Linoleoyl tryptophan, its method of synthesis and use

Abstract

The application provides linoleoyl tryptophan, a structure of which is shown in formula I: the application also provides a preparation method and use of the linoleoyl tryptophan.The linoleoyl tryptophan is a colorectal cancer candidate compound with development potential, and the mechanism of action is related to affecting ACSL family proteins and then interfering with cell cycle progression.Experiments prove that the linoleoyl tryptophan has obvious anti-tumor activity and small toxicity.

Description

Technical Field

[0001] This invention relates to linoleyltryptophan, its synthesis method, and its uses. Background Technology

[0002] Colorectal cancer is a malignant tumor that occurs in the colon and rectum, also known as colorectal cancer. It ranks third in incidence and fifth in mortality among malignant tumors in my country, with a significantly higher incidence in urban areas than in rural areas. Etiology involves environmental factors (high-fat diet, insufficient dietary fiber), genetic factors (familial adenomatous polyposis), and high-risk factors (colorectal adenoma, ulcerative colitis). Early symptoms include rectal bleeding, changes in bowel habits, abdominal pain, and anemia; later stages may present with intestinal obstruction and metastatic symptoms. Diagnosis mainly relies on colonoscopy, fecal occult blood test, and imaging evaluation. Pathological classification is primarily adenocarcinoma, while molecular classification includes microsatellite instability (MSI) testing. Treatment involves surgical resection combined with radiotherapy and chemotherapy. Targeted therapy requires selection of anti-EGFR monoclonal antibodies based on KRAS, NRAS, and BRAF gene mutation status. Immunotherapy is effective for MSI-H type patients. Screening is recommended for individuals over 45 years of age for fecal DNA testing or colonoscopy, with high-risk individuals requiring screening as early as 40 years of age. Preventive measures include increasing dietary fiber, controlling weight, and quitting smoking.

[0003] Numerous studies have reported on the role of tryptophan and its metabolites in the treatment of colorectal cancer (CRC). However, it is noteworthy that not all tryptophan-related metabolites possess anticancer activity. In fact, the metabolic pathway of tryptophan in the body is highly complex, and different metabolites play drastically different, even opposing, roles in CRC; some act as a protective shield against tumors, while others may become accomplices in tumor growth. Furthermore, linoleic acid regulates gut microbiota and promotes the development and progression of colorectal cancer (Yin Pei-hao, Research on the Mechanism of Linoleic Acid Regulating Gut Microbiota to Promote the Development and Progression of Colorectal Cancer, Anhui Medical University, May 2023). Summary of the Invention

[0004] This invention provides a method for synthesizing linoleyltryptophan and its metal salts, as well as their uses.

[0005] This invention provides linoleyltryptophan, with the structural formula shown in Formula I:

[0006] Formula I.

[0007] It is prepared by the following synthetic route:

[0008]

[0009] The synthesis of linoleyltryptophan employed an N,N'-dicyclohexylcarbodiimide (DCC)-mediated active esterification method. The synthetic route primarily involved the preparation of the active ester, amidation, and a multi-step purification process. First, linoleic acid reacted with N-hydroxysuccinimide in ethyl acetate in the presence of DCC to generate N-hydroxysuccinimide linoleate. This active ester intermediate underwent amidation with L-tryptophan in an ethanol-water mixed solvent under triethylamine catalysis. The crude product was obtained after extraction, acidification, and drying. The crude product was then fractionated and purified sequentially by normal-phase silica gel column chromatography (dichloromethane / methanol gradient elution) and reversed-phase preparative high-performance liquid chromatography (Xtimate C18 column, methanol-water = 80:20, isocratic elution). The final product was confirmed by area normalization analysis under 254 nm UV using HPLC, confirming its chemical structure and purity of 95%.

[0010] This invention also provides a metal salt form, the general structural formula of which is shown in Formula II and Formula III.

[0011]

[0012] Formula II

[0013]

[0014] (Formula III)

[0015] M is a monovalent metal cation or NH4+. + Preferably, M in formulas (II) and (III) is selected from K. + Na + or NH4 + .

[0016] The present invention provides a compound composition for treating rectal cancer, which contains the aforementioned linoleyltryptophan.

[0017] This invention discloses a novel compound, linoleyltryptophan, with antitumor activity. The invention successfully synthesized and characterized linoleyltryptophan, and systematically evaluated its in vitro and in vivo anti-colorectal cancer activity and potential mechanism. First, linoleyltryptophan was synthesized via an active ester method, achieving a purity of 95% by HPLC analysis. Its structure was confirmed by 1H NMR spectroscopy, 1C NMR spectroscopy, and high-resolution mass spectrometry. In vitro activity studies showed that this compound exhibited significant inhibitory activity against the proliferation of human (HCT116) and mouse (CT26) colorectal cancer cells, with half-maximal inhibitory concentrations (IC50) of 5.941 μg / mL and 3.032 μg / mL, respectively. Simultaneously, it showed low toxicity to normal intestinal epithelial cells NCM460 (IC50 >100 μg / mL), demonstrating good safety. In vivo experiments in a CT26 tumor-bearing mouse model further confirmed that linoleyltryptophan effectively inhibited tumor growth, with an inhibition rate exceeding 60% after 7 days of administration, and had no significant effect on mouse body weight, indicating its clear in vivo antitumor activity and good safety profile. Molecular docking was performed with the classic target of linoleic acid, the ACSL family proteins. The results showed that linoleyltryptophan had stronger binding activity with ACSL1, ACSL3, ACSL4 and ACSL5 compared with linoleic acid.

[0018] In summary, linoleyltryptophan is a promising candidate compound for treating colorectal cancer, and its mechanism of action is related to affecting ACSL family proteins and thus interfering with cell cycle progression. Experiments have demonstrated that the linoleyltryptophan of this invention exhibits significant antitumor activity with low toxicity. Attached Figure Description

[0019] Figure 1 Synthetic route of linoleyltryptophan;

[0020] Figure 2 Chemical structure of linoleyltryptophan;

[0021] Figure 3 Linoleyl tryptophan 1 H nuclear magnetic resonance spectrum;

[0022] Figure 4 Linoleyl tryptophan 13 C10 NMR spectrum;

[0023] Figure 5 LC-ESI-HRMS total ion chromatogram of linoleyl tryptophan in positive ion mode;

[0024] Figure 6 Solubility of linoleyltryptophan in different pH buffer solutions;

[0025] Figure 7 Apparent lipid-water partition coefficients of linoleyltryptophan in different pH buffer solutions;

[0026] Figure 8 Drug-time curves of linoleyltryptophan administered by gavage and intravenous injection in SD rats (Note: (n=3, mean±SD) (A: gavage administration; B: intravenous administration));

[0027] Figure 9 MTT assay results and IC50 values ​​of linoleyltryptophan on HCT116, CT26 and NCM460 cells 50 value;

[0028] Figure 10 Comparison of the activities of linoleyltryptophan, linoleic acid, and tryptophan in HCT116 and CT26 cells;

[0029] Figure 11 Flow cytometry analysis of cell cycle results for HCT116 and CT26 cells;

[0030] Figure 12 Effects of linoleyltryptophan on tumor growth and body weight in a colorectal cancer-bearing model;

[0031] Figure 13 Visualization of docking between linoleic acid, linoleyltryptophan and classic target molecules of linoleic acid;

[0032] Figure 14 Evaluation of the toxic effects of linoleyltryptophan on mice (Note: (A) Curve of mouse body weight change during acute toxicity test (B) Average daily food intake during acute toxicity test (C) Body weight change of male mice during subacute toxicity test (D) Body weight change of female mice during subacute toxicity test (E) Organ index of male and female mice in reproductive toxicity test (F) Serum hormone levels of male and female mice in reproductive toxicity test (G) HE-stained histopathological images, including heart, liver, spleen, lung and kidney tissues in acute toxicity test, and testis and ovary tissues in reproductive toxicity test). Detailed Implementation

[0033] Example 1: Preparation of linoleyltryptophan according to the present invention

[0034] like Figure 1As shown, linoleyltryptophan was prepared using the active ester method: A 100 mL round-bottom flask was filled with linoleic acid (2.23 mL) and hydroxysuccinimide (0.813 g), followed by 40 mL of ethyl acetate. The mixture was stirred thoroughly with a magnetic stirrer until completely dissolved. N,N'-dicyclohexylcarbodiimide (DCC, 1.46 g) was added to the well-stirred mixture, and the reaction was continued for 24 hours at room temperature. After the reaction was complete, the white byproduct N,N'-dicyclohexylurea (DCU) was removed by filtration. The filtrate was transferred to a separatory funnel and extracted with ultrapure water. After standing for 10 minutes to separate the layers, the organic layer was collected and evaporated under reduced pressure to remove ethyl acetate, yielding a yellow, transparent, oily liquid, namely hydroxysuccinimide linoleic acid. In another 100 mL round-bottom flask, 1 g L-tryptophan, 2.7 mL triethylamine, and 40 mL 65% ethanol solution were added. The mixture was stirred at room temperature until completely dissolved. Then, the prepared hydroxysuccinimide linoleic acid was added, and the reaction was continued at room temperature for 48 hours with stirring. Thin-layer chromatography (TLC) was used to monitor the reaction until complete. After the reaction, impurities and insoluble matter were removed by filtration. The filtrate was then evaporated under reduced pressure to remove ethanol and water. 40 mL of ethyl acetate was added to the concentrated mixture for extraction twice. During the second extraction, the pH was adjusted to 2 with 1 mol / L hydrochloric acid solution. The lower aqueous layer was discarded. The ethyl acetate layer was then mixed thoroughly with anhydrous sodium sulfate granules. After the newly added anhydrous sodium sulfate no longer clumped, the mixture was allowed to stand for 10 minutes. The anhydrous sodium sulfate was removed by filtration. The filtrate was evaporated under reduced pressure until anhydrous and ethyl acetate were removed, yielding a yellow to brown oily substance, which was crude linoleyltryptophan, with a yield of approximately 80%.

[0035] Purification was then performed. 1-2 µL of the linoleyltryptophan sample solution was spotted onto a thin-layer chromatography plate activated with distilled water for 30 minutes. Polarity screening was conducted using methanol, ethanol, ethyl acetate, dichloromethane, and petroleum ether as single developing solvents to determine the suitable elution system. A glass chromatography column was selected and wet-packed with silica gel powder using dichloromethane as the initial solvent. 4 g of linoleyltryptophan was mixed thoroughly with 8 g of silica gel powder and dichloromethane, and the mixture was rotary evaporated under reduced pressure until powdery. The powder was then dissolved in 10 mL of dichloromethane and loaded onto the column using a dry method. A 2 cm layer of anhydrous sodium sulfate was placed at the top of the column. Gradient elution was performed using the selected elution system. The eluent was collected, confirmed by TLC, and then rotary evaporated under reduced pressure to obtain a yellow, transparent oily substance. 2 g of the oily substance was further dissolved in 4 mL of methanol and passed through a normal-phase silica gel column at a flow rate of 2 min / mL. The solution was then purified using a high-performance liquid chromatography (HPLC) preparative system. The chromatographic conditions were: reversed-phase Xtimate C18 semi-preparative column (10 mm × 250 mm, 5.0 µm), column temperature 30℃, flow rate 15 mL / min, mobile phase methanol-water (80:20), detection wavelength 254 nm, injection volume 4000 µL per injection. After collecting the target peak, the solution was rotary evaporated under reduced pressure to obtain high-purity linoleyltryptophan with a purity higher than 95%. The final pure product yield was 45%, and it was a light yellow oily substance. Its SMILES representation is: CCCCC / C=C / C / C=C / CCCCCCCC(=O)NC(CC1=CNC2=CC=CC=C21)C(=O)O. The structural formula is: C 29 H 42 N₂O₃, with a relative molecular mass of 466.7 g / mol, has the following chemical structure diagram: Figure 2 As shown.

[0036] Linoleyl tryptophan is a white gel in appearance, and its structure is characterized as follows: Figure 3-5As shown: ¹H NMR (400MHz, DMSO-d6): δ 12.53 (s, ¹H), 10.82 (d, J=2.4Hz, ¹H), 8.03 (d, J=7.9Hz, ¹H), 7.52 (d, J=7.8Hz, ¹H), 7.32 (d, J=8.0Hz, ¹H), 7.12 (d, J=2.4Hz, ¹H), 7.05 (ddd, J=8.2, 7.0, 1.2Hz, ¹H), 7.01–6.92 (m, ¹H), 5.40–5.24 ( m, 4H), 4.46 (td, J=8.4, 5.0Hz, 1H), 3.15 (dd, J=14.8, 4.2Hz, 2H), 2.98 (dd, J=14.6, 8.8Hz, 1H), 2.73 (t, J=6.3 Hz, 2H), 2.11-1.93 (m, 6H), 1.40 (p, J=7.3Hz, 2H), 1.35-1.13 (m, 15H), 1.14 (s, 1H), 0.85 (t, J=6.8Hz, 3H).13C NMR (101MHz, DMSO-d6) δ174.07, 172.61, 136.55, 130.23, 128.23, 128.20, 127.69, 123.94, 121.32, 118.75, 118.64, 111.79, 11 0.51, 53.29, 49.07, 35.55, 31.37, 29.52, 29.20, 29.17, 29.03, 27.61, 27.13, 27.08, 26.14, 25.69, 25.63, 22.44, 14.39.HR-ESI MS (m / z): calcd for: C29H42N2O3, [MH]-:467.4.

[0037] Example 2 Physicochemical constants of linoleyltryptophan

[0038] 1. Equilibrium solubility and apparent lipid-water partition coefficient

[0039] The solubility and apparent lipid-water partition coefficient of linoleyltryptophan were determined by a combination of shake-flask method and HPLC. The solubility of linoleyltryptophan in water was 0.038 mg / mL. The solubility variation of linoleyltryptophan at different pH values ​​is shown below. Figure 6 The apparent lipid-water partition coefficients of linoleyltryptophan in different pH buffer solutions are shown in the figure. Figure 7 The solubility of linoleyltryptophan increases significantly above pH 7, suggesting that the presence of a double bond in the linoleyltryptophan molecule may enhance its polarity or alter its molecular packing. logP reaches its maximum value in the pH range of 7-9, indicating that this compound is more likely to dissolve in the oil phase in practical applications.

[0040] 2. Stability Assessment

[0041] 2.1 Experiment on Factors Affected by Strong Light

[0042] To investigate the stability of linoleyltryptophan under light conditions, it was placed in a strong light environment with an intensity of 4500 Lx ± 500 Lx. Samples were taken on days 1, 3, 5, and 10. After sampling, the appearance of the samples was observed, and HPLC was used to analyze whether decomposition occurred. The results are shown in Table 1, indicating that linoleyltryptophan is unstable under strong light within the corresponding time periods set in this experiment, and impurities begin to form after day 3. Therefore, this type of compound should be stored away from light.

[0043] Table 1. Experimental results on the high photostability of linoleyl tryptophan (n=3)

[0044]

[0045] 2.2 Experiment on the Effect of High Temperature

[0046] To investigate the stability of linoleyltryptophan under high-temperature conditions, it was placed in a high-temperature environment of 60℃±5℃. Samples were taken on days 1, 3, 5, and 10. After sampling, the appearance of the samples was observed, the content was determined, and HPLC was used to analyze whether decomposition occurred. The results are shown in Table 2. The results show that the morphology of linoleyltryptophan did not change significantly within the corresponding time periods set in this experiment; however, a small amount of impurities was detected from day 3 onwards, and the impurity content further increased after day 5, indicating that linoleyltryptophan gradually decomposes under these experimental conditions. Therefore, this type of compound needs to be stored at low temperatures.

[0047] Table 2. Results of high-temperature stability experiments of linoleyltryptophan (n=3)

[0048]

[0049] 2.3 Experiment on the Effects of High Humidity

[0050] To assess the tolerance of linoleyltryptophan (LIT) to humid conditions, we investigated in detail the changes in its morphological characteristics and key content under high humidity. LIT was placed in an environment of 25°C and 90% ± 5% relative humidity, and samples were taken on days 1, 3, 5, and 10 to observe its appearance, determine its content, and analyze for decomposition using HPLC. The results (see Table 3) showed that the morphology of LIT remained stable under the above experimental conditions and time range. However, from day 3 onwards, a large amount of impurities were detected, indicating that LIT gradually decomposed under these conditions, suggesting its instability under high humidity. Subsequently, further experiments were conducted according to the relevant evaluation criteria in the pharmacopoeia appendix: the compound was stored in a saturated sodium chloride solution (25°C, 75% ± 5% humidity) for 10 days, and the same indicators were examined. The results showed that under these experimental conditions and within the corresponding time period, impurities were generated in LIT, but the impurity content was low.

[0051] Table 3. Results of high humidity stability test of linoleyltryptophan (25℃, relative humidity 90% ± 5%) (n=3)

[0052]

[0053] In summary, linoleyltryptophan is relatively stable in its physical form at room temperature, but its chemical stability is significantly affected by environmental factors. Strong light, high temperature, and high humidity conditions all accelerate its decomposition, leading to a decrease in content and the generation of impurities, especially under high temperature and high humidity environments where decomposition is more rapid. Furthermore, the solubility of this compound increases significantly under alkaline conditions (pH>7). Therefore, it is recommended that linoleyltryptophan be stored, transported, and used in a cool, dry place, protected from light, to prevent degradation of its active ingredients.

[0054] Example 3: Pharmacokinetic Parameter Testing Experiment of Linoleyltryptophan

[0055] Twelve SD rats were randomly divided into four groups of four (n=4 per group, both male and female). Rats were fasted for 12 hours prior to the experiment but had free access to water. Linoleyltryptophan was administered by gavage at a dose of 100 mg / kg and intravenously at a dose of 50 mg / kg. Mean plasma concentration-time curves are shown below. Figure 8 The pharmacokinetic parameters of SD rats after drug administration are shown in the table.

[0056] Table 4. Pharmacokinetic parameters of linoleyltryptophan administered by gavage and intravenous injection to SD rats (n=3, mean±SD).

[0057]

[0058] The Cmax of linoleyltryptophan administered by gavage was (747.40±329.48) ng / mL at (6.00±3.00) h, with AUC(0-t) and AUC(0-∞) of (5424.41±1269.61) ng / mL∙h and (5676.13±1397.11) ng / mL∙h, respectively, indicating a low concentration of linoleyltryptophan absorbed into the bloodstream via gavage. The C0 of linoleyltryptophan administered intravenously was (6895.72±1269.97) ng / mL, with t1 / 2 of 6.21±1.15 and 1.89±0.99 h, respectively, resulting in a calculated bioavailability F of 20.78%.

[0059] The following pharmacodynamic tests demonstrate the beneficial effects of this invention.

[0060] Experimental Example 1: In vitro antitumor activity of linoleyltryptophan in this invention

[0061] 1. MTT assay for the inhibitory effect of linoleyltryptophan on the proliferation of colorectal cancer cells.

[0062] 1.1 Cell Culture

[0063] Human colorectal cancer cells HCT116, mouse colorectal cancer cells CT26, and normal human intestinal epithelial cells NCM460 were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator and passaged using 0.25% trypsin digestion.

[0064] 1.2 MTT Detection

[0065] Logarithmic growth phase cells were seeded into 96-well plates. After complete cell adhesion, the original culture medium was discarded, and 100 μL / well of complete culture medium containing serial concentrations of linoleyltryptophan, linoleic acid, and tryptophan was added to each well, with 6 replicates per group. After culturing for 48 h, 10 μL of MTT working solution (5 mg / mL) was added to each well, and the plates were incubated in the dark for 4 h. The supernatant was discarded, and 100 μL of LDMSO was added to each well. The plates were shaken for 10 min until the formazan crystals were completely dissolved. The absorbance (OD) of each well was measured at 490 nm using a microplate reader. 490 Cell viability calculation formula: Cell viability (%) = ( The half-maximal inhibitory concentration (IC50) of linoleyltryptophan on HCT116, CT26, and NCM460 cells was calculated by multiplying the result by 100%. 50 ).

[0066] 2. Experimental Results

[0067] 2.1 Linoleyltryptophan inhibits the proliferation of colorectal cancer cells HCT116 and CT26.

[0068] The results of the MTT experiment show that ( Figure 9 This compound exhibits significant inhibitory activity against the proliferation of colorectal cancer cells HCT116 and CT26 in vitro (IC50). 50 The concentrations were 5.941 μg / mL and 3.032 μg / mL, respectively. The MTT assay results for linoleyltryptophan on normal human intestinal epithelial cells NCM460 showed (…). Figure 9 At concentrations of 10 μg / mL and 50 μg / mL, the relative survival rate of NCM460 cells showed no significant difference compared to the control group, suggesting that the compound had no significant inhibitory effect on normal intestinal epithelial cells at the same dose. When the compound concentration was ≥100 μg / mL, the cell survival rate decreased significantly in a concentration-dependent manner (P<0.001). This confirms that linoleyltryptophan has a good anti-proliferative effect on tumor cells, while exhibiting extremely low toxicity to normal intestinal epithelial cells, possessing potential anti-tumor activity and safety.

[0069] 2.2 Linoleyl tryptophan exhibits superior antitumor activity compared to its raw materials, linoleic acid and tryptophan.

[0070] The MTT experimental results show that ( Figure 10 Compared with the linoleic acid group, the cell viability of HCT116 and CT26 cells in the linoleic acid group, tryptophan group, and their physical mixture group was significantly restored. Experiments showed that at the same concentration (5 μg / mL), linoleic acid inhibited the activity of HCT116 cells by 89.7%, significantly higher than that of the raw material linoleic acid (39.18%, P < 0.01), tryptophan (29.75%, P < 0.001), and their simple mixture (42.7%, P < 0.01), with activities approximately 2.2, 3, and 2 times higher than the latter, respectively. In CT26 cells, the inhibition rate of linoleic acid was 72.33%, significantly higher than that of the raw material linoleic acid (28.73%, P < 0.001), tryptophan (5.5%, P < 0.001), and their simple mixture (16.7%, P < 0.001), with activities approximately 2.5, 13, and 4.3 times higher than the latter, respectively. The above results indicate that the newly synthesized compound exhibits better antitumor activity than both active pharmaceutical ingredients.

[0071] 3. Effects of linoleyltryptophan on the cell cycle of HCT116 and CT26 cells:

[0072] 2.1 Flow cytometry detection of cell cycle distribution

[0073] Logarithmically grown HCT116 and CT26 cells were seeded into 6-well plates. After cell attachment, the plates were treated with linoleyltryptophan at different concentration gradients (0, 1.25, 2.5, 5 μg / mL) for 24 h. Cells were digested with 0.25% trypsin and collected, then fixed with 70% ethanol by slow dropwise addition. After washing twice with PBS, the cells were incubated with a staining working solution containing 50 μg / mL propidium iodide (PI) and 100 μg / mL RNase A for 30 min in the dark to allow PI to fully insert into the DNA double strand. Flow cytometry was then used to select the PI channel for detection. Cell cycle fitting analysis was performed using Novo. software, with PI fluorescence intensity as the x-axis and cell number as the y-axis. The software automatically calculated the percentage distribution of the cell population in G0 / G1, S, and G2 / M phases.

[0074] 2.2 Experimental Results

[0075] Cell cycle was detected by flow cytometry. Results are shown below. Figure 11 In HCT116 cells, linoleyltryptophan at concentrations of 2.5 and 5 μg / mL downregulated the proportion of cells in the G0 / G1 phase (P<0.01, P<0.05) and upregulated the proportion in the S phase (P<0.05, P<0.05), while at a concentration of 1.25 μg / mL, it upregulated the proportion in the G2 / M phase (P<0.05). In CT26 cells, linoleyltryptophan at concentrations of 1.25, 2.5, and 5 μg / mL upregulated the proportion in the S phase (P<0.05, P<0.01, P<0.001). These results indicate that linoleyltryptophan can significantly arrest the tumor cell cycle.

[0076] Experimental Example 2: In vivo antitumor activity study of linoleyltryptophan

[0077] 1. Experimental Methods

[0078] 1.1 Preparation of tumor cell suspension

[0079] Log-grown CT26 cells were washed with PBS to remove residual culture medium, then digested with 0.25% trypsin, centrifuged, counted, and the cell concentration was adjusted to 5 × 10⁻⁶ cells with PBS. 6 The tumor cell suspension is obtained by mixing the cells at a density of 1 cell / mL.

[0080] 1.2 Establishment and grouping of mouse model

[0081] The hair under the right armpit of mice was removed using a small animal electric shaver and disinfected with 75% ethanol. 0.2 mL of tumor cell suspension was then injected subcutaneously into the mouse axilla to establish a tumor-bearing mouse model. Seven days later, wheal-like tumors were visible in the mouse axilla, indicating successful model establishment. Individuals without tumor formation or with abnormally large tumors were excluded, and the mice were divided into the following groups: ① Model group; ② Subcutaneous injection group (20 mg / kg); ③ Tail vein injection group (20 mg / kg). Linoleyltryptophan was administered on day 8 post-inoculation, once every two days for a total of four times.

[0082] 1.3 Tumor growth monitoring

[0083] Starting on day 7 after subcutaneous injection of CT26 cells, mouse body weight was recorded daily, and the long axis (a) and short axis (b) of the mouse tumor were precisely measured using calipers. The tumor volume was calculated using the formula V=a×b² / 2. On day 15, the tumor was removed from the mouse and its wet weight was measured using an electronic balance.

[0084] 2. Experimental Results

[0085] like Figure 12 As shown, in the CT26 tumor-bearing mouse model in vivo, tumor growth was gradually inhibited with increasing administration days. Starting on day 5, compared to the model group, tumor growth in the tail vein injection group was significantly inhibited (day 5, P<0.05; day 6, P<0.01). On day 7, tumor growth was inhibited in both the subcutaneous injection and tail vein injection groups (P<0.05). After tumor removal, tumor weight was measured; compared to the model group, the tumor inhibition rate in both the subcutaneous and tail vein injection groups reached 60% (P<0.05). Simultaneously, weight monitoring showed a stable trend in weight distribution among the groups, with no significant differences. Furthermore, the mice in all groups maintained good mental condition without signs of emaciation or lethargy, indicating that linoleyltryptophan possesses good antitumor activity and safety in vivo.

[0086] Experimental Example 3: Docking of linoleyl tryptophan and linoleic acid with the classical target molecules of linoleic acid

[0087] 1.1 Experimental Methods

[0088] Molecular docking technology was employed to predict the binding affinity of linoleyltryptophan (LIT) and linoleic acid (LLA) to the target protein. First, the SMILE numbers and 3D structures of LIT and LLA were obtained from the PubChem database. Hydrogenation was then performed using PyMOL, followed by energy minimization. The target protein crystal structure was downloaded from the RCSB PDB (https: / / www.rcsb.org / ) database. Water molecules, redundant ligands, and heteroatoms were removed using PyMOL. Polar hydrogen addition and charge calculations were performed using AutoDock Tools. Semi-flexible docking was then performed using AutoDock Vina software to obtain the docking score. Subsequently, key interactions, such as hydrogen bonds and hydrophobic interactions, in the optimal conformation were analyzed using PyMOL and Discovery Studio to clarify the binding mode.

[0089] 1.2 Experimental Results

[0090] As shown in Table 1, the docking fractions of linoleic acid with ACSL1, ACSL3, ACSL4, and ACSL5 were -7.5 kcal / mol, -6.6 kcal / mol, -7 kcal / mol, and -7.8 kcal / mol, respectively; the docking fractions of linoleyltryptophan with ACSL1, ACSL3, ACSL4, and ACSL5 were -7.5 kcal / mol, -7.8 kcal / mol, -7.9 kcal / mol, and -7.9 kcal / mol, respectively; all docking fractions were less than -7 kcal / mol. A lower docking fraction indicates a stronger binding affinity between the ligand and receptor, and a tighter interaction. Therefore, the binding of linoleyltryptophan to the above four proteins is stable. Further visualization analysis of the above eight complex systems was performed using Pymol and Discovery Studio. Figure 13The 3D results show that linoleic acid did not form hydrogen bonds with ACSL1, but formed 1, 2, and 1 hydrogen bonds with ACSL3, ACSL4, and ACSL5, respectively. Linoleyl tryptophan formed 5, 7, 3, and 4 hydrogen bonds with ACSL1, ACSL3, ACSL4, and ACSL5, respectively. The more hydrogen bonds, the stronger the stability of the complex system; therefore, linoleyl tryptophan exhibits stronger binding stability with the aforementioned proteins. The 2D results show that linoleic acid can form van der Waals forces, C-H bonds, and alkyl interactions with the amino acid residues of ACSL1, ACSL3, ACSL4, and ACSL5. In addition to these interactions, linoleyl tryptophan can also form π-cation and π-σ interactions with the amino acid residues of ACSL1, ACSL3, ACSL4, and ACSL5. These aromatic ring-related interactions significantly enhance the binding affinity between the ligand and the protein. Molecular docking results showed that linoleyltryptophan had a stronger binding affinity to ACSL1, ACSL3, ACSL4 and ACSL5 compared to linoleic acid.

[0091] Table 5 Molecular docking results

[0092]

[0093] Example 4: Safety evaluation of linoleyltryptophan (toxicity test)

[0094] 1. Toxicity test

[0095] 1.1 Acute toxicity test

[0096] Thirty-six six-week-old Kunming mice were randomly divided into three groups of 12 mice each, with half males and half females: two experimental groups and one control group. The experimental groups were administered 10,000 mg / kg linoleyltryptophan by gavage, using corn oil as a solvent, while the control group was administered an equal volume of corn oil by gavage. The mice were fasted for 12 hours prior to the experiment but allowed free access to water. The administration volume was 0.1 mL / 10 g body weight. Clinical symptoms were observed during the administration period, and HE staining was performed 14 days later.

[0097] 1.2 Subacute toxicity test

[0098] Eighty-four 6-week-old Kunming mice (half male and half female) were randomly divided into seven groups of 12 mice each: a high-dose group, a medium-dose group, a low-dose group, and a saline control group. The high-, medium-, and low-dose groups were administered linoleyltryptophan at 400, 200, and 100 mg / kg via gavage, respectively, while the control group was administered an equal volume of corn oil via gavage. Administration was once daily for 28 days, with a dosage of 0.1 mL / 10 g body weight. Clinical symptoms were observed in the mice during the treatment period.

[0099] 1.3 Reproductive toxicity test

[0100] Forty-eight six-week-old Kunming mice (half male and half female) were randomly divided into eight groups of six each: a high-dose group, a medium-dose group, a low-dose group, a negative control group, and a positive control group. The high-dose, medium-dose, and low-dose groups were administered 400, 200, and 100 mg / kg linoleyltryptophan, respectively, via gavage. The negative control group was administered an equal volume of physiological saline via gavage, and the positive control group was administered 50 mg / kg cyclophosphamide via gavage. Administration was once daily for 30 days, with a dosage of 0.1 mL / 10 g. After one month of continuous administration, the mice were sacrificed, and the reproductive organ index was calculated to observe for any lesions in the reproductive organs.

[0101] 2. Clinical observation

[0102] Daily cage-side observations were conducted during the drug administration period, focusing on monitoring the mice's mental state, physical characteristics, fur condition, behavior, fecal characteristics, and mortality. Weight changes and food intake were recorded during the drug administration period. Acute experiments were observed for 14 days, while subacute and reproductive experiments were observed throughout the entire drug administration cycle.

[0103] 3. HE staining

[0104] After drug administration, mice were anesthetized with chloral hydrate, and their hearts were perfused with physiological saline until the outflow was bloodless, followed by fixation with 4% paraformaldehyde. Heart, liver, spleen, lung, kidney, and brain tissues (testes, epididymis, ovaries, and uterus were additionally collected for reproductive toxicity experiments) were removed and fixed in 4% paraformaldehyde for 24 h. Tissue blocks were trimmed to approximately 3 mm thickness and rinsed with running water for 20 min. The tissue blocks were dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Sections (4-5 μm thick) were spread in a 40℃ water bath, retrieved from anti-detachment slides, and baked at 60℃ for 2 h. Sections were dewaxed sequentially with xylene I and II, rehydrated with graded ethanol, and soaked in distilled water for 1 min. Hematoxylin staining; hydrochloric acid alcohol differentiation; eosin staining. Finally, xylene clearing, neutral resin mounting, and microscopic observation and image acquisition were performed.

[0105] 4. Serum sex hormone level measurement (reproductive toxicity test)

[0106] Blood was collected from the eyes of mice, centrifuged at 3000 r / min for 10 min at 4℃, and the supernatant serum was stored at -80℃. Serum testosterone (T) and estradiol (E2) levels were measured using ELISA, following the kit instructions. The optical density was measured at 450 nm using a microplate reader, and the sample concentration was calculated using a standard curve.

[0107] result

[0108] 4.1 Toxicity Test

[0109] All mice survived without death. During the experiment, the mental state, behavior, fur luster, and fecal characteristics of the mice in the drug-treated group showed no significant abnormalities compared to the control group (see Table 4-1). Figure 14 As shown in AB, there was no statistically significant difference in body weight changes among the groups of mice. During the first three days of drug administration, mouse body weight tended to decrease, and food intake also decreased. After three days, the mice gradually recovered, but the difference was not statistically significant. Mice were sacrificed after drug administration, and anatomical examinations of the heart, liver, spleen, lungs, kidneys, and brain revealed no obvious congestion, edema, or necrosis. HE staining results showed that the structures of all organs and tissues were intact, cell morphology was normal, and no obvious pathological damage was observed. Figure 14 As shown in G. The above results indicate that the median lethal dose (LD50) of linoleyltryptophan in mice is greater than 10,000 mg / kg, and its safety profile is good.

[0110] To further observe the long-term toxicity of the compound in mice, a subacute toxicity experiment was conducted. During the observation period, all mice survived without death and were in good general condition. No signs of poisoning such as piloerection, tremors, or respiratory distress were observed. The body weight of mice in all groups steadily increased over time. Figure 14 As shown in CD, there was no statistically significant difference in body weight between the NAL group and the control group. These results indicate that NAL has no significant effect on the general condition and weight gain of mice.

[0111] Further observation was conducted on the effects of the compound on the reproductive organs of mice. For example... Figure 14 As shown in Figure E, there were no significant differences in testicular and ovarian indices among the treated mice compared to the negative control group. Further ELISA results indicated that there were no significant differences in testosterone and estradiol levels among the treated mice compared to the negative control group. Figure 14 F. For example Figure 14 As shown in Figure G, HE staining results indicated that, compared to the control group, the spermatogenic epithelium in the NAL group mice was intact, morphologically normal, and structurally complete. HE staining of the ovaries in each group showed that, compared to the control group, the ovarian cortex in the NAL group mice was structurally intact, with uniform follicle distribution, tightly packed cells, intact nuclei, and uniform staining. These results indicate that linoleyltryptophan administered continuously for 30 days within a dose range of 100-400 mg / kg has no significant reproductive toxicity.

[0112] Table 6. General Observation Items in Acute Toxicity Tests

[0113]

Claims

1. Linoleyl tryptophan, characterized in that: The structural formula is shown in Formula I: 。 2. The method for preparing linoleyltryptophan according to claim 1, characterized in that, It uses the active ester method to prepare linoleyl tryptophan, and the synthetic route is shown below: 。 3. The method for preparing linoleyltryptophan according to claim 2, characterized in that, Includes the following steps: a. In the presence of the coupling agent N,N'-dicyclohexylcarbodiimide (DCC), linoleic acid and N-hydroxysuccinimide (NHS) are reacted in an organic solvent to prepare N-hydroxysuccinimide linoleate intermediate; b. The intermediate is subjected to an amidation reaction with L-tryptophan under alkaline conditions to obtain the crude target product; c. The crude product is purified to obtain linoleyltryptophan.

4. The method for preparing linoleyltryptophan according to claim 3, characterized in that, The organic solvent in step a is ethyl acetate; the alkaline conditions in step b are the reaction in the presence of triethylamine; the purification in step c includes silica gel column chromatography and high performance liquid chromatography.

5. Use of the linoleyltryptophan according to claim 1 in the preparation of an anti-colorectal cancer drug.

6. A pharmaceutical composition for treating colorectal cancer, characterized in that, It comprises the linoleyltryptophan or metal salt as described in claim 1, and pharmaceutically acceptable excipients.

7. The pharmaceutical composition for treating colorectal cancer according to claim 6, characterized in that, The structure of the metal salt is shown in Formula II or Formula III, where M is a monovalent metal cation or NH4⁺; 。 8. The pharmaceutical composition for treating colorectal cancer according to claim 7, characterized in that, The M is selected from Na + K + or NH4 + .

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