Phthalocyanine compound, preparation method and application thereof

Abstract

The invention provides a phthalocyanine compound as well as a preparation method and application thereof, and relates to the technical field of medicinal chemistry. The structure of the phthalocyanine compound is A-X-B. In the formula, A is a structure containing phthalocyanine and salts thereof; x is a divalent organic structure which enables A and B to be bonded through a chemical bond; and B contains a first polypeptide structure which specifically binds and / or recognizes urease. The phthalocyanine compound disclosed by the invention combines the effects of a photosensitizer and targeting, and has good targeting and antibacterial activity on helicobacter pylori.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry and relates to a phthalocyanine compound, a preparation method and application thereof. Background Art

[0002] Helicobacter pylori (H. pylori) infection is closely associated with the development of gastritis and gastric cancer. Urease decomposes urea in the host's gastric juice to produce ammonia and carbon dioxide, thereby forming a local alkaline microenvironment and neutralizing gastric acid, allowing H. pylori to survive in the highly acidic environment of the stomach. Therefore, inhibition of urease is detrimental to the survival of H. pylori. Urease inhibitors, such as acetohydroxamic acid (AHA) and flutolanil, have shown potential in disrupting H. pylori colonization in vitro. However, their clinical application is limited by low bioavailability, off-target effects, and toxicity.

[0003] Photodynamic antimicrobial therapy (PDAT) can generate reactive oxygen species (ROS) through light irradiation to destroy a variety of molecular targets (such as proteins, lipids, and nucleic acids), offering significant advantages in eliminating pathogens. Compared to traditional antibiotic therapy, PDAT has the advantage of being reusable without inducing adverse drug resistance.

[0004] The core of photodynamic antimicrobial therapy is photosensitizer. Summary of the Invention

[0005] The inventors discovered that compounds formed by chemically bonding a polypeptide that specifically recognizes and / or binds to urease to phthalocyanine and its salts can accumulate in the stomach and exhibit good photosensitivity. Upon exposure to light, they effectively kill Helicobacter pylori in the stomach. Based on this, the present invention provides a phthalocyanine compound, a preparation method, and its application.

[0006] The technical solutions of the present invention are as follows:

[0007] A phthalocyanine compound having the structure shown in the following formula (1):

[0008] AXB(1)

[0009] Wherein, A is a structure containing phthalocyanine and its salt; X is a divalent organic structure that allows A and B to be bonded via a chemical bond; and B contains a first polypeptide structure that specifically binds to and / or recognizes urease.

[0010] Preferably, the A is a structure containing one or a combination of two or more of zinc phthalocyanine, magnesium phthalocyanine, copper phthalocyanine, iron phthalocyanine, aluminum phthalocyanine and calcium phthalocyanine.

[0011] More preferably, the A is a structure containing zinc phthalocyanine.

[0012] Preferably, X is an amide-substituted C1-C6 divalent alkyl group.

[0013] Preferably, the structure of B is as shown in the following formula (2):

[0014]

[0015] wherein R is absent, a C1-C10 unsubstituted divalent alkyl group, or a C2-C40 substituted divalent alkyl group.

[0016] More preferably, R is selected from C2-C10 unsubstituted divalent alkyl groups.

[0017] More preferably, the R comprises a structure consisting of 1-4 amino acids.

[0018] More preferably, the structure of R is as shown in the following formula (3) or (4):

[0019]

[0020] A method for preparing the phthalocyanine compound according to any one of the above embodiments, wherein the phthalocyanine compound is obtained by condensing a carboxyl-substituted phthalocyanine and a salt thereof with a second polypeptide represented by the following formula (5):

[0021] NH2-B(5)

[0022] The meaning of B is as above.

[0023] Preferably, the carboxyl-substituted phthalocyanine and its salt are monocarboxyl-substituted phthalocyanine and its salt;

[0024] Alternatively, the phthalocyanine compound is obtained by condensing an amino-substituted phthalocyanine and a salt thereof with a third polypeptide represented by the following formula (6):

[0025] HOOC-B(6)

[0026] The meaning of B is as above.

[0027] Preferably, the amino-substituted phthalocyanine and its salt are monoamino-substituted phthalocyanine and its salt.

[0028] A phthalocyanine compound according to any of the above embodiments or a phthalocyanine compound prepared by the method for preparing the phthalocyanine compound according to the above embodiments is used as a detection reagent or treatment reagent for Helicobacter pylori infection, or for preparing a detection reagent or treatment reagent for Helicobacter pylori infection.

[0029] The beneficial effects of the present invention are:

[0030] (1) The phthalocyanine compounds of the present invention can specifically recognize and / or bind to the urease of Helicobacter pylori, thereby being enriched in the stomach infected with Helicobacter pylori, with high recognition and good targeting. Furthermore, by using medical optical fiber to transmit light, such as LED lights, the phthalocyanine compounds can quickly and easily kill Helicobacter pylori in the stomach, with excellent antibacterial properties.

[0031] (2) The preparation method of the phthalocyanine compound of the present invention is simple and can be obtained through a condensation reaction such as an amidation reaction.

[0032] (3) The phthalocyanine compound of the present invention has high photoactivity and can significantly kill a variety of Helicobacter pylori including drug-resistant strains under light irradiation. It has broad-spectrum antibacterial properties and can prevent the formation of drug-resistant Helicobacter pylori strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of testing the antibacterial activity of two phthalocyanine compounds against H. pylori (a) ATCC 43504, (b) ATCC 26695 and (c) MDR-H. pylori.

[0034] Figure 2 To test the ability of three phthalocyanine compounds to produce reactive oxygen species under light.

[0035] Figure 3 Fluorescence imaging of PS1 in mouse gastrointestinal tissue (left) and other organs (heart, liver, spleen, lung, and kidney, right) after four oral administrations.

[0036] Figure 4 To test the results of five PS1-based photodynamic antibacterial treatments. DETAILED DESCRIPTION

[0037] Definition of terms :

[0038] The term "urease" refers to an enzyme having urea amidohydrolase (EC 3.5.1.5) enzymatic activity, either naturally occurring or obtained, for example, by recombinant nucleic acid technology and / or chemical synthesis. Urease can also include fusion proteins comprising the complete urease, subunits, or fragments thereof, and / or ureases that retain the urea amidohydrolase activity of a peptide by amino acid substitution, deletion, or addition.

[0039] The term "polypeptide" refers to a biological polymer composed of 3-50 amino acid or amino acid analog subunits, typically some or all of the 20 common L-amino acids found in biological proteins, connected by peptide intersubunit bonds or other intersubunit bonds.

[0040] The term "short peptide" refers to a biopolymer composed of 2-10 amino acid or amino acid analog subunits, typically some or all of the 20 common L-amino acids found in biological proteins, connected by peptide intersubunit bonds or other intersubunit bonds.

[0041] The term "specific binding" refers to the process by which biological molecules can accurately recognize and selectively bind based on structural and functional complementarity.

[0042] The term "specific recognition" refers to the ability of immune cells or molecules to accurately identify specific substances from a large number of complex substances.

[0043] The term "targeting" refers to the process by which a specific drug or treatment selectively acts on a specific biological target molecule or cell.

[0044] The technical solution of the present invention is further illustrated and described below through specific implementation methods.

[0045] In order to achieve efficient killing of Helicobacter pylori, including drug-resistant Helicobacter pylori strains, the present invention provides a phthalocyanine compound having a structure shown in the following formula (1):

[0046] AXB(1)

[0047] Wherein, A is a structure containing phthalocyanine and its salt; X is a divalent organic structure that allows A and B to be bonded via a chemical bond; and B contains a first polypeptide structure that specifically binds to and / or recognizes urease.

[0048] Phthalocyanine and its salts have good photosensitivity, and the structural formulas are shown in (A-1) and (A-2).

[0049]

[0050] In formula (A-2), M represents a metal ion, such as Mg 2+ 、Zn 2+ 、Al 3+ 、Na 2+ 、Fe 3+ , Ca 2+ Phthalocyanine and its salts can be excited to produce singlet oxygen under light irradiation, which has important applications in photodynamic therapy.

[0051] In the phthalocyanine compound shown in the above formula (1) of the present invention, the A structure provides good photosensitivity for the phthalocyanine compound and excites the production of singlet oxygen under light irradiation, thereby killing microorganisms; the B structure contains a polypeptide structure that specifically binds and / or recognizes Helicobacter pylori urease, so that the phthalocyanine compound can recognize and bind to urease. Urease is mainly present in the gastrointestinal tract of the human body, especially in the site of Helicobacter pylori infection, with high recognition and good targeting. Therefore, the phthalocyanine compound can be enriched in the gastrointestinal tract of the human body, especially in the site of Helicobacter pylori infection, and plays a better and more concentrated role in identifying and / or killing microorganisms (such as Helicobacter pylori); the role of X is to chemically bond A and B, so that the phthalocyanine compound has good stability in the gastrointestinal tract (such as an acidic environment), and exerts the effects of photodynamic and antibacterial sterilization. From the perspective of reactivity, in the structure of X, it is preferred that the side chain does not contain a large-volume group such as a benzene ring or a cycloalkyl group to avoid generating a large steric hindrance.

[0052] In the present invention, A is a structure containing phthalocyanine and its salts, which may be phthalocyanine, zinc phthalocyanine, copper phthalocyanine, magnesium phthalocyanine, calcium phthalocyanine, etc. Therefore, in some embodiments, A is a structure containing one or a combination of two or more of zinc phthalocyanine, copper phthalocyanine, iron phthalocyanine, aluminum phthalocyanine, and calcium phthalocyanine. For example, A is a structure containing zinc phthalocyanine. Furthermore, A is a phthalocyanine and its salt structure, such as a zinc phthalocyanine structure.

[0053] In some embodiments, X is an amide-substituted C1-C6 divalent alkyl group. The amide structure is stable and easy to prepare and form bonds. For example, X can be -CONH-, -CH2CONH-, -CONHCH2-, -CH2CH2CONH-, -CH2CONHCH2-, -CH2CH2CONHCH2-, etc., which can be obtained by condensation reaction of the corresponding carboxyl group and amino group.

[0054] In some embodiments, the structure of B is shown in Formula (2),

[0055]

[0056] wherein R is absent, a C1-C10 unsubstituted divalent alkyl group, or a C2-C40 substituted divalent alkyl group. In the structure B represented by formula (2), the structure to the right of R is a fourth polypeptide structure (the structure within the dotted box in the structure represented by formula (2), and the structure of the fourth polypeptide corresponding to the fourth polypeptide structure is represented by formula (A-3) below). This structure has specific recognition and binding capabilities for urease and serves as the fundamental structure for enabling the specific recognition and binding of the phthalocyanine compound of the present invention to urease.

[0057]

[0058] However, the side chain of the leftmost C atom of the fourth polypeptide represented by formula (A-3) contains a phenol structure, which creates significant steric hindrance to the adjacent amino group (indicated by the arrow in the structure represented by formula (A-3)). This results in low reactivity of this amino group, making it difficult to chemically bond with phthalocyanine and its salts through chemical reactions (such as amidation reactions), or the yield of the chemical reaction is very low. By introducing the R structure, the structure represented by formula (A-3) is more likely to chemically bond with phthalocyanine and its salts through chemical reactions, thereby improving the reaction efficiency and reaction degree.

[0059] In some embodiments, R is selected from a C2-C10 unsubstituted divalent alkyl group, for example, R can be -(CH2)3-, -(CH2)4-, -(CH2)6-, -(CH2)8-, etc. Due to the low steric hindrance between the phthalocyanine and its salt structure and the second polypeptide structure, it does not affect or has little effect on the above-mentioned chemical reactions, such as the amidation reaction.

[0060] In some embodiments, R comprises a structure composed of 1-4 amino acids. In this case, the phthalocyanine compound has better hydrophilicity, biocompatibility, biological activity, etc. The phthalocyanine compound can better specifically recognize and bind to urease and produce a good function of killing microorganisms under light irradiation, such as Helicobacter pylori, including drug-resistant Helicobacter pylori strains.

[0061] In some embodiments, the structure of R is as shown in formula (3) or (4).

[0062]

[0063] When the structure of R is as shown in formula (3) or (4), the corresponding structure of B can be as shown in the following formulas (A-4) and (A-5).

[0064]

[0065]

[0066] Taking the structure A as zinc phthalocyanine (code name PS) and the structures B as the structures shown in formula (A-4) and formula (A-5) as examples, the corresponding phthalocyanine compounds can be shown as the following formulas (A-6) and (A-7) respectively.

[0067]

[0068] On the other hand, the present invention also provides a method for preparing the phthalocyanine compound described in any of the above embodiments, which can be obtained by the following method 1 or method 2:

[0069] Method 1: Carboxyl-substituted phthalocyanine and its salt are subjected to condensation reaction with the second polypeptide represented by the following formula (5).

[0070] NH2-B(5)

[0071] The meaning of B is as above.

[0072] In some embodiments, the carboxyl-substituted phthalocyanine and its salts are monocarboxyl-substituted phthalocyanine and its salts;

[0073] In this case, the carboxyl group of the carboxyl-substituted phthalocyanine and its salt undergoes an amidation reaction with the NH2 group of the second polypeptide to produce a phthalocyanine compound. For example, the phthalocyanine compound represented by formula (A-6) can be obtained by reacting monocarboxyl zinc phthalocyanine with a polypeptide (A-9) as shown in formula (A-8).

[0074]

[0075] The phthalocyanine compound represented by the above formula (A-7) can be obtained by reacting monocarboxyphthalocyanine zinc with a polypeptide (A-11) as shown in the following formula (A-10).

[0076]

[0077] The reaction method represented by the above formula (A-8) is well known to those skilled in the art, such as the reaction under the action of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA). The above polypeptide (A-9) can be obtained by amino acid condensation reaction of the fourth polypeptide represented by formula (A-3) and the short peptide represented by the following formula (A-12), such as liquid phase synthesis (e.g., using DCC as a condensing agent), solid phase synthesis (e.g., microwave-assisted solid phase synthesis), enzyme-catalyzed synthesis, etc., which are well known to those skilled in the art.

[0078]

[0079] Similarly, the above-mentioned polypeptide (A-11) can be obtained by subjecting the fourth polypeptide represented by formula (A-3) and the corresponding short peptide to an amino acid condensation reaction, which is well known to those skilled in the art.

[0080] For the reaction of formula (A-8), in addition to directly reacting the two raw materials—zinc phthalocyanine and polypeptide (A-9)—to obtain the product, polypeptide (A-9) can also be loaded onto Wang resin before reacting with zinc phthalocyanine. This facilitates post-processing. After post-processing, the loaded connection between the product and Wang resin is removed to obtain the phthalocyanine compound represented by formula (A-6). The above-mentioned Wang resin loading method is well known to those skilled in the art.

[0081] Method 2: The product is obtained by condensing an amino-substituted phthalocyanine and its salt with the third polypeptide represented by the following formula (6).

[0082] HOOC-B(6)

[0083] The meaning of B is as above.

[0084] In some embodiments, the amino-substituted phthalocyanine and its salts are monoamino-substituted phthalocyanine and its salts.

[0085] The method for obtaining a phthalocyanine compound by condensing an amino-substituted phthalocyanine and its salt with the third polypeptide represented by formula (6) (amidation reaction) is also well known to those skilled in the art, for example, the reaction occurs under the action of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA).

[0086] In the above method 2, the raw materials and reaction methods can refer to the description of the above method 1.

[0087] On the other hand, the present invention also proposes the use of a phthalocyanine compound described in any of the above embodiments or a phthalocyanine compound prepared by the preparation method of the phthalocyanine compound described in the above embodiments as a detection reagent or treatment reagent for Helicobacter pylori infection, or for preparing a detection reagent or treatment reagent for Helicobacter pylori infection.

[0088] Because the phthalocyanine compound of the present invention has photosensitivity and the ability to specifically recognize and bind to urease, it can be enriched in the stomach and intestines, especially the location of Helicobacter pylori, and play a role in detecting or killing Helicobacter pylori infection, especially having good killing performance against drug-resistant Helicobacter pylori strains. It can be directly used as a detection reagent or treatment reagent for Helicobacter pylori infection, or as one of the components of a detection reagent or treatment reagent for Helicobacter pylori infection.

[0089] The technical solution of the present invention is further described and illustrated below based on various embodiments.

[0090] Example 1

[0091] This example is to prepare the phthalocyanine compound represented by the above formula (A-6)

[0092] To a round-bottom flask, add 2 mL of anhydrous dimethylformamide, followed by monocarboxyphthalocyanine zinc (24.88 mg, 0.04 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (30.5 mg, 0.08 mmol), and N,N-diisopropylethylamine (0.04 mL). Stir at room temperature for 30 min. Weigh Wang resin loaded with the peptide (A-9) (192 mg, 0.04 mmol) and add to the solution. Stir at room temperature for 24 h.

[0093] After the reaction is completed, the supernatant is removed by centrifugation (8000 rpm, 10 min) to obtain a solid precipitate. The filtered solid precipitate is washed 3 times with pure DMF and methanol respectively until the washing liquid is colorless. At this time, the solid product is a photosensitizer connected to the resin. The solid product is placed in a freeze dryer for freeze drying.

[0094] The solid product was added to 2 ml of 95% trifluoroacetic acid aqueous solution and shaken at room temperature for 3 hours to remove the side-chain protecting groups from the Wang resin. The filtrate was then collected using a Buchner funnel. 10 volumes of anhydrous ether, precooled to 2°C, was added to the filtrate. After 7 minutes, the precipitate was collected and dried to obtain the product phthalocyanine compound, referred to as PS-1.

[0095] Example 2

[0096] This example differs from Example 1 in that the Wang resin loaded with the polypeptide (A-9) in Example 1 was replaced with a Wang resin loaded with the polypeptide (A-11), with the polypeptide (A-11) and polypeptide (A-9) in equimolar amounts. The remaining steps remained unchanged. The phthalocyanine compound represented by the formula (A-7) was obtained, referred to as PS-2.

[0097] Performance Testing

[0098] Test 1: Minimum inhibitory concentration (MIC) of phthalocyanine compounds against H. pylori

[0099] The minimum inhibitory concentration (MIC) was determined using the two-fold dilution method. The bacterial solution containing the test compound was diluted two-fold using BHI medium containing 10% FBS, and the MIC value of the drug was determined. The experimental steps are as follows: the stock solution of the test compound was diluted to different concentrations using BHI medium, and then 100 μL of bacterial solution (bacterial concentration is approximately 10 6 CFU / mL). Two groups of control wells were set up, one without bacterial solution (containing only culture medium) and one without test compound (containing only bacterial solution and culture medium) to eliminate the interference of culture medium and operation process on experimental results. At the same time, control wells containing only bacterial solution and culture medium were set up to verify the normal growth ability of the strain. Three parallel experiments were set up for each group. The illumination group was given 680nm wavelength, 54J / cm 2 The cells were cultured under microaerophilic conditions at 37°C and 150 rpm for 24 hours. Bacterial growth was determined by observing changes in the culture medium. When the liquid became clear and there was no flocculent or lumpy white precipitate at the bottom, bacterial growth was absent. If no bacterial growth was observed in the culture medium, the corresponding compound concentration was the minimum inhibitory concentration (MIC). The results are shown in Table 1.

[0100] Table 1 MIC test results

[0101]

[0102] The experimental results showed that under light conditions, the MIC of phthalocyanine compounds against standard H.pylori was significantly lower than that of photosensitizers used alone. Among them, the MIC value of PS1 and PS2 against the standard strain was 25μM, which was 4 times lower than the MIC value of PS (phthalocyanine zinc) (100μM). This shows that phthalocyanine compounds can significantly enhance the inhibitory effect on H.pylori after being combined with peptides targeting urease as photosensitizers. In addition, the sensitivity of multidrug-resistant strains isolated from the clinic (MDR H.pylori in Table 1) to phthalocyanine compounds is basically consistent with that of the standard strain, proving that phthalocyanine compounds also have the same inhibitory effect on antibiotic-resistant bacteria.

[0103] Test 2: Antibacterial activity of phthalocyanine compounds against Helicobacter pylori

[0104] In a clean bench, use a pipette tip to gently pick up a small amount of glycerol bacteria (ATCC 43504, ATCC 26922, MDRH.pylori) on the blood plate. Use a sterile inoculation loop to streak. The inoculated blood plate was revived and cultured under microaerobic conditions for 3 days. After 3 days, the blood plate was removed, and the surface colonies were scraped with a sterile inoculation loop and added to the liquid culture medium. It was placed in a sealed culture tank and continued to expand the culture in a 37°C shaker at a shaking speed of 190 rpm. The bacterial solution in the logarithmic phase was diluted 1000 times with PBS (the bacterial solution concentration was about 10 5 Take 100 μL of 25 μM phthalocyanine compound and 100 μL of H. pylori suspension and incubate them in a 96-well plate at room temperature for 10 min. The illumination group was illuminated for 20 min (680 nm, 45 mW / cm 2 ), the control group was wrapped in tin foil to protect from light throughout the entire process. After the end of the light exposure, the cells were serially diluted and 100 μL was spread on blood agar plates. The cells were cultured in sealed culture jars at 37°C for 3 days and then the colonies were counted.

[0105]

[0106] The calculation formula of antibacterial rate is shown in formula (7), where N A Refers to the number of bacterial colonies on the blood plate after the test compound is co-incubated with H. pylori, N B It refers to the number of bacterial colonies on the blood agar plate after PBS and H. pylori are co-incubated.

[0107] The results are as attached Figure 1Figures 2 and 3 show the antibacterial activity of phthalocyanine compounds against H. pylori (a) ATCC 43504, (b) ATCC 26695, and (c) MDR-H. pylori. (d) shows the results of blood agar plate culture. Under 20 minutes of light exposure, phthalocyanine compounds at a concentration of 25 μM reduced the colony-forming units of H. pylori, including multidrug-resistant strains, by 3 logs (i.e., a bacterial kill rate of 99.9% or greater). This result demonstrates that PS1 and PS2 exhibit significant bactericidal activity under light-activated conditions, particularly against multidrug-resistant strains.

[0108] Test 3: Detection of reactive oxygen species produced by phthalocyanine compounds under light

[0109] To evaluate the ability and types of ROS generated by phthalocyanine compounds under light, 2,7-dichlorofluorescein diacetate (DCFH-DA) was first used as a probe for detection. DCFH-DA is oxidized to the fluorescent DCF under the action of ROS, and its fluorescence intensity (Ex = 488 nm, Em = 525 nm) is proportional to the amount of ROS generated. Furthermore, to distinguish between different types of ROS, NaN3 (final concentration 30 mM) was added to specifically quench singlet oxygen. As a control, a PBS group without quencher was set up to assess the total amount of ROS generated. The phthalocyanine compounds to be tested (final concentration 10 μM) were dissolved in PBS containing 20 μM DCFH-DA, with a total volume of 200 μL per group. Subsequently, the solutions were illuminated with a planar LED light source (wavelength 680 nm) for 2 minutes each, and the fluorescence intensity changes of the solutions were recorded after each illumination period.

[0110] In order to further verify that phthalocyanine compounds generate singlet oxygen ( 1 The singlet oxygen fluorescence probe SOSG (Singlet Oxygen Sensor Green) was also used for detection. A phthalocyanine solution (final concentration 10 μM) and PBS were added to a 96-well plate and illuminated under a flat LED light (wavelength 680 nm, duration 5 minutes). The fluorescence signal intensity (Ex = 504 nm, Em = 525 nm) was then measured using a microplate reader.

[0111] The results are as attached Figure 2 The photodynamic effect of photosensitizer is mainly the process of generating ROS under the excitation of light of a specific wavelength. SOSG probe and DCFH-DA probe were used to detect PS1 and PS2 under light (680nm, 45mW / cm 2 ) under light conditions. Under light conditions, both PS1 and PS2 can generate 1O2, and the fluorescence signal rose rapidly, reaching a plateau within 2 minutes. No significant fluorescence signal was observed in the non-illumination group and the blank control group. In addition, the fluorescence signal of the ROS probe DCFH-DA was observed to increase steadily over time, indicating that the generation of ROS continued during the experiment. Hydroxyl radicals (·OH), as a component of ROS, can be quenched by NaN3. It was observed that in the presence of NaN3, the fluorescence intensity of PS1 and PS2 decreased to varying degrees, but the generation of fluorescence signals was still detected. This shows that PS1 and PS2 can simultaneously generate type I and type II ROS under the action of light.

[0112] Test 4: Fluorescence imaging of PS1 in mouse gastrointestinal tissue (left) and other organs (heart, liver, spleen, lung, and kidney, right) after oral administration

[0113] Six-week-old ICR mice (weighing approximately 18-20 g) were selected to establish an in vivo H. pylori infection model. The experiment was conducted after one week of adaptive feeding. After fasting for 12 hours, each mouse was orally administered with an H. pylori suspension (concentration of 10 8 CFU / 200μL), once every other day, for a total of 5 inoculations. After the inoculation, the mice were kept for 2 weeks to simulate the gastric colonization process of H. pylori. After fasting for 12 hours, the infected model mice and healthy control mice (n=5 / group) were given oral administration of PS1 solution (final dose 0.6mg / kg, 200μL). After 12 hours, the mice were sacrificed and the stomach, small intestine, large intestine and other major organs (such as heart, liver, kidney, spleen, lung, etc.) were collected for fluorescence imaging and quantification of fluorescence intensity.

[0114] The results are as attached Figure 3 As shown. Taking PS1 as an example, 12 hours after oral administration, fluorescence imaging analysis was performed on the main organs of mice in the infected and uninfected groups. The results showed that after 12 hours, the fluorescence signal of PS1 in the gastric tissue of the infected mice was significantly stronger than that of the uninfected mice. In addition, in other major organs (right side, such as heart, liver, spleen, lung, and kidney), there was no significant difference in the distribution of fluorescence signals between the two groups of mice, indicating that PS1 had less adverse effects on other organs. The experimental results show that PS1 can attach to the surface of the gastric mucosa of infected mice, and the high retention rate of PS1 in gastric tissue can increase its utilization rate and achieve better antibacterial effect.

[0115] Test 5: PS1-based photodynamic antibacterial therapy detection

[0116] Mice infected with H.pylori were randomly divided into four groups, including a negative control group, a positive control group using triple therapy with commonly used clinical antibiotics, a compound PS1 light-avoidance group, and a PS1-based PDT group. The mice in the triple therapy group were orally administered with omeprazole (138 mg / kg) every day, and 30 minutes later, they were orally administered with clarithromycin (14.3 mg / kg) and amoxicillin (28.5 mg / kg) for three consecutive days. The mice in the PS1 light-avoidance group and the PDT group were orally administered with a single dose of PS1 (0.89 mg / kg). After the administration of PS1, the mice in the PDT group were treated with a medical fiber-coupled laser system (wavelength 670 nm, light dose 15 J / cm 2 ) The stomach was illuminated. 48 hours after treatment, the mouse stomachs were removed, opened longitudinally along the greater curvature, and the gastric contents removed. The stomach cavity was rinsed with sterile PBS and ground with a tissue grinder to collect a bacterial suspension. Urease activity was assayed using a urease assay kit. The collected suspension was diluted 10-fold and plated on Columbia blood agar plates. The plates were incubated under microaerophilic conditions for 2-3 days. Changes in H. pylori colonization in each treatment group were assessed by counting colony-forming units.

[0117] The results are as attached Figure 4 As shown. In the photodynamic therapy group, irradiation was performed through a medical fiber optic system 0.5h after oral administration. The fiber optic was fixed by an animal gavage needle with a diameter of about 1.0mm. The device was portable and easy to adjust. When treating H.pylori-infected mice, a current of 4A was used, corresponding to a power of about 40mW. After the treatment, the in vivo therapeutic effect was evaluated by re-culture of gastric tissue and pathological analysis. In the PS1 photodynamic therapy group, the H.pylori load was 3.90log 10 CFU, which is comparable to that of the triple therapy group. This result shows that PS1 combined with photodynamic therapy can effectively inhibit H. pylori, and its antibacterial effect can replace traditional antibiotic treatment. In addition, by collecting gastric tissues of mice in each treatment group for urease activity detection, it was found that the urease activity of the PS1 photodynamic therapy group and the triple therapy treatment group was significantly reduced. The experimental results further confirmed the effectiveness of peptide-modified phthalocyanine compounds against H. pylori and showed their potential as an alternative strategy to antibiotics. In addition, the weight of mice in each group remained stable during the experiment, indicating that the experimental treatment did not significantly affect their diet and digestive function, further verifying the safety and feasibility of PS1 photodynamic therapy.

[0118] Therefore, the phthalocyanine compounds of the present invention combine the efficient reactive oxygen species generation capability of zinc phthalocyanine as a photosensitizer with a polypeptide that specifically recognizes and / or binds to urease, resulting in excellent targeting and antibacterial activity. In vitro experiments have shown that at the minimum inhibitory concentration, the phthalocyanine compounds of the present invention can achieve a 99.9% bactericidal rate and maintain good biosafety at this concentration.

[0119] As described above, the basic principles, main features, and advantages of the present invention are shown and described. Those skilled in the art should understand that the present invention is not limited to the above-described embodiments. The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A phthalocyanine compound, characterized in that It has the structure shown in the following formula (1), AXB(1) Wherein, A is a structure containing phthalocyanine and its salt; X is a divalent organic structure that allows A and B to be bonded via a chemical bond; and B contains a first polypeptide structure that specifically binds to and / or recognizes urease.

2. The phthalocyanine compound according to claim 1, wherein The A is a structure containing one or a combination of two or more of phthalocyanine zinc, phthalocyanine magnesium, phthalocyanine copper, phthalocyanine iron, phthalocyanine aluminum and phthalocyanine calcium.

3. The phthalocyanine compound according to claim 2, characterized in that The A is a structure containing zinc phthalocyanine.

4. The phthalocyanine compound according to claim 1, characterized in that The X is an amide-substituted C1-C6 divalent alkyl group.

5. The phthalocyanine compound according to claim 1, characterized in that The structure of B is shown in the following formula (2): wherein R is absent, a C1-C10 unsubstituted divalent alkyl group, or a C2-C40 substituted divalent alkyl group. The phthalocyanine compound according to claim 5, characterized in that The R is selected from C2-C10 unsubstituted divalent alkyl groups.

7. The phthalocyanine compound according to claim 5, characterized in that The R comprises a structure consisting of 1 to 4 amino acids.

8. The phthalocyanine compound according to claim 7, characterized in that The structure of R is shown in the following formula (3) or (4), 9. A method for preparing the phthalocyanine compound according to any one of claims 1 to 8, characterized in that: The phthalocyanine compound is obtained by condensing carboxyl-substituted phthalocyanine and its salt with the second polypeptide represented by the following formula (5): NH2-B(5) The meaning of B is as above. Preferably, the carboxyl-substituted phthalocyanine and its salt are monocarboxyl-substituted phthalocyanine and its salt; Alternatively, the phthalocyanine compound is obtained by condensing an amino-substituted phthalocyanine and a salt thereof with a third polypeptide represented by the following formula (6): HOOC-B(6) The meaning of B is as above. Preferably, the amino-substituted phthalocyanine and its salt are monoamino-substituted phthalocyanine and its salt.

10. Use of the phthalocyanine compound according to any one of claims 1 to 8 or the phthalocyanine compound prepared by the method for preparing the phthalocyanine compound according to claim 9, characterized in that: As a detection reagent or treatment reagent for Helicobacter pylori infection, or for preparing a detection reagent or treatment reagent for Helicobacter pylori infection.

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