AU2016213759B2

AU2016213759B2 - Transdermal delivery systems of peptides and related compounds

Info

Publication number: AU2016213759B2
Application number: AU2016213759A
Authority: AU
Inventors: Chongxi Yu
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-11-08
Filing date: 2016-08-10
Publication date: 2018-03-29
Anticipated expiration: 2026-11-08
Also published as: AU2016213759A1

Abstract

TRANSDERMAL DELIVERY SYSTEMS OF PEPTIDES AND RELATED COMPOUNDS Abstract RXF.- Y, Xj N R z n Structure 1 The novel positively charged pro-drugs of peptides and related compounds in the general formula (1) 'Structure 1' were designed and synthesized. The compounds of the general formula (1) 'Structure 1' indicated above can be prepared by standard peptide synthesis protocols. The positively charged amino group of the pro-drug of peptides not only makes the drugs soluble in water, but also bonds to the negative charge on the phosphate head group of membranes and pushes the pro-drug into the cytosol. The lipophilic portion (by modification of the polar groups with lipophilic alkyl groups) of the prodrugs will facilitate the entering of the drugs into the skin membrane. The results suggest that the pro-drugs diffuses through human skin -100 1000 times faster than do peptides and related compounds. In plasma, more than 40% of these pro-drugs can change back to the parent drugs in a few minutes. The prodrugs can be used medicinally in treating any peptides and related compounds-treatable conditions in humans or animals. The prodrugs can be administered transdermally for any kind of medical treatments and avoid proteolysis by proteolytic enzymes in the GI tract. Controlled transdermal administration systems of the prodrug enables peptides and related compounds to reach constantly optimal therapeutic blood levels to increase effectiveness and reduce the side effects of peptides and related compounds. Another great benefit of transdermal administration of these pro-drugs is that administering medication, especially to children, will be much easier.

Description

TRANSDERMAL DELIVERY SYSTEMS OF PEPTIDES AND

RELATED COMPOUNDS

Technical Field [1] The present invention relates to transdermal delivery systems of peptides and related compounds by transferring peptides to positively charged and water-soluble pro-drugs and their medicinal use in treating conditions that are treatable by peptides and related compounds in humans or animals. More specifically, the present invention is to enable fast skin penetration of peptides and related compounds and make them ad ministrable transdermally.

Background Art [2] All peptides are polymers, and the monomers that combine to make them are amino acids. Chains containing 2 to 50 amino acid residues are collectively referred to as peptides. If the chains are more than 50 amino acid residues, they are called proteins. Peptides play an enormous variety of roles in all living matter. Peptide hormone is the largest group of hormones. An ever increasing number of peptide hormones are being identified and synthesized. They have a fascinating role in processes that control life. One nanogram of hyrotropin-releasing hormone injected into a mouse increases the uptake of iodide from the blood into the thyroid gland (R.L. Kisliuk, Principles of Medicinal Chemistry, 4^th Ed., W.O. Foye, et al. Eds., Williams & Wilkins, 4^th Ed. 1995, p. 606). Tuftsin (Thr-Lys-Pro-Arg) stimulates phagocytosis and promotes antibody-dependent cellular cytotoxicity (V.A. Najjar, Mol. Cell. Biochem. 41, 1, 1981), Met-enkephaline (Tyr-Gly-Gly-Phe-Met) isolated from brain and small intestine, acts as morphine does, in that it binds to the same receptor and has analgesic activity (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.B. Sandberg, Annu, Rep. Med. Chem., 18. 31, 1983), somatostatin (A.V. Schally, et al., Annu. Rev. Biochem., 47. 89, 1978), dynorphin (M.G. Weisskopf, et al., Nature, 362. 423, 1993), endothelin (A.M. Doherty, J. Med. Chem., 35. 1493, 1992), secretin (E. Jorper, Gastroenterology, 55. 157, 1968), calcitonin (M.V.L. Ray, et al., Biotechnology, IT, 64, 1993), insulin (F. Sanger, Br. Med. Bull.,

16. 183, 1960), and many other are peptides whose structures have been deduced and they are used for the treatment of many diseases.

2016213759 02 Mar 2018 [3] Unfortunately, peptides and related compounds are rapidly proteolysized by proteolytic enzymes. When peptides are taken orally, they are destroyed in a few minutes . In the case of injection, the administration of peptides is painful, and in many cases requires frequent and costly office visits to treat chronic conditions.

[4] One alternative method of administering drugs is topical delivery. Topical drug ₅ delivery has several advantages. This method helps to avoid inactivation of a drug caused by first pass metabolism in the liver and gastro-intestmal tract. It can provide local delivery of appropriate concentrations of a drug to the intended site of action without systemic exposure. Fishman (Fishman; Robert, U.S. Pat. No. 7,052,715) indicated that an additional problem associated with oral medications, is that the concentration levels which must be achieved in the bloodstream must be significant in ₁₀ order to effectively treat distal areas of pain or inflammation. These levels are often much higher than would be necessary if it were possible to accurately target the particular site of pain or injury. Yeager has tried to use penetration enhancer to deliver PGE] for the treatment of male erectile dysfunction (Yeager, James L, U.S. Pat. No. 6,693,135). Susan Milosovich, et al. designed and prepared testosteronyl4-dimethylaminobutyrate.HCl (TSBH), which has a lipophilic portion and a tertiary j amine groups that exists in the protonated form at physiological pH. They found that the prodrug (TSBH) diffuses through human skin —60 times faster than does the drug (TS) itself [Susan Milosovich, et al., J. Pharm. Sci., 82, 227(1993).

Disclosure of Invention

Technical Problem [5] Peptides play an enormous variety of roles in all living matter and they are used for treatment of many diseases, [6] Unfortunately, peptides and related compounds are rapidly proteolysized by proteolytic enzymes. When peptides are taken orally, they are destroyed a few minutes. In the case of injection, the administration of peptides is painful, and in many cases requires frequent and costly office visits to treat chronic conditions.

Summary of the Invention

A first aspect of the invention provides for a compound of the general formula (1) 'Structure Γ, ^Yx2-^Rx2 \

Structure

2a

2016213759 09 Mar 2018 wherein, X represents O or S; Xi and X_n each represents CO, SO, SO2, NO, or nothing; Z_n and Z_ni each represents H, CH₃, C2H5, C3H7, CF₃, C2F₅, or C₃F₇; R_nrepresents one of any aliphatic side chains of amino acids, hydroxyl or sulfur containing side chains of amino acids, aromatic side chains of amino acids, amino, imidazolyl, or quanidino group-containing side chains of amino acids, or carboxyl or carboxamide group-containing side chains of amino acids, one of any alkyl or alkyloxyl residues having 1 to 10 carbon atoms, alkenyl or aikynyl residues having 2 to 10 carbon atoms, aryl or heteroaryl residues having 5 to 12 carbon atoms, or nothing; Y_xi, Y_x2, and Y_n each represents H, ^R|t5 , and nothing; R_xi, R_x2,

R_x3, R_x4, R_X5 and R_sri each represents H, OH, one of alkyl or alkyloxy residues having 2 to 10 carbon atoms, alkenyl or aikynyl residues having 2 to 10 carbon atoms, aryl or heteroaryl residues having 5 to 12 carbon atoms, or nothing; A represents Cl', Br', F', Γ, AcO', citrate, or any negative ions; n=l, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein all R groups are branched or straight chains, wherein the compound has a lipophilic portion and one or two primary, secondary, or tertiary amine groups, guanidino, or monoprotected guanidino groups at the N-terminal or C-terminal position that are protonatable at physiological pH; and the compound has no unprotected carboxyl group; and

A I

R_X4 wherein at least one of Y_xi or Y^ is

A second aspect of the invention provides for a use of a compound of the general formula (1) 'Structure 1', according to the first aspect of the invention for the manufacture of a medicament for treating any condition treatable by peptides and related compounds in humans or animals, wherein the medicament is formulated for oral or transdermal administration.

A third aspect of the invention provides for a method for treating any condition in a human or an animal that is treatable by peptides and related compounds, comprising administering transdermally to any part of the human or animal body a composition comprising of at least one compound of the general

2b

2016213759 02 Mar 2018 formula (1) 'Structure 1' according to the first aspect of the invention, wherein the compound or composition is in the form of a solution, spray, lotion, ointment, emulsion or gel.

A fourth aspect of the invention provides for a transdermal therapeutic 5 application system comprising a compound of the general formula (1) 'Structure 1' according to the first aspect of the invention when used for treating any condition in a human or an animal treatable by peptides and related compounds.

Technical Solution [71 This invention relates to the preparation of novel positively charged pro-drugs of peptides and related compounds and their medicinal use. The pro-drugs of peptides and io related compounds have the general formula (1) 'Structure Γ:

2016213759 10 Aug 2016

Υχ2

Structure 1

Wherein, X represents O, S, or NH; X or X represents CO, SO, SO , PO (OR), NO,

In 2 or nothing; Z or Z represents H, CH ,CH CH CF CF orCF;R represents n nl 3 2 5, 3 7, 3, 2 5, 3 7 n one of any aliphatic side chains of amino acids, hydroxyl- or sulfur-containing side chains of amino acids, aromatic side chains of amino acids, amino, imidazolyl, or quanidino group-containing side chains of amino acids, or carboxyl or carboxamide group-containing side chains of amino acids, H, one of any alkyl, alkyloxyl, alkenyl or alkynyl residues having 1 to 12 carbon atoms, aryl or heteroaryl residues, or nothing; Y , Y , or Y represents H, one of any alkyl, alkyloxyl, alkenyl or alkynyl residues xl x2 n having 1 to 12 carbon atoms, aryl or heteroaryl residues, or the following group,

Rx3

A ® NRx/f

RxS or nothing; R , R R , R , R or R represents Η, O, one of any alkyl, alkyloxyl, xl x2, x3 x4 x5, xn alkenyl or alkynyl residues having 1 to 12 carbon atoms, aryl or heteroaryl residues, or nothing; A' represents Cl, Br, F, I, AcO, citrate, or any negative ions; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ...; All R, -(CH ) - or -(CH ) - groups are branched or straight chains n 2 m and may include C, Η, O, S, or N atoms and may have single, double, and triple bonds. Any CH₂ groups may be replaced with O, S, or NH. One or more amino acids residues within the sequence can be substituted by non-natural amino acids, such as, β-amino acids, 2-naphthylalanine, and one of any alkyl, alkyloxyl, alkenyl or alkynyl, aryl or heteroaryl residues. The amino and carboxyl functions of amino acids on the peptide chain may form lactam bridges to form homodetic cyclic peptides. The thiol groups of cysteine, homecysteine, or other amino acids may form disulfide bridges to form heterodetic cyclic peptides.

[8] The principles for designing these prodrugs of peptides or related compounds are:

1. The prodrug must have a lipophilic portion and a primary, secondary, or tertiary

2016213759 10 Aug 2016 [9] amine group, a quanidino, or monoprotected quanidino group that exists in the protonated form (hydrophilic portion) at physiological pH. 2. Every prodrug of a peptide should have only one or two (preferably one) primary, secondary, or tertiary amine group, a quanidino, or monoprotected quanidino group that exists in the protonated form (hydrophilic portion) at physiological pH. 3. The primary, secondary, or tertiary amine group, a quanidino, or monoprotected quanidino groups can be on the N-terminal, C-terminal, or side-chain of peptides. The N-terminal or C-terminal position is preferable. 4. Carboxyl groups, amino groups, guanidine groups or other hy drophilic groups can be protected with an alkyl, aryl, or heteroaryl ester or amide group to make the peptide lipophilic.

The following are examples of these prodrugs:

Structure 2

2016213759 10 Aug 2016

Structure 4

2016213759 10 Aug 2016

Structure 5

Wherein, R represents H, a branched or straight chain, -(CH ) -, wherein n=0, 1, 2, 3, n

4, 5, 6, 7, 8, 9, 10,..., aryl or heteroaryl residue; X₄ X₅ X^ X? X_g or X^ represents CO, SO , PO(OR), NO, or nothing; R,R,R R,R,R R R orR represents Η, O, one

1 2 3, 4 5 6, 7, 8, 9 of any alkyl, alkyloxyl, alkenyl or alkynyl residues having 1 to 12 carbon atoms, aryl or heteroaryl residues; A' represents Cl, Br, F, I, AcO, citrate, or any negative ions; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Ar represents phenyl, 2'-naphthyl, 4-iodophenyl, or other ary or heteroaryl residue; All R, -(CH ) - or -(CH ) -groups are branched or n 2 m straight chains and may include C, Η, O, S, or N atoms and may have single, double, and triple bonds; Any CH₂ groups may be replaced with O, S, or NH.

2016213759 10 Aug 2016

Structure 8

2016213759 10 Aug 2016

Structure 9

Structure 10 [10] Drug absorption, whether from the gastrointestinal tract or other sites, requires the passage of the drug in a molecular form across the barrier membrane. The drug must first dissolve, and if the drug possesses the desirable biopharmaceutical properties, it will pass from a region of high concentration to a region of low concentration across the membrane into the blood or general circulation. All biological membranes contain lipids as major constituents. The molecules that play the dominant roles in membrane formation all have phosphate-containing highly polar head groups, and, in most cases, two highly hydrophobic hydrocarbon tails. Membranes are bilayers, with the hydrophilic head groups facing outward into the aqueous regions on either side. Very hydrophilic drugs (most peptides) cannot pass the hydrophobic layer of membrane and very hydrophobic drugs will stay in the hydrophobic layer as part of the membrane due

2016213759 10 Aug 2016 to their similarities and therefore cannot enter the cytosol on the inside efficiently.

[11] The goal of this invention is to make peptides and related compounds administrable transdermally (topical application) by increasing their penetration rate through the membrane and skin barrier. These novel pro-drugs of peptides and related compounds have two structural features in common: they have a lipophilic portion (which can be formed by using lipophilic alcohols to protect the carboxyl groups and lipophilic acids to protect amino, hydroxyl, or guanidine groups or other hydrophilic groups of peptides) and a primary, secondary, or tertiary amine group, a quanidino group, or a monoprotected quanidino group that exists in the protonated form (hydrophilic part) at physiological pH. Such a hydrophilic-lipophilic balance is required for efficient passage through the membrane barrier [Susan Milosovich, et al., J. Pharm. Sci., 82, 227(1993)]. The positively charged amino groups largely increase the solubility of the drugs in water and the lipophilic portion will help the prodrugs enter the lipophilic membrane and skin barrier. When these new pro-drugs are administered transdermally in a dosage form such as a solution, spray, lotion, ointment, emulsion or gel, they will dissolve in moisture of the skin surface immediately. The positive charge on the amino groups of these pro-drugs will bond to the negative charge on the phosphate head group of membrane. Thus, the local concentration of the outside of the membrane will be very high and will facilitate the passage of these pro-drugs from a region of high concentration to a region of low concentration. When these pro-drugs enter the membrane, the hydrophilic part will push the pro-drug into the cytosol, a semi-liquid concentrated aqueous solution or suspension. The penetration rates of some prodrugs through human skin were measured in vitro by using modified Franz cells, which were isolated from human skin tissue (360-400 pm thick) of the anterior and posterior thigh areas. The receiving fluid consisted of 2 ml of 2% bovine serum albumin in normal saline and was stirred at 600 rpm. The cumulative amounts of these prodrugs and their parent drugs penetrating the skin versus time were determined by a specific highperformance liquid chromatography method. The results using a donor consisting of a 10% solution of some of the prodrugs and peptides in 0.2mL of pH 7.4-phosphate buffer (0.2M) are shown in Figure 1. Apparent flux values of 0.52 mg, 0.55 mg, 0.46 mg, 0.34 mg, 0.50 mg, 0.60 mg, 0.001 mg, 0.001 mg, 0.001 mg, and 0.001 mg/cm²/h were calculated for Ac-Tyr(Ac)-Gly-Gly-Phe-Met-OCH CH N(CH CH )₂.HC1, HC1.(CH ) NCH CH CH CO-Tyr(Ac)-Gly-Gly-Phe-Met-OCH CH CH CH , cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCHCH₂N(CHCH)₂.HCl, cyclo(l,6)-Ac-Nle-Asp-His-D-Phe(4-I)-Arg(Ac)-Trp-Lys-NH.HCl, cyclo(l,6)-Ac-Nle-Asp-His-D-Ala(2-naphthyl)-Arg-Trp-Lys-NH₂.HCl, AcVal-Pro-Gly-Pro-Arg(di Ac)-OCIICHN(CHCH) ₂.HC1, AcTyr-Gly-Gly-Phe-Met-OH, cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg-Trp-Lys-OH,

2016213759 10 Aug 2016 cyclo(l,6)-Ac-Nle-Asp-His-D-Phe(4-I)-Arg-Trp-Lys-NH₂ and HVal-Pro-Gly-Pro-Arg-OH diffuse through human skin. The results suggest that the pro drugs diffuse through human skin 340-600 times faster than do peptides and related compounds. The results suggest that the positive charge on the dialkyaminoethyl group has a very important role in the passage of the drug across the membrane and skin barrier.

[12] A good prodrug should change back to the drug itself once in plasma. We found out that the prodrugs of peptides in this invention can change back to the parent peptide very quickly in good yield in human plasma. 20 mg of AcTyr(Ae)-Gly-Gly-Phe-Met-OCH CH N(CH CH ^.HCl in 1 ml of whole blood was incubated for 30 min at 37°C. The mixture was analysized by HPLC. 3% AcTyr(Ac)-Gly-Gly-Phe-Met-OCH CH N(CH CH ) .HCI, 2% AcTyr-Gly-Gly-Phc-Mct-OCHCHN(CUCU ₃)₂-HCl, 8% Ac-Tyr-Gly-Gly-Phe-Met-OH, 60% H-Tyr-Gly-Gly-Phe-Met-OH, 27% of other side products (amino acids, dipeptides, tripeptides, tetrapeptides) were observed. For UC1.(CU ) NCU CU CU CO-Tyr(Ac)-Gly-Gly-Phe-Met-OCH CH CH CH , 5% HC1.(CH ) NCH CH CH COTyr(Ac)-Gly-Gly-Phe-Met-OCH CH CH CH 6% of (CH ) NCH CH CH CO^{J v J J} 2223 3 2 2 2 2

Tyr-Gly-Gly-Phe-Met-OCH CH CH CH , 10% of (CH ) NCH CH CH COTyr-Gly-Gly-Phe-Met-OH, 55% H-Tyr-Gly-Gly-Phe-Met-OH, and 24% of other side products were observed . For cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCHCH ₂N(CHCH )₂.HC1, 4% of cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCHCH N(CH CH )₂.HC1, 8 % of cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(Ac)-Trp-Lys-OH, 10% of cyclo(l,6)-Nle-Asp-His-Phe-Arg-Trp-Lys-OH, 45% cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg-Trp-Lys-OH, and 33% of other side products were observed . The results show that most of the prodrug of peptides was changed back to the parent peptides, so the transdermal delivery system of peptides is successful.

[13] Enterostatins [Val-Pro-Asp-Pro-Arg (VPDPR), Val-Pro-Gly-Pro-Arg (VPGPR), and Ala-Pro-Gly-Pro-Arg (APGPR)] are pentapeptides derived from the Nil -terminus of procolipase after tryptic cleavage and belong to the family of gut-brain peptides. They regulate fat intake and may be used for the treatment of obesity (Erlanson-Albertsson C, York D, Obes. Rev. 1997 Jul; 5(4): 360-72 and Sorhede M, Mei J, Erlanson-Albertsson C., J Physiol. 87:273-275 ,1993). H- ValPro-Asp-Pro-Arg -OH produced a dose-dependent reduction in food intake when injected intraperitoneally into Osborne-Mendel rats that had been starved overnight. This inhibition of feeding was observed when the rats were fed a high-fat diet but not in rats fed a high-carbohydrate, low-fat diet (Okada S. et al. Physiol Behav., 1991 Jun;

2016213759 10 Aug 2016

49(6): 1185-9). Rats (n=5) were administered 5 mg/kg of Ac- ValPro-Asp(OEt)-Pro-Arg (diAc)- OCH CH N(CH CH ) ₂.HC1 transdermally in 0.5 ml of water that was applied to the backs of rats (n=5 each, both overnight-fasted rats presented with food and in ad-lib-fed rats ) at the beginning of the dark-onset feeding period . This selective inhibition of fat intake was observed .

[14] Melanocortin II is a cyclic lactam peptides Cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg-Trp-Lys-OH. It is the Palatin's (AMEX:PTN) novel drug candidate for the treatment of male and female sexual dysfunction. First in a new class of therapies called melanocortin agonists, melanocortin Π has shown promise in effectively treating erectile dysfunction (ED) and female sex dysfunction without the cardiovascular effects found in ED drugs currently available. Melanocortin II works through a mechanism involving the central nervous system rather than directly on the vascular system. As a result, it may offer significant safety and efficacy benefits over currently available products. The novel prodrugs in the invention can diffuse through human skin in very high rate (-0.3-0.5 mg/h/cm ) and can provide almost side-effects-free methods of treating erectile dysfunction or enhancing female sexual arousal. 2 mg/kg cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCH₂CH N(CH CH )₂.HC1 (peptide A) and cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(NO₂)-Trp-Lys-OCHCH N(CH CH ) Ή<21 (peptide B) in 0.2 ml of pH 7.0 phosphate buffer (0.1 M) was applied to the back of male rats (30 rats ) once per day for 5 days. The results showed a 5 fold increase for peptide A and 6 fold increase for peptide B in solicitation and 3 fold increase for both of peptide A and B in copulation in rats that were given the peptide A or peptide B compared to those that were not. When the same amount of cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCH₂CH N(CII CH ) .HCI (peptide A) and cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(NO₂)-Trp-Lys-OCH₂CH N(CII CH ) .HCI (peptide B) in 0.2 ml of pH 7.0 phosphate buffer (0.1 M) were applied to the back of both male rats (30 rats) and female rats (30 rates) once per day for 5 days, the results showed a 6 fold increase in solicitation and 5 fold increase in copulation in rats that were given the peptide A or peptide B compared to those that were not.

[15] Opioid peptides, such as, Met-enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH), Leuenkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH), H-Tyr-D-Ala-Gly-N-Me-Phe-Met(O)-OL, H-Tyr-D-Ala-Gly-Phe-Leu-OH, and many other exert morphine-like analgesic action. The number of writhings that occurred when mice were administered an acetic acid solution intraperitoneally were counted, and the rate of inhibition based on the control group was calculated. HCl.H-Tyr(Ac)-D-Ala-Gly-Phe-Leu-OCH₂(CH₂) ₄CII (10 mg/ kg, B), Ac-Tyr(Ac)-D-Ala-Gly-Phe-Leu-OCHCHN(CHCH J.IICI (10 mg/kg, C), and HCl.H-Tyr(Ac)-D-Ala-Gly-Phe-Met(O)-OL (10 mg/kg, D) were administered transdermally the neck of mice 30 minutes before the acetic acid solution was administered. The group A is the control group. The results are shown in Table 1.

[16] Table 1. The rate of writhings inhibition by the prodrugs of enkephalin and related compounds.

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Group	Dose (mg/kg)	No. of Writhings	%
A	0	35.0	-
B	10	8.6	75
C	10	5.2	85
D	10	3.2	91

The results show that the transdermal delivery system of using the peptide prodrugs works very well for the treatment of obesity and pain, and for treatment of male and female sexual dysfunction.

[17] Peptides and related compounds are very hydrophilic and they cannot penetrate the skin membrane barrier. When peptides are taken orally, peptides and related compounds are rapidly proteolysized by proteolytic enzymes in the GI tract in a few minutes . In the case of injection, administration of peptides is painful and in many cases requires frequent and costly office visits to treat chronic conditions . When the prodrugs of peptides are topically applied to skin, they will dissolve in the moisture of t he skin immediately. The positive charge on the amino groups of these pro-drugs will bond to the negative charge on the phosphate head group of the skin membrane. Thus, the local concentration of the outside of the membrane will be very high and will facilitate the passage of these pro-drugs from a region of high concentration to a region of low concentration. When these pro-drugs enter the membrane, the hydrophilic part will push the pro-drug into the cytosol.

[18] The compounds of the general formula (1) 'Structure Γ indicated above can be prepared by standard peptide synthesis protocols. The preparation of cyclopeptides and related compound of the general formula (4-C) 'Structure 4-C', wherein the peptide chain can be synthesized by standard peptide synthesis protocols, the side chain of lycine was protected by 2-, or 4-Pyoc protecting group and the cyclization of the peptide was done on resin.

Advantageous Effects [19] These prodrugs of peptides and related compounds in the present invention have a lipophilic portion and a hydrophilic portion (the amine groups that exist in the protonated form at physiological pH). The positively charged amino groups of these pro-drugs have two major advantages. First, it makes prodrugs soluble in water; when these new pro-drugs are administered transdermally in a dosage form such as a

2016213759 10 Aug 2016 [20] [21] solution, spray, lotion, ointment, emulsion or gel, they will mix with moisture on the skin, eye, genital area, mouth, nose, or other part of the body immediately. Second, the positive charge on the amino group of these pro-drugs will bond to the negative charge on the phosphate head group of membrane. Thus, the local concentration outside of the membrane will be very high and will facilitate the passage of these pro-drugs from a region of high concentration to a region of low concentration. The lipophilic portion (by modification of the polar groups with lipophilic alkyl groups) of the prodrugs will facilitate the entering of the drugs into the skin membrane. When these pro-drugs enter the membrane, the hydrophilic part will push the pro-drugs into the cytosol, a semiliquid concentrated aqueous solution or suspension. Due to the short stay on the skin, eye, genital area, mouth, nose, or other part of the body, the pro-drugs will not cause itching, burning or pain to the skin, eye, genital area, mouth, nose, or other part of the body. Experiment results show that more than 40% of the pro-drugs were changed back to the parent peptides in a few minutes. The pro-drugs can even penetrate the blood-brain barrier easily. The transdermal delivery systems of peptides and related compounds can make peptide hormones usable medicinally. Another great benefit of transdermal administration of these pro-drugs is that administering medication, especially to children, will be much easier.

Description of Drawings

Figure 1: Cumulative amounts of Ac-Tyr(Ac)-Gly-Gly-Phe-Met-OCH₂CH₂N(CH₂CH ) .HCI, HC1.(CH ) NCH CH CH CO-Tyr(Ac)-Gly-Gly-Phe-Met-OCH CH CH CH , cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCHCH N(CHCH) .HCI, cyclo(l ,6)-Ae-Nle-Asp-His-D-Phe(4-I)-Arg(Ae)-Trp-Lys-NH.HCl, cyclo(l,6)-Ac-Nle-Asp-His-D-Ala(2-naphthyl)-Arg-Trp-Lys-NH₂.HCl, Ac- ValPro-Gly-Pro-Arg (diAc)- OCH₂CH₂N(CH₂CH ) ₂.HC1, Ac-Tyr-Gly-Gly-Phe-Met-OH, cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg-Trp-Lys-OH, cyclo(l,6)-Ac-Nle-Asp-His-D-Phe(4-I)-Arg-Trp-Lys-NH₂ andH- ValPro-Gly-Pro-Arg - OH, crossing isolated human skin tissue in Franz cells (n=5). In each case, the vehicle was pH 7.4 phosphate buffer (0.2 M).

Figure 2: Structure 1. Wherein, X represents O, S, or NH; X or X represents CO, n

SO, SO , PO(OR), NO, or nothing; Z or Z represents H, CH , C H C H CF C F n nl 3 2 5, 3 7, 3, 2 5, or C F ; R represents one of any aliphatic side chains of amino acids, hydroxyl- or

7 n sulfur-containing side chains of amino acids, aromatic side chains of amino acids, amino, imidazolyl, or quanidino group-containing side chains of amino acids, or carboxyl or carboxamide group-containing side chains of amino acids, one of any alkyl, alkyloxyl, alkenyl or alkynyl residues having 1 to 12 carbon atoms, aryl or heteroaryl residues, or nothing; Y , Y , or Y represents H, one of any alkyl, xl x2 n alkyloxyl, alkenyl or alkynyl residues having 1 to 12 carbon atoms, aryl or heteroaryl

2016213759 10 Aug 2016 residues, R R R N A'or nothing; R , R R , R , R or R represents Η, O, one of x3 x4 x5 xl x2, x3 x4 x5, xn any alkyl, alkyloxyl, alkenyl or alkynyl residues having 1 to 12 carbon atoms, aryl or heteroaryl residues, or nothing; A' represents Cl, Br, F, I, AcO, citrate, or any negative ions; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ...; All R, -(CH ) - or -(CH ) - groups n 2 m are branched or straight chains and may include C, Η, O, S, or N atoms and may have single, double, and triple bonds. Any CH₂ groups may be replaced with O, S, or NH. One or more amino acids residues within the sequence can be substituted by nonnatural amino acids, such as, β-amino acids, 2-naphthylalanine, and one of any alkyl, alkyloxyl, alkenyl or alkynyl, aryl or heteroaryl residues. The amino and carboxyl functions of amino acids on the peptide chain may form lactam bridges to form homodetic cyclic peptides. The thiol groups of cysteine, homecysteine, or other amino acids may form disulfide bridges to form heterodetic cyclic peptides.

Best Mode

Preparation of Ac-VaI-Pro-Asp(OEt)-Pro-Arg(diAc)-OCH CH N(CH CH )

2 2 3 2 .HCI [22]

1. Preparation of H-Arg(diAc)-OCH CH N(CH CH ) 30.8 g of Z-Arg-OH was dissolved in 500 ml of acetone. 200 ml of 20% NaOH was added into the reaction mixture. 40 g of acetic anhydride was added into the reaction mixture drop by drop. The mixture was stirred for 2 h at RT. The solvent was evaporated off. The residue was extracted with 500 ml of ethyl acetate. The ethyl acetate solution was washed with water (3 x 100 ml). Ethyl acetate layer was dried over sodium sulfate. Ethyl acetate solution was evaporated to dryness. The residue (Z-Arg(diAc)-OH, 30 g) was dissolved in 300 ml of acetonitrile. The mixture was cooled into 0°C with ice-water bath. 12 g of Ν,Ν-Diethylaminoethanol, 2g of 4-dimethylaminopyridine, and 22 g of

1,3-dicyclohexylcarbodiimide were added into the reaction mixture. The reaction mixture is stirred for 1 hour at 0°C and overnight at RT. The solid was removed by filtration and the solution was evaporated to dryness. The residue was extracted with ethyl acetate (2 x 250 ml). The ethyl acetate solution was washed with 5% sodium bicarbonate (1 x 500 ml) and water (3 x 100 ml). The ethyl acetate solution was dried over sodium sulfate. The solution is evaporated to dryness. The residue [Z-Arg(diAc)-OCH₂CH₂N(CH₂CU ) 28 g] was dissolved in 300 ml of methanol. 2 g of 10% Pd/C was added into the solution. The mixture was stirred for 10 h under hydrogen at RT. Pd/C was removed by filtration. The solution was evaporated to dryness. Yielded 22 g of H-Arg(diAc)-OCH CH N(CH CH ) .

2 2 3 2

2016213759 10 Aug 2016

2. Preparation of Boc-Asp(OEt)-Pro-OSu. 15 g of L-proline was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 36 g of BocAsp(OEt)-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. The mixture was washed with ether (1 x 300 ml). 500 ml of ethyl acetate was added into the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCI. The ethyl acetate layer was collected and washed with water (3 x 300 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. 25 g of the residue (Boc-Asp(OEt)-Pro-OH ) and 11 g of N-hydroxysuccinimide were dissolved in 300 ml of dichloromethlene. The mixture was cooled to 0°C. 16 g of 1,3-dicyclohexylcarbodiimide was added into the reaction mixture. The mixture was stirred for 1 hour at 0°C. The solid was removed by filtration.

The dichloromethlene solution is washed with 5% sodium bicarbonate (lx 200 ml) and water ( 3 x 200 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. Yield 28 g BocAsp(OEt)-Pro-OSu.

3. Preparation of H-Asp(OEt)-Pro-Arg(diAc)-OCH CH N(CH CH ) .2TFA. 22 g of H-Arg(diAc)-O CH CH N(CH CH ) was dissolved in 300 ml of 5% NallCO . 24 g of Boc-Asp(OEt)-Pro-OSu in 150 ml of acetone was added into the reaction mixture. The mixture was stirred for 5 h at RT. 500 ml of ethyl acetate was added into the mixture. The ethyl acetate solution was washed with water (3 x 100 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. The residue was dissolved in 250 ml of dichloromethlene. 250 ml of trifluoroacetic acid was added into the mixture and the mixture was stirred for 30 min. The mixture was evaporated to dryness. Yield 32 g of H-Asp(OEt)-Pro-Arg(diAc)-OCH CH N(CH CH ) .2TFA.

4. Preparation of Ac-Val-Pro-OSu. 15 g of L-proline was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 26 g of Ac-Val-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. The mixture was washed with ether (1 x 300 ml). 500 ml of ethyl acetate was added into the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCI. The ethyl acetate layer was collected and washed with water (3 x 300 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. 20 g of the residue (Ac-Val-Pro-OH) and 11 g of N-hydroxysuccinimide were dissolved in 300 ml of dichloromethlene. The mixture was cooled to 0°C. 16 g of

1,3-dicyclohexylcarbodiimide was added into the reaction mixture. The

2016213759 10 Aug 2016 mixture was stirred for 1 hour at 0°C and 1 hour at RT. The solid was removed by filtration. The dichloromethlene solution is washed with 5% sodium bicarbonate (lx 200 ml) and water (3 x 200 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. Yield g Ac-Val-Pro-OSu.

5. Preparation of Ac-Val-Pro-Asp(OEt)-Pro-Arg(diAc)-OCHCHN(CU ₂CH ) .HCI. 31 g of H-Asp(OEt)-Pro-Arg(diAc)-OCH CH N(CH CH ) .2TFA was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 15 g of Ac-Val-Pro-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. 500 ml of ethyl acetate was added into the mixture. The organic layer is washed with water (3 x 100 ml). The ethyl acetate layer was dried over sodium sulfate. Sodium sulfate is removed by filtration. 3.5 g of HCI gas in dioxane (50 ml) is added into the solution. The solid is collected and washed with ether (3 x 50 ml). After drying, it yielded 20 g of the desired hygroscopic product; Solubility in water: 150 mg/ml; Elementary analysis: C H C1N O ; MW: 872.45. Calculated % C: 53.69; H: 7.62; Cl: 4.06; N: 14.45; O: 20.17; Found % C: 53.61; H: 7.67; Cl: 4.10; N: 14.40, O: 20.22. MS: m/e: 836.4; m/e+1: 836.4.

Mode for Invention

Preparation of Ac-Tyr(Ac)-Gly-Gly-Phe-Met-OCH CH N(CH CH ) .HCI

2 2 3 2 [23]

1. Preparation of H-Met-OCH CH N(CH CH ) TFA. 25 g of Boc-Met-OH was dissolved in 300 ml of dichloromethlene. The mixture was cooled into 0°C with ice-water bath. 12 g of Ν,Ν-Diethylaminoethanol, 2g of 4-dimethylaminopyridine, and 22 g of 1,3-dicyclohexylcarbodiimide were added into the reaction mixture. The reaction mixture is stirred for 1 hour at 0°C and overnight at RT. The solid was removed by filtration and the dichloromethlene solution was washed with 5% sodium bicarbonate (lx 500 ml) and water (3 x 100 ml). The ethyl acetate solution was dried over sodium sulfate. The solution is evaporated to dryness. The residue [Boc-Met-OCH₂CH N(CH CH ) 30 g] was dissolved in 250 ml of dichloromethlene. 250 ml

2 3 2, of trifluoroacetic acid was added into the mixture and the mixture is stirred for 30 min.. The solution was evaporated to dryness. Yielded 26 g of HMet-OCH CH N(CH CH ) TFA.

2 2 3 2

2. Preparation of Boc-Gly-Phe-OSu. 20 g of L-phenylalanine was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 28 g of BocGly-OSu were added into the reaction mixture. The mixture was stirred for 5

2016213759 10 Aug 2016 h at RT. The mixture was washed with ether (1 x 300 ml). 500 ml of ethyl acetate was added into the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCI. The ethyl acetate layer was collected and washed with water (3 x 300 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. 22 g of the residue (Boc-Gly-Phe-OH) and 10 g of N-hydroxysuccinimide were dissolved in 300 ml of dichloromethlene. The mixture was cooled to 0°C. 15 g of

1,3-dicyclohexylcarbodiimide was added into the reaction mixture. The mixture was stirred for 1 hour at 0°C. The solid was removed by filtration.

The dichloromethlene solution is washed with 5% sodium bicarbonate (lx 200 ml) and water (3 x 200 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. Yield 25 g of BocGly-Phe-OSu.

3. Preparation of H-Gly-Phe-Met-OCH CH₂N(CH₂CH ) ₂-TFA. 25 g of HMet-OCH CH N(CH CH ) TFA was dissolved in 300 ml of 5% NaHCO . 22

2 2 3 2. 3 g of Boc-Gly-Phe-OSu in 150 ml of acetone was added into the reaction mixture. The mixture was stirred for 5 h at RT. 500 ml of ethyl acetate was added into the mixture. The ethyl acetate solution was washed with water (3 x 100 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. The residue was dissolved in 250 ml of dichloromethlene. 200 ml of trifluoroacetic acid was added into the mixture and the mixture was stirred for 30 min. The mixture was evaporated to dryness. Yield 25 g of H-Gly-Phe-Met-OCH₂CH₂N(CH₂CH ) ₂-TFA.

4. Preparation of Ac-Tyr(Ac)-Gly-OSu. 11 g of L-glycine was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 36 g of AcTyr(Ac)-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. The mixture was washed with ether (1 x 300 ml). 500 ml of ethyl acetate was added into the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCI. The ethyl acetate layer was collected and washed with water (3 x 300 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. 28 g of the residue (Ac-Tyr(Ac)-Gly-OH) and 13 g of N-hydroxysuccinimide were dissolved in 300 ml of dichloromethlene. The mixture was cooled to 0°C. 18 g of 1,3-dicyclohexylcarbodiimide was added into the reaction mixture. The mixture was stirred for 1 hour at 0°C. The solid was removed by filtration.

The dichloromethlene solution is washed with 5% sodium bicarbonate (lx 200 ml) and water (3 x 200 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. Yield 20 g of Ac18

2016213759 10 Aug 2016

Tyr(Ac)-Gly-OSu.

5. Preparation of Ac-Tyr(Ac)-Gly-Gly-Phe-Met-OCH CH N(CH CH ) .HCI. 24 g of H-Gly-Phe-Met-OCH CH N(CH CH ) .TFA was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 15 g of Ac-Tyr(Ac)-Gly-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. 500 ml of ethyl acetate was added into the mixture. The organic layer is washed with water (3 x 100 ml). The ethyl acetate layer was dried over sodium sulfate. Sodium sulfate is removed by filtration. 3.5 g of HCI gas in dioxane (50 ml) is added into the solution. The solid is collected and washed with ether (3 x 50 ml). After drying, it yielded 18 g of the desired hygroscopic product; Solubility in water: 200 mg/ml; Elementary analysis: C^H CIN O S; MW: 793.37. Calculated % C: 56.01; H: 6.73; Cl: 4.47; N: 10.59; O: 18.15; S: 4.04. Found % C: 55.96; H: 6.76; Cl: 4.52; N: 10.54, O: 18.19; S: 4.03. MS: m/e: 757.4; m/e+1: 758.4.

Preparation of Ac-VaI-Pro-Gly-Pro-Arg(diAc)-OCH CH N(CH CH ) .HCI

2 2 3 2 [24]

1. Preparation of Boc-Gly-Pro-OSu. 15 g of L-proline was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 27.2 g of Boc-Gly-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. The mixture was washed with ether (1 x 300 ml). 500 ml of ethyl acetate was added into the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCI. The ethyl acetate layer was collected and washed with water (3 x 300 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. 21 g of the residue (Boc-Gly-Pro-OH ) and 11 g of N-hydroxysuccinimide were dissolved in 300 ml of dichloromethlene. The mixture was cooled to 0°C. 17 g of

The dichloromethlene solution is washed with 5% sodium bicarbonate (lx 200 ml) and water ( 3 x 200 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. Yield 23 g BocGly-Pro-OSu.

2. Preparation of H-Gly-Pro-Arg(diAc)-OCH CH N(CH CH ) .2TFA. 22 g of H-Arg(diAc)-O CH CH N(CH CH ) was dissolved in 300 ml of 5% NaHCO

2 2 3 2 3 . 20 g of Boc-Gly-Pro-OSu in 150 ml of acetone was added into the reaction mixture. The mixture was stirred for 5 h at RT. 500 ml of ethyl acetate was added into the mixture. The ethyl acetate solution was washed with water (3 x 100 ml). The organic solution was dried over sodium sulfate. Sodium sulfate

2016213759 10 Aug 2016 is removed by filtration. The solution was evaporated to dryness. The residue was dissolved in 250 ml of dichloromethlene. 250 ml of trifluoroacetic acid was added into the mixture and the mixture was stirred for 30 min. The mixture was evaporated to dryness. Yielded 28 g of HGly-Pro-Arg(diAc)-OCH CH N(CH CH ) .2TFA.

3. Preparation of Ac-Val-Pro-Gly-Pro-Arg(diAc)-OCHCHN(CHCH )₂.HC1.

g of H-Gly-Pro-Arg(diAc)-OCH CH N(CH CH ) .2TFA was dissolved in 300 ml of 10% sodium bicarbonate. 150 ml of acetone and 15 g of AcVal-Pro-OSu were added into the reaction mixture. The mixture was stirred for 5 h at RT. 500 ml of ethyl acetate was added into the mixture. The organic layer is washed with water (3 x 100 ml). The ethyl acetate layer was dried over sodium sulfate. Sodium sulfate is removed by filtration. 3.5 g of HCI gas in dioxane (50 ml) is added into the solution. The solid is collected and washed with ether (3 x 50 ml). After drying, it yielded 18 g of the desired hygroscopic product; Solubility in water: 150 mg/ml; Elementary analysis: C^H

C1N O ; MW: 786.36. Calculated % C: 53.46; H: 7.69; Cl: 4.51; N: 16.03;

9 9

O: 18.31; Found % C: 53.43; H: 7.73; Cl: 4.55; N: 16.01, O: 18.29. MS: m/e: 750.4; m/e+1:751.4.

Preparation of Cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCH CH N(CH CH ) .HCI.

2 3 2 [25]

1. Preparation of Ac-Nle-Asp(OFm)-OH. 43 g of H-Asp(OFm)-OH.TFA and 27 g of Ac-Nle-OSu were suspended in 300 ml of acetone. 300 ml of 5%

NaHCO was added into the reaction mixture. The mixture was stirred for overnight at RT. The mixture was washed with ether (1 x 300 ml). 500 ml of ethyl acetate was added into the aqueous layer. The pH of the mixture was adjusted to 2.4-2.5 with ice-cooled 3N HCI. The ethyl acetate layer was collected and washed with water (3 x 300 ml). The organic solution was dried over sodium sulfate. The solution was evaporated to dryness. Yielded 42 g of Ae-Nle-Asp(OFm)-OH.

2. Preparation of Fmoc-Trp-Lys(4-Pyoc)-OH. H-Lys(4-Pyoc)-OH was preparated according to reference (H. Kunz and S. Bimbach, Tetrahedron Lett., 25, 3567, 1984; H. Kunz and R. Barthels, Angew. Chem., Int. Ed. Engl., 22. 783, 1983). 33 g of H-Lys(4-Pyoc)-OH was suspended in 300 ml of 5% NaHCO . 300 ml of acetone and 52 g of Fmoc-Trp-OSu were added into the reaction mixture. The mixture was stirred for overnight at RT. The mixture was washed with ether (lx 500 ml). 500 ml of ethyl acetate was added into

2016213759 10 Aug 2016 the mixture and the pH of the mixture is adjusted to 2.2-2.3 with 3N HCI. The ethyl acetate layer is collected and washed with water. The organic solution was dried over sodium sulfate. The organic solution was evaporated to dryness. Yielded 55 g of Fmoc-Trp-Lys(4-Pyoc)-OH.

3. Preparation of Cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OH. 100 g of Wang resin was suspended in 700 ml of DMF. 50 g of FmocTrp-Lys(4-Pyoc)-OH, 13 g of 1-Hydroxybenzotriazole, 2 g of 4-dimethylaminopyridine, and 12 g of Ν,Ν'-diisopropylcarbodiimide. The mixture was stirred overnight at RT. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of 20% piperidine was added into the resin. The mixture was stirred for 30 min. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of DMF, 48 g of Fmoc-Arg(diAc)-OH, 13 g of 1-Hydroxybenzotriazole, 35 ml of triethylamine, and 38 g of O(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium were added into the resin. The mixture was stirred for 2 hours at RT. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of 20% piperidine was added into the resin. The mixture was stirred for 30 min. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of DMF, 39 g of Fmoc-Phe-OH, 13 g of 1-Hydroxybenzotriazole, 35 ml of triethylamine, and 38 g of O(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium were added into the resin. The mixture was stirred for 2 hours at RT. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of 20% piperidine was added into the resin. The mixture was stirred for 30 min. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of DMF, 60 g of FmocHis(Fmoc)-OH, 13 g of 1-Hydroxybenzotriazole, 35 ml of triethylamine, and 38 g of O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium were added into the resin. The mixture was stirred 2 hours at RT. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of 20% piperidine was added into the resin. The mixture was stirred for 30 min. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of DMF, 60 g of Ac21

2016213759 10 Aug 2016

Nle-Asp(OFm)-OH, 13 g of 1-Hydroxybenzotriazole, 35 ml of triethylamine, and 38 g of O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium were added into the resin. The mixture was stirred 2 hours at RT. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). The peptided resin was suspended in 700 ml of DMF. 50 g of Mel was added into the reaction mixture. The mixture was stirred for 1 h at RT and lh at 50°C. The resin is collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of 30% piperidine was added into the resin. The mixture was stirred for 60 min. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 700 ml of DMF, 13 g of 1-Hydroxybenzotriazole, 35 ml of triethylamine, and 38 g of O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium were added into the resin. The mixture was stirred 10 hours at RT. The resin was collected by filtration and washed with DMF (3 x 400 ml), methanol (3 x 400 ml), and dichloromethlene (3 x 400 ml). 500 ml of trifluoroacetic acid was added into the resin and the mixture was stirred for 1 hour at RT. The resin was removed by filtration and the solution is evaporated to dryness. The residue was washed with ether ( 3 x 100 ml).

4. Preparation of Cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OCH₂CH N(CH CH ).HC1. 10 g of

2 3

Cyclo(l,6)-Ac-Nle-Asp-His-Phe-Arg(diAc)-Trp-Lys-OH was dissolved in 300 ml of DMF. The mixture was cooled into 0°C with ice-water bath. 12 g of Ν,Ν-Diethylaminoethanol, 2g of 4-dimethylaminopyridine, and 22 g of

1,3-dicyclohexylcarbodiimide were added into the reaction mixture. The reaction mixture is stirred for 1 hour at 0°C and overnight at RT. The solid was removed by filtration and the dichloromethlene solution was washed with 5% sodium bicarbonate (1 x 500 ml) and water (3 x 100 ml). The ethyl acetate solution was dried over sodium sulfate. 2 g of HC1 in dioxane (20 ml) was added into the solution. The solid was collected and washed with ether ( 3 x 30 ml). Yield 8 g of.

Industrial Applicability [26] The pro-drugs of the general formula (1) 'Structure Γ can penetrate the skin barrier easily. The transdermal delivery systems of peptides and related compounds can make peptide hormones useful in treating many diseases in humans or animals, such as, pains from rheumatoid arthritis and osteoarthritis, fever, male erectile dysfunction and female sex dysfunction, systemic blood pressure, hypotensive control, inhibition of platelet aggregation, pulmonary diseases, gastrointestinal disease, inflammation,

2016213759 10 Aug 2016 shock, reproduction, fertility etc..

Sequence List Text [27]

2016213759 09 Mar 2018

Claims

Claims:

1. A compound of the general formula (1) 'Structure Γ, ^Yx2-^Rx2 \

^rYx1

Structure 1 wherein, X represents O or S; Xi and X_n each represents CO, SO, SO2, NO, or nothing; Z_nand Z_ni each represents H, CH₃, C2H5, C3H7, CF₃, C2F5, or C₃F₇; R_n represents one of any aliphatic side chains of amino acids, hydroxyl or sulfur containing side chains of amino acids, aromatic side chains of amino acids, amino, imidazolyl, or quanidino group-containing side chains of amino acids, or carboxyl or carboxamide group-containing side chains of amino acids, one of any alkyl or alkyloxyl residues having 1 to 10 carbon atoms, alkenyl or alkynyl residues having 2 to 10 carbon atoms, aryl or heteroaryl residues having 5 to 12 carbon ^Rx3 atoms, or nothing; Y_xb Y_x2, and Y_n each represents H, ^R*⁵ , and nothing; R_xb

R_X2, Rx3, Rx4, Rx5 and R_xn each represents H, OH, one of alkyl or alkyloxy residues having 2 to 10 carbon atoms, alkenyl or alkynyl residues having 2 to 10 carbon atoms, aryl or heteroaryl residues having 5 to 12 carbon atoms, or nothing; A represents Cl, Br, F, I, AcO, citrate, or any negative ions; n=l, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein all R groups are branched or straight chains, wherein the compound has a lipophilic portion and one or two primary, secondary, or tertiary amine groups, guanidino, or monoprotected guanidino groups at the N-terminal or C-terminal position that are protonatable at physiological pH; and the compound has no unprotected carboxyl group; and

2016213759 02 Mar 2018

A |

Εχ4 wherein at least one of Y_xi or Y_X2 is
2 The compound according to claim 1, wherein

a) amino acids are independently from each other in the L-form or D-form;

and/or

5 b) one or more amino acids residues within the sequence are substituted by nonnatural amino acids, β-amino acids, 2-naphthylalanine, and one substituent selected from the group consisting of alkyl residues, alkyloxy residues, alkenyl residues, alkynyl residues, aryl residues and heteroaryl residues; and/or

c) the amino and carboxyl functions of amino acids on the peptide chain form

10 lactam bridges to form homodetic cyclic peptides; and/or

d) the thiol groups of cysteine, homocysteine, or other amino acids form disulphide bridges to form heterodetic cyclic peptides.
3. Use of a compound according to Claim 1 or Claim 2, for the manufacture of a medicament for treating any condition treatable by peptides and related compounds in is humans or animals, wherein the medicament is formulated for oral or transdermal administration.
4. The use according to claim 3, wherein the condition is selected from the group consisting of glaucoma or ocular hypertension, male erectile dysfunction and female sex dysfunction, systemic blood pressure, abortion, hypotensive control, inhibition of platelet

20 aggregation, pulmonary diseases, gastrointestinal disease, inflammation, shock, reproduction, fertility and obesity.
5. A method for treating any condition in a human or an animal that is treatable by peptides and related compounds, comprising administering transdermally to any part of the human or animal body a composition comprising at least one compound according to

25 Claim 1 or Claim 2, wherein the compound or composition is in the form of a solution, spray, lotion, ointment, emulsion or gel.

2016213759 02 Mar 2018
6. A transdermal therapeutic application system comprising at least one compound according to Claim 1 or Claim 2 when used for treating any condition in a human or an animal treatable by peptides and related compounds.
7. The transdermal therapeutic application system of claim 6, wherein the system is a 5 bandage or a patch comprising one active substance-containing matrix layer and an impermeable backing layer.
8. The transdermal therapeutic application system of claim 6 or claim 7, wherein the system has a permeable bottom facing the skin.
9. The transdermal therapeutic application system of any one of claims 6 to 8, wherein io the system has a controlling means controlling the rate of release, enabling the peptides and related compounds to reach constantly optimal therapeutic blood levels to increase effectiveness and reduce the side effects of peptides and related compounds.

Chongxi Yu

Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON

1/1

2016213759 10 Aug 2016

Fig. 1

Fig. 2

Structure 1