Prodrug designed based on tumor LAT1 transporter and preparation method thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a prodrug designed based on a tumor LAT1 transporter and a preparation method of the prodrug, in particular to a gemcitabine analogue prodrug taking LAT1 as a target spot and a preparation method of the gemcitabine analogue prodrug. The invention relates to a derivative shown as a total general formula and sub general formulas 1-3, and pharmaceutically acceptable salt, hydrate, solvate or prodrug structure thereof, wherein X, Y, Dug and Amino acid are defined in the specification and claims. The series of prodrugs provided by the invention can realize synchronous delivery of chemotherapeutic drugs and lactic acid transport inhibitors in tumors through LAT1 mediation. The lactic acid transport inhibitor is used for inducing apoptosis of hypoxic cells and sensitizing chemotherapeutic drugs, and the lactic acid transport inhibitor and the chemotherapeutic drugs have a synergistic effect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, relates to a class of new compounds, and specifically relates to prodrugs designed targeting tumor LAT1 transporter, and their preparation and application. Background Art

[0002] Currently, chemotherapy is still one of the main treatment methods, but it has problems such as a narrow therapeutic window and poor tumor targeting, which easily lead to poor efficacy and unbearable side effects. To improve the anti-tumor effect, combination therapy is mostly used clinically. The combined drugs act on different targets to achieve the purpose of synergistically treating cancer with a lower dosage. For example, the combination of lactate transporter inhibitors and chemotherapeutic drugs for anti-cancer has become a research hotspot. However, simple sequential administration cannot change the non-specific distribution of the drug itself, resulting in side effects on normal tissues. Moreover, after drugs with different pharmacokinetic characteristics enter the body, it cannot be guaranteed that the two enter the tumor tissue in the optimal dosage ratio, resulting in a significant reduction in the synergistic anti-tumor effect.

[0003] Tumors express a variety of transporters, such as oligopeptide transporter 1 (PEPT1) and large neutral amino acid transporter 1 (LAT1), which are used to uptake amino acids required by tumors, and thus are also ideal targets for drug delivery. Targeting transporters, connecting specific groups to the parent drug structure according to the requirements of their substrate structure, so that they are recognized by the targeted transporters during in vivo transport, to change their membrane permeability, and then improve the targeted delivery of drugs. Some scholars connected doxorubicin with Gly-Gly-Gly (3 peptides) for targeting the liver tumor PEPT1 transporter. Compared with the doxorubicin group, the doxorubicin tripeptide prodrug significantly increased tumor distribution under the mediation of tumor PEPT1, greatly reduced cardiotoxicity, and increased the tumor inhibition rate (40.33% vs 32.35%). Obviously, combining existing anti-tumor drugs with transporters highly expressed in tumors or organs to design small molecule prodrugs can improve selectivity, prolong the residence time in vivo, reduce systemic side effects, and enhance drug efficacy.

[0004] During tumor growth, due to insufficient angiogenesis, there are oxygen-rich areas and hypoxic areas within the tumor. The hypoxic areas can cause cell cycle arrest, enhanced DNA repair enzyme activity, inhibition of apoptosis, etc., and thus lead to tumor chemotherapy resistance through multiple pathways. There are obvious diversities in the metabolism of tumor cells. Cells farther away from blood vessels (cells in the hypoxic area) produce energy through glycolysis, and the lactate produced is excreted into the stroma via monocarboxylate transporter 4 (MCT4), while cells closer to blood vessels (cells in the oxygen-rich area) take up lactate from the stroma via monocarboxylate transporter 1 (MCT1) for energy production through oxidative phosphorylation. The two types of cells coordinate with each other and coexist harmoniously through the lactate shuttle mechanism, that is, metabolic symbiosis. If a lactate transporter inhibitor is used to act on tumor cells, the oxygen-rich area will be unable to take up lactate and instead take up more glucose for aerobic glycolysis. A large amount of lactate produced by glycolysis cannot be excreted from the cells, resulting in the gradual apoptosis of cells in the oxygen-rich area due to excessive acidification. In addition, the sudden reduction in glucose supply will also lead to the death of tumor cells in the hypoxic area. Summary of the Invention

[0005] The object of the present invention is to construct a small molecule carrier prodrug targeting the tumor LAT1 transporter. Through LAT1 mediation, synchronous delivery of a chemotherapeutic drug and a lactate transporter inhibitor within the tumor is achieved. The lactate transporter inhibitor induces apoptosis of hypoxic cells and sensitizes the chemotherapeutic drug, and the two work synergistically. To achieve this object, the general formula (i.e., the total formula) of the prodrug designed based on the tumor LAT1 carrier protein and lactate transport and the structural general formulas of its control drugs (i.e., sub-formulas 1, 2, and 3) are as follows:

[0006]

[0007] Wherein: X and Y are O and NH; Drug is an antitumor drug residue containing an amino group or a hydroxyl group;

[0008] The present invention specifically provides compounds shown by the general formula (total formula), sub-formulas 1, 2, and 3, geometric isomers, and the structural formulas of their pharmaceutically acceptable salts, hydrates, solvates, or prodrugs selected from:

[0009]

[0010] Wherein: X and Y are O and NH; Drug is an antitumor drug residue containing an amino group or a hydroxyl group.

[0011] The antitumor drugs containing an amino group or a hydroxyl group described in the present invention are selected from gemcitabine, decitabine, cytarabine, 5-fluorouracil, cladribine, fludarabine, azacitidine, and their derivatives.

[0012] The MCTs Inhibitor (i.e., lactate transporter inhibitor) in the present invention is α-cyano-4-hydroxycinnamic acid (α-Cyano-4-hydroxycinnamic acid, α-AHC).

[0013] The Amino acid (i.e., amino acid) in the present invention is L-phenylalanine and L-lysine.

[0014] The preparation method of the small molecule carrier prodrug targeting tumor LAT1 transporter in the present invention:

[0015] (1) Dissolve amino acid (2eq) in N,N-dimethylformamide (DMF), add succinic anhydride (2eq) and triethylamine (1.4eq), and react at 50 °C overnight to prepare amino acid-succinate ester;

[0016] (2) Dissolve the lactate transporter inhibitor (1eq) and the anti-tumor drug (1eq) in DMF, add 1-hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBT, 1.4eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI, 1.4eq) and triethylamine (1.4eq), and react overnight at 50 °C under nitrogen protection to form the anti-tumor drug-lactate transporter inhibitor monoamide;

[0017] (3) Dissolve the anti-tumor drug-α-AHC monoamide (1eq) and the amino acid-succinate ester (2eq) in DMF, add HOBT (1.4eq), EDCI (1.4eq) and triethylamine (1.4eq), and carry out esterification at 50 °C under nitrogen protection, and then remove the protection through CH 2 Cl 2 / TFA or Pd / C, H 2 to generate the target product (general formula structure I);

[0018] The preparation methods of the compounds of sub-general formula 1 and sub-general formula 2 are as follows:

[0019] Dissolve the lactate transporter inhibitor (1eq) and different anti-tumor drugs (1eq) in DMF, add HOBT (1.4eq), EDCI (1.4eq) and triethylamine (1.4eq), and react overnight at 50 °C under nitrogen protection to generate the target products (sub-general formula 1 structure II) and (sub-general formula 2 structure III).

[0020] The preparation method of the general formula 3 compound is as follows:

[0021] (1) Dissolve the amino-protected amino acid (2eq) in DMF, add succinic anhydride (2eq) and triethylamine (1.4eq) to prepare the amino acid-succinate ester;

[0022] (2) Dissolve different anti-tumor drugs (1eq) and the amino acid-succinate ester (2eq) in DMF, add HOBT (1.4eq), EDCI (1.4eq) and triethylamine (1.4eq), and react overnight under the conditions of 50 °C and nitrogen protection. After CH 2 Cl 2 / TFA or Pd / C, H 2 Remove the protection to generate the target product (general formula 3 structure IV);

[0023] The amino-protected amino acid described in the present invention is selected from Boc-4-amino-L-phenylalanine and Cbz-L-lysine.

[0024] A pharmaceutical composition according to the present invention, characterized in that it comprises the compound, geometric isomer, and pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof described in any one of claims 2-4 and a pharmaceutically acceptable carrier.

[0025] Use of the compound, geometric isomer, and pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof or a pharmaceutical composition thereof described in the present invention in the preparation of anti-tumor drugs.

[0026] The anti-tumor described above includes anti-breast cancer, prostate cancer and colon cancer.

[0027] Compared with the prior art, the advantages of the present invention are as follows:

[0028] In the present invention, an anti-tumor drug, a lactate transporter inhibitor and a targeting group are connected through an amide bond or an ester bond to synthesize an LAT1-targeted small molecule prodrug, which has increased in vivo metabolic stability, reduced toxicity and improved efficacy compared with the anti-tumor drugs used in clinical applications.

[0029] The LAT1-targeted small molecule prodrug constructed in the present invention combines the regulation of lactate transport and chemotherapy while ensuring synchronous transport. The inhibition of lactate transport induces tumor apoptosis while correcting hypoxia, overcomes drug resistance, and sensitizes chemotherapeutic drugs. The two cooperate with each other to maximize the anti-tumor effect. Description of the Drawings

[0030] Figure 1.a: Cytotoxicity experiments of gemcitabine and α-cyano-4-hydroxycinnamic acid at different ratios on 3D 4T1 Spheroids; b: Cytotoxicity experiments of different prodrugs (the ratios of gemcitabine and α-cyano-4-hydroxycinnamic acid are 1:1 and 1:2 respectively) on 3D 4T1 Spheroids. mean±SD(n=3), *P<0.05 versus control versus control.

[0031] Figure 2 . Combination index of prodrug 1 and prodrug 2.

[0032] Figure 3 . Toxicity of prodrugs to 2D 4T1 cells (Figure a) and cytotoxicity to 3D 4T1 Spheroids (Figure b). mean±SD(n=3), *P<0.05 versus control versus control, **P<0.01 versus control versus control.

[0033] Figure 4 . Live / dead cell staining of 2D / 3D 4T1 cells treated with prodrug 1-6.

[0034] Figure 5 . a: Effects of temperature on the uptake of gemcitabine, α-AHC, non-targeted prodrug (prodrug1) and LAT1-targeted prodrug (prodrug6) by 4T1 (LAT1(+)); b: Effects of prodrug1, prodrug6, gemcitabine and α-AHC on the cellular uptake of L-phenylalanine; c: Effects of L-phenylalanine on the uptake of prodrug1, prodrug6, gemcitabine and α-AHC. mean±SD(n=3), *P<0.05 versus control and **P<0.01 versus control.

[0035] Figure 6 . a: Effects of gemcitabine, α-AHC, non-targeted prodrug (prodrug1) and LAT1-targeted prodrug (prodrug6) on glucose consumption by 3D 4T1 Spheroids (outside); b: Effects of gemcitabine, α-AHC, non-targeted prodrug (prodrug1) and LAT1-targeted prodrug (prodrug6) on lactate consumption by 3D 4T1 Spheroids (outside). (n=3, mean±SD, *: p<0.05 vs Control).

[0036] Figure 7.a: Effects of gemcitabine, α-AHC, non-targeted prodrug (prodrug1), and LAT1-targeted prodrug (prodrug6) on intracellular lactate transport in 3D 4T17 Spheroids cells; b: Effects of gemcitabine, α-AHC, non-targeted prodrug (prodrug1), and LAT1-targeted prodrug (prodrug6) on intracellular pH value in 3D 4T17 Spheroids cells. (n = 3, mean ± SD, *: p < 0.05 vs Control).

[0037] Figure 8 . Tissue distribution curves of LA1-targeted prodrug, non-targeted prodrug, gemcitabine, and α-AHC. a, b are the curves at 1 h, c, d are the curves at 4 h (n = 3, mean ± SD, #P < 0.05).

[0038] Figure 9 . a: Tumor volume change curves of tumor-bearing mice in different treatment groups; b: Tumor weights after 14 days of treatment in different treatment groups; c: Representative tumor images after 14 days of treatment in different treatment groups; d: Body weight change curves of tumor-bearing mice in different treatment groups (n = 5, mean ± SD, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).

[0039] Figure 10 . H&E and TUNEL staining images of tumor tissue sections after 14 days of treatment in different treatment groups.

[0040] Figure 11 . H&E staining images of important tissue sections after 14 days of treatment in different treatment groups.

[0041] Figure 12 . Body weight change curve (a) and analysis of blood biochemical indexes (b-i) of healthy KM mice after continuous intravenous administration for 14 days. Indexes of liver function include aspartate aminotransferase (AST) (b) and alanine aminotransferase (ALT) (c); indexes of renal function include serum creatinine (CR) (d) and blood urea nitrogen (BUN) (e); inflammatory factors include white blood cells (WBC) (f); anemia-related indexes include red blood cells (RBC) (g), hemoglobin (HGB) (h), and platelets (PLT) (i) (n = 3).

[0042] Figure 13 . H&E staining images of organ tissue sections of healthy KM mice after continuous intravenous administration for 14 days.

[0043] Figure 14 . Chemical structural formulas of gemcitabine series prodrugs 1-6. Detailed implementation manners

[0044] The present invention is further illustrated by the following examples, but is not limited thereto.

[0045] Example 1 Preparation of Gemcitabine-α-AHC Prodrug (Prodrug 1)

[0046] Precisely weigh gemcitabine (2.4 g, 9.12 mmol), α-AHC (1.76 g, 9.32 mmol), HOBT (1.32 g, 8.88 mmol), and EDCI (1.76 g, 9.16 mmol) and sequentially add them into a clean 100 ml eggplant-shaped flask. Using DMF (40 ml) as the reaction solvent, slowly add triethylamine (0.96 ml, 9.52 mmol) dropwise under the conditions of room temperature and stirring. After dropping, evacuate and protect with nitrogen, and react at 50 °C for about 1 h. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a reddish-brown oily substance. Column chromatography gives a yellow solid with a yield of 46.7%. MS (ESI): m / z = 433 [M-H]. - The HPLC purity is 97.5%. 1 1H NMR (500 MHz, DMSO-d6): δ 8.31 (s, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 6.4 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 6.49 (s, 1H), 6.22 (s, 1H), 5.80 (d, J = 7.5 Hz, 1H), 4.56 (d, J = 3.7 Hz, 2H), 4.27 (s, 1H), 4.12 (dt, J = 7.6, 3.4 Hz, 1H).

[0047] Example 2 Preparation of α-AHC-Gemcitabine-α-AHC Prodrug (Prodrug 2)

[0048] Precisely weigh gemcitabine (2.40 g, 9.12 mmol), α-AHC (1.76 g, 9.32 mmol), HOBT (1.32 g, 8.88 mmol), and EDCI (1.76 g, 9.16 mmol) and sequentially add them into a clean 100 ml eggplant-shaped flask. Using DMF (40 ml) as the reaction solvent, slowly add triethylamine (0.96 ml, 9.52 mmol) dropwise under the conditions of room temperature and stirring. After dropping, evacuate and protect with nitrogen, and react at 50 °C for about 1 h. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a reddish-brown oily substance. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a reddish-brown oily substance. Column chromatography gives a yellow solid with a yield of 27.4%. MS (ESI): m / z = 604 [M-H]. - The HPLC purity is 94.11%. 11H NMR (500 MHz, DMSO-d6): δ 8.33 (s, 1H), 8.28 (s, 1H), 7.99 (dd, J = 8.8, 3.9 Hz, 4H), 7.66 (d, J = 7.5 Hz, 1H), 7.48 (d, J = 11.3 Hz, 2H), 6.92 (d, J = 8.4 Hz, 4H), 6.35 (s, 2H), 5.82 (d, J = 7.5 Hz, 1H), 5.69 (s, 2H), 4.70 - 4.65 (m, 1H), 4.62 (d, J = 9.9 Hz, 2H).

[0049] Example 3 Preparation of Gemcitabine-L-Lysine Prodrug (Prodrug 3)

[0050] First step: Weigh accurately Cbz-L-Lysine (0.98 g, 3.50 mmol) and succinic anhydride (0.36 g, 3.50 mmol) and add them into a 100 ml eggplant-shaped flask. Using DMF (30 ml) as the reaction solvent, slowly add triethylamine (0.49 ml, 4.90 mmol) dropwise under the conditions of room temperature and stirring. After the addition, evacuate and protect with nitrogen, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0051] Second step: Take the reaction product of the first step (about 0.58 g) into a 100 ml eggplant-shaped flask. Weigh accurately gemcitabine (0.20 g, 0.76 mmol), HOBT (0.15 g, 1.10 mmol), and EDCI (0.22 g, 1.10 mmol) and add them successively into the above 100 ml eggplant-shaped flask. Using DMF (20 ml) as the reaction solvent, evacuate and protect with nitrogen, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0052] Third step: Take the reaction product of the second step (about 0.60 g) into a 100 ml eggplant-shaped flask. Weigh accurately Pd / C (0.65 g) and add it into the above clean 100 ml eggplant-shaped flask. Using methanol (10 ml) as the reaction solvent, introduce hydrogen, evacuate and replace the air 3 times to protect the reaction with hydrogen. Then react at 25 °C under stirring conditions for 2 hours; monitor the reaction by TLC until it is complete. Transfer the reaction solution to a 50 ml centrifuge tube, use a centrifuge (rotation speed 4000 rpm, time 6 min) to separate the reaction solution from Pd / C, and use methanol as the solvent to repeat the above centrifugation operation 3 times. Collect the supernatant obtained by centrifugation and remove the solvent under reduced pressure; obtain a white powder by column chromatography with a yield of 63.8%. MS (ESI): m / z = 490 [M - H] - . HPLC purity is 98.72%. 11H NMR (500 MHz, DMSO-d6): δ 8.22 (d, J = 7.9 Hz, 1H), 7.90 (t, J = 5.9 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.58 (s, 1H), 7.02 (s, 1H), 6.45 (s, 1H), 6.11 (t, J = 8.1 Hz, 1H), 6.01 (d, J = 7.3 Hz, 1H), 4.44 (q, J = 7.3 Hz, 1H), 4.15 (td, J = 12.7, 8.3 Hz, 1H), 3.76 (t, J = 11.7 Hz, 2H), 3.65–3.58 (m, 1H), 2.99 (q, J = 6.5 Hz, 2H), 2.22 (d, J = 5.9 Hz, 4H), 1.71–1.53 (m, 2H), 1.37 (dt, J = 12.1, 6.6 Hz, 2H), 1.28 (h, J = 8.0 Hz, 2H).

[0053] Example 4 Preparation of Gemcitabine-L-Phenylalanine Prodrug (Prodrug 4)

[0054] Step 1: Accurately weigh Boc-4-amino-L-phenylalanine (0.98 g, 3.50 mmol) and succinic anhydride (0.36 g, 3.50 mmol) and add them to a 100 ml eggplant-shaped flask. Using DMF (30 ml) as the reaction solvent, slowly add triethylamine (0.49 ml, 4.90 mmol) dropwise at room temperature under stirring. After dropping, evacuate and protect with nitrogen, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0055] Step 2: Take the reaction product of the first step (about 0.58 g) in a 100 ml eggplant-shaped flask. Accurately weigh gemcitabine (0.20 g, 0.76 mmol), HOBT (0.15 g, 1.10 mmol), and EDCI (0.22 g, 1.10 mmol) and add them to the above 100 ml eggplant-shaped flask in sequence. Using DMF (20 ml) as the reaction solvent, evacuate and protect with nitrogen, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0056] Step 3: Take the reaction product of the second step in a 100 ml eggplant-shaped flask, add CH 2 Cl 2 / TFA, and then react at 25 °C under stirring for 2 hours; monitor the reaction by TLC until it is complete. Perform column chromatography to obtain a pale yellow powder with a yield of 72.5%. MS (ESI): m / z = 524 [M-H] - . The HPLC purity is 91.5%. 11H NMR (500 MHz, DMSO-d6): δ 9.91 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 8.1 Hz, 3H), 7.26 (t, J = 8.1 Hz, 4H), 7.18–7.11 (m, 2H), 6.35 (s, 1H), 6.18 (t, J = 7.3 Hz, 2H), 5.39–5.23 (m, 1H), 4.39–4.32 (m, 1H), 4.19 (td, J = 12.8, 8.4 Hz, 2H), 3.89 (dd, J = 8.4, 3.3 Hz, 1H), 3.81 (d, J = 12.6 Hz, 1H), 3.69–3.58 (m, 3H), 2.95 (dd, J = 13.7, 3.9 Hz, 1H), 2.68 (dd, J = 13.9, 11.0 Hz, 1H), 1.90 (s, 2H).

[0057] Example 5 Preparation of α-AHC-Gemcitabine-L-Lysine Prodrug (Prodrug 5)

[0058] Step 1: Weigh accurately Cbz-L-Lysine (0.98 g, 3.50 mmol) and succinic anhydride (0.36 g, 3.50 mmol) and add them to a 100 ml eggplant-shaped flask. Using DMF (30 ml) as the reaction solvent, slowly add triethylamine (0.49 ml, 4.90 mmol) dropwise under stirring at room temperature. After the addition, evacuate and protect with nitrogen, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0059] Step 2: Take the reaction product from the first step (about 0.58 g) in a 100 ml eggplant-shaped flask. Weigh accurately Gemcitabine-α-AHC prodrug (Prodrug 1) (0.33 g, 0.76 mmol), HOBT (0.15 g, 1.10 mmol), and EDCI (0.22 g, 1.10 mmol) and add them successively to the above 100 ml eggplant-shaped flask. Using DMF (20 ml) as the reaction solvent, evacuate and protect with nitrogen, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0060] Step 3: Take the reaction product of the second step (about 0.60 g) in a 100 ml eggplant-shaped flask. Weigh Pd / C (0.65 g) precisely and add it to the above 100 ml eggplant-shaped flask. Use methanol (10 ml) as the reaction solvent, introduce hydrogen gas, displace air under reduced pressure 3 times, protect the reaction with hydrogen gas, and then react at 25 °C under stirring conditions for 2 hours; monitor the reaction by TLC until it is complete. Transfer the reaction solution to a 50 ml centrifuge tube, use a centrifuge (rotation speed 4000 rpm, time 6 min) to separate the reaction solution from Pd / C, and use methanol as the solvent to repeat the above centrifugation operation 3 times. Collect the supernatant obtained by centrifugation and remove the solvent under reduced pressure; obtain a white powder by column chromatography with a yield of 34.1%. MS (ESI): m / z = 490 [M-H] - . The HPLC purity is 99%. 1 H NMR (500 MHz, DMSO-d6): δ 8.26 (s, 1H), 7.99 (d, J = 8.5 Hz, 2H), 7.91 (t, J = 5.5 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.51 (s, 1H), 7.47 (s, 1H), 6.96 (d, J = 8.6 Hz, 2H), 6.29 (s, 1H), 5.82 (d, J = 7.5 Hz, 1H), 5.51 (s, 1H), 4.56 (qd, J = 12.2, 4.1 Hz, 2H), 4.45 (dt, J = 8.1, 4.0 Hz, 1H), 3.21 (t, J = 6.0 Hz, 2H), 3.17 (s, 1H), 2.99 (q, J = 6.4 Hz, 3H), 2.69–2.58 (m, 2H), 2.40 (t, J = 7.5 Hz, 2H), 1.71 (ddt, J = 14.8, 10.4, 5.3 Hz, 1H), 1.66–1.57 (m, 1H), 1.40–1.26 (m, 5H), 1.23 (s, 3H).

[0061] Example 6 Preparation of α-AHC-gemcitabine-L-phenylalanine prodrug (prodrug 6)

[0062] Step 1: Weigh Boc-4-amino-L-phenylalanine (0.98 g, 3.50 mmol) and succinic anhydride (0.36 g, 3.50 mmol) precisely and add them to a 100 ml eggplant-shaped flask. Use DMF (30 ml) as the reaction solvent, slowly add triethylamine (0.49 ml, 4.90 mmol) dropwise under stirring at room temperature, evacuate and protect with nitrogen gas after dropping, and react at 50 °C for 6 hours. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0063] Step 2: Take the reaction product of the first step (about 0.58 g) in a 100 ml eggplant-shaped flask. Weigh accurately gemcitabine-α-AHC prodrug (prodrug 1) (0.33 g, 0.76 mmol), HOBT (0.15 g, 1.10 mmol), and EDCI (0.22 g, 1.10 mmol) and add them successively to the above 100 ml eggplant-shaped flask. Use DMF (20 ml) as the reaction solvent, evacuate and protect with nitrogen, react at 50 °C for 6 hours, and monitor the reaction completion by TLC. Concentrate the reaction solution under reduced pressure to obtain a yellowish-brown oily substance.

[0064] Step 3: Take the reaction product of the second step in a 100 ml eggplant-shaped flask, add CH 2 Cl 2 / TFA, and then react at 25 °C under stirring conditions for 2 hours; monitor the reaction completion by TLC. Perform column chromatography to obtain a pale yellow powder with a yield of 31.3%. MS (ESI): m / z = 524 [M-H] - . The HPLC purity is 99.82%. 1 H NMR (500 MHz, Methanol-d4): δ 8.27 (s, 1H), 7.99 (dd, J = 8.8, 3.7 Hz, 2H), 7.60 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 6.97–6.90 (m, 2H), 6.27 (t, J = 8.1 Hz, 1H), 5.99 (d, J = 7.7 Hz, 1H), 4.62 (s, 2H), 4.33–4.24 (m, 1H), 4.16 (dt, J = 8.5, 3.0 Hz, 1H), 3.31 (t, J = 1.6 Hz, 3H), 3.15 (dd, J = 14.0, 6.1 Hz, 1H), 2.93 (dd, J = 13.9, 8.5 Hz, 1H), 2.63 (s, 4H), 1.37–1.25 (m, 5H).

[0065] Example 7 Screening of Different Ratios of Drugs

[0066] In order to synthesize small molecule prodrugs at the optimal combination ratio of drugs, we investigated the cytotoxicity of different drug ratios of free gemcitabine / α-AHC combinations, as well as prodrug 1 and prodrug 2 (prodrugs synthesized from gemcitabine and α-AHC at a ratio of 1:1 and 1:2 respectively) against 3D 4T1, observed the cell viability, and thus optimized the optimal drug ratio.

[0067] From Figure 1It can be seen that in the free drug group, when the ratio of gemcitabine to α-AHC is 1:1, the cell survival rate is the lowest and the cytotoxic effect is the best. In the prodrug group, prodrug 1 (the ratio of gemcitabine to α-AHC is 1:1) has a better cytotoxic effect than prodrug 2 (the ratio is 1:2), probably because the drug release rate of prodrug 1 is faster than that of prodrug 2. The above results show that the optimal drug ratio in the free drug group and the prodrug group is the same, both 1:1.

[0068] To further study the synergistic therapeutic effect of gemcitabine and α-AHC, we performed a combination index analysis. The calculation formula of the combination index is as follows:

[0069] Combination index CI = D 1 / Dm 1 +D 2 / Dm 2

[0070] D1 and D2 respectively represent the concentrations of drug 1 and drug 2 when the two drugs are used in combination to achieve a certain effect (such as 50% inhibition rate), and Dm 1 and Dm 2 respectively represent the concentrations of drug 1 and drug 2 when used alone to achieve the same effect. CI less than 1, equal to 1, and greater than 1 represent that the combination of the two drugs has synergistic, additive, and antagonistic effects respectively.

[0071] We used the CI value as the ordinate and the ICx of the two-proportion prodrug as the abscissa to make Figure 2 , after the prodrug acts on 3D4T1Spheroids cells for 48 hours, most of the data points are below the line of CI = 1, indicating that the combination of the two drugs has a synergistic effect ( Figure 2 ), probably because gemcitabine and α-AHC have different mechanisms of action. Gemcitabine mainly binds to DNA to kill tumor cells, while α-AHC acts on the MCTs transporter, blocks lactate transport, induces apoptosis, and sensitizes chemotherapeutic drugs. When the two are used in combination, a high-efficiency synergistic anti-tumor effect can be achieved with a relatively low drug concentration. The above research results show that the synergistic anti-tumor effect of the prodrug synthesized by gemcitabine and α-cyano-4-hydroxycinnamic acid in a ratio of 1:1 is better.

[0072] Example 8 Study on the cell viability of gemcitabine series prodrugs 1-6

[0073] We used 4T1 cells as a model to investigate the cytotoxic effects of gemcitabine, α-AHC, and prodrugs. To further investigate the effect of prodrugs on cells in the hypoxic area, we used 4T1 cells as a model and constructed 3D 4T1 cell spheres (including hypoxic and oxygen-rich areas) using the three-dimensional culture method.

[0074] The results are asFigure 3 as shown in a and Figure 3 b, in the 2D 4T1 planar cell model, the cytotoxicity difference between the Prodrug 1 group and the gemcitabine raw drug group was relatively small. However, in the 3D 4T1 cell spheroid model, the cytotoxicity difference between the two was more obvious. This may be because in the 3D model, the environment of the central cells is closer to the real tumor tissue environment. At this time, α-AHC inhibits the lactate uptake of aerobic tumor cells, changes the type of cell glucose metabolism (from oxidative phosphorylation supplied by lactate to glycolysis), consumes a large amount of glucose, and the amount of lactate generated surges, over-acidifying the cells and causing cell death. In addition, the hypoxic tumor cells also die due to lack of glucose supply. It shows that α-AHC can change the type of glucose metabolism of tumor tissue cells, disrupt the metabolic symbiosis between tumor cells, and at the same time induce excessive acidification of cells, achieving the effect of sensitizing chemotherapeutic drugs and synergistically anti-tumor. At the same time, compared with the gemcitabine raw drug group, the cytotoxicity of the Prodrug 3 and 4 groups was significantly improved. It is worth noting that in the 3D model, the difference between the two was further manifested. The author believes that this is because after grafting large and medium-sized amino acid molecules, the prodrug molecules can be actively taken up by cells through the LAT1 transporter, increasing the uptake efficiency of tumor cells for the prodrug molecules. Compared with the gemcitabine raw drug group, the cytotoxicity of the Prodrug 5 and 6 groups was more significantly improved, and the best cytotoxicity was shown in both the 4T1 and 3D 4T1 cell spheroid models. It is worth noting that in the 3D model, the cell viability of the maximum dose of the Prodrug 6 group was only 4.25%, much lower than 23.14% of the gemcitabine raw drug group. In addition, by comparing the Prodrug 3 and 4 groups, among the effects of the two amino acid targets, L-phenylalanine was better than L-lysine. Similarly, the same conclusion can be obtained by comparing the Prodrug 5 and 6 groups.

[0075] Example 9: Live and dead cell staining analysis of the preferred prodrug

[0076] According to the results of the cytotoxicity experiment, Prodrug 1, 4, and 6 were selected as the preferred structures for live and dead cell staining studies. The cells in different administration groups were stained using a live / dead cell staining kit (Calcein AM, EthD-I method; Cat No: PFO0008), and then the live and dead cells in each group were observed and photographed using a fluorescence inverted microscope. As Figure 4As shown, from left to right are Ⅰ. Blank group, Ⅱ. Gemcitabine group, Ⅲ. Prodrug 1 group, Ⅳ. Prodrug 4 group, Ⅴ. Prodrug 6 group. It can be clearly seen that from left to right, the proportion of red fluorescence gradually increases. It is worth noting that in the 3D model, the red fluorescence of Ⅴ. Prodrug 6 group almost occupies the entire cell spheroid model, indicating that in the 3D structure model, with the dual blessing of large and medium-sized amino acid target heads and lactate transport inhibitors, the therapeutic effect of gemcitabine has been greatly improved.

[0077] Example 10 Cellular uptake studies of preferred prodrugs

[0078] The process of LAT1-mediated drug transport is active transport, so temperature has a great influence on transport efficiency. We first used 4T1 (LAT1 (+)) cells as a model to study the cellular uptake of prodrugs at 37°C and 4°C. Figure 5 As shown in a, compared with 37°C, the cellular uptake of Prodrug 6 (LAT1-targeted prodrug) at 4°C was significantly decreased (about 4 times), while the cellular uptake of Prodrug1 (non-targeted prodrug), gemcitabine and α-AHC was slightly reduced. The above research results indicate that prodrug 6 enters 4T1 cells through active transport. In order to further determine the cellular uptake mechanism of the prodrug, we used L-Phe as a known substrate and co-incubated gemcitabine, α-AHC, Prodrug 1 and Prodrug 6 with it to examine the effects of different prodrugs on the uptake of L-Phe. The results are shown in Figure 5 As shown in Figure b, with the addition of Prodrug 6, the uptake of L-Phe was inhibited and reduced by about 73%. The results show that the targeted prodrug can compete with L-Phe to bind to LAT1 and reduce the cellular uptake of L-Phe. At the same time, we used L-Phe as a competitive substrate, co-incubated gemcitabine, α-AHC, Prodrug 1 and Prodrug 6 in 4T1 cells, and investigated the effect of LAT1 competitive substrate on prodrug uptake. The results are shown in Figure 2. Figure 5 As shown in Figure c, when L-Phe was added, the cellular uptake of Prodrug 6 was significantly inhibited, which was reduced by about 3.3 times compared with the group without L-Phe, while the cellular uptake of Prodrug 1 (non-targeted prodrug), gemcitabine and α-AHC did not change significantly. The above results indicate that the prodrug enters 4T1 cells through LAT1-mediated active transport.

[0079] Example 11 Effect of Prodrug on Lactate and Glucose

[0080] In order to investigate the effect of prodrug on glucose metabolism and lactate transport in tumor cells, we measured the changes in intracellular and extracellular glucose and lactate levels after incubation of prodrug with cells.

[0081] We first measured the utilization of glucose by tumor cells. As Figure 6 shown in a, through research, we found that the glucose consumption in the control group (without inhibitor) only increased slightly. We then co-incubated the cells with a medium containing lactate but no glucose and measured the utilization of lactate by the cells. The results showed that as time extended, the lactate content in the matrix gradually decreased. In contrast, in the groups with the addition of the lactate transporter inhibitor (α-AHC), the non-targeted prodrug (Prodrug1), and the LAT1-targeted prodrug (prodrug 6), the amount of lactate in the matrix remained basically unchanged. This indicates that when there is no glucose in the matrix, the cells can still take up lactate for sugar metabolism ( Figure 6 b). Thus, it can be seen that under normal circumstances, tumor cells take up lactate for oxidative phosphorylation rather than taking up glucose for aerobic glycolysis. When the lactate transporter inhibitor (α-AHC), prodrug1, and prodrug6 were added, the glucose consumption of the cells increased significantly compared with that before inhibition. This shows that the metabolic type of the cells has changed, possibly from oxidative phosphorylation using lactate as a raw material to glycolysis using glucose as a raw material.

[0082] Furthermore, we investigated the effects of gemcitabine, α-AHC, and the prodrugs on intracellular lactate transport. As Figure 7 shown in a, when the lactate transporter inhibitor α-AHC, prodrug6, and the non-targeted prodrug prodrug1 were co-incubated with the cells, we found that the concentration of lactate in the cells gradually increased and reached a peak at 6 h. Moreover, the pH value in the cells also decreased from 7.5 to 6.7 ( Figure 7 b). This indicates that the tumor cells underwent glycolysis and produced a large amount of lactate. Since the lactate transporter was inhibited, the newly produced lactate remained entirely in the tumor cells, resulting in an increase in intracellular lactate and a decrease in pH. In contrast, the lactate level in the control group cells remained basically at a low level, indicating that the cells carried out sugar metabolism through oxidative phosphorylation, maintained a neutral or weakly alkaline environment inside the cells, and maintained the normal physiological functions of the cells. Gemcitabine did not interfere with lactate transport. Comparing the inhibitory effects of the two prodrugs on lactate, it shows that the LAT1-targeted prodrug prodrug6 and the non-targeted prodrug prodrug1 can be rapidly taken up by the cells and activate the release of gemcitabine and α-AHC, thereby playing a role in inhibiting lactate transport.

[0083] The above research shows that lactate transporter inhibitors can change the sugar metabolism type of cells (oxidative phosphorylation - glycolysis), and then inhibit lactate transport to anti-tumor. Prodrugs can enter the cells through LAT1-mediated and rapidly activate the release of gemcitabine and α-AHC, thereby playing a role in inhibiting lactate transport.

[0084] Example 12 Tissue Distribution Study of Prodrugs

[0085] To investigate the in vivo behavior of the prodrugs, we conducted tissue distribution studies. Nude mice bearing 4T1 tumors were randomly divided into groups of 6. The LAT1-targeted prodrug 6, non-targeted prodrug 1, gemcitabine, and α-AHC were administered via intravenous injection at a dose equivalent to 10 mg / kg of gemcitabine. After administration, the animals were sacrificed at 1 and 4 h, respectively, and the heart, liver, spleen, lung, kidney, brain, and tumor were removed. The drug concentrations of the LAT1-targeted prodrug, non-targeted prodrug, gemcitabine, and α-AHC were determined by UPLC-MS / MS. To facilitate comparison of the tissue distribution differences between the prodrugs and the parent drugs, the amounts of the prodrugs measured in the tissues were converted into gemcitabine and α-AHC, respectively. As Figure 8 shown, after intravenous administration, the non-targeted prodrug, gemcitabine, and α-AHC were mainly distributed in the kidney, brain, spleen, and other sites. The LAT1-targeted prodrug had the highest distribution in the tumor, and the differences among the groups were obvious. At 1 h, the distribution of the LAT1-targeted prodrug in the tumor was 3 times that of gemcitabine and 2.2 times that of α-AHC, and 1.9 times that of the non-targeted prodrug. At 4 h, they were 4 times that of gemcitabine and 3 times that of α-AHC, and 2 times that of the non-targeted prodrug, respectively. This may be related to the high expression of the LAT1 transporter in the tumor. The targeted prodrug significantly increased its distribution in the tumor through the mediation of the LAT1 transporter.

[0086] In vivo antitumor activity study of the preferred prodrugs in Example 13

[0087] A 4T1 tumor-bearing mouse model was constructed by subcutaneous injection of 4T1 cells. When the tumor volume of the tumor-bearing mice reached 100 mm 3 , they were treated with intravenous injection of normal saline, gemcitabine, Prodrug 1, Prodrug 4, and Prodrug 6, respectively, once every 24 h for a total of four times. Then, the tumor volume and mouse body weight were monitored every two days, and the tumor volume change graph and mouse body weight change graph were drawn as follows Figure 9 . As can be seen from Figure 9 a, Prodrug 6 induced the most effective anticancer effect. Specifically, 14 days after administration, the average tumor size of the mice treated with Prodrug 6 was 55.97 mm 3 , much smaller than that of the normal saline group and the gemcitabine group. The average body weight of the mice treated with Prodrug 6 on the 14th day also showed a reduced toxicity and higher biosafety, being nearly 1.21 times higher than its initial average body weight ( Figure 9 d). Subsequently, these mice were sacrificed, and their tumors were collected and weighed. The results showed that the average tumor weight of the mice treated with Prodrug 6 was 0.072 g, much smaller than that of the normal saline group and the gemcitabine group ( Figure 9 b, Figure 9c). Finally, the anti-tumor activity of Prodrug6 was further confirmed by H&E and TUNEL staining ( Figure 10 ).

[0088] Biological safety study of the preferred prodrugs in Example 14

[0089] The H&E results of important tissues and organs after 14 days of tumor treatment showed no obvious pathological changes, indicating good biological safety of the prodrug ( Figure 11 ). In addition, we analyzed the biological safety of the preferred prodrugs using a healthy KM mouse model. Healthy KM mice were treated by tail vein injection with normal saline, gemcitabine, Prodrug 1, Prodrug 4, and Prodrug6, once every 24 h for a total of seven times. Then the body weight of the mice was monitored every two days, and the liver and kidney functions and related blood routine biochemical indexes were evaluated 14 days after drug administration. As Figure 12 shown, there were no obvious changes in the body weight of the mice treated with Prodrug 6 and the blood parameters of the mice treated with different drugs. In addition, the hematoxylin and eosin (H&E) staining of the organ tissue sections 14 days after drug administration was as Figure 13 shown, and no obvious morphological abnormalities were observed in the main tissues and organs of the mice treated with Prodrug 6. The above results indicate that Prodrug6 has good biological safety.

[0090] The structural formulas of prodrugs 1 to 6 described in the present invention are as shown in the structure in Figure 14 .

Claims

1. A targeted drug for multimodal tumor treatment, characterized in that: The targeted drug targets the tumor LAT1 transporter, improves the synchronous delivery of chemotherapy drugs and lactate transport inhibitors in the tumor, and then induces hypoxic cell apoptosis through the lactate transport inhibitor, sensitizes the chemotherapy drugs, and the two synergize. The targeted drug is a compound, geometric isomer, and pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof as shown in the general formula; wherein, the general formula 1, the general formula 2 and the general formula 3 are the reference compound structures of the general formula: Wherein: X, Y are O, NH; Drug is an antitumor drug residue containing amino or hydroxyl groups.

2. The targeted drug according to claim 1, characterized in that The structural formula of the targeted drug is selected from: Wherein: X and Y are O or NH; Drug is an antitumor drug residue containing amino or hydroxyl groups.

3. The targeted drug according to claim 1, characterized in that The structural formula of the targeted drug is selected from:

4. The targeted drug according to claim 1, characterized in that The amino- or hydroxyl-containing antitumor drug is selected from gemcitabine, decitabine, cytarabine, 5-fluorouracil, cladribine, fludarabine, azacitidine and their derivatives.

5. The targeted drug according to claim 1, characterized in that The lactate transport inhibitor is α-cyano-4-hydroxycinnamic acid (α-Cyano-4-hydroxycinnamic acid, α-AHC).

6. The targeted drug according to claim 1, characterized in that The amino acid (i.e. amino acid) is L-phenylalanine or L-lysine.

7. The method for preparing the targeted drug according to any one of claims 1 to 6 is as follows: (1) dissolving the amino acid in N,N-dimethylformamide (DMF), adding succinic anhydride and triethylamine, reacting at 50°C overnight to prepare amino acid-succinate; (2) α-AHC and different anti-tumor drugs were dissolved in DMF, 1-hydroxybenzotriazole (HOBT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and triethylamine were added, and the mixture was reacted overnight at 50°C under nitrogen protection to generate anti-tumor drug-α-AHC monoamide and α-AHC-anti-tumor drug-α-AHC monoamide; (3) Dissolve the anticancer drug-α-AHC monoamide and amino acid-succinate in DMF, add HOBT, EDCI and triethylamine, and esterify at 50°C under nitrogen protection, and then remove the protection by CH2Cl2 / TFA or Pd / C, H2 to generate the target product (general structure I); The preparation methods of the compounds of general formula 1 and general formula 2 are as follows: α-AHC and different anti-tumor drugs were dissolved in DMF, HOBT, EDCI and triethylamine were added, and the mixture was reacted overnight at 50° C. under nitrogen protection to generate target products (General Formula 1 Structure II) and (General Formula 2 Structure III). The preparation method of the compound of general formula 3 is as follows: (1) dissolving an amino-protected amino acid in DMF, adding succinic anhydride and triethylamine to prepare amino acid-succinate; (2) Different antitumor drugs and amino acid monosuccinates were dissolved in DMF, HOBT, EDCI and triethylamine were added, and the reaction was carried out overnight at 50°C under nitrogen protection. The protection was removed by CH2Cl2 / TFA or Pd / C, H2 to generate the target product (Formula 3 Structure IV).

8. The preparation method according to claim 7, characterized in that: The amino-protected amino acid is selected from Boc-4-amino-L-phenylalanine and Cbz-L-lysine.

9. Use of the compound, geometric isomer, and pharmaceutically acceptable salt, hydrate, solvate or prodrug of any one of claims 1 to 6 in the preparation of anti-tumor drugs.

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