N-benzyl benzamide compounds, pharmaceutical compositions and uses thereof

Abstract

This invention discloses an N-benzylbenzamide compound, its pharmaceutical composition, and its applications. The compound has the structure of Formula I and also contains a pharmaceutically acceptable salt. It effectively releases the parent drug in vitro and in vivo, exhibits excellent tumor-suppressive activity, and significantly reduces the toxicity of the parent drug through a prodrug strategy. It also demonstrates good safety and shows promise as a potential drug.

Description

Technical Field

[0001] This invention relates to an N-benzylbenzamide compound, pharmaceutical compositions thereof, and applications thereof, and more particularly to an N-benzylbenzamide compound that can be prepared as a prodrug for a microtubule inhibitor, pharmaceutical compositions thereof, and applications thereof. Background Technology

[0002] Microtubules are a major component of the cytoskeleton and play a crucial role in maintaining cell morphology, cell division, and signal transduction. Therefore, tubulin is a promising target for novel chemotherapeutic drugs. Tubulin inhibitors can prevent the excessive proliferation of tumor cells and are an important class of anti-tumor therapeutics. Currently, clinically used microtubule inhibitors mainly include drugs that inhibit microtubule depolymerization, such as paclitaxel, and drugs that inhibit microtubule aggregation, such as vinca alkaloids. The colchicine binding site is one of the most studied sites for microtubules, and inhibitors acting on this site are usually structurally simple, such as colchicine and Combretastatin A-4. Furthermore, inhibitors acting on this site can also disrupt blood vessels in tumor tissue; therefore, research on microtubule inhibitors acting on the colchicine site has become a hot topic in current anti-tumor research. However, these drugs have drawbacks such as significant toxicity, poor water solubility, and a tendency to develop drug resistance. Summary of the Invention

[0003] Purpose of the invention: The first purpose of this invention is to provide an N-benzylbenzamide compound, the second purpose is to provide a pharmaceutical composition comprising the compound, and the third purpose is to provide the use of the compound and the pharmaceutical composition thereof in the preparation of microtubule inhibitor drugs.

[0004] Technical solution: The N-benzylbenzamide compounds of this invention have the structure of Formula I and also contain their pharmaceutically acceptable salts:

[0005]

[0006] in:

[0007] R1 is selected from hydrogen, hydroxyl, mercapto, halogen, cyano, carboxyl, C1-C3 ester, C1-C3 amide, hydroxymethyl, aldehyde, nitro, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 cycloalkyl, C1-C6 alkylamino, C6-C 10 Aryl, C3-C 10 Contains a heteroaryl group with one nitrogen, oxygen, and sulfur group; C4-C8 heterocyclic groups containing one nitrogen, oxygen, and sulfur group.

[0008] R2 is selected from hydrogen, hydroxyl, mercapto, halogen, cyano, carboxyl, C1-C3 ester, C1-C3 amide, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 cycloalkyl, C3-C 10 Contains a heteroaryl group with one nitrogen, oxygen, and sulfur group; C4-C8 heterocyclic groups containing one nitrogen, oxygen, and sulfur group.

[0009] R3 is selected from hydrogen, C1-C8 alkyl, C1-C8 unsaturated alkyl containing one double or triple bond, C3-C8 cycloalkyl, C6-C 10 Aryl, C3-C 10 Contains a heteroaryl group with one nitrogen, oxygen, and sulfur group; C4-C8 heterocyclic groups containing one nitrogen, oxygen, and sulfur group.

[0010] X is selected from CH2, NH, O, S, C4-C8 azaspirocyclic, C4-C6 azaarospirocyclic, C1-C8 chain alkyl, C1-C3 chain ether, and C1-C3 chain ester.

[0011] Preferably, in the structure:

[0012] R1 is selected from hydrogen, halogen, nitro, C1-C8 alkoxy, C1-C6 alkylamino, C6-C 10 Aryl, C3-C 10 It contains a heteroaryl group consisting of nitrogen, oxygen, and sulfur;

[0013] R2 is selected from hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 cycloalkyl, C3-C 10 Contains a heteroaryl group with one nitrogen, oxygen, and sulfur group; C4-C8 heterocyclic groups containing one nitrogen, oxygen, and sulfur group.

[0014] R3 is selected from hydrogen, C1-C8 alkyl, C1-C8 unsaturated alkyl containing one double or triple bond, C3-C8 cycloalkyl, C6-C 10 Aryl, C3-C 10 Contains a heteroaryl group with one nitrogen, oxygen, and sulfur group; C4-C8 heterocyclic groups containing one nitrogen, oxygen, and sulfur group.

[0015] X is selected from CH2, NH, O, S, C4-C8 azaspirocyclic rings, C4-C6 azaarospirocyclic rings, and C1-C8 chain alkyl groups.

[0016] Further preferably, in the structure:

[0017] R1 is selected from hydrogen, halogen, nitro, C1-C8 alkoxy, and C1-C6 alkylamino;

[0018] R2 is selected from hydrogen, C1-C8 alkyl, C1-C8 alkoxy, and C4-C8 heterocyclic groups containing one nitrogen, oxygen, or sulfur group;

[0019] R3 is selected from hydrogen, C1-C8 alkyl, C1-C8 unsaturated alkyl containing one double or triple bond, C6-C 10 Aryl;

[0020] X is selected from CH2, NH, O, S, C4-C8 azaspirocyclic compounds, and C4-C6 azaaromatic spirocyclic compounds.

[0021] Further preferred, in the structure:

[0022] R1 is selected from hydrogen, fluorine, chlorine, bromine, nitro, methoxy, ethoxy, ethylamino, and N,N-dimethylamino.

[0023] R2 is selected from hydrogen, methyl, ethyl, methoxy, ethoxy, piperazine, morpholine, and piperidine;

[0024] R3 is selected from hydrogen, methyl, ethyl, allyl, propyne, 1-butyn-3-yl, 3-methyl-1-butyn-3-yl, 1-buten-3-yl, benzyl, 4-propynoxybenzyl, 3-propynoxybenzyl, 2-propynoxybenzyl, 4-allyloxybenzyl, 3-allyloxybenzyl, 2-allyloxybenzyl;

[0025] X is selected from NH, O, and S.

[0026] Specifically, the N-benzylbenzamide compounds described in this invention are selected from any of the following compounds:

[0027]

[0028]

[0029] Bioorthogonal reactions are a class of chemical reactions that can occur in biological systems without interfering with natural biochemical processes. Traditional bioorthogonal reactions are primarily linkage reactions involving the formation of new chemical bonds. In recent years, a new type of reaction—bioorthogonal shearing reactions based on the breaking of chemical bonds—has gradually developed and is finding increasingly widespread applications in the "release," "activation," and "manipulation" of molecules. Focusing on medicinal chemistry, bioorthogonal chemistry is widely used to reduce drug toxicity, improve drug-like properties, and enhance therapeutic efficacy.

[0030] This invention is the first to combine a microtubule inhibitor with a colchicine binding site with a transition metal-catalyzed bioorthogonal scission strategy, providing a microtubule inhibitor prodrug molecule with low toxicity, high selective tumor killing power, and good safety, which is expected to achieve precision treatment of tumors.

[0031] The prodrugs described in this invention have weak or even no activity, but after administration, they are converted into their corresponding biologically active forms under physiological conditions (e.g., through metabolism, solvation, or other means). Compounds of Formula I can exist in non-solventized forms and in solvated forms containing pharmaceutically acceptable solvents (e.g., water, ethanol, etc.); compounds of Formula I can contain asymmetric or chiral centers, and therefore can exist in different stereoisomers; all stereoisomers of this invention, including but not limited to diastereomers, enantiomers, and transisomers, as well as mixtures thereof (e.g., racemic mixtures), are included within the scope of this invention.

[0032] Wherein, the pharmaceutically acceptable salt of the present invention is a salt formed by the compound and an acid selected from any of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0033] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0034] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0035] The compounds described in this invention can also be used as solvates.

[0036] This invention includes various deuterated forms of the compound. Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom.

[0037] The compounds of general formula I of this invention can be prepared by the following methods:

[0038]

[0039] The definitions of R1, R2, and R3 are the same as those described above.

[0040] The specific synthesis steps are as follows:

[0041] Starting with 2-fluorobenzoic acid with different substitutions at the 5-position, an esterification reaction was carried out under concentrated sulfuric acid and ethanol conditions. This was followed by a nucleophilic substitution reaction with piperazine / morpholine / thiomorpholine, and then esterification under alkaline conditions to obtain intermediate 4. The hydroxyl group of a 3-hydroxybenzonitrile with different substitutions at the 4-position was protected by reacting it with benzyl bromide, followed by reduction of the cyano group with borane-tetrahydrofuran to obtain intermediate 7. Intermediates 4 and 7 underwent amide condensation to give intermediate 8, which was then hydrogenated to remove the benzyl protecting group, yielding parent compound 9. Finally, nucleophilic substitution reactions with bromoalkane with different substituents were carried out to obtain bioorthogonal prodrug compound 10.

[0042] The pharmaceutical composition of the present invention comprises the N-benzylbenzamide compound of the present invention and a pharmaceutically acceptable carrier.

[0043] Preferably, the dosage form of the pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, and patches. The carriers that can be mixed in any way can be changed depending on the dosage form, administration method, etc. Examples of carriers include excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, coloring agents, or sweeteners.

[0044] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0045] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0046] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0047] The N-benzylbenzamide compounds or pharmaceutical compositions described in this invention are used in the preparation of drugs that inhibit tubulin activity.

[0048] Preferably, the drug is an anti-tumor drug used for colon cancer, leukemia, liver cancer, breast cancer, etc.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0050] These compounds can effectively release the parent drug in vitro and in vivo and significantly inhibit tubulin inhibitory activity. They exhibit excellent tumor inhibitory activity (cellular level inhibitory activity reaches nanomolar concentration level, and animal level tumor inhibition rate reaches more than 50%). Furthermore, the toxicity of the parent drug is significantly reduced through a prodrug strategy, and the safety profile is good, showing promising potential for drug development. Attached Figure Description

[0051] Figure 1 The results show the effects of some compounds of the present invention on mouse body weight (*P<0.05, **P<0.01 compared with the blank group). Detailed Implementation

[0052] The technical solution of the present invention will be further described below with reference to the embodiments.

[0053] Example 1

[0054]

[0055] 5-Fluoro-N-(4-methoxy-3-(prop-2-yn-1-oxy)benzyl)-2-morpholinobenzamide

[0056] Step 1: 2,5-Difluorobenzoic acid (1) (3.0 g, 19.0 mmol) was mixed in 20 mL of ethanol, and a catalytic amount of concentrated sulfuric acid was added. The mixture was refluxed for 12 h. The solvent was removed by rotary evaporation, the residue was diluted with water, and extracted with DCM (3 × 50 mL). The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain intermediate 2 (2.82 g, 80%).

[0057] Step 2: Intermediate 2 (2.82 g, 15.2 mmol) was mixed in 20 mL of DMSO, and morpholine (6.63 mL, 75.7 mmol) was added. The mixture was stirred at 120 °C for 6 h. After cooling, the reaction solution was diluted with 200 mL of water and extracted with ethyl acetate (3 × 50 mL). The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain intermediate 3 (2.80 g, 73%).

[0058] Step 3: Intermediate 3 (2.80 g, 11.06 mmol) was mixed with 10 mL of methanol, and 10 mL of 10% NaOH aqueous solution was added. The mixture was stirred at 80 °C for 30 min, and the methanol was removed by rotary evaporation. The pH was then adjusted to 3 with 10% hydrochloric acid. The solid was collected by vacuum filtration, the filter cake was washed with water, and dried to obtain intermediate 4 (2.21 g, 90%).

[0059] Step 4: Dissolve 3-hydroxy-4-methoxybenzonitrile (2.5 g, 16.8 mmol) in acetonitrile (25.0 mL). Then add potassium carbonate (4.65 g, 33.5 mmol) and benzyl bromide (3.35 mL, 29.4 mmol). Reflux the resulting mixture for 1.5 h, cool, and filter. Concentrate the filtrate, dilute with ethyl acetate, and wash with water. Dry the organic phase with anhydrous sodium sulfate, evaporate to obtain the residue, and purify by column chromatography to give intermediate 6 (3.73 g, 93%).

[0060] Step 5: Intermediate 6 (2 g, 8.36 mmol) was mixed in a tetrahydrofuran borane solution and refluxed with stirring under argon protection for 10 hours. The solvent was removed by rotary evaporation, and intermediate 7 (1.18 g, 58%) was purified by column chromatography.

[0061] Step 6: Intermediate 4 (370 mg, 1.64 mmol), intermediate 7 (800 mg, 3.29 mmol), HATU (633.45 mg, 1.97 mmol), and triethylamine (274 μL, 1.97 mmol) were added to 10 mL of anhydrous acetonitrile solution. The mixture was stirred for 3 hours and extracted with DCM (3 × 25 mL). The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue, which was purified by column chromatography to obtain intermediate 8 (533.3 mg, 72%).

[0062] Step 7: Dissolve intermediate 8 (500 mg, 1.11 mmol) in 20 mL of ethanol and add palladium on carbon (125 mg). Stir the reaction under a hydrogen atmosphere for 2 h, filter, concentrate the filtrate under vacuum to obtain the residue, and purify by column chromatography to obtain intermediate 9 (328 mg, 82%).

[0063] Step 8: Intermediate 9 (150 mg, 416.22 μmol), bromopropyne (297.08 mg, 2.5 mmol), and anhydrous potassium carbonate (517.71 mg, 3.75 mmol) were dissolved in acetone and heated under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a white solid with a yield of 72.7%.

[0064] 1H NMR (300MHz, DMSO-d6) δ9.92(t,J=5.7Hz,1H),7.63–7.52(m,1H),7.42–7.27(m,2H),7.09(d,J=1.6Hz,1H),7.00(d,J=2.3Hz,2H) ,4.77(d,J=2.4Hz,2H),4.44(d,J=5.6Hz,2H),3.77(s,3H),3.54(t,J=2.4Hz,1H),3.49–3.40(m,4H),2.83(dd,J=5.6,3.3Hz,4H). 13 C NMR(100MHz,DMSO-d6)δ164.89,149.07,147.55,147.52,146.96,131.53,131.37,123.58,123.50,121.95, 118.67,118.45,116.90,116.67,114.84,112.67,79.76,78.64,66.46,56.56,56.13,53.32,43.02.ESI-MS m / z:399.2[M+H] + .

[0065] Example 2

[0066]

[0067] N-(3-(but-3-yne-2-oxy)-4-methoxybenzyl)-5-fluoro-2-morpholinobenzamide

[0068] Intermediate 9 (150 mg, 416.22 μmol), 3-butyn-2-ol (35.01 mg, 499.47 μmol), and triphenylphosphine (218.34 mg, 832.44 μmol) were dissolved in anhydrous dichloromethane. The mixture was cooled to 0 °C under nitrogen protection, and diisopropyl azodicarbonate (126.25 mg, 624.33 μmol) was added dropwise. The mixture was reacted at room temperature for 3 hours, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a white solid with a yield of 34.3%.

[0069] 1H NMR(400MHz, DMSO-d6)δ9.89(t,J=5.7Hz,1H),7.56(dd,J=9.5,2.8Hz,1H),7.41–7.27(m,2H),7.10(d,J=1.8Hz,1H),7.04–6.88(m,2H) ,5.00(qd,J=6.5,2.0Hz,1H),4.43(t,J=5.8Hz,2H),3.76(s,3H),3.44(p,J=3.0,2.6Hz,5H),2.85–2.78(m,4H),1.54(d,J=6.5Hz,3H). 13 C NMR (100MHz, DMSO-d6) δ164.90,149.47,147.54,147.51,146.50,131.51,123.55,123.47,122.18,118.66 ,118.45,116.90,116.66,116.54,112.77,83.73,76.95,66.46,64.32,56.14,53.33,42.96,22.53.ESI-MS m / z:413.2[M+H] + .

[0070] Example 3

[0071]

[0072] 5-Fluoro-N-(4-methoxy-3-((2-methylbut-3-yn-2-yl)oxo)benzyl)-2-morpholinobenzamide

[0073] Intermediate 9 (100 mg, 277.48 μmol), 3-chloro-3-methyl-1-butyne (170.75 mg, 1.66 mmol), and anhydrous cesium carbonate (362.86 mg, 1.66 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a yellow solid with a yield of 68.4%.

[0074] 1H NMR(400MHz, DMSO-d6)δ9.87(t,J=5.7Hz,1H),7.59–7.49(m,1H),7.40–7.25(m,3H),7.09(dd,J=8.3,2.1Hz,1H ),6.99(d,J=8.4Hz,1H),4.42(d,J=5.7Hz,2H),3.73(s,3H),3.53–3.42(m,5H),2.84–2.78(m,4H),1.55(s,6H). 13 CNMR(100MHz,DMSO-d6)δ164.91,152.29,147.47,144.37,131.14,124.07,123.46,123.38,123.10,118.64,11 8.42,116.90,116.66,113.03,86.50,76.57,73.73,66.46,56.08,53.28,42.84,29.57.ESI-MSm / z:427.2[M+H] + .

[0075] Example 4

[0076]

[0077] N-(3-(allyloxy)-4-methoxybenzyl)-5-fluoro-2-morpholinobenzamide

[0078] Intermediate 9 (150 mg, 416.22 μmol), bromopropylene (302.12 mg, 2.50 mmol), and anhydrous potassium carbonate (517.71 mg, 3.75 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a yellowish-brown solid with a yield of 82.1%.

[0079] 1H NMR (300MHz, DMSO-d6) δ9.95 (t, J=5.7Hz, 1H), 7.64 (ddd, J=9.5, 2.7, 0.9Hz, 1H), 7. 49–7.34(m,2H),7.12(s,1H),7.03(d,J=1.2Hz,2H),6.13(ddt,J=17.3,10.5,5.3Hz ,1H),5.47(dq,J=17.3,1.7Hz,1H),5.32(dq,J=10.5,1.5Hz,1H),4.62(dt,J=5.4,1 .5Hz,2H),4.51(d,J=5.7Hz,2H),3.85(s,3H),3.57–3.48(m,4H),2.95–2.85(m,4H). 13 CNMR(100MHz,DMSO-d6)δ164.91,148.89,148.06,147.51,134.29,131.69,131.43,131.36,123.51,123.43,121 .14,118.63,118.41,117.91,116.88,116.64,114.29,112.59,69.48,66.44,56.12,53.31,43.06,38.70.ESI-MS m / z:401.2[M+H] + .

[0080] Example 5

[0081]

[0082] N-(3-(but-3-en-2-oxy)-4-methoxybenzyl)-5-fluoro-2-morpholinobenzamide

[0083] Intermediate 9 (100 mg, 277.48 μmol), 3-chloro-1-butene (150.76 mg, 1.66 mmol), and anhydrous potassium carbonate (230.09 mg, 1.66 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a yellow liquid with a yield of 69.4%.

[0084] 1H NMR(300MHz,DMSO-d6)δ9.86(t,J=5.7Hz,1H),7.62–7.51(m,1H),7.40–7.26(m,2H), 7.01(s,1H),6.94(d,J=1.1Hz,2H),5.88(ddd,J=17.0,10.5,6.1Hz,1H),5.23(dt,J=1 7.3,1.5Hz,1H),5.09(dt,J=10.5,1.4Hz,1H),4.84(p,J=6.3Hz,1H),4.41(dd,J=5.7, 3.9Hz, 2H), 3.76 (s, 3H), 3.48–3.40 (m, 4H), 2.85–2.76 (m, 4H), 1.35 (d, J = 6.3Hz, 3H). 13 C NMR(100MHz,DMSO-d6)δ164.54,148.25,148.12,147.66,134.62,131.89,131.25,131.01,125.5 4,124.52,122.47,119.28,118.84,117.55,116.97,116.54,114.25,112.64,68.44,66.32,56.75 53.43,42.52,38.62.20.65.ESI-MS m / z:415.2[M+H] + .

[0085] Example 6

[0086]

[0087] N-(3-(benzyloxy)-4-methoxybenzyl)-5-fluoro-2-morpholinobenzamide

[0088] Following the procedure in Example 1, the target compound, intermediate 8, was obtained as a white solid with a yield of 87.3%.

[0089] 1 H NMR(300MHz, DMSO-d6)δ9.89(t,J=5.6Hz,1H),7.57(dd,J=8.9,2.2Hz,1H),7.50–7.26(m,7H),7.13(s,1H), 6.97(s,2H),5.07(s,2H),4.44(d,J=5.6Hz,2H),3.78(s,3H),3.43(t,J=4.5Hz,4H),2.79(t,J=4.5Hz,4H). 13C NMR(100MHz,DMSO-d6)δ167.84,152.55,149.71,148.12,147.83,144.71,136.62,130.42,128.65,12 8.39,127.64,124.16,122.87,119.42,118.85,117.25,116.52,116.04,109.15,71.24,66.32,56.15 52.93,44.41.ESI-MS m / z:451.2[M+H] + .

[0090] Example 7

[0091]

[0092] 5-Fluoro-N-(4-methoxy-3-((4-(prop-2-yn-1-oxy)benzyl)oxo)benzyl)-2-morpholinobenzamide

[0093] Intermediate 9 (70 mg, 194.24 μmol), 1-(bromomethyl)-4-(prop-2-yn-1-oxy)benzene (264.67 mg, 1.17 mmol), and anhydrous cesium carbonate (254 mg, 1.17 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a yellow solid with a yield of 71.6%.

[0094] 1 H NMR(300MHz,Chloroform-d)δ10.19(d,J=6.2Hz,1H),7.88(dd,J=9.7,2.7Hz,1 H),7.26(d,J=8.5Hz,2H),7.14–6.98(m,2H),6.86(d,J=1.9Hz,1H),6.86–6.72( m,3H),6.74(s,1H),4.98(s,2H),4.48(d,J=2.4Hz,2H),4.43(d,J=5.5Hz,2H),3 .79(s,3H),3.27(d,J=4.9Hz,3H),2.61(t,J=4.5Hz,4H),2.40(t,J=2.4Hz,1H). 13C NMR(100MHz,Chloroform-d)δ167.54,156.71,152.59,149.94,148.67,147.72,144.63,136.62,13 0.42,129.36,122.53,119.58,117.71,116.37,114.28,109.78,78.21,76.91,71.14,66.41,56.18 52.87,44.48.ESI-MS m / z:505.2[M+H] + .

[0095] Example 8

[0096]

[0097] 5-Fluoro-N-(4-methoxy-3-((3-(prop-2-yn-1-oxy)benzyl)oxo)benzyl)-2-morpholinobenzamide

[0098] Intermediate 9 (70 mg, 194.24 μmol), 1-(bromomethyl)-3-(prop-2-yn-1-oxy)benzene (264.67 mg, 1.17 mmol), and anhydrous cesium carbonate (254 mg, 1.17 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a white solid with a yield of 68.5%.

[0099] 1 H NMR(400MHz,Chloroform-d)δ10.28(t,J=5.4Hz,1H),7.97(dd,J=9.7,2.9Hz,1H),7. 24–7.10(m,3H),7.05(dd,J=7.0,1.5Hz,2H),6.97–6.90(m,2H),6.88(d,J=7.9Hz,1H ),6.83(dt,J=8.2,1.4Hz,1H),5.14(s,2H),4.66(d,J=2.4Hz,2H),4.53(d,J=5.5Hz, 2H), 3.91 (s, 3H), 3.39 (t, J = 4.6Hz, 4H), 2.74 (t, J = 4.6Hz, 4H), 2.51 (t, J = 2.4Hz, 1H). 13C NMR(100MHz,Chloroform-d)δ167.81,160.43,152.85,149.46,148.74,147.58,144.74,136.72,13 0.17,129.63,122.58,119.43,117.52,115.85,114.61,109.42,79.41,76.32,70.42,66.54,55.86 52.37,44.26.ESI-MS m / z:505.2[M+H] + .

[0100] Example 9

[0101]

[0102] 5-Fluoro-N-(4-methoxy-3-((2-(prop-2-yne-1-oxy)benzyl)oxo)benzyl)-2-morpholinobenzamide

[0103] Intermediate 9 (70 mg, 194.24 μmol), 1-(bromomethyl)-2-(prop-2-yn-1-oxy)benzene (264.67 mg, 1.17 mmol), and anhydrous cesium carbonate (254 mg, 1.17 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a pink solid with a yield of 74.1%.

[0104] 1 H NMR(400MHz,Chloroform-d)δ10.28(d,J=6.0Hz,1H),7.98(dd,J=9.7,3.0Hz,1H),7.54–7.47(m,1H),7.27–7.10(m,3H),7.02–6.90(m,4H),6.89(d,J= 8.1Hz,1H),5.23(s,2H),4.76(d,J=2.4Hz,2H),4.53(d,J=5.3Hz,2H),3.92 (s,3H),3.40(t,J=4.5Hz,4H),2.78(t,J=4.6Hz,4H),2.51(t,J=2.4Hz,1H). 13C NMR(100MHz,Chloroform-d)δ167.47,156.74,152.36,149.75,148.45,147.26,144.59,136.21,130.52,129. 47,122.49,119.24,117.48,115.13,114.94,109.43,79.66,76.27,70.28,66.87,55.3252.57,44.51.ESI-MS m / z:505.2[M+H] + .

[0105] Example 10

[0106]

[0107] N-(3-((4-(allyloxy)benzyl)oxo)-4-methoxybenzyl)-5-fluoro-2-morpholinobenzamide

[0108] Intermediate 9 (100 mg, 277.48 μmol), 1-(allyloxy)-4-(bromomethyl)benzene (378.10 mg, 1.66 mmol), and anhydrous cesium carbonate (362.86 mg, 1.66 mmol) were dissolved in acetone and reacted under reflux for 12 hours. The solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to obtain the target product as a pink solid with a yield of 66.4%.

[0109] 1 H NMR(300MHz,Chloroform-d)δ10.28(d,J=6.4Hz,1H),8.28–6.51(m,10H),6.03(ddt,J=17.2,10.5,5.2Hz,1H),5.40(dq,J=17.3,1.7Hz,1H),5.2 9(dt,J=10.5,1.5Hz,1H),5.09(s,2H),4.55(d,J=5.5Hz,2H),4.43(dt,J =5.2,1.6Hz,2H),3.90(s,3H),3.43–3.34(m,4H),2.73(t,J=4.5Hz,4H). 13C NMR(100MHz,Chloroform-d)δ167.82,157.42,152.66,149.34,148.37,147.82,145.53,134.25,13 0.74,128.58,123.65,119.73,118.26,115.58,114.62,111.68,82.58,76.27,72.69,66.34,55.64 52.92,44.51.ESI-MSm / z:507.2[M+H] + .

[0110] Example 11: Tablets

[0111]

[0112] Take the above formula and prepare it into tablets using conventional methods.

[0113] Example 12: Evaluation of antiproliferative activity

[0114] 1. Experimental Methods

[0115] (1) Cells were digested, counted, and prepared to a concentration of 3.5 × 10⁻⁶. 4 Add 100 μl of cell suspension per well to a 96-well plate;

[0116] (2) The 96-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 24 hours.

[0117] (3) Dilute the drug to the required concentration with complete culture medium, add 100 μL of the corresponding drug-containing culture medium to each well, and set up a negative control group at the same time;

[0118] (4) The 96-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 72 hours.

[0119] (5) CCK-8 method:

[0120] ① The 96-well plate was stained with CCK-8, λ=450nm, and the OD value was measured.

[0121] ② Add 10 μL of CCK-8 to each well and continue incubation in an incubator for 2-3 hours;

[0122] ③ Shake gently for 10 minutes to mix; λ=450nm, read the OD value of each well with an ELISA reader, and calculate the inhibition rate.

[0123]

[0124] 2. Experimental Results

[0125] Table 1. IC50 values ​​of some compounds of the present invention against four human cancer cell lines and one human normal cell line for antiproliferation. 50 Value (nM) and IC50 of the compound and parent drug 50 The ratio

[0126]

[0127] As shown in Table 1, some of the bio-orthogonal prodrug compounds of the present invention exhibit significantly reduced cytotoxicity against four cancer cell lines and one normal cell line compared to the parent drug, indicating that these bio-orthogonal prodrug compounds have good safety and fewer toxic side effects during treatment.

[0128] Example 13: Evaluation of the growth inhibitory activity of some bio-orthogonal prodrug compounds released by palladium resin catalysis on K562 cells

[0129] 1. Experimental Methods

[0130] The compounds were prepared into stock solutions according to the relevant requirements, and then diluted to the final concentration for subsequent experiments. Different concentrations of the bio-orthogonal prodrug compounds and different doses of palladium resin were co-incubated with K562 cells in 96-well plates for 72 hours, and the inhibition rate was then measured and calculated using the CCK-8 assay.

[0131] 2. Experimental Results

[0132] Table 2. Experimental results of the growth inhibition of K562 cells by some compounds of the present invention after being catalytically released by palladium resin.

[0133]

[0134] As can be seen from Table 2, the cytotoxicity of some of the bio-orthogonal prodrug compounds of the present invention was effectively restored after co-incubation with palladium resin, indicating that these bio-orthogonal prodrug compounds can effectively inhibit the growth of tumor cells after being activated and released by palladium resin, and play a highly effective and low-toxicity therapeutic role.

[0135] Example 14: Evaluation of the inhibitory activity of some bioorthogonal prodrug compounds on tubulin aggregation before and after release.

[0136] 1. Experimental Methods

[0137] The compounds were prepared as stock solutions according to the corresponding requirements and diluted to the final concentrations for subsequent experiments. Five concentrations were set, with each concentration repeated three times. 2 mg / mL of tubulin (cytoskeleton) was resuspended in PEM buffer [80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl2, and 15% glycerol] and then pre-incubated on ice with the compound or solvent DMSO for 5 minutes. Before detecting the tubulin polymerization reaction, PEG containing GTP was added to a final concentration of 3 mg / mL. The absorbance was measured at 340 nm after 30 minutes using a Berthold LB941 microplate reader. The IC50 of different compounds was calculated using Graphpad by setting up a blank control group. 50 Results are in μM.

[0138] 2. Experimental Results

[0139] Table 3. Experimental results on the inhibition of tubulin aggregation by some compounds of the present invention before and after release catalyzed by palladium resin.

[0140] Example <![CDATA[IC before release 50 > <![CDATA[IC after release 50 > <![CDATA[IC 50(释放前) / IC 50(释放后) ]]> 1 275 2.53 109 2 361 3.03 119 3 251 2.56 98 4 284 2.22 128 5 289 1.87 155 6 301 2.45 123 7 233 2.49 94 8 201 1.88 107 9 249 2.58 97 10 219 2.09 105

[0141] As can be seen from Table 3, some of the bioorthogonal prodrug compounds of the present invention have low inhibitory activity against tubulin aggregation. However, when the compounds are co-incubated with palladium resin, their inhibitory activity against tubulin aggregation is restored, thereby inhibiting cell proliferation and demonstrating good safety.

[0142] Example 15: In vivo safety evaluation of some compounds

[0143] 1. Experimental Methods

[0144] Twenty-one 4-week-old female ICR mice were provided by Shanghai Silex Laboratory Animal Co., Ltd. The animals were randomly divided into three groups of three: a blank control group, a mother drug group, and a prodrug group. The blank control group was injected with DMF:Tween 80:9% saline solution = 10:2:88 (V:V:V). The mother drug group and the prodrug group received intravenous administration of 40 mg / kg, 80 mg / kg, and 160 mg / kg of DMF:Tween 80:9% saline solution = 10:2:88 (V:V:V) via tail vein, respectively. Administration continued for 21 days, and body weight was measured every two days.

[0145] 2. Experimental Results

[0146] Depend on Figure 1 It can be seen that the compounds in the representative embodiments of the present invention do not significantly affect the body weight of mice at high doses, while the parent drug significantly reduces the body weight of mice at high doses. This indicates that the bioorthogonal prodrug compounds of the present invention have superior in vivo safety.

[0147] Example 16: Evaluation of the in vivo antitumor activity of some compounds

[0148] 1. Experimental Methods

[0149] Twenty-four 4-week-old female ICR mice were provided by Shanghai Silex Laboratory Animal Co., Ltd. A suspension of cultured mouse hepatocellular carcinoma cells (H22 cells) at a concentration of 1×10⁻⁶ was collected. 7 The xenograft was administered subcutaneously to the right forelimb axilla of mice at a dose of 0.1 ml per mouse. The diameter of the xenograft was measured using calipers; tumors were considered xenografts when they reached 100 mm in size. 3 Animals were randomly divided into four groups of six each: a blank control group, a palladium resin control group, a mother drug group, and a prodrug + palladium resin group. The blank control group received an injection of DMF:Tween 80:9% saline solution in a ratio of 10:2:88 (V:V:V). Both the palladium resin control group and the prodrug + palladium resin group received intratumoral injection of Pd resin (1 mg in 50 μL of PBS) before administration. The blank control group and palladium resin control group received no administration. The mother drug group and the prodrug + palladium resin group received the drug via tail vein at a dose of 20 mg / kg, with DMF:Tween 80:9% saline solution in a ratio of 10:2:88 (V:V:V), for 21 consecutive days. Mice were sacrificed after 21 days of administration, and tumor masses were surgically removed and weighed. The tumor growth inhibition rate (%) was calculated, and the results were analyzed using SPSS 17.0. Statistical analysis between groups was performed using a t-test, with the calculation formula as follows:

[0150]

[0151] 2. Experimental Results

[0152] Table 4. In vivo antitumor activity of some compounds

[0153]

[0154] As can be seen from Table 4, some of the bioorthogonal prodrug compounds of the present invention can be released in situ at the tumor site, producing a higher concentration of the parent drug. Therefore, their in vivo tumor growth inhibition effect is stronger than that of the parent drug, and the therapeutic effect is better.

Claims

1. A kind N -Benzylbenzamide compounds, characterized in that... It has the structure of Formula I and also contains its pharmaceutically acceptable salt: I， in: R1 is selected from halogens; R2 is selected from methoxy groups; R3 is selected from allyl, propargyl, 1-butyn-3-yl, 3-methyl-1-butyn-3-yl, 1-buten-3-yl, benzyl, 4-propargyloxybenzyl, 3-propargyloxybenzyl, 2-propargyloxybenzyl, and 4-allyloxybenzyl. X is selected from O.

2. As described in claim 1 N -Benzylbenzamide compounds, characterized in that... In the structure: R1 is selected from fluorine; R2 is selected from methoxy groups; R3 is selected from hydrogen, methyl, ethyl, allyl, propyne, 1-butyn-3-yl, 3-methyl-1-butyn-3-yl, 1-buten-3-yl, benzyl, 4-propynoxybenzyl, 3-propynoxybenzyl, 2-propynoxybenzyl, 4-allyloxybenzyl. X is selected from O.

3. As described in claim 1 N -Benzylbenzamide compounds, characterized in that... Compounds selected from any of the following: 。 4. As described in claim 1 N -Benzylbenzamide compounds, characterized in that... The pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the claims 1 to 4. N - Benzylbenzamide compounds and pharmaceutically acceptable carriers.

6. A single claim 1 to 4 N The use of a benzylbenzamide compound or the pharmaceutical composition of claim 5 in the preparation of a drug that inhibits microtubule activity, wherein the drug is an antitumor drug and the tumor is colon cancer, leukemia, liver cancer, or breast cancer.

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