Preparation method and application of novel lipophilic gallium complex with activity

Abstract

The invention relates to the technical field of antibacterial drugs, and particularly discloses a preparation method and application of a novel lipophilic gallium complex with activity. A preparation method of a novel lipophilic gallium complex with activity comprises the following steps: firstly, mixing Ga (NO3) 3.9 H2O and 2-hydroxy-3-methyl-1, 4-naphthoquinone in a 90% ethanol solution containing K2CO3, then stirring at room temperature for 3 hours for synthesis reaction, then filtering and collecting red solid filter residues, then washing with ethanol for 3 times, and drying in a vacuum oven to obtain the novel lipophilic gallium complex with activity. The molecular formula of the complex is C33H21O9Ga. The novel lipophilic gallium complex with activity provided by the invention can be used for preparing a medicine for resisting gram-negative bacteria, and has the advantages of efficient antibacterial activity and capability of effectively inhibiting the formation of a biological membrane and eradicating a mature biological membrane; in addition, the preparation method disclosed by the invention has the advantages of simplicity and convenience in operation, mild conditions and high product purity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibacterial drugs, more particularly, it relates to a preparation method and application of a new type of active lipophilic gallium complex. BACKGROUND

[0002] As a metal element of the same group as aluminum, the trivalent ion (Ga 3+ ) of gallium (Ga) exhibits unique properties in biological systems. Because its ionic radius, charge, and coordination number are similar to those of the trivalent iron ion (Fe 3+ ), it can competitively interfere with iron acquisition and metabolic pathways in cells, thereby exerting antibacterial, anticancer, and anti-inflammatory activities. For example, clinical phase II trials have shown that gallium nitrate can effectively treat chronic lung infections caused by Pseudomonas aeruginosa in patients with cystic fibrosis, highlighting its potential as an antibacterial agent. However, Ga 3+ has a serious hydrolysis problem in aqueous solution: when dissolved, it forms a hexahydrate [Ga(H2O)6] 3+ However, at neutral pH, due to the high charge radius ratio and hard Lewis acid properties, Ga 3+ is highly prone to hydrolysis to form insoluble hydroxide or oxyhydroxide, both of which have very low solubility at physiological pH; this not only significantly reduces the bioavailability of Ga 3+ , limiting its application in oral or intravenous injection, but also leads to the accumulation of insoluble deposits in the kidneys, causing renal toxicity, which is the main safety obstacle in clinical application.

[0003] To overcome the hydrolysis problem, related technologies usually use ligand stabilization strategies, such as adding citrate to gallium nitrate formulations to form soluble complexes to prevent the formation of precipitates. However, such methods have limited effectiveness: citrate can stabilize Ga 3+ in vitro, but it has little effect on pharmacokinetics in vivo. Studies have found that the proportion of citrate complex to total gallium in animal models is less than 1%, and stronger ligands cannot completely prevent the dissociation of Ga 3+ and the formation of hydrolysis products. This not only affects the delivery efficiency of Ga 3+ , but also weakens its antibacterial activity, especially when combating gram-negative bacteria. The decrease in bioavailability and toxicity risk caused by hydrolysis limit the therapeutic effect. SUMMARY

[0004] To solve the problem of weakened antibacterial activity and limited therapeutic effect caused by the use of ligand stabilization strategies and the addition of citrate to gallium nitrate formulations to form soluble complexes, the present application provides a preparation method and application of a new type of active lipophilic gallium complex.

[0005] The application provides a preparation method of a new type of active lipophilic gallium complex, which adopts the following technical scheme:

[0006] The preparation method of the new type of active lipophilic gallium complex comprises the following steps: firstly, Ga(NO3)3·9H2O is mixed with 2-hydroxy-3-methyl-1,4-naphthoquinone in a 90% ethanol solution containing K2CO3, then a synthesis reaction is stirred at room temperature for 3 hours, then red solid filter residues are collected, then the red solid filter residues are washed with ethanol for 3 times and dried in a vacuum oven, and finally a complex with a molecular formula of C 33 H 21 O9Ga is obtained, and the reaction formula is as follows:

[0007]

[0008] Through the above technical scheme, the 90% ethanol solvent system balances the solubility of the gallium salt and the ligand through moderate polarity, and ensures that the reaction is homogeneous; and the room temperature condition avoids the decomposition of the ligand or the hydrolysis of the gallium ion caused by high temperature, and the chelation reaction of Ga 3+ and the ligand is fully completed within 3 hours of stirring, so that a stable octahedral coordination structure is formed; then the filter step separates the red solid product precipitated due to the disappearance of the molecular polarity, and the ethanol washing effectively removes the unreacted impurities, and then the vacuum drying removes the solvent molecules, so that the high-purity target complex is finally obtained.

[0009] Preferably, the chemical formula of the 2-hydroxy-3-methyl-1,4-naphthoquinone is C 11 H8O3.

[0010] Through the above technical scheme, the ortho-hydroxyl group and the carbonyl group in the C 11 H8O3 molecule form a bidentate coordination unit, the rigid naphthoquinone skeleton provides spatial matching during the complexation process, and the three ligands form octahedral coordination bonds with Ga 3+ through six oxygen donors, the hydrophobic naphthalene ring directionally wraps the metal center, and the lipophilic feature of the complex is endowed, and the specific molecular structure is the structural basis for the complex to penetrate the bacterial cell membrane and maintain stability.

[0011] Preferably, the addition amount of the K2CO3 is 1.5 mmol based on 1.0 mmol of Ga(NO3)3·9H2O.

[0012] Through the above technical scheme, 1.5 mmol of K2CO3 provides an alkaline environment to deprotonate the ligand hydroxyl group, which corresponds to the neutralization requirement of the acidic sites of 3 equivalent ligands; and the carbonate ion avoids competing for the metal binding site through weak coordination, while maintaining the pH value in the range of 7-8, which not only inhibits the hydrolysis and precipitation of Ga 3+ , but also ensures that the ligand is completely converted into an active anion form to participate in the complexation.

[0013] Preferably, cold deionized water is added to induce precipitation after the reaction is completed.

[0014] By adopting the technical scheme, cold deionized water with a temperature of ≤10°C is added, and then the solubility of the complex is reduced by a solvent polarity mutation, so that the intermolecular hydrophobic interaction is enhanced to direct crystallization; because the low-temperature condition inhibits the occurrence of side reactions, the water molecules do not participate in coordination as a precipitation medium, and the product is ensured to be precipitated in the form of an intact crystal, which is convenient for subsequent separation and purification.

[0015] Preferably, the vacuum drying condition is overnight drying.

[0016] By adopting the technical scheme, the embedded ethanol and water molecules in the crystal lattice are removed through a continuous negative pressure gradient in a vacuum environment combined with a drying time of 8-12 hours; this drying parameter avoids the destruction of the crystal structure caused by high temperature, maintains the stability of the intramolecular hydrogen bond network, and reduces the water content of the product.

[0017] Preferably, the complex belongs to the rhombohedral system, has a space group R3c, and has a unit cell parameter of a=b=16.0686A, c=39.771A, and γ=120°.

[0018] By adopting the technical scheme, the rhombohedral system parameter a=b=16.0686A reflects the symmetrical arrangement of three ligands to form a propeller configuration, the axial length c=39.771A corresponds to the stacking distance of the naphthalene ring of the ligand, and the γ=120° angle confirms the D3 point group symmetry; this crystal packing pattern maximizes the van der Waals force to reduce the molecular polarity, and is the structural basis of lipophilic antibacterial activity.

[0019] Preferably, the application of the preparation method of the novel lipophilic gallium complex with activity according to any one of claims 1-6 in the preparation of an anti-Gram-negative bacteria drug.

[0020] By adopting the technical scheme, the complex replaces the iron absorption pathway of bacteria: the lipophilic ligand carries Ga 3+ penetrates the outer membrane of Gram-negative bacteria, and Ga 3+ competitively replaces Fe 3+ destroys the function of respiratory chain enzymes, leading to the inhibition of planktonic bacteria growth and the disintegration of biofilms.

[0021] Preferably, the Gram-negative bacteria is Pseudomonas aeruginosa, including drug-sensitive Pseudomonas aeruginosa and multi-drug-resistant Pseudomonas aeruginosa.

[0022] By adopting the technical scheme, for Pseudomonas aeruginosa, the complex penetrates into the dormant bacteria by destroying the extracellular polysaccharide matrix of the biofilm at a concentration of 32μM, inactivates the iron-dependent enzymes in the metabolic pathway of drug-resistant bacteria, and overcomes the efflux pump mechanism to achieve bactericidal activity.

[0023] In summary, the present application has the following beneficial effects:

[0024] 1. Since the present application adopts the preparation method of stirring Ga(NO3)3·9H2O and 2-hydroxy-3-methyl-1,4-naphthoquinone in a 90% ethanol solution containing K2CO3 at room temperature for 3 hours, the 90% ethanol solvent balances the solubility of gallium salt and ligand through moderate polarity, ensuring homogeneous reaction, while the room temperature condition avoids ligand decomposition or gallium ion hydrolysis caused by high temperature, and the 3-hour stirring time makes Ga 3+ and the ligand fully chelate to form a stable octahedral coordination structure, obtaining a high-purity, lipophilic C 33 H 21 O9Ga complex, thereby achieving the effects of effectively penetrating the cell membrane of gram-negative bacteria, realizing planktonic bacteria growth inhibition and biofilm disintegration.

[0025] 2. In the present application, 2-hydroxy-3-methyl-1,4-naphthoquinone is preferably used, because the rigid naphthoquinone skeleton of the ligand provides a bidentate coordination unit and spatial matching, and in the complexing process, six oxygen donors form a stable octahedral bond with Ga 3+ , the hydrophobic naphthalene ring directionally wraps the metal center, giving the complex lipophilic characteristics, so as to achieve enhanced membrane permeability and intracellular stability, enable the complex to competitively replace bacterial iron-dependent enzymes, overcome the efflux pump mechanism, and realize efficient sterilization of multiple drug-resistant Pseudomonas aeruginosa.

[0026] 3. In the method of the present application, after the reaction, cold deionized water is added to induce precipitation, red solids are collected by filtration, ethanol is washed three times, and vacuum drying is performed overnight, because the cold deionized water reduces the solubility of the complex by solvent polarity mutation, promotes hydrophobic directional crystallization, and vacuum drying removes solvent molecules embedded in the crystal lattice, maintaining the stability of the crystal structure, so as to obtain biofilm eradication ability, and realize the synergistic effect of reducing the FIC index in the synergistic application with meropenem, enhance the antibiotic efficacy, and further remove mature biofilms. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A schematic diagram of the molecular structure of a new type of active lipophilic gallium complex proposed in the present application;

[0028] Figure 2 A schematic diagram of the bacterial colony logarithm of a new type of active lipophilic gallium complex proposed in the present application;

[0029] Figure 3 A schematic diagram of the MDRPA antibacterial activity of a new type of active lipophilic gallium complex proposed in the present application;

[0030] Figure 4 A schematic of biofilm inhibition by a new active lipophilic gallium complex proposed in the present application;

[0031] Figure 5 A schematic of biofilm removal by a new active lipophilic gallium complex proposed in the present application. DETAILED DESCRIPTION

[0032] The present application is further described in detail by the following drawings and examples.

[0033] TECHNICAL CONCEPT

[0034] Related art typically employs ligand stabilization strategies, such as the addition of citrate to gallium nitrate formulations to form soluble complexes that prevent precipitation. However, such methods have limited efficacy: citrate can stabilize Ga 3+ in vitro, but has little effect on pharmacokinetics in vivo, with studies finding that less than 1% of the total gallium in animal models is in the form of citrate complexes, and that stronger ligands cannot completely prevent Ga 3+ dissociation and the formation of hydrolysis products. This not only affects the delivery efficiency of Ga 3+ , but also weakens its antibacterial activity, especially against gram-negative bacteria. The decrease in bioavailability and the risk of toxicity caused by hydrolysis limit the therapeutic effect.

[0035] A new lipophilic 2-hydroxy-3-methyl-1,4-naphthoquinone Ga(III) complex was designed and synthesized in the present application, which has clear activity against P. aeruginosa planktonic and biofilm bacteria, and has potential oral bioavailability. 2-hydroxy-3-methyl-1,4-naphthoquinone is a naturally occurring hydroxynaphthoquinone derivative and also a structural analogue of vitamin K, which provides a unique skeleton for metal coordination. This chelating ligand has both hydroxyl and carbonyl donor groups, which are easy to form stable complexes with hard Lewis metal ions, and these complexes usually exhibit higher water stability and bioavailability.

[0036] Example 1

[0037] Preparation of Ga(phthiocolate)3

[0038] First, in a 50 mL beaker, 2-hydroxy-3-methyl-1,4-naphthoquinone (3.0 mmol) was dissolved in 20 mL of an ethanol solution containing Na2CO3(1.5 mmol) while stirring. Then, a solution of Ga(NO3)3(1.0 mmol) in 5 mL of ethanol was added dropwise to the reaction mixture at room temperature; stirring was continued for 3 hours, and after the reaction was completed, 10 mL of cold deionized water was quickly added to induce precipitation; the resulting bright red solid was collected by vacuum filtration and washed with 50% ethanol three times. The crude product was dried in a vacuum oven overnight to obtain Ga(phthiocolate)3as a red solid with a yield of 76%. The purity of the product was >98% as determined by elemental analysis and HPLC; elemental analysis results for Ga(phthiocolate)3: calculated C (62.79%), H (3.35%), Ga (11.05%). Found: C (62.88%), H (3.32%), Ga (11.13%).1H-NMR (CHCl3-d6, 500 MHz) δ 8.17 (ddd, 3H), 7.84 (td, 3H), 7.65 (td, 3H), 7.32 (s, 3H), 2.19 (s, 9H). Slow evaporation of CHCl3at room temperature for more than a week resulted in a gem-red cubic single crystal suitable for X-ray analysis.

[0039] The specific reaction process is as follows:

[0040]

[0041] Example 2

[0042] Single crystal X-ray structure determination of Ga(phthiocolate)3

[0043] A single crystal was grown from chloromethane using the solvent evaporation method; for X-ray examination and data collection, a suitable dark red plate-like crystal (size about 0.171 x 0.154 x 0.052 mm) was mounted in a loop with Paratone-N oil and transferred to the goniometer head immersed in a stream of cold nitrogen; intensity data were collected on a Bruker APEX-II CCD diffractometer using Mo Kα radiation Intensity data were collected at 150 K; data frames were processed using the SAINT program; the data were corrected for decay, Lorentz and polarization effects, and absorption and beam divergence; the structure was solved by a combination of direct methods and difference Fourier techniques in the SHELX program suite, and reflections on F2were refined using full-matrix least squares, with a resolution of The space group R-3c was fitted to the structural fragments; this resulted in overall structural disorder in the ligand and required extensive constraints to ensure a reasonable model and an overly large weighting scheme; further refinement of the structure as a twinned disordered twin in the space group R3c [Flack parameter = 0.46(3)] was chosen without any constraints; the non-hydrogen atoms were then refined with anisotropic displacement parameters, with chloroform and water crystallized in the lattice at 3-fold symmetry positions, and a second independent molecule showing disorder in several ring atoms and O3; however, a multi-component disorder model could not be refined reasonably; at this point, the disordered chloroform Cl atoms were refined with a two-component model, with anisotropic displacement parameters constraints imposed on the solvent Cl atoms, while the H atoms were calculated and treated with a riding model; hydrogen atom isotropic displacement parameters were defined as a*Ueq of the adjacent atoms (a = 1.5 for methyl groups and a = 1.2 for other atoms); the crystallographic agreement factors after refinement were R1 = 4.31%, wR2 = 10.40% for 3135 reflections with I > 2σ(I) (R1 = 6.39%, wR2 = 11.66% for all data) and 288 variable parameters.

[0044] Referring to Figure 1 At the molecular level, single-crystal X-ray diffraction analysis revealed that Ga(phthiocolate)3 adopts a geometry derived from an octahedral coordination environment with the Ga(III) ion at the center and the metal center coordinated to six oxygen donor atoms from three bidentate ligands; this coordination induces the originally planar ligand to form a three-dimensional propeller-like arrangement, resulting in a structure with approximate D3 point group symmetry, and the symmetric arrangement of the ligands leads to a net zero molecular dipole moment; and the Ga(III) ion is completely encapsulated in the hydrophobic cavity formed by the ligands, which can shield its ionic character, this structural feature contributes to the overall neutrality of the complex and enhances its membrane permeability; the results of crystal data and structure refinement are summarized in Table 1, and the atomic coordinates are listed in Table 2.

[0045] Table 1 Crystal data and structure refinement of Ga(pthiocolate)3

[0046]

[0047]

[0048]

[0049] Table 2 Atomic coordinates [x 104] and equivalent isotropic displacement parameters of Ga(pthiocolate)3

[0050]

[0051]

[0052] Example 3

[0053] Antibacterial activity test

[0054] To further evaluate the antiplankton efficacy of Ga(phthiocolate)3, we used colony forming unit (CFU) counting to assess the dose-dependent response of DSPA and MDRPA strains. These results were compared with those obtained using gallium nitrate (Ga(NO3)3)3 and gallium maltol (Ga(maltolate)3)3. Figure 2 As shown, compared with gallium nitrate (Ga(NO3)3) and gallium maltolate (Ga(maltolate)3), Ga(phthiocolate)3 exhibited enhanced anti-plankton activity. At a concentration of 8 μM (1×MIC), Ga(phthiocolate)3 reduced the CFU of DSPA by 3 logs (99.9%) and MDRPA by more than 2 logs (99%). When the concentration was increased to 16 μM (2×MIC), DSPA was reduced by nearly 4 logs (99.99%) and MDRPA by more than 3 logs (99.9%), thus effectively eliminating these two bacterial strains. This dose-dependent bactericidal effect persisted at concentrations of 126 μM (4 × MIC) and 256 μM (5 × MIC), with CFU reductions of DSPA and MDRPA by 6 logs, respectively. While Ga(NO3)3 also exhibited bactericidal activity against these two strains, approximately 3-4 times the molar concentration of Ga(phthiocolate)3 was required to achieve a comparable reduction effect. In stark contrast, Ga(maltolate)3 showed negligible antibacterial activity within the tested concentration range (4-256 μM), particularly against the MDRPA strain. Furthermore, based on colony forming unit (CFU) counts, the minimum bactericidal concentration (MBC) was determined to be 10 μg / mL for DSPA and 20 μg / mL for DRPA. Therefore, the MBC / MIC ratio of Ga(phthiocolate)3 can be calculated as 2 for DSPA and 4 for MDRPA, indicating bactericidal activity against both strains. According to established standards, an MBC / MIC ratio ≤ 4 indicates bactericidal activity, while an MBC / MIC ratio > 4 indicates bacteriostatic activity. Figure 2 In the study, Ga(phthiocolate)3 showed growth inhibition against both DSPA (left) and MDRPA (right) compared to Ga(NO3)3 and Ga(maltolate)3. Data are presented as mean ± standard deviation (sd) based on three biological replicates (n=3). Clinically, bactericides are often the first choice for treating immunocompromised patients or severe infections because they actively eliminate pathogens rather than simply inhibiting their growth.

[0055] Referring to Figure 3 To evaluate the potential synergy of Ga(phthiocolate)3 with meropenem against planktonic MDRPA, a checkerboard test was performed. The results showed that the MIC of meropenem alone was 2 pg / mL, which was significantly reduced to 0.25 pg / mL in the presence of Ga(phthiocolate)3. In contrast, the MIC of Ga(phthiocolate)3 alone was 5 pg / mL, which was reduced to 1.25 pg / mL when combined with meropenem. The fractional inhibitory concentration (FIC) index calculated was 0.375, indicating synergy (FIC < 0.5). These results suggest that Ga(phthiocolate)3 can enhance the antibacterial activity of meropenem against MDRPA, thereby improving the antibiotic efficacy of meropenem. This synergy highlights the potential of Ga(phthiocolate)3 as an adjuvant to overcome drug resistance in Gram-negative pathogens.

[0056] Referring to Figure 4 To investigate the antibacterial effect of Ga(phthiocolate)3 and meropenem against multidrug-resistant P. aeruginosa (MDRPA, ATCC BAA-2108), the biofilm inhibition and eradication effects were quantitatively evaluated by colony-forming units (CFU). In the inhibition model, bacterial suspensions were statically incubated in the presence of Ga(phthiocolate)3 at a concentration range of 8 to 128 pM for 24 hours. A clear dose-dependent inhibition of biofilm formation was observed. At 8 pM, the viable cell count decreased by at least 1 log unit, which was defined as the minimum biofilm inhibitory concentration (MBIC). The MBIC was essentially the same as the minimum inhibitory concentration (MIC) for planktonic cells. This result indicates that Ga(phthiocolate)3 can prevent biofilm formation without the need for a higher concentration than that effective against planktonic bacteria.

[0057] Referring to Figure 5 To evaluate the eradication effect of biofilms, mature 24-hour biofilms were subsequently treated with Ga(phthiocolate)3 or meropenem (8-128 pM) for 24 hours:

[0058] Ga(phthiocolate)3exhibited a remarkable biofilm disruption effect, reducing the number of viable bacteria associated with the biofilm by >3 logs at a concentration of 32 μΜ, which is equivalent to the minimum biofilm eradication concentration (MBEC). Notably, this MBEC value is only four times higher than the MIC, indicating that the complex maintains strong bactericidal activity and excellent penetration in the biofilm matrix. Furthermore, a 4-log reduction was achieved at a concentration of 128 μΜ, resulting in the eradication of mature biofilms. In comparison, the clinically common broad-spectrum β-lactam antibiotic meropenem was significantly less effective in this case. Although its MIC against MDR PA was also determined to be 8 μΜ, a concentration of more than 256 μΜ, i.e. more than 32 times the MIC, was required to clear mature biofilms. This large difference highlights the limitations of the relevant antibiotics: they are usually effective against planktonic cells, but often lose activity against bacteria within biofilms, which are often protected by the extracellular polymeric substance (EPS) matrix, exposed to low oxygen and low pH conditions, and often enter a dormant state, which confers tolerance to antibiotics.

[0059] The specific embodiments are only illustrative of the application and are not intended to limit the scope of the application. Any modifications of the application made during the life of the application derived from the disclosure herein are to be considered as being within the scope of the application as defined by the claims of the application.

Claims

1. A method for the preparation of a new lipophilic gallium complex with activity, characterized by, The method comprises the following steps: Ga(NO3)3-9H2O was first mixed with 2-hydroxy-3-methyl-1,4-naphthoquinone in a 90% ethanol solution containing K2CO3, followed by a 3-hour synthesis reaction with stirring at room temperature, and then the red solid filter residue was collected, washed with ethanol 3 times and dried in a vacuum oven to obtain a complex of molecular formula C 33 H 21 O9Ga, as shown in the following reaction formula:

2. A method of preparing a novel lipophilic gallium complex having activity according to claim 1, characterized in that, The chemical formula of the 2-hydroxy-3-methyl-1,4-naphthoquinone is C11H8O3.

3. A method of preparing a novel lipophilic gallium complex having activity according to claim 1, characterized by, The added amount of the K2CO3 is 1.5 mmol based on 1.0 mmol of Ga(NO3)3·9H2O.

4. A method of preparing a novel lipophilic gallium complex having activity according to claim 1, characterized by, After the reaction is completed, cold deionized water is added to induce precipitation.

5. A method of preparing a novel lipophilic gallium complex having activity according to claim 1, characterized by, The vacuum drying condition is overnight drying.

6. A method of preparing a novel lipophilic gallium complex having activity according to claim 1, characterized by, The complex belongs to the rhombohedral system, space group R3c, and the cell parameters are: a=b=16.0686A, c=39.771A, γ=120°.

7. The use of a method for preparing the novel active lipophilic gallium complex according to any one of claims 1-6 in the preparation of a drug against gram-negative bacteria.

8. Use according to claim 7, characterized in that, The gram-negative bacteria are Pseudomonas aeruginosa, including drug-sensitive Pseudomonas aeruginosa and multi-drug resistant Pseudomonas aeruginosa.

9. Use according to claim 7, characterized in that, Meropenem and C 33 H 21 O9Ga were co-administered, wherein the combination exhibited a fractional inhibitory concentration (FIC) index of 0.375 or less.

10. Use according to claim 7, characterized in that, contacting the biofilm with an effective amount of C 33 H 21 O9Ga, wherein the minimum biofilm eradication concentration (MBEC) is no more than four times the minimum inhibitory concentration (MIC).

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