A geldanamycin derivative and its use in the imaging of malignant tumors

Abstract

The present application relates to the field of medical chemistry synthesis and tumor treatment, in particular to a geldanamycin derivative and its application in malignant tumor imaging. The geldanamycin derivative is combined with Hsp90 to inhibit the chaperone function, induce guest protein degradation, and achieve the anti-tumor effect. The geldanamycin derivative provided by the present application has a good radio-labeling site, is suitable for 125 I、 124 I, etc. The obtained iodine-labeled geldanamycin derivative has tumor targeting and radioactivity functions, and is suitable for PET or SPECT imaging and tumor radiotherapy. The in vitro experimental results show that the geldanamycin derivative has good inhibitory activity on Raji cells and LS-174T cells, the iodine-labeled geldanamycin derivative still has anti-tumor effect, and has the development potential as a diagnosis and treatment integrated drug.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical chemical synthesis and tumor treatment applications, specifically to a geldmycin derivative and its application in malignant tumor imaging. Background Technology

[0002] In the global public health field, malignant tumors have become a major disease that seriously threatens human life and health, with their incidence and mortality rates continuing to rise. Data from the 2020 Global Cancer Burden Report released by the International Agency for Research on Cancer (IARC) of the World Health Organization shows that there were 19.29 million new cancer cases and 9.96 million cancer deaths worldwide that year.

[0003] Currently, commonly used clinical cancer treatments include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Surgery is mainly suitable for early-stage tumors and is difficult to cure mid-to-late-stage cases; chemotherapy drugs lack targeting and have significant side effects; radiotherapy, while able to locally control tumors, causes significant damage to surrounding normal tissues; targeted and immunotherapies, although highly specific, are only suitable for certain patients, and long-term use easily leads to drug resistance. Furthermore, tumor cells themselves exhibit significant heterogeneity, with different subpopulations showing varying sensitivities to the same treatment, making it difficult for single therapies to completely eliminate tumor cells, resulting in a high likelihood of recurrence and metastasis. Therefore, developing novel, highly effective, low-toxicity, and highly targeted anti-tumor treatments has become a research hotspot in this field.

[0004] Geldanamycin (GA) is a natural benzoquinone-ansamycin antibiotic derived from Streptomyces, exhibiting potent antitumor activity. Its mechanism of action focuses on the highly conserved molecular chaperone protein heat shock protein 90 (Hsp90). By competitively binding to the N-terminal ATP-binding pocket of Hsp90, it inhibits its ATPase activity, disrupting its ability to stabilize tumor-related proteins, thereby inducing the degradation of client proteins (such as Akt, HER2, EGFR, and C-Met), thus achieving its antitumor effect. Studies have shown that Hsp90 derived from tumor cells has a significantly higher binding affinity for GA-like molecules than that from normal cells, making Hsp90 a highly promising tumor target.

[0005] Various GA-based derivatives have been developed to improve their physicochemical properties and clinical applicability. For example, 17-AAG, 17-DMAG, and 7-AG have entered clinical trials. Among them, 17-AAG showed good activity in various tumor models, but its Phase III clinical trial was terminated due to hepatotoxicity and patent issues. 17-DMAG and 7-AG also failed to enter clinical application due to high toxicity, poor water solubility, and low bioavailability. Despite some progress, no ideal GA derivative has yet achieved clinical translation, and challenges remain regarding water solubility, targeting capabilities, and imaging / therapeutic efficacy.

[0006] In the fields of precision oncology and nuclear medicine, radioactive iodine isotopes such as 124 I, 131 I, 125 Iodine, due to its combined imaging and therapeutic capabilities, has been widely used. Positron and gamma rays can be used in imaging technologies such as PET / SPECT to achieve precise localization of tumor lesions; while beta rays can release high energy locally to destroy tumor cell DNA, achieving targeted radiotherapy. By labeling radioactive iodine with tumor-affinity-based target molecules such as antibodies and small molecule inhibitors, their accumulation efficiency in tumor tissues can be significantly improved, reducing radiation damage to normal tissues, thereby realizing an integrated "diagnosis + treatment" (theranostics) strategy.

[0007] Therefore, by combining the technical approach of galdromycin molecules with radioactive iodine labeling, a novel iodine-labeled galdromycin derivative with both high Hsp90 affinity and radiotherapy capability can be constructed. This is expected to overcome the bottlenecks of traditional GA molecules in terms of pharmacokinetics, bioavailability, and imaging / therapeutic targeting, and provide a new solution for the precision diagnosis and treatment of malignant tumors. Summary of the Invention

[0008] To achieve the above objectives, this invention proposes a geldamicin derivative and its application in malignant tumor imaging. The technical solution adopted by this invention is as follows:

[0009] (1) Structural design of geldmycin derivatives

[0010] The 17-p-hydroxyphenylethylamine geldamicin described in this invention is a geldamicin derivative with a p-hydroxyphenylethylamine group introduced into the geldamicin molecular structure. This structural design retains the high affinity of geldamicin for heat shock protein 90 (Hsp90) while introducing a usable iodine isotope. 125 I, 131 I or 124 I-labeled electron-dense sites are beneficial for achieving efficient and stable radiolabeling.

[0011] (2) Preparation method of Gerdmycin derivatives

[0012] Using geldemycin as the starting material, geldemycin was dissolved in one of the following solutions at a mass:volume ratio of 1 g:volume:200 mL: chloroform, dichloromethane, ethyl acetate, ethanol, or dimethyl sulfoxide. Hydroxyphenethylamine, dissolved in ethanol or methanol at a mass:volume ratio of 1 g:volume:volume:100 mL, was then added dropwise. The reaction was carried out under nitrogen or argon protection at 20°C–25°C for 8–12 hours. After the reaction was complete, the solvent was removed by rotary evaporation and the solution was concentrated to dryness to obtain the target compound. This method is simple to operate, yields stable results, and is suitable for further large-scale preparation.

[0013] (3) Iodine isotope labeling method

[0014] Radioactive iodine isotopes were introduced into the benzene ring and benzoquinone ring sites of the 17-p-hydroxyphenethylaminogeldmycin, and then labeled using the chloramine-T method. This method involves the oxidation of Na by chloramine-T. 124 I, Na 131 I or Na 125 I, will iodide ions (I - ) is oxidized to iodide ions (I) + The iodine-labeled derivative then undergoes an electrophilic substitution reaction with the target compound to complete the labeling. The reaction is terminated with sodium metabisulfite, and the product is purified to obtain a high-purity, stable iodine-labeled derivative. This method has mild reaction conditions and is suitable for the preparation of radiopharmaceuticals. The specific steps are as follows:

[0015] Radioactive sodium iodide (Na) 124 I, Na 131 I or Na 125 One of them was dissolved in pure water at a mass:volume ratio of 1g:200mL;

[0016] The geldmycin derivative was dissolved in ethanol at a mass:volume ratio of 1g:200mL;

[0017] At 0℃–25℃, the solutions obtained from steps M1 and M2 were mixed 1:1, and chloramine solution was added to maintain the concentration of chloramine-T in the system at 0.5–2.0 mg / mL for 1–2 minutes.

[0018] Sodium metabisulfite (NA2S2O5) solution was added to the reaction system to terminate the reaction and prevent excessive oxidation and side reactions.

[0019] The reaction solution was separated and purified to remove unreacted chloramine-T, sodium iodide and sodium metabisulfite residues, to obtain iodine-labeled 17-p-hydroxyphenethylamine geldamycin;

[0020] (3) Pharmaceutical composition

[0021] The pharmaceutical composition comprises 17-p-hydroxyphenethylamine geldromycin or iodine-labeled 17-p-hydroxyphenethylamine geldromycin with a pharmaceutically acceptable carrier, diluent or excipient.

[0022] (4) Application

[0023] The above-mentioned pharmaceutical composition can be used to prepare drugs for treating a variety of malignant tumors, including but not limited to leukemia, lymphoma, breast cancer, lung cancer, colorectal cancer, liver cancer, stomach cancer, ovarian cancer, prostate cancer, and melanoma.

[0024] When the pharmaceutical composition comprises iodine-labeled 17-p-hydroxyphenethylamine geldromycin as described in claim 2, it can be further used for SPECT or PET / CT imaging diagnosis of malignant tumors, including but not limited to lymphoma, breast cancer, lung cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, prostate cancer, and melanoma.

[0025] (5) Mechanism of action and administration method

[0026] Gerdemycin derivative, namely 17-p-hydroxyphenethylaminogerdemycin, binds to the Hsp90 molecular chaperone protein with high affinity, inhibiting its ATPase activity. This leads to the degradation of various oncogenic client proteins such as HER2, AKT, and EGFR, thereby disrupting tumor cell signaling pathways, inducing cell cycle arrest and apoptosis, and achieving an anti-tumor effect. Further introduction of radioactive iodine labeling can release radiation at the tumor site, causing DNA ionization damage, enhancing local killing effects, and achieving a synergistic anti-tumor mechanism of targeted therapy and radiodestruction.

[0027] Iodine-labeled 17-p-hydroxyphenethylamine geldromycin causes ionizing radiation damage to tumor cells in vivo by releasing β-rays and γ-rays or positrons, thereby destroying the DNA structure of tumor cells, inhibiting tumor cell proliferation, and inducing apoptosis.

[0028] The drug composition can be administered in various ways, including but not limited to intravenous injection, oral administration, local injection, and intratumoral injection.

[0029] Compared with the prior art, the present invention has the following significant advantages:

[0030] 1. Possesses significant antitumor activity: The synthesized 17-p-hydroxyphenethylamine geldamycin (17-Tyr-GA) showed superior or near-superior cell proliferation inhibition ability compared to geldamycin and 17-AAG in tumor cells such as Raji (human lymphoma) and LS-174T (human colon adenocarcinoma), EC50. 50 The values ​​were 5.386 µg / mL and 1.412 µg / mL, respectively, indicating that the compound possesses broad-spectrum and highly effective antitumor potential.

[0031] 2. Iodine labeling retains its biological activity, making it suitable for integrated diagnosis and treatment: radioactive iodine is labeled as... 131 I, 124 After I-labeling the benzene and benzoquinone rings of the compound, the resulting 17-Tyri-GA still exhibited detectable inhibitory activity in in vitro cell experiments, indicating that the labeling process did not significantly disrupt its Hsp90 binding ability, and it has the potential for dual application in tumor imaging and radiotherapy.

[0032] 3. Targeting Hsp90 with a clear mechanism and high specificity: The parent compound in this invention specifically binds to the ATP-binding pocket of heat shock protein 90 (Hsp90), promoting the ubiquitination and degradation of its client proteins, thereby inhibiting cancer cell signaling pathways and inducing apoptosis. It has the characteristics of a clear mechanism, high targeting, and minimal impact on normal tissues.

[0033] 4. The structural design facilitates radioactive labeling, resulting in high stability and reaction efficiency: The benzene and benzoquinone rings in the 17-p-hydroxyphenylethylamine structure have high electron densities, enabling highly efficient electrophilic substitution reactions of iodine isotopes. The chloramine-T method for labeling has mild reaction conditions, high labeling efficiency, and the purity of the finished product can reach a high level as detected by HPLC, facilitating standardized production and subsequent formulation development.

[0034] 5. Possesses dual value in nuclear medicine imaging and targeted radiotherapy: the iodine-labeled derivative, under appropriate isotope selection, such as... 124 I is used for PET imaging. 131 I-ray therapy can be widely used in the imaging and radiotherapy of various malignant tumors, especially suitable for tumors with high Hsp90 expression, such as breast cancer, lung cancer, lymphoma, and colorectal cancer, providing a new strategy for achieving integrated tumor diagnosis and treatment. Attached Figure Description

[0035] Figure 1 This is a synthetic route diagram for 17-p-hydroxyphenethylamine geldamycin;

[0036] Figure 2 Schematic diagram for the preparation of 17-p-hydroxyphenylethylamine geldamycin iodine-labeled products;

[0037] Figure 3 The HPLC chromatogram of 17-p-hydroxyphenethylamine geldromycin;

[0038] Figure 4 HPLC chromatogram of 17-p-hydroxyphenylethylamine geldamycin iodine-labeled product;

[0039] Figure 5 The mass spectrum of 17-p-hydroxyphenethylamine geldromycin;

[0040] Figure 6 The chromatogram of 17-p-hydroxyphenethylamine geldromycin;

[0041] Figure 7 Table of mass spectrometry fragment peak data for 17-p-hydroxyphenethylamine geldromycin;

[0042] Figure 8 LC-MS mass spectrum of 17-p-hydroxyphenylethylamine geldamycin iodine-labeled product;

[0043] Figure 9 Chromatogram of 17-p-hydroxyphenylethylamine gerdromycin iodine-labeled product;

[0044] Figure 10 A table showing the mass spectrometry ion peak assignments for iodine-labeled 7-p-hydroxyphenethylamine geldamycin;

[0045] Figure 11 The killing effects of geldamicin, 17-allylaminogeldamicin, and 17-p-hydroxyphenethylaminogeldamicin and their iodides on Raji cells were investigated.

[0046] Figure 12 The killing effects of geldamicin, 17-allylaminogeldamicin, and 17-p-hydroxyphenethylaminogeldamicin and their iodides on LS-174T cells were investigated.

[0047] Figure 13 The effects of geldromycin, 17-allylaminogeldromycin, and 17-p-hydroxyphenethylaminogeldromycin and their iodides on umbilical cord endothelial cells (HUVECs);

[0048] Figure 14 PET / CT results after injecting 17-Tyri-GA into tumor-bearing mice. Detailed Implementation

[0049] To enable those skilled in the art to understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0050] The present invention will now be described in detail with reference to the accompanying drawings:

[0051] Example 1 (refer to) Figure 1 , Figure 3 and Figures 5-7 This embodiment provides a method for synthesizing 17-p-hydroxyphenethylamine geldromycin, and the specific implementation method is as follows:

[0052] The raw materials and reagents used in this experiment are shown in the table below:

[0053]

[0054] This experiment used geldamicin as the starting material to synthesize 17-p-hydroxyphenethylamine geldamicin via a nucleophilic substitution reaction. The structures of the reactants and products are detailed in [reference needed]. Figure 1 In life science research, nucleophilic substitution reactions are a common method for constructing complex bioactive molecules, and the successful execution of this reaction is of great significance for in-depth exploration of the biological functions of target compounds.

[0055] Reaction Procedure: 0.250 g of galdromycin (GA) was accurately weighed into a 250 mL three-necked flask equipped with a magnetic stirrer and 50 mL of chloroform was added. To prevent GA from being oxidized by oxygen in the air, high-purity nitrogen or argon gas was bubbled into the flask to fully displace the air. The magnetic stirrer was then turned on, and stirring was continued until the GA was completely dissolved. While stirring continuously, 50 mL of ethanol solution containing 0.500 g of p-hydroxyphenethylamine was slowly added. Five minutes into the reaction, the solution color changed from yellow to red, possibly due to the formation of an intermediate product with a specific conjugated structure in the early stages of the reaction. After one hour, the solution turned wine-red, and after two hours, it deepened to a dark red, after which the color change stabilized. The reaction was continued at room temperature in the dark for 17 hours. High-performance liquid chromatography (HPLC) analysis showed that GA had been completely converted to 17-p-hydroxyphenethylamine galdromycin (17-Tyr-GA). HPLC, a commonly used separation and analysis technique in life sciences, can accurately monitor the reaction progress and product purity.

[0056] Removal of unreacted reagents: Transfer the reaction solution from the three-necked flask to a 500 ml separatory funnel, add 150 g of pure water, and then add 1.0 ml of 5 mole / L hydrochloric acid solution to the upper aqueous phase. The principle of this operation is to use the acid to convert p-hydroxyphenylethylamine (tyramine) into water-soluble tyramine hydrochloride, thereby achieving separation from the target product. After tightening the ground glass stopper, shake the liquid in the funnel vigorously up and down for 3-5 minutes, and let it stand to separate the layers. Carefully open the valve at the bottom of the separatory funnel, slowly transfer the chloroform layer solution to another 250 ml separatory funnel, add 100 ml of pure water, shake vigorously for 3-5 minutes, let it stand for 1 minute, and finally collect the lower chloroform solution into a round-bottom flask.

[0057] Concentration process: Install the flask onto the rotary evaporator, turn on the cooling water, set the rotation speed to 20 RPM, and start the water ring vacuum pump to slowly create a vacuum, gradually increasing the vacuum level. After confirming that there is no boiling over, begin rotary evaporation. Evaporate the trichloromethane in 15 minutes, and continue rotary evaporation for another 20 minutes to ensure complete solvent removal. Rotary evaporation is a highly efficient solvent removal method that effectively prevents the target product from degrading due to high temperatures.

[0058] Product collection: Carefully scrape the contents of the flask to collect 261 mg of the solid synthesized product, labeled as 17-Tyr-GA, and then freeze it at a temperature below -20°C. Low-temperature storage can effectively maintain the chemical stability of the target product and prevent it from undergoing chemical reactions such as oxidation and hydrolysis.

[0059] Purity testing: An appropriate amount of 17-Tyr-GA, concentrated into powder, was dissolved in ethanol and analyzed using a Shimadzu LC20AT high-performance liquid chromatograph with a C18 reversed-phase column at a wavelength of 334 nm. The results showed that the main peak accounted for 96.6% of the purity. Figure 3 The results were obtained by Waters UPLC-MS liquid chromatography-mass spectrometry. Figure 5 Analysis revealed that the molecular weight of the compound was 665.3.

[0060] This synthesis experiment uses chloroform as the solvent primarily because it can effectively separate the target product from water-soluble impurities. After the reaction is complete, excess hydrochloric acid is added to promote the conversion of p-hydroxyphenethylamine into water-soluble tyramine hydrochloride, thereby removing tyramine. In addition, ethyl acetate, butyl acetate, ethanol, and methanol can also be used as solvents to dissolve geldamicin and p-hydroxyphenethylamine. The choice of different solvents may have a certain impact on the reaction rate, product yield, and purity; in actual research, optimization and adjustment can be made according to specific needs.

[0061] In this reaction, 250 mg of GA (MW 560.63) was added, and 261 mg of 17-Tyr-GA (MW 667.78) product was collected, with a yield of 104% by weight; the yield was 88% by molar number.

[0062] Example 2 (refer to) Figure 2 , Figure 4 and Figures 8-10 This embodiment provides a method for synthesizing 17-p-hydroxyphenethylamine geldamicin iodide, and the specific implementation method is as follows:

[0063] For detailed structures of reactants and products, please refer to [link / reference]. Figure 2Take three 20 ml neutral borosilicate glass bottles, weigh them on an analytical balance and remove the tare, then accurately weigh 24 mg, 39 mg, and 29 mg of NaI, chloramine T trihydrate, and sodium metabisulfite, respectively. Then add 4.8 ml, 7.9 ml, and 5.8 ml of pure water to the bottles, respectively, tighten the caps, and allow the three compounds to dissolve completely at room temperature.

[0064] Remove the 17-Tyr-GA prepared in Example 1 from the -20℃ freezer and allow it to return to room temperature. Weigh 10 mg and dissolve it in 2.0 ml of ethanol to a concentration of 5 mg / ml. Accurately pipette 1.0 ml of this solution into a 15 ml centrifuge tube, and add 1.0 ml of sodium iodide solution and 1.0 ml of chloramine T solution sequentially. Mix thoroughly on a vortex mixer. At this point, the solution changes from purplish-red to pale yellow, and the liquid becomes clear and transparent. This color change may reflect the formation of intermediate products in the early stage of the reaction. After reacting for 2 minutes, immediately add 2.0 ml of sodium metabisulfite solution and mix quickly and thoroughly. The liquid quickly changes from clear and transparent to having a purple precipitate, indicating that the target iodide has begun to form. After reacting for 5 minutes, mix thoroughly again on a vortex mixer. Take another 15 ml screw-cap centrifuge tube, add an equal weight of pure water, and place the two centrifuge tubes diagonally on a benchtop centrifuge. Centrifuge at 4000 RPM for 5 minutes to allow the precipitate to settle at the bottom of the tube. Carefully aspirate the liquid from the tube using a pipette, then wash the precipitate twice with 4.0 ml of pure water. After each wash, centrifuge at 4000 RPM for 5 minutes and discard the washing water. Finally, dissolve the precipitate in 1.0 ml of 95% ethanol and filter through a 0.22 μm sterile filter. HPLC analysis showed that the main component content was 95.6%. Figure 4 .

[0065] Detection by WATERS UPLC-MS revealed that the synthesized 17-p-hydroxyphenethylamino-19-iodobenzoquinone iodide was 17-o-diiodo-p-hydroxyphenethylamino-19-iodobenzoquinone iodide (17-Tyri-GA), containing three iodine atoms, with the corresponding molecular formula C0. 36 H 44 N3O9I3, with a molecular weight of 1043.46. See the mass spectrum below. Figures 6-10 .

[0066] In this experiment, NaI was used instead of radioactive iodine isotopes such as... 131 I, 125 I, 124 I can be used in simulation experiments. In clinical applications, the corresponding sodium iodine isotope salt can be used to replace NaI to achieve radioactive labeling of the target compound.

[0067] Example 3 (refer to) Figures 11-14 This embodiment provides the application of 17-p-hydroxyphenethylamine geldromycin and 17-p-hydroxyphenethylamine geldromycin iodide in inhibiting the activity of tumor cells, and the specific implementation method is as follows:

[0068] Gerdom and its derivatives can bind to HSP90, thereby blocking tumor cells from taking up ATP for energy metabolism. HSP90 is a molecular chaperone of many tumor cell proteins, and inhibition of HSP90 function will lead to tumor cell apoptosis or death.

[0069] This study used galdromycin (GA) and 17-allylaminogaldromycin (17-AAG) as control drugs to detect the tumor-killing activity of 17-Tyr-GA and 17-Tyri-GA on various tumor cells and normal cells. The aim was to determine the difference in tumor-killing ability between the newly synthesized compound and GA and 17-AAG.

[0070] The inhibitory effects of 17-p-hydroxyethylamine geldromycin and its iodide on the proliferation of Raji, LS174T, and HUVEC cells were detected using CCK-8 assay.

[0071] (1) Sample dilution: GA, 17-AAG, 17-Tyr-GA and 17-Tyri-GA samples were diluted with cell culture medium containing 10% fetal bovine serum to a final concentration of 200µg / ml for later use.

[0072] (2) Take three 96-well cell culture plates and number them 1-3. Add RPMI 1640 culture medium with 10% fetal bovine serum to plate 1, except for wells A1-H1. Add DMEM culture medium with 10% fetal bovine serum to plate 2, except for wells A1-H1. Add ECM culture medium with 10% fetal bovine serum to plate 3, except for wells A1-H1. Add 100µl to each well. Add 200 µl of the diluted sample to each well (A1-H1) of three cell culture plates. Transfer 100 µl from each well (A1-H1) to each well (A2-H2), mix thoroughly, and then perform a 2-fold serial dilution from left to right, for a total of 11 dilutions. Discard the last 100 µl. The corresponding concentrations for each well are 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, and 0.10 μg / ml. Column 12 is used as a blank control without any added reagents.

[0073] Raji, LS174T, and HUVEC cells in logarithmic growth phase were diluted to 1.0 × 10⁻⁶. 5 cells / ml, at 1.0 × 10 4The samples were seeded at a density of 0.1 ml per well in a 96-well plate. Each concentration was replicated, and the plates were incubated at 37°C with 5% CO2 for 48 hours.

[0074] The absorbance values ​​of the four drugs inhibiting Raji cell growth are shown in the table below:

[0075]

[0076] Forty-eight hours later, the 96-well cell culture plate was removed from the incubator, and 10 μl of CCK-8 solution was added to each well. The plate was then incubated at 37°C for another 5 hours. The absorbance at 450 nm was then measured using a microplate reader. According to A... 450 The dose-response curve for calculating cell viability was derived from the drug concentration and the half-maximal inhibitory concentration (EC50). 50 The absorbance values ​​of the four drugs on the inhibitory response of Raji and LS-174T cells are shown in Tables 1 and 2, respectively. The dose-response curves are shown in Tables 1 and 2. Figures 11-14 .

[0077] The absorbance values ​​of the four drugs inhibiting the growth of LS-174T cells are shown in the table below:

[0078]

[0079] Human umbilical vein endothelial cells (HUVECs) are normal cells that can be used within a limited number of passages. Table 3 shows that the four drugs had no effect on the growth of umbilical vein endothelial cells. There was also no dose-response relationship; their nearly horizontal dose-response curves are shown in Table 3. Figure 9 .

[0080] CCK-8 assay results showed that 17-Tyr-GA and 17-o-diiodo-1,9-iodobenzoquinone-17-Tyri-GA had inhibitory effects on Raji and LS-174T cells comparable to 17-AAG, indicating promising potential for development. All four drugs had no effect on the growth of umbilical vein endothelial cells (HUVECs). No dose-response relationship was also observed, demonstrating that 17-Tyri-GA and its derivatives do not affect normal cells. The binding of 17-Tyri-GA to HSP90 in tumor cells is specific, exhibiting clear targeting.

[0081] The absorbance values ​​of the four drugs on the inhibitory response of umbilical vein endothelial cells are shown in the table below:

[0082]

[0083] In summary, this invention successfully prepared 17-p-hydroxyphenethylaminogeldemycin (17-Tyr-GA) and its iodide, 17-o-diiodo-p-hydroxyphenethylamino-19-iodobenzoquinone geldemycin (17-Tyri-GA), through an innovative synthetic route and labeling method. The products exhibit high purity and good biological activity, demonstrating potential application value in the field of antitumor therapy. Further research will delve deeper into the mechanism of action, pharmacokinetics, and toxicology of 7-Tyri-GA, providing a more solid theoretical foundation and experimental evidence for its clinical application.

[0084] Example 4 (refer to) Figure 10 This embodiment provides the application of 17-o-diiodo-p-hydroxyphenethylamino-19-iodobenzoquinone geldamycin (17-Tyri-GA) imaging in tumor-bearing mice, and the specific implementation method is as follows:

[0085] 17-Tyr-GA Iodine Isotope Labeling: Weigh two 10 ml neutral borosilicate glass vials on an analytical balance, removing the tare. Accurately weigh 39 mg of chloramine T trihydrate and 29 mg of sodium metabisulfite, respectively. Add 7.9 ml and 5.8 ml of pure water to the vials, respectively. Tighten the caps and allow to dissolve completely at room temperature. Take Na… 124 170.2 MBq of 17-Tyr-GA, prepared according to Example 1, was placed in a radiation-proof cabinet and removed from a -20°C freezer. After returning to room temperature, 2 mg was weighed and added to a 5 ml centrifuge tube. It was dissolved in 0.4 ml of ethanol, followed by the addition of 0.4 ml of Na... 124 Mix solution I and 0.4 ml of chloramine T solution thoroughly in a vortex mixer.

[0086] At this point, the solution changed from purplish-red to pale yellow, and the liquid became clear and transparent. This color change likely reflects the formation of intermediate products in the early stages of the reaction. After 2 minutes of reaction, 1.2 ml of sodium metabisulfite solution was immediately added and quickly mixed thoroughly. The liquid rapidly changed from clear and transparent to having a purple precipitate, indicating that the target iodide had begun to form. After 5 minutes of reaction, the mixture was again thoroughly mixed on a vortex mixer. Another 5 ml centrifuge tube was taken, and an equal weight of pure water was added to balance the centrifuge. The two centrifuge tubes were placed diagonally on a benchtop centrifuge and centrifuged at 10,000 RPM for 2 minutes to allow the precipitate to settle at the bottom of the tube. The liquid in the tube was carefully aspirated with a pipette, and the precipitate was washed twice with 2.0 ml of pure water. After each wash, the mixture was centrifuged at 10,000 RPM for 2 minutes, and the pure water used for washing was aspirated. 17-o-diiodo-p-hydroxyphenethylamino-19-iodobenzoquinone geldamycin (17-Tyri-GA) was prepared.

[0087] Finally, 17-Tyri-GA was dissolved in 1.0 ml of 95% ethanol, and then 5 ml of water for injection was added. The liquid in the centrifuge tube was drawn into a 5 ml sterile syringe and filtered through a 0.22 μm sterile filter to obtain the drug composition.

[0088] Eight tumor-bearing nude mice that had been inoculated with SW1116 cells for 4 weeks were used in the experiment.

[0089] Inject the drug composition into the tail vein of nude mice bearing tumors: Disinfect the tail of the mouse with iodine, wipe with alcohol to fully expose the blood vessels, and use a microsyringe to draw up the drug composition and inject it into the tail vein. After injection, apply pressure to the injection site with an alcohol swab to prevent extravasation of the marker and infection.

[0090] Nude mice were kept under SPF conditions, and their activity was observed. Nude mice were examined at 4, 12, and 24 hours post-injection. After anesthetizing the mice with 10% ursodeoxycholic acid via intraperitoneal injection, the animals were placed in a prone position on an Inveon PET / CTscanner (Siemens Healthcare Global) sliding bed for imaging. Figure 10 The PET / CT results after injecting the drug composition into one of the tumor-bearing mice show that the subcutaneous tumor was precisely located.

[0091] As can be seen from the above embodiments, the present invention...

[0092] 1. Simple Synthetic Process and High Yield: The synthetic route for 17-p-hydroxyphenethylaminogeldmycin (17-Tyr-GA) described in Example 1 employs a nucleophilic substitution reaction under mild operating conditions (room temperature, ambient pressure, and light protection), requiring no complex catalytic system. The solvents used in this method, such as chloroform and ethanol, are readily available and inexpensive. In terms of actual yield, the molar yield of 17-Tyr-GA reaches 88%, and the gravimetric yield reaches 104%, indicating few side reactions and stable target product. Compared with traditional methods for geldmycin derivatization, this invention has significant advantages in both reaction simplicity and yield.

[0093] 2. High product purity and well-defined structure: HPLC results showed that the purity of the 17-Tyr-GA main peak reached 96.6%, and the molecular weight detected by UPLC-MS was in good agreement with the theoretical value, specifically 665.3 (actual value) and 667.78 (theoretical value). High purity means greater feasibility for subsequent formulation development and in vivo studies, and reduces the safety risks posed by impurities.

[0094] 3. Further preparation of radiolabeled iodine derivatives was achieved: In Example 2, 17-Tyr-GA was successfully converted into 17-Tyri-GA containing three iodine atoms via a chloramine-T-mediated iodination reaction. The product purity was 95.6%, and the molecular weight was in perfect agreement with the theoretical calculation. This result demonstrates that the method of the present invention can not only achieve structural modification of the geldmycin molecule but also obtain stable radiolabeled derivatives, providing a new chemical basis for tumor imaging and radiotherapy.

[0095] 5. It exhibits good activity in inhibiting tumor cells and has low toxicity to normal cells:

[0096] Raji cell (lymphoma cell) experimental results: GA EC 50 The concentrations were 3.191 µg / ml for 17-Tyr-GA, 5.386 µg / ml for 17-Tyri-GA, and 7.448 µg / ml for 17-Tyri-GA, all within the same order of magnitude as the clinical candidate drug 17-AAG, indicating that they possess considerable antitumor activity.

[0097] HUVEC normal cell assay results: 17-Tyr-GA and 17-Tyri-GA showed no significant toxicity to normal cells, and the dose-response curves were nearly horizontal. This indicates that these derivatives have higher selectivity and potentially better safety.

[0098] 6. Broad application prospects: Since HSP90 is a key molecular chaperone in many tumor types, this type of derivative can not only be used for the development of chemotherapy drugs, but also for tumor imaging diagnosis through radioactive iodine labeling, and may be combined with targeted radiotherapy to broaden its clinical application areas.

[0099] It should be particularly noted that the various embodiments listed in this specification and accompanying drawings are intended to illustrate the technical solutions and advantages of the present invention, and not to limit the scope of protection of the present invention. Without departing from the core ideas and technical effects of the present invention, those skilled in the art can make any form of improvement, substitution, combination, or modification to the structural arrangement, process parameters, material selection, control logic, etc., of the described embodiments; any obvious changes based on the same concept should be considered equivalent solutions of the present invention and should be included within the scope of protection defined by the claims of the present invention. The actual scope of protection of the present invention is determined by the appended claims and should be correctly understood in conjunction with the specification and accompanying drawings.

Claims

1. A germamycin derivative, characterized in that, The derivative is 17-p-hydroxyphenethylamine geldromycin, and its chemical structural formula is as follows: ， The derivative has a high affinity for Hsp90 and can be used for the treatment of malignant tumors.

2. The germamycin derivative according to claim 1, characterized in that, The derivative 17-p-hydroxyphenethylamino group can be labeled with iodine isotopes to form iodine-labeled 17-p-hydroxyphenethylaminogeldmycin, wherein the iodine isotope is... 125 I, 131 I or 124 One of them, its chemical structural formula is as follows: ， The iodine-labeled 17-p-hydroxyphenethylamine geldromycin can be used for the treatment and imaging diagnosis of malignant tumors.

3. A geldamicin derivative according to claim 1, characterized in that, The preparation method includes the following steps: S1. Dissolve geldromycin in one of the following solutions: chloroform, dichloromethane, ethyl acetate, ethanol, or dimethyl sulfoxide, at a mass:volume ratio of 1g:200mL. S2. Dissolve p-hydroxyphenylethylamine in ethanol or methanol at a mass:volume ratio of 1g:100mL, then add it dropwise to the solution obtained in step S1, and stir the reaction under a nitrogen or argon protective atmosphere at 20℃-25℃ for 8-12 hours. S3. After the reaction is complete, the solvent is removed by rotary evaporation and the product is concentrated to obtain the target product.

4. A germamycin derivative according to claim 2, characterized in that, The iodine isotope labeling method employs the chloramine-T method, comprising the following steps: M1. Radioactive sodium iodide (Na) 124 I, Na 131 I or Na 125 One of them was dissolved in pure water at a mass:volume ratio of 1g:200mL; M2. Dissolve the geldamicin derivative in ethanol at a mass:volume ratio of 1g:200mL; M3. At 0℃–25℃, mix the solutions obtained in step M1 and step M2 at a ratio of 1:1, add chloramine-T solution, maintain the concentration of chloramine-T in the system at 0.5–2.0 mg / mL, and react for 1–2 minutes. M4. Add sodium metabisulfite (NA2S2O5) solution to the reaction system to terminate the reaction and prevent excessive oxidation and side reactions. M5. The reaction solution was separated and purified to remove unreacted chloramine-T, sodium iodide and sodium metabisulfite residues, to obtain iodine-labeled 17-p-hydroxyphenethylamine geldromycin.

5. A pharmaceutical composition, characterized in that, The composition comprises 17-p-hydroxyphenethylamine geldromycin or iodine-labeled 17-p-hydroxyphenethylamine geldromycin with a pharmaceutically acceptable carrier, diluent or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition can be used to treat various malignant tumors, including leukemia, lymphoma, breast cancer, lung cancer, colorectal cancer, liver cancer, stomach cancer, ovarian cancer, prostate cancer, and melanoma.

7. The pharmaceutical composition according to claim 5, characterized in that, When the pharmaceutical composition comprises iodine-labeled 17-p-hydroxyphenethylamine geldromycin as described in claim 2, it can be further used for SPECT or PET / CT imaging diagnosis of malignant tumors, including lymphoma, breast cancer, lung cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, prostate cancer, and melanoma.

8. The pharmaceutical composition according to claim 5, characterized in that, The administration methods of the pharmaceutical composition include, but are not limited to, intravenous injection and oral administration.

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