阅读说明：本技术 一种七甲川菁近红外染料及其应用 (Heptamethine cyanine near-infrared dye and application thereof ) 是由 顾月清 李昌盛 王丽 代亚雪 蔡惠明 于 2021-06-04 设计创作，主要内容包括：本发明公开了一种七甲川菁近红外染料及其应用；该七甲川菁近红外染料具有更好的体内代谢特点,在肝脏、肠等器官滞留时间短,在肿瘤中的滞留时间几乎不变,靶向作用依旧保持,从而更适合用于肿瘤精准诊断,具备一定的临床应用前景,应用于外科肿瘤切除过程中手术导航。(The invention discloses a heptamethine cyanine near-infrared dye and application thereof; the heptamethine cyanine near-infrared dye has the advantages of better in-vivo metabolism characteristics, short retention time in organs such as liver, intestine and the like, almost constant retention time in tumor, and maintained targeting effect, so that the heptamethine cyanine near-infrared dye is more suitable for accurate diagnosis of tumor, has certain clinical application prospect, and is applied to surgical navigation in surgical tumor resection.)

1. A heptamethine cyanine near-infrared fluorescent dye shown in a structural formula I,

wherein X is selected from SCH₂CH₂COOH、SCH₂COOH、SCH₂CH₂CONH(CH₂CH₂O)_n-CH₂CH₂NH₂Or SCH₂CH₂CONH(CH₂CH₂O)_mCH₂CH₂COOH, n and m are selected from 0-10.

2. The heptamethine cyanine near-infrared fluorescent dye according to claim 1, wherein the near-infrared fluorescent dye is selected from the group consisting of I-1 or I-2:

3. the use of the heptamethine cyanine near-infrared fluorescent dye or the pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of a precise diagnostic reagent for tumors.

4. Use according to claim 3, wherein the pharmaceutically acceptable salt is a sodium, potassium or calcium salt.

5. The use of claim 3 or 4, wherein the tumor is liver cancer, breast cancer, lung cancer or cervical cancer.

Technical Field

The invention belongs to the technical field of nonspecific molecular tumor targeted diagnosis reagents, and particularly relates to a heptamethine cyanine near-infrared dye which has the advantages of tumor aggregation, long retention time and rapid removal of normal tissues such as liver, intestine and the like, and an application thereof.

Background

With the improvement of living standard of residents, the change of living conditions, the aggravation of environmental pollution and the change of eating habits, malignant tumors are more and more common, and most of the malignant tumors deprive countless patients of lives because the malignant tumors cannot be cured every year, so that the cure of the malignant tumors still remains to be a huge challenge. In recent years, the incidence and mortality of malignant tumors in our country have been on the rise year by year, with the attendant high treatment costs and deprivation of lives of countless patients. For clinical treatment of malignant tumor, surgical resection and chemotherapy are mainly used; chemotherapy mainly adopts chemotherapeutic drugs to kill tumors, can prolong the short-term survival time of patients, but is accompanied by serious adverse reactions, and causes irreparable physical and psychological damage to the patients; with the advent of various advanced devices, such as nuclear magnetic resonance imaging, ultrasonic radiography, spectra/CT and the like, tumors can be accurately positioned, the focus position can be found, and then the tumor position and normal tissues can be better distinguished by using near-infrared imaging equipment and a near-infrared fluorescent probe and by using the near-infrared imaging equipment, so that the tumors can be more accurately resected, and the normal tissues can be reserved.

Indocyanine green (ICG) is a near-infrared fluorescent dye approved by FDA and used for clinical intraoperative navigation, and has good safety, the surgical doctor basically performs intravenous injection administration one day before surgery in the operation using process, after one day of metabolism, the surgery is performed after the metabolism of tissues such as liver, intestine and the like is completed, and the ICG tumor targeting mechanism is EPR passive targeting aggregation at a tumor part. NIR-04(CN 111196896A) is an ICG derivative obtained by modifying on the basis of an ICG mother nucleus, the living body verification shows that the targeting effect of the derivative is stronger than that of ICG, the tumor retention time is greatly prolonged compared with that of ICG, and meanwhile, the research on in-vivo safety, in-vivo metabolism and tumor targeting of tumor-bearing mice is carried out by taking the NIR-04 as a candidate compound, and the NIR-04 is found to accumulate in the liver and is difficult to metabolize and clear, so that the derivative is not suitable for clinical use.

Therefore, the NIR-04 is further modified on the basis of no structural change, and the aim is to develop a near-infrared fluorescent dye which does not stay in the liver or has short retention time.

Disclosure of Invention

In view of the deficiencies of the prior art, a first object of the present invention is to provide a heptamethine cyanine near-infrared dye; the dye has better metabolism in vivo, is not retained in organs such as liver for a long time and has tumor specific targeting property; the second purpose of the invention is to provide the application of the near-infrared fluorescent dye.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a heptamethine cyanine near-infrared fluorescent dye shown in a structural formula I,

wherein X is selected from SCH₂CH₂COOH、SCH₂COOH、SCH₂CH₂CONH(CH₂CH₂O)_n-CH₂CH₂NH₂Or SCH₂CH₂CONH(CH₂CH₂O)_mCH₂CH₂COOH, n and m are selected from 0-10.

The structural formula I is abbreviated as YQ-04-X below, and X is selected from SCH₂CH₂COOH、SCH₂COOH、SCH₂CH₂CONH(CH₂CH₂O)_n-CH₂CH₂NH₂Or SCH₂CH₂CONH(CH₂CH₂O)_mCH₂CH₂COOH, n and m are selected from 0-10.

Preferably, the near-infrared fluorescent dye is selected from the following I-1 or I-2:

the YQ-04-SCH₂CH₂The synthesis method of COOH (also called I-1) is as follows: in a dipolar aprotic solvent, a compound II (also called NIR-04) reacts with 3-mercaptopropionic acid and alkali to obtain YQ-04-SCH₂CH₂COOH。

The YQ-04-SCH₂CH₂CONH(CH₂CH₂O)_nCH₂CH₂NH₂The synthesis method of (also named I-2) comprises the following steps:

a) in a dipolar aprotic solvent, a compound II reacts with 3-mercaptopropionic acid and alkali to obtain YQ-04-SCH₂CH₂COOH；

b) YQ-04-SCH reaction in the presence of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), a base and a polar solvent₂CH₂COOH, and NH (CH)₂CH₂O)_nCH₂CH₂NH₂Or NH (CH)₂CH₂O)_nCH₂CH₂NHBoc reaction to obtain YQ-04-SCH₂CH₂CONH(CH₂CH₂O)_nCH₂CH₂NH₂Or YQ-04-SCH₂CH₂CONH(CH₂CH₂O)_nCH₂CH₂NH₂Boc(n＝0-10)；

c) Subjecting the YQ-04-SCH of step b)₂CH₂CONH(CH₂CH₂O)_nCH₂CH₂NH₂Boc (n-0-10) is removed under the action of TFA to obtain YQ-04-SCH₂CH₂CONH(CH₂CH₂O)_nCH₂CH₂NH₂。

The invention also protects the sodium salt, potassium salt and calcium salt forms of the heptamethine cyanine near-infrared fluorescent dye YQ-04-X.

The invention also protects the application of the heptamethine cyanine near-infrared fluorescent dye or the pharmaceutically acceptable salt thereof in preparing accurate tumor diagnosis reagents.

Preferably, the pharmaceutically acceptable salt is a sodium salt, a potassium salt or a calcium salt.

Preferably, the tumor is liver cancer, breast cancer, lung cancer or cervical cancer.

The invention also provides a pharmaceutical preparation which comprises the heptamethine cyanine near-infrared fluorescent dye or other prodrug forms as the active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier is a pharmaceutical carrier which is conventional in the pharmaceutical art and refers to one or more inert, non-toxic solid or liquid fillers, diluents, adjuvants and the like which do not interact with the active compound or the patient.

The dosage form of the pharmaceutical preparation can be common pharmaceutical dosage forms such as tablets, capsules, pills, suppositories, soft capsules, oral liquid, suspensions, injections and the like.

The pharmaceutical preparations of the present invention can be prepared according to methods well known in the pharmaceutical art.

Advantageous effects

The invention modifies on the basis of NIR-04 structure, replaces chlorine atoms, introduces carboxyl, PEG and the like, and under the condition of not changing key groups and solubility, the defect that NIR-04 is retained in liver and is not eliminated is improved, the uptake and targeting of the NIR-04 in tumor are not influenced, and a more proper connection site is provided for the subsequent application of NIR-04 as a near infrared fluorescent dye development fluorescent probe or radionuclide marking; in the research process, the key problem of the invention is to solve the problem that NIR-04 liver cannot be metabolically cleared and simultaneously the targeting capability of the NIR-04 liver is not changed.

Drawings

FIG. 1 shows YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂The synthetic route of (1);

FIG. 2 shows ICG, NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂The in vivo metabolism condition, wherein the graph A is the in vivo distribution condition of each dye mouse, and the graph B is the in vitro organ distribution condition of the dye mouse at different time;

FIG. 3 shows NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Fluorescence data, in turn, fig. A, B, C;

FIG. 4 shows ICG, NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Imaging a breast cancer Mcf7 tumor-bearing mouse;

FIG. 5 shows ICG, NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Imaging a 7721 liver cancer tumor-bearing mouse;

FIG. 6 shows ICG, NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Developing a cervical cancer Hela tumor-bearing mouse;

FIG. 7 shows ICG, NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Imaging a hepatoma Hepg2 tumor-bearing mouse;

FIG. 8A is a mass spectrum of YQ-04-SCH2CH2COOH, and B is a nuclear magnetic hydrogen spectrum thereof;

FIG. 9A is YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Mass spectrum, B is nuclear magnetic hydrogen spectrum;

FIG. 10 shows YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Boc mass spectrum.

Detailed Description

The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.

Example 1 YQ-04-SCH₂CH₂Synthesis of COOH

NIR-04(200mg,1.0eq)、HSCH₂CH₂COOH (55mg,2.0eq) and triethylamine (26mg, 1.0eq) were reacted in dimethyl sulfoxide (1ml) away from light for about 3 hours at room temperature, and the reaction was monitored by HPLCAnd (3) after the reaction is completely shown, dropping the reaction liquid into ethyl acetate to separate out dark green flocculent solid, filtering, purifying to obtain dark green solid (100mg), and storing in a dark place, wherein mass spectrum and nuclear magnetic hydrogen spectrum data are as follows: 847.47, m/z is equal to,¹h NMR (300MHz, DMSO) δ 8.78(dd, J ═ 23.1,14.1Hz,2H),7.74(s,1H),7.62(dd, J ═ 8.3,4.6Hz,2H),7.53(d, J ═ 8.0Hz,1H),7.41(dd, J ═ 15.2,7.9Hz,2H),7.27(t, J ═ 7.4Hz,1H),6.49(t, J ═ 14.5Hz,2H), 4.46-4.24 (m,4H),2.93(t, J ═ 10.9Hz,2H),2.67(s,4H),2.59(t, J ═ 6.7Hz,4H), 2.31-2.17 (m,2H),2.04(t, J ═ 10.6, 4H), potassium iodide (q, 1H), potassium iodide, sodium, potassium iodide, etc. 1, etc. 5-5 Hz,2H, etc. 5H, 2H, etc. and potassium iodide, etc. are used in the compounds, etc. to make up in the compounds, etc. to be used in the compounds, to be added to make up the reaction to₂CH₂COOH reaction to obtain.

Example 2 YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂To synthesize

YQ-04-SCH₂CH₂COOH(50mg,1.0eq),HATU(17mg，2.0eq)，NH₂(CH₂CH₂O)_2-CH₂CH₂NH₂(18mg, 2.0eq) and triethylamine (6mg, 1.0eq) in 300ul of dimethyl sulfoxide, reacting for 1 hour in the dark at room temperature, monitoring by HPLC, dropping the reaction solution into ethyl acetate after the reaction is completed, separating out dark green flocculent solid, filtering, purifying to obtain dark green solid (30mg), storing in the dark, confirming the structure by mass spectrum and nuclear magnetic hydrogen spectrum, wherein the mass spectrum data m/z is 977.35, m/z is 977.35,¹h NMR (300MHz, DMSO) δ 8.78(dd, J ═ 23.1,14.1Hz,2H),7.74(s,1H),7.62(dd, J ═ 8.3,4.6Hz,2H),7.53(d, J ═ 8.0Hz,1H),7.41(dd, J ═ 15.2,7.9Hz,2H),7.27(t, J ═ 7.4Hz,1H),6.49(t, J ═ 14.5Hz,2H), 4.46-4.24 (m,4H),2.93(t, J ═ 10.9Hz,2H),2.67(s,4H),2.59(t, J ═ 6.7Hz,4H), 2.31-2.17 (m,2H),2.04(t, J ═ 10.6, 4H), potassium iodide (q, 1H), potassium iodide, sodium, potassium iodide, etc. 1, etc. 5-5 Hz,2H, etc. 5H, 2H, etc. and potassium iodide, etc. are used in the compounds, etc. to make up in the compounds, etc. to be used in the compounds, to be added to make up the reaction to₂CH₂COCONH(CH₂CH₂O)₂CH₂CH₂NH₂And (3) reacting to obtain the compound.

Example 3 YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Synthesis of Boc

YQ-04-SCH₂CH₂COOH(50mg,1.0eq),HATU(17mg，2.0eq)，NH₂(CH₂CH₂O)_2-CH₂CH₂NH₂Boc (18mg, 1.2eq) and triethylamine (12mg, 2.0eq) in 300ul dimethyl sulfoxide were reacted at room temperature in the dark for 1 hour, HPLC was monitored, after the reaction was completed, the reaction solution was dropped into ethyl acetate to precipitate a dark green flocculent solid, which was then purified to obtain a dark green solid (50mg), which was stored in the dark.

Example 4 YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Synthesis of (2)

YQ-04-SCH₂CH₂COOH(50mg,1.0eq),HATU(17mg，2.0eq)，NH₂(CH₂CH₂O)_2-CH₂CH₂NH₂Boc (18mg, 1.2eq), triethylamine (12mg, 2.0eq), in 300ul dimethyl sulfoxide under dark room temperature for 1 hour, HPLC monitoring, after the reaction is completed, dropping the reaction solution into ethyl acetate, separating out dark green flocculent solid, purifying to obtain dark green solid (50mg), storing in dark, and mass spectrum data: and (3) adding TFA (100ul) into the obtained dark green solid, reacting for 30min at the dark room temperature, monitoring by HPLC (high performance liquid chromatography), drying trifluoroacetic acid after the reaction is completed, purifying to obtain the dark green solid (20mg), and storing in the dark.

Performance testing

1.ICG、NIR-04、YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)_2-CH₂CH₂NH₂Tumor-bearing mouse model imaging contrast inoculated to different human cell lines

To investigate NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂In vivo distribution, for ICG, NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂The four dyes are used for researching the metabolism conditions of in vivo and in vitro organs of a BCR (B cell receptor) white mouse, simultaneously constructing four subcutaneous tumor-bearing mouse models (inoculated in armpits), and monitoring ICG, NIR-04 and YQ-04-SCH in real time by a near-infrared fluorescence imaging technology₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Dynamic distribution in Mcf7 (breast cancer), 7721 (liver cancer), cervical cancer (Hela) and Hepg2 (liver cancer). In the experimental process, 785nm excitation is adopted, a received signal is selected to be in a wave band of 800nm-900nm (the imaging equipment adopts FL-10A of Nanjing Noyuan medical instrument Co., Ltd.), each dye is administrated according to ICG 1.25mg/kg, and the dye is respectively in a corresponding sodium salt form.

The experimental results showed that NIR-04 and YQ-04-SCH are shown from the 0, 2, 4, 6, 9, 12, 24, and 48 hour metabolism patterns in ICR mice, and the data and anatomical results of mice in vivo₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Compared with ICG, the metabolism is similar, the medicine can be completely discharged from the body after 24 hours, NIR-04 tail vein administration has strong fluorescence signal in the liver after 48 hours, and YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂Can be rapidly metabolized from liver after tail vein administration, is discharged from body via intestine, and does not stay in liver to influence the false positive of the subsequent excision in operation process, but modified NIR-04, YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH₂NH₂And the like show longer tumor retention time (up to 48 hours and even longer) in tumor-bearing mice of breast cancer (Mcf7, as shown in figure 4), cervical cancer (Hela, as shown in figure 6), liver cancer (7721, as shown in figure 5, and Hepg2, as shown in figure 7). Modified compound YQ-04-SCH₂CH₂COOH、YQ-04-SCH₂CH₂CONH(CH₂CH₂O)₂CH₂CH_2-NH2 and the like are taken 24 hours after tail vein administration, only the tumor part in the tumor-bearing mouse is left with fluorescence signals, and no fluorescence signals exist in liver, intestine, lung and the like, while NIR-04 is taken 24 hours after tail vein administration, except the tumor part with fluorescence signals, the liver part also has strong fluorescence signals, thus being not beneficial to the application in the aspect of later clinical transformation.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.