A prodrug conjugate and its preparation method

Abstract

The present invention relates to the technical field of pesticide formulations, and provides a prodrug conjugate and a preparation method thereof. A drug molecule is combined with a fluorine-containing nonionic surfactant, and the release rate of fenhexamid can be regulated by controlling the pH value. The larger the pH value, the faster the phenolic ester bond in the prodrug conjugate breaks, and the faster fenhexamid is released. Moreover, the remaining part after release can be used as a surfactant to improve the solubility, wetting and spreading properties of fenhexamid.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide formulations, and particularly relates to a prodrug conjugate and a preparation method thereof. Background Art

[0002] As a fungicide, fenhexamid has a short photolysis half-life and low utilization rate. At present, the improved method is to use the physical encapsulation method to load fenhexamid in a nanocarrier for use. However, due to the cumbersome preparation process of the nanocarrier pesticide formulation and the low loading amount, the practical application of fenhexamid is limited.

[0003] Prodrug conjugates usually have optimized physicochemical properties, such as high solubility, strong light stability, and low volatility. In addition, due to the differences in the internal environments between target organisms and non-target organisms, the toxicity of prodrug conjugates to non-target organisms may be reduced. Moreover, no organic solvents need to be added during the use of prodrug conjugates, which can effectively reduce environmental pollution caused by the use of organic solvents.

[0004] However, existing prodrug conjugates are generally formed by connecting a drug molecule with a hydrophilic segment. Once the two are disconnected during use, the drug molecule will no longer adsorb to the target organism through the hydrophilic segment, thereby reducing the efficacy of the drug molecule. In addition, existing prodrug conjugates cannot achieve on-demand release, and the hydrophilic segments generated after release will no longer have other applications, unable to rationally utilize resources. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the related art. For this purpose, the present invention provides a prodrug conjugate, and the structural formula of the prodrug conjugate is shown as follows:

[0006] ;

[0007] wherein, R is , , , , or -(CH2)6-;

[0008] Z is one of H or F;

[0009] Both m and n are integers, 1 ≤ m ≤ 90, 1 ≤ n ≤ 6.

[0010] According to the prodrug conjugate provided by the present invention, the critical micelle concentration of the prodrug conjugate is 0.2 - 0.9 g / L; the surface tension of the prodrug conjugate is 26 - 38 mN / m; the particle size of the prodrug conjugate is 100 - 250 nm.

[0011] The present invention also provides a method for preparing a prodrug conjugate as described above, comprising the following steps:

[0012] S1: Place in an organic solvent, stir to obtain a first mixed solution, and add to the first mixed solution, stir, and reflux for 8 - 16 h to obtain a second mixed solution;

[0013] S2: Add to the second mixed solution, and reflux for 8 - 20 h to obtain the prodrug conjugate.

[0014] According to the method for preparing a prodrug conjugate provided by the present invention, the organic solvent is one of tetrahydrofuran, chloroform, toluene, or 1,2 - dichloroethane.

[0015] According to the method for preparing a prodrug conjugate provided by the present invention, the is one of hexamethylene diisocyanate, cyclohexane - 1,4 - diisocyanate, p - phenylene diisocyanate, m - phenylene diisocyanate, isophorone diisocyanate, and 4,4'-methylenebis(phenyl isocyanate).

[0016] One or more of the above - mentioned technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0017] 1. The prodrug conjugate provided by the present invention combines a drug molecule with a fluorinated non - ionic surfactant, has excellent surface activity, can not only achieve the on - demand release of the fungicide fenhexamid, but also the remaining part after release can be used as a surfactant to improve the solubility, wetting, and spreading properties of fenhexamid.

[0018] 2. The present invention provides a method for preparing a prodrug conjugate, which has a short synthesis route, easy - to - control reaction conditions, is easy to develop industrially, and has low cost.

[0019] 3. The prodrug conjugate provided by the present invention has good wettability, can self - assemble into nanoparticles in an aqueous solution, and the particle size can be controlled within the range of 100 - 250 nm.

[0020] 4. The prodrug conjugate provided by the present invention has glutathione - responsive release performance, and can control the release rate of fenhexamid by controlling the pH value. The larger the pH value, the faster the cleavage rate of the phenolic ester bond in the prodrug conjugate, and the faster the release of fenhexamid.

[0021] 5. Compared with the original drug of fenhexamid, the prodrug conjugate provided by the present invention has excellent comprehensive properties, higher biological activity, significantly enhanced utilization rate, and has good application prospects.

[0022] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned by practice of the present invention. Detailed Description

[0023] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments of the present invention fall within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0024] In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be construed as indicating or implying relative importance.

[0025] In the description of this specification, the descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without conflict, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0026] The present invention provides a prodrug conjugate, and the structural formula of the prodrug conjugate is shown as follows:

[0027] ;

[0028] Wherein, R is , , , , or -(CH2)6-;

[0029] Z is one of H or F;

[0030] Both m and n are integers, 1 ≤ m ≤ 90, 1 ≤ n ≤ 6.

[0031] According to the present invention, a prodrug conjugate is provided, and the critical micelle concentration of the prodrug conjugate is 0.2 to 0.9 g / L; the surface tension of the prodrug conjugate is 26 to 38 mN / m; the particle size of the prodrug conjugate is 100 to 250 nm.

[0032] The present invention also provides a preparation method of a prodrug conjugate as described above, comprising the following steps:

[0033] S1: Place in an organic solvent, stir to obtain a first mixed solution, and add to the first mixed solution, stir, and reflux for 8 to 16 h to obtain a second mixed solution;

[0034] S2: Add to the second mixed solution, and reflux for 8 to 20 h to obtain the prodrug conjugate.

[0035] Among them, the reaction equation of step S1 is as follows:

[0036] .

[0037] The reaction equation of step S2 is as follows:

[0038] .

[0039] According to the preparation method of a prodrug conjugate provided by the present invention, the organic solvent is one of tetrahydrofuran, chloroform, toluene or 1,2-dichloroethane.

[0040] According to the preparation method of a prodrug conjugate provided by the present invention, the is one of hexamethylene diisocyanate, cyclohexane-1,4-diisocyanate, p-phenylene diisocyanate, m-phenylene diisocyanate, isophorone diisocyanate and 4,4'-methylenebis(phenyl isocyanate).

[0041] The following describes a prodrug conjugate and its preparation method provided by the present invention according to Examples 1-8.

[0042] First of all, it should be noted that:

[0043] (1) Determination method of the surface tension of fluorinated nonionic surfactants: Prepare a series of fluorinated surfactants with different concentrations, and use the pendant ring method to measure the surface tension by a JK99M full-automatic static surface tension meter (outer diameter 20.30 mm, platinum wire 0.30 mm, perimeter 61.89 mm, density 0.998 g / cm 3 , temperature 25 ± 1 °C). The instrument is calibrated with pure water before and after the test.

[0044] (2) The microscopic morphology of the prodrug conjugate prepared based on the fluorinated nonionic surfactant and fenhexamid was characterized using a field emission transmission electron microscope (TEM) with a working voltage of 200 kV. The ultrasonicated aqueous nano-pesticide solution was gently dropped onto a 300-mesh carbon support film using a pipette with a measuring range of 10 μL. After the sample was dried, the microscopic morphology was observed.

[0045] (3) The cumulative release rate of fenhexamid in the prodrug conjugate was determined under the conditions that the pH of the PBS buffer solution containing the prodrug conjugate was 5.0, 7.4, or 9.0 using the dialysis method. First, 5.0 mL of the PBS buffer solution (30% methanol) of the prodrug conjugate was placed in a dialysis bag with a molecular cut-off of 500, and then the dialysis bag was placed in a beaker containing 95 mL of PBS buffer solution. The beaker was placed on a stirrer with a rotation speed of 250 r / min and released under dark conditions at ambient temperature. At 1 h, 5.0 mL of the sustained-release medium (ethanol: water = 70:30) was collected, and at the same time, 5.0 mL of the sustained-release medium was replenished. The obtained sample was filtered through a 0.45 μm microporous membrane and analyzed by an HPLC system. The cumulative release rate of fenhexamid was calculated according to the standard curve of fenhexamid and the cumulative release rate formula as follows:

[0046]

[0047] (4) The particle size of the 1.0 g / L aqueous solution of the prodrug conjugate was measured. 1.5 mL of the sample was pipetted into a cuvette, and the particle size of the aqueous solution of the prodrug conjugate was measured using a Mastersizer 3000E particle size analyzer at a test temperature of 25 ± 1 °C.

[0048] (5) A certain amount of the test samples of fenhexamid technical, fluorinated nonionic surfactant, and prodrug conjugate were dissolved in an aqueous solution of Tween-80 with a concentration of 0.1% to prepare a series of solutions for measuring the antibacterial effect. In vitro tests were carried out with the test target being Magnaporthe oryzae.

[0049] (6) The preparation method of the fluorinated nonionic surfactant refers to Chinese Patent CN 114276537 A.

[0050] Example 1

[0051] The fluorinated nonionic surfactant , where m is 22, n is 3, and Z is F. It is dissolved in the solvent 1,2-dichloroethane, and then hexamethylene diisocyanate (the molar ratio of the fluorinated nonionic surfactant to hexamethylene diisocyanate is 1:1.2) is added. After reacting at 90 °C for 12 h, fenhexamid is added, and the reaction continues at 90 °C for 12 h to obtain the prodrug conjugate, which is labeled C1.

[0052] After testing, the critical micelle concentration of the prodrug conjugate C1 prepared in this example is 0.71 g / L, and the corresponding surface tension is 30.3 mN / m.

[0053] Example 2

[0054] The fluorinated nonionic surfactant , where m is 1, n is 2, and Z is H, is dissolved in the solvent chloroform, and then cyclohexane-1,4-diisocyanate (the molar ratio of the fluorinated nonionic surfactant to cyclohexane-1,4-diisocyanate is 1:0.8) is added. After reacting at 70 °C for 8 h, fenhexamid is added, and the reaction continues at 70 °C for 8 h to obtain the prodrug conjugate, which is labeled C2.

[0055] After testing, the critical micelle concentration of the prodrug conjugate C2 prepared in this example is 0.9 g / L, and the corresponding surface tension is 38 mN / m.

[0056] Example 3

[0057] The fluorinated nonionic surfactant , where m is 90, n is 6, and Z is H, is dissolved in the solvent toluene, and then p-phenylene diisocyanate (the molar ratio of the fluorinated nonionic surfactant to p-phenylene diisocyanate is 1:1.1) is added. After reacting at 120 °C for 16 h, fenhexamid is added, and the reaction continues at 120 °C for 16 h to obtain the prodrug conjugate, which is labeled C3.

[0058] After testing, the critical micelle concentration of the prodrug conjugate C3 prepared in this example is 0.2 g / L, and the corresponding surface tension is 26 mN / m.

[0059] Example 4

[0060] The fluorinated nonionic surfactant , where m is 44, n is 3, and Z is F, is dissolved in the solvent toluene, and then m-phenylene diisocyanate (the molar ratio of the fluorinated nonionic surfactant to m-phenylene diisocyanate is 1:1.0) is added. After reacting at 120 °C for 16 h, fenhexamid is added, and the reaction continues at 120 °C for 16 h to obtain the prodrug conjugate, which is labeled C4.

[0061] After testing, the critical micelle concentration of the prodrug conjugate C4 prepared in this example was 0.69 g / L, and the corresponding surface tension was 32.7 mN / m.

[0062] Example 5

[0063] Dissolve the fluorinated nonionic surfactant , where m is 11, n is 4, and Z is H, in the solvent tetrahydrofuran, then add isophorone diisocyanate (the molar ratio of the fluorinated nonionic surfactant to isophorone diisocyanate is 1:0.9), react at 60 °C for 12 h, add fenhexamid, and continue to react at 60 °C for 12 h to obtain the prodrug conjugate, which is labeled C5.

[0064] After testing, the critical micelle concentration of the prodrug conjugate C5 prepared in this example was 0.74 g / L, and the corresponding surface tension was 34.8 mN / m.

[0065] Example 6

[0066] Dissolve the fluorinated nonionic surfactant , where m is 4, n is 5, and Z is F, in the solvent toluene, then add 4,4'-methylenebis(phenyl isocyanate), where the molar ratio of the fluorinated nonionic surfactant to 4,4'-methylenebis(phenyl isocyanate) is 1:1), react at 120 °C for 14 h, add fenhexamid, and continue to react at 120 °C for 14 h to obtain the prodrug conjugate, which is labeled C6.

[0067] After testing, the critical micelle concentration of the prodrug conjugate C6 prepared in this example was 0.82 g / L, and the corresponding surface tension was 36.9 mN / m.

[0068] Example 7

[0069] The prodrug conjugates prepared in Examples 1-6 were respectively subjected to performance comparison according to the above test method, as shown in Tables 1-4 specifically.

[0070] Table 1 Comparison table of surface tensions of prodrug conjugates prepared in Examples 1-6

[0071]

[0072] It can be seen from Table 1 that the prodrug conjugates C1-C6 provided by the present invention all have relatively small surface tensions, indicating that the prodrug conjugates C1-C6 provided by the present invention all have good wetting and spreading properties. In addition, the prodrug conjugates C1-C6 provided by the present invention can self-assemble into nanoparticles in aqueous solution, and the size can be controlled within the range of 100-250 nm.

[0073] Surface tension (mN / m) of the prodrug conjugate in PBS buffer at different pH values in Table 2

[0074]

[0075] As can be seen from Table 2, for the prodrug conjugates C1 - C6 provided by the present invention, the surface tension does not change significantly under acidic, neutral or alkaline conditions, indicating that the prodrug conjugates C1 - C6 provided by the present invention all have good stability. The change in pH value does not affect the wetting and spreading properties of C1 - C6, and the applicability is wider.

[0076] Release rate (%) of fenhexamid in the prodrug conjugate at different pH values after 46 h of accumulation in Table 3

[0077]

[0078] As can be seen from Table 3, for the prodrug conjugates C1 - C6 provided by the present invention, as the pH value increases, the release rate of fenhexamid becomes larger. This is because the higher the pH value, the faster the cleavage rate of the phenol ester bond in the prodrug conjugates C1 - C6 provided by the present invention, so the release of fenhexamid is faster. Thus, it can be realized that by controlling the pH value, the fungicide fenhexamid can be released as needed. The remaining part after release is , which can be used as a surfactant to improve the solubility, wetting and spreading properties of fenhexamid.

[0079] Release rate (%) of fenhexamid in the prodrug conjugate at p = 7.4 and different glutathione contents after 46 h of accumulation in Table 4

[0080]

[0081] As can be seen from Table 4, under the conditions of the same pH value and different glutathione contents, for the prodrug conjugates C1 - C6 provided by the present invention, as the glutathione content increases, the release rate of fenhexamid becomes larger, indicating that C1 - C6 has glutathione-responsive release performance. Further, it shows that the prodrug conjugates C1 - C6 provided by the present invention can control the release rate of fenhexamid by controlling the glutathione content.

[0082] Example 8

[0083] Take a certain amount of fenhexamid, fluorosurfactant and prodrug conjugate C3 in Example 3, and prepare aqueous solutions of Tween-80 with different mass concentrations of 0.1%, and measure their inhibitory effects on Magnaporthe oryzae.

[0084] After testing, the inhibitory effects of fenhexamid, fluorosurfactant and prodrug conjugate C3 in Example 3 on Magnaporthe oryzae are shown in Table 5.

[0085] Table 5 Inhibitory effects of fenhexamid, fluorosurfactant and prodrug conjugate at different concentrations on Magnaporthe oryzae

[0086]

[0087] As can be seen from Tables 1 to 4, the release rate of fenhexamid of the prodrug conjugate C3 prepared in Example 3 is the lowest. Therefore, C3 was selected to determine its inhibitory effect on Magnaporthe oryzae. As can be seen from Table 5, compared with the original fenhexamid drug and the mixture of fenhexamid + fluorosurfactant, the inhibitory effect of the prodrug conjugate on Magnaporthe oryzae was significantly enhanced. It can be expected that the inhibitory effects of C1, C2, C4, C5 and C6 on Magnaporthe oryzae are all better than that of C3, indicating that the prodrug conjugates C1 - C6 prepared in the present invention all have excellent comprehensive properties, higher biological activities, significantly enhanced utilization rates, and have good application prospects.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A prodrug conjugate, characterized in that, The structural formula of the prodrug conjugate is as follows: ； wherein, R is , , , , or one of -(CH2)6-; Z is one of H or F; Both m and n are integers and 1 ≤ m ≤ 90, 1 ≤ n ≤ 6.

2. The prodrug conjugate according to claim 1, wherein The critical micelle concentration of the prodrug conjugate is 0.2 - 0.9 g / L; the surface tension of the prodrug conjugate is 26 - 38 mN / m; the particle size of the prodrug conjugate is 100 - 250 nm.

3. A method for preparing a prodrug conjugate according to any one of claims 1-2, characterized in that, It includes the following steps: S1: Place in an organic solvent, stir to obtain a first mixed solution, and add to the first mixed solution, stir, and carry out a reflux reaction for 8 to 16 h to obtain a second mixed solution; S2: Add to the second mixed solution and carry out a reflux reaction for 8 - 20 h to obtain the prodrug conjugate.

4. The preparation method of a prodrug conjugate according to claim 3, characterized in that, The organic solvent is one of tetrahydrofuran, chloroform, toluene or 1,2-dichloroethane.

5. The preparation method of a prodrug conjugate according to claim 3, characterized in that, The said is one of hexamethylene diisocyanate, cyclohexane-1,4-diisocyanate, p-phenylene diisocyanate, m-phenylene diisocyanate, isophorone diisocyanate, and 4,4'-methylenebis(phenyl isocyanate).

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