthesis container in the order of R3, R4, R1 and R2 to perform a coupling reaction to obtain a product E1 with an Fmoc group, and after removing the Fmoc protecting group, obtaining a product E2, S3, pour the product E2 into a cleavage bottle for peptide resin cleavage and deprotection, filter out the resin to obtain a crude product cleavage solution, S4. The crude product lysate is concentrated, centrifuged and purified to finally obtain the target solid peptide.
6. The method for preparing the novel SS-31 derivative according to claim 5, characterized in that: In step S1, DCM and DMF are used for washing alternately, and DCM or a mixture of DMF and DCM is used for swelling.
7. The method for preparing the novel SS-31 derivative according to claim 5, characterized in that: In step S2, the HBTU / DIEA condensation system is used, and the ratio of Fmoc-amino acid-OH:HBTU:DIEA is 3:3:
6.
8. The method for preparing the novel SS-31 derivative according to claim 5, characterized in that: In step S3, the lysis solution is TFA:Tips:water=90:5:
5.
9. The method for preparing the novel SS-31 derivative according to claim 5, characterized in that: Step S4, the crude product lysate is first purged and concentrated with pure nitrogen, then precipitated with pre-cooled anhydrous ether, and the mixture is centrifuged to obtain a precipitated crude peptide; the crude peptide is then washed with ether and vacuum dried to obtain a powdered crude peptide; the crude peptide is then dissolved in a CH3CN / H2O solvent containing 0.1% TFA; finally, it is purified by semi-preparative RP-HPLC and freeze-dried for 24-48 hours to obtain the target solid peptide.
10. Application of novel SS-31 derivatives, characterized in that: Use of the novel SS-31 derivative as claimed in any one of claims 1 to 4 in the preparation of a medicament for treating nervous system diseases.