Oligonucleotide-modified podophyllotoxin derivatives, methods of making and using the same

Abstract

The application discloses an oligonucleotide modified podophyllotoxin derivative, a preparation method and application thereof, and introduces an oligonucleotide aptamer with special functions, such as targeting tumor microenvironment, reversing T cell exhaustion, and improving inflammatory factors such as IFN-gamma and IL-2, into an azacyclic derivative of podophyllotoxin to obtain a podophyllotoxin derivative shown in formula (II) which has significantly improved antitumor activity, reduced toxic side effects, and has the double antitumor effect of immunotherapy and chemotherapy. In vitro tumor cell activity inhibition experiments show that the antitumor activity of the compound shown in formula (II) is significantly improved compared with that of the azacyclic podophyllotoxin, and the safety of the azacyclic podophyllotoxin is also significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of podophyllotoxin derivatives, specifically relating to oligonucleotide-modified podophyllotoxin derivatives and their preparation methods. This invention also relates to the use of the oligonucleotide-modified podophyllotoxin derivatives in the preparation of antitumor drugs. Background Technology

[0002] Podophyllotoxin is a lignan compound isolated and extracted from natural Podophyllum peltatum, possessing antiviral and antitumor activities. The structure of podophyllotoxin is as follows: Figure 19 As shown in Chinese formula (Ⅰ), researchers have focused on effectively modifying the podophyllotoxin nucleus to obtain podophyllotoxin derivatives that retain or even enhance the original antitumor activity while having fewer toxic side effects. For example, by substituting a nitrogen heterocycle at the 4-position of the C ring, a derivative, 5-amino-indazole-podophyllotoxin, was obtained, further improving the antitumor activity of podophyllotoxin. However, due to its strong toxic side effects and poor bioavailability, its clinical application is limited. Summary of the Invention

[0003] One objective of this invention is to provide a class of oligonucleotide-modified podophyllotoxin derivatives with good safety profiles;

[0004] A second objective of this invention is to provide a method for preparing the oligonucleotide-modified nitrogen-substituted podophyllotoxin derivatives;

[0005] A third objective of this invention is to apply the oligonucleotide-modified nitrogen heterocyclic-substituted podophyllotoxin derivative to the preparation of clinical antitumor drugs.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] A class of oligonucleotide-modified nitrogen-heterocyclic substituted podophyllotoxin derivatives or pharmaceutically acceptable salts thereof with antitumor activity, wherein the structural formula of the podophyllotoxin derivative is shown in (II):

[0008]

[0009] Wherein, R1 is selected from:

[0010]

[0011] Where * represents the attachment points of these structures to nitrogen atoms;

[0012] R2 is: # represents the attachment point between R2 and the nitrogen atom;

[0013] R3 is an oligonucleotide sequence.

[0014] The oligonucleotide used in this invention is a flexible chain compound with a defined nucleotide sequence and good water solubility. It can target galactolectin 1 in the tumor microenvironment, with the sequence AP-74M545. Using it as a modifying group is beneficial to improve the water solubility and targeting of podophyllotoxin derivatives, thereby improving the safety of podophyllotoxin derivatives by targeting tumor tissue.

[0015] The nucleotide sequence AP-74M545 is shown in SEQ ID No. 1.

[0016] The method for preparing the compound shown in formula (II) provided by the present invention includes the following steps:

[0017] Step (1) involves protecting the hydroxyl terminus of 2-hydroxyethyl disulfide with a TBS group to obtain compound 1:

[0018]

[0019] Step (2) involves a condensation reaction in which the N-formylimidazole of N,N-carbonyldiimidazole is attached to the hydroxyl group of compound 1 to obtain compound 2:

[0020] Step (3): In organic solvent three, an organic base catalyst is added, and the imidazole of compound 2 is replaced by a reactive monomer to obtain compound 3. The organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene and 4-dimethylaminopyridine, and the organic solvent is acetonitrile. The reactive monomer is selected from one of 5-nitro-indazole, 6-nitro-indazole, 7-nitro-indazole, 5-nitro-indole, 6-nitro-indole, and 7-nitro-indole. Compound 3 is... One of them;

[0021] Step (4) uses a hydrogenation reaction to convert the nitro group of compound 3 into an amino group to obtain compound 4. The catalyst used is palladium on carbon.

[0022] Step (5): Iodine is introduced into the 4-position of the C ring of podophyllotoxin through an iodine substitution reaction to obtain iodopodophyllotoxin; compound 4 is introduced into the 4-position of the C ring of iodopodophyllotoxin through an affinity substitution reaction to obtain compound 5.

[0023] Step (6): Remove the TBS group of compound 5 with HCl to expose the free hydroxyl group and obtain compound 6;

[0024] Step (7): Compound 6 is dissolved in pyridine, and p-toluenesulfonyl chloride is added to react with it, so that the p-toluenesulfonyl group replaces the hydroxyl group of compound 6 to obtain compound 7;

[0025] Step (8): Compound 7 is dissolved in N,N-dimethylacetamide, and sodium azide is added to replace the p-toluenesulfonyl group with the azide group to obtain compound 8;

[0026] Step (9): Compound 8 and the oligonucleotide modified with DBCO at the 5' end are dissolved in a biphasic solution and copper-free click chemistry is performed to obtain compound 9, i.e., the compound shown in formula (II), wherein the biphasic solution is an equal volume mixture of phosphate buffer and dimethyl sulfoxide.

[0027] The synthesis of the conjugate requires modification of both ends of the sulfur bridge with oligonucleotides and nitrogen-containing heterocyclic podophyllotoxin derivatives. To increase yield, the addition of nucleotides is performed as the final step in the synthesis. Therefore, at the beginning of the synthesis, in the presence of imidazole, one end of the sulfur bridge is protected with a TBS group to obtain compound 1; compound 2 is obtained by substituting the other end of the hydroxyl group of compound 1 with CDI; compound 3 is generated by substituting compound 2 with a 5-nitro-indazole module in the same manner; to link compound 3 to iodophyllotoxin via a C-NH bond, the nitro group in compound 3 is reduced to an amino group by Pd / C to obtain compound 4. Compound 4 is then subjected to TEA and BaCO3 catalysis to obtain compound 5, using triethylamine or pyridine as the acid-binding agent. The TBS protecting group in compound 5 is then rapidly removed with HCl, exposing the free hydroxyl group (compound 6); the hydroxyl group is converted to an N3 group by a TOS group and NaN3. The final product 9 is obtained by the N3 group via a click reaction with a DBCO-modified oligonucleotide in phosphate buffer.

[0028] In this invention, the reaction steps can be carried out under the following conditions: (1) 2-hydroxyethyl disulfide is dissolved in an organic reagent, then imidazole is added, mixed well, and then tert-butyldimethylchlorosilane is added. The solvent can be tetrahydrofuran, etc.; (2) The product of the previous step is dissolved in an organic reagent, N,N-carbonyldiimidazole is added, and the mixture is stirred at room temperature. Solvents can be dichloromethane, etc.; (3) Dissolve 5-nitro-indazole and the product of the previous step in an organic solvent, add an organic base catalyst, and stir at room temperature. The organic base can be 1,8-diazabicyclo[5.4.0]undec-7-ene and 4-dimethylaminopyridine. The organic solvent can be acetonitrile, etc.; (4) In the presence of methanol, mix the product of the previous step with palladium on carbon and reduce the nitro group in a hydrogen atmosphere; (5.1) Dissolve podophyllotoxin in an organic reagent and add sodium iodide. The organic reagent can be boron trifluoride ether and acetonitrile; (5.2) Mix the products of the previous two steps in an organic solvent, add a catalyst, and the organic reagent can be... The catalyst is barium carbonate, and the acid-binding agent is triethylamine or pyridine; (6) Place the product of the previous step in a solvent and stir overnight. The solvent is 1M HCl / THF with a volume ratio of 1; (7) Dissolve the product of the previous step in an organic solvent and add p-toluenesulfonyl chloride. The organic solvent can be pyridine; (8) Dissolve the product of the previous step in N,N-dimethylacetamide, add sodium azide and stir for 4 hours; (9) Dissolve the product of the previous step and the DBCO-modified oligonucleotide in a biphasic solution and carry out a copper-free click chemical reaction. The biphasic solution is an equal volume mixture of phosphate buffer and dimethyl sulfoxide.

[0029] In this invention, the molar ratio between the reactants in all reactions can be 1:1 to 10.

[0030] In this invention, all reaction temperatures can be from -20 to 50°C.

[0031] Furthermore, in this invention, compound 1 is prepared at 0°C, while the reaction temperature for the preparation of other compounds is room temperature.

[0032] The above preparation method also includes: pouring the reaction solution after steps (1), (2), and (3) into 20 to 50 times the volume of deionized water, extracting with dichloromethane, and drying to obtain crude products of compounds 2, 3, and 4.

[0033] Furthermore, the method includes separating the crude product sequentially using silica gel column chromatography and gel column chromatography to obtain purified nitrogen-substituted podophyllotoxin derivative products.

[0034] Preferably, the silica gel column chromatography separation method includes: (1) the silica gel column chromatography is normal phase silica gel column chromatography or reversed phase silica gel column chromatography. The normal phase silica gel is packed into the column after being mixed with a low polarity organic solvent and equilibrated with an eluent. The eluent is preferably composed of petroleum ether and ethyl acetate in a volume ratio of 20:1. The reversed phase silica gel is packed into the column after being mixed with methanol and equilibrated with an eluent. The eluent is preferably composed of methanol and water in a volume ratio of 60:1. (2) the sample to be separated and purified is dissolved in the eluent, loaded for adsorption, then eluted with the eluent, the eluent is collected, the sample is evaporated and recrystallized.

[0035] In vitro tests on HepG2 cell activity inhibition showed that the antitumor activity of the compound of formula (II) prepared in this invention is basically equivalent to that of 5-amino-indazole-podophyllotoxin. Tests on proliferation inhibition using the most active 5-amino-indazole-podophyllotoxin-oligonucleotide conjugate (AP74-IZP) showed that the compound of formula (II) prepared in this invention has superior antiproliferative capacity compared to 5-amino-indazole-podophyllotoxin and the marketed drugs etoposide and oxaliplatin. In vitro micro-thermal surge tests showed that the compound of formula (II) from the vegetation of this invention can target the oligonucleotide receptor protein galactoglucosinolate-1. In vivo HepG-2 subcutaneous xenograft tumor assays showed that the compound of formula (II) of this invention has superior in vivo antitumor activity compared to 5-amino-indazole-podophyllotoxin and the marketed drugs etoposide and oxaliplatin. The results of the in vivo Hepa1-6 subcutaneous xenograft tumor assay showed that the compound of formula (II) of this invention has superior in vivo antitumor activity compared to 5-amino-indazole-podophyllotoxin and the marketed drug etoposide, and exhibits the ability to reverse T cell depletion and produce immunotherapeutic effects. The above test results prove that the compound of formula (II) of this invention can be prepared into an antitumor drug and clinically applied to antitumor treatment.

[0036] The antitumor pharmaceutical composition provided by this invention comprises an effective amount of a compound of formula (II) or a salt thereof and a pharmaceutically acceptable carrier, that is, a pharmaceutically acceptable amount of the compound of formula (II) is combined with a pharmaceutically acceptable carrier and prepared into any suitable pharmaceutical composition according to conventional formulation methods in the art. This pharmaceutical composition is generally suitable for oral and injectable administration, and is also suitable for other administration methods, such as transdermal administration. The pharmaceutical composition can be in the form of tablets, capsules, powders, granules, lozenges, suppositories, or liquid formulations such as oral solutions or sterile parenteral suspensions. The composition can also be in the form of large or small volume injections, lyophilized powder for injection, sterile powder repackaging, etc. To achieve consistent dosing, the pharmaceutical composition of this invention is preferably in single-dose form. Single-dose forms for oral administration may be tablets and capsules, and may contain conventional excipients such as binders, such as syrup, gum arabic, gelatin, sorbitol, astragalus gum, or polyvinylpyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, such as magnesium stearate; disintegrants, such as starch, polyvinylpyrrolidone, sodium starch glycolate, or microcrystalline cellulose, or pharmaceutically acceptable wetting agents, such as sodium dodecyl sulfate. Attached Figure Description

[0037] Figure 1 The cloning of HepG2 cells with compound 9;

[0038] Figure 2 Compound 9 induces cell cycle arrest in HepG2 cells;

[0039] Figure 3 Distribution of compound 9 in small animals (various organs);

[0040] Figure 4 The therapeutic effects of compound 9 on a mouse model of HepG2 subcutaneous xenograft tumor;

[0041] Figure 5 The therapeutic effects of compound 9 on a mouse model of HepG2 subcutaneous xenograft tumor;

[0042] Figure 6 H&E staining of HepG2 tumors after administration of compound 9, etc.;

[0043] Figure 7 Compound 9 and others were administered to HepG2 tumors and resulted in Ki67-IHC.

[0044] Figure 8 Changes in body weight in mice with HepG2 tumors after administration of compound 9, etc.;

[0045] Figure 9Changes in organ coefficients after administration of compound 9 and other drugs to HepG2 tumor mice; the bar data for each organ in the figure correspond from left to right to Normalmice, Vehicle, AP74, Oxaliplatin, Etoposide, IZP, and AP74-IZP.

[0046] Figure 10 Changes in blood counts in mice with HepG2 tumors after administration of compound 9, etc.;

[0047] Figure 11 Changes in blood biochemical parameters in mice with HepG2 tumors after administration of compound 9 and other substances;

[0048] Figure 12 Organ damage (H&E) in mice with HepG2 tumors treated with compound 9, etc.

[0049] Figure 13 The effect of compound 9 on tumor volume reduction in C57 mice with Hepa1-6 tumors;

[0050] Figure 14 The effect of compound 9 on tumor mass reduction in C57 mice with Hepa1-6 tumors;

[0051] Figure 15 The effect of compound 9 on the CD4 / CD8 ratio in Hepa1-6 tumor tissue;

[0052] Figure 16 The effect of compound 9, etc., on the CD4 / CD8 ratio in Hepa1-6 tumor tissue;

[0053] Figure 17 The effects of compound 9 on the expression levels of various cytokines in Hepa1-6 tumor tissues;

[0054] Figure 18 The preparation process of compound 9;

[0055] Figure 19 It is the molecular formula of 5-amino-indazole-podophyllotoxin. Detailed Implementation

[0056] The technical solution of the present invention will be further described below through specific embodiments.

[0057] Unless otherwise specified, the raw materials and equipment used in this invention can be purchased from the market or are commonly used in the field. Unless otherwise specified, the methods in the embodiments are conventional methods in the field.

[0058] Test materials

[0059] 1. Podophyllotoxin: All were purchased from Xi'an Helin Bioengineering Co., Ltd.;

[0060] 2, 5-nitro-indazole, tert-butyldimethylchlorosilane, N,N-carbonyldiimidazole, imidazole, 1,8-diazabicyclo[5.4.0]undec-7-ene were all purchased from Shanghai Bid Pharmaceutical Co., Ltd.

[0061] 3. The DBCO-modified oligonucleotides were synthesized by Shanghai Bioengineering Co., Ltd.

[0062] Example 15: Synthesis and purification of the amino-indazole-podophyllotoxin-oligonucleotide conjugate (compound 1)

[0063] (1) Synthesis of 5-amino-indazole-podophyllotoxin-oligonucleotide conjugate:

[0064] Synthetic intermediate 1

[0065] 10 mmol of di(hydroxyethyl) disulfide was dissolved in 40 mL of tetrahydrofuran and stirred at 0 °C. Imidazole was then added, and the mixture was kept under nitrogen protection and stirred at room temperature for 30 min. TBSCl (tert-butyldimethylchlorosilane) was then added, and the reaction was allowed to proceed for 8 h to protect one of the hydroxyl groups of the di(hydroxyethyl) disulfide. This synthesis was performed in four parallel groups, with a total of 40 mmol of di(hydroxyethyl) disulfide added. After the reaction was complete, the product was extracted three times with 200 mL of DCM. The organic phase was collected, washed three times with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, concentrated, and purified by 200-300 mesh silica gel column chromatography (PE:EA = 15:1, v:v) to obtain a colorless oily liquid product 1-1. Figure 18 Compound 1 was obtained in a total of 7.5 g, with a yield of 69.9%.

[0066] Synthetic intermediate 2

[0067] Compound 1 (27.9 mmol) was dissolved in 100 mL of dichloromethane in a 250 mL flask. N,N-carbonyldiimidazole (41.8 mmol, 6.8 g) was added at room temperature, and the mixture was stirred magnetically for 3 h. After TLC analysis, the mixture was extracted three times with 200 mL of DCM. The organic phase was collected, washed three times with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, and concentrated to obtain a pale yellow oily liquid product 1-2. Figure 18 Compound 2 was obtained in a total of 9.4 g, with a yield of 93.0%.

[0068] Synthetic intermediate 3

[0069] Compound 2 (3.32 mmol) was dissolved in 10 mL of acetonitrile in a 25 mL round-bottom flask. At room temperature, the monomer 5-nitroinazole (4.93 mmol) was added, followed by 1,8-diazabicycloundec-7-ene (DBU 0.24 mmol). After TLC analysis, all reaction mixtures were collected and extracted three times with 100 mL of DCM. The organic phase was collected, washed three times with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, concentrated, and purified by 200-300 mesh silica gel column chromatography (PE:EA = 30:1, v:v) to obtain pale yellow waxy solid products 1-3. Figure 18 Compound 3 was present in a total of 1.3 g, with a yield of 21.4%.

[0070] Synthetic intermediate 4

[0071] Take 3600 mg of the above compound into a 25 mL flask, add 5 mL of methanol and palladium on carbon catalyst, seal with a rubber stopper, remove air using a vacuum pump, and insert a hydrogen balloon. The reaction is confirmed to be complete by TLC. The filtrate is dried over anhydrous Na₂SO₄, concentrated, and then purified by 200-300 mesh silica gel column chromatography (PE:EA = 3:1, v:v) to obtain a pale yellow oily liquid product 1-4. Figure 18 Compound 4 was collected in a total of 217 mg, with a yield of 38.7%.

[0072] Synthetic intermediate 5

[0073] 2 mmol of PTOX (Podophyllotoxin) was placed in a 50 mL round-bottom flask, 3.0 mmol of NaI was added, and 10 mL of acetonitrile was added to dissolve it. 6.0 mmol of BF3·Et2O (boron trifluoride diethyl ether) was added under ice bath conditions. The reaction was continued under ice bath conditions for 1 h. After removing the acetonitrile by rotary evaporation, the product was redissolved in tetrahydrofuran, and 3.0 mmol of compound 4 was added. Barium carbonate was added as a catalyst and triethylamine as an acid-binding agent under stirring at room temperature. The reaction was continued at room temperature for 8 h. After the reaction was completed, the mixture was filtered, and the filtrate was extracted three times with 50 mL of water and 100 mL of DCM. The organic phase was collected, washed three times with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, concentrated, and purified by 200-300 mesh silica gel column chromatography (PE:EA = 3:1, v:v) to obtain pale yellow solid products 1-5. Figure 18 Compound 5 was collected in a total of 825 mg, with a yield of 50.1%.

[0074] Synthetic intermediate 6

[0075] 800 mg of compound 5 was dissolved in a 1:1 (v / v) mixture of tetrahydrofuran and HCl (2 M), and stirred at room temperature. After the reaction was complete as determined by TCL, 300 mL of water and 50 mL of ethyl acetate were added for extraction three times. The organic phase was collected, washed three times with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, concentrated, and then purified by 200-300 mesh silica gel column chromatography (DCM:MeOH = 150:1, v:v) to obtain pale yellow solid products 1-6. Figure 18 Compound 6 was extracted in a total of 613 mg, with a yield of 88.9%.

[0076] Synthetic intermediate 7

[0077] 6200 mg of the compound was dissolved in 4 mL of pyridine, and p-toluenesulfonyl chloride was added with stirring. The reaction progress was monitored by TLC until the reaction was complete. The mixture was extracted with EA (2 × 30 mL), the organic compound was washed with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, evaporated under reduced pressure, and purified by column chromatography (PE:EA = 3:1) to obtain a pale yellow solid powder. Figure 18 Compound 7 was collected in a total of 86 mg, with a yield of 43%.

[0078] Synthetic intermediate 8

[0079] 7100 mg of the compound was dissolved in 3 mL of LDM, and 70 mg of sodium azide was added with stirring at room temperature. The reaction was monitored by TLC plate overnight until complete. 100 mL of water was added, and the mixture was extracted three times with 50 mL of EA. The organic phase was collected, washed three times with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, concentrated, and then separated and purified by 200-300 mesh silica gel column chromatography (PE:EA = 3:1, v:v) to obtain pale yellow solid powder products 1-9. Figure 18 Compound 8 was obtained in a total of 77.3 mg, with a yield of 86.10%.

[0080] Synthetic product 9

[0081] The DBCO (dibenzocyclooctyne)-modified oligonucleotide (20 nmol) was mixed with compound 8 (25 nmol) in 0.8 mL of 20 mM phosphate-buffered saline (PBS) (pH 7.2) in a 10 mL flask. The mixture was stirred overnight at room temperature. The reaction progress was then analyzed by liquid chromatography. After the reaction was complete, the final product was obtained by HPLC separation using a triethylamine acetic acid system. Figure 18 The pale yellow powder compound 9.

[0082] The oligonucleotide sequence is AP-74M545, as shown in SEQ ID No. 1. SEQ ID NO: 1: 5′-DBCO-TTACAGTCGGCTGTAATTTAGTGTATGTACCGGTGTGTGTACGAT--3′.

[0083] (2) Separation and purification of AP74-IZP,

[0084] Separation and purification were prepared using silica gel column chromatography and HPLC.

[0085] (A) Separation was performed using a normal-phase silica gel column (normal-phase silica gel: Qingdao Ocean Chemical Co., Ltd., China, HG / T2354-92; separation system: Buchi isocratic rapid chromatography system; chromatographic column: Buchi glass column C-690, 460 mm long, 15 mm inner diameter) or a similar polar column; the eluents for the separation and purification of the above 8 compounds were (1) petroleum ether: ethyl acetate = 15:1. (3) petroleum ether: ethyl acetate = 30:1. (4) petroleum ether: ethyl acetate = 3:1. (5) petroleum ether: ethyl acetate = 3:1. (6) dichloromethane: methanol = 150:1. (7) petroleum ether: ethyl acetate = 3:1. (8) petroleum ether: ethyl acetate = 3:1. The sample loading volume for the separation and purification of the above 8 compounds was 2 ml, and the flow rate was constant at 1.0 ml / min; each 2 ml of eluent was collected as a fraction. Each fraction was examined using normal-phase silica gel thin-layer chromatography (Mokk high-efficiency silica gel thin-layer chromatography, Germany) or similar polarity thin-layer chromatography; fractions with an Rf value of 0.5 were combined using a petroleum ether:ethyl acetate 3:1 system as the developing solvent; the combined samples were vacuum dried and stored in a refrigerator at 4°C under light-protected conditions as samples for purification.

[0086] (B) Separation was performed using high performance liquid chromatography (column: C18; length 280 mm, inner diameter 0.5 mm); the elution was carried out with methanol, and the triethylamine acetic acid: methanol gradient elution conditions were as follows: the sample to be purified was diluted with double-distilled water, loaded for adsorption at a flow rate of 0.6 ml / min, and then subjected to gradient elution. The target product eluted at 10.2 min. 5 mL of the eluent was eluted for every 90 μL, and the eluent was lyophilized to obtain the final product.

[0087] Compound 12-(2-(tert-butyldimethylsiloxy)ethyl)dithioyl)acetyn-1-ol: a colorless oily liquid, C 10 H 24 O2S2Si.

[0088] 1 HNMR (600MHz, CDCl3, 298K): δ (ppm) = 3.91-3.86 (m, 4H), 2.84-2.88 (m, 4H), 0.92 (s, 9H), 0.10 (s, 6H).13 CNMR (151MHz, CDCl3298K): δ (ppm) = 61.83, 60.18, 41.27, 41.23, 25.97, 18.30, -5.26.ESI-ToF: [C 10 H 24 O2S2Si+Na] + 291.0863 (Calculated for C) 10 H 24 O2S2Si268.0987).

[0089] Compound 22-((2-(tert-butyldimethylsiloxy)ethyl)dithio)ethyl 1H-imidazolium-1-carboxylic acid ester: pale yellow oily liquid, C 14 H 26 N2O3S2Si.

[0090] 1 HNMR (600MHz, CDCl3, 298K): δ (ppm) = 8.17 (s, 1H), 7.46 (t, J = 1.4Hz, 1H), 7.10 (dd, J = 1.5, 0.8Hz, 1H), 4.70 (t ,J=6.6Hz,2H),3.89(t,J=6.5Hz,2H),3.07(t,J=6.6Hz,2H),2.88(t,J=6.5Hz,2H),0.92(s,9H),0.10(s,6H). 13 CNMR (151MHz, CDCl3298K): δ (ppm) = 148.45, 137.13, 130.70, 117.14, 65.90, 61.54, 41.69, 36.46, 25.85, 18.30, -5.27.ESI-ToF[C 14 H 26 N2O3S2Si+Na]+385.1526(CalculatedforC 14 H 26 N2O3S2Si362.1154).

[0091] Compound 32-((2-(tert-butyldimethylsiloxy)ethyl)dithio)ethyl 5-nitro-1H-indazole-1-carboxylic acid ester: pale yellow waxy solid, C 18 H 27 N3O5S2Si.

[0092] 1HNMR (600MHz, CDCl3, 298K): δ (ppm) = 8.74 (d, J = 1.8Hz, 1H), 8.48 (dd, J = 9.3, 1.8Hz, 1H), 8.42 (d, J = 9.3Hz, 1H), 8.41 (s,1H),4.86(t,J=6.8Hz,2H),3.91(s,2H),3.18(t,J=6.8Hz,2H),2.91(t,J=6.5Hz,2H),0.92(s,9H),0.10(s,6H). 13 CNMR (151MHz, CDCl3, 298K): δ (ppm) = 149.88, 144.69, 142.14, 141.01, 125.62, 124 .22,118.03,115.17,66.22,61.69,41.58,36.42,25.88,18.33,-5.24.ESI-ToF:[C 18 H 27 [N3O5S2Si+H] + 458.1590 (Calculated for C) 18 H 27 N3O5S2Si457.1161).

[0093] Compound 42-((2-(tert-butyldimethylsiloxy)ethyl)dithio)ethyl 5-amino-1H-indazole-1-carboxylic acid ester: pale yellow oily liquid, C 18 H 29 N3O3S2Si.

[0094] 1 HNMR (600MHz, CDCl3, 298K): δ (ppm) = 8.03–7.99 (m, 2H), 6.97 (dd, J = 8.9, 2.2Hz, 1H), 6.93 (d, J = 2.1Hz, 1H), 4.75 (t,J=6.9Hz,2H),3.87(t,J=6.6Hz,2H),3.13(t,J=7.0Hz,2H),2.87(t,J=6.6Hz,2H),0.89(s,9H),0.07(s,6H). 13 CNMR (151MHz, CDCl3, 298K): δ (ppm) = 150.36, 143.17, 139.78, 134.21, 127. 10,119.68,115.16,104.03,41.53,36.63,25.88,18.32,-5.26.ESI-ToF:[C 18 H 29 [N3O3S2Si+H] +428.1485 (Calculated for C) 18 H 29 N3O3S2Si427.1420).

[0095] Compound 52-((2-(tert-butyldimethylsiloxy)ethyl)dithio)ethyl 5-amino-1H-indazole-podophyllotoxin: pale yellow solid, C 40 H 49 N3O3S2Si.

[0096] 1HNMR (600MHz, CDCl3, 298K): δ (ppm) = 8.04 (d, J = 8.9Hz, 1H), 8.02 (s, 1H), 6.87 (dd, J = 9.0, 2.3Hz, 1H), 6 .75(s,1H),6.50(s,1H),6.31(s,2H),5.93(dd,J=12.0,1.1Hz,2H),4.73(t,J=6.9Hz,2H),4.72–4.69(m, 1H),4.57(d,J=4.9Hz,1H),4.41(t,J=8.0Hz,1H),4.13(d,J=5.8Hz,1H),3.97(dd,J=10.7,8.6Hz,1H),3 .85(t,J=6.6Hz,2H),3.78(s,3H),3.73(s,6H),3.16(dd,J=14.1,5.0Hz,1H),3.11(t,J=6.9Hz,2H),3.08 –3.00(m,1H),2.85(t,J=6.6Hz,2H),0.87(s,9H),0.05(s,6H).13CNMR(151MHz,CDCl3,298K):δ(ppm)=1 73.59,151.65,149.28,147.38,146.71,143.65,138.70,136.34,134.00,130.80,129.30,126.11,117.4 0,114.74,108.99,108.03,107.32,100.60,98.38,67.78,64.43,60.74,59.75,55.29,52.26,42.57,40. 93,40.53,37.66,35.60,17.31,0.00,-6.26.ESI-ToF:[C40H49N3O3S2Si+H]+846.4382(CalculatedforC 40 H 49 N3O3S2Si823.2629).

[0097] Compound 6,2-((ethyl)dithio)ethyl 5-amino-1H-indazole-podophyllotoxin: pale yellow solid, C 34 H 35 N3O 10 S2.

[0098] 1 HNMR (600MHz, CDCl3, 298K): δ (ppm) = 8.07 (d, J = 8.9Hz, 1H), 8.05 (s, 1H), 6.89 (dd, J = 9.0, 2.1Hz, 1H), 6.76 (s, 1H) ,6.73(d,J=1.9Hz,1H),6.54(s,1H),6.33(s,2H),5.97(d,J=8.4Hz,2H),4.78(t,J=6.7Hz,2H),4.72(d,J=3.9Hz,1 H),4.62(d,J=4.9Hz,1H),4.44(t,J=8.0Hz,1H),4.00(dd,J=10.6,8.7Hz,1H),3.91(t,J=5.8Hz,2H),3.82(s,3H) ,3.77(s,6H),3.21–3.18(m,1H),3.17(t,J=6.6Hz,2H),3.06(tdd,J=11.2,7.2,3.7Hz,1H),2.94(t,J=5.8Hz,2H). 13 CNMR (151MHz, CDCl3, 298K): δ (ppm) = 174.67, 152.67, 150.34, 148.40, 147.72, 144.76, 139.84, 137.35, 135.04, 131.82, 130.30, 127.12, 118.56, 115.73,110.01,109.08,108.34,101.63,99.40,68.82,65.53,60.78,60 .28,56.32,53.25,43.59,41.96,41.82,38.68,36.60,29.70.ESI-ToF:[C 34 H 35 N3O 10 S2+H] + 710.1800 (Calculated for C) 34 H 35 N3O 10 S2709.1764).

[0099] Compound 72-(2-Toluenesulfonyloxy)ethyldithioethyl-5-amino-1H-indazole-podophyllotoxin: pale yellow solid, C 41 H41 N3O 12 S3.

[0100] 1HNMR (600MHz, CDCl3, 298K): δ (ppm) = 7.91 (s, 1H), 7.34 (d, J = 8.8Hz, 1H), 6.78 (s, 1H) ),6.77(dd,J=8.9,2.1Hz,1H),6.71(d,J=1.5Hz,1H),6.53(s,1H),6.35(s,2H),5.95 (d,J=11.3Hz,2H),4.70(d,J=4.0Hz,1H),4.60(d,J=5.0Hz,1H),4.44(t,J=8.0Hz,1H ),4.04(dd,J=10.6,8.7Hz,1H),3.82(s,3H),3.76(s,6H),3.22(dd,J=14.0,5.0Hz,1 H),3.09–3.01(m,1H).13CNMR(151MHz,CDCl3,298K):δ(ppm)=173.74,151.57,149.2 0,147.21,146.56,144.15,143.89,138.92,136.16,134.17,131.61,130.70,129.38 ,128.96,126.90,126.15,117.47,114.55,108.83,108.16,107.26,100.55,98.33,67.84,66.67,64.04,59.70,55.22,51.98,42.55,41.25,40.84,37.67,35.79,20.63.

[0101] Compound 82-(azidoethyl)dithioethyl-5-amino-1H-indazole-podophyllotoxin: pale yellow solid, C 34 H 34 N6O9S2.

[0102] 1HNMR (600MHz, CDCl3, 298K): δ (ppm) = 8.07 (d, J = 8.9Hz, 1H), 8.05 (s, 1H), 6.89 (dd, J = 8.9, 2.1Hz, 1H), 6.77 (s, 1H), 6.73(d,J=1.7Hz,1H),6.54(s,1H),6.33(s,2H),5.97(d,J=9.0Hz,2H),4.77(t,J=6.7Hz,2H),4.72(t,J=4.5Hz,1H) ,4.61(d,J=4.9Hz,1H),4.44(t,J=8.0Hz,1H),4.08(d,J=5.5Hz,1H),4.00(dd,J=10.6,8.7Hz,1H),3.82(s,3H),3.7 7(s,6H),3.61(t,J=6.8Hz,2H),3.21–3.18(m,1H),3.17(t,J=5.5Hz,2H),3.10–3.02(m,1H),2.92(t,J=6.8Hz,2H). 13 CNMR (151MHz, CDCl3, 298K): δ (ppm) = 174.63, 152.66, 150.28, 148.38, 147 .70,144.75,139.84,137.33,135.04,131.81,130.31,127.15,118.46,115 .69,109.99,109.07,108.32,101.62,99.40,68.81,65.23,60.77,60.40,5 6.30,53.23,49.95,43.59,41.94,38.68,37.54,36.82,14.20.ESI-ToF:[C 34 H 34 N6O9S2+H] + 735.1872 (Calculated for C) 34 H 35 N3O 10 S2734.1829).

[0103] Compound 95-amino-indazole-podophyllotoxin-oligonucleotide conjugate (AP74-IZP): pale yellow powder.

[0104] [M] - 16365.24; found 16362.1689.

[0105] Experimental Example 1: Activity test of the compound prepared in the embodiments of the present invention to inhibit tumor cells.

[0106] I. Experimental Materials

[0107] 1. Test compound: Compound 9 from Example

[0108] 2. Control compounds: 5-aminoinzazole-podophyllotoxin, synthesized in our laboratory, purity 98%; etoposide (purchased from Shanghai Bide Pharmaceutical); oxaliplatin (purchased from Shanghai Bide Pharmaceutical);

[0109] 3. Cell lines: HepG2, Huh7, PLC / PRF / 5 and SNU387 cell lines were purchased from Wuhan Pronosai Biotechnology Co., Ltd.

[0110] II. Test Methods

[0111] HepG2, Huh7, PLC / PRF / 5, and SNU387 cell lines in logarithmic growth phase were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in an appropriate amount of culture medium. The cell concentration was adjusted to 7000-10000 cells / well, and the cells were seeded in 96-well culture plates. The following experimental groups were set up:

[0112] One negative control group (1‰ DMSO, i.e., solvent control group); the experimental group (i.e., compound 9); and three control groups: 5-aminoinazole-podophyllotoxin group, etoposide group, and oxaliplatin group.

[0113] 0.10 mL of cells were added to each well using RPMI 1640 medium containing 10% fetal bovine serum (FBS). Cells were cultured at 37°C, 5% CO2, and saturated humidity for 12 h until adherence. The culture medium was then discarded. For the experimental groups, 0.10 M DMEM medium containing the same amount of compound 9 (10% FBS) was added. For the 5-aminoindazole-podophyllotoxin, etoposide, and oxaliplatin groups, 0.10 M DMEM medium containing 5-aminoindazole-podophyllotoxin, etoposide, and oxaliplatin (10% FBS) was added, respectively. The amounts of 5-aminoindazole-podophyllotoxin, etoposide, and oxaliplatin were exactly the same as those of compound 9 in the experimental groups. The negative control group was treated with 0.5% DMSO. Each group was divided into three replicates and cultured for another 48 h. 10 μL of 5 mg / mL MTT was added to each well, and the cells were incubated at 37°C for 4 h. Add 100 μL LDMSO to each well, shake on a shaker at 37°C for 30 min, measure absorbance (OD) at 492 nm, and calculate the MTT ratio as: OD value of drug group / OD value of negative control group.

[0114] III. Test Results

[0115] The experimental results are shown in Table 1. As can be seen from Table 1, compound 9 has significantly improved antitumor activity against HepG2, Huh7, PLC / PRF / 5 and SNU387 cell lines compared with 5-aminoinazole-podophyllotoxin, as well as etoposide, a podophyllotoxin derivative that is currently marketed as an antitumor drug, and oxaliplatin, an anti-liver cancer drug that is already on the market; and its water solubility is significantly better.

[0116] Table 1. IC50 of oligonucleotide-substituted 5-aminoindazole-podophyllotoxin against in vitro tumor lines. 50 value

[0117]

[0118] After activity evaluation, HepG2 cells were selected as the cells with the best activity and cell colony formation experiments were performed.

[0119] Depend on Figure 1 It can be seen that compound 9 exhibits the same anti-cell proliferation ability as 5-aminoinzazole-podophyllotoxin, with a significant reduction in cell clonal clusters, and its anti-proliferative ability is significantly improved compared with etoposide and oxaliplatin.

[0120] After evaluating antiproliferative capacity, cell cycle arrest experiments were conducted on compound 9, 5-aminoinzazole-podophyllotoxin, etoposide, and oxaliplatin.

[0121] Depend on Figure 2 It is known that compound 9 exhibits a cell cycle arrest ability similar to that of 5-aminoinzazole-podophyllotoxin, and can almost completely arrest HepG2 cells in the G2 / M phase, which is significantly stronger than etoposide and oxaliplatin.

[0122] After evaluating the periodicity blocking effect, in vivo distribution experiments were conducted on compound 9.

[0123] Depend on Figure 3 It can be seen that compound 9 is mainly distributed in the liver, kidney and tumor sites. Compared with the MCF-7 tumor model with low galactoglobin expression, it shows a clear preference for HepG2 tumors with high galactoglobin expression. Moreover, as time goes on, the proportion in the liver and kidney gradually decreases, but the proportion in the tumor tissue gradually increases.

[0124] After evaluation of in vivo distribution, in vivo efficacy experiments were conducted on compound 9, etoposide, oxaliplatin, and 5-aminoinzol-podophyllotoxin.

[0125] Depend on Figure 4 , 5As shown in 6 and 7, 5-aminoinzol-podophyllotoxin (5 mg / kg) and etoposide (20 mg / kg) exhibited similar tumor-killing effects, with tumors significantly smaller than the untreated group (Vehicle), tumor collapse prevented, and tumor cell proliferation protein expression decreased. Compound 9 further increased this difference.

[0126] After efficacy evaluation in nude mice, the in vivo safety of compound 9, etoposide, oxaliplatin, and 5-aminoinazole-podophyllotoxin was evaluated in mice.

[0127] Depend on Figure 8 , 9 As shown in 10, 11, and 12, the conjugation of oligonucleotides significantly improved the toxicity of 5-aminoinzazole-podophyllotoxin, and compared with etoposide and oxaliplatin, it showed low organ toxicity and delayed decrease in blood cell count.

[0128] After safety evaluation in nude mice, the efficacy of compound 9, etoposide, and 5-aminoinazole-podophyllotoxin in C57 mice was evaluated.

[0129] Depend on Figure 13 It can be seen that the experimental group of compound 9 showed a significant ability to alleviate T cell exhaustion in the tumor microenvironment. Compound 9 includes an oligonucleotide modified with DBCO (dibenzocyclooctylene) and 5-aminoindazole-podophyllotoxin, meaning that the two exerted a synergistic anti-tumor effect, which was significantly better than the 5-aminoindazole-podophyllotoxin group and etoposide alone.

[0130] In this embodiment, the 5-nitroindazole monomer used in the synthesis of intermediate 3 can also be replaced with one of 6-nitroindazole, 7-nitroindazole, 5-nitroindole, 6-nitroindole, or 7-nitroindole. Therefore, the synthesized compound 3 will correspondingly become...

[0131] The final synthesized compound 9 is

[0132] Wherein, R1 is selected from:

[0133]

[0134] * indicates the attachment points of these structures to nitrogen atoms;

[0135] R2 is: # represents the attachment point between R2 and the nitrogen atom;

[0136] R3 is the oligonucleotide sequence shown in SEQ ID No. 1, which can target galactolectin 1 in the tumor microenvironment.

Claims

1. A compound of formula (II) or a pharmaceutically acceptable salt thereof: Equation (II) in, R1 is selected from: Where * represents the attachment points of these structures to nitrogen atoms; R2 is: # represents the attachment point between R2 and the nitrogen atom; R3 is an oligonucleotide sequence as described in SEQ ID No. 1, which can target galactolectin 1 in the tumor microenvironment.

2. A method for preparing the compound of claim 1, characterized in that, Includes the following steps: Step (1) involves protecting the hydroxyl terminus of 2-hydroxyethyl disulfide with a TBS group to obtain compound 1: ； Step (2) involves a condensation reaction in which the N-formylimidazole of N,N-carbonyldiimidazole is attached to the hydroxyl group of compound 1 to obtain compound 2: ; Step (3): In organic solvent three, an organic base catalyst is added, and the imidazole group of compound 2 is replaced with a reactive monomer to obtain compound 3. The organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene and 4-dimethylaminopyridine, and the organic solvent is acetonitrile. The reactive monomer is selected from one of 5-nitro-indazole, 6-nitro-indazole, 7-nitro-indazole, 5-nitro-indole, 6-nitro-indole, and 7-nitro-indole. Compound 3 is... , , , , , One of them; Step (4) uses a hydrogenation reaction to convert the nitro group of compound 3 into an amino group to obtain compound 4. The catalyst used is palladium on carbon. Step (5): Iodine is introduced into the 4-position of the C ring of podophyllotoxin through an iodine substitution reaction to obtain iodopodophyllotoxin; compound 4 is introduced into the 4-position of the C ring of iodopodophyllotoxin through a nucleophilic substitution reaction to obtain compound 5. Step (6): Remove the TBS group of compound 5 with HCl to expose the free hydroxyl group and obtain compound 6; Step (7): Compound 6 is dissolved in pyridine, and p-toluenesulfonyl chloride is added to react with it, so that the p-toluenesulfonyl group replaces the hydroxyl group of compound 6 to obtain compound 7; Step (8): Compound 7 is dissolved in N,N-dimethylacetamide, and sodium azide is added to replace the p-toluenesulfonyl group with the azide group to obtain compound 8; Step (9): Compound 8 and the oligonucleotide modified with DBCO at the 5' end are dissolved in a biphasic solution and copper-free click chemistry is performed to obtain compound 9, i.e., the compound shown in formula (II), wherein the biphasic solution is an equal volume mixture of phosphate buffer and dimethyl sulfoxide.

3. The method according to claim 2, characterized in that, In step (5), the molar ratio of iodopodophyllotoxin to compound 4 is 1:1~10.

4. The method according to claim 2, characterized in that, The reaction temperature for steps (2) to (9) is -20 to 50°C.

5. The method according to claim 4, characterized in that, Compound 1 was prepared at 0°C, while the other compounds were prepared at room temperature.

6. The method according to claim 2, characterized in that, Also includes: The reaction solution after steps (1), (2), and (3) was poured into 20 to 50 times its volume of deionized water, extracted with dichloromethane, and dried to obtain crude products of compounds 2, 3, and 4.

7. The method according to claim 6, characterized in that, Also includes: The crude product was sequentially separated using silica gel column chromatography to obtain the purified product from each step.

8. Use of the compound of claim 1 or a salt thereof in the preparation of an antitumor drug.

9. An antitumor drug composition, characterized in that, It includes an effective amount of the compound of claim 1 or a salt thereof and a pharmaceutically acceptable carrier.

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