PEG-leuprolide conjugate and preparation method thereof

A technology for leuprolide and conjugates, applied in the field of PEG-leuprolide conjugates and their synthesis, can solve the problems of large losses, many synthesis steps, etc., and achieves small losses, easy commercialization, and synthetic routes short effect

CN110639022AInactive Publication Date: 2020-01-03NANKAI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2020-01-03
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention provides a novel PEG-leuprolide conjugate and a preparation method thereof. The preparation method comprises the steps that a methoxy-polyethylene glycol succinimide active ester directly reacts with unprotected leuprolide to obtain imidazolyl-modified PEG-leuprolide conjugate on the histidine 2-D-histidine. The conjugate retains leuprolide activity, a half-life period is extended, plasma clearance is reduced, and pharmacokinetic properties are improved.
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Description

technical field

[0001] The invention relates to the field of medicinal chemistry, in particular to a PEG-leuprolide conjugate and a synthesis method thereof. Background technique

[0002] Leuprolide is a polypeptide composed of nine amino acids, and its amino acid sequence is: GLP-HIS-TRP-SER-TYR-DLEU-LEU-ARG-PRO-NHET, which is a synthetic gonadotropin-releasing hormone ( GnRH, also known as luteinizing hormone releasing hormone, LHRH) analogs, its activity is much higher than natural GnRH. In short-term use, due to its agonistic effect on gonadotropin-releasing hormone receptors, it can promote the secretion of gonadotropins from the pituitary gland, and the secretion of steroid hormones from the testes or ovaries. When used for a long time, due to the influence of receptor down-regulation, the secretion of gonadotropins is inhibited, thereby inhibiting the secretion of steroid hormones by reproductive organs. Therefore, leuprolide is clinically used to treat or alleviate...

Examples

preparation example Construction

[0028] The present invention provides a new PEG-leuprolide conjugate and its synthesis method, through the direct reaction of monomethoxypolyethylene glycol succinimide active ester with unprotected leuprolide to obtain leuprolide PEG-leuprolide conjugate (I or II) modified by the imidazole group on the 2-position histidine of propylidene, the reaction formula is shown in the figure below:

[0029]

[0030] In the present invention, the leuprolide includes leuprolide and leuprolide acetate. The structure of the active ester of monomethoxy polyethylene glycol succinimide is CH 3 O-(CH 2 CH 2 O)x-(CH 2 CH 2 )y-NHS; x is an integer value of 10 to 1300, the structure of polyethylene glycol includes linear structure and branched structure, and the molecular weight is 400-60KDa; y is an integer value of 0, 0.5, 1 to 5, including monomethyl Carbonate, acetate, propionate, succinate, valerate, etc. of oxypolyethylene glycol succinimide. In the reaction, the molar ratio of leu...

Embodiment 1

[0035] Preparation of PEG-leuprolide conjugate (PEG2K-LEU):

[0036] 12.6 mg (0.01 mmol) of leuprolide acetate was dissolved in 5 mL of distilled water, and an equimolar amount of CH was added 3 O-(CH 2 CH 2 O) 45 -CH 2 CH 2 -NHS, reacted at 37°C for 4 hours, purified by cation exchange chromatography, purification conditions: 20mM pH 6.5PBS, sodium chloride gradient: 0-0.5M, collected target peaks for dialysis, and freeze-dried to obtain PEG2K-LEU. MALDI-TOF mass spectrometry detection shows that the molecular weight is 3235.6, which is exactly equal to the sum of the molecular weights of leuprolide and PEG, indicating that only one PEG chain is attached to each leuprolide molecule ( figure 1) . Dissolve PEG2K-LEU in deuterated water, do 1 H NMR spectrum ( figure 2); Compared with leuprolide, the chemical shift of the hydrogen on the imidazole group moves to the high field, indicating that the PEG segment is connected to the imidazole group of the 2-position histidi...

Embodiment 2

[0038] Preparation of PEG-leuprolide conjugate (PEG5K-LEU):

[0039] 12.6 mg (0.01 mmol) of leuprolide acetate was dissolved in 5 mL of distilled water, and an equimolar amount of CH was added 3 O-(CH 2 CH 2 O) 110 -CH 2 CH 2 -NHS, reacted at 37°C for 6 hours, purified by cation exchange chromatography, purification conditions: 20mM pH6.5PBS, sodium chloride gradient: 0-0.5M, collected target peaks for dialysis, and freeze-dried to obtain PEG5K-LEU. MALDI-TOF mass spectrometry detection shows that the molecular weight is 6144.2, which is exactly equal to the sum of the molecular weights of leuprolide and PEG, indicating that only one PEG chain is attached to each leuprolide molecule ( figure 1) . Dissolve PEG5K-LEU in deuterated water, do 1 H NMR spectrum ( figure 2) ; Compared with leuprolide, the chemical shift of the hydrogen on the imidazole group moves to the high field, indicating that the PEG segment is connected to the imidazole group of the 2-position histid...