A tea amino acid macrocyclic derivative, and a preparation method and use thereof

Abstract

The application discloses a theanine macrocycle derivative, a preparation method and application thereof, relates to the technical field of peptide compounds, and particularly relates to a derivative of theanine and application thereof.The theanine macrocycle derivative is synthesized by adopting a convergent solid-phase peptide synthesis strategy on a CTC resin to synthesize a linear precursor peptide, and then synthesizing a macrocycle compound through cyclization.The derivative of the application expands the theanine derivative category, has a remarkable effect on reversing the multidrug resistance of a K562 / ADR cell line mediated by P-gp, improves the pharmacokinetics and physicochemical properties, and expands the dosage form and application scenarios.

Description

Technical Field

[0001] This invention relates to the field of peptide compound technology, and in particular to a macrocyclic derivative of theanine and its uses. Background Technology

[0002] Multidrug resistance (MDR) in tumors refers to the phenomenon where tumor cells develop resistance to one chemotherapeutic drug while simultaneously developing cross-resistance to other drugs with completely different structures and mechanisms of action. The mechanisms of MDR are complex, but overexpression of the P-glycoprotein (P-gp, encoded by the MDR1 gene) is currently considered the most crucial factor. However, existing tetrahydroisoquinoline cyclic compound P-gp inhibitors are difficult to further develop into drugs due to their inherent toxicity or side effects such as altering the pharmacokinetics of chemotherapeutic drugs. In contrast, natural products, due to their structural diversity and good biocompatibility, represent a current research direction for developing novel, low-toxicity, and highly effective MDR reversal agents.

[0003] Theanine is one of the main active ingredients in tea. As a non-protein amino acid, long-term consumption does not significantly cause toxic side effects. Furthermore, theanine exhibits good biocompatibility, and its metabolites are considered non-toxic. It demonstrates significant pharmacological activity in areas such as anti-tumor multidrug resistance. When used in combination with the anticancer drug doxorubicin, theanine enhances the anticancer efficacy of doxorubicin. The mechanism of action lies in theanine's ability to prevent tumor cells from effluxing the drug, thereby improving the chemotherapy effect. Existing research shows that theanine can be chemically modified to obtain derivatives that can enhance biological activity and improve target selectivity. Currently, publicly available theanine derivatives are mainly a few glycosides, salts, and dipeptides, lacking skeletal diversity. Therefore, it is necessary to expand the types of theanine derivatives and enrich the molecular skeleton to obtain compounds with enhanced activity or novel pharmacological functions, improve pharmacokinetic and physicochemical properties, and expand dosage forms and application scenarios. Summary of the Invention

[0004] This invention provides a macrocyclic derivative of theanine, which yields new pharmacological results and expands its applications in pharmaceuticals, food, and daily chemicals.

[0005] A macrocyclic theanine derivative, characterized by the following structural formula:

[0006] .

[0007] A method for preparing a macrocyclic theanine derivative, characterized in that the method includes the following steps:

[0008] Step S1: After connecting Fmoc-GABA to CTC Resin, Fmoc-Theanine, Fmoc-Ahx-OH, and Fmoc-Phe-OH were sequentially coupled under reagent conditions to obtain the compound. The compound was then cleaved from CTC Resin at room temperature, filtered, and the compound precipitated in ice-cold ether. After centrifugation, the compound was purified by reversed-phase HPLC on a C18 preparative column to obtain the pure intermediate Compd 1.

[0009] In step S2, the intermediate Compd 1 obtained in step S1 is dissolved in a reagent and reacted, precipitated in ice-cold ether, centrifuged, dried, and then the theanine macrocyclic derivative TMD-1 is prepared by reverse phase reaction.

[0010] Furthermore, in step S1:

[0011] The reagent conditions for Fmoc-GABA to be ligated with CTC Resin were DIPEA (6 eq) and DCM;

[0012] The reagent conditions for ligating Fmoc-Theanine with H-GABA-CTC Resin were HBTU (2.85 eq), DIPEA (6 eq), and DMF.

[0013] The reagent conditions for the ligation of Fmoc-Ahx-OH with H-Theanine-GABA-CTC Resin were HBTU (2.85 eq), DIPEA (6 eq), and DMF.

[0014] The reagent conditions for the linkage of Fmoc-Phe-OH with H-Ahx-Theanine-GABA-CTC Resin are HOBt (3 eq), DIC (3 eq), DIEPA (6 eq), and DMF;

[0015] In step S2:

[0016] Intermediate Compd 1 was dissolved in DMF, and then the temperature was adjusted to 0°C. EDC (1 eq) and DIPEA (6 eq) were added and reacted at 0°C.

[0017] Furthermore, during the coupling process in step S1, the Fmoc protecting groups of the amino acids are cleaved using a mixed solution with a volume ratio of Pip:DMF = 1:4.

[0018] Studies have shown that multidrug resistance (MDR) is one of the main causes of chemotherapy failure in cancer patients, and theanine exhibits anti-MDR properties and is widely recognized as a safe substance. Existing first- and third-generation P-gp inhibitors have been difficult to further develop into drugs due to inherent toxicity and other side effects in clinical trials. This invention utilizes theanine as a natural amino acid backbone, introducing a macrocyclic structure to retain the high safety of theanine while enhancing its anti-MDR activity. Analysis of the anti-MDR mechanism of TMDs indicates that the compounds of this invention help address the clinical problem of chemotherapy failure caused by multidrug resistance in cancer patients. Attached Figure Description

[0019] Figure 1 This is a molecular structure diagram of the compound TMD-1 of this invention.

[0020] Figure 2 This is a synthetic route diagram for the compound TMD-1 of this invention.

[0021] Figure 3 The hydrogen spectrum of compound TMD-1 prepared in Example 1.

[0022] Figure 4 The carbon spectrum of compound TMD-1 prepared in Example 1.

[0023] Figure 5 The mass spectrum of compound TMD-1 prepared in Example 1 is shown.

[0024] Figure 6 The liquid chromatogram of compound TMD-1 prepared in Example 1 is shown.

[0025] Figure 7 The figure shows the results of the efflux experiment of compound TMD-1 Rhodamine-123 prepared in Example 1.

[0026] Figure 8 This is a molecular docking diagram of theanine and its macrocyclic derivatives. Detailed Implementation

[0027] Example 1: A theanine macrocyclic derivative was synthesized using a convergent solid-phase peptide synthesis strategy on CTC resin to synthesize a linear precursor peptide, followed by cyclization to synthesize a macrocyclic compound. The specific process is as follows:

[0028] Step S1: Synthesize the linear intermediate Compd 1;

[0029] Step S1-1: Connect Fmoc-GABA with CTC Resin under DIPEA (6 eq) and DCM conditions to obtain H-GABA-CTC Resin;

[0030] Steps S1-2: Fmoc-Theanine and H-GABA-CTC Resin are connected under HBTU (2.85 eq), DIPEA (6 eq), and DMF conditions to obtain H-Theanine-GABA-CTC Resin;

[0031] Steps S1-3: Fmoc-Ahx-OH was linked to H-Theanine-GABA-CTC Resin under HBTU (2.85 eq), DIPEA (6 eq), and DMF conditions to obtain H-Ahx-Theanine-GABA-CTC Resin;

[0032] Steps S1-4: Fmoc-Phe-OH and H-Ahx-Theanine-GABA-CTC Resin were linked under the conditions of HOBt (3 eq), DIC (3 eq), DIPEA (6 eq), and DMF to obtain H-Phe-Ahx-Theanine-GABA-CTC Resin;

[0033] Steps S1-5: The compound obtained in step S1-4 was lysed from CTC Resin for 1 h at room temperature using HFIP reagent; subsequently filtered, the filtrate was precipitated in ice-cold diethyl ether, and the precipitate was centrifuged to obtain the compound, which was then analyzed by reversed-phase HPLC at CTC Resin. 18 The intermediate Compd 1 was purified on a preparative column.

[0034] In step S1:

[0035] The HPLC reagent used was 0.1% (v / v) TFA-acetonitrile;

[0036] During the coupling process, the Fmoc protecting groups of the amino acids were cleaved using a 20% Pip / DMF solution.

[0037] Step S2: Synthesis of macrocyclic compounds;

[0038] The intermediate Compd 1 (1 eq) obtained in step 1 was dissolved in 150 mL DMF, and then the temperature was adjusted to 0 °C. EDC (1 eq) and DIPEA (6 eq) were added and reacted at 0 °C for 24 hours. After the reaction was completed, 500 mL of ice-cold diethyl ether was added, the solid precipitated, centrifuged and dried, and the theanine macrocyclic derivative 1 (TMD-1) was prepared by reverse phase reaction.

[0039] In this embodiment, the yield of TMD-1 was 85%, which was verified by LC-MS. The chemical formula of TMD-1 is C1.26 H 39 N5O5, structure as follows Figure 1 As shown, the molecular weight is 501.6280. The proton and carbon spectral data are as follows:

[0040] Hydrogen spectrum data: 1 H NMR (500 MHz, DMSO-d6) δ H 8.07 (d, J = 8.8 Hz, 1H), 7.92(m, 2H), 7.78 (t, J = 5.6 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.24 (t, J = 7.5Hz, 3H), 7.21 – 7.12 (m, 4H), 4.42 (td, J = 9.3, 5.1 Hz, 1H), 3.96 (q, J =7.1 Hz, 1H), 3.21 (m, 1H), 3.16 – 2.99 (m, 4H), 2.99 – 2.81 (m, 2H), 2.71 (m,1H), 2.20 – 1.90 (m, 5H), 1.92 – 1.70 (m, 2H), 1.59 (q, J = 12.1, 9.7 Hz, 2H), 1.50 – 1.23 (m, 4H), 1.23 – 1.10 (m, 1H), 0.98 (t, J = 7.1 Hz, 3H);

[0041] Carbon spectrum data: 13 C NMR (125 MHz, DMSO) δ C 172.4, 172.3, 171.9, 171.0, 170.8, 138.5, 128.9, 128.0, 126.1, 54.1, 53.2, 38.6, 37.1, 36.9, 34.7, 33.3, 32.1, 32.0, 28.6, 26.9, 26.0, 25.6, 24.6, 14.7.

[0042] As shown in Table 1 below, the compound TMD-1 prepared in this embodiment, at a non-cytotoxic concentration of 10 μM, achieved a maximum reversal fold of 138.8 against P-gp-mediated MDR in the K562 / ADR cell line. This is significantly higher in reversal ability and safety compared to the positive control group verapamil (reversal fold of 45.8) and theanine (reversal fold of 48.1). The macrocyclic derivative of theanine significantly enhances its biological activity compared to theanine, while retaining the safety of theanine.

[0043] Studies have shown that drug resistance in resistant cells is due to overexpression of P-gp (overexpression of the MDR1 gene). The compound TMD-1 of this invention reverses multidrug resistance in tumors by inhibiting the transport function of the P-gp protein. Rho-123 efflux assays also demonstrated that TMD-1 inhibits the function of the P-gp transporter. Molecular docking results between the compound and a P-gp homology protein model (PDB code: 3G5U) revealed the interaction pattern between the compound and P-gp.

[0044] Table 1. Effect of compound TMD-1 (10 μM) on the reversal of P-gp-mediated MDR in the K562 / ADR cell line.

[0045] .

[0046] Table 2. Evaluation of the toxic side effects of compound TMD-1 on K562 / ADR cells

[0047] .

[0048] In this embodiment, the K562 / ADR and K562 cells used for testing were grown in RPMI-1640 medium containing 10% fetal bovine serum, manufactured by Zhejiang Tianhang Biotechnology Co., Ltd. The cells were cultured at 37˚C with 5% carbon dioxide. To maintain their MDR (metastatic descent rate), K562 / ADR cells were induced with 8.6 μM doxorubicin once a month.

[0049] This embodiment uses the MTT assay to detect the reversal effect of TMD-1 on drug resistance in K562 / ADR cells. The specific process is as follows:

[0050] The study included a blank control group, a negative control group, and an experimental group, with the experimental group receiving verapamil, theanine, and a theanine macrocyclic derivative (TMD-1), respectively. K562 / ADR cells in logarithmic growth phase were seeded into corresponding multi-well plates for the negative and experimental groups at a concentration of 80 μL per well and cultured for 12 h. An equal volume of serum-free RPMI 1640 medium was added to the blank control group. The final concentration of the compound added to the corresponding wells in the verapamil, theanine, and theanine macrocyclic derivative experimental groups was 10 μM. One hour after addition, ADR at concentrations of 1 μM, 10 μM, 20 μM, and 100 μM were added to the corresponding wells in each experimental group, with an administration volume of 10 μL / well. Four replicates were set for each concentration. After 48 hours of cell-drug interaction, 50 μL of 1 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 hours. Then, 100 μL of DMSO was added to each well, and the cells were cultured overnight at 37˚C with 5% CO2. The next day, the cells were removed, cooled to room temperature, and the absorbance (OD value) was measured at 570 nm using a microplate reader. The cell growth inhibition rate was calculated using the following formula:

[0051] Cell growth inhibition rate (%) = (OD value of negative group - OD value of experimental group) / (OD value of negative group - OD value of blank group) × 100%;

[0052] Computing IC 50 The value is calculated using the following formula to determine the reversal factor:

[0053] Reversal factor = IC50 of drug-resistant cells before the use of reversal agent 50 Value / IC50 of drug-resistant cells after use of reversal agent 50 The value was determined, and the experiment was repeated three times in parallel.

[0054] The screening process for non-toxic doses (safety assessment) of TMD-1 is as follows:

[0055] The study included a blank control group, a negative control group, and an experimental group, with verapamil, theanine, and theanine macrocyclic derivative (TMD-1) as the experimental compounds. K562 / ADR cells in logarithmic growth phase were seeded into corresponding multi-well plates for the negative and experimental groups at 90 μL per well, and cultured for 12 h. An equal volume of serum-free RPMI 1640 was added to the blank control group. Verapamil, theanine, and theanine macrocyclic derivative (TMD-1) at a final concentration of 25 μM were added to each experimental group at 10 μL per well, with four replicates for each concentration. After 48 h of cell-drug interaction, 50 μL of 1 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 h. Then, 100 μL of DMSO was added to each well, and the cells were cultured overnight at 37˚C with 5% CO2. The next day, the cells were removed, cooled to room temperature, and the absorbance (OD) was measured at 570 nm using a microplate reader. Calculate the cell growth inhibition rate using the following formula:

[0056] Cell growth inhibition rate (%) = (OD value of negative group - OD value of experimental group) / (OD value of negative group - OD value of blank group) × 100%.

[0057] The procedure for determining Rhodamine-123 efflux is as follows:

[0058] Cells were treated with verapamil, theanine, and a theanine macrocyclic derivative at 37 °C for 3 hours, followed by incubation with 5 μM Rho-123 for 1 hour. Cell images were taken under a fluorescence microscope (Olympus Optical, Tokyo, Japan), and the cells were analyzed using an Accuri C6 Plus instrument (BD Biosciences, San Jose, CA, USA). Results are as follows: Figure 7 As shown, Figure 7 In the diagram, "control" represents the control group's fluorescence spectrum. The absence of fluorescent dots indicates that P-gp effluxes the fluorescent substance Rho-123 into the extracellular space, demonstrating that P-gp has normal transport function. "TMD-1" represents the fluorescence spectrum of the theanine macrocyclic derivative of this invention. The cells in the spectrum exhibit fluorescence, indicating that the fluorescent substance Rho-123 was not effluxed by P-gp, proving that P-gp's transport function was inhibited. The positive control drug verapamil also exhibits fluorescence, indicating that the fluorescent substance Rho-123 was not effluxed by P-gp into the extracellular space, further demonstrating that P-gp's transport function was inhibited.

[0059] Molecular docking diagrams show that the protein structures were downloaded from the RCSB PDB database (PDB ID: 3G5U) and processed using Maestro 13.5. Ligand optimization was performed using Gaussian 16 via the PM6D3 method. Promising P-gp compounds were docked using the Glide SP method with the Schrodinger 2023-1 software package. The final docking was performed using Maestro 13.5, and the results are as follows. Figure 8 As shown, theanine relies on "charge clamps," with its two charged ends binding Arg1197 and Glu1204 through a salt bridge to complete recognition. Its function is relatively singular and highly hydrophilic. After macrocyclic modification with the introduction of polycyclic rings such as benzene rings, it additionally obtains aromatic π–π stacking, a wider hydrogen bond network, and a larger hydrophobic buried surface, allowing the ligands to be embedded deeper and wider into the pocket, binding more tightly and with higher specificity. This is the basis for the improved activity of TMD-1 relative to theanine.

Claims

1. A macrocyclic theanine derivative, characterized in that, The structure is as follows: 。 2. The method for preparing a theanine macrocyclic derivative as described in claim 1, characterized in that, The method includes the following steps: Step S1: After linking Fmoc-GABA to CTC Resin, Fmoc-Theanine, Fmoc-Ahx-OH, and Fmoc-Phe-OH were sequentially coupled under reagent conditions to obtain the compound. The compound was then cleaved from CTC Resin at room temperature, filtered, and precipitated in ice-cold ether. After centrifugation, the precipitate was analyzed by reversed-phase HPLC at C100°C. 18 The intermediate Compd 1 was purified by column chromatography. In step S2, the intermediate Compd 1 obtained in step S1 is dissolved in a reagent and reacted, precipitated in ice-cold ether, centrifuged, dried, and the theanine macrocyclic derivative TMD-1 is prepared by reverse phase reaction.

3. The method for preparing a theanine macrocyclic derivative as described in claim 2, characterized in that, In step S1: The reagent conditions for Fmoc-GABA to be ligated with CTC Resin are 6 eq of DIPEA and DCM; The reagent conditions for ligating Fmoc-Theanine with H-GABA-CTC Resin were 2.85 eq of HBTU, 6 eq of DIPEA, and DMF. The reagent conditions for the linkage of Fmoc-Ahx-OH with H-Theanine-GABA-CTC Resin are 2.85 eq of HBTU, 6 eq of DIPEA, and DMF; The reagent conditions for the linkage of Fmoc-Phe-OH with H-Ahx-Theanine-GABA-CTC Resin are 3 eq of HOBt, 3 eq of DIC, 6 eq of DIPEA, and DMF. In step S2: Compd 1 was dissolved in DMF, and then the temperature was adjusted to 0°C. 1 eq of EDC and 6 eq of DIPEA were added and reacted at 0°C.

4. The method for preparing a theanine macrocyclic derivative as described in claim 2, characterized in that, During the coupling process in step S1, the Fmoc protecting groups of the amino acids are cleaved using a mixed solution with a volume ratio of Pip:DMF = 1:

4.

5. The theanine macrocyclic derivative as described in claim 1 for reversing P-gp-mediated MDR in the K562 / ADR cell line.

6. A pharmaceutical composition comprising the theanine macrocyclic derivative of claim 1 and a pharmaceutically acceptable carrier and excipients.

7. The use of the theanine macrocyclic derivative or pharmaceutical composition thereof as described in claim 1 or 6 in the preparation of a drug that reverses tumor multidrug resistance.

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