CN116640182A - Transdermal delivery system for polypeptides and related compounds - Google Patents
Transdermal delivery system for polypeptides and related compounds Download PDFInfo
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- CN116640182A CN116640182A CN202310537602.XA CN202310537602A CN116640182A CN 116640182 A CN116640182 A CN 116640182A CN 202310537602 A CN202310537602 A CN 202310537602A CN 116640182 A CN116640182 A CN 116640182A
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Abstract
The present application relates to the design and synthesis of prodrugs of novel positively charged polypeptides and related compounds having the structure of the general formula (1) 'Structure 1'. The compound represented by the general formula (1) 'Structure 1' can be prepared by standard polypeptide synthesis methods. The positively charged amino groups of the polypeptide prodrug molecule not only render the drug soluble in water, but also bind to the negative charge on the phosphate head group of the biofilm to push the drug into the cytoplasm. The lipophilic moiety in the prodrug molecule (by modification of the polar group with a lipophilic alkyl group) will facilitate entry of the drug into the skin membrane. Experimental results show that more than 40% of the prodrug can switch back to the parent peptide structure in a few minutes. The prodrug can even easily cross the blood brain barrier. Transdermal drug delivery systems for polypeptides and related compounds may enable the use of polypeptide hormones in the medical field. Another great benefit of transdermal administration of these prodrugs is that administration is more convenient, particularly to children.
Description
The application is a divisional application of Chinese application patent application No. 201510599254.4. The application date of the original application is 11/08 in 2006, and the application is named as a transdermal drug delivery system of the polypeptide and related compounds.
Technical Field
The present invention relates to transdermal drug delivery systems for polypeptides and related compounds by converting the polypeptides into positively charged water-soluble prodrugs and the therapeutic use of these prodrugs in the treatment of conditions treatable by polypeptides and related compounds in humans or animals. In particular, the invention allows polypeptides and related compounds to penetrate the skin rapidly and allow them to be administered transdermally.
Background
All polypeptides are polymers and the monomers that make up the polypeptide by binding are amino acids. Peptide chains containing 2-50 amino acid residues are collectively referred to as polypeptides. If the peptide chain has more than 50 amino acid residues, it is referred to as a protein. Polypeptides play a very diverse role in all living bodies. Polypeptide hormones are the largest class of hormones. New polypeptide hormones are continually being discovered and synthesized. They play a very important role in the life control process. Injection of 1 nanogram of thyroid stimulating hormone releasing hormone into mice can enhance the uptake of iodide by the thyroid gland from the blood (r.l. kisliuk, principles of Medicinal Chemistry,4th Ed., w.o. foye, et al eds., williams & Wilkins,4th Ed.1995,p.606). The phagocytosis agent (Tuftsin, thr-Lys-Pro-Arg) stimulates phagocytosis and promotes antibody-dependent cellular cytotoxicity (V.A.Najjar, mol.Cell.Biochem.41,1, 1981), methionine enkephalin (Tyr-Gly-Gly-Phe-Met) was isolated from brain and small intestine, which acts identically to morphine in that it binds to the same receptor and has analgesic effects (J.R. Jaffe and W.R. Martin, in Pharmacological Basis of Therapeutics, A.G. Gilman, et al, eds., new York, pergamon Press,1990, p.481). Oxytocin (Pierce et al, j.biol. Chem.199, 929, 1952), vasopressin (Kamm et al, j.am. Chem. Soc.50, 573, 1928), angiotensin (j.c. garrison and m.j. peach, in Pharmacological Basis of Therapeutics, a.g. gilman, et al, eds, new York, pergamon Press,1990, p.749), gastrin (p.c. emson and b.e. sandberg, annu, rep.Med. Chem.,18, 31, 1983), somatostatin (a.v. schaliy, et al, annu. Rev. Biochemim, 47, 89, 1978), dynorphin (m.g. Weisskf, et al, nature,362, 423, 1993), endothelin (A.M.Doherty, J.Med.Chem.,35, 1992), endothelin (1990, 35, 1998), and other polypeptides known as such polypeptides, and many of the present invention are known in the art, and are known in the biological arts (p.c. 15, 1993, 1998), and in the biological arts, and the biological arts (13, 35, 55, and the polypeptides are known as many of the polypeptides.
However, polypeptides and related compounds are rapidly cleaved by proteolytic enzymes. The polypeptide is destroyed within minutes when orally administered. The administration of the injected polypeptides is painful, and many times, the administration of the polypeptides is often carried out in hospitals for the treatment of chronic diseases, and the cost is increased.
An alternative mode of administration is topical administration. Administration by external use has several advantages. This method can avoid drug deactivation due to first pass effects of liver and gastrointestinal tract. It delivers the drug locally to the site of action of interest and achieves the proper local concentration without systemic drug exposure. Another problem with the oral administration of fisheman (fisheman; robert, U.S. patent No. 7,052,715) is that the concentration of the drug in the blood circulation must be high in order to effectively treat pain or inflammation at the distal site. These concentrations are often far higher than actually required assuming that the drug is directly targeted to the pain or injury site. Yeager attempts to administer PGE via permeation enhancers 1 Is used for treating male erectile dysfunction (Yeager, james L. U.S. Pat. No. 6,693,135). Susan Milcovich et al designed and synthesized testosterone 4-dimethylaminobutyrate hydrochloride (TSBH) having a lipid-soluble moiety and a protonated form at physiological pH The tertiary amine structure present in the formula. They found that this prodrug (TSBH) penetrated human skin at a rate nearly 60 times that of the parent drug (TS) [ Susan Milosovich et al, j.pharm.sci.,82, 227 (1993)]。
Disclosure of Invention
Technical problem
Polypeptides play a very diverse role in life and are used to treat a variety of diseases.
However, polypeptides and related compounds are rapidly hydrolyzed by proteolytic enzymes. Polypeptides are destroyed within minutes when taken orally. The administration of the injected polypeptides is painful, and many times, the administration of the polypeptides is often carried out in hospitals for the treatment of chronic diseases, and the cost is increased.
Solution scheme
The present invention relates to the preparation of novel positively charged polypeptides and related compounds and their therapeutic uses. Prodrugs of these polypeptides and related compounds have the general formula (1) 'Structure 1':
wherein X represents O, S or NH; x is X 1 Or X n Represents CO, SO 2 PO (OR), NO, OR none; z is Z n Or Z is n1 Represents H, CH 3 ，C 2 H 5 ，C 3 H 7 ，CF 3 ，C 2 F 5 Or C 3 F 7 ；R n Represents any aliphatic side chain of an amino acid, any side chain containing a hydroxyl group or a sulfur atom of an amino acid, any side chain containing an aryl group of an amino acid, any side chain containing an amino group, an imidazolyl group or a guanidyl group of an amino acid, or any side chain containing a carboxyl group or an amide group of an amino acid, H, any alkyl group of 1 to 12 carbon atoms, an alkoxy group of 1 to 12 carbon atoms, an alkenyl group of 1 to 12 carbon atoms, an alkynyl group of 1 to 12 carbon atoms, an aryl group or a heteroaryl group, or none; y is Y x1 ，Y x2 Or Y n Represents H, an alkyl group of any 1 to 12 carbon atoms, an alkoxy group of 1 to 12 carbon atomsAlkenyl, alkynyl of 1 to 12 carbon atoms, aryl or heteroaryl, or a group,
or none; r is R x1 ，R x2 ，R x3 ，Rx 4 ，R x5 Or R is xn Represents H, O, alkyl of any one of 1 to 12 carbon atoms, alkoxy of 1 to 12 carbon atoms, alkenyl of 1 to 12 carbon atoms, alkynyl of 1 to 12 carbon atoms, aryl or heteroaryl, or none; a is that - Represents Cl - ，Br - ，F - ，I - ，AcO - Citrate or any other negative ion; n=0, 1,2,3,4,5,6,7,8,9, 10 … …; all R, - (CH) 2 ) n -or- (CH) 2 ) m The group may be branched or straight and may contain C, H, O, S or N atoms, and may have single, double and triple bonds. Any CH 2 The groups may be substituted with O, S or NH. One or more amino acid residues in the polypeptide sequence may be substituted with an unnatural amino acid, e.g., a β -amino acid, a 2-naphthylalanine, and any alkyl, alkoxy, alkenyl, or alkynyl group, aryl or heteroaryl group. Amino and carboxyl groups of amino acids on polypeptide chains can form a homocyclic peptide via a lactam bridge (homodetic cyclic peptides). The sulfhydryl groups on cysteines, homocysteines, or other amino acids may form heterocyclic peptides through disulfide bridges (heterodetic cyclic peptides).
These prodrugs of designed polypeptides and related compounds have the following principles: 1. the prodrug must have a lipophilic moiety and a primary, secondary or tertiary amine structure, a guanidino group, or a mono-protected guanidino group (hydrophilic moiety) that exists in protonated form at physiological pH. 2. Each polypeptide prodrug should have only one or two (preferably one) primary, secondary or tertiary amine groups, guanidines, or mono-protected guanidines (hydrophilic moieties) present in protonated form at physiological pH. 3. The primary, secondary or tertiary amine, guanidine, or mono-protected guanidine group can be at the N-terminus, C-terminus, or side chain of the polypeptide. The N-terminus or the C-terminus is the preferred position. 4. Carboxyl, amino, guanidino or other hydrophilic groups may be protected with alkyl, aryl or heteroaryl groups in the form of ester or amide bonds to enhance the lipid solubility of the polypeptide.
The following are some examples of these prodrugs:
wherein R represents H, branched or straight chain, - (CH) 2 ) n -wherein n = 0,1,2,3,4,5,6,7,8,9, 10 … …; aryl or heteroaryl; x is X 4 、X 5 、X 6 、X 7 、X 8 Or X 9 Represents CO, SO 2 PO (OR), NO OR none; r is R 1 ，R 2 ，R 3 ，R 4 ，R 5 ，R 6 ，R 7 ，R 8 Or R is 9 Represents H, O, alkyl of any one of 1 to 12 carbon atoms, alkoxy of 1 to 12 carbon atoms, alkenyl of 1 to 12 carbon atoms, alkynyl of 1 to 12 carbon atoms, aryl or heteroaryl; a is that - Represents Cl - ，Br - ，F - ，I - ，AcO - Citrate or any other negative ion; n=0, 1,2,3,4,5,6,7,8,9, 10; ar represents phenyl, 2' -naphthyl, 4-iodophenyl, or other aryl or heteroaryl groups; all R, - (CH) 2 ) n -or- (CH) 2 ) m The group may be branched or linear and may contain C, H, O, S or N atoms, and may have single, double and triple bonds; any CH 2 The groups may be substituted with O, S or NH.
Drugs, whether absorbed through the gastrointestinal tract or other pathways, need to traverse the barrier membrane in molecular form. The drug must first dissolve and if the drug has the desired biopharmaceutical properties, it will pass through the high concentration region to the low concentration region across the cell membrane into the blood or systemic circulatory system. All biological membranes contain lipids as the main component. The dominant molecules in the biofilm structure all have a highly polar head structure containing phosphate and, in most cases, two highly hydrophobic hydrocarbon tails. The biofilm has a bilayer structure with a hydrophilic head structure facing the aqueous phase region on both sides. Very hydrophilic drugs (most polypeptides) cannot pass through the hydrophobic layer of the biofilm and very hydrophobic drugs stay in it as part of the biofilm for similar compatibility reasons and thus cannot effectively enter the inner cytoplasm.
The object of the present invention is to make it possible to administer polypeptides and related compounds transdermally (for external use) by increasing the rate at which they cross biological membranes and skin barriers. The novel prodrugs of these polypeptides and related compounds share two structural similarities: they all have a lipophilic moiety which can be formed by a lipophilic alcohol to protect the carboxyl group and by a lipophilic acid to protect the amino, hydroxyl or guanidino group or other hydrophilic group on the polypeptide) and a primary, secondary or tertiary amine structure, guanidino group, or mono-protected guanidino group (hydrophilic moiety) which exists in protonated form at physiological pH. Such a water-oil-soluble balance is that the drug is able to effectively cross the biological membraneNecessary conditions [ Susan Miosovich, et al, J.Pharm.Sci.,82, 227 (1993) ]]. The positively charged amino groups greatly increase the solubility of the drug in water and the lipophilic moiety can assist the prodrug in entering the lipophilic biofilm and skin barrier. When these novel prodrugs are transdermally administered in the form of a solution, spray, emulsion, ointment, emulsion or gel, they dissolve rapidly in the moisture of the skin surface. The positive charge on the amino group in these pro-drug molecules will bond with the negative charge of the phosphate head group of the cell membrane. Thus, the local concentration of these prodrugs outside of the biofilm is high to facilitate passage of these prodrugs from high concentration regions to low concentration regions. After these pro-drug molecules enter the biofilm, the hydrophilic moiety pushes the pro-drug into the cytoplasm, a semi-liquid concentrated aqueous solution or suspension. The penetration rate of certain prodrugs through human skin is measured in vitro by a modified Franz cell, wherein human skin is isolated from human skin tissue (360-400 μm thick) in front of or behind the thigh area. The receiving solution consisted of 2ml of physiological saline containing 2% bovine serum globulin and was stirred at 600 rpm. The cumulative total amount of these prodrugs and their parent drugs that pass through the skin is determined by specific high performance liquid chromatography. The results are shown in FIG. 1 with 0.2ml of phosphate buffer solution (0.2M) pH 7.4 containing 10% of these prodrugs and polypeptides as donor solution. Calculated, acetyl-tyrosine (acetyl) -glycine-phenylalanine-methionine diethylaminoethyl hydrochloride, dimethylaminobutylcarbonyl-tyrosine (acetyl) -glycine-phenylalanine-methionine n-butyl hydrochloride, cyclo (1, 6) -acetyl-norleucine-aspartic acid-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylaminoethyl hydrochloride, cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-D-phenylalanine (4-iodo) -arginine (acetyl) -tryptophan-lysine amide hydrochloride, cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-D-alanine (2-naphthyl) -arginine-tryptophan-lysine amide hydrochloride, acetyl-valine-proline-glycine-proline-arginine (diacetyl) diethylaminoethyl hydrochloride Apparent penetration values of acid-phenylalanine-methionine, cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine-tryptophan-lysine, cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-D-phenylalanine (4-iodo) -arginine-tryptophan-lysamide and valine-proline-glycine-proline-arginine through human skin were 0.52mg, 0.55mg, 0.46mg, 0.34mg, 0.50mg, 0.60mg, 0.001mg and 0.001mg/cm, respectively 2 And/h. The results demonstrate that the pro-drugs permeate human skin 340-600 times faster than the polypeptides and related compounds. The results demonstrate that the positive charge on the dialkylaminoethyl group is very important for the drug to cross the biological membrane and skin barrier.
A good prodrug should be able to return to the parent drug in plasma. We have found that the polypeptide prodrugs of the invention can be returned rapidly to the structure of the parent polypeptide in human plasma in higher yields. 1ml of whole blood containing 20mg of acetyl-tyrosine (acetyl) -glycine-phenylalanine-methionine diethylaminoethyl hydrochloride was incubated at 37℃for 30 minutes. The mixture was analyzed by HPLC. Experiments have found 3% acetyl-tyrosine (acetyl) -glycine-phenylalanine-methionine diethylaminoethyl hydrochloride, 2% acetyl-tyrosine-glycine-phenylalanine-methionine diethylaminoethyl hydrochloride, 8% acetyl-tyrosine-glycine-phenylalanine-methionine, 60% tyrosine-glycine-phenylalanine-methionine and 27% other by-products (amino acids, dipeptides, tripeptides, tetrapeptides). 5% of the hydrochloride dimethylaminobutylcarbonyl-tyrosine (acetyl) -glycine-phenylalanine-methionine butyl ester, 6% of the dimethylaminobutylcarbonyl-tyrosine-glycine-phenylalanine-methionine butyl ester, 10% of the dimethylaminobutylcarbonyl-tyrosine-glycine-phenylalanine-methionine, 55% of the tyrosine-glycine-phenylalanine-methionine and 24% of other by-products were measured in the hydrochloride dimethylaminobutylcarbonyl-tyrosine (acetyl) -glycine-phenylalanine-methionine butyl ester experiment. In the experiment of cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylaminoethyl ester hydrochloride, 4% cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylaminoethyl ester hydrochloride, 8% cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (acetyl) -tryptophan-lysine, 10% cyclo (1, 6) -norleucine-aspartic acid-histidine-phenylalanine-arginine-tryptophan-lysine, 45% cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine-tryptophan-lysine and 33% other by-products were measured. The results show that most of the polypeptide prodrugs returned to the structure of the parent peptide, indicating that transdermal delivery systems for the polypeptides were successful.
Enterostatin (Enterostatins) [ valine-proline-aspartic acid-proline-arginine (VPDPR), valine-proline-glycine-proline-arginine (VPGPR) and alanine-proline-glycine-proline-Arginine (APGPR)]Is a pentapeptide derived from NH of a trypsin-cleaved colipase (procaspase) 2 And (c) a polypeptide belonging to the entero-brain polypeptide family. They regulate fat intake and are useful in the treatment of obesity (Erlanson-Albertsson C, york D, obes. Rev.1997Jul;5 (4): 360-72and Sorhede M,Mei J,Erlanson-Albertsson C., J physiol.87:273-275, 1993). Injection of valine-proline-aspartic acid-proline-arginine into the abdominal cavity of osborn-Mendel rats starved overnight resulted in a dose-dependent reduction in ingestion. Ingestion inhibition was observed in high fat diet fed rats, but not in high carbohydrate low fat diet fed rats (Okada S.et al Physiol Behav.,1991 Jun;49 (6): 1185-9). At the beginning of the dark feeding period, 5mg/kg of acetyl-valine-proline-aspartic acid (oxyethyl) -proline-arginine (diacetyl) -diethylaminoethyl ester hydrochloride dissolved in 0.5ml of water was transdermally administered to the backs of the rats (5 rats per group, one group fed food after starvation overnight, and the other group fed free). Selective inhibition of fat uptake was experimentally observed.
Melanocyte corticosteroid II (melanocortin II) is a lactam-type cyclic peptide (cyclic acta)m peptides), i.e. cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine-tryptophan-lysine. It is a novel candidate for the treatment of male and female sexual dysfunction by palatine Ding Gongsi (Palatin, AMEX: PTN). As the first drug of this new class of drugs, melanocortin agonists, melanocortin II is expected to be effective in treating male Erectile Dysfunction (ED) and female sexual dysfunction without the cardiovascular side effects common to other ED drugs. Melanocortin II acts through a mechanism of action associated with the central nervous system, rather than directly on the vascular system. Therefore, the safety and effectiveness of the product are obviously superior to those of the current products. The novel prodrugs of the invention are capable of penetrating the human skin at very rapid rates (-0.3-0.5 mg/h/cm) 2 ) And provides a method for treating erectile dysfunction or enhancing female sexual arousal with little or no side effects. Ring (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylaminoethyl hydrochloride (peptide A) dissolved in 0.2ml of phosphate buffer solution (0.1M) at pH 7.0 and ring (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (nitro) -tryptophan-lysine diethylaminoethyl hydrochloride (peptide B) dissolved in 0.2ml of phosphate buffer solution (0.1M) at pH 7.0 were applied to the back of male rats (30) at a dose of 2mg/kg, five days a day continuously. The experimental results show that: the sexual impulse was increased 5-fold and 6-fold, respectively, in rats administered peptide a and in rats administered peptide B, and the number of sexual intercourse was increased 3-fold, respectively, compared to rats not administered peptide a or peptide B. Equivalent amounts of cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylaminoethyl hydrochloride (peptide A) and cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (nitro) -tryptophan-lysine diethylaminoethyl hydrochloride (peptide B) dissolved in 0.2ml of phosphate buffer solution (0.1M) at pH 7.0 were transdermally administered to the backs of male rats (30) and female rats (30) once a day for 5 days, and the results showed rats without drug administration Compared with the rats to which the peptide A or the peptide B was administered, the sexual impulse was increased by 6 times, and the sexual intercourse frequency was increased by 5 times.
Opioid peptides, such as methionine-enkephalin (tyrosine-glycine-phenylalanine-methionine), leucine-enkephalin (tyrosine-glycine-phenylalanine-leucine), tyrosine-D-alanine-glycine-N-methyl-phenylalanine-egg-inol (oxy) (H-Tyr-D-Ala-Gly-N-Me-Phe-Met (O) -OL), tyrosine-D-alanine-glycine-phenylalanine-leucine and many other polypeptides, all exhibit morphine-like analgesic effects. The number of writhing occurring after the intraperitoneal injection of acetic acid solution was counted, and the inhibition rate was calculated based on the blank group. Tyrosine (acetyl) -D-alanine-glycine-phenylalanine-leucine n-hexyl ester hydrochloride (10 mg/kg, B), acetyl-tyrosine (acetyl) -D-alanine-glycine-phenylalanine-leucine diethylaminoethyl ester hydrochloride (10 mg/kg, C) and tyrosine (acetyl) -D-alanine-glycine-phenylalanine-egg-inolin (oxy) hydrochloride (10 mg/kg, D) were transdermally administered to the neck of the mice 30 minutes prior to injection of the acetic acid solution. Group a is a control group. The results are shown in Table 1.
TABLE 1 inhibition of writhing by prodrugs of enkephalin and related compounds
| Group of | Dosage (mg/kg) | Number of times of twisting body | Percentage (%) |
| A | 0 | 35.0 | - |
| B | 10 | 8.6 | 75 |
| C | 10 | 5.2 | 85 |
| D | 10 | 3.2 | 91 |
The above results show that transdermal drug delivery systems for polypeptide prodrugs are effective in treating obesity and pain, as well as in treating male and female sexual dysfunction.
Polypeptides and related compounds are highly hydrophilic and they are difficult to cross the skin and membrane barrier. When the polypeptide is orally administered, the polypeptide and related compounds are rapidly decomposed by proteolytic enzymes in the gastrointestinal tract within a few minutes. The administration of the injected polypeptides is painful, and many times, the administration of the polypeptides is often carried out in hospitals for the treatment of chronic diseases, and the cost is increased. When the polypeptide prodrugs are topically applied to the skin, they are rapidly dissolved in the moisture on the skin surface. The positive charge on the amino groups of these prodrug molecules can bond with the negative charge of the phosphate head group of the dermal membrane. Thus, the local concentration of the drug outside the membrane is high, which is advantageous for the passage of the drug from the high concentration region to the low concentration region. After these prodrug molecules enter the biological membrane, the hydrophilic portion of the prodrug molecule will push the prodrug into the cytoplasm.
The compound represented by the above general formula (1) 'Structure 1' can be synthesized by a standard polypeptide synthesis method. In the preparation of cyclic peptides and related compounds of the general formula (4-C) 'Structure 4-C', the polypeptide chain can be synthesized by standard polypeptide synthesis methods, the leucine side chain can be protected by a 2-, or 4-Pyoc protecting group, and cyclization of the polypeptide can be accomplished on the resin.
Advantages are that
The pro-drug structures of the polypeptides and related compounds of the invention each have a lipid soluble moiety and a water soluble moiety (amine groups present in protonated form at physiological pH). The positively charged amino groups of these prodrugs have two major benefits. First, it renders these prodrugs water-soluble; when these novel prodrugs are transdermally administered in a dosage form such as a solution, spray, emulsion, ointment, emulsion or gel, they rapidly mix with moisture on the surface of the skin, eyes, genital area, mouth, nose or other parts of the body. Second, the positive charges on these pro-drug amino groups can bond with the negative charges of the phosphate head group of the biofilm. Thus, the local concentration outside the biofilm is high to facilitate passage of these prodrugs from high concentration regions to low concentration regions. The lipophilic portion of the prodrug molecule (through modification of the polar group with a lipophilic alkyl group) facilitates entry of the drug into the dermal membrane. When these pro-drug molecules enter the biological membrane, the hydrophilic moiety pushes the pro-drug molecules into the cytoplasm, a semi-liquid concentrated aqueous solution or suspension. The prodrug does not cause itching, burning or pain in the skin, eyes, genital area, mouth, nose or other parts of the body due to the short residence time in the skin, eyes, genital area, mouth, nose or other parts of the body. Experimental results show that more than 40% of the prodrug can switch back to the parent peptide structure in a few minutes. The prodrug can even easily cross the blood brain barrier. Transdermal drug delivery systems for polypeptides and related compounds may enable the use of polypeptide hormones in the medical field. Another great benefit of transdermal administration of these prodrugs is that administration is more convenient, particularly to children.
Drawings
Fig. 1: acetyl-tyrosine (acetyl) -glycine-phenylalanine-methionine-diethylamine ethyl hydrochloride, dimethylaminobutylcarbonyl-tyrosine (acetyl) -glycine-phenylalanine-butyl methionine, cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylamine ethyl hydrochloride, cyclo (1, 6) acetyl-norleucine-aspartic acid-histidine-D-phenylalanine (4-iodo) -arginine (acetyl) -tryptophan-lys amine hydrochloride, cyclo (1, 6) acetyl-norleucine-aspartic acid-histidine-D-alanine (2-naphthyl) -arginine-tryptophan-lys amine hydrochloride, acetyl-valine-proline-glycine-proline-arginine (diacetyl) diethylamine ethyl hydrochloride, acetyl-tyrosine-glycine-phenylalanine-methionine, cyclo (1, 6) acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine-lysine-arginine hydrochloride of human skin tissue isolated in Franz cell (n=5), the cumulative total of cyclo (1, 6) acetyl-norleucine-aspartic acid-histidine-D phenylalanine (4-iodo) -arginine-tryptophan-lys amide and valine-proline-glycine-proline-arginine. In each case, the carrier solution was phosphate buffer solution (0.2M) at pH 7.4.
Fig. 2: structural formula 1. Wherein X represents O, S or NH; x is X 1 Or X n Represents CO, SO 2 PO (OR), NO, OR none; z is Z n Or Z is n1 Represents H, CH 3 ，C 2 H 5 ，C 3 H 7 ，CF 3 ，C 2 F 5 Or C 3 F 7 ；R n Represents any aliphatic side chain of an amino acid, any side chain containing a hydroxyl group or a sulfur atom of an amino acid, any aryl side chain of an amino acid, any side chain containing an amino group, an imidazolyl group or a guanidino group of an amino acid, or any side chain containing a carboxyl group or an amide group of an amino acid, any alkyl group containing 1 to 12 carbon atoms, an alkoxy group containing 1 to 12 carbon atoms, an alkenyl group containing 1 to 12 carbon atoms, an alkynyl group containing 1 to 12 carbon atoms, an aryl group or a heteroaryl group, or none; y is Y x1 ，Y x2 Or Y n Represents H, any alkyl group having 1 to 12 carbon atoms, alkoxy group having 1 to 12 carbon atoms, alkenyl group having 1 to 12 carbon atoms, alkynyl group having 1 to 12 carbon atoms, aryl or heteroaryl group, R x3 R x4 R x5 N + A - Or none; r is R x1 ，R x2 ，R x3 ，R x4 ，R x5 Or R is xn Represents H, O, alkyl of any one of 1 to 12 carbon atoms, alkoxy of 1 to 12 carbon atoms, alkenyl of 1 to 12 carbon atoms, alkynyl of 1 to 12 carbon atoms, aryl or heteroaryl or none; a is that - Represents Cl - ，Br - ，F - ，I - ，AcO - Citrate or any other negative ion; n=0, 1,2,3,4,5,6,7,8,9, 10 … …; all R, - (CH) 2 ) n -or- (CH) 2 ) m The group may be branched or linear and may contain C, H, O, S or N atoms, and may have single, double and triple bonds; any CH 2 The groups may be substituted with O, S or NH; one or more amino acid residues in the polypeptide sequence may be substituted with an unnatural amino acid, e.g., a β -amino acid, 2-naphthylalanine, and any alkyl, alkoxy, alkenyl or alkynyl group, aryl or heteroaryl group. Amino and carboxyl groups of amino acids on a polypeptide chain may form a homocyclic peptide via a lactam bridge. The sulfhydryl groups on cysteines, homocysteines, or other amino acids may form heterocyclic peptides through disulfide bridges.
Best mode for carrying out the invention
Synthesis of acetyl-valine-proline-aspartic acid (oxyethyl) -proline-arginine (diacetyl) diethylaminoethyl hydrochloride
1. Synthesis of arginine (diacetyl) -diethylaminoethyl ester. 30.8. 30.8g Z-arginine was dissolved in 500ml acetone. 200ml of 20% sodium hydroxide solution was added to the reaction mixture. 40g of acetic anhydride was added dropwise to the mixture. The mixed solution was stirred at room temperature for 2 hours. The solvent was evaporated to dryness. The residue was extracted with 500ml of ethyl acetate. The ethyl acetate solution was washed three times with 100ml of water each time. The ethyl acetate layer was dried over anhydrous sodium sulfate. The ethyl acetate solution was evaporated to dryness. The residue (Z-arginine (diacetyl), 30 g) was dissolved in 300ml of acetonitrile. The mixture was cooled to 0 ℃ with an ice-water bath. 12g of N, N-diethylaminoethanol, 2g of 4-dimethylaminopyridine, and 22g of 1, 3-dicyclohexylcarbodiimide were added to the reaction mixture. The mixture was stirred at 0 ℃ for 1 hour and then at room temperature overnight. The solids were removed by filtration and the solution was evaporated to dryness. The residue was extracted twice with 250ml of ethyl acetate. Ethyl acetate solution Wash once with 5% sodium bicarbonate solution, 500ml each time, and three times with water, 100ml each time. The ethyl acetate solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. Residue [ Z-arginine (diacetyl) diethylaminoethyl ester, 28g]Dissolved in 300ml of methanol. 2g of 10% Pd/C was added to the solution. The mixture was stirred at room temperature for 10 hours under a hydrogen atmosphere. Pd/C was removed by filtration. The solution was evaporated to dryness to give 22g of arginine (diacetyl) diethylaminoethyl ester (H-Arg (di-Ac) -OCH 2 CH 2 N(CH 2 CH 3 ) 2 )。
2. Synthesis of t-butoxycarbonyl-aspartic acid (oxyethyl) -proline N-succinimide (Boc-Asp (OEt) -Pro-OSu). 15g L-proline was dissolved in 300ml of 10% sodium bicarbonate solution. 150ml of acetone and 36g of t-butoxycarbonyl-aspartic acid (oxyethyl) -succinimide were then added to the mixture. The mixed solution was stirred at room temperature for 5 hours. The reaction solution was washed once with 300ml of diethyl ether. 500ml of ethyl acetate was added to the aqueous layer. The pH of the mixture of ethyl acetate and water was adjusted to 2.4-2.5 with ice-cooled 3N HCl. The ethyl acetate layer was collected and washed three times with 300ml each. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. 25g of the residue (t-butoxycarbonyl-aspartic acid (oxyethyl) -proline) and 11g N-hydroxysuccinimide are dissolved in 300ml of dichloromethane. The mixed solution was cooled to 0 ℃. 16g of 1, 3-dicyclohexylcarbodiimide was then added to the reaction mixture. The mixture was stirred at 0℃for 1 hour. The solid by-product was removed by filtration. The dichloromethane solution was washed once and three times with 200ml of 5% sodium bicarbonate solution, 200ml each time. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. 28g of t-butoxycarbonyl-aspartic acid (oxyethyl) -proline-N-succinimide were obtained.
3. Synthesis of aspartic acid (oxyethyl) -proline-arginine (diacetyl) -diethylaminoethyl ester. 22g of arginine (diacetyl) -diethylaminoethyl ester were dissolved in 300ml of 5% aqueous sodium bicarbonate solution. To the reaction mixture was added 150ml of acetone containing 24g of t-butoxycarbonyl-aspartic acid (oxyethyl) -proline-N-succinimide. The mixed solution was stirred at room temperature for 5 hours. MixingTo the solution was added 500ml of ethyl acetate. The ethyl acetate layer was washed three times with 100ml of water. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. The residue was dissolved in 250ml of dichloromethane. 250ml of trifluoroacetic acid was added to the mixed solution, and the mixed solution was stirred for 30 minutes. The mixed solution was evaporated to dryness. 32g of aspartic acid (oxyethyl) -proline-arginine (diacetyl) -diethylaminoethyl ester were obtained, bis trifluoroacetate (H-Asp (OEt) -Pro-Arg (di-Ac) -CH 2 CH 2 N(CH 2 CH 3 ) 2 .2TFA)。
4. Synthesis of acetyl-valine-proline N-succinimide. 15g L-proline was dissolved in 300ml of 10% sodium bicarbonate solution. To the reaction mixture was added an acetone solution in which 26g of acetyl-valine-N-hydroxysuccinimide was dissolved. The mixture was stirred at room temperature for 5 hours. The mixture was washed once with 300ml of diethyl ether. 500ml of ethyl acetate was added to the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCl. The ethyl acetate layer was collected and washed three times with 300ml each. The organic solution was dried over anhydrous sodium sulfate. The organic solution was evaporated to dryness. 20g of the residue (acetyl-valine-proline) and 11g of N-hydroxysuccinimide are dissolved in 300ml of dichloromethane. The mixed solution was cooled to 0 ℃. 16g of 1, 3-dicyclohexylcarbodiimide was added to the reaction mixture. The mixed solution was stirred at 0℃for 1 hour and at room temperature for 1 hour. The solid by-product was removed by filtration. The dichloromethane solution was washed once with 200ml of 5% aqueous sodium bicarbonate and three times with 200ml of water. The organic solution was dried over anhydrous sodium sulfate. The solvent was evaporated to dryness. 20g of acetyl-valine-proline N-succinimide (Ac-Val-Pro-OSu) was obtained.
5. Synthesis of acetyl-valine-proline-aspartic acid (oxyethyl) -proline-arginine (diacetyl) diethylaminoethyl ester hydrochloride (Ac-Val-Pro-Asp (OEt) -Pro-Arg (di-Ac) -OCH 2 CH 2 N(CH 2 CH 3 ) 2 HCl). 31g of aspartic acid (oxyethyl) -proline-arginine (diacetyl) diethylaminoethyl ester bistrifluoroacetate were dissolved in 300ml of 10% aqueous sodium bicarbonate solution. 150ml of acetone and 15g of acetyl-valine-proline N-succinimide were added to the reaction mixture. The mixture was stirred at room temperature for 5 hours.500ml of ethyl acetate was added to the mixture. The organic layer was washed three times with 100ml of water. The ethyl acetate layer was dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration. Dioxane (50 ml) containing 3.5g of hydrochloric acid gas was added to the ethyl acetate solution. The solid was collected by filtration and washed three times with 50ml of diethyl ether each. After drying, 20 g of the target product which is easy to absorb moisture is obtained. Solubility in water: 150mg/ml. Elemental analysis: c (C) 39 H 66 ClN 9 O 11 The method comprises the steps of carrying out a first treatment on the surface of the MW:872.45. theoretical value (%): c:53.69; h:7.62; CI:4.06; n:14.45; o:20.17; measured value (%) 53.61; h:7.67; cl:4.10; n:14.40, O:20.22. mass spectrometry: m/e:836.4; m/e+1:836.4.
description of the embodiments
Synthesis of acetyl-tyrosine (acetyl) -glycine-phenylalanine-methionine diethylaminoethyl ester hydrochloride (Ac-Tyr (Ac) -Gly-Gly-Phe-Met-OCH) 2 CH 2 N(CH 2 CH 3 ) 2 .HCl).
1. Methionine diethylaminoethyl ester trifluoroacetate (H-Met-OCH) 2 CH 2 N(CH 2 CH 3 ) 2 TFA). 25g of t-butoxycarbonyl-methionine were dissolved in 300ml of methylene chloride. The mixture was cooled to 0 ℃ with an ice-water bath. To the reaction mixture were added 12g of N, N-diethylaminoethanol, 2g of 4-dimethylaminopyridine and 22g of 1, 3-dicyclohexylcarbodiimide. The reaction mixture was stirred at 0 ℃ for 1 hour and then at room temperature overnight. The solid by-product was removed by filtration, and the dichloromethane solution was washed once with 500ml of 5% sodium bicarbonate and three times with 100ml of water. The ethyl acetate solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. Residue [ tert-Butoxycarbonyl-methionine diethylaminoethyl ester, 30g]Dissolved in 250ml of dichloromethane. 250ml of trifluoroacetic acid was added to the mixture, and the mixture was stirred for 30 minutes. The solution was evaporated to dryness. 26g of methionine diethylaminoethyl ester trifluoroacetate was obtained.
2. Preparation of t-butoxycarbonyl-glycine-phenylalanine N-succinimide. 20g L-phenylalanine was dissolved in 300ml of a 10% aqueous sodium hydrogencarbonate solution. 150ml of acetone and 28g of t-butoxycarbonyl-glycine-N-succinimide were added to the reaction mixture. The mixture was stirred at room temperature for 5 hours. The mixture was washed once with 300ml of diethyl ether. 500ml of ethyl acetate was added to the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCl. The ethyl acetate layer was collected and washed three times with 300ml each. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. 22g of the residue (tert-butoxycarbonyl-glycine-phenylalanine) and 10g N-hydroxysuccinimide were dissolved in 300ml of dichloromethane. The mixture was cooled to 0 ℃.15 g of 1, 3-dicyclohexylcarbodiimide was added to the reaction mixture. The mixture was stirred at 0℃for 1 hour. The solid by-product was removed by filtration. The dichloromethane solution was washed once with 200ml of 5% sodium bicarbonate solution and three times with water, 200ml each. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. 25g of t-butoxycarbonyl-glycine-phenylalanine N-succinimide was obtained.
3. Preparation of glycine-phenylalanine-methionine-diethylaminoethyl trifluoroacetate. 25g of methionine-diethylaminoethyl ester trifluoroacetic acid are dissolved in 300ml of 5% sodium bicarbonate solution. To the reaction mixture was added 150ml of an acetone solution containing 22g of t-butoxycarbonyl-glycine-phenylalanine N-hydroxysuccinimide. The mixed solution was stirred at room temperature for 5 hours. 500ml of ethyl acetate was added to the mixture. The ethyl acetate solution was washed three times with 100ml of water each time. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. The residue was dissolved in 250ml of dichloromethane. 200ml of trifluoroacetic acid was added to the mixture, and stirred for 30 minutes. The mixed solution is evaporated to dryness. 25g of glycine-phenylalanine-methionine-diethylaminoethyl ester trifluoroacetate are obtained.
4. Preparation of acetyl-tyrosine (acetyl) -glycine N-succinimide. 11-g L-Glycine was dissolved in 300ml of 10% sodium bicarbonate solution. 150ml of acetone and 36g of acetyl-tyrosine (acetyl) -N-hydroxysuccinimide were added to the reaction mixture. The mixed solution was stirred at room temperature for 5 hours. The mixed solution was washed once with 300ml of diethyl ether. The aqueous layer was added 500ml of ethyl acetate. The pH of the mixed solution was adjusted to 2.4-2.5 with ice-cooled 3N HCl. The ethyl acetate layer was collected and washed three times with 300ml each. The organic solution was dried over anhydrous sodium sulfate. The organic solution was evaporated to dryness. 28g of the residue (acetyl-tyrosine (acetyl) -glycine) and 13g N-hydroxysuccinimide were dissolved in 300ml of dichloromethane. The mixed solution was cooled to 0 ℃.18g of 1, 3-dicyclohexylcarbodiimide was added to the reaction mixture. The mixture was stirred at 0℃for 1 hour. The solid by-product was removed by filtration. The dichloromethane solution was washed once with 200ml of 5% sodium bicarbonate and three times with water, 200ml each. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. 20g of acetyl-tyrosine (acetyl) -glycine N-succinimide are obtained.
5. Synthesis of acetyl-tyrosine (acetyl) -glycine-phenylalanine-methionine diethylaminoethyl ester hydrochloride. 24g glycine-phenylalanine-methionine diethylaminoethyl ester trifluoroacetate was dissolved in 300ml10% sodium bicarbonate solution. 150ml of acetone and 15g of acetyl-tyrosine (acetyl) -glycine N-succinimide were added to the reaction mixture. The mixture was stirred at room temperature for 5 hours. 500ml of ethyl acetate was added to the mixed solution. The organic layer was washed three times with 100ml of water. The ethyl acetate layer was dried over sodium sulfate. Sodium sulfate was removed by filtration. A dioxane solution (50 ml) containing 3.5g of hydrochloric acid gas was added to the ethyl acetate solution. The solid was collected and washed three times with 50ml of diethyl ether each. After drying, 18g of the target product which is hygroscopic is obtained. Solubility in water: 200mg/ml; elemental analysis: c (C) 37 H 53 ClN 6 O 9 S, S; molecular weight: 793.37. theoretical value (%): c:56.01; h:6.73; cl:4.47; n:10.59; o:18.15; s:4.04. measured value (%): c:55.96; h:6.76; cl:4.52; n:10.54, O:18.19; s:4.03. mass spectrometry: m/e:757.4; m/e+1:758.4.
preparation of acetyl-valine-proline-glycine-proline-arginine (diacetyl) diethylaminoethyl ester hydrochloride (Ac-Val-Pro-Gly-Pro-Arg (diAc) -OCH) 2 CH 2 N(CH 2 CH 3 ) 2 .HCl)
1. Preparation of t-butoxycarbonyl-glycine-proline N-succinimide. 15g L-proline was dissolved in 300ml of 10% sodium bicarbonate solution. 150ml of acetone and 27.2g of t-butoxycarbonyl-glycine N-succinimide were added to the reaction mixture solution. The mixture was stirred at room temperature for 5 hours. The mixture was washed once with 300ml of diethyl ether. The aqueous layer was added 500ml of ethyl acetate. The pH of the mixed solution was adjusted to 2.4-2.5 with ice-cooled 3N HCl. The ethyl acetate layer was collected and washed three times with 300ml each. The organic solution was dried over anhydrous sodium sulfate. The solvent was evaporated to dryness. 21g of the residue (t-butoxycarbonyl-glycine-proline) and 11g N-hydroxysuccinimide were dissolved in 300ml of dichloromethane. The mixture was cooled to 0 ℃. 17g of 1, 3-dicyclohexylcarbodiimide was added to the reaction solution. The mixture was stirred at 0℃for 1 hour. The solid by-product was removed by filtration. The dichloromethane solution was washed once with 200ml of 5% aqueous sodium bicarbonate solution and three times with water, 200ml each. The organic solution was dried over anhydrous sodium sulfate. The solvent was evaporated to dryness. 23g of t-butoxycarbonyl-glycine-proline N-succinimide was obtained.
2. Synthesis of glycine-proline-arginine (diacetyl) -diethylaminoethyl bistrifluoroacetate. 22g of arginine (diacetyl) diethylaminoethyl ester were dissolved in 300ml of 5% sodium bicarbonate solution. To the reaction mixture was added 150ml of an acetone solution containing 20g of t-butoxycarbonyl-glycine-proline N-succinimide. The mixed solution was stirred at room temperature for 5 hours. To the mixed solution was added 500ml of ethyl acetate. The ethyl acetate solution was washed three times with 100ml of water each time. The organic solution was dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration. The solvent was evaporated to dryness. The residue was dissolved in 250ml of dichloromethane. 250ml of trifluoroacetic acid was added to the mixed solution and stirred for 30 minutes. The mixed solution was evaporated to dryness. 28g of glycine-proline-arginine (diacetyl) diethylaminoethyl bistrifluoroacetate were obtained.
3. Synthesis of acetyl-valine-proline-glycine-proline-arginine (diacetyl) -diethylaminoethyl ester hydrochloride. 26g of glycine-proline-arginine (diacetyl) diethylaminoethyl ester bistrifluoroacetate are dissolved in 300ml of 10% sodium bicarbonate solution. 150ml of acetone and 15g of acetyl-valine-proline-N-succinimide were added to the reaction mixture. The mixed solution was stirred at room temperature for 5 hours. 500ml of ethyl acetate was added to the mixed solution. The organic layer was washed three times with 100ml of water. The ethyl acetate layer was dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration. Will contain3.5g of dioxane (50 m 1) with hydrochloric acid gas was added to the ethyl acetate solution. The solid was collected and washed three times with 50ml of diethyl ether each. After drying, 18g of the target product which is hygroscopic is obtained. Solubility in water: 150mg/ml; elemental analysis: c (C) 35 H 60 ClN 9 O 9 The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 786.36. theoretical value (%) C:53.46; h:7.69; cl:4.51; n:16.03; o:18.31; measured value (%) C:53.43; h:7.73; cl:4.55; n:16.01, O:18.29. mass spectrometry: m/e:750.4; m/e+1:751.4.
synthesis of Cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine diethylamine ethyl ester hydrochloride (Cyclo (1, 6) -Ac-Nle-Asp-His-Phe-Arg (diAc) -Trp-Lys-OCH 2 CH 2 N(CH 2 CH 3 ) 2 .HCl)
1. Synthesis of acetyl-norleucine-aspartic acid (9-fluorenylmethanol). 43g of aspartic acid (9-fluorenylmethanol) trifluoroacetate (H-Asp (OFm) -OH. TFA) and 27g of acetyl-norleucine N-succinimide were suspended in 300ml of acetone. 300ml of 5% sodium bicarbonate solution was added to the reaction mixture. The mixed solution was stirred at room temperature overnight. The mixed solution was washed once with 300ml of diethyl ether. 500ml of ethyl acetate was added to the aqueous layer. The pH of the mixed solution was adjusted to 2.4-2.5 with ice-cooled 3N HCl. The ethyl acetate layer was collected and washed three times with 300ml each. The organic solution was dried over anhydrous sodium sulfate. The solution was evaporated to dryness. 42g of acetyl-norleucine-aspartic acid (9-fluorenylmethanol) were obtained [ Ac-Nle-Asp (OFm) -OH ].
Synthesis of 9-fluorenylmethoxycarbonyl-tryptophan-lysine (4-Pyoc). According to the reference (h.kunz and s.birnbach, tetrahedron lett,25，3567，1984；H.Kunz and R.Barthels，Angew.Chem.，Int.Ed.Engl.，22preparation of lysine (4-Pyoc) [ H-Lys (4-Pyoc) -OH, 783, 1983)].33g of lysine (4-Pyoc) were suspended in 300ml of 5% sodium bicarbonate solution. 300ml of acetone and 52g of fluorenylmethoxycarbonyl-tryptophan-N-succinimide were added to the reaction mixture solution. The mixture was stirred at room temperature overnight. The mixed solution was washed once with 500ml of diethyl ether. 500ml of ethyl acetate was added to the mixture, and the pH of the mixture was ice-cooled The temperature of the 3N hydrochloric acid is adjusted to 2.2-2.3. The ethyl acetate layer was collected and washed with water. The organic solution was dried over anhydrous sodium sulfate. The organic solution was evaporated to dryness. 55g of fluorenylmethoxycarbonyl-tryptophan-lysine (4-Pyoc) [ Fmoc-Trp-Lys (4-Pyoc) -OH were obtained]。
3. Synthesis of cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine. 100g king-resin was suspended in 700ml dimethylformamide. 50g of fluorenylmethoxycarbonyl-tryptophan-lysine (4-Pyoc), 13g of 1-hydroxybenzotriazole, 2g of 4-dimethylaminopyridine and 12g of N, N-diisopropylcarbodiimide were used. The mixed solution was stirred at room temperature overnight. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. 700ml of 20% piperidine were added to the resin. The mixture was stirred for 30 minutes. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, 400ml each time and three times with dichloromethane, 400ml each time. To the resin was added 700ml dimethylformamide, 48g fluorenylmethoxycarbonyl-arginine (diacetyl), 13g 1-hydroxybenzotriazole, 35ml triethylamine and 38g O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea. The mixed solution was stirred at room temperature for 2h. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. 700ml of 20% piperidine were added to the resin. The mixture was stirred for 30 minutes. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, 400ml each time and three times with dichloromethane, 400ml each time. 700ml dimethylformamide, 39g fluorenylmethoxycarbonyl-phenylalanine, 13g 1-hydroxybenzotriazole, 35ml triethylamine and 38g O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea were added to the resin. The mixed solution was stirred at room temperature for 2 hours. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. 700ml of 20% piperidine were added to the resin. The mixture was stirred for 30 minutes. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time and three times with dichloromethane, 400ml each time. 700ml dimethylformamide, 60g fluorenylmethoxycarbonyl-histidine (fluorenylmethoxycarbonyl), 13g 1-hydroxybenzotriazole, 35ml triethylamine and 38g O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea were added to the resin. The mixed solution was stirred at room temperature for 2 hours. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. 700ml of 20% piperidine were added to the resin. The mixture was stirred for 30 minutes. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time and three times with dichloromethane, 400ml each time. 700ml dimethylformamide, 60g acetyl-norleucine-aspartic acid (9-fluorenylmethanol), 13g 1-hydroxybenzotriazole, 35ml triethylamine and 38g O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea were added to the resin. The mixed solution was stirred at room temperature for 2 hours. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. The peptide-bearing resin was suspended in 700ml dimethylformamide. 50g of methyl iodide (MeI) was added to the reaction mixture. The mixed solution was stirred at room temperature for 1 hour and at 50℃for 1 hour. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. 700ml of 30% piperidine were added to the resin. The mixture was stirred for 60 minutes. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, 400ml each time and three times with dichloromethane, 400ml each time. 700ml dimethylformamide, 13g 1-hydroxybenzotriazole, 35ml triethylamine and 38g O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethylurea were added to the resin. The mixed solution was stirred at room temperature for 10 hours. The resin was collected by filtration and washed three times with 400ml each time, three times with methanol, three times with 400ml each time, three times with dichloromethane, 400ml each time. 500ml of trifluoroacetic acid was added to the resin, and the mixed solution was stirred at room temperature for 1 hour. The resin was removed by filtration and the organic solution was evaporated to dryness. The residue was washed three times with 100ml of diethyl ether each.
4. Preparation of cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine-diethylaminoethyl ester hydrochloride. 10g of cyclo (1, 6) -acetyl-norleucine-aspartic acid-histidine-phenylalanine-arginine (diacetyl) -tryptophan-lysine were dissolved in 300ml of dimethylformamide. The mixed solution was cooled to 0 ℃ with an ice-water bath. 12g of N, N-dimethylaminoethanol, 2g of 4-dimethylaminopyridine and 22g of 1, 3-dicyclohexylcarbodiimide were added to the reaction mixture. The reaction solution was stirred at 0℃for 1 hour and at room temperature overnight. The solid by-products were removed by filtration, and the dichloromethane solution was washed once with 500ml of 5% sodium bicarbonate solution and three times with water, each 100ml. The ethyl acetate solution was dried over anhydrous sodium sulfate. Dioxane (20 ml) containing 2g of hydrochloric acid gas was added to the ethyl acetate solution. The solid was collected and washed three times with 100ml of diethyl ether each. 8g were obtained.
Industrial applicability
The prodrugs represented by the general formula (1) 'Structure 1' can easily cross the skin barrier. Transdermal administration of polypeptides and related compounds may allow the use of polypeptide hormones in the treatment of a number of conditions in humans or animals, such as pain in rheumatoid arthritis and osteoarthritis, fever, male and female erectile dysfunction, systemic blood pressure, hypotension control, inhibition of platelet aggregation, lung disease, gastrointestinal disorders, inflammation, shock, reproductive disorders, infertility and the like.
Claims (10)
1. A compound represented by the following structural formula,
wherein X is 1 Represents CH 3 SCH 2 CH 2 -，CH 3 SOCH 2 CH 2 -or (CH) 3 ) 2 CHCH 2 -；X 2 Represents H or CH 3 ；X 3 Represents H or CH 3 ；R 2 、R 4 Represents an alkyl group of any 1 to 6 carbon atoms; a is that - Represents Cl - ，Br - ，F - ，I - ，AcO - Or citrate; the amino acid may be in the L-or D-configuration.
2. The compound of claim 1, wherein X 1 Representative (CH) 3 ) 2 CHCH 2 -；X 2 Represents CH 3 ；X 3 Represents H; r is R 2 Represents an alkyl group of any 1 to 6 carbon atoms; r is R 4 Represents CH 3 CH 2 -；A - Represents Cl - ，Br - ，F - ，I - ，AcO - Or citrate; the amino acid may be in the L-or D-configuration.
3. A compound represented by the following structural formula,
wherein X is 1 Represents CH 3 SCH 2 CH 2 -，CH 3 SOCH 2 CH 2 -or (CH) 3 ) 2 CHCH 2 -；X 2 Represents H or CH 3 ；X 3 Represents H or CH 3 ；R 2 Represents an alkyl group of any 1 to 6 carbon atoms; a is that - Represents Cl - ，Br - ，F - ，I - ，AcO - Or citrate; the amino acid may be in the L-or D-configuration.
4. A compound represented by the following structural formula,
wherein R represents a branched or linear chain, - (CH) 2 ) n -, wherein n=1 or 2; x represents CO; x is X 1 Representative ofCH 3 SCH 2 CH 2 -or (CH) 3 ) 2 CHCH 2 -；X 2 Represents H or CH 3 ；X 3 Represents H or CH 3 ；R 1 ，R 2 ，R 4 Or R is 5 Represents an alkyl group of any 1 to 2 carbon atoms; r is R 3 Represents H; a is that - Represents Cl - ，Br - ，F - ，I - ，AcO - Or citrate; the amino acid may be in the L-or D-configuration.
5. A compound selected from:
tyrosine (acetyl) -D-alanine-glycine-phenylalanine-leucine n-hexyl ester hydrochloride;
acetyl-tyrosine (acetyl) -D-alanine-glycine-phenylalanine-leucine diethylaminoethyl ester hydrochloride;
Tyrosine (acetyl) -D-alanine-glycine-phenylalanine-alaninol (oxy) hydrochloride.
6. Use of a compound according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of dental pain, headache, arthritis, inflammation, fever, cancer, menstrual pain and acute migraine.
7. Use of a compound according to any one of claims 1 to 5 or a composition comprising at least one compound according to any one of claims 1 to 5 as active ingredient for the preparation of a medicament for the treatment of a condition treatable by a polypeptide of interest and a compound of interest in a human or animal by transdermal administration of any part of the human or animal in solution, spray, emulsion dosage form to a therapeutically effective concentration.
8. Transdermal therapeutic application system comprising a compound according to any one of claims 1 to 5 or a composition comprising at least one compound according to any one of claims 1 to 5 as active ingredient.
9. The transdermal therapeutic application system as claimed in claim 8, wherein the compounds or compositions are administered transdermally via a solution dosage form.
10. The transdermal therapeutic application system of claim 8, wherein the system stabilizes the polypeptide and related compounds at optimal therapeutic blood levels by controlling release rates to increase efficacy and reduce side effects of the polypeptide and related compounds.
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