Liver-targeting alkylating agents and uses thereof

Abstract

The application belongs to the technical field of drug research and development, and particularly relates to a cyclophosphamide or ethylene imine prodrug compound, and further discloses the use thereof for preparing a liver-targeting alkylating agent. The compound disclosed in the application is based on traditional nitrogen mustard and derivatives thereof and ethylene imine alkylating agents, and is metabolically activated by a prodrug form through liver enzymes, further enhances the selectivity of the related compounds, reduces the influence on normal cells, and is metabolically activated by cytochrome P450 (CYP450) to release active ingredients, and has a good application prospect in treating liver-related cancers.

Description

Technical Field

[0001] This invention belongs to the field of drug development technology, specifically relating to a cyclophosphamide or ethylene imine prodrug compound, and further disclosing its use in the preparation of liver-targeting alkylating agents. Background Technology

[0002] In recent years, the incidence of malignant tumors has been on the rise, becoming one of the leading causes of death in humans. Chemotherapy is currently a crucial treatment for tumors, and alkylating agents, as an important class of chemotherapeutic drugs, form compounds with active electrophilic groups in the body. These compounds then covalently bind to electron-rich groups (such as amino, thiol, hydroxyl, carboxyl, and phosphate groups) in cellular macromolecules (such as DNA, RNA, and enzymes), causing them to lose activity or break DNA molecules, ultimately leading to tumor cell death. However, many alkylating agents currently in clinical trials suffer from poor selectivity, damaging normal human cells while killing tumor cells. For example, cyclophosphamide and ifosfamide alkylating agents have been reported to cause bone marrow suppression and damage to the gastrointestinal tract and kidneys, limiting the clinical application of traditional alkylating agents.

[0003] Therefore, enhancing the selectivity of alkylating agents and thus reducing their toxic side effects during application has always been a goal of drug developers. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a novel compound and its derivatives, stereoisomers, tautomers, pharmaceutically acceptable salts, amorphous substances, isotopes, polymorphs or solvates.

[0005] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned compound in the preparation of liver-targeting alkylating agents.

[0006] To address the aforementioned problems, the present invention provides a compound of formula (I) or (II) and its derivatives, stereoisomers, tautomers, pharmaceutically acceptable salts, amorphous substances, isotopes, polymorphs, or solvates:

[0007]

[0008] in,

[0009] The A1 and A2 are independently selected from haloalkane groups, or the A1 and A2 form a heterocyclic group with the N group attached to them;

[0010] The R1-R6 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic aryl, or ester group as shown in -R7OCOR8;

[0011] The substitution may be made by alkyl, cycloalkane, heterocycloalkane, or halogen groups.

[0012] It should be noted that the compounds of the present invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. Unless otherwise specified, all stereoisomeric forms of the compounds of the present invention, as well as mixtures thereof, including racemic mixtures, are part of the present invention.

[0013] This invention includes all pharmaceutically acceptable isotopically labeled compounds of formula (I) or (II), wherein one or more atoms are replaced by atoms having the same number of atoms but different atomic masses or mass numbers from those commonly found in nature. Substitution with heavier isotopes such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and may therefore be preferred in certain situations.

[0014] Specifically, the compound is selected from the structures shown in (A)-(D) below:

[0015]

[0016] Specifically, in the compound, R1-R6 are independently selected from H, substituted or unsubstituted C1-C3 alkyl groups, substituted or unsubstituted C6-C8 aromatic groups, substituted or unsubstituted C6-C8 N-heterocyclic aromatic groups, or ester groups represented by -R7OCOR8; wherein R7 and R8 are independently selected from C1-C5 alkyl groups or C3-C5 cycloalkyl groups;

[0017] The substitution may be made by C1-C3 alkyl groups, C3-C5 cycloalkane groups, C3-C5 O heterocyclic alkane groups, or halogens.

[0018] Specifically, in the compound, R1-R6 are independently selected from H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, or ester group represented by -R7OCOR8; wherein R7 and R8 are independently selected from C2-C5 alkyl or C3-C5 cycloalkyl.

[0019] The substitution may be an O-heterocyclic alkane group of C3-C5 or a halogen.

[0020] Specifically, in the aforementioned compound, among R1-R3, there is one and only one that is not H;

[0021] Preferably, R2 is H, and only one of R1 and R3 is not H.

[0022] Specifically, in the aforementioned compound, among R4-R6, there is one and only one H;

[0023] Preferably, R4 is not H, and only one of R5 and R6 is H.

[0024] In the compounds defined in this invention, the term "alkyl" as used refers to the formula -C n H (2n+1) Straight-chain or branched monovalent hydrocarbon groups, non-limiting examples of which include methyl, ethyl, propyl, butyl, 2-methyl-propyl, 1,1-dimethylethyl, pentyl, and hexyl.

[0025] In the compounds defined in this invention, the term "cycloalkyl" as used means a compound of formula -C containing at least three carbon atoms. n H (2n-1) The cyclic monovalent hydrocarbon group, non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0026] In the compounds defined in this invention, the term "alkoxy" as used means an alkyl substituent linked by an oxygen atom, and non-limiting examples include methoxy, ethoxy, propoxy, and butoxy.

[0027] In the compounds defined in this invention, the term halogen refers to F, Cl, Br, and I.

[0028] As used in this application, the term "heterocyclic" refers to alicyclic and aromatic heterocyclic compounds having 4 to 7 carbon atoms, wherein one or more of the cyclomethylene (-CH2-) groups of the alicyclic heterocyclic compound are replaced by groups selected from -O-, -S-, or -N-, wherein the valence of -N- is required to be satisfied by H or by a group acting as a linker; aromatic heterocyclic compounds refer to 5- or 6-membered heterocyclic systems that conform to Hückel's rule and contain 1 to 2 O, S, or N atoms.

[0029] In the compounds defined in this invention, aryl refers to a functional group or substituent derived from a simple aromatic ring (such as a benzene ring, naphthalene ring, etc.) that does not contain heteroatoms.

[0030] Specifically, the compound is selected from one of the following compounds (1)-(15):

[0031]

[0032] Specifically, the compound is selected from one of the following compounds:

[0033]

[0034] Preferably, the compound is selected from one of the following compounds:

[0035]

[0036] More preferably, the compound is selected from one of the following compounds:

[0037]

[0038] This invention also discloses the use of the compounds of formula (I) or (II) and their derivatives, stereoisomers, tautomers, pharmaceutically acceptable salts, amorphous forms, isotopes, polymorphs, or solvates for the preparation of medicaments for treating liver-related cancers. The compounds of this invention can be administered alone or in combination with other drugs, including but not limited to: checkpoint inhibitors, small molecule targeted therapies, and other similar drugs.

[0039] This invention also discloses the use of the compounds of formula (I) or (II) and their derivatives, stereoisomers, tautomers, pharmaceutically acceptable salts, amorphous forms, isotopes, polymorphs, or solvates for the preparation of DNA alkylating agents, particularly liver-targeting alkylating agents. The compounds of this invention can be administered alone or in combination with other drugs, including but not limited to: checkpoint inhibitors, small molecule targeted therapies, and other similar drugs.

[0040] The compounds of this invention, based on traditional nitrogen mustard and its derivatives, as well as ethylene imine alkylating agents, are further enhanced in their selectivity for related compounds and reduced in their impact on normal cells through prodrugation and activation by hepatic drug-metabolizing enzymes. The compounds release their active ingredients through cytochrome P450 (CYP450) metabolic activation and can be used to treat liver-related cancers (such as hepatocellular carcinoma, cholangiocarcinoma, liver metastases, hepatoblastoma, and hepatic angiosarcoma), showing promising application prospects in the treatment of liver-related cancers.

[0041] The DNA alkylating agent of this invention activates the alkylating agent as a prodrug through an enzyme system specifically expressed by tumor cells. By introducing a novel structural compound into a nitrogen mustard molecule, which is specifically metabolically activated by cytochrome P450 (CYP450), the liver targeting of the molecule is enhanced, achieving selectivity of the alkylating agent and effectively reducing the toxicity of this type of drug. The alkylating agent has good application prospects in the treatment of liver-related cancers. Detailed Implementation

[0042] To better illustrate the purpose, technical solution, and advantages of this invention, the following will provide further explanation of this application in conjunction with specific embodiments.

[0043] In the following embodiments of the present invention, the general abbreviations used include: ADP for adenosine diphosphate; ATP for adenosine triphosphate; CDCl3 for deuterated chloroform; CO2Et for ethyl carboxylate; DCM for dichloromethane; DIPEA for N,N-diisopropylethylamine; DMF for dimethylformamide; DMSO for dimethyl sulfoxide; EtOAc for ethyl acetate; H or h or hr for hours; HEPES for 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; KCl for potassium chloride; Min for minutes; MgCl2 for magnesium chloride; NaHCO3 for sodium bicarbonate; Na2SO4 for sodium sulfate; NADH for nicotinamide adenine dinucleotide (reduced form); NAD + Nicotinamide adenine dinucleotide (oxidized form); PEP is phosphoenolpyruvate; RT or rt is room temperature; TCEP is tris(2-carboxyethyl)phosphine; TFA is trifluoroacetic acid; THF is tetrahydrofuran.

[0044] Example 1

[0045] This embodiment is used to prepare compound 1 having the following structure: 2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphanecyclopropane 2-oxide.

[0046]

[0047] It includes two isomers, namely (2S,4R)-2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphanecyclopropane 2-oxide (compound 1a below) and (2R,4R)-2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphanecyclopropane 2-oxide (compound 1b below):

[0048]

[0049] The preparation method of compound 1 (including compound 1a and compound 1b) described in this embodiment includes the following steps:

[0050] (1) Methyl 3-(3-chlorophenyl)-3-oxopropionate

[0051]

[0052] At room temperature, take a 250 mL dry reaction flask, add 100 mL of toluene and sodium hydride (5.17 g, 129.37 mmol), and under nitrogen protection, stir at room temperature for 10 min. Then add 3-chloroacetophenone (5.00 g, 32.34 mmol) and dimethyl carbonate (8.74 g, 97.03 mmol). Heat to 110 °C and react for 3 hours. After the reaction is complete, cool down by 0-10 °C, quench the reaction dropwise with water, add 80 mL of water, adjust the pH to 4-5 with 4 M HCl (aq.), extract with ethyl acetate (100 mL × 2), combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify the residue by silica gel column chromatography (PE:EA = 10:1) to give a yellow oily target product (6.0 g, yield 87%).

[0053] (2) 1-(3-chlorophenyl)propane-1,3-diol

[0054]

[0055] Take a 250 mL reaction flask, add sodium borohydride (4.40 g, 116.6 mmol) and 45 mL methanol, cool to 0-10 °C, take methyl 3-(3-chlorophenyl)-3-oxopropionate (3.10 g, 14.6 mmol) diluted in 10 mL methanol, add it dropwise to the reaction flask, react at room temperature for 20 min after the addition is complete, then heat to 60 °C and react overnight; after the reaction is complete, remove methanol under reduced pressure, add 60 mL water, extract three times with 80 mL × 3 ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 1:1) to obtain a colorless oily liquid target product (2.61 g, yield 96%).

[0056] (3) 4-(3-chlorophenyl)-2,2,8,8-tetramethyl-3,7-dioxo-2,8-disilonane

[0057]

[0058] Take a 50 mL reaction flask, add 1-(3-chlorophenyl)propane-1,3-diol (2.00 g, 10.72 mmol), 10 mL tetrahydrofuran, and hexamethyldisilazane (4.30 g, 26.80 mmol), under nitrogen protection, and add 2-4 drops of trimethylsilyl trifluoromethanesulfonate. React at room temperature for 1 hour, add 50 mL of water, extract once with 100 mL of ethyl acetate, dry the organic phase with anhydrous sodium sulfate, concentrate, and give a colorless oily crude product of the target compound (3.62 g), which was used directly in the next step without further purification.

[0059] (4)(2S,6S,7S,10R)-2-(3-chlorophenyl)-7-isopropyl-10-methyl-1,5-dioxypyrrole[5.5]undecane

[0060]

[0061] Take a 100 mL dry reaction flask, add 3.55 g (10.72 mmol) of 4-(3-chlorophenyl)-2,2,8,8-tetramethyl-3,7-dioxo-2,8-disilonane and 20 mL of dichloromethane, under nitrogen protection, cool to -40 °C (dry ice-acetonitrile bath), add (2S,5R)-2-isopropyl-5-methylcyclohexanone (1.98 g, 12.86 mmol), and add trimethylsilyl trifluoromethanesulfonate (285 mg, 1.29 mmol) dropwise. Maintain the reaction at -20 to -40 °C for 3 hours. Add 6 mL of pyridine to quench the reaction, add 100 mL of petroleum ether and 50 mL of water, separate the layers, dry the organic phase with anhydrous sodium sulfate, concentrate the organic phase, and precipitate by silica gel column chromatography (triethylamine-based silica gel) (PE:DCM = 5:1) to obtain a colorless oily target compound (1.68 g, yield 48%).

[0062] (5)(S)-1-(3-chlorophenyl)propane-1,3-diol

[0063]

[0064] At room temperature, take a 50 mL reaction flask, add (2S,6S,7S,10R)-2-(3-chlorophenyl)-7-isopropyl-10-methyl-1,5-dioxyllorhex[5.5]undecane (1.68 g, 5.20 mmol), 10 mL methanol, and 3 mL concentrated hydrochloric acid, and react overnight at room temperature. Remove methanol by vacuum concentration, add 30 mL water, and extract with ethyl acetate (50 mL × 3). Combine the organic phases, dry to anhydrous sodium sulfate, concentrate the filtrate, and purify by column chromatography (PE:EA = 1:1) to obtain a colorless oily liquid target compound (820 mg, yield 84.4%).

[0065] (6) Di(2-chloroethyl)dichlorophosphamide

[0066]

[0067] Take a 50 mL reaction flask, add bis(2-chloroethyl)amine hydrochloride (3.00 g, 16.81 mmol) and 10 mL of phosphorus oxychloride, heat to 100 °C and react for 40 hours; cool to room temperature, concentrate under reduced pressure to remove phosphorus oxychloride, and obtain a grayish-white solid crude target compound (5.0 g), which was used directly in the next step without further purification.

[0068] (7) (2S,4R)-2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphanecyclopropane 2-oxide (compound 1a) and (2R,4R)-2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphanecyclopropane 2-oxide (compound 1b).

[0069]

[0070] Take a 50 mL dry three-necked reaction flask, and under N2 protection, add (S)-1-(3-chlorophenyl)propane-1,3-diol (300 mg, 1.61 mmol) and 10 mL tetrahydrofuran. Cool to -70 °C, and add n-butyllithium (1.6 mL, 4.02 mmol, 2.5 M) dropwise. The mixture was stirred at low temperature for 20 minutes with bis(2-chloroethyl)dichlorophosphoramide (416 mg, 1.61 mmol) added, and reacted at -70 °C for 2 hours. Then, it was allowed to warm to room temperature overnight. The reaction was quenched with 10 mL of saturated ammonium chloride aqueous solution, and 30 mL of water was added. The mixture was extracted three times with 50 mL × 3 ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE:EA = 5:1-1:1) to obtain (2S,4R)-2-(bis(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphane propane 2-oxide (compound 1a, 80 mg, 13%) and (2R,4R)-2-(bis(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphane propane 2-oxide (compound 1b, 90 mg, 15%).

[0071] The characterization data of the (2S,4R)-2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphazenecyclopropane 2-oxide (compound 1a) prepared above are as follows:

[0072] 1 H NMR (500MHz, CDCl3) δ7.44 (s, 1H), 7.41–7.30 (m, 3H), 5.44 (ddd, J = 10.3, 6.8, 3.2Hz, 1H), 4.56–4. 37(m,2H),3.75–3.56(m,4H),3.45(dt,J=14.1,7.1Hz,4H),2.62–2.48(m,1H),2.18–2.10(m,1H);

[0073] MS(ESI,pos.ion)m / z:371.9[M+H] + ;

[0074] The characterization data of the (2R,4R)-2-(di(2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphazenecyclopropane 2-oxide (compound 1b) prepared above are as follows:

[0075] 1 H NMR (500MHz, CDCl3) δ7.37–7.30(m,3H),7.24–7.18(m,1H),5.65(d,J=11.1Hz,1H),4.73(t,J=12.0Hz,1H),4.40(dddd,J=23 .5,11.5,4.7,1.4Hz,1H),3.64(t,J=6.9Hz,4H),3.57–3.42(m,4H),2.20(dtd,J=16.9,12.4,4.8Hz,1H),2.05–1.97(m,1H);

[0076] MS(ESI,pos.ion)m / z:371.9[M+H] + .

[0077] The above identification results prove that the structure of the synthesized product in this embodiment is correct.

[0078] Example 2

[0079] This embodiment is used to prepare compound 11 having the following structure: (1,3-dioxolane-2-yl)ethane-(bis(2-chloroethyl)amino)(phenoxy)phosphoryl)-(S)-1-amine.

[0080]

[0081] The preparation method of compound 11 described in this embodiment includes the following steps:

[0082]

[0083] (1) Benzyl(S)-(1-oxopropan-2-yl)carbamate

[0084]

[0085] Take a 50 mL dry three-necked reaction flask, add benzyl (S)-(1-hydroxypropane-2-yl)carbamate (1.00 g, 4.78 mmol), add 15 mL dichloromethane, under nitrogen protection, cool to 0-5 °C, add Dys-Martin reagent (3.04 g, 7.17 mmol), allow to return to room temperature, and react at room temperature for 4 hours; after the reaction is complete, add 10 mL saturated sodium thiosulfate solution to quench the reaction, add 25 mL water, separate the layers, extract three times with 30 mL × 3 ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate the organic phase, and perform silica gel column chromatography (PE:EA = 2:1) to obtain a colorless oily liquid target compound (780 mg, yield 78%).

[0086] The MS (ESI, pos.ion) m / z of the product prepared in this step was determined to be 208.1 [M+H]. + .

[0087] (2) Benzyl(S)-(1-(1,3-dioxolane-2-yl)ethyl)carbamate

[0088]

[0089] Take a 100 mL reaction flask, add benzyl (S)-(1-oxopropane-2-yl)carbamate (700 mg, 3.38 mmol), 10 mL toluene, 1 mL ethylene glycol, and p-toluenesulfonic acid (116 mg, 0.68 mmol), and heat to 90 °C overnight. After the reaction is complete, concentrate under reduced pressure and perform silica gel column chromatography (PE:EA = 3:1) to obtain a colorless oily liquid of the target compound (520 mg, yield 61%).

[0090] The MS (ESI, pos.ion) m / z of the product prepared in this step was determined to be 252.1 [M+H]. + .

[0091] (3)(S)-1-(1,3-dioxapentan-2-yl)ethane-1-amine

[0092]

[0093] Take a 50 mL reaction flask, add benzyl(S)-(1-(1,3-dioxolane-2-yl)ethyl)carbamate (520 mg, 2.07 mmol), 5 mL methanol, and 100 mg 5% Pd / C. Replace the air three times with hydrogen using a hydrogen balloon, and heat to 50 °C overnight. After the reaction is complete, filter through a diatomaceous earth liner, wash the filter cake with a small amount of methanol, and concentrate under reduced pressure to obtain a colorless oily liquid of the target compound (226 mg, crude product yield 93%). Note that the product in this step is easily swept away during concentration under reduced pressure.

[0094] The MS (ESI, pos.ion) m / z of the product prepared in this step was determined to be 118.1 [M+H]. + .

[0095] (4) (1,3-Dioxolane-2-yl)ethane-(bis(2-chloroethyl)amino)(phenoxy)phosphoryl)-(S)-1-amine

[0096]

[0097] Take a 100 mL dry three-necked reaction flask, protect it with N2, add di(2-chloroethyl)dichlorophosphamide (1100 mg, 4.25 mmol), 10 mL dichloromethane, and triethylamine (860 mg, 8.50 mmol), cool to -60 °C, add phenol (400 mg, 4.25 mmol), and stir the reaction at -60 °C for 0.5 hours; then add (S)-1-(1,3-dioxapentan-2-yl)ethane-1-amine (498 mg, 4.25 mmol) and triethylamine (430 mg, 4.25 mmol) to the reaction system, and react the resulting mixture at -60 °C for 1 hour, then allow it to return to room temperature overnight. Add water (100 mL) to the reaction solution, extract with dichloromethane (80 mL × 3), combine the organic phases, dry with anhydrous sodium sulfate, concentrate the organic phase, and perform silica gel column chromatography (PE:EA = 6:1-3:1) to give the title compound as a white solid (180 mg, 11%).

[0098] The characterization data of the products obtained in the above preparation are as follows:

[0099] MS(ESI,pos.ion)m / z:397.0[M+H] + ;

[0100] 1 H NMR (500MHz, CDCl3) δ7.20(t,J=7.7Hz,2H),7.10(ddd,J=7.5,4.6,1.0Hz,2H),7.02(td,J=7.2,1.1Hz,1H),4.70(ddd,J= 14.0, 2.3, 0.9Hz, 1H), 3.94–3.70 (m, 4H), 3.55–3.27 (m, 9H), 2.76 (dt, J=39.5, 10.5Hz, 1H), 1.07 (dd, J=11.1, 6.8Hz, 3H).

[0101] The above identification results prove that the structure of the synthesized product in this embodiment is correct.

[0102] Example 3

[0103] This embodiment is used to prepare compound 6 having the following structure: (4S)-2-(azacyclopropane-1-yl)-4-(3-chlorophenyl)-1,3,2-dioxophosphacyclopropane 2-oxide.

[0104]

[0105] The preparation method of compound 6 in this embodiment includes the following steps:

[0106] (1)(4S)-2-((2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphine-2-oxide

[0107]

[0108] Take a 100 mL dry three-necked reaction flask, add (S)-1-(3-chlorophenyl)propane-1,3-diol (500 mg, 2.68 mmol), 10 mL dichloromethane, and triethylamine (1356 mg, 13.40 mmol). Under N2 protection, cool to 0-5 °C, add phosphorus oxychloride (452 ​​mg, 2.95 mmol), and react at low temperature for 20 minutes. Then add 2-chloroethane-1-amine hydrochloride (342 mg, 2.95 mmol), react at low temperature for 30 minutes, and then naturally warm to room temperature for 2 hours. Add 30 mL of water, extract three times with 20 mL × 3 dichloromethane, combine the organic phases, dry to anhydrous sodium sulfate, concentrate the organic phase, and perform silica gel column chromatography (PE:EA = 1:2) to obtain a colorless oily liquid of the target compound (340 mg, yield 40.9%).

[0109] The MS (ESI, pos.ion) m / z of the product prepared in this step was determined to be 309.9 [M+H]. + .

[0110] (2)(4S)-2-(azacyclopropane-1-yl)-4-(3-chlorophenyl)-1,3,2-dioxophosphazene 2-oxide

[0111]

[0112] Take a 100 mL dry three-necked reaction flask, add (4S)-2-((2-chloroethyl)amino)-4-(3-chlorophenyl)-1,3,2-dioxophosphine 2-oxide (340 mg, 1.10 mmol), 20 mL toluene, tetrabutylammonium iodide (81 mg, 0.22 mmol), sodium hydroxide (44 mg, 1.10 mmol), and potassium carbonate (303 mg, 2.19 mmol), and react at room temperature for 3 hours; collect the reaction product and filter it, wash the filter cake with 20 mL toluene, take the filtrate, add 40 mL of water, separate the liquid, extract the aqueous phase with dichloromethane (30 mL × 3), combine the organic phases, dry with anhydrous sodium sulfate, concentrate the organic phase, and perform silica gel column chromatography (DCM:MeOH = 30:1) to obtain a colorless oily liquid target compound (70 mg, yield 23.3%).

[0113] The characterization data of the products obtained in the above preparation are as follows:

[0114] MS(ESI,pos.ion)m / z:273.9[M+H] + ;

[0115] 1 H NMR(500MHz, CDCl3)δ7.35(s,1H),7.28–7.18(m,3H),5.59(dd,J=11.7,1.9Hz,1H),4.72–4.59(m, 1H), 4.39 (dddd, J=22.2, 11.2, 4.7, 1.6Hz, 1H), 2.26–2.18 (m, 3H), 2.17 (s, 2H), 1.95–1.88 (m, 1H).

[0116] The above identification results prove that the structure of the synthesized product in this embodiment is correct.

[0117] Example 4

[0118] This embodiment is used to prepare compound 10 having the following structure: ((bis(2-chloroethyl)amino)(phenoxy)phosphoryl)-L-propionic acid isopropyl ester.

[0119]

[0120] The preparation method of compound 10 described in this embodiment includes the following steps:

[0121]

[0122] (1) Bis(2-chloroethyl)dichlorophosphamide

[0123]

[0124] At room temperature, take a 100 mL dry reaction flask, add bis(2-chloroethyl)amine hydrochloride (0.50 g, 2.82 mmol), under nitrogen protection, add phosphorus oxychloride (10 mL), heat and reflux with stirring for 5 hours; after the reaction is complete, cool to room temperature, and directly evaporate to dryness to obtain a white solid crude product (0.47 g), which was used directly in the next step without further purification.

[0125] (2)((bis(2-chloroethyl)amino)(phenoxy)phosphoryl)-L-propionic acid isopropyl ester

[0126]

[0127] Take a 25 mL reaction flask, add bis(2-chloroethyl)dichlorophosphamide (200.0 mg, 0.77 mmol) and 5 mL tetrahydrofuran, add triethylamine (155.4 mg, 1.54 mmol) and phenol (79.8 mg, 0.84 mmol) with stirring at room temperature, and react at room temperature for 1.0 h. Then add L-alanine isopropyl ester (110.0 mg, 0.84 mmol), and stir the reaction at room temperature for another 2.0 h. Add 50 mL of water to the reaction solution, extract with ethyl acetate (40 mL × 3), combine the organic phases, dry with anhydrous sodium sulfate, concentrate the organic phase, and perform silica gel column chromatography (PE:EA = 1:1) to give a white solid target compound (36.0 mg, yield 11.3%).

[0128] The characterization data of the products obtained in the above preparation are as follows:

[0129] MS(ESI,pos.ion)m / z:411.0[M+H] + ;

[0130] 1 H NMR(500MHz, CDCl3)δ7.38-7.41(m,2H),7.15–7.18(m,3H),4.49–4.32(m, 1H),3.56-3.30(m,5H),2.80-2.76(m,4H),1.21(s,3H),1.20-1.09(m,6H).

[0131] The above identification results prove that the structure of the synthesized product in this embodiment is correct.

[0132] Experimental Example

[0133] 1. Inhibitory effect of the compound on the proliferation of mouse primary hepatocytes

[0134] After anesthetizing C57BL / 6 mice, the abdomen was opened, the thoracic cavity was cut open, the superior vena cava was clamped, the inferior vena cava was separated, and an indwelling venous catheter was inserted into the renal branch of the inferior vena cava. 25 mL of perfusion solution 1 (137 mM NaCl, 5.4 mM KCl, 0.64 mM NaH2PO4·H2O, 0.85 mM Na2HPO4, 9.9 mM HEPES, 4.2 mM NaHCO3, 0.5 mM EGTA, 5 mM M glucose, pH 7.4) was slowly infused, preheated to 42°C. At the same time, the portal vein was cut to bleed the mice. Replace the preheated (42℃) perfusion buffer 2 containing 0.5 mg / mL type IV collagenase, 1% DNase I (1 mg / mL) (137 mM NaCl, 5.4 mM KCl, 0.64 mM NaH2PO4·H2O, 0.85 mM Na2HPO4, 9.9 mM HEPES, 4.2 mM NaHCO3, 3.8 mM CaCl2·2H2O, pH 7.4) and continue perfusion for another 25 mL. After perfusion, remove the liver and place it in a culture dish containing 10 mL of preheated (42℃) Buffer 1 (142 mM NaCl, 6.7 mM KCl, 10 mM HEPES, 0.23 mM BSA, pH 7.4), and transfer to a clean bench.

[0135] The liver capsule was punctured using sterile pointed forceps to obtain scattered hepatocytes. The cell suspension was passed through a 200 μm cell sieve (30 mL of Buffer 1 was used to rinse the sieve). The suspension was collected and centrifuged at 100 g for 3 min at 4 °C, and the cell pellet was collected. 40 mL of Buffer 2 (137 mM NaCl, 4.7 mM KCl, 0.66 mM MgSO4, 1.6 mM CaCl2, 2.4 mM HEPES, 0.23 mM BSA, pH 7.4) was added for resuspending. The pellet was centrifuged at 100 g for 3 min at 4 °C, and the cell pellet was collected. 5 mL of the above Buffer 2 was added for resuspending. The cell suspension was slowly added to a 15 mL centrifuge tube containing 5 mL of 75% Percoll to cover the cells. The tube was centrifuged at 300 rpm for 15 min at 4 °C, and the stop was adjusted to 1.

[0136] Collect the product, discard the supernatant, and take the intermediate layer cells. Resuspend the cells in 2 mL of DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin DMEM), count the cells, and calculate cell purity using 0.4% trypan blue. Adjust the cell density to 102. 5The concentration of each compound was seeded at 100 μL per well in a 96-well plate and incubated overnight at 37°C in a 5% CO2 cell culture incubator. The 96-well plate was then removed, the supernatant was discarded, and 100 μL of each of eight gradient concentrations of test compound / positive compound / negative control working solution was added sequentially, with two replicates per concentration. The plates were then incubated for 24 hours. The 96-well plate was then removed, the supernatant was discarded, and 100 μL of DMEM medium containing 10% CCK-8 was added to each well. After incubation for 1 hour, the absorbance (A) of each well was measured at 450 nm using a microplate reader.

[0137] The cell proliferation inhibition rate of each compound was calculated based on the measured A value. The cell proliferation inhibition rate (IR) = (1 - experimental group (Ai) value / control group (Ao) value) × 100%. The data processing software calculated the IC50 of each compound at 24 h as shown in Table 1 below. 50 The values ​​are shown in Table 1 below. For details of the operation process, please refer to Ref. Mederacke I, Dapito DH, Affò S, et al. High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers[J]. Nat Protoc, 2015, 10(2): 305-315.

[0138] Table 1. Inhibitory effects of some compounds of the present invention on the proliferation of mouse primary hepatocytes.

[0139] compound <![CDATA[IC 50 (μM)]]> In Example 1, 1a 0.86 Example 1b 6.58 Example 2 1.62 Example 3 7.55 Example 4 3.83 Cisplatin 14.72 Cyclophosphamide 5781

[0140] It is evident that the alkylating agent with the structure described in this invention has an inhibitory effect on the proliferation of primary mouse hepatocytes.

[0141] 2. Inhibitory effect of the compound on the proliferation of HepG2 and HEK293 cells

[0142] Collect cells in the logarithmic growth phase (HepG2, HEK293). When the cells reach 85%-95% confluence, digest and collect the cells using standard passage methods, count the cells, and adjust the cell concentration to 10⁻⁶. 5Cells were seeded at a density of 100 μL / mL in 96-well plates, with 100 μL of cell suspension added to each well. The plates were incubated overnight at 37°C in a 5% CO2 incubator. After removing the plates and discarding the supernatant, 100 μL of each of the eight gradient concentrations of test compound / positive compound / negative control working solution was added to each well, with two replicates per concentration. The plates were then incubated for 24 hours. After removing the plates and discarding the supernatant, 100 μL of DMEM medium containing 10% CCK-8 was added to each well. The plates were incubated for 1 hour, and the absorbance (A) of each well was measured at 450 nm using a microplate reader. The cell proliferation inhibition rate (IR) of each compound was calculated based on the measured A values. The inhibition ratio (IR) was calculated as (1 - experimental group (Ai) value / control group (Ao) value) × 100%. The IC50 of each compound at 24 h was calculated using data processing software. 50 Values, specific compounds and results are shown in Table 2 below.

[0143] Table 2. Inhibitory effects of some compounds of the present invention on the proliferation of HepG2 and HEK293 cells.

[0144]

[0145]

[0146] As can be seen from the above data, the compound of the present invention can effectively inhibit cell growth in cells rich in cytochrome P450 enzyme system, with better effects than cisplatin, while having less impact on cells with low cytochrome P450 enzyme system content, thus exhibiting good selectivity and showing great promise for therapeutic applications.

[0147] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A compound of formula: ###0001### and tautomers, pharmaceutically acceptable salts, amorphous forms thereof; said compound being selected from one of the following compounds: ###0002### ###0003### 。 2. The compound of claim 1, wherein said compound being selected from one of the following compounds: ###0004### 。 3. Use of a compound according to any one of claims 1-2, and tautomers, pharmaceutically acceptable salts, amorphous forms thereof, for the manufacture of a liver-targeted alkylating agent.

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