CN112225731A - FAP alpha specific recognition methylene blue derivative with tumor diagnosis and treatment functions and preparation method and application thereof - Google Patents

FAP alpha specific recognition methylene blue derivative with tumor diagnosis and treatment functions and preparation method and application thereof Download PDF

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CN112225731A
CN112225731A CN201910582379.4A CN201910582379A CN112225731A CN 112225731 A CN112225731 A CN 112225731A CN 201910582379 A CN201910582379 A CN 201910582379A CN 112225731 A CN112225731 A CN 112225731A
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pharmaceutically acceptable
dimethylamino
benzyl
bis
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赵春顺
徐从军
罗勇
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Sun Yat Sen University
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Abstract

The invention relates to a methylene blue derivative specifically identified by FAP alpha shown in a general formula I, and pharmaceutically acceptable salts, hydrates or prodrugs thereof and a preparation method thereof. Wherein the groups X and R have the meanings given in the description. The invention also relates to compounds of the general formula I and pharmaceutically acceptable salts, hydrates or prodrugs thereofThe application in FAP alpha high expression solid tumor diagnosis and photodynamic therapy such as breast cancer, in particular to the application in preparing medicines for diagnosing and treating cancer.

Description

FAP alpha specific recognition methylene blue derivative with tumor diagnosis and treatment functions and preparation method and application thereof
Invention book abstract
The invention relates to a methylene blue derivative specifically identified by FAP alpha shown in a general formula I, and pharmaceutically acceptable salts, hydrates or prodrugs thereof and a preparation method thereof. Wherein the groups X and R have the meanings given in the description. The invention also relates to application of the compound shown in the general formula I and pharmaceutically acceptable salts, hydrates or prodrugs thereof in diagnosis and photodynamic therapy of FAP alpha high-expression solid tumors such as breast cancer, in particular to application in preparing medicines for diagnosing and treating cancers.
Figure BSA0000185274170000011
FAP alpha specific recognition methylene blue derivative with tumor diagnosis and treatment functions and preparation method and application thereof
Technical Field
The invention relates to a novel FAP alpha specific recognition compound containing a methylene blue structure and pharmaceutically acceptable salts, hydrates or prodrugs thereof, a preparation method thereof and a pharmaceutical composition containing the compound. The invention also relates to a tumor diagnosis effect of the compound and a stronger photodynamic therapy effect of a tumor part, and also relates to application of the compound and pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof in preparing medicaments for diagnosing and treating solid tumors with high expression of FAP alpha, in particular to application in preparing medicaments for diagnosing and treating solid tumors with high expression of FAP alpha.
Background
The medical problem of cancer in the global scope is a serious refractory disease threatening the life and health of human beings, the death rate of the disease is even higher than that of cardiovascular and cerebrovascular diseases, and the disease is the first of all. At present, the defects of poor sensitivity and the like still exist in tumor diagnosis, the problem of poor targeting or poor characteristics also exists in common tumor treatment medicines, and meanwhile, a real-time effective treatment and detection means is also lacked in tumor treatment. Therefore, it is necessary to establish convenient, sensitive and accurate tumor diagnosis and treatment methods.
Methylene Blue (MB) has attracted considerable attention as an organic near-infrared fluorescent dye for use in fluorescent diagnosis of tumors. Furthermore, methylene blue is also a phenothiazine photosensitizer and has been used clinically in photodynamic therapy. However, the clinical application of methylene blue is limited by the defects of poor affinity to tumor, short retention time of tumor tissues and the like.
Figure BSA0000185274170000021
Fibroblast Activation Protein (FAP) is a membrane serine protease expressed by activated tumor-associated fibroblasts, plays an important role in degradation and reconstruction of tumor-host interface matrix, participates in growth, infiltration and metastasis of tumors, and is hardly expressed in normal tissues. The FAP comprises two subunits of FAP alpha and FAP beta, wherein the FAP alpha has collagenase activity, dipeptide kinase activity and endopeptidase activity. FAP α has gradually evolved as an effective anti-tumor target due to its ability to specifically recognize and hydrolyze peptide bonds formed between N-terminally blocked glycylproline and other amino acids or small molecule amino groups.
According to literature reports and the basis of previous work, the inventor finds that when nitrogen atoms in mother nucleus of methylene blue phenothiazine have other substituents, fluorescence is weakened and photodynamic activity is lost. Therefore, the inventor designs and synthesizes a series of novel small molecules for diagnosing and treating the tumor stroma by carrying out tumor FAP alpha targeted fracture modification on the parent nuclear nitrogen atom based on methylene blue as a medicine molecule for diagnosing and photodynamic treating. The molecule has no fluorescence property and photodynamic activity, and the targeted tumor part in the organism is sheared by FAP alpha to expose amino and then is self-cracked to form methylene blue fluorescence to recover and exert photodynamic activity. In vitro activity screening shows that the compounds have better tumor targeting property and diagnosis and treatment effect.
Disclosure of Invention
The invention mainly aims to provide a novel compound which can be used for diagnosing and performing photodynamic therapy on a structure shown in a formula I by specifically targeting tumor FAP alpha, in particular relates to a preparation method of the compound, a composition comprising the compound and isomers thereof, and application of the compound, pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof in preparing medicines for diagnosing and treating tumors with high expression of FAP alpha.
The invention relates to a compound of a general formula I, an optical isomer and pharmaceutically acceptable salt, hydrate or prodrug thereof,
Figure BSA0000185274170000031
wherein the content of the first and second substances,
x is optionally selected from O, NH and S;
y is methylene or
Figure BSA0000185274170000032
R is hydrogen,
Figure BSA0000185274170000033
R1And R2Are each independently selected from C1-C10Alkyl radical, C3-C7Cycloalkyl radical, C3-C7Heterocycloalkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl, C6-C10Aryl, 5-to 10-membered heteroaryl, fused ring aryl, C6-C10Heterocyclyl, C optionally substituted with one or more hydroxy, amino, halogen, cyano, nitro, sulfonyl, aromatic, heteroaromatic and fused ring aryl groups1-C6An alkyl group;
Figure BSA0000185274170000037
represents a substituent attachment;
the invention preferably relates to the compounds of the general formula I, optical isomers and pharmaceutically acceptable salts, hydrates or prodrugs thereof, wherein,
wherein the content of the first and second substances,
x is optionally selected from O and NH;
y is methylene or
Figure BSA0000185274170000034
R is hydrogen,
Figure BSA0000185274170000035
R1And R2Are each independently selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C3-C5Cycloalkyl radical, C3-C7Heterocycloalkyl radical, C2-C8Alkenyl radical, C2-C4Alkynyl, C6-C10Aryl, 5-to 10-membered heteroaryl, fused ring aryl, C6-C10Heterocyclyl, C optionally substituted with one or more hydroxy, amino, halogen, cyano, nitro, aromatic, heteroaromatic or fused ring aryl groups1-C6An alkyl group;
Figure BSA0000185274170000038
represents a substituent attachment;
the invention particularly preferably relates to a compound of a general formula I, an optical isomer and a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein,
x is optionally selected from O and NH;
y is methylene or
Figure BSA0000185274170000036
R is hydrogen,
Figure BSA0000185274170000041
R1And R2Are each independently selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C2-C8Alkenyl radical, C2-C4Alkynyl, C6-C10Aryl, 5-10 membered heteroaryl, fused ring aryl, C optionally substituted with one or more hydroxy, amino, halogen, cyano, nitro, aromatic ring, aromatic heterocycle or fused ring aryl1-C6An alkyl group;
Figure BSA0000185274170000042
represents a substituent attachment;
the invention also relates to a compound, an optical isomer and a pharmaceutically acceptable salt, hydrate or prodrug thereof, which are shown in the general formula I,
x is optionally selected from O;
y is
Figure BSA0000185274170000043
R is hydrogen,
Figure BSA0000185274170000044
R1And R2Are each independently selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C2-C6Alkenyl radical, C2-C4Alkynyl, phenyl, pyridine, indole, thiophene, quinoline, C optionally substituted with one or more amino, halogen, benzene ring, pyridine ring, quinoline, indole ring1-C6An alkyl group;
the compounds of the general formula I, optical isomers and pharmaceutically acceptable salts, hydrates or prodrugs thereof according to the present invention are preferably the following compounds but these compounds are not meant to limit the present invention in any way:
[4- (glycylprolamido) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- (N-acetylglucosaminyl prolyl) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- (N-propionylglycylprolylamino) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (2-methacryloyl) glycylprolylamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazin-10-carboxylic acid ester;
[4- [ N- (2-dimethylaminoacetyl) glycylprolamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- (phenylalanyl glycyl prolyl amido) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (1H-indole-2-propionyl) glycylprolylamino ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (tert-butoxycarbonyl) glycylprolylamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (benzyloxycarbonyl) glycylprolylamino ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazin-10-carboxylic acid ester;
[4- [ N- [1- (pyridin-4-yl) methoxycarbonyl ] glycylprolamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazin-10-carboxylic acid ester;
a pharmaceutical composition comprising a compound of any one of claims 1 to 5, an optical isomer, and a pharmaceutically acceptable salt, solvate or prodrug thereof as an active ingredient together with a pharmaceutically acceptable excipient.
Furthermore, the compounds of formula I and the optical isomers of the present invention may form pharmaceutically acceptable salts thereof with acids according to conventional methods in the art to which the present invention pertains. The acid may include inorganic or organic acids, and salts with the following acids are particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, tartaric acid, benzenesulfonic acid, benzoic acid, p-toluenesulfonic acid, and the like.
The term "optical isomer" used in the present invention includes R-type isomer and S-type isomer. The anti-tumor activity of the compound of the general formula I is obtained by testing a racemate thereof.
The invention also relates to a compound of the general formula I and an optical isomer thereof, which have strong diagnosis and treatment effects on FAP alpha targeted tumors, and also relates to applications of the compound and pharmaceutically acceptable salts and hydrates thereof in preparation of diagnosis and treatment of FAP alpha high-expression tumors.
The following schemes 1-4 describe the preparation of compounds of formula I of the present invention, all starting materials are prepared by the methods described in the schemes, by methods well known to those of ordinary skill in the art of organic chemistry or are commercially available. All of the final compounds of the present invention are prepared by the methods described in the synthetic schemes or by methods analogous thereto, which are well known to those of ordinary skill in the art of organic chemistry. All of the variables used in the synthetic routes are as defined below or in the claims.
The derivatives of formula I according to the present invention can be obtained from methylene blue by reduction, amidation, acylation, hydrolysis and acylation according to the method of scheme 1.
Figure BSA0000185274170000051
Drawings
FIG. 1 shows the fluorescence intensity change of different target compounds after incubation with tumor homogenates highly expressing FAP alpha.
Detailed Description
The examples are intended to illustrate, but not to limit, the scope of the invention. NMR of the compounds was measured using BrukeraRx-300 and Mass Spectroscopy was measured using Agilent 100 LC/MSD; all reagents used were analytically or chemically pure.
The general preparation method comprises the following steps:
step A3, 7-dimethylamino-10H-phenothiazine-10-carbonyl chloride blue (III)
Dissolving methylene blue in a mixed solvent of 10mL of water and 30mL of dichloromethane at room temperature, adding sodium carbonate, stirring for 5min at 40 ℃, dissolving sodium dithionite in 15mL of water, adding the reaction solution, stirring for 1h at 40 ℃, changing the solution from blue to brown yellow, showing that the reduction reaction is finished, monitoring by TLC to obtain an intermediate II, carrying out unpurified reaction, cooling the reaction solution to an ice bath at 0 ℃, slowly adding 10mL of triphosgene solution in dichloromethane to the ice bath, dropwise adding the triphosgene solution to the reaction solution under stirring of the ice bath, maintaining the ice bath condition, continuously stirring for 2h, carrying out the reaction, pouring the reaction solution into 100mL of ice water to quench the reaction solution, carrying out suction filtration on kieselguhr, washing a filter cake by dichloromethane, collecting a filtrate, placing the filtrate in a separating funnel, extracting DCM (50mL of 3), washing by saturated saline (60 x 3), drying by anhydrous sodium sulfate, concentrating, purifying by column chromatography, a white solid was obtained.
Step B N-Boc-glycylproline derivative (IV)
Dissolving BOC-glycine-L-proline in DMF10mL at room temperature, adding HATU and DIPEA, placing the reaction solution in an ice bath condition, slowly dropwise adding 5mL of corresponding amine DMF solution into the reaction solution at 0 ℃, maintaining the ice bath and stirring for 30min, continuously stirring for 4h at room temperature, wherein the solution is brown, after the reaction is finished, rotationally evaporating the solvent, adding 20mL of water into the residue, extracting with dichloromethane (50mL of 3), washing with saturated saline (50mL of 3), drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain a yellow solid.
Step C Synthesis of intermediate V
Compound III is dissolved in 10ml of dichloromethane at room temperature, DMAP and Na are added2CO3Placing the reaction solution in an ice bath condition at 0 ℃ for 10min, dissolving a compound IV in 5mL of dichloromethane, dropwise adding the mixture into the reaction solution under stirring in the ice bath, reacting at room temperature overnight, and monitoring by TLC after the reaction is finished. And (4) carrying out suction filtration on the reaction solution, filtering out insoluble substances, evaporating the filtrate to dryness, and carrying out column chromatography purification to obtain a blue solid.
Step D Synthesis of intermediate VI
At room temperature, compound V was dissolved in DCM, TFA was added dropwise in an ice bath, and after addition, the reaction was carried out at room temperature for 2h, TLC monitored for completion, pH was adjusted to 12 with 5% NaOH solution, the organic layer was separated, washed with saturated brine (50ml × 3), dried over anhydrous sodium sulfate, and evaporated to dryness to give a blue solid.
Step E Synthesis of the target product I
The method comprises the following steps: and (3) dissolving the compound VI in 10mL of dichloromethane at room temperature, adding corresponding anhydride and DMAP, continuously stirring at room temperature overnight, monitoring by TLC (thin layer chromatography) after the reaction is finished, concentrating the reaction liquid, and purifying by column chromatography to obtain a light blue solid.
The second method comprises the following steps: at room temperature, compound VI is dissolved in 10mL of dichloromethane and the corresponding acid chloride (or
Figure BSA0000185274170000061
) And DIPEA, stirring at room temperature overnight, monitoring by TLC, concentrating the reaction liquid, and purifying by column chromatography to obtain the target compound.
The compound of example 1 (see table one) was prepared separately according to the general procedure.
Table one:
Figure BSA0000185274170000062
Figure BSA0000185274170000071
Figure BSA0000185274170000081
specific FAP alpha targeting and fluorescence recovery (diagnostic) experimental screening was performed on the compounds of formula I according to the invention.
(1) Tissue homogenate buffer (50mM tris-HCl, 100mM NaCl): 3.03g Tris base, 2.92g NaCI were weighed out in ddH2And (4) metering the volume of the O to a 500mL volumetric flask, adjusting the pH value to 7.4 by using hydrochloric acid, and storing at a low temperature of 4 ℃.
(2) Preparing FAP-MB-1-10 prodrug stock solution: accurately weighing a proper amount of prodrug, dissolving the prodrug in a proper amount of DMSO to prepare a prodrug stock solution with the concentration of 1mM, and storing at a low temperature of 4 ℃.
After the FAP alpha tumor with high expression grows to a proper size (100-3(tumor volume)), killing tumor-bearing mice, dissecting out tumor tissues, placing the tumor-bearing mice in a culture dish, shearing the tissues as much as possible under the ice bath condition, pouring the tissues into a homogenizer, adding 800uL of tissue homogenization buffer solution into every 200mg of tissues, fully grinding in ice bath until no tissue blocks can be seen by naked eyes, centrifuging at 3500rpm for 15min at low temperature, centrifuging to obtain upper-layer tissue homogenate, adding 400uL of tumor homogenate into every 1.5mL of EP tube, simultaneously adding 400uL of blank buffer solution into every tube of a negative control group, adding 3 uL of FAP-MB-1-10 prodrug stock solution into every tube, placing the EP tube seal in a constant temperature shaking table at 37 ℃ for incubating for 12h at 200rpm, adding 1mL of acetonitrile into each tube to precipitate protein, vortexing to the upper and lower layers, centrifuging at 10000rpm for 15min, taking supernatant, adding 0.45um of organic filter membrane insoluble substances after centrifugation, filtering out of the obtained solution, adding 1mL of acetonitrile, preserving in a dark place, to be subjected to fluorescence spectrum scanningAnd (6) testing.
Fluorescence spectrum scanning experiment:
the fixed excitation wavelength λ ex is 633 nm; the emission wavelength lambda em is 650-720 nm; the slit width was 5 scans of each prodrug incubated tumor homogenate and blank buffer. The fluorescence curve and the fluorescence value for each compound were recorded and compared.

Claims (8)

1. A compound of general formula I, optical isomer and pharmaceutically acceptable salt, hydrate or prodrug thereof,
Figure FSA0000185274160000011
wherein the content of the first and second substances,
x is optionally selected from O, NH and S;
y is methylene or
Figure FSA0000185274160000012
R is hydrogen,
Figure FSA0000185274160000013
R1And R2Are each independently selected from C1-C10Alkyl radical, C3-C7Cycloalkyl radical, C3-C7Heterocycloalkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl, C6-C10Aryl, 5-to 10-membered heteroaryl, fused ring aryl, C6-C10Heterocyclyl, C optionally substituted with one or more hydroxy, amino, halogen, cyano, nitro, sulfonyl, aromatic, heteroaromatic and fused ring aryl groups1-C6An alkyl group;
Figure FSA0000185274160000014
represents a substituent connection site.
2. The compound, optical isomer, and pharmaceutically acceptable salt, hydrate, or prodrug thereof according to claim 1,
wherein the content of the first and second substances,
x is optionally selected from O and NH;
preferably, the first and second electrodes are formed of a metal,
x is O.
3. The compound, optical isomer, and pharmaceutically acceptable salt, hydrate, or prodrug thereof according to claim 1,
wherein the content of the first and second substances,
R1and R2Are each independently selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C2-C8Alkenyl radical, C2-C4Alkynyl, C6-C10Aryl, 5-10 membered heteroaryl, fused ring aryl, C optionally substituted with one or more hydroxy, amino, halogen, cyano, nitro, aromatic ring, aromatic heterocycle or fused ring aryl1-C6An alkyl group;
preferably, the first and second electrodes are formed of a metal,
R1and R2Are each independently selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C2-C6Alkenyl radical, C2-C4Alkynyl, phenyl, pyridine, indole, thiophene, quinoline, C optionally substituted with one or more amino, halogen, benzene ring, pyridine ring, quinoline, indole ring1-C6An alkyl group.
4. A compound, optical isomer and pharmaceutically acceptable salt, hydrate or prodrug thereof of the following general formula I:
[4- (glycylprolamido) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- (N-acetylglucosaminyl prolyl) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- (N-propionylglycylprolylamino) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (2-methacryloyl) glycylprolylamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazin-10-carboxylic acid ester;
[4- [ N- (2-dimethylaminoacetyl) glycylprolamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- (phenylalanyl glycyl prolyl amido) benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (1H-indole-2-propionyl) glycylprolylamino ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (tert-butoxycarbonyl) glycylprolylamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester;
[4- [ N- (benzyloxycarbonyl) glycylprolylamino ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazin-10-carboxylic acid ester;
[4- [ N- [1- (pyridin-4-yl) methoxycarbonyl ] glycylprolamido ] benzyl ] -3, 7-bis (dimethylamino) -10H-phenothiazine-10-carboxylic acid ester.
5. A pharmaceutical composition comprising a compound of any one of claims 1 to 4, an optical isomer, and a pharmaceutically acceptable salt, solvate or prodrug thereof as an active ingredient together with a pharmaceutically acceptable excipient.
6. The process for preparing a compound, optical isomer, and pharmaceutically acceptable salt, hydrate, or prodrug thereof according to claim 1,
Figure FSA0000185274160000021
wherein X, Y and R are as defined in claim 1.
7. Use of a compound, optical isomer, and pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1-4 or a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the diagnosis and treatment of cancer.
8. Use of a compound, optical isomer, and pharmaceutically acceptable salt, solvate or prodrug thereof of any of claims 1-4 or a pharmaceutical composition of claim 5 for the preparation of a medicament for the diagnosis and treatment of cancers highly expressing FAP α.
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CN113387973A (en) * 2021-05-24 2021-09-14 云南师范大学 Double-recognition fluorescent probe molecule and preparation method and application thereof
CN116102521A (en) * 2023-02-17 2023-05-12 西安交通大学 Multifunctional prodrug delivery compound based on in-vivo active oxygen response, preparation method and application

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