AU2022338581A1

AU2022338581A1 - N,N-DIPHENYLAMINO-MODIFIED β-CARBOLINE INDOLIUM SALT, PREPARATION METHOD AND USE

Info

Publication number: AU2022338581A1
Application number: AU2022338581A
Authority: AU
Inventors: Qian Ding; Peng Li; Yong Ling; Chi MENG; Daoxin MIAO; Jianqiang QIAN; Zhongyuan Xu; Yumin Yang; Yan'an ZHANG; Yuting Zhang
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-09-03
Filing date: 2022-06-09
Publication date: 2023-06-15
Anticipated expiration: 2042-06-09
Also published as: WO2023029638A1; CN113717169B; CN113717169A; AU2022338581B2

Abstract

The present invention relates to the field of biological medicines, and relates to an N,N-diphenylamino-modified β-carboline indolium salt, a preparation method and the use. The β-carboline indolium salt has a structure as represented by general formula (I), can be activated in the acidic microenvironment of tumor tissue by the ICT principle, and selectively and rapidly generates pH-sensitive near-infrared fluorescence at a tumor site. The specific implementation method comprises: spraying or locally injecting a solution of the β-carboline indoliumm salt of the present invention onto/into a tumor lesion site and the surrounding tissue, and performing rapid and selective fluorescence imaging and tracing on the tumor lesion tissue by means of using a fluorescence endoscope or an in vivo imager. The method has a relatively high tumor tissue fluorescence imaging selectivity and a relatively low background fluorescence interference, and can be used for accurately detecting tumors.

Description

The preparation method and application of N, N-diphenylamino modified p-Carboline indolyl onium salt

Description: The preparation method and application of N, N-diphenylamino modified p Carboline indolyl onium salt Technical field: The invention relates to the field of biomedicine, and relates to preparation method and Application of a class of N, N-diphenylamino modified P- Carboline indolyl onium salt and specifically, it relates to a class of preparation method and application of p Carboline indolyl onium which is modified by N, N-diphenylamino. Background technology: Cancer is one of the most fatal diseases. According to the statistics of the World Health Organization in 2018, there are 18.1 million new cancer cases and 9.6 million cancer deaths worldwide. However, if imaging diagnosis can be carried out at the early stage of tumor development, it will greatly reduce the mortality of cancer. The common imaging techniques in clinic, such as CT, MRI and PET, are difficult to guide the accurate diagnosis of tumors due to their limited spatial resolution. In contrast, fluorescence imaging technology has attracted more and more attention due to its advantages of high sensitivity, high spatial resolution and real-time imaging diagnosis. In the technology of tumor fluorescence imaging diagnosis, the key is to design a fluorescence probe that can respond quickly and accurately to tumor markers. Tumor cells mainly rely on aerobic glycolysis to provide energy. The lactic acid produced in this process is discharged out of the cell, thus creating an acidic tumor microenvironment. Some literature reports that low pH is used to design tumor diagnostic agents. The previous reported pH-active probes are often based on acid sensitive bonds to achieve 'on-off' switch, such as controlling the cleavage of acid sensitive hydrazone bond, imine bond or acetal to control fluorescence. However, these kind of pH probes have obvious defects. First, the covalent bond breaking process requires a certain amount of time, so it is unable to carry out rapid and real-time diagnosis of tumor by spraying; second, it does not have fluorescence reversibility, so it cannot dynamically observe tumors and normal tissues with different pH. Compared with ultraviolet visible fluorescence, near-infrared (NIR) fluorescence probe is more suitable for imaging in vivo because of its advantages such as less damage to organisms, good penetration to tissues, and less interference with tissue autofluorescence. Therefore, in order to get real-time, accurate and fast imaging diagnosis of tumors, we modified the p-Carboline. Contents of the invention: p-Carboline compounds are a large class of natural indole alkaloids, which have a planar three ring structure skeleton of pyridine [3,4-b] indole similar to carbazole, and have low toxicity. There is an introduction of electron donor group N, N diphenylamino group at the 6-position of carbaline in P- carboline so that it can quickly generate stable pH sensitive fluorescence. There is an introduction of vinyl which connects different substituted indolyl salts or benzoindolylium salts with various splicing forms at the 6-position of carbaline in P- carboline, so it can enhance the electron donating ability to obtain fluorescent compounds with longer fluorescence wavelength and larger Stokes shift. There is an introduction of water-soluble side chains such as morpholinyl and piperazinyl at the 9-position of nitrogen in P- carboline, so it can improve the lipid-water partition coefficient of the whole molecule and obtain NIR fluorescent molecules based on 'donor - receptor' with large stokes shift. Unlike the traditional pH fluorescence probe which is activated by acid sensitive hydrazone bond or acetal group, this fluorescence probe can rapidly realize pH sensitive fluorescence through intramolecular charge transfer (ICT) and has the fluorescence reversibility of acid pH regulation. Our invention has the characteristics of pH sensitive near-infrared fluorescence, and the fluorescence is reversible with the change of pH. It can be prepared into spray, which can be sprayed or locally injected on the surface of tumor cells or tissues to realize fast, real-time and selective fluorescence imaging. At present, no fluorescent agent has been used to spray tumor tissue to realize fast, real-time and selective fluorescence imaging. Our invention will be a good supplement and expansion for this field of clinical application. The specific technical scheme of the invention is as follows: A class of pH sensitive, N, N - diphenylamino group modified p-Carboline indolyl onium salt and it has the general formula I:

Y N+

\ N N

Wherein, Ri is selected from H, C1 - C6 alkyl or C1 - C6 linear alkyl morpholine; R2 is selected from H, F, Cl, Br orI; Y represents halogen anion, hexafluorophosphate anion, p-toluenesulfonic acid anion or methanesulfonic acid anion; n is 0 or 1. Furthermore, a class of pH sensitive N, N-diphenylamino group modified p-Carbaline indolyl onium salts have following features: in the general formula I, Ri, R2, Y and n are selected from the following combinations: R2,,y

Ri=CH3 , R2=I, Y=I, n=O, = R2

0 /_

Or Ri= R2=H, Y=CH3SO3 , n=O, =

R2-L

Or R1=CH3CH2, R2=H, Y=Br, n=1, = | R2

. I Qb N

/ Or Ri=CH3, R2=I, Y=PF6, n=l, =

The preferred structures of the compounds with the above general formula are shown in Table 1: Table 1. Codes of part general formula I compounds and their corresponding structures Compd. RI R n Y Product

11 CH3 1 0 I

12 H 0 CH3SO 3 CHy9OI

13 CH3 H 1 Br Or

14 CH 3 I I PF6

Another object of the invention is to provide the following preparation method for the compound of general formula I: The route of the preparation method is as follows:

0 0 N 'Ph 0 Br / Br o H Ph-N OH N NaH t-BuONu __H \~N\,- /N NLIH N R 1I or R1 Br N Pd(dBa) 3 N THF H DMF P(t-Bu) 3 1 2 3

Ph HO Ph H Ph-N Ph-N 0

N DM -N N DCM N

4 5

OH ,Ph ,Ph HO'I S H Ph N Ph-N OH Ph-N H N\3O." Pd(PPh3) 4 P 12 MoCN K2 C0 3 \ N N R1, R1 3 6 7

Pth H Ph 15)= ph-N 0 ph- PhP -N L

. /N \,N E10H s n /NY,

N N NN R7 5 R1

Wherein, Ri is selected from H, Cl - C6 alkyl or Cl - C6 linear alkyl morpholine; R2 is selected from H, F, Cl, Br orI; Y represents halogen anion, hexafluorophosphate anion, p-toluenesulfonic acid anion or methanesulfonic acid anion; n is 0 or 1. The preparation method comprises the following steps: Sl. Preparation of intermediate 5: 6-Bromine- 3-Carboline 1 reacts with halohydrocarbon RiBr or RiI through NaH to generate compound 2; S2. Compound 2 was reacted with diphenylamine under the condition of sodium tert butoxide via Pd(dBa)3 and P(t-Bu)3 catalysts to obtain amination product 3; S3. The carboxyl group of aminated product 3 which is reduced by LiAlH4 produces alcohol intermediate 4; The alcohol intermediate 4 is oxidized by DMP to obtain aldehyde intermediate 5; S4. Under the catalysis of K3P04 andI2, the amination product 3 was decarboxylated and iodized to obtain compound 6, and then Suzuki coupling with formyl thiophene borate under the catalysis of K2C03and Pd(PPh)4 toobtain intermediate 7; S5. Aldehyde intermediate 5 or intermediate 7 is reacted with indolyl salt or benzoindolyl salt with catalytic amount of piperidine in heating reflow and compound I is obtained by Knoevenagel reaction. The invention also provides the application of p-carbaline which modified by N, N diphenylamino in the preparation of pH responsive fluorescent agent. Furthermore, the fluorescent agent is a selective fluorescent imaging agent for tumor tissues or tumor cells in vivo and in vitro. Furthermore, the specific application is that the N, N - diphenylamino modified p-Carboline indolyl onium salt is dissolve with cosolvent/surfactant/solvent system and get sprayable solution. Furthermore, in the cosolvent/surfactant/solvent system, the content of the cosolvent is 1-30% and the content of the surfactant is 1-30% by volume percentage; the cosolvent is 1,2 - propylene glycol, DMSO or ethanol; the solvent is water; the surfactant is Tween 20, Tween 40 or Tween 80. Furthermore, the tumor is one of liver cancer, colon cancer, breast cancer, lung cancer and cervical cancer. Compared with the present technology, the invention has the following application effects: The compound of the invention uses ICT principle to be activated in the acidic microenvironment of tumor tissue, selectively and rapidly generate pH sensitive near-infrared fluorescence at tumor sites. The specific implementation method is to spray or locally inject the compound solution on the tumor focus site and surrounding tissues before or during the operation and use fluorescence endoscopy or living imaging instrument to image and trace of tumors with fast and selective fluorescence. It has high tumor tissue fluorescence imaging selectivity and low background fluorescence interference and it can accurately diagnose tumors to guide surgery and/or drug treatment.

Description of attached drawings: Figure 1 shows the UV and fluorescence spectra of compounds in the invention, wherein Figure 1A shows the UV absorption spectrum of Ii, Figure 1B shows the fluorescence spectrum of Ii, Figure IC shows the UV absorption spectrum of14, and Figure ID shows the fluorescence spectrumof14; Figgure 2 shows the selective fluorescence imaging of compound I in liver cancer cells; Figgure 3 shows the fluorescence imaging of compound12 in lung cancer cells; Figure 4 shows the selective fluorescence imaging of compound I on the isolated breast cancer tissue; Figure 5 shows the selective fluorescence imaging of compound I on clinical colon cancer tissue; Figure 6 shows the selective fluorescence imaging of compound14 on clinical colon cancer tissue.

Specific embodiments: In order to further clarify the invention, a series of embodiments are given below. These embodiments are purely illustrative and they are only used to describe the invention in detail and should not be interpreted as restrictions on the invention. Example 1: (E)-2-(2-(6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-7-iodo 1,1,3-trimethyl-1H-benzo[e]indol-3-iodate (compound I1) (1) Preparation of 6-bromo-9-methyl-9H-pyridine methyl[3,4-b] indole-3-carboxylate (compound 2) A solution of compound 1 (500 mg, 1.57 mmol) was dissolved in anhydrous

DMF at 0 °C and NaH (301 mg, 12.56 mmol) was added and then CH3I (1 ml, 15.7 mmol) was added to the mixture and was stirred for 1 h at room temperature. After the reaction completed which was monitored through TLC, the mixture was poured into ice water and adjusted pH to 7 with 0.1 mol hydrochloric acid solution. The mixture was filtered and the precipitation was dried to obtain compound 2 with yield of 85%. 'H NMR (400 MHz, DMSO-d) 612.21 (s, 1H, NH), 8.84 (s, 1H, ArH), 8.66 (d, J= 1.7 Hz, 1H, ArH), 7.70 (dd, J=8.7, 1.9Hz, 1H, ArH), 7.61 (d, J=8.7Hz, 1H, ArH), 3.90 (s, 3H, OCH3), 2.81 (s, 3H, CH3). (2) Preparation of 6-(diphenylamino)-1,9-dimethyl-9H- pyridino[3,4-b] indole 3-carboxylic acid (compound 3) To a solution of compound 2 (480 mg,1.45 mmol), diphenylamine (1.47 g,8.7 mmol), Pd(dba)2 (83.375 g,0.145 mmol) and sodium tert-butoxide (430 mg,5.8 mmol) in 0.1 mL tri-tert-butylphosphine was added 4 mL toluene and stirred at 110 °C for12 h with N2 protection. Monitoring the completed reaction through TLC, the solution was concentrated and was finally purified by column chromatography to get compound 2 with yield of 76%. H NMR (400 MHz, DMSO-d) 610.8 (s, 1H, COOH), 8.70 (s, 1H, ArH), 8.19 (s, 1H, ArH), 6.98 (dd, J=10.7, 7.7Hz, 6H, 2CH=C, 4ArH), 4.21 (s, 3H, CH3), 3.08 (s, 3H, CH3). (3) 6-(diphenylamino)-1,9-dimethyl-9H-pyridino[3,4-b]indole-3-methanol (compound 4) A solution of compound 3 (530 mg, 1.3 mmol) was dissolved in 10 mL anhydrous THF at 0 °C for 10 min and then LiAlH4 (741 mg, 19.5 mmol) was slowly added. The reaction was stirred for 30 min. Then the mixture was stirred for 3h at room temperature. Monitoring the completed reaction through TLC. Methanol was added until no bubbles could be observed and then 1 mL H20 was added. Then the mixture was filtered and washed with methanol. The collecting filtrate was concentrated under reduced pressure to afford compound 4 with yield of 80%. H NMR (400MHz, DMSO-d) 68.70 (s, 1H, ArH), 8.19 (s, 1H, ArH), 7.79 (d, J=8.9 Hz, 1H, ArH), 7.42 (dd, J=8.8, 1.9Hz, 1H, ArH), 7.27 (t, J=7.9 Hz, 4H, 4ArH), 6.98 (dd, J=10.7,7.7Hz, 6H, 2CH=C, 4ArH), 5.21 (m, 2H, CH2),4.21 (s, 3H, CH3),3.89 (s, 1H, OH), 3.08 (s, 3H, CH3). (4) Preparation of 6-(diphenylamino)-1,9-dimethyl-9H-pyridino[3,4-b]indole-3 formaldehyde (compound 5) To a solution of compound 4 (500 mg,1.27 mmol) in 10 mL DCM was added DMP (1.076 g, 2.54 mmol). About 2 h later, 50 mL sodium bicarbonate and sodium thiosulfate were slowly added to the mixture. Filter the mixture and afford compound with yield of 67%. H NMR (400 MHz, DMSO-d) 6 9.75 (s, 1H, CHO), 8.70 (s, 1H, ArH), 8.19 (s, 1H, ArH), 7.79 (d, J=8.9 Hz, 1H, ArH), 7.42 (dd, J=8.8, 1.9 Hz, 1H, ArH), 7.27 (t, J=7.9 Hz, 4H, 4ArH), 6.98 (dd, J=10.7,7.7 Hz, 6H, 2CH=C, 4ArH), 4.21 (s, 3H, CH3),3.08 (s, 3H, CH3). (5) Preparation of (E)-2-(2-(6-(diphenylamino)-1,9-dimethyl-9H-pyridino [3,4-b] indole-3-yl) vinyl)-7-iodine-1,1,3-trimethyl-1H-benzo [e] indole -3-iodate (compound

I1) To a solution of compound 5 (480 mg, 1.22 mmol) in 2 mL anhydrous ethanol was added 7-Iodine-1,1,2,3-Tetramethyl-1H-Benzo[e]Indole-3-Iodine salt (379 mg, 1.22 mmol) and stirred for 0.5 h. After the reaction was finished, the mixture was filtered and afforded compound I with yield of 85%. 1H NMR (400 MHz, CDC3) 6 8.13 (d, J=8.5 Hz, 1H, ArH), 8.08 (d, J=8.9 Hz, 2H, 2ArH), 7.99 (d, J=8.1 Hz, 2H, 2ArH), 7.84 (m, 1H, CH=C), 7.66 (s, 2H, 2ArH), 7.59 (d, J=7.9 Hz, 2H, 2ArH), 7.43 (dd, J=10.1, 8.4 Hz, 3H, 3ArH), 7.04 (d, J=7.7 Hz, 5H, ArH), 6.95 (t, J=7.4 Hz, 3H, 2ArH, CH=C), 4.45 (s, 3H, CH3), 4.18 (s, 3H, CH 3), 2.06 (s, 6H, 2CH3), 1.92 (s, 3H, CH3).

Example 2: (E)-2-(2-(6-(6-(diphenylamino)-1-methyl-9-(2-morpholinoethyl)-9H pyridino[3,4-b]indole-3-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indole-3-sulfonate (compound 12) Referring to the preparation method of 2 in Example 1, the compound iodomethane was substituted for bromoethyl morpholine in the method to obtain dark red product 12 with yield of 76%. 'H NMR (400 MHz, DMSO-d) 6 8.67 (s, 1H, ArH), 8.23 (d, J=2.1 Hz, 1H, ArH), 8.23 (m, 2H, 2ArH), 8.06 (m, 2H, 2ArH), 7.82 (d, J=8.9 Hz, 1H, ArH), 7.54 (m, 3H, 3ArH), 7.44 (d, J=2.1 Hz, 1H, ArH), 7.30 (m, 4H, 4ArH), 6.99 (t, J=8.2 Hz, 6H, 6ArH), 4.78 (dt, 2H, CH2), 3.54 (m, 4H, 2CH2), 3.11 (s, 3H, CH3), 2.75 (m, 2H, CH2),2.47 (m, 4H, 2CH2),2.06 (s, 6H, 2CH3),1.9 (s, 3H, CH3). Example 3: (E)-2-(2-(5-(6-(diphenylamino)-1,9-dimethyl-9H-pyridino [3,4-b] indole-3-base) thiophene-2-base) vinyl)-1,3,3-trimethyl-3H-indole -1- bromide (compound 13) (1) Preparation of 3-iodo-1-methyl-N,N-diphenyl-9H-pyridino[3,4-b] indole -6-amine (compound 6) To a solution of compound 3 (200 mg, 0.508 mmol), 12 (280 mg, 1.103 mmol) and K3P04 (186 mg, 0.876 mmol) was added 2 mL acetonitrile in seal pipe and stirred at 110 °C for18 h under N2 protection. After reaction, the mixture was added 1.2 mL triethylamine and stirred at 150 °C for 4 h. The solution was concentrated and was finally purified by column chromatography to get compound 6 with yield of 65%. H NMR (400 MHz, DMSO-d) 6 8.70 (s, 1H, ArH), 8.19 (s, 1H, ArH), 7.79 (d, J=8.9 Hz, 1H, ArH), 7.42 (dd, J=8.8, 1.9 Hz, 1H, ArH), 7.27 (t, J=7.9 Hz, 4H, 4ArH), 6.98 (dd, J=10.7, 7.7Hz, 6H, 2CH=C, 4ArH), 4.21 (s, 3H, CH3), 3.08 (s, 3H, CH3). (2) Preparation of 5-(6-(diphenylamino)-1,9-dimethyl-9H-pyridino[3,4-b]indole-3-yl) thiophene-2- formaldehyde (compound 7) To a solution of compound 6 (170 mg, 0.730 mmol), K2C03 (202 mg, 1.46 mmol), Pd(PPh3)4(69.44 mg, 0.073 mmol), thiophene borate aldehyde (163 mg, 1.46 mmol) was added 4 mL toluene in seal pipe and stirred at 110 °C for 3 h under N2 protection. After reaction, yellow fluorescence was seen on the spot plate. The mixture was finally purified by column chromatography (EA : PE = 1:2)to afford compound 7 as yellow solid with yield of 82%. 'H NMR (400 MHz, DMSO-d) 6 9.84 (s, 1H, CHO), 8.13 (s, 1H, ArH), 8.00 (d, 1H, CH=C), 7.37-7.20 (m, 6H, 6ArH), 6.81 (m, 2H, ArH), 6.73-6.63 (m, 4H, 4ArH), 6.12

(m, 1H, ArH), 3.82 (s, 3H, CH3), 2.89 (s, 3H, CH3). (3) Preparation of (E)-2-(2-(5-(6-(diphenylamino)-1,9-dimethyl-9H-pyridino[3,4-b] indole-3-base)thiophene-2-base)vinyl)-1,3,3-trimethyl-3H-indole-1-bromide (compound 13) To a solution of compound 7 (121 mg, 0.256 mmol) in 2 mL anhydrous ethanol was added 1,2,3,3-tetramethyl-3H-indole bromide (58.3 mg, 0.256 mmol) and stirred for 0.5 h. After the reaction was completed, the reaction solution was filtered to obtain product 13 with yield of 78%. H NMR (400 MHz, DMSO-d) 6 8.92 (d, 1H, ArH), 8.13 (s, 1H, ArH), 8.02 (m, 1H, CH=C), 7.73 (d, 1H, ArH), 7.37-7.20 (m, 9H, 8ArH, CH=C), 6.81 (m, 2H, 2ArH), 6.65 (m, 5H, 4ArH, CH=C), 6.20 (m, 1H, ArH), 5.67 (m, 1H, ArH), 3.82 (s, 3H, CH3), 2.89 (s, 3H, CH3), 2.06 (s, 6H, 2CH3), 1.92 (s, 3H, CH3). Example 4: Preparation of (E)-2-(2-(5-(6-(diphenylamino)-1,9-dimethyl-9H-pyridino

[3,4-b]indole-3-base)thiophene-2-base)vinyl)-7-iodo-1,1,3-trimethyl-1H-benzo[e] indole-3-hexafluorophosphate (compound 14) Referring to the preparation method of 13 in Example 3, compound 7-iodine-1,1,2,3-tetramethyl-1H-benzo[e]indole-3-hexafluorophosphate replaces 1,2,3,3-tetramethyl-3H-indole-1-bromine salt in the method to obtain dark red product 14 with yield of 78%. H NMR (400 MHz, DMSO-d) 6 8.92 (d, 1H, ArH), 8.13 (s, 1H, ArH), 8.02 (m, 3H, 3ArH), 7.73 (d, 1H, ArH), 7.54 (d, 2H, 2ArH), 7.37-7.20 (m, 9H, 9ArH), 6.81 (m, 2H, 2ArH), 6.65 (m, 5H, 4ArH, CH=C), 6.20 (m, 1H, ArH), 5.67 (m, 1H, ArH), 3.82 (s, 3H, CH3), 2.89 (s, 3H, CH3), 2.06 (s, 6H, 2CH3), 1.92 (s, 3H, CH3). Example 5: Ultraviolet absorption spectrum test of the compound I and 14 in the invention The fluorescent compound was dissolved in aqueous solution containing 1% DMSO to prepare 5 - 20 M test solution. The ultraviolet spectrophotometer was used to test ultraviolet absorption spectrum at pH= 3-8, and the results showed that the maximum ultraviolet absorption wavelength of the fluorescent compound was within 520 650nm. Among them, the maximum UV absorption wavelengths of compounds I and 14 are about 592 and 583 nm respectively (Figure 1A and IC). Example 6: PH sensitive fluorescence test of the compound of I and 14 in the invention The fluorescent compound in the invention was dissolved in aqueous solution containing 1% DMSO to obtain 5 - 20 M test solution. The fluorescence spectrum at pH 3-8 were measured with fluorescence spectrometer and the results were shown in Figure 1. Note: Figure 1A was the UV absorption spectrum of Ii, Figure lB was the fluorescence spectrum of I, Figure IC was the UV absorption spectrum of 14, and Figure ID was the fluorescence spectrum ofI4. The results showed that the maximum emission wavelength of the fluorescent compound was 680 - 750 nm. The maximum emission wavelengths of compounds I1 and 14 at pH=3.5-6.5 were about 740 and 692 nm respectively and the fluorescence wavelength reached the near-infrared region, while there was almost no fluorescence at neutral pH. In addition, the Stokes shift of the compound reached 150-200 nm and had good fluorescent characteristics. In addition, the fluorescent peak of compound I at about 740 nm was increased with the decrease of pH, on the contrary, its fluorescence peak was decreased with the increase of pH and its difference was 8 times (Figure 1B). Example 7: Selective fluorescent imaging test of compounds I andI2on tumor cells Cell uptake and localization were performed using confocal laser scanning microscope (Leica TCS SP8) with 40X objective. Human hepatoma cell (HepG2), normal hepatocyte (L02) and human lung cancer cell (A549) were cultured in 1 mL medium with density of 1 x 10' cells in confocal dish at 37 °C for 24 h. However, HepG2 and L02 cell culture media was replaced with 1-100 M fresh medium of Ii, and A549 cell culture medium was replaced with 1-100 M fresh mediumof12 and then incubated at 37 °C for 10 to 30 min and washed the cells with PBS for three times. Finally, the confocal laser scanning microscope was used to obtain the image of cell fluorescence imaging, and the results were shown in Figure 2 (compound I1i) and Figure 3 (compound2). In Figure 2 and Figure 3, the left figure was the light field and the right figure was the fluorescence imaging graph. The imaging results in Figure 2 and Figure 3 showed that compound I1 (10 M) could perform clear fluorescence imaging of HepG2 after 4 h while the fluorescence imaging of L02 was weak. According to the quantification of intracellular fluorescence, the fluorescence intensity of HepG2 was 6.5 times that of L02 cells, indicating that the compound of the invention had the capability of fluorescence imaging selectively in liver tumor cells. Meanwhile, compound 12 (50 M) could perform clear fluorescence imaging on A549 cells after 2h. These results showed that the compound of the invention had the ability of selective fluorescence imaging for tumor cells. Example 8: The fluorescence imaging test of the compound in the invention through spraying on the tumor tissue in vitro The nude mice with human breast cancer cells (MDA - MB - 231) were killed and the breast cancer tumors and main organs were taken out for spray imaging analysis. The prepared compound I solution (10~ 100 M) was sprayed on the tissue for 3-5 times, washed with PBS and dried it with cotton. As shown in Figure 4, the fluorescence intensity of breast cancer tissue was significantly higher than other organ and tissues, while the normal organ and tissues hardly had fluorescence. This showed that the compound of the invention could selectively and rapidly image by spraying on tumor tissue to achieve rapid detection of clinical tumor tissue. Example 9: Fluorescence imaging test of the compound of the invention on clinical tumor tissue On this basis, the selective imaging ability of the compound of the invention to clinical tumor tissue was further studied. Clinical colon cancer tissue and paracarinoma tissues was selected for spray imaging analysis and compound I1 and14 solution (10-100 M) was sprayed on colon cancer tissue and paracarinoma tissues for 1-3 times. Then washed off the excess solution on the surface with appropriate saline after 3-10 minutes and used the live imaging instrument to perform fluorescence imaging. The results were shown in Figure 5 (compound I1) and Figure 6

(compoundI4). The fluorescence imaging showed that the compounds Ii and 14 could selectively and rapidly light the clinical colon cancer tissue, but the surrounding normal tissue was not lighted or weak (Figure 5-6). This further confirmed that the compound of the invention had the ability of selective imaging to clinical liver tumor tissue. The embodiments of the invention are described in detail above, but the contents described are only the preferred embodiments of the invention and cannot be considered to limit the scope of implementation of the invention. All equal changes and improvements made according to the application scope of the invention shall still fall within the scope of the patent of the invention.

Claims

1. A class of pH sensitive N, N - diphenylamino group modified p-Carboline indolyl onium salt has the structure shown in the general formula I:

Y +N

/\N S n --- / \R2 \N N

Wherein, Ri is selected from H, C1 - C6 alkyl or C1 - C6 linear alkyl morpholine; R2 is selected from H, F, Cl, Br or I; Y represents halogen anion, hexafluorophosphate anion, p-toluenesulfonic acid anion or methanesulfonic acid anion; n is 0 or 1.

2. According to claim 1, a class of pH sensitive N, N-diphenylamino group modified p-Carbaline indolyl onium salts have the features as follows: Ri is selected from H, methyl, ethyl or ethyl morpholine; R2 is selected from H or I; Y represents halogen anion, hexafluorophosphate anion, p-toluenesulfonic acid anion or methanesulfonic acid anion; n is 0 or 1.

3. According to claim 1, a class of pH sensitive N, N-diphenylamino group modified p-Carbaline indolyl onium salts have the features as follows: in the structure shown in the general formula I, Ri, R2, Y and n are selected from the following combinations: R2

N'

R1= CH3, R2= I,Y=I, n=0, \ =\;

N N/ N

05_NN N+

Or Ri= '- R2 H, Y CH3S3,n , \ =

R2 <y'

~ N N Or Ri CH3 CH2, R2 H, Y Br, n= 1, = ; R2 yZ, &

&N N Or Ri=CH3, R2= I, Y = PF6, n= 1, =

4. The preparation method of a class of pH sensitive N, N-diphenylamino group modified p-Carbaline indolyl onium salts, the route of the preparation method is as follows:

0 0 N Ph 0 Br 0" Br IH Ph-N 'OH NaH t-BuONa NLA - \N - -. \N LLIH N R 1 1 or R1Br N Pd(dBa)3 N THF H DMF I P(t-Bu)a

1 2 3

Ph HO Ph H Ph-N Ph - N

N DMP N N N R1 R1 4 5 OH Ph 0 Ph HO-Bi S H Ph \ Ph-N OH Ph-N 0 H KPPO \ d(PPh PN N\ \ ,MeCN N K 2C0 3 N

R1 R1 R1 3 6 7

0 R2 Ph H Ph Ph-N 0o Ph-N N.

/ \: NN N Ns N - -N

7 R

Wherein, Ri is selected from H, Cl - C6 alkyl or Cl - C6 linear alkyl morpholine; R2 is selected from H, F, Cl, Br orI; Y represents halogen anion, hexafluorophosphate anion, p-toluenesulfonic acid anion or methanesulfonic acid anion; n is 0 or 1. The preparation method comprises the following steps: S1. Preparation of intermediate 5: 6-Bromine- p-Carboline 1 reacts with halohydrocarbon RiBr or RiI through NaH to generate compound 2; S2. Compound 2 was reacted with diphenylamine under the condition of sodium tert butoxide via Pd(dBa)3 and P(t-Bu)3 catalysts to obtain amination product 3; S3. The carboxyl group of aminated product 3 which is reduced by LiAlH4 produces alcohol intermediate 4; the alcohol intermediate 4 is oxidized by DMP to obtain aldehyde intermediate 5; S4. Under the catalysis of K3P04 and 12, the amination product 3 was decarboxylated and iodized to obtain compound 6, and then Suzuki coupling with formyl thiophene borate under the catalysis of K2C03 and Pd(PPh)4 to obtain intermediate 7; S5. Aldehyde intermediate 5 or intermediate 7 is reacted with indolyl salt or benzoindolyl salt with catalytic amount of piperidine in heating reflow and compound I is obtained by Knoevenagel reaction.

5. According to any one of claim 1-3, the P-Carbaline indolyl onium salt which is modified by N, N-diphenylamino or the p-Carbaline indolyl onium salt which is modified by N, N-diphenylamino according to claim 4 is applied to preparation of pH-reactive fluorescent agent.

6. According to the application of claim 5, the characteristic is that the fluorescent agent is a selective fluorescent imaging agent for tumor tissues or tumor cells in vivo and in vitro.

7. According to the application of claim 5, the characteristic is that the N, N-diphenylamino group modified p-Carboline indolyl onium salt is dissolved with cosolvent/surfactant/solvent system and get sprayable solution.

8. According to the application of claim 7, thecharacteristic is that in the cosolvent/surfactant/solvent system, the content of the cosolvent is 1-30% and the content of the surfactant is 1-30% by volume percentage; the cosolvent is 1,2 propylene glycol, DMSO or ethanol; the solvent is water; the surfactant is Tween 20, Tween 40 or Tween 80.

9. According to the application of claim 6, the characteristic is that the tumor is one of liver cancer, colon cancer, breast cancer, lung cancer and cervical cancer.