CN112121173B - Fatty acid modified immunosuppressant MMF and FK506 nano preparation and preparation method and application thereof - Google Patents
Fatty acid modified immunosuppressant MMF and FK506 nano preparation and preparation method and application thereof Download PDFInfo
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- CN112121173B CN112121173B CN202010852776.1A CN202010852776A CN112121173B CN 112121173 B CN112121173 B CN 112121173B CN 202010852776 A CN202010852776 A CN 202010852776A CN 112121173 B CN112121173 B CN 112121173B
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Images
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
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Abstract
The invention discloses a fatty acid coupled prodrug and a preparation method thereof, wherein the preparation method comprises the following steps: under the action of a condensing agent and a catalyst, tacrolimus or mycophenolate mofetil and saturated or unsaturated fatty acid corresponding to R are subjected to esterification reaction to obtain the fatty acid coupled prodrug. The nano preparation in the form of the invention obviously improves the water solubility of MMF and FK506, avoids using auxiliary materials such as solubilizer and the like, and further improves the drug-loading rate; meanwhile, the saturated or unsaturated fatty acid used for modification is a substance required by human body, has good biocompatibility, is convenient for clinical transformation, and has better application prospect. More importantly, the nano preparation is assembled by the pegylated MMF-saturated or unsaturated fatty acid coupled prodrug and the FK506, the pharmacokinetic property of the drug is obviously improved, the in vivo circulation time of the drug is prolonged, the MMF and the FK506 can be simultaneously delivered to a target site to play a drug effect, and the effect of resisting transplant rejection is better compared with that of clinical MMF/FK 506.
Description
Technical Field
The invention belongs to the field of immunosuppressive drugs, and particularly relates to synthesis of an immunosuppressant MMF and FK506 prodrug, a preparation method and application of an MMF prodrug and an FK506 nano preparation.
Background
Tacrolimus (FK506) is the first immunosuppressant after organ transplantation, and causes various dose-dependent adverse reactions after long-term use. Clinically, the traditional Chinese medicine composition is usually combined with Mycophenolate Mofetil (MMF) to prevent and treat acute rejection after organ transplantation so as to improve adverse reactions caused by high medicine dosage. Because FK506 and MMF can significantly reduce postoperative rejection and transplanted organ failure and effectively improve in vivo side effects, the combined treatment mode is included in Clinical Practice Guidelines 2016 (manufactured by European Liver disease society), and becomes the most conventional combined immunosuppressive treatment scheme after Liver transplantation. However, the FK506 and MMF are extremely poor in water solubility, are mainly orally taken clinically, greatly reduce the bioavailability of the medicament in vivo due to the liver first-pass effect and have a narrow treatment window. Particularly, when FK506 is absorbed by gastrointestinal tract, P-glycoprotein on epithelial cell membrane of gastrointestinal tract is easy to be pumped from serosa side to mucous membrane side and then is discharged from intestinal cavity, and the drug absorption rate is obviously reduced, so that the bioavailability is only 17-22%. Therefore, the improvement of the bioavailability of FK506 and MMF in vivo and the improvement of the treatment effect thereof are problems which are urgently needed to be solved at present.
The nanometer preparation technology provides a new idea for improving the in vivo bioavailability of insoluble drugs. At present, the water solubility of the MMF and the FK506 is mainly increased by preparing the MMF and the FK506 into nano preparations, but the single-medicine nano preparation cannot well play the synergistic effect of the MMF and the FK506, so that the treatment effect is limited.
Disclosure of Invention
The invention provides a prodrug and a preparation based on MMF/FK 506-saturated or unsaturated fatty acid coupling, a preparation and a preparation method and application thereof, and the method has simple steps and can be industrially produced in large scale; animal transplantation model experiments show that the drug delivery system assembled by the MMF-saturated or unsaturated fatty acid coupled prodrug and FK506 has better anti-transplantation rejection effect compared with clinical MMF/FK 506.
A fatty acid coupled prodrug having one or more of the structures shown in the following formulas:
wherein R represents a saturated or unsaturated fatty acyl group; or R is derived from saturated or unsaturated fatty acid.
Preferably, R represents a saturated C1-C24 alkanoyl group; or C1-C24 unsaturated fatty acyl with 1-6 double bonds. Or R is from saturated C1-C24 alkyl carboxylic acid; or C1-C24 unsaturated fatty carboxylic acid with 1-6 double bonds.
The saturated fatty acid structure is shown as the following formula:
n＝0～24。
the unsaturated fatty acid is omega-9, omega-6 and omega-3 series unsaturated fatty acid. Further R is selected from acetyl, n-heptanoyl, stearoyl, tetracosanyl, oleoyl, ricinoleic, palmitoleic, linoleoyl, linolenoyl, arachidonoyl, eicosapentaenoyl, docosahexenoyl. Or R is selected from acyl obtained by removing hydroxyl from acetic acid, n-heptanoic acid, stearic acid, tetracosanoic acid, oleic acid, ricinoleic acid, palmitoleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.
In a method for preparing the fatty acid coupled prodrug according to any one of the above technical schemes, under the action of a condensing agent and a catalyst, tacrolimus or mycophenolate mofetil is subjected to esterification reaction with saturated or unsaturated fatty acid corresponding to R to obtain the fatty acid coupled prodrug, which is shown in the formula.
The condensing agent is 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), and the catalyst is 4-Dimethylaminopyridine (DMAP). Preferably, the reaction solvent is dichloromethane or chloroform. Preferably, the reaction temperature is 40 to 50 ℃. The reaction time is 2-8 hours. Preferably, the molar ratios of the catalyst, the condensing agent, the unsaturated or saturated fatty acid and the tacrolimus or mycophenolate mofetil are (1-2): (1-2): (1-1.5): 1.
a fatty acid coupled prodrug self-assembly nano preparation can be a liposome preparation, a self-assembly preparation or a high molecular polymer micelle preparation:
the method comprises the following steps: one or more of the fatty acid conjugated prodrugs according to any of the previous embodiments;
or comprises the following steps: (1) one or more of the fatty acid conjugated prodrugs of any of the above; (2) tacrolimus;
or comprises the following steps: (1) one or more of the fatty acid conjugated prodrugs of any of the above; (2) tacrolimus or a fatty acid-coupled prodrug corresponding to tacrolimus; (3)1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ].
Preferably, the method comprises the following steps: (1) one or more of the fatty acid conjugated prodrugs of any of the above; (2) when the tacrolimus is used, the content ratio of mycophenolate mofetil to tacrolimus is 20: 0.5 to 5; alternatively, when included, the method comprises: (1) one or more of the fatty acid conjugated prodrugs of any of the above; (2) tacrolimus or a fatty acid-coupled prodrug corresponding to tacrolimus; (3)1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ], wherein the content ratio of mycophenolate mofetil to tacrolimus is 20: 0.5-5: 1 to 10. Preferably, the content ratio of the two is 20: 1; the content ratio of the three is 20:1: 2.
a preparation method of the fatty acid coupling prodrug self-assembly nanometer preparation comprises the steps of injecting an organic solvent of the corresponding fatty acid coupling prodrug into a water phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nanometer particles.
Specifically, the method comprises the following steps:
a self-assembling nanoformulation of an MMF-saturated or unsaturated fatty acid coupled prodrug comprising only the MMF-saturated or unsaturated fatty acid coupled prodrug.
The invention provides a preparation method of a self-assembly nano preparation of an MMF-saturated or unsaturated fatty acid coupling prodrug, which comprises the steps of injecting an organic solvent dissolved with the MMF-saturated or unsaturated fatty acid coupling prodrug into a water phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nano particles; preferably, the organic solvent is dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 9.
a self-assembled nano-preparation of FK 506-saturated or unsaturated fatty acid coupling prodrug only comprises the FK 506-saturated or unsaturated fatty acid coupling prodrug.
The invention provides a preparation method of an FK 506-saturated or unsaturated fatty acid coupling prodrug self-assembly nano preparation, which comprises the steps of injecting an organic solvent dissolved with the FK 506-saturated or unsaturated fatty acid coupling prodrug into a water phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nano particles; preferably, the organic solvent is a dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 9.
a co-assembled nano-preparation of MMF-saturated or unsaturated fatty acid coupled prodrug and FK506 comprises the MMF-saturated or unsaturated fatty acid coupled prodrug and FK 506.
The invention provides a preparation method of a nanometer preparation formed by co-assembling an MMF-saturated or unsaturated fatty acid coupling prodrug and FK506, which comprises the steps of injecting an organic solvent in which the MMF-saturated or unsaturated fatty acid coupling prodrug and the FK506 are dissolved into an aqueous phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nanometer particles; preferably, the mass ratio of the MMF-saturated or unsaturated fatty acid coupled prodrug to FK506 is 20:1, calculated on the mass of MMF. Preferably, the organic solvent is dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 9.
a co-assembled nano-preparation of a pegylated MMF-saturated or unsaturated fatty acid coupled prodrug and FK506 comprises the MMF-saturated or unsaturated fatty acid coupled prodrug, FK506 and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ].
Preferably, the MMF-saturated or unsaturated fatty acid conjugated prodrug is reacted with FK506 and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) based on the mass of MMF]The mass ratio of (A) to (B) is 20:1: 2. Preferably, the 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol)]Is DSPE-PEG2k。
The invention provides a preparation method of a co-assembled nano preparation of a pegylated MMF-saturated or unsaturated fatty acid coupled prodrug and FK506, which comprises the following steps: dissolving MMF-saturated or unsaturated fatty acid coupled prodrug, FK506 and DSPE-PEG2kThe organic solvent is injected into the water phase under the ultrasonic condition, and uniformly dispersed nano particles are obtained through dialysis;
the preparation method comprises coupling MMF-saturated or unsaturated fatty acid to prodrug, FK506 and DSPE-PEG2kRespectively dissolving in organic solvent, mixing the organic solvent, injecting the mixed organic solvent into water phase under ultrasonic condition, and dialyzing to obtain uniformly dispersed nanoparticles. Preferably, the organic solvent is dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 9.
a co-assembly nano-formulation of MMF/FK 506-saturated or unsaturated fatty acid conjugated prodrug comprises the MMF-saturated or unsaturated fatty acid conjugated prodrug and FK 506-saturated or unsaturated fatty acid conjugated prodrug.
The invention provides a method for preparing a nanometer preparation by co-assembling an MMF/FK 506-saturated or unsaturated fatty acid coupling prodrug, which comprises the steps of injecting an organic solvent in which the MMF-saturated or unsaturated fatty acid coupling prodrug and the FK 506-saturated or unsaturated fatty acid coupling prodrug are dissolved into a water phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nanometer particles;
preferably, the mass ratio of the MMF-saturated or unsaturated fatty acid coupled prodrug to the FK 506-saturated or unsaturated fatty acid coupled prodrug is 20:1, calculated on the mass of MMF, FK 506. Preferably, the organic solvent is dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 9.
a co-assembled nano-formulation of a pegylated MMF/FK 506-saturated or unsaturated fatty acid-conjugated prodrug comprises the MMF-saturated or unsaturated fatty acid-conjugated prodrug, FK 506-saturated or unsaturated fatty acid-conjugated prodrug and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ].
Preferably, the MMF-saturated or unsaturated fatty acid conjugated prodrug is combined with FK 506-saturated or unsaturated fatty acid conjugated prodrug and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) based on the mass of MMF and FK506]The mass ratio of (A) to (B) is 20:1: 2. Preferably, the 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol)]Is DSPE-PEG2k。
The invention provides a preparation method of a pegylated MMF/FK 506-saturated or unsaturated fatty acid coupled prodrug co-assembled nano preparation, which comprises the following steps: dissolving MMF-saturated or unsaturated fatty acid coupled prodrug, FK 506-saturated or unsaturated fatty acid coupled prodrug and DSPE-PEG2kThe organic solvent is injected into the water phase under the ultrasonic condition, and uniformly dispersed nano particles are obtained through dialysis;
the preparation method comprises coupling MMF-saturated or unsaturated fatty acid prodrug, FK 506-saturated or unsaturated fatty acid prodrug and DSPE-PEG2kRespectively dissolving in organic solvent, mixing with organic solvent, and mixingAnd (3) injecting the uniformly mixed organic solvent into the water phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nano particles. Preferably, the organic solvent is dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 9.
the invention provides a particle size distribution diagram and a scanning electron microscope diagram of the self-assembly nano preparation based on the MMF-saturated or unsaturated fatty acid coupling prodrug, the MMF-saturated or unsaturated fatty acid coupling prodrug and FK506 co-assembly nano preparation, the polyethylene glycol MMF-saturated or unsaturated fatty acid coupling prodrug and FK506 co-assembly nano preparation, wherein the average particle size is within the range of 100-200 nm. The present invention provides a method for preparing a liposomal formulation of an MMF/FK 506-saturated or unsaturated fatty acid conjugate prodrug comprising: dissolving MMF-saturated or unsaturated fatty acid coupled prodrug, FK 506-saturated or unsaturated fatty acid coupled prodrug, lecithin, cholesterol and DSPE-PEG2kThe lipid mixture is dissolved in an organic solvent, and the mixed solution is injected into a water phase under the ultrasonic condition or prepared by a film method to obtain the evenly dispersed drug-loaded liposome.
The medicine has medicine carrying amount of 1-20%, and is prepared with lecithin, cholesterol and DSPE-PEG2kThe lipid mixture with different mass ratios is dissolved in ethanol, and the MMF/FK 506-saturated or unsaturated fatty acid prodrug is mixed with the lipid mixture and then mixed with water to obtain the lipid particles.
The invention provides a preparation method of an MMF/FK 506-saturated or unsaturated fatty acid coupled prodrug coating and polymer micelle, which comprises the following steps: dissolving the MMF-saturated or unsaturated fatty acid coupling prodrug, the FK 506-saturated or unsaturated fatty acid coupling prodrug and polyethylene glycol-polylactic acid, polyethylene glycol-polylactic acid-glycolic acid copolymer or polyethylene glycol-polycaprolactone mixture in an organic solvent, and injecting the mixed solution into a water phase under the ultrasonic condition to prepare the uniformly dispersed drug-loaded polymer micelle. Use of a fatty acid conjugated prodrug of any of the above in the manufacture of a medicament for the anti-rejection of a transplanted organ. Preferably, the transplanted organ comprises a liver transplant.
In order to better utilize the advantages of the nano-preparation to enhance the synergistic effect of MMF and FK506, the invention constructs the self-assembly nano-preparation of the MMF-saturated or unsaturated fatty acid coupling prodrug, the co-assembly nano-preparation of the MMF-saturated or unsaturated fatty acid coupling prodrug and the fatty acid coupling prodrug corresponding to FK506 or FK506, and the co-assembly nano-preparation of the pegylated MMF-saturated or unsaturated fatty acid coupling prodrug and the fatty acid coupling prodrug corresponding to FK506 or FK506 by utilizing the strategy of modifying the prodrug by using saturated or unsaturated fatty acid. The prodrug can be wrapped in liposome and polymer micelle, so that the water solubility of MMF and FK506 is obviously improved. The MMF and FK506 nanometer preparation has the advantages that the pharmacokinetic properties of the MMF and FK506 are better improved, the in vivo circulation time of the MMF and FK506 nanometer preparation is prolonged, the bioavailability of the MMF and FK506 nanometer preparation in vivo is improved, the MMF and FK can be simultaneously delivered to a target site to play a synergistic effect, and the MMF and FK506 nanometer preparation has a better anti-transplant rejection effect compared with clinical MMF/FK 506.
Compared with the prior art, the invention has the beneficial effects that:
1) the saturated or unsaturated fatty acid used for modifying the MMF/FK506 is a substance required by a human body, has good biocompatibility and is convenient for clinical transformation;
2) the invention effectively improves the water solubility of the medicine by modifying the saturated and unsaturated fatty acids by the immunosuppressant, and reduces the toxicity by reducing the release of the medicine in blood;
3) the MMF/FK 506-saturated or unsaturated fatty acid coupling prodrug self-assembly nano preparation, the MMF-saturated or unsaturated fatty acid coupling prodrug and the FK506 or fatty acid coupling prodrug corresponding to the FK506 are assembled into the nano preparation, so that the water solubility of the MMF and the FK506 is obviously improved, auxiliary materials such as a solubilizer, a surfactant and the like are avoided, and the drug loading capacity is further improved;
4) the polyethylene glycol MMF-saturated or unsaturated fatty acid coupling prodrug and FK506 co-assembled nano preparation constructed by the invention can simultaneously deliver MMF and FK506 to a target site to play a drug effect, and has a better anti-transplant rejection effect compared with clinical MMF/FK 506;
5) the synthesized saturated or unsaturated fatty acid coupled prodrug increases the affinity and compatibility with carriers (such as liposome and polymer micelle) commonly used in pharmaceutics, so that a water-soluble drug preparation delivery system is constructed by the auxiliary materials of the drugs.
Drawings
FIG. 1 is a scheme showing the synthesis of Ac-MMF conjugated prodrug of example 1;
FIG. 2 is a scheme for the synthesis of a Hep-MMF conjugated prodrug of example 2;
FIG. 3 is a scheme showing the synthesis of the Ste-MMF conjugated prodrug of example 3;
FIG. 4 is a scheme showing the synthesis of the t-cos-MMF conjugated prodrug of example 4;
FIG. 5 is a scheme for the synthesis of the PA-MMF conjugated prodrug of example 5;
FIG. 6 is a scheme showing the synthesis of OA-MMF conjugated prodrug of example 6;
FIG. 7 is a scheme for the synthesis of the RA-MMF conjugated prodrug of example 7;
FIG. 8 is a scheme showing the synthesis of the LA-MMF conjugated prodrug of example 8;
FIG. 9 is a scheme showing the synthesis of the LNA-MMF conjugate prodrug of example 9;
FIG. 10 is a scheme showing the synthesis of a GLA-MMF conjugated prodrug of example 10;
FIG. 11 is a scheme showing the synthesis of the AA-MMF conjugated prodrug of example 11;
FIG. 12 is a scheme for the synthesis of EPA-MMF conjugate prodrug of example 12;
FIG. 13 is a scheme for the synthesis of a DHA-MMF conjugated prodrug of example 13;
FIG. 14 is a scheme showing the synthesis of Ac-FK506 conjugate prodrug of example 14;
FIG. 15 is a scheme showing the synthesis of a Hep-FK506 conjugate prodrug of example 15;
FIG. 16 is a scheme showing the synthesis of Ste-FK506 conjugated prodrug of example 16;
FIG. 17 is a scheme showing the synthesis of t-cos-FK506 conjugate prodrug of example 17;
FIG. 18 is a scheme showing the synthesis of a PA-FK506 conjugate prodrug of example 18;
FIG. 19 is a scheme showing the synthesis of OA-FK506 conjugated prodrug of example 19;
FIG. 20 is a scheme showing the synthesis of RA-FK506 conjugate prodrug of example 20;
FIG. 21 is a scheme showing the synthesis of a prodrug of LA-FK506 conjugate in example 21;
FIG. 22 is a scheme showing the synthesis of LNA-FK506 conjugate prodrug of example 22;
FIG. 23 is a scheme showing the synthesis of a prodrug of GLA-FK506 conjugate in example 23;
FIG. 24 is a scheme showing the synthesis of AA-FK506 conjugate prodrug of example 24;
FIG. 25 is a scheme showing the synthesis of EPA-FK506 conjugate prodrug of example 25;
FIG. 26 is a scheme showing the synthesis of a DHA-FK506 conjugate prodrug of example 26;
FIG. 27 is a nuclear magnetic spectrum of a Hep-MMF conjugated prodrug of example 2;
FIG. 28 is a nuclear magnetic spectrum of the Ste-MMF conjugated prodrug of example 3;
FIG. 29 is a nuclear magnetic spectrum of the OA-MMF conjugate prodrug of example 6;
FIG. 30 is a nuclear magnetic spectrum of the LA-MMF conjugate prodrug of example 8;
FIG. 31 is a nuclear magnetic spectrum of the DHA-MMF conjugate prodrug of example 13;
FIGS. 32 to 36 are particle size distributions of the nano-formulations of examples 2, 3, 6, 8 and 13;
FIGS. 37-41 are particle size distributions of the nano-formulations of examples 2, 3, 6, 8, 13;
FIGS. 42-46 are particle size distributions of the nano-formulations of examples 2, 3, 6, 8, 13;
FIGS. 47-51 are TEM images of the nano-formulations of examples 2, 3, 6, 8 and 13;
FIGS. 52-56 are TEM images of the nano-formulations of examples 2, 3, 6, 8 and 13;
FIGS. 57-61 are TEM images of the nano-formulations of examples 2, 3, 6, 8 and 13;
FIG. 62 is the survival rate test results obtained in example 50;
FIG. 63 shows the results of the body weight change test in example 50.
Detailed Description
The following specific embodiments are intended to further illustrate the invention, but the invention is not limited thereto.
Example 1 synthesis of Ac-MMF conjugate prodrug, as shown in figure 1:
MMF (43.2g, 0.10mmol), glacial acetic acid (6.0mg, 0.10mmol) and DMAP (12.6mg,0.10mmol) were added sequentially to a 100mL round bottom flask, dissolved in 4mL dry dichloromethane and EDC (16.6mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 6 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 1(20.5mg, 43.0% yield, [ C%25H33NO8]+[M+H]+＝476.2279)。
Example 2 synthesis of Hep-MMF conjugate prodrug, as shown in figure 2:
MMF (73.6mg, 0.17mmol), n-heptanoic acid (23.4mg, 0.18mmol) and DMAP (20.6mg, 0.17mmol) were added sequentially to a 100mL round bottom flask, dissolved in 5mL dry dichloromethane and EDC (26.9mg, 0.17mmol) was added dropwise rapidly. After stirring at 43 ℃ for 4 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 20:1) gave product 2(37.5mg, 40.5% yield).
Of product 21The H NMR nuclear magnetic data is as follows, and the nuclear magnetic spectrum is shown in FIG. 27:
1H NMR(400MHz,CDCl3):δ5.14(s,2H),5.10(t,1H,J＝13.4Hz),4.17(t,2H,J＝11.7Hz),3.78(s,3H),3.69(t,4H.J＝9.3Hz),3.34(d,2H,J＝7.6Hz),2.68(t,2H,J＝15.3Hz),2.58(t,2H,J＝11.9Hz),2.47(t,4H,J＝9.3Hz),2.36-2.41(m,2H),2.27-2.31(m,2H),2.22(s,3H),1.75-1.81(m,5H),1.44(t,2H,J＝15.3Hz),1.31-1.36(m,4H),0.90(t,3H,J＝13.0Hz).
example 3 synthesis of a Ste-MMF conjugate prodrug, as shown in figure 3:
MMF (45.1mg, 0.10mmol), stearic acid (31.4mg, 0.11mmol) and DMAP (14.6mg, 0.12mmol) were added sequentially to a 100mL round bottom flask, dissolved in 5mL dry dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise quickly. After stirring at 43 ℃ for 4 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 3(36.4mg, 52% yield).
Of product 31The H NMR nuclear magnetic data is as follows, and the nuclear magnetic spectrum is shown in FIG. 28:
1H NMR(400MHz,CDCl3):δ5.14(s,2H),5.10(t,1H,J＝13.7Hz),4.18(t,2H,J＝11.8Hz),3.78(s,3H),3.70(t,4H.J＝9.3Hz),3.34(d,2H,J＝6.7Hz),2.67(t,2H,J＝15.2Hz),2.60(t,2H,J＝11.8Hz),2.49(t,4H,J＝9.1Hz),2.37-2.41(m,2H),2.27-2.30(m,2H),2.22(s,3H),1.77-1.80(m,5H),1.22-1.36(m,28H),0.88(t,3H,J＝13.6Hz).
example 4 synthesis of t-cos-MMF conjugate prodrug, as shown in figure 4:
MMF (45.1mg, 0.10mmol), tetracosanoic acid (40.6mg, 0.11mmol) and DMAP (14.6mg, 0.12mmol) were added sequentially in a 100mL round bottom flask, dissolved in 5mL dry dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise quickly. After stirring at 43 ℃ for 4 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 4(32.3mg, 41.2% yield, [ C%47H77NO8]+[M+H]+＝784.5722)。
Example 5 synthesis of PA-MMF conjugate prodrug, as shown in figure 5:
MMF (43.2mg, 0.10mmol), palmitoleic acid (24.5mg, 0.10mmol), and DMAP (12.6mg,0.10mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane, and EDC (16.6mg, 0.11mmol) was added dropwise rapidlymmol). After stirring at 43 ℃ for 6 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 5(27.0mg, 40.2% yield, [ C%39H59NO8]+[M+H]+＝670.4313)。
Example 6 synthesis of OA-MMF conjugate prodrug, as shown in fig. 6:
MMF (46.8mg, 0.11mmol), oleic acid (31.6mg, 0.11mmol) and DMAP (15.8mg, 0.13mmol) were added sequentially to a 100mL round bottom flask, dissolved in 5mL dry dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise quickly. After stirring at 43 ℃ for 5 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 50:1) gave product 6(35.5mg, 46.3% yield).
Of product 61The H NMR nuclear magnetic data is as follows, and the nuclear magnetic spectrum is shown in FIG. 29:
1H NMR(400MHz,CDCl3):δ5.35(t,2H,J＝9.9Hz),5.14(s,2H),5.10(t,1H,J＝17.2Hz),4.17(t,2H,J＝11.9Hz),3.78(s,3H),3.69(t,4H.J＝9.2Hz),3.34(d,2H,J＝6.7Hz),2.67(t,2H,J＝15.0Hz),2.59(t,2H,J＝12.0Hz),2.49(t,4H,J＝9.5Hz),2.39(t,2H,J＝14.7Hz),2.28(t,2H,J＝15.0Hz),2.22(s,3H),1.99-2.04(m,4H),1.77-1.81(m,5H),1.27-1.34(m,20H),0.88(t,3H,J＝13.2Hz).
example 7 synthesis of RA-MMF conjugate prodrug, as shown in figure 7:
MMF (46.8mg, 0.11mmol), ricinoleic acid (32.8mg, 0.11mmol) and DMAP (15.8mg, 0.13mmol) were added sequentially to a 100mL round bottom flask, dissolved in 5mL anhydrous dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 5 hours, the reaction was observed by thin layer chromatography. When the reaction is substantially complete, coolCooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 50:1) gave product 7(32.4mg, 41.2% yield, [ C%41H63NO9]+[M+H]+＝714.4576)。
Example 8 synthesis of LA-MMF conjugate prodrug, as shown in fig. 8:
MMF (65mg, 0.15mmol), linoleic acid (42.1mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round bottom flask, dissolved in 4mL dry dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise quickly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 20:1) gave product 8(47mg, 45.1% yield).
Of product 81The H NMR nuclear magnetic data is as follows, and the nuclear magnetic spectrum is shown in FIG. 30:
1H NMR(400MHz,CDCl3):δ5.30-5.42(m,4H),5.14(s,2H),5.10(t,1H,J＝13.3Hz),4.16(t,2H,J＝11.8Hz),3.78(s,3H),3.68(t,4H.J＝9.4Hz),3.34(d,2H,J＝6.9Hz),2.78(t,2H,J＝13.3Hz),2.68(t,2H,J＝15.4Hz),2.57(t,2H,J＝12.0Hz),2.47(t,4H,J＝9.2Hz),2.36-2.41(m,2H),2.26-2.31(m,2H),2.22(s,3H),2.03-2.08(m,4H),1.75-1.81(m,5H),1.25-1.36(m,14H),0.89(t,3H,J＝13.4Hz).
example 9 synthesis of LNA-MMF coupled prodrugs as shown in figure 9:
MMF (65mg, 0.15mmol), linolenic acid (42.1mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round bottom flask, dissolved in 4mL dry dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise quickly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; organic phaseDrying with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 20:1) gave product 9(47.1mg, 45.2% yield, [ C%41H59NO8]+[M+H]+＝694.4313)。
Example 10 synthesis of GLA-MMF conjugate prodrug, as shown in figure 10:
MMF (65mg, 0.15mmol), linolenic acid (42.1mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round bottom flask, dissolved in 4mL dry dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise quickly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 20:1) gave product 10(44.2mg, 42.4% yield, [ C%41H59NO8]+[M+H]+＝694.4313)。
Example 11 synthesis of AA-MMF coupled prodrug, as shown in fig. 11:
MMF (65mg, 0.15mmol), arachidonic acid (45.7mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round bottom flask, dissolved in 4mL dry dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise quickly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 20:1) gave product 11(46.9mg, 43.4% yield, [ C%43H61NO8]+[M+H]+＝720.4470)。
Example 12 synthesis of EPA-MMF conjugate prodrug, as shown in figure 12:
MMF (65mg, 0.15mmol), eicosapentaenoic acid (45.4mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added successively to a 100mL round-bottomed flask and dissolved in 4mLAnhydrous dichloromethane, followed by rapid dropwise addition of EDC (23.3mg, 0.15 mmol). After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 12(46.1mg, 42.8% yield, [ C%43H59NO8]+[M+H]+＝718.4313)。
Example 13 synthesis of DHA-MMF conjugated prodrug, as shown in figure 13:
to a 100mL round bottom flask were added sequentially MMF (43.2mg, 0.10mmol), cis-4, 7,10,13,16, 19-docosahexaenoic acid (34.5mg, 0.10mmol) and DMAP (12.6mg,0.10mmol), dissolved in 4mL dry dichloromethane, and EDC (16.6mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 6 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 13(32.7mg, 43.9% yield).
Of product 131The H NMR nuclear magnetic data is as follows, and the nuclear magnetic spectrum is shown in FIG. 31:
1H NMR(400MHz,CDCl3):δ5.29-5.50(m,12H),5.15(s,2H),5.10(t,1H,J＝13.6Hz),4.17(t,2H,J＝12.0Hz),3.79(s,3H),3.70(t,4H,J＝9.3Hz),3.34(d,2H,J＝6.7Hz),2.79-2.89(m,10H),2.76(t,2H,J＝15.2Hz),2.55-2.66(m,4H),2.49(t,4H,J＝9.3Hz),2.36-2.41(m,2H),2.26-2.31(m,2H),2.22(s,3H),2.04-2.11(m,2H),1.77(s,3H),0.97(t,3H,J＝15.1Hz).
example 14 synthesis of Ac-FK506 conjugate prodrug, as shown in figure 14:
FK506(80.4mg, 0.10mmol), glacial acetic acid (6.0mg, 0.10mmol) and DMAP (12.6mg,0.10mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane and EDC (16.6mg, 0.11mmol) was added dropwise rapidly. Stirring the mixture for 6 hours at the temperature of 43 ℃,the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 14(39.0mg, 46.1% yield, [ C%46H71NO13]+[M+H]+＝846.4998)。
Example 15 synthesis of Hep-FK506 conjugate prodrug, as shown in figure 15:
FK506(136.7mg, 0.17mmol), n-heptanoic acid (23.4mg, 0.18mmol) and DMAP (20.6mg, 0.17mmol) were added sequentially to a 100mL round bottom flask, dissolved in 5mL anhydrous dichloromethane and EDC (23.7mg, 0.15mmol) was added dropwise rapidly. After stirring at 43 ℃ for 4 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 15(65.0mg, 41.7% yield, [ C%51H81NO13]+[M+H]+＝916.5781)。
Example 16 synthesis of Ste-FK506 conjugated prodrug, as shown in figure 16:
FK506(80.4mg, 0.10mmol), stearic acid (31.4mg, 0.11mmol) and DMAP (14.6mg, 0.12mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 5mL anhydrous dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 4 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 16(50.6mg, 47.3% yield, [ C%62H103NO13]+[M+H]+＝1070.7502)。
Example 17 synthesis of t-cos-FK506 conjugate prodrug, as shown in FIG. 17:
FK506(80.4mg, 0.10mmol), tetracosanoic acid (40.6mg, 0.11mmol) and DMAP (14.6mg, 0.12mmol) were added sequentially in a 100mL round bottom flask, dissolved in 5mL dry dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 4 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 17(54.2mg, 46.9% yield, [ C%68H115NO13]+[M+H]+＝1154.8441)。
Example 18 synthesis of PA-FK506 conjugate prodrug, as shown in fig. 18:
FK506(80.4mg, 0.10mmol), palmitoleic acid (25.5mg, 0.10mmol), and DMAP (12.6mg,0.10mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane, and EDC (16.6mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 6 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 18(43.8mg, 42.1% yield, [ C%60H97NO13]+[M+H]+＝1040.7033)。
Example 19 synthesis of OA-FK506 conjugated prodrugs as shown in fig. 19:
FK506(88.4mg, 0.11mmol), oleic acid (31.6mg, 0.11mmol) and DMAP (15.8mg, 0.13mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 5mL anhydrous dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 5 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; by column chromatographyAfter chromatographic purification (DCM: MeOH ═ 50:1), product 19(47.4mg, 40.3% yield, [ C ] was obtained62H101NO13]+[M+H]+＝1068.7346)。
Example 20 synthesis of RA-FK506 conjugate prodrug, as shown in figure 20:
FK506(88.4mg, 0.11mmol), ricinoleic acid (32.8mg, 0.11mmol) and DMAP (15.8mg, 0.13mmol) were added successively to a 100mL round-bottomed flask, dissolved in 5mL anhydrous dichloromethane and EDC (17.5mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 5 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 50:1) gave product 20(46.1mg, 38.6% yield, [ C%62H101NO14]+[M+H]+＝1084.7295)。
Example 21 synthesis of LA-FK506 conjugate prodrug, as shown in figure 21:
FK506(120.6mg, 0.15mmol), linoleic acid (42.1mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round bottom flask, dissolved in 4mL anhydrous dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise rapidly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 21(63.2mg, 39.5% yield, [ C)62H99NO13]+[M+H]+＝1066.7189)。
Example 22 synthesis of LNA-FK506 conjugate prodrug, as shown in fig. 22:
FK506(120.6mg, 0.15mmol), linolenic acid (42.1mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane, and EDC (23.3mg, 0.15mmol) was added dropwise rapidly. Stirring for 3 hours at the temperature of 43 DEG CThen, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 22(65.3mg, 40.9% yield, [ C%62H97NO13]+[M+H]+＝1064.7033)。
Example 23 synthesis of GLA-FK506 conjugate prodrug, as shown in figure 23:
FK506(120.6mg, 0.15mmol), linolenic acid (42.1mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane, and EDC (23.3mg, 0.15mmol) was added dropwise rapidly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 23(66.1mg, 41.4% yield, [ C%62H97NO13]+[M+H]+＝1064.7033)。
Example 24 synthesis of AA-FK506 conjugate prodrug, as shown in figure 24:
FK506(120.6mg, 0.15mmol), arachidonic acid (45.7mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added sequentially to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise rapidly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 24(60.2mg, 36.8% yield, [ C%64H99NO13]+[M+H]+＝1090.7189)。
Example 25 synthesis of EPA-FK506 conjugate prodrug, as shown in fig. 25:
FK506(120.6mg, 0.15mmol), eicosapentaenoic acid (45.4mg, 0.15mmol) and DMAP (18.3mg, 0.15mmol) were added successively to a 100mL round-bottomed flask, dissolved in 4mL anhydrous dichloromethane and EDC (23.3mg, 0.15mmol) was added dropwise rapidly. After stirring at 43 ℃ for 3 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 25(74.5mg, 45.6% yield, [ C%64H97NO13]+[M+H]+＝1088.7033)。
Example 26 synthesis of a DHA-FK506 conjugated prodrug, as shown in figure 26:
to a 100mL round-bottomed flask were added FK506(80.4mg, 0.10mmol), cis-4, 7,10,13,16, 19-docosahexaenoic acid (32.8mg, 0.10mmol) and DMAP (12.6mg,0.10mmol) in that order, dissolved in 4mL anhydrous dichloromethane, and EDC (16.6mg, 0.11mmol) was added dropwise rapidly. After stirring at 43 ℃ for 6 hours, the reaction was observed by thin layer chromatography. When the reaction is almost finished, cooling the reaction solution, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate and saturated saline solution respectively; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 40:1) gave product 26(48.2mg, 42.6% yield, [ C)67H103NO13]+[M+H]+＝1130.7502)。
EXAMPLE 27 preparation of Hep-MMF self-assembled Nanodiulation
Preparing Hep-MMF self-assembly nanometer preparation with MMF final concentration of 1 mg/ml. Based on the mass of MMF, a corresponding amount of Hep-MMF (prepared in example 2) was dissolved in 100. mu.L of dimethyl sulfoxide solution and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 32 and 47.
EXAMPLE 28 preparation of Ste-MMF self-assembled Nanodiulation
Ste-MMF self-assembled nano-formulations were prepared with a final MMF concentration of 1 mg/ml. Calculated on the mass of MMF, the corresponding amount of Ste-MMF (prepared in example 3) was dissolved in 100. mu.L of dimethyl sulfoxide solution and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscope thereof are shown in FIGS. 33 and 48.
Example 29 preparation of OA-MMF self-assembled Nanodiulation
OA-MMF self-assembly nano-preparation with MMF final concentration of 1mg/ml is prepared. A corresponding amount of OA-MMF (prepared in example 8) was dissolved in 100. mu.L of dimethyl sulfoxide solution, calculated on the mass of MMF, and then rapidly injected into 900. mu.L of water under ultrasonic conditions. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 34 and 49.
Example 30 preparation of LA-MMF self-assembled Nanodisclosed
Preparing the LA-MMF self-assembly nano preparation with the MMF final concentration of 1 mg/ml. Calculated on the mass of MMF, the corresponding amount of LA-MMF (prepared in example 6) was dissolved in 100. mu.L of DMSO solution and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 35 and 50.
Example 31 preparation of DHA-MMF self-assembled Nanodisclosures
Preparing the DHA-MMF self-assembly nano preparation with the final concentration of MMF of 1 mg/ml. A corresponding amount of DHA-MMF (prepared in example 13) was dissolved in 100. mu.L of dimethyl sulfoxide solution, calculated on the mass of MMF, and then rapidly injected into 900. mu.L of water under ultrasonic conditions. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 36 and 51.
Example 32 preparation of Hep-FK506 self-assembled Nano-formulations
Preparation of Hep-FK506 self-assembled nano-preparation with final concentration of FK506 of 1mg/ml. Based on the mass of FK506, the corresponding amount of Hep-FK506 (prepared in example 15) was dissolved in 100. mu.L of dimethyl sulfoxide solution and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
Example 33 preparation of Ste-FK506 self-assembled Nanormulation
Ste-FK506 self-assembling nano-formulations with a final concentration of FK506 of 1mg/ml were prepared. The corresponding amount of Ste-FK506 (prepared in example 16) was dissolved in 100. mu.L of dimethyl sulfoxide solution by mass of FK506 and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
Example 34 preparation of LA-FK506 self-assembled Nano-formulations
Preparing the LA-FK506 self-assembly nano preparation with the final concentration of FK506 of 1 mg/ml. Corresponding amounts of LA-FK506 (prepared in example 21) were dissolved in 100. mu.L of dimethyl sulfoxide solution, calculated on the mass of FK506, and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
Example 35 preparation of DHA-FK506 self-assembled Nano-formulations
Preparing the DHA-FK506 self-assembly nano preparation with the final concentration of FK506 of 1 mg/ml. The corresponding amount of DHA-FK506 (prepared in example 26) was dissolved in 100. mu.L of dimethyl sulfoxide solution by mass of FK506 and rapidly injected into 900. mu.L of water under sonication. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
EXAMPLE 36 preparation of Hep-MMF/FK506 Co-assembled Nanopropulation
Preparing a Hep-MMF/FK506 co-assembled nano-preparation with the final concentration of MMF of 1 mg/ml. Based on the mass of the MMF, the Hep-MMF and the FK506 are mixed in 100 mu L of dimethyl sulfoxide solution according to the mass ratio of 20:1, and then are quickly injected into 900 mu L of water under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 37 and 52Shown in the figure.
Example 37 preparation of Ste-MMF/FK506 Co-assembled Nanodiulation
Ste-MMF/FK506 co-packaged nano-formulations were prepared with a final concentration of MMF of 1 mg/ml. Calculated by the mass of MMF, Ste-MMF and FK506 are mixed in 100 mu L of dimethyl sulfoxide solution according to the mass ratio of 20:1, and then are quickly injected into 900 mu L of water under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 38 and 53.
Example 38 preparation of OA-MMF/FK506 Co-assembled NanoPrepration
OA-MMF/FK506 co-packaged nano-formulations with a final concentration of MMF of 1mg/ml were prepared. Calculated by the mass of MMF, OA-MMF and FK506 are mixed in 100 mu L of dimethyl sulfoxide solution according to the mass ratio of 20:1, and then are quickly injected into 900 mu L of water under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 39 and 54.
Example 39 preparation of LA-MMF/FK506 Co-assembled NanoPrepration
Preparing LA-MMF/FK506 co-assembled nano-preparation with MMF final concentration of 1 mg/ml. Calculated by the mass of MMF, LA-MMF and FK506 are mixed in 100 mu L of dimethyl sulfoxide solution according to the mass ratio of 20:1, and then are quickly injected into 900 mu L of water under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 40 and 55.
Example 40 preparation of DHA-MMF/FK506 Co-packaged Nano formulations
Preparing DHA-MMF/FK506 co-assembled nano-preparation with MMF final concentration of 1 mg/ml. Based on the mass of the MMF, the DHA-MMF and the FK506 are mixed in 100 mu L of dimethyl sulfoxide solution according to the mass ratio of 20:1, and then are quickly injected into 900 mu L of water under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 41 and 56.
EXAMPLE 41 preparation of LA-MMF/LA-FK506 Co-assembled Nanowormulations
Preparing LA-MMF/LA-FK506 co-assembled nano-preparation with MMF final concentration of 1 mg/ml. Calculated by the mass of MMF and FK506, LA-MMF and LA-FK506 are mixed in 100 mu L of dimethyl sulfoxide solution according to the mass ratio of 20:1, and then are quickly injected into 900 mu L of water under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
Example 42 preparation of DHA-MMF/DHA-FK506 Co-assembled Nanodisclosure
Preparing a DHA-MMF/DHA-FK506 co-assembled nano-preparation with the MMF final concentration of 1 mg/ml. Calculated according to the mass of MMF and FK506, LA-MMF and LA-FK506 are mixed in dimethyl sulfoxide solution of 100 mu L according to the mass ratio of 20:1, and then are quickly injected into water of 900 mu L under the ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
EXAMPLE 43 preparation of Pegylated Hep-MMF/FK506 Co-assembled Nanopropulation
Preparing the PEGylated Hep-MMF/FK506 co-assembled nano-preparation with the MMF final concentration of 1 mg/ml. Based on the mass of MMF, Hep-MMF, FK506 and DSPE-PEG2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 42 and 57.
EXAMPLE 44 preparation of Pegylated Ste-MMF/FK506 Co-assembled Nanodiulation
A pegylated Ste-MMF/FK506 co-packaged nano-formulation was prepared with a final concentration of MMF of 1 mg/ml. Calculating by mass of MMF, Ste-MMF, FK506 and DSPE-PEG2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscope thereof are shown in FIGS. 43 and 58.
EXAMPLE 45 preparation of Pegylated LA-MMF/FK506 Co-assembled Nanodisclosure
Preparation of PEGylated LA-containing drugs with MMF final concentration of 1mg/mlMMF/FK506 co-packaged nano-formulations. Calculating by mass of MMF, LA-MMF, FK506 and DSPE-PEG2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 44 and 59.
EXAMPLE 46 preparation of a Pegylated OA-MMF/FK506 Co-assembled NanoPrepration
Preparing the PEGylated OA-MMF/FK506 co-assembled nano-preparation with the MMF final concentration of 1 mg/ml. Calculating OA-MMF, FK506 and DSPE-PEG according to the mass of MMF2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 45 and 60.
EXAMPLE 47 preparation of a Pegylated DHA-MMF/FK506 Co-assembled NanoPrep
Preparing the PEGylated DHA-MMF/FK506 co-assembled nano-preparation with the MMF final concentration of 1 mg/ml. Calculating DHA-MMF, FK506 and DSPE-PEG based on MMF mass2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles. The particle size distribution and transmission electron microscopy are shown in FIGS. 46 and 61.
EXAMPLE 48 preparation of Pegylated LA-MMF/LA-FK506 Co-assembled NanoPrepration
Preparing the co-assembled nano preparation of the pegylated LA-MMF/LA-FK506 with the MMF final concentration of 1 mg/ml. Calculating the mass of MMF and FK506, and mixing LA-MMF, LA-FK506 and DSPE-PEG2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
Example 49 preparation of a Pegylated DHA-MMF/DHA-FK506 Co-assembled NanoPrepration
Preparing the co-assembled nano preparation of the polyethylene glycol DHA-MMF/DHA-FK506 with the MMF final concentration of 1 mg/ml. Calculating the mass of the MMF and FK506, and adding DHA-MMF, DHA-FK506 and DSPE-PEG2kMixing the mixture in a mass ratio of 20:1:2 in 100 mu L of dimethyl sulfoxide solution, and quickly injecting the mixture into 900 mu L of water under an ultrasonic condition. Final dialysis (M)W3500) The organic solvent is removed to obtain uniformly dispersed nanoparticles.
EXAMPLE 50 preparation of LA-MMF prodrug liposomes
To prepare a 4% drug loading final drug concentration of LA-MMF prodrug liposome, lecithin, cholesterol and DSPE-PEG were first mixed2kDissolving the lipid mixture with the mass ratio of 35:5:8 in 0.9mL of ethanol, mixing 0.1mL of the LA-MMF prodrug dissolved in the ethanol and 10mg/mL of the lipid mixture with 0.9mL of the lipid mixture, and directly injecting the mixture into 10mL of water to obtain the liposome particles. Centrifuging at 100000g × 10min with ultra-high speed centrifuge to remove organic solvent, collecting precipitate to obtain high purity lipid particles, diluting, and measuring concentration with high performance liquid. The liposomes were then diluted to 1mg/mL according to the measured liposome concentration in the HPLC. The liposome solution can be lyophilized to obtain lyophilized powder.
EXAMPLE 51 preparation of DHA-MMF prodrug liposomes
To prepare a 4% drug loading DHA-MMF prodrug liposome with a final drug concentration of 1mg/mL, lecithin, cholesterol and DSPE-PEG were first mixed2kDissolving the lipid mixture with the mass ratio of 35:5:8 in 0.9mL of ethanol, mixing 0.1mL of the DHA-MMF prodrug dissolved in the ethanol and 10mg/mL of the lipid mixture with 0.9mL of the lipid mixture, and directly injecting the mixture into 10mL of water to obtain the liposome particles. Centrifuging at 100000g × 10min with ultra-high speed centrifuge to remove organic solvent, collecting precipitate to obtain high purity lipid particles, diluting, and measuring concentration with high performance liquid.
EXAMPLE 52 preparation of LA-MMF Polymer Nanodisclositions
In order to prepare the LA-MMF prodrug polymer micelle drug-loaded particles with drug-loading capacity of 5% and final drug concentration of 1mg/mL, firstly, a mixture of polyethylene glycol-polylactic acid (molecular weight is 5k-10k)/LA-MMF (1mg) in a mass ratio of 20:1 is dissolved in 1mL of acetone, and then the acetone is dissolved and slowly injected into 10mL of water to obtain the micelle particles. Removing the organic solvent, and concentrating to obtain LA-MMF (1mg/mL) drug-loaded nanoparticles. The polymer micelle solution can be freeze-dried to obtain freeze-dried powder.
Example 53 construction of liver transplantation model and Combined immunosuppressive therapy experiment
Constructing an acute rejection rat liver transplantation model, taking a DA rat and a Lewis rat as a donor and an acceptor respectively, and performing liver transplantation operation according to a 'double-sleeve technology' established by Kamada and Calne. DA rats were anesthetized and then heparinized systemically, the donor liver was isolated and briefly placed in Ringer's equilibrium at 4 ℃ and subsequently transplanted in situ into Lewis rats. The reconstruction of the hepatic superior and inferior vena cava is performed by a suture method, and the reconstruction of the hepatic inferior vena cava and portal vein is performed by a sleeve method. The administration was started the next day after the liver transplantation operation, and was performed 1 time every two days for 7 times (the dose was shown in FIGS. 62 and 63). The nano-drug group is administrated in a tail vein mode, the clinical administration forms of MMF and FK506 are orally administrated, the change situation of the body weight of rats is recorded within 15 days after the administration, and the survival time of Lewis rats is used as the evaluation basis of the treatment effect of the drug. As shown in fig. 62 and 63 (n is the number of experimental mice), MMF and FK506 (i.e., TAC) clinical drug form (Combo) and nano drug form (SAIC, prepared in example 45) both effectively prolonged median survival of liver transplant recipients (Lewis rats) compared to Saline (salt). Compared with clinical administration forms of MMF and FK506, the nano-drug group has more remarkable treatment effect.
In conclusion, the nano preparation in the form of the invention obviously improves the water solubility of MMF and FK506, avoids using auxiliary materials such as solubilizer and the like, and further improves the drug-loading rate; meanwhile, the saturated or unsaturated fatty acid used for modification is a substance required by human body, has good biocompatibility, is convenient for clinical transformation, and has better application prospect. More importantly, the nano preparation is assembled by the pegylated MMF-saturated or unsaturated fatty acid coupled prodrug and the FK506, the pharmacokinetic property of the drug is obviously improved, the in vivo circulation time of the drug is prolonged, the MMF and the FK506 can be simultaneously delivered to a target site to play a drug effect, and the effect of resisting transplant rejection is better compared with that of clinical MMF/FK 506.
Claims (9)
1. A fatty acid coupled prodrug characterized by one or more of the structures shown in the following formulas:
wherein R is selected from acyl obtained by removing hydroxyl from n-heptanoic acid, stearic acid, tetracosanoic acid, oleic acid, ricinoleic acid, palmitoleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.
2. A process for the preparation of the fatty acid conjugated prodrug of claim 1, wherein the fatty acid conjugated prodrug is obtained by esterification of mycophenolate with a saturated or unsaturated fatty acid corresponding to R in the presence of a condensing agent and a catalyst.
3. A self-assembled nano-preparation of a fatty acid coupled prodrug, characterized in that: comprising one or more of the fatty acid conjugated prodrugs of claim 1.
4. The co-assembled nano preparation of the fatty acid coupled prodrug is characterized in that:
the method comprises the following steps: (1) one or more of the fatty acid coupled prodrugs of claim 1; (2) tacrolimus;
or comprises the following steps: (1) one or more of the fatty acid coupled prodrugs of claim 1; (2) tacrolimus; (3)1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ].
5. The co-assembled nanoformulation of fatty acid coupled prodrugs according to claim 4, comprising: (1) one or more of the fatty acid coupled prodrugs of claim 1; (2) when tacrolimus is used, the content ratio of the tacrolimus to the nano preparation is 20: 0.5 to 5; or, when comprising: (1) one or more of the fatty acid conjugate prodrugs of claim 1; (2) tacrolimus; (3)1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ], wherein the content ratio of the three is 20: 0.5-5: 1 to 10.
6. The co-assembled nanoformulation of fatty acid coupled prodrug of claim 5, wherein the ratio of the two is 20: 1; the content ratio of the three is 20:1: 2.
7. a method for preparing the self-assembled nano preparation of the fatty acid coupling prodrug as claimed in claim 3, wherein the organic solvent of the corresponding fatty acid coupling prodrug is injected into the water phase under the ultrasonic condition, and the nano preparation is dialyzed to obtain uniformly dispersed nano particles.
8. A method for preparing a co-assembled nano-formulation of a fatty acid coupled prodrug of any one of claims 4 to 5, wherein the organic solvent of the corresponding fatty acid coupled prodrug and tacrolimus, or the organic solvent of the corresponding fatty acid coupled prodrug, tacrolimus and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] is injected into an aqueous phase under ultrasonic conditions, and then dialyzed to obtain uniformly dispersed nano-particles.
9. Use of the fatty acid conjugate prodrug of claim 1 and a water-soluble preparation thereof for preparing a graft-versus-rejection drug.
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