CN108641075B

CN108641075B - Rapamycin and double short-chain polyethylene glycol prodrug of rapamycin derivative and application of rapamycin and double short-chain polyethylene glycol prodrug

Info

Publication number: CN108641075B
Application number: CN201810178155.2A
Authority: CN
Inventors: 周新基; 张毅; 李飞
Original assignee: Thousands Of Jiangsu Kang Biological Medicine Technology Co Ltd
Current assignee: Thousands Of Jiangsu Kang Biological Medicine Technology Co Ltd
Priority date: 2018-03-05
Filing date: 2018-03-05
Publication date: 2019-12-06
Anticipated expiration: 2038-03-05
Also published as: CN108641075A

Abstract

The water solubility of the compound is more than 10mg/mL, so that the problem of poor water solubility of RAPA is solved; the molecular weight of the short-chain MPEG is 400-600, which avoids the safety problem possibly brought by the high molecular weight MPEG, has better safety and is suitable for injection administration.

Description

Rapamycin and double short-chain polyethylene glycol prodrug of rapamycin derivative and application of rapamycin and double short-chain polyethylene glycol prodrug

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a double short-chain polyethylene glycol prodrug of rapamycin and a derivative thereof and application thereof.

Background

Rapamycin (Rapamycin, RAPA) is a novel macrolide immunosuppressant, has high immunosuppressive activity, and is clinically used for the treatment of organ transplantation rejection and autoimmune diseases. Rapamycin was first marketed in the united states in oral liquid form in 1999, after which tablets prepared by Elan using the nano-crystallization technique were also marketed in the united states. The rapamycin oral liquid is a self-emulsifying concentrated solution containing absolute ethyl alcohol, surfactant and oil, the preparation is mainly an ethanol solution of the medicine, after oral administration, in a water-soluble medium of a digestive tract, the medicine can be rapidly separated out and aggregated to form large solid medicine particles which are difficult to be directly absorbed by the digestive tract, and although a large amount of surfactant (Tween 80) in the preparation can block the separation of the medicine to a certain extent and promote the absorption of the medicine particles by the digestive tract, the bioavailability in a human body is low and is about 14%. In addition, the oral liquid is diluted with water in vitro and then stirred vigorously to form an emulsion, which is inconvenient for patients to take and quantitatively concentrate, and the preparation needs to be refrigerated at low temperature, needs to be used within one month after being unsealed, and is not suitable for carrying. The bioavailability of the rapamycin nanocrystal tablet prepared by the grinding method is about 16 percent. The preparation of the lappaconitine tablets by the grinding method requires a ball mill to grind for 5 days at 50 percent of critical speed, and the production period is long. The rapamycin oral preparation also has the problems of large individual difference and the like, and the clinical application of the rapamycin oral preparation is influenced.

In order to improve the water solubility of rapamycin, researchers at home and abroad have conducted a great deal of research, and among them, RAPA derivatives Everolimus (Everolimus) and Temsirolimus (Temsirolimus) have been approved by FDA in the united states as antitumor drugs and have been on the market, but the water solubility thereof is still unsatisfactory, and a non-aqueous solvent is used for Temsirolimus injection.

The technology of polyethylene glycol (PEG) modification of drugs is a new technology rapidly developed in recent years, namely, the existing proteins, polypeptides and some drug ingredients are coupled with activated PEG to form PEG-drug conjugates. The applicant finds that the water solubility of the RAPA-PEG-4000 derivative (A) can be obviously improved by adopting succinic acid as a connecting chain and connecting the glycine derivative of RAPA with PEG-4000, but the molecular weight of the derivative reaches about 6000, and the oral bioavailability is not improved. Glycine derivatives of RAPA are connected with MPEG-2000 to obtain compound B with molecular weight of about 3000, and water solubility is obviously improved. However, further research shows that B is difficult to absorb when taken orally, and is directly injected, so that MPEG-2000 has a large molecular weight and has a safety problem. The polyethylene glycol with small molecular weight such as PEG-400, PEG-500 and the like can be clinically used as an auxiliary material for injection, and has good safety. However, by linking the glycine derivative of RAPA to MPEG-500, Compound C was obtained with a molecular weight of about 1600 without significant improvement in water solubility. Suggesting that too short a PEG chain is not beneficial for improving the water solubility of RAPA-PEG.

To compromise the safety and water solubility of the RAPA derivatives, applicants propose linking multiple short chains of PEG to the RAPA molecule. A plurality of hydroxyl groups exist in RAPA molecules, but only the hydroxyl group on the side chain has better reactivity. Therefore, the applicant uses glutamic acid and succinic acid as connecting chains, 1 molecule of RAPA is coupled with 2 molecules of short-chain MPEG-500 to form the water-soluble MPEG-500-RAPA derivative (D), and the water-soluble MPEG-500-RAPA derivative (D) is expected to improve the water solubility and have better safety.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a double short-chain polyethylene glycol prodrug of rapamycin and a derivative thereof and application thereof. The drug is formed by coupling 1 molecule of RAPA or derivative thereof with 2 molecules of short-chain MPEG to form a water-soluble prodrug. The water solubility of the compound is more than 10mg/mL, so that the problem of poor water solubility of RAPA is solved; the molecular weight of the short-chain MPEG is 400-600, which avoids the safety problem possibly brought by the high molecular weight MPEG, has better safety and is suitable for injection administration. Can be used for preparing anti-rejection medicines for organ transplantation, autoimmune diseases, and antitumor medicines.

The technical scheme is as follows: rapamycin and double short-chain polyethylene glycol prodrug of rapamycin derivative, the structure conforms to the general formula (I)

Wherein: m is 0 or 1, n is 0, 1, 2, 3 or 4, R1 is-H, alkyl of 1-4 carbon atoms or benzyl, x is 0 or 1, PEG has a molecular weight of 400-.

Preferably any one of the following compounds 1 to 12:

The application of the compound or the pharmaceutically acceptable salt thereof in preparing the immunosuppressant.

An immunosuppressant contains the above compound or its pharmaceutically acceptable salt as effective component.

The compound or the pharmaceutically acceptable salt thereof can be applied to the preparation of antitumor drugs.

An antitumor drug contains the above compound or its pharmaceutically acceptable salt as effective component.

The water solubility of the compound shown by the invention is more than 10mg/mL, and the problem of poor water solubility of RAPA is solved. In vivo, the compounds of the invention are capable of releasing RAPA.

Has the advantages that: the water solubility of the compound is more than 10mg/mL, so that the problem of poor water solubility of RAPA is solved; the molecular weight of the short-chain MPEG is 400-600, which avoids the safety problem possibly brought by the high molecular weight MPEG, has better safety and is suitable for injection administration.

Drawings

FIG. 1 is a schematic diagram of the chemical structure of a target compound of the present invention.

FIG. 2 shows a scheme for synthesizing the objective compound (1).

FIG. 3 shows a scheme for synthesizing the objective compound (6).

Detailed Description

The following examples are given to enable a person skilled in the art to fully understand the invention, but do not limit it in any way. The amino acid to be used in the present invention may be an L-form or D-form amino acid, or may be a racemic amino acid.

Example 1: synthesis of Methyleneglycol 500-glutamic acid-succinic acid-rapamycin conjugate (Compound (1))

The synthetic route is shown in figure 2

(1) Synthesis of Boc protected glutamic acid and polyethylene glycol monomethyl ether 500 conjugate (1-2)

MPEG-5004.4 g (8.8mmol) and 10mL of anhydrous methylene chloride were put into a 100mL reaction flask and dissolved by stirring. A50 mL Erlenmeyer flask was charged with Boc-Glu (1-1)1.0g (4mmol), DMAP 0.14g (1.14mmol), EDCI 1.93g (10.3mmol) and dry dichloromethane 10mL, and the mixture was dissolved with stirring. The reaction was carried out at room temperature for 8h, and the disappearance of the starting material BocGlu (1-1) was monitored by TLC. The mixture was washed by adding 10 wt.% aqueous KHSO4 solution and brine, collecting the organic layer and drying over anhydrous Na2SO 4. DCM/MeOH (40: 1-30: 1, v/v) was purified by flash column chromatography to give 3.6g of colorless oil (5) in 75% yield. 1H NMR (300MHz, CDCl3) δ 4.53(s,1H),4.34-4.28(M,2H),4.25(d, J ═ 4.6Hz,2H),3.75-3.71(M,4H),3.67(d, J ═ 5.8Hz,82H),3.57(dd, J ═ 5.6,3.8Hz,4H),3.40(s,6H),2.46(dt, J ═ 18.8,9.4Hz,4H),1.46(s,9H), ms esi) M/z:1156.6,1200.3,1244.7,1288.1,1332.2, { [ M + H + ] }.

(2) Synthesis of glutamic acid polyethylene glycol 500 monomethyl ether conjugate (1-3)

A100 mL reaction flask was charged with 3.6g (3mmol) of Boc-protected glutamic acid-polyethylene glycol monomethyl ether 500 conjugate (1-2) and 20mL of anhydrous dichloromethane, and the mixture was dissolved by stirring. Trifluoroacetic acid (2.74 g, 24mmol) was added dropwise stepwise to an eggplant type bottle. Stir at rt for 2h and TLC monitored the disappearance of the starting colorless oil (5). The reaction was stopped by adding excess methanol, and the solvent was distilled off under reduced pressure to give 3.16g of glutamic acid polyethylene glycol 500 monomethyl ether conjugate (1-3) as a colorless oil in a yield of 95%. 1H NMR (300MHz, CDCl3) δ 4.42(s,2H),4.16(d, J ═ 7.1Hz,2H),3.81(s,4H),3.74-3.62(M,82H),3.61-3.56(M,4H),3.41(s,6H),3.36(s,1H),2.68(s,2H),2.41(t, J ═ 13.8Hz,2H). (ms esi) M/z:1056.3,1100.2,1144.5,1188.1,1232.2, { [ M + H + ] }.

(3) Synthesis of Compound (1-4)

A100 mL reaction flask was charged with 2.2g (2mmol) of glutamic acid polyethylene glycol 500 monomethyl ether conjugate (1-3) and 20mL of anhydrous tetrahydrofuran, and the mixture was dissolved by stirring. 0.24g (2.4mmol) of succinic anhydride was weighed into a reaction flask, and 0.5mL of triethylamine was added. TLC monitored the disappearance of the starting glutamic acid polyethylene glycol 500 monomethyl ether conjugate (1-3). DCM/MeOH (20: 1-10: 1, v/v) was purified by flash column chromatography to give 2.1g of colorless oily compound (1-4) in 87% yield. 1H NMR (300MHz, CDCl3) δ 5.18(s,1H),4.36-4.24(M,3H),4.20(d, J ═ 5.2Hz,2H),3.68(dd, J ═ 4.9,3.6Hz,4H),3.65-3.58(M,82H),3.53(dd, J ═ 5.3,3.5Hz,4H),3.35(s,6H),2.60(M,2H),2.47-2.37(M,2H),2.16(dd, J ═ 13.1,6.2Hz,1H),2.05(s,1H),2.01-1.86(M,2H), ms (esi) M/z:1156.3,1200.2,1244.6,1288.1,1332.1, { [ M + H + ].

(4) Synthesis of target Compound (1)

A100 mL reaction flask was charged with 0.914g (1mmol) of RAPA and 20mL of anhydrous dichloromethane, and the mixture was dissolved with stirring. 1.4g (1.2mmol) of the compound (1-4), 0.035g (0.25mmol) of DMAP and 0.22g (1.1mmol) of DCC were weighed out, and the mixture was put into a 50mL eggplant type flask, followed by addition of 10mL of anhydrous dichloromethane and dissolution by stirring. The mixed solution is dripped into a 100mL eggplant-shaped bottle, and the mixture reacts for 2 hours at the temperature of 0 ℃ and then reacts for 8 hours at room temperature. TLC monitored the disappearance of raw RAPA. The solvent was removed under reduced pressure and flash column chromatography purification was carried out with DCM/MeOH (60: 1-20: 1, v/v) to give the desired compound (1) as a white solid (1)1.83g, 86% yield.

H NMR(300MHz,CDCl)δ9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45 (dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d, 2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,82H),3.53(dd,4H),3.43(d,1H),3.36(s,6H), 3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd,3H),2.27-1.77(m,6H),1.72 (s,3H),1.62(s,3H),1.56(d,4H),1.44-1.11(m,6H),0.97(d,3H),0.84(dd,5H),0.74(dd, 4H),0.67-0.51(m,1H).

Example 2: synthesis of Methyleneglycol 500-glutamic acid-malonic acid-rapamycin conjugate (Compound (2))

The reference compound (1) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, malonic acid and rapamycin as raw materials. White solid, 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,82H),3.53(dd,4H),3.45-3.39(m,3H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd, 7H), 1.7 (d,1H), 1.11H), 1.7 (d,1H), 3.7 (d,1H), 3H), 3.7 (d,1H), 0.84(dd,5H),0.74(dd,4H),0.67-0.51(m,1H).

Example 3: synthesis of Methyleneglycol 500-glutamic acid-glutaric acid-rapamycin conjugate (Compound (3))

The reference compound (1) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, glutaric acid and rapamycin as raw materials. White solid, 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,82H),3.53(dd,4H),3.43(d,1H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H), 2.45-36 (m, 7.7H), 1H), 1, 11.7.7 (d, 6H), 3.7.7, 1H), 0.84(dd,5H),0.74(dd,4H),0.67-0.51(m,1H).

Example 4: synthesis of Methyleneglycol 500-glutamic acid-oxalic acid-rapamycin conjugate (Compound (4))

The reference compound (1) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, oxalic acid and rapamycin as raw materials. White solid, 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,82H),3.53(dd,4H),3.43(d,1H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd, 3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(d, 1H), 3.84H), 3.7 (d,1H), 3.72, 1H), 1.7 (d, 6H), 5H) 0.74(dd,4H),0.67-0.51(m,1H).

Example 5: synthesis of Methyleneglycol 500-glutamic acid-adipic acid-rapamycin conjugate (Compound (5))

The reference compound (1) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, adipic acid and rapamycin as raw materials. White solid, 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,82H),3.53(dd,4H),3.43(d,1H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.38 (m, 38H), 1H), (1.7.11-7.11H), (1H, 1H), (7.72-1H), (1H, 1H), (7.11-7.72H), (1H, 1H), 6H) 0.97(d,3H),0.84(dd,5H),0.74(dd,4H),0.67-0.51(m,1H).

Example 6: synthesis of Methyleneglycol 500-glutamic acid-succinic acid-glycine-rapamycin conjugate (Compound (6))

The synthetic route is shown in figure 3

(1) Synthesis of Compound (6-1)

Compound (1-4) (1.56g, 1.25mmol) was dissolved in 30m L dichloromethane and then cooled to about 1 ℃ in an ice bath. N-hydroxysuccinimide (NHS) (1.44g, 12.5mmol) was dissolved in 20ml of N, N-dimethylformamide and added dropwise to the above solution; finally, N' -Dicyclohexylcarbodiimide (DCC) (2.58g, 12.5mmol) was dissolved in 40m L dichloromethane and added dropwise to the reaction solution until the reaction solution was clear. The mixture is reacted for 2 hours under ice bath condition and then placed at room temperature for 12 hours. After the reaction is finished, white precipitate is removed by filtration, filtrate is washed with saturated NaCl for 2 times, anhydrous Na2SO4 is dried, the filtrate is filtered, after the filtrate is dried in a spinning mode, 30mL of dichloromethane is added, stirring and dissolving are carried out, the filtrate is filtered again, the filtrate is concentrated again and then added into a large amount of anhydrous ether, and white pasty precipitate is separated out to be the compound (6-1).

(2) Synthesis of Compound (6)

A100 mL reaction flask was charged with 0.46g (0.5mmol) of RAPA and 20mL of anhydrous dichloromethane, and the mixture was dissolved with stirring. 1.0g (0.8mmol) of the compound (6-1), 35mg (0.25mmol) of DMAP and 220mg (1.1mmol) of DCC were weighed, and the weighed mixture was put into a 50mL eggplant-type bottle, followed by addition of 10mL of anhydrous dichloromethane and dissolution by stirring. The mixture was slowly dropped into a 100mL eggplant-shaped flask, reacted at 0 ℃ for 2 hours, and then reacted at room temperature for 8 hours. TLC monitored the disappearance of raw RAPA. The solvent was removed under reduced pressure and flash column chromatography purification was carried out with DCM/MeOH (60: 1-20: 1, v/v) to give 1.83g of a white solid with 86% yield. 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.02-3.98(m,4H),3.92(d,1H),3.62(t,82H),3.53(dd,4H),3.43(d,1H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd,3H), 2.28 (s,6H),3.14(s,3H),3.04 (d,2H), 3.76 (d,1H), 3.7 (d, 6H), 3.7H), 1H), 3.6H), 1.7 (d,1H), 3.6H), 3.11, 1H), 3.6H), 1H, 11, 6H, 1H), 0.74(dd,4H),0.67-0.51(m,1H).

Example 7: synthesis of Methyleneglycol 500-glutamic acid-succinic acid-valine-Everolimus conjugate (Compound (7))

The reference compound (6) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, succinic acid, valine and everolimus as raw materials.

H NMR(300MHz,CDCl)δ9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45 (dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d, 2H),4.13(s,1H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,86H),3.53(dd,4H),3.43(d,1H), 3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd,3H),2.27-1.77 (m,7H),1.72(s,3H),1.62(s,3H),1.56(d,4H),1.44-1.11(m,6H),0.97(d,3H),0.89-0.82 (dd,11H),0.74(dd,4H),0.67-0.51(m,1H).

Example 8: synthesis of Methyleneglycol 500-glutamic acid-succinic acid-leucine-Everolimus conjugate (Compound (8))

The reference compound (6) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, succinic acid, leucine and everolimus as raw materials.

H NMR(300MHz,CDCl)δ9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45 (dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.41-4.24(m,4H),4.20(d, 2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,86H),3.53(dd,4H),3.43(d,1H),3.36(s,6H), 3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd,3H),2.27-1.52(m,9H),1.72 (s,3H),1.62(s,3H),1.56(d,4H),1.44-1.11(m,6H),1.06-0.95(m,9H),0.84(dd,5H),0.74 (dd,4H),0.67-0.51(m,1H).

Example 9: synthesis of Polyoxyethylene Monomethylether 400-glutamic acid-glutaric acid-isoleucine-Everolimus conjugate (Compound (9))

The reference compound (6) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, glutaric acid, isoleucine and everolimus as raw materials.

H NMR(300MHz,CDCl)δ9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45 (dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d, 2H),4.03-4.00(m,3H),3.92(d,1H),3.65-3.55(m,67H),3.53(dd,4H),3.43(d,1H),3.36 (s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.45-2.36(m,7H),2.27-1.77 (m,8H),1.72(s,3H),1.62(s,3H),1.56(d,4H),1.53-1.49(m,1H),1.44-1.11(m,8H),0.99- 0.90(m,9H),0.84(dd,5H),0.74(dd,4H),0.67-0.51(m,1H).

Example 10: synthesis of polyethylene glycol monomethyl ether 600-glutamic acid-malonic acid-phenylalanine-everolimus conjugate (Compound (10))

The reference compound (6) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, malonic acid, phenylalanine and everolimus as raw materials.

H NMR(300MHz,CDCl)δ9.21(s,1H),7.29-7.19(m,5H),6.46-6.29(m,2H),6.28- 6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.65-4.40(m,2H),4.36- 4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,105H),3.53(dd,4H), 3.45-3.39(m,3H),3.36(s,6H),3.28(s,6H),3.19-2.91(m,7H),2.76(dd,4H),2.37(dd, 3H),2.27-1.77(m,2H),1.72(s,3H),1.62(s,3H),1.56(d,4H),1.44-1.11(m,6H),0.97(d, 3H),0.84(dd,5H),0.74(dd,4H),0.67-0.51(m,1H).

Example 11 Synthesis of Methyleneglycol 500-glutamic acid-succinic acid-Everolimus conjugate (Compound (11))

The reference compound (1) is synthesized by taking polyethylene glycol monomethyl ether 500, glutamic acid, maleic anhydride and rapamycin as raw materials.

A white solid. 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,3H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,86H),3.53(dd,4H),3.43(d,1H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd,3H), 2.27.7 (dd,1H), 2.7 (s,6H),3.14(s,3H),3.04 (d,2H), 1.76 (d,4H), 3.37 (dd, 13H), 2H), 1H), 1.84 (m,6H), 3.7, 1H), 0.74(dd,4H),0.67-0.51(m,1H).

Example 12 Synthesis of Methyleneglycol 500-glutamic acid-butenedioic acid-alanine-Everolimus conjugate (Compound (12))

A white solid. 1H NMR (300MHz, CDCl3) delta 9.21(s,1H),6.46-6.29(m,2H),6.28-6.02(m,5H),5.45(dd,1H),5.22(d,1H),5.09(d,1H),4.93(d,1H),4.55(d,1H),4.36-4.24(m,3H),4.20(d,2H),4.01(s,2H),3.92(d,1H),3.65-3.55(m,87H),3.53(dd,4H),3.43(d,1H),3.36(s,6H),3.28(s,6H),3.14(s,3H),3.04(s,2H),2.76(dd,4H),2.37(dd,3H), 2.27.7 (dd,1H), 2.7 (s,6H),3.14(s,3H),3.04 (d,2H), 2.76(dd,4H), 2H), 3.37 (d, 3.7H), 3.7 (d,1H), 3.7 (m,1H), 1.7 (d, 6H), 3.7, 1H), 3.6H), 1.11, 1H), 0.74(dd,4H),0.67-0.51(m,1H).

EXAMPLE 13 solubility test of target Compound in pure Water

Each target compound was precisely weighed at 10mg at 20 ℃ and added to 1mL of water. The target compounds are all completely dissolved, and the water solubility of the target compounds is measured to be more than 10mg/mL and is improved by more than 3000 times compared with the solubility (2.6mg/L) of rapamycin. The water solubility of the compound is promoted to be well improved.

TABLE 1 solubility test (mg/mL) of target Compounds in purified Water

Target compound

1

2

3

4

5

6

7

8

9

10

11

12

Test results

Complete solution

Conclusion of the test

＞10

EXAMPLE 14 bioavailability assay of the target Compound

Chromatographic conditions the column was an Agilent Eclipse Plus C18 column (50 mm. times.2.1 mm, 1.8 μm); the mobile phase is ultrapure water-acetonitrile; the flow rate is 0.4mL min < -1 >; the column temperature was 40 ℃; the sample injection amount is 5 mu L; the autosampler temperature was 4 ℃. 0-1 min 10% acetonitrile, 1-3 min 10% -90% acetonitrile, 3-7 min 90% acetonitrile, 7-8 min 90% -10% acetonitrile.

Mass spectrum conditional electrospray ionization (ESI) using positive ion mode detection; multiple Reaction Monitoring (MRM); the used gas is high-purity nitrogen; the air curtain air is 35 psi; the ion source voltage is 5500V and the temperature is 500 ℃; atomizing gas (GS1) pressure was 55 psi; supplemental heating gas (GS2) pressure was 55 psi; the collision gas is Medium; the residence time is 3.53 min; rapamycin mass spectrometry monitoring channels and part of the mass spectrometry parameters are shown in the following table.

Sample processing method Compound (1) was dissolved in physiological saline for injection to prepare a stock solution of 2 mg/mL. 18 ICR mice, weighing about 20g, were randomly divided into 3 groups of 6 mice each, and compound (1) was administered into the tail vein at a dose of 40 mg. kg-1. Mice were fasted for 12h without water deprivation and had free access to water during the experiment. Sufficient blood was drawn from the orbital venous plexus at 15min, 30min, 1h in time after tail vein administration into centrifuge tubes previously treated with heparin, respectively, and the mice were then sacrificed immediately.

In a 1.5mL centrifuge tube, 100. mu.L of whole blood was accurately added followed by 300. mu.L of methanol as a precipitant. Mixing in vortex oscillator for 1min, and centrifuging in a centrifuge at 12000r min-1 for 10 min. And sucking 150. mu.L of the supernatant, adding 50. mu.L of ultrapure water, taking 100. mu.L of the supernatant, and finally injecting 5. mu.L of the supernatant.

Finally, the concentration of rapamycin in whole blood was measured to be 502ng/mL and 267ng/mL at 15min and 30min, respectively.

The in vivo drug concentrations of the other compounds of interest after administration were determined in a similar manner and the results are shown in Table 2

TABLE 2 drug concentration (ng/mL) after administration of the target Compound

Preliminary in vivo pharmacokinetics studies show that the compound (1) can be metabolized to generate rapamycin in vivo, and the compound (7) can be metabolized to generate everolimus in vivo, so that the prodrug can increase water solubility and release active drugs after entering the body, and the active drugs have relatively stable blood concentration.

Claims

1. Application in preparing immunosuppressant.

2. Application in preparing antitumor drugs.