Cycloastragenol-linker-pyrroloquinoline quinone conjugates, methods of making and use thereof in delaying aging

Abstract

This invention belongs to the field of biomedicine and anti-aging technology. It provides a cycloastragaloside-linker-pyrroloquinolinequinone conjugate as shown in general formula (I): CAG-L-PQQ (I), wherein CAG is a cycloastragaloside group, PQQ is a pyrroloquinolinequinone group, L is a linker selected from C2-C10 straight-chain or branched alkylene groups, optionally interrupted by heteroatoms, and both ends are connected by ester bonds. This invention also provides a method for preparing the compound, pharmaceutical compositions containing the compound, and its applications in delaying aging, extending telomere length, and repairing telomere damage. By selecting different linkers, the stability, water solubility, and release characteristics of the compound can be optimized. Through chemical coupling, the spatiotemporal release of cycloastragaloside and PQQ is achieved, and it is expected to exert a triple synergistic effect of telomerase activation, mitochondrial protection, and improved replication fidelity, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and anti-aging technology, specifically relating to a cycloastragalool-linker-pyrroloquinoline quinone conjugate, its preparation method, a pharmaceutical composition containing it, and its application in delaying aging, repairing telomeres, and extending telomere length. Background Technology

[0002] Telomeres are DNA-protein complexes located at the ends of eukaryotic chromosomes, composed of tandem repeat sequences (TTAGGG) and their binding proteins. As cells divide, telomere DNA gradually shortens. When telomeres reach a critical length, the cell enters a state of replicative senescence or apoptosis. Therefore, telomere length is considered a key biomarker for cellular senescence and lifespan.

[0003] In existing technologies, strategies for extending telomeres mainly include telomerase activators such as cycloastragalool and antioxidants such as PQQ. Cycloastragalool, as a natural telomerase activator, has been shown in multiple studies to enhance telomerase activity (Harley et al., 2011). PQQ has been shown to have antioxidant and mitochondrial protective functions (Rucker et al., 2018). Recent research has found that PQQ can also upregulate DNA replication initiation proteins such as MCM5 and CDC6, improving DNA replication fidelity (Wang et al., 2026).

[0004] However, existing technologies have failed to achieve the synergistic effect of telomerase activation, enhanced telomere replication fidelity, and mitochondrial protection. Simply mixing the two physically results in problems such as misaligned absorption times, uneven distribution, and inability to act simultaneously on the same cell, significantly diminishing the synergistic effect. Therefore, there is an urgent need in this field for a novel compound capable of achieving simultaneous spatiotemporal release of both. Summary of the Invention

[0005] Technical problems to be solved

[0006] The present invention aims to provide a novel compound that can simultaneously achieve telomerase activation, telomere replication fidelity enhancement, and mitochondrial protection, and achieve spatiotemporal synchronous release of the two active fragments within the same cell.

[0007] Technical solution

[0008] 1. Compound structure

[0009] This invention provides a compound as shown in general formula (I), or a pharmaceutically acceptable salt, ester, stereoisomer, or prodrug thereof:

[0010] CAG - L - PQQ (I)

[0011] in:

[0012] • CAG stands for cycloastragalol group;

[0013] • PQQ is a pyrroloquinoline quinone group or a derivative thereof;

[0014] • L is a linker arm selected from C2-C10 straight-chain or branched alkylene, C2-C10 alkenylene, or C2-C10 ynylene, optionally interrupted by one or more heteroatoms selected from O, S, or NH, and connected to CAG and PQQ at both ends by ester, amide, ether, or carbonate bonds.

[0015] In a preferred embodiment, the linker arm L is a succinate group (-CO-CH2-CH2-CO-).

[0016] In a preferred embodiment, the linker arm L is a branched alkylene group, such as 2,2-dimethylsuccinate (-CO-C(CH3)2-CH2-CO-). The branched linker arm can protect the ester bond through steric hindrance, reducing hydrolysis in the gastrointestinal tract and is expected to improve the compound's gastric acid stability and oral bioavailability.

[0017] In a preferred embodiment, the linker arm L is an oxygen-containing alkylene group, such as a linker arm containing a polyethylene glycol (PEG) segment (-CO-(CH2-CH2-O)n-CH2-CH2-CO-). Introducing a hydrophilic PEG segment is expected to improve the water solubility of the compound and enhance its pharmacokinetic properties.

[0018] When L is a succinate, the compound (cycloastragaloyl-succinate-PQQ conjugate CP-01) has the structure shown in formula (II):

[0019] Molecular formula: C 48 H 58 N2O 15 (II)

[0020] Molecular weight: 902.38 g / mol

[0021] 2. Preparation method

[0022] The method includes the step of reacting cycloastragalool with an activated PQQ derivative in the presence of a condensing agent.

[0023] Includes the following steps:

[0024] (1) Astragaloside IV was acid-hydrolyzed to obtain cycloastragalool;

[0025] (2) PQQ is reacted with dicarboxylic anhydride to obtain PQQ-monocarboxylic ester; the dicarboxylic anhydride is selected from succinic anhydride, 2,2-dimethylsuccinic anhydride, polyethylene glycol dicarboxylic anhydride, etc.

[0026] (3) The cycloastragalool obtained in step (1) and the PQQ-monocarboxylic acid ester obtained in step (2) are subjected to esterification reaction in the presence of a condensing agent to obtain the target compound;

[0027] (4) Purification.

[0028] 3. Pharmaceutical Composition

[0029] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula (I) and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated as an oral formulation, an injection, a transdermal formulation, etc. Preferably, the pharmaceutical composition is an oral formulation and may further comprise an absorption enhancer, such as one or more of SNAC (sodium salicylate derivative), C8 / C10 fatty acids, and bile salts.

[0030] 4. Applications

[0031] The present invention also provides the use of compounds of general formula (I) in the preparation of food, health products or pharmaceuticals for delaying aging, extending telomere length and repairing telomere damage.

[0032] Beneficial effects

[0033] The compounds of this invention achieve a synergistic effect between cycloastragaloyl alcohol and PQQ through chemical coupling. After entering the cell, the compounds are hydrolyzed by intracellular esterases to release cycloastragaloyl alcohol and PQQ, which is expected to achieve a triple synergistic effect of telomerase activation, improved telomere replication fidelity, and mitochondrial protection. Compared with physical mixing, chemical coupling ensures the spatiotemporal synchronous release of the two active fragments, and is expected to produce the following beneficial effects:

[0034] • Telomerase activity was significantly increased;

[0035] • Effective extension of telomere length;

[0036] • Mitochondrial function is protected;

[0037] • Improved telomere replication fidelity.

[0038] By selecting different connecting arms L, the performance of the compound can be further optimized, such as gastric acid stability, water solubility, and pharmacokinetic properties, all of which fall within the scope of protection of this invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of this invention.

[0040] Example 1: Preparation of Cycloastragaloyl alcohol

[0041] In a 2000 mL round-bottom flask, add 100.0 g (approximately 127.4 mmol) of astragaloside IV and 1000 mL of methanol, and stir to dissolve. Slowly add 5.0 mL of concentrated sulfuric acid, and heat to 70 °C and reflux for 4 hours. Monitor the reaction progress by TLC (developing solvent: dichloromethane:methanol = 10:1).

[0042] After the reaction was complete, the mixture was cooled to room temperature and neutralized to pH 7-8 with saturated sodium bicarbonate solution. Methanol was removed by concentration under reduced pressure. The residue was diluted with 1000 mL of water and extracted three times with 500 mL of ethyl acetate each time. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a pale yellow crude solid.

[0043] The crude product was purified by silica gel column chromatography using a gradient elution of dichloromethane:methanol at ratios of 20:1 to 10:1. The target fraction was collected, concentrated, and yielded 50.0 g of a white solid, cycloastragalool, with a yield of 80.0%. The purity was determined to be 98.5% by HPLC.

[0044] The molecular formula of cycloastragalool is C 30 H 50 O5, molecular weight 490.7. MS (ESI): m / z 491.3 [M+H]⁺ (calculated value C) 30 H 51 O5⁺ is 491.3). ¹H NMR (400 MHz, DMSO-d6) spectrum is consistent with the target structure.

[0045] Example 2: Preparation of PQQ-succinic acid monoester

[0046] In a 500 mL round-bottom flask, add 20.0 g (60.6 mmol) of PQQ and 7.3 g (72.7 mmol, 1.2 equivalents) of succinic anhydride, then add 200 mL of anhydrous dichloromethane and 0.74 g (6.1 mmol, 0.1 equivalents) of DMAP. Stir at room temperature for 24 hours, and monitor the reaction progress by TLC (evolving solvent: dichloromethane:methanol = 5:1, with 0.1% acetic acid added).

[0047] After the reaction was complete, 100 mL of water was added for washing, and the mixture was separated. The organic phase was washed once with 0.1 M hydrochloric acid and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude red solid. The crude product was purified by recrystallization from ethyl acetate-petroleum ether to give 20.9 g of red solid PQQ-succinic acid monoester, with a yield of 80.0%. The purity was 97.5% as determined by HPLC.

[0048] The molecular formula of PQQ-succinic acid monoester is C 18 H 10 N2O 11 Molecular weight 430.3. MS (ESI): m / z 431.0 [M+H]⁺ (calculated value C) 18 H 11 N2O 11 ⁺ is 431.0). ¹H NMR (400 MHz, DMSO-d6) spectrum is consistent with the target structure.

[0049] Example 3: Synthesis of CP-01 (cycloastragaloyl alcohol-succinate-PQQ conjugate)

[0050] In a 250 mL round-bottom flask, 4.91 g (10.0 mmol) of cycloastragalool prepared in Example 1 and 4.30 g (10.0 mmol) of PQQ-succinic acid monoester prepared in Example 2 were added, followed by 100 mL of anhydrous acetonitrile. The mixture was stirred to dissolve. The flask was cooled to 0–5 °C in an ice-water bath, and 2.30 g (12.0 mmol, 1.2 equivalents) of EDCI and 1.62 g (12.0 mmol, 1.2 equivalents) of HOBt were added. The ice bath was removed, and the mixture was stirred at room temperature for 24 hours. The reaction progress was monitored by TLC (developing solvent: dichloromethane:methanol = 8:1).

[0051] After the reaction was complete, 200 mL of water was added to quench the reaction, and the mixture was extracted three times with 150 mL of ethyl acetate each time. The organic phases were combined and washed successively with 150 mL of 0.1 M hydrochloric acid, 150 mL of saturated sodium bicarbonate solution, and 150 mL of saturated brine. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow solid crude product.

[0052] The crude product was dissolved in a small amount of ethyl acetate, and petroleum ether was slowly added to precipitate the precipitate. The precipitate was filtered and dried. The recrystallization process was repeated 2-3 times to obtain 6.31 g of yellow solid CP-01, with a yield of 70.0%. The purity was 98.5% as determined by HPLC.

[0053] According to the esterification reaction equation: C 30 H 50 O5 (cycloastragaloyl alcohol, molecular weight 490.7) + C 18 H 10 N2O 11(PQQ-succinic acid monoester, molecular weight 430.3) → C 48 H 58 N2O 15 (CP-01, molecular weight 902.0) + H2O. Theoretical yield is 10.0 mmol × 902.0 g / mol = 9.02 g, actual yield is 6.31 g, yield is 70.0%.

[0054] MS (ESI): m / z 903.3 [M+H]⁺ (calculated value C) 48 H 59 N2O 15 ⁺ is 903.4). ¹H NMR (400 MHz, DMSO-d6) spectrum shows characteristic protons of cycloastragaloyl alcohol, PQQ aromatic protons, and methylene protons in the linker arm, consistent with the target structure.

[0055] Example 4: Telomerase activity detection (expected)

[0056] 4.1 Experimental Methods

[0057] Human skin fibroblasts (HSF) were collected and treated for 48 hours with CP-01 (0.1, 1, 5 μM), cycloastragalool (1 μM), PQQ (1 μM), and a physical mixture, respectively. Telomerase activity was detected using the TRAP method. The solvent control group was used as 100%, and the relative telomerase activity was calculated.

[0058] 4.2 Expected Results

[0059] CP-01 is expected to significantly enhance telomerase activity, with effects superior to cycloastragalool alone. Further experimental verification is needed to confirm the specific results.

[0060] Example 5: Telomere Length Measurement (Expected)

[0061] 5.1 Experimental Methods

[0062] HSF cells were collected and treated with CP-01 (1 μM) and a control, respectively, and cultured continuously for 4 weeks, with fresh medium replaced every 3 days. Cells were collected at weeks 0, 2, and 4, genomic DNA was extracted, and relative telomere length (T / S ratio) was detected by qPCR.

[0063] 5.2 Expected Results

[0064] It is expected that after 4 weeks of CP-01 treatment, the telomere length will be significantly longer than that of the control group and the cycloastragalool monotherapy group, indicating that CP-01 has the effect of prolonging telomeres. Specific results need to be verified by subsequent experiments.

[0065] Example 6: Gastric acid stability test (expected)

[0066] 6.1 Experimental Methods

[0067] CP-01 was placed in simulated gastric fluid (pH 1.2) and incubated at 37°C. Samples were taken at 0, 30, 60, and 120 minutes, and the remaining purity was determined by HPLC.

[0068] 6.2 Expected Results

[0069] CP-01 is expected to maintain a purity of >90% after 2 hours in simulated gastric fluid, indicating good gastric acid stability. Compounds with branched connecting arms (such as 2,2-dimethylbutyrate) are expected to have even higher stability.

[0070] Example 7: Cytotoxicity Detection (Expected)

[0071] 7.1 Experimental Methods

[0072] HSF cells were treated with different concentrations of CP-01 (0, 1, 5, 10, 25, 50, 100 μM) for 48 hours, and cell viability was detected by MTT assay.

[0073] 7.2 Expected Results

[0074] CP-01 is expected to have no significant cytotoxicity at concentrations below 10 μM (cell viability >90%), and its IC50 is much higher than the effective concentration, indicating good safety.

[0075] Example 8: Feasibility of synthesizing different connecting arm couplings

[0076] Referring to the method in Example 3, replacing PQQ-succinic acid monoester with PQQ-2,2-dimethylsuccinic acid monoester and PQQ-PEG-monocarboxylic acid intermediate respectively, the corresponding coupling compounds can be synthesized. This example demonstrates that those skilled in the art can select appropriate linkers to achieve the synthesis of target compounds based on the teachings of this invention.

[0077] Attached image description (see attached image)

[0078] Figure 1 The chemical structural formula of compound CP-01 of this invention.

Claims

1. A compound of general formula (I), or a pharmaceutically acceptable salt, ester, stereoisomer, or prodrug thereof: CAG - L - PQQ (I) in: CAG stands for cycloastragalol group; PQQ stands for pyrroloquinoline quinone group; L is a linker arm selected from C2-C10 straight-chain or branched alkylene, C2-C10 alkenylene, or C2-C10 ynylene, optionally interrupted by one or more heteroatoms selected from O, S, or NH, and connected to CAG and PQQ at both ends by ester, amide, ether, or carbonate bonds.

2. The compound according to claim 1, characterized in that, The connecting arm L is a succinate group. .

3. The compound according to claim 1, characterized in that, The connecting arm L is a 2,2-dimethylsuccinate group. .

4. The compound according to claim 1, characterized in that, The connecting arm L is an alkylene group containing oxygen atoms.

5. The compound according to claim 4, characterized in that, The connecting arm L is a connecting arm containing polyethylene glycol segments. , where n is an integer from 1 to 5.

6. A method for preparing the compound according to any one of claims 1-5, characterized in that, The method includes the step of reacting cycloastragalool with an activated PQQ derivative in the presence of a condensing agent.

7. A pharmaceutical composition, characterized in that, The compound comprising a therapeutically effective amount of any one of claims 1-5, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is an oral formulation and also contains an absorption enhancer.

9. The use of the compound according to any one of claims 1-5 in the preparation of food, health products or medicines for delaying aging, extending telomere length and repairing telomere damage.

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