HU200480B - Process for producing linear and cyclic analogues of alpha-melanotropin - Google Patents

Abstract

The present invention relates to a novel α-melanotropin derivative of the formula R1 -Nle-R2-His-3-Arg-Trp-Ra-Rs (I) of formula (I) and their 5-10 ring analogues, wherein R1 is Ac-Ser- Tyr-Ser or Ac, R2 Glu or Asp, R3 Phe or D-Phe, R1 -GyY-Pro-Val-NH2 or -NH2, and R4 Lys, or when R5 NH2 and R1 Ac, Dab, Dpr, Om or Gly can be. The process comprises cleaving the protecting groups and / or the resin from at least one peptide known in the peptide chemistry and / or from a peptide of formula (I) bound to a known resin and optionally ring-forming the resulting open-chain compound. The scope of the description is 16 pages, with no o o o o CM 3 -1-

Description

The present invention relates to a process of formula (I)

-Nle-R ¹ R 2 3-Arg-His-Trp-R-R (I) α-melanotropin derivatives and 5-10 cyclic analogues thereof, wherein

R 1 is Ac-Ser-Tyr-Ser or Ac,

R2 is Glu or Asp,

Rí Phe or D-Phe,

R1 is -Gly-Pro-Val-NH2 or -NH2, and

R4 is Lys or ha

R5 may be NH2 and R1 may also be Ac, Dab, Dpr, Om or Gly.

The process involves cleaving the protecting groups and / or resin of at least one peptide of formula (I) having known protecting groups known in peptide chemistry and / or bound to a known resin and, if desired, cyclizing the resulting open chain compound.

Scope of the description: 16 pages, without figure a

o o

o o

CM

-1HU 200480 Β

The present invention relates to a process of formula (I)

R1-Nle-R1-His-R5-Arg-Trp-Ra-R1 (I) for the preparation of novel α-melanotropin derivatives and their 5-10 ring analogs, wherein

R 1 is Ac-Ser-Tyr-Ser or Ac,

R? Glu or Asp,

R3 is Phe or D-Phe,

Rí Gly-Pro-Val-NH? or -NH ?, and

R4 is Lys or ha

Rí -NH? and R1, Ac, R4 are Dab, Dpr, Om or Gly.

With that in mind. that the funding for the present invention was provided in part by the United States Public Health Service and the National Science Foundation, pursuant to Section 35 of United States Act 202, you may assign rights to the method in this description.

The present invention relates to a process for preparing a novel group of previously unknown, open-chain and cyclic fragments of α-melanotropin and to pharmaceutical compositions containing these compounds. The transdermal (transdermal) use of these analogs can stimulate the activity of melanocytes (dye producing cells called melanin) in vertebrates.

The color and other features of the skin, coat and plumage of vertebrates are determined by the number and distribution of certain dye-containing cells, such as melanocytes. however, the number and distribution of such cells is genetically regulated. In mammals, melanocytes occur in the lower part of the epidermis, at the junction of the epidermis and the scrotum, and in the hair follicles of the hair. The synthesis of dye melanin in nielanocytes is regulated by the enzyme tyrosinase, which is found in a premelanosome in a cellular formation. As a result of the activating action of the tyrosinase enzyme, a dye called eumelanin (brown-black) or phaeomelanin (yellow-red) is deposited in this formation (organellum); after completion of nielanin formation, premelanosome to melanosome. and, in particular, is called either a eumelanosome or a phaeomelanosome, depending on its color (see Fitzpatrick, T.I., Y. Hori, K. Toda, M. Seiji, Jap. J. Derm .. 79, 278 (1969)]. The melanosomes then enter the keratinocytes that surround the melanocytes in the skin or the cells that make up the growing hair cap, a process called cytocrine secretion.

Although melanin synthesis and coat color distribution are genetically regulated, melanogenesis (formation of melanin) in hair follicles in some mammals and hair color changes with α-melanotropin (also known as a-melanocyte stimulating hormone),

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Ara-Trp-Gly-Lys-Pro-Val-NH?

formula. with open-chain tridecapeptide can be regulated hormonally.

This hormone can be derived from a high molecular weight protein called proopio-melanocortin, selected by the pituitary middle lobe. Α-Melanotropin enhances the activity of the adenylate cyclase and tyrosinase enzymes and thereby enhances the production of melanin [see Hadley. Μ. E .. C. B. Ho>

Ward, V. J. Hruby, T. K. Sawyer and Y. C. S. Younc, Pigment Cell, 6, 323 (1980)].

In humans, α-melanotropin appears to occur only in the fetal pituitary gland and not in adults. In adults, there is a certain amount of melanin production that is genetically regulated and continuous. Above this basic level, different levels of melanin synthesis are directly under ultraviolet light. depends, for example, on the stimulating effect of sunlight; strong sunlight triggers an increased production of melanin, which results in a tan. This response may be the result of adaptation during core development to protect the individual from the aging and mutagenic effects of ultraviolet light. If the skin is exposed to lower levels of ultraviolet radiation, the rate of melanin formation in the skin slows down, the color of the skin fades, the effect of light penetration is reduced, and the skin can absorb more light. Although adults do not produce α-melanotropin in their pituitary gland, human melanocytes respond to this hormone and to the racemized preparation of it.

Less than normal dye production in human skin results from local deficiencies in melanin production in melanocytes, but the cause of many of these dysfunctions due to diminished dye production is not yet known.

It is estimated that some 1% of the world's population is affected by reduced toner formation disorders. Although it is known that α-melanotropin and some of its analogues, when administered subcutaneously (subcutaneously), can darken the skin of amphibians, and that α-melanotropin administered intramuscularly (muscle) darkens the skin of adrenal glands [Lemer. A. B. and J. S. McGuire, N. E. J. Med. 270, 539-546. (1964)], these modes of administration do not meet the goals of repeated administration necessary to achieve and maintain the desired effect. Prior to the present invention, there were no suitable means for treating such dysfunctions due to reduced toner production.

It has been found that certain analogs of α-melanotropin can be administered transcutaneously (transdermally) with good activity, and these compounds, in their active form, reach melanocytes and stimulate melanin production. Thus, pharmaceutical compositions containing the α-melanotropin analogs of the present invention can be simply and conveniently topically applied to eliminate disorders resulting from reduced dye production, such as post-inflammatory and other dye deficiencies (e.g., pityriasis alba. tinea versiocolor, vitiligo, unknown). hypomelanosis and nevus depigmentosus). In addition, topical application of alpha-melanotropin analogues may result in the regrowth of aged hair as a result of aging. Furthermore, the value of commercially valuable animal fur can also be increased by darkening the color of the fur by topical application of said analogs. In addition, it can be achieved. tanning the skin even in the absence of sunlight or ultraviolet light.

-2HU 200480 Β

All previous studies with α-melanotropin-related peptides isolated from the pituitary have shown that all of these peptides still contain the Met ⁴ -Glu ⁵ -His ⁶ -Phe ⁷ -Arg ⁸ -Trp ⁹ -Gly ¹⁰ sequence responsible for the effect (i.e., methionine). glutamic acid histidine phenylalanine arginine tryptophan glycine). Previous results have suggested that this heptapeptide is responsible for the effect in the hormones derived from proopio-melanocortin.

Examination of the structure-biological relationships of α-melanotropin and its analogs and fragments in the in vitro (extracellular) system on frog skin reveals that the portion responsible for the action of these compounds is common and is appropriately modified by Ac-Met-Glu-His -Phe-Arg-Trp-Gly-NHz structural element. This fragment (designated a-MSH4-io) was synthetically produced and tested for its ability to stimulate melanocyte function in the frog's skin. This fragment has been shown to be a weak agonist (having the same effect) as the natural, full hormone. However, prior to the present invention, the effect of (in the original numbering of α-melanotropin) a glutamic acid at position 5 of the peptide chain and a different amino acid at position 10 of glycine has not been studied. Previous studies (see U.S. Patent Nos. 4,457,864 and 4,485,039), which also serve as a basis for researching compounds of the present invention, have found that when the methionine is replaced by norleucine in the open-chain heptapeptide responsible for its activity, it is obtained. In addition, the exchange of phenylalanine at the 7-position of the peptide chain to the antipode (enantiomer) D-phenylalanine has led to a more biologically active analogue which has a time-delayed effect according to both of the bioassays used (frog skin and lizard skin). . Some results from these previous studies are summarized in the following table:

Biological activity of certain fragments of α-melanotropin as determined on skin of frog (Rana pipiens) and lizard (Anolis carolinensis)

PEPTID Relative efficiency frog lizard

a-MSH 1.0 1.0 Ac- [Nle ⁴ , D-Phe ⁷ ] -α-MSH 1 - 13 NH 2 60.0 5.0 Ac-a-MSH4-ioNH2 0.0003 0,004 Ac- (Nle ⁴ ) -α-MSH ⁴ -ioNH 2 0,002 0.06 Ac- [Nle ⁴ ] -α-MSH 4-1HN 2 0,002 1.0 Ac- [Nle ⁴ , D-Phe ⁷ ] -α-MSH 4 -10NH 2 0.02 10.0 Ac- [Nle ⁴ , D-Phe ⁷ ] -α-MSH 4 -1NH 2 0.16 8.0

See the list of examples above for the meaning of the abbreviations in the table above.

This examination of the structure-activity relationships yielded two important results:

1. methionine containing an oxidizable side chain to norleucine insensitive to oxidation. so the biological effect of the peptide fragment thus obtained, when replaced by one of the methionine isosteres, is about 10 times that of the original,

2. By incorporating D-phenylalanine at position 7 of the peptide chain in position 7 of the peptide chain and retaining lysine at position 11, the effect of α-melanotropin can be further enhanced.

In addition to open-chain analogs of α-melanotropin, certain fixed conformation analogues of α-melanotropin have been previously prepared and their biological activity investigated. The first such analogue tested was the compound represented by the formula [Cys ⁴ , Cys ¹⁰ ] -a-MSH 1 -13 NH 2 (i.e., analogs of α-melanotropin which are peptide chain 4 and

Containing 10 cysteine, these two cysteines are linked by oxidative disulfide bridges to form a ring structure), which has an effect of about 10-20 times that of the natural α-melanotropin on the skin of the frog, and measured about twice as much. These earlier cyclic analogs of α-melanotropin have been designed and constructed with the consideration that the middle portion of the portion responsible for the effect in α-melanotropin (His ⁶ -Phe ⁷ -Arg ⁸ -Trp ⁹ ) is a so-called "beta-nucleus". helical bend), and this conformational arrangement is essential for biological activity.

From these classical studies of structure-activity relationships of cyclic analogs of α-melanotropin, several conclusions can be drawn:

1. the activity of the melanocytes is increased by at least ten times as measured by the frog's skin and by at least two times the size of the lizard's skin by ring closure between position 4 (methionine) and position 10 (glycine) of the peptide chain,

2. incorporation of D-phenylalanine in position 7 of the phenylalanine in these cyclic analogs results in a doubled effect on the frog's skin and four times its effect on the lizard's skin,

3. analogs containing lysine at position 11 are always more potent than compounds containing other amino acids at this site, and

4. if analogs with higher or lower number of ring members are prepared instead of the 23-membered ring formed by the disulfide bridge, the biological activity of these compounds will be substantially reduced.

The results of these previous studies with these cyclic analogs of α-melanotropin are summarized in the following table:

Relative biological activity of cyclic analogs of α-melanotropin as determined by in vitro studies on frog skin and lizard skin

The peptide analog Efficiency frog F.A.Q Effect of amino acid substitution a-MSH 1.0 1.0 [Cys ⁴ , Cys ¹⁰ ] -α-MSH 1 - 13 NH 2 10.0 2.0 Ac- [Cys ⁴ , Cys ^{1 ()} ] -α-MSH 4 - 13 NH 2 30.0 0.6 3

HU 200480 Β

Peptide Analog Efficiency * Frog lizard

Effect of amino acid substitution

Ac- [Cys ^4, D-Phe ^7, Cys' °] -a-MSH4-i3NH2 6.0 6.0 Ac- [Cys ^4, Cys ^{* Ac 1} °] -a-MSH4-i NH _{2 2} 10.0 1.5

Ac- [Cys ⁴ , D-Phe ⁷ , Cys ¹⁰ ] -q-MSH 4 -12 NH 2 20.0 6.0 Ac- [Cys ⁴ , Cys ¹⁰ ] -α-MSH ⁴ -i NH 2 0.16 0.07

Ac- [Cys ^4, D-Phe ^{7, 1} ° Cys] q-i-1NH2 MSH4 2.5 3.0 Ac- [Cys ^4, Cys ^lo] -MSH4-ioNH2 0.06 0.003

Ac- [Cys ⁴ , D-Phe ⁷ , Cys ^lo ] -q-MSH 4 -10 NH ₂ 0.75 0.5

The peptide analog Efficiency* frog lizard

Effect of ring size ** Ac- [Cys ⁴ , Cys 10] -q-MSH 4 - 13 NH ₂ 30.0 0.60 [MpÉcCysj-q-MSHa-3NH2 30.0 1.0 [Earth ⁴ Cys ¹⁰ 10q-MSH ₄ -13NH ₂ 0.06 0.06 Ac- [Hcy ⁴ , Cys ⁴ ] -q-MSH ⁴ - 13 NH ₂ 0.06 0.70

= Biological activity versus q-melanotropin, linear dose response curve.

= Ring Size: Number of rings forming a ring formed by the peptide chain.

See the list of examples above for the meaning of abbreviations in the table above.

To further investigate the structure-activity relationships of the 4-10 fragments of q-melanotropin (q-MSHt-10), we investigated in detail the structural requirements for melanotropic activity in a series of previously unknown open-chain (q-MSHt-10) analog fragments. , in particular the role of the amino acids introduced at positions 5 and 10 of the peptide chain.

Subsequently, we investigated the fixed conformation of another type of q-melanotropin analogs with open-chain analogs of q-melanotropin. In short, 4-10 fragments of the analogue q-melanotropin that were

Ac- [Nle ⁴ -XXX ⁵ , D-Phe ⁷ , Yyy ¹⁰ ] -q-MSH 4 -10NH _{2 may} be represented by the following general formula:

Xxx is an amino acid, namely glutamic acid or aspartic acid, i.e. a monoaminodicarboxylic acid, and

Yyy is a basic amino acid such as lysine, omitin, 2,4-diaminobutyric acid or 2,3-diaminopropionic acid.

The open-chain analogs of q-melanotropin, characterized by the general formula Ac-q-MSH4-10 listed below, are prepared by solid phase peptide synthesis on a p-methylbenzhydrylamine-type resin as a support and the products are prepared by q -melanotropin and its analogs are purified by methods similar to those used previously.

THE; structure of α-melanotropin analogs

number

Primary sequence

First

Second

Third

4th

5th

6th

7th

8. 9.

10th

11th

12th

13th

14th

15th

2 3 4 5 6 7 8 9 10 11 12 13

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂ Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg- Trp-Lys-Gly-Pro-Val-NH ₂ Ac-Ser-Tyr-Ser-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH ₂

Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH ₂

Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH ₂

Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH ₂

Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-NH ₂

Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-NH ₂

Ac-Nle-Glu-His-D-Phe-Arg-Trp-Orn-NH ₂

Ac-Nle-Asp-His-D-Phe-Arg-Trp-Orn-NH ₂

Ac-Nle-Glu-His-D-Phe-Arg-Trp-Dab-NH ₂

Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dab-NH ₂

Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dpr-NH ₂

Ac-Nle-Glu-His-Phe-Arg-Trp-Lys-NH ₂

Ac-Nle-Asp-His-Phe-Arg-Trp-Lys-NH ₂

-4EN 200480 Β

ANALOGUE FORMULAS First a-MSH Second Ac- [Nle ^4, D-Phe ^7, Lys ^10, Gly ^1I] -a-MSHi i3NH ₂ Third Ac- [Nle ⁴ Asp ⁵ , D-Phe ⁷ .Lys ¹⁰ , Gly ^II ] -α-MSHi-13NH ₂ 4th Ac- [Nle ⁴ D-Phe ⁷ , Ly s ¹ °, Gly ¹¹ ] -OC-MSH 4-13NH ₂ 5th Ac- [Nle ⁴ . Asp- \ D-Phe ⁷ , Ly s ¹ °, Gly ¹ '] -a-MSH 4 -I 3 NH ₂ 6th Ac- [Nle ^4. Asp ^s. D-Phe ⁷ ] -α-MSH 4 -10 NH ₂ 7th Ac- [Nle ⁴ .D-Phe ⁷ , Lys ^lo ] -α-MSH 4 -10 NH ₂ 8th Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Lys ^lo ] -a-MSH 4 -oNH ₂ 9th Ac- [Nle ⁴ , D-Phe ⁷ , Orn ¹⁰ ] -α-MSH 4 -10 NH ₂ 10th Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Om ¹⁰ ] -α-MSH 4 -oNH ₂ 11th Ac- [Nle ⁴ D-Phe ⁷ , Dab ¹⁰ ] -α-MSH _{4 - 10} NH ₂ 12th Ac- [Nle ⁴ , Asp ⁵ , D-Phe ^7. Dab ^lo ] -a-MSH 4 -oNH ₂ 13th Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Dpr ¹ °] -α-MSH 4 -NH ₂ 14th Ac- [Nle ⁴ , Lys ¹ °] -α-MSH 4 -NH ₂ 15th Ac- [Nle ⁴ Asp ⁵ .Lys' °] -α-MSH 4 -10 NH ₂ The following abbreviations are used throughout this specification: α-MSH α-melanotropic hormone, α-melanotropin, α-melanocyte stimulating hormone Ac acetyl for CHO Boc a tert.-butoxycarbonyl group pMBHA p-methylbenzhydrylamine resin Bzl benzyl Z benzyloxycarbonyl group 2C1-Z 2-chloro-benzyloxycarbonyl group 2Br-Z 2-bromo-benzyloxycarbonyl group DCC dicyclohexylcarbodiimide HOBt 1-hydroxybenzotriazole DMF dimethylformamide N ^lm the nitrogen atom of the imidazole ring (in histidine) N ¹ nitrogen of the indole ring system (contained in tryptophan) NS nitrogen of the guanidino group (in arginine) Tos tosyl = p-toluenesulfonyl below alanine Asn asparagine asp aspartic acid Arg arginine Cys cysteine Dab 2,4-diaminobutyric acid Dpr 2,4-diaminopropionic acid Gin gl utam in Glu glutamic acid Gly glycine Hcy homocysteine His histidine Ile isoleucine Leu leucine Lys lysine Maa 2-mercaptoacetic acid mpa 2-mercaptopropionic acid Met methionine Nie norleucine Nva norvaline

HU 200480 Β

Orn ornithine Phe phenylalanine pCl-Phe p-chloro-phenylalanine Pro proline Ser serine Thr threonine Trp tryptophan Tyr tyrosine With valine

The preparation of open-chain analogue fragments of α-melanotropin in accordance with the present invention will now be illustrated by the following non-limiting examples. 15

Example 1

Ac- [Nie ⁴ , D-Phe ⁷ , Lys ^l0 , Cly ' ^! 3NH2

For the preparation of the title compound, first 2 g of p-methylbenzhydrylamine resin (containing 0.7 mmol of amino groups per gram) were tert-butoxycarbonyl on the a-amino group by solid phase peptide synthesis. Valine is coupled with a triple excess of amino acid. After one and a half hours, the resin was washed with dichloromethane, neutralized and the amino group was megacetylated with a mixture of acetic anhydride and pyridine. After a half-hour reaction time, the ninhydrin probe can no longer detect reactive amino groups on the resin.

Each amino acid is sequentially linked to the resin-linked peptide chain by the following procedure:

1. Wash the resin with 4 x 30 mL dichloromethane for 2-2 min.

2. cleaving the tert-butoxycarbonyl group twice with 48% trifluoroacetic acid in dichloromethane containing 2% anisole, the first 40 treatments lasting 5 minutes and the second 20 minutes,

3. Wash the resin with 4 x 30 mL dichloromethane for 2-2 min.

4. Neutralize the resin with 2 x 30 mL of 10% diisopropylethylamine in dichloromethane for 2-2 minutes.

5. wash the resin with 2 x 30 mL dichloromethane for 2-2 minutes,

6. Add to the resin a triple excess of t-butoxycarbonyl-protected amino acid (in this case 2.1 mmol) in 5 mL of dichloromethane, then add 2.4 mL of solution of N-hydroxybenzotriazole in dimethylformamide (except for the histidine protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the imidazole ring), and finally 2.4 ml of a 1 mM solution of dicyclohexylcarbodiimide in dimethylformamide.

The mixture is then shaken for 2-3 hours (dimethylformamide used as coupling solvent for tryptophan, arginine and histidine),

7. After coupling (when the ninhydrin test gives a negative result), the resin is washed with dichloromethane (30 ml) three times for 2-2 minutes, 65 tert-butoxycarbonyl tert-butoxycarbonyl tert-butoxycarbonyl8. the resin was washed with 3 mL of 100% ethanol for 2 minutes and finally

9. Wash the resin with 4 x 30 mL dichloromethane for 1-1 min.

The title peptide is prepared in protected and resin-bound form by linking the following suitably protected amino acids to the resin-bound and deprotected valine stepwise (one by one) in the following order:

- proline protected by a-amino group,

- glycine protected by the a-amino group,

- on the α-amino group

and lysine protected by a 2-chlorobenzyloxycarbonyl group on the epsilon amino group,

- tryptophan protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the indole ring system,

- arginine protected on the α-amino group by the tert-butoxycarbonyl group and on the guanidino group by the p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group, and

- histidine protected on the α-amino group by the tert-butoxycarbonyl group and on the nitrogen atom of the imidazole ring by the p-toluenesulfonyl group.

The resulting Boc-His (N ^im- Tos) -D-Phe-Arg (NS-Tos) -Trp (N'-For) -Lys (N ^E-2 Cl-Z) -Gly-Pro-VaI-p The protected peptide chain resin of the formula -MBHA is divided into four portions.

Then, to one portion of 1.0 g (about 0.5 mmol), the following protected amino acids are coupled:

- gamma-benzyl ester of glutamic acid protected at the α-amino group with a tert-butoxycarbonyl group,

- norleucine protected on the α-amino group by the tertiary butoxycarbonyl group,

- serine-O-benzyl ether protected on the α-amino group by a tert-butoxycarbonyl group,

- tyrosine protected on the α-amino group by the tertiary butoxycarbonyl group and on the phenolic hydroxyl group by the 2-bromobenzyloxycarbonyl group, and

- a serine-O-benzyl ether protected on the α-amino group by a tert-butoxycarbonyl group.

After coupling with the last amino acid, the t-butoxycarbonyl group is cleaved from the α-amino group, the resulting compound is neutralized and the protected, resin-bound peptide is dissolved in 20 ml of dichloromethane with 10-fold excess of N-acetylimidazole. megacetylation at the α-amino group. That's how it got. the resin bearing the protected peptide chain is dried under reduced pressure (1.0 g). The protected pep-61

HU 200480 Β is cleaved from the resin using liquid hydrofluoric acid. After cleavage, the volatiles were removed by distillation under reduced pressure at 0 DEG C., the residue was dried, washed three times with 3 ml of diethyl ether, three times with 30 ml of 30% aqueous acetic acid and freeze-dried. The 530 mg peptide thus obtained as a powdery substance was divided into two portions weighing 260 mg each. One portion was dissolved in 1.5 ml of ammonium acetate buffer pH 4.5, filtered through a pad and applied to the top of a 2.0 x 30.0 cm column filled with carboxylmethyl cellulose. The column was eluted with 250-250 mL of 0.01 M (pH 4.5), 0.1 M (pH 6.8) and 0.2 M ammonium acetate (pH 6.8). The major product is detected at 280 nm, which is between the end of the 0.1 M ammonium acetate solution and the first half of the 0.2 M ammonium acetate solution. These fractions were freeze-dried to give 142.3 mg of a white powder. A portion of this peptide (80.0 mg) was purified by preparative high performance liquid chromatography. The fractions containing the main component were combined and freeze-dried to give 63 mg of the title peptide.

[?] 546 ²³ = -51.6 ° (c = 0.31, 10% acetic acid), Rp = 0.34 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 2

Ac- [Nle ⁴ As 1 D-Phe ⁷ Jys ^{, 0} , Gly ^{, 1} ] -α-MSH 1 - 13 NH 2 The title compound is prepared by

1.0 2 (about 0.5 mmol)

N ^a -Boc-His (N ^im -Tos) -D-Phe-Arg (N? -Tos) -Trp (N'-For) -Lys (N ^£ -2-ClZ) -Gly-Pro-Val-p -The following amino acids are coupled to the resin-bound protected peptide of the formula -MBHA by the stepwise method:

- N-benzyl ester of aspartic acid protected on the α-amino group by tert-butoxycarbonyl,

- norleucine protected on the α-amino group by the tertiary butoxycarbonyl group,

- serine-O-benzyl ether protected on the α-amino group by a tert-butoxycarbonyl group,

- tyrosine protected on the α-amino group by the tertiary butoxycarbonyl group and on the phenolic hydroxyl group by the 2-bromobenzyloxycarbonyl group, and

- a serine-O-benzyl ether protected on the α-amino group by a tert-butoxycarbonyl group.

Coupling with all amino acids is carried out as described above. The resin-bound protected tridecapeptide, after cleavage and neutralization of the tert-butoxycarbonyl group at the N-terminus, is acetylated in dichloromethane with ten times the excess of N-acetylimidazole (reaction time: 5 hours). The product was dried under reduced pressure to give 1.8 g

Ac-Ser (O-Bzl) -Tyr (O-2-BrZ) -Ser (O-Bzl) -Nle-Asp (P-Bzl) -His (N'-Tos) -D-Phe-Arg- (NS -Tos) -Trp (N ¹ -For) -Lys (N ² -2 -Cl 2) -Gly-Pro-Val-p-MBHA resin bearing the protected peptide chain is obtained.

1.0 g of the protected peptide chain resin thus obtained is treated with liquid hydrofluoric acid and the volatiles are distilled off. the residue was washed three times with 30 ml of diethyl ether, the peptide was extracted three times with 30 ml of 30% aqueous acetic acid, and the solution was freeze-dried. A 200 mg portion of the crude tridecapeptide was dissolved in 1.5 mL of ammonium acetate buffer, pH 4.5, filtered through a pad and applied to a 2.0 x 30.0 cm column filled with carboxymethylcellulose. Top. The column was eluted with ammonium acetate as described in Example 1, using a stepwise gradient elution. In this way, the corresponding fractions were freeze-dried to isolate 152 mg of the peptide. A 100 mg portion of this crude peptide was subjected to high performance liquid chromatography! further purification afforded 67 mg of the title peptide.

[α] 546 - -52.3 ° (c = O, 33, 10% acetic acid), Rf = O, 32 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)]

Example 3

The title peptide of Ac- [Nle ⁴ , D-Phe ⁷ .Lys ¹⁰ , Gly ¹¹ ] -α-MSH 4 - / 3NH 2 was prepared by the solid phase method so as to obtain 1.0 g (0.5 mmol).

Boc-His (N ^im -Tos) -D-Phe-Arg (NS-Tos) -Trp (N * -for) -Lys (N ^E -2-ClZ) -Gly-resin of formula Pro-Val-p-MBHA The stepwise method is followed by the coupling of a gamma-benzyl ester of glutamic acid protected on the α-amino group with a tert-butoxycarbonyl group followed by a tert-butoxycarbonyl group on the α-amino group. The coupling with both amino acids was carried out as described in the examples above, except that the N-terminus was acetylated with twice the excess of a 1: 1 mixture of acetic anhydride and pyridine in dichloromethane for 1 hour. Peptide 4 was purified in the same manner as Example 2 to give a white powder in 22% yield.

[a] 546 ²³ = -49; 14 ° (c = 0.35, 10% acetic acid)

Rp = 0.43 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 4

Ac- [Nle ⁴ Asp ⁵ , D-Phe ⁷ d.ys ¹¹⁾ .Gly ^{, l} ] -a.-MSH 4 -13NH 2

The title peptide was prepared by 1.0 g (about 0.5 mmol), prepared as described in Example 1.

Boc-His (N ' ^m -Tos) -D-Phe-Arg (N 8 -Tos) -Trp (N ⁱ -For) -Lys- (N ^E -2 -Cl 2) -Gly-Pro-Val-p-MBHA The resin-bound protected peptide of formula (II) is first stepwise coupled with a t-butoxycarbonyl-protected aspartic acid p-benzyl ester on its α-amino group followed by tert-butoxycarbonyl protected on its α-amino group. . The resulting resin-bound protected peptide was processed as described in Example 1. The crude product was first chromatographed on carboxymethylcellulose as in Example 1. and the resulting product (130 mg) was further purified by high performance liquid chromatography. This gives 53 mg of pure peptide.

[?] 546 ²³ = -50.0 ° (c = 0.32, 10% acetic acid)

R f = 0.41 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 5

Ac-1Nle ⁴ .D-Phe ⁷ , Lys 1f ⁾ 1α-MSH 4 -10NH 2

2.7 g of p-methylbenzhydrylamine resin (containing 0.7 mmol of amino groups per gram)

The mixture is suspended in dichloromethane and washed three times with 30 ml of dichloromethane each time. The washed resin was shaken with dichloromethane for 2 hours and filtered. The p-methylbenzhydrylamine-type resin thus swollen is neutralized and the following amino acids are coupled, as described in Example 1, in the order given herein;

- D-lysine protected on the α-amino group by the tertiary butoxycarbonyl group and on the ε-amino group by the 2-chlorobenzyloxycarbonyl group,

- tryptophan protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the indole ring system,

- arginine protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the guanidino group by the p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group,

- histidine protected on the α-amino group by the tert-butoxycarbonyl group and on the nitrogen atom of the imidazole ring by the p-toluenesulfonyl group.

The father also received

Boc-His (N ^im -Tos) -D-Phe-Arg (N 1 -Tos) -Trp (N ¹ -For) -Lys (Ne-2-ClZ) -p-MBHA resin bearing the protected peptide chain in two portions we share. To one of the moieties is added first glutamic acid gamma-benzyl ester protected on the α-amino group and then on norleucine protected on the α-amino group by tert-butoxycarbonyl. The resulting N-terminus of the resin-bound peptide is deprotected and the free N-terminus is megacetylated in dichloromethane with twice the excess of a 1: 1 mixture of acetic anhydride and pyridine. The resulting resin bearing the protected peptide chain was washed with dichloromethane and dried under reduced pressure to give 2.1 g of product. A portion of the protected peptide resin, weighing 1.7 g, contains 17 ml of anhydrous liquid hydrofluoric acid, 2 ml of anisole and 1 ml

Treat with 1,2-ethanedithiol. The volatiles were evaporated, the residue was dried, washed three times with 30 ml of anhydrous diethyl ether and extracted three times with 30 ml of 30% aqueous acetic acid each time. The aqueous extract of the peptide was freeze-dried to give 700 mg of crude peptide. A sample of this crude peptide (300 mg) was dissolved in 2 mL of ammonium acetate buffer (pH 4.5) and filtered through a pad on top of a column filled with carboxymethylcellulose. The fractions containing the main product were combined and freeze-dried to give 172 mg of the peptide as a white powder. The resulting peptide (110 mg) was further purified by high performance liquid chromatography to give 50 mg of the pure title peptide.

[α] 546 ²³ = -50.0 ° (c = 0.12, 10% acetic acid),

Ri-0.35 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 6

Ac-INIc ⁴ .Axi> \ D-Phe ⁷ .Lys ^lo ] -a-MSH4-loNH2 The title compound is prepared in a resin-bound protected form by 1.8 g

Boc-His (N ^'m -Tos) -D-Phe-Arg (NS-Tos) -Trp (N-For) -Lys ^(N? -2-ClZ) -p-MBHA formula, the peptide resin was stepwise method coupling, first, the p-benzyl ester of aspartic acid protected on the α-amino group with tert-butoxycarbonyl and then on the α-amino group protected with tert-butoxycarbonyl. Each linkage is as described above, with a triple excess of the t-butoxycarbonyl protected amino acid (or derivative) on the α-amino group, 2.4 times the excess of dicyclohexylcarbodiimide, and 2,4-dimethyl-1-hydroxybenzotriazole. times the excess. After attachment of the latter amino acid, the α-amino protecting group, i. E. . So it got,

Ac-Nle-Asp (P-BzL) -His (N ^im -Tos) -D-Phe-Arg- (Ng-Tos) -Trp (N'-For) -Lys (N ^e- 2-Cl 2) -p The resin bearing the protected peptide -MHBA was washed with dichloromethane and dried under reduced pressure to give 2.1 g. A 1.5 g portion of this protected peptide resin was treated with liquid hydrofluoric acid and subsequently

Work up as described in Example 5. Chromatography on carboxymethylcellulose afforded 112 mg of product, which was further purified by high performance liquid chromatography. This gives 64 mg of pure peptide.

[α] ²⁵ D ²³ = -28.0 ° (c = H, 05, 10% acetic acid),

R f = 0.36 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 7

Ac- [Nle ⁴ , Asp ⁵ , D-Phc ^7. Dah ^lri ] -a.-MSH 4 -oNH 2

The protected, resin-bound form of the title compound was prepared as described in Example 5, except that it used a tert-butoxycarbonyl group on the a-amino group and a 2-group on the epsilon-amino group. lysine protected by chlorobenzyloxycarbonyl and glutamic acid gamma-benzyl protected on the α-amino group by tert-butoxycarbonyl instead of ester on the α-amino group and, on the gamma amino group, 2,4-diaminobutyric acid protected with a benzyloxycarbonyl group and a benzyl ester of aspartic acid protected with a t-butoxycarbonyl group on the α-amino group. In this way, the

Ac-Nle-Asp (p-Bzl) -His (N ' ^m -Tos) -D-Phe-Arg (NS-Tos) -Trp- (Ni-For) -Dab (NS ^amma -Z) -p-MBHA yields a protected peptide resin of formula IIa. This product was cleaved as described in Example 5 and further processed to give peptide 12 as a white powder in 36% yield.

[aw ³ = -32.7 ° (c = 0:11, 10% acetic acid);

R f = 0.43 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

HU 200480 Β

Example 8

Ac- [Nle @ ^4, Asp \ D-Phc ', Dpr ^0] -a-MSH4-ioNH2

The protected, resin-bound form of the title compound was prepared as described in Example 5, except that it used a tert-butoxycarbonyl group on the a-amino group and a 2-group on the epsilon-amino group. instead of lysine protected by a chlorobenzyloxycarbonyl group, 2,3-diaminopropionic acid protected on the α-amino group by a tertiary butoxycarbonyl group and on the β-amino group by a benzyloxycarbonyl group in this example; instead of the t-butoxycarbonyl-gamma-benzyl ester protected on the α-amino group in Example 5, the aspartic acid on the α-amino group on the α-amino group is protected in the present example by ester. In this way it is

Ac-Nle-Asp (3-Bzl) -His (N ' ^m -Tos) -D-Phe-Arg- (N-Tos) -Trp (N'-For) -Dpr (NP-Z) -p- A peptide-bearing resin of the formula MBHA is obtained. The peptide is then cleaved from the resin, hereinafter referred to as

Work up as described in Example 5. In this way, the desired peptide was obtained in 36% yield as a white powder.

[α] 546 ²³ = -53.7 ° (c = 0.03, 10% acetic acid),

R f = 0.41 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 9

Ac- [Nle ⁴ , Lys ^lo ] -α-MSH 4 -10NH 2

The protected, resin-bound form of the title compound was prepared as described in Example 5, except that the D-phenylalanine protected on the α-amino group protected by the t-butoxycarbonyl group was used in this example. the α-amino group used is phenyl-allyl protected by a tert-butoxycarbonyl group. In this way it is

Ac-Nle-Glu (gamma-Bzl) -His (N ^im -Tos) -Phe-Arg (NS-Tos) -Tip (N'-For) -Lys (N ^£ -2-ClZ) -p-MBHA The peptide is then cleaved and further processed to give the title compound in 40% yield.

[α] 54 D ²³ = -25.0 ° (c = 0.02, 10% acetic acid),

Rp = 0.34 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Example 10

Ac-INLc ⁴ AstP .Lys ^{, ()} ] -a-MSH4-0.0NH2 The protected, resin-bound form of the title compound was prepared as described in Example 5, except that the α Instead of D-phenylalanine protected on t-butoxycarbonyl group on the amino-amino group, t-butoxycarbonyl-protected phenylalanine is used on the alpha-amino group in this example and the a- Instead of the glutamic acid gamma-benzyl ester protected on the amino group with tert-butoxycarbonyl, the t-butoxycarbonyl-protected aspartic acid benzyl ester on the α-amino group is used in this example. In this way it is

Ac-Nle-Asp (-Bzl) -Phe-Arg (NS-Tos) -Trp (N'-For) -Lys (N ^t '-2-ClZ) -p-MBHA has a protected peptide chain-bearing resin. The peptide is then cleaved from the resin, deprotected and the title compound purified as described above. This gave the product as a white powder in 37% yield.

[α] 546 ²³ = -24.6 ° (c = 0.16, 10% acetic acid),

R f = 0.32 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

The biological activity of the open-chain analogs prepared as described in the examples above is summarized in the table below, with "P" in the table representing a prolonged effect ("+" = yes, = no) and "NV" being sample was not tested.

analog number Biological effect Residual effect Frog F.A.Q Frog F.A.Q First 1.0 1.0 PH p (-) Second 6.0 8.0 P (+) P (-) Third 9.0 8.0 p (+) PH 4th 0.8 8.0 p (+) P (+) 5th 1.0 10.0 p (+) P (+) 6th 0.1 8.0 P (-) p (-) 7th 0.2 8.0 p (-) p (-) 8th 0.7 8.0 p (-) P (-) 9th 0.6 8.0 P (-) PH 10th 0.9 10.0 NV P (-) 11th 0.9 10.0 P (±) P (±) 12th 0.9 50.0 P (-) P (-) 13th 0.2 8.0 p H P (-) 14th 0,001 0.08 p (+) P (-)  15th 0,004 0.08 p (+) PH

Based on the results obtained with α-melanotropin fixed conformation, open-chain analogs, we investigated a number of other types of α-melanotropin cyclic analogs, also of fixed conformation.

Cyclic peptide analogs of melanotropin are prepared by conventional methods known in the art for solid phase peptide synthesis. In summary, a t-butoxycarbonyl-protected amino acid or a derivative thereof, a tert-butoxycarbonyl-protected amino acid, is sequentially coupled to a p-methylbenzhydrylamine-type resin. used in triple excess and as an excipient

A 2.4-fold excess of N-hydroxybenzotriazole in the form of a 1 mM solution in dimethylformamide (except histidine) and a 2.4-fold excess of 1 mM in dicyclohexylcarbodiimide in dimethylformamide are used. . The coupling reactions are carried out in dichloromethane for a reaction time of 1 to 3 hours and the reaction is monitored by the ninhydrin and / or chloroanilic assay. If necessary, the coupling reactions are repeated.

The reactive groups in the amino acid side chain are protected with the following protecting groups:

- the epsilon-amino group of lysine with 2,4-dichlorobenzyloxycarbonyl;

- ornithine. The β-amino group of 2,4-diaminobutyric acid and 2,3-diaminopropionic acid with a benzyloxycarbonyl group;

-9HU 200480 Β

- the nitrogen of the indole ring system of tryptophan with a formyl group;

- the nitrogen of the guanidino group of arginine with p-toluenesulfonyl;

- the nitrogen atom of the imidazole ring of histidine with ß-p-toluenesulfonyl; and

- the gamma-carboxyl group of glutamic acid and the β-carboxyl group of aspartic acid in the form of the benzyl ester.

The tert-butoxycarbonyl group, as a protecting group for the amino group, was removed by treatment twice with a 48% solution of trifluoroacetic acid in dichloromethane containing 2% anisole. minutes and the second takes 20 minutes. 1

Each amino acid is sequentially linked to the resin-bound peptide chain by the following procedure:

1. Wash the resin with 4 x 30 mL dichloromethane for 1-1 2 min.

2. cleaving the tert-butoxycarbonyl group twice with 48% trifluoroacetic acid in dichloromethane containing 2% anisole, first 5 minutes, second 20 minutes, 2

3. the resin is washed twice with 30 ml of dichloromethane,

4. Neutralize the resin with 2 x 30 mL of 10% diisopropylethylamine in dichloromethane for 3 to 3 minutes.

5. wash the resin three times with 30 ml of dichloromethane for 2 to 2 minutes,

6. Add to the resin a solution of the tert-butoxycarbonyl protected amino acid derivative in 20 ml dichloromethane (except tryptophan, arginine and histidine, in which case, due to solubility problems, dimethyl is substituted for dichloromethane). formamide), then 1-hydroxybenzotriazole is added followed by dicyclohexylcarbodiimide and the resulting mixture is shaken for 4 to 3 hours,

7. The resin is washed three times with 30 ml of dichloromethane for 2-2 minutes and the resin is washed three times with 30 ml of 100% ethanol for 2-2 minutes.

The progress of the coupling reaction is followed as described above and the tert-butoxycarbonyl group attached to the α-amino group is cleaved after attachment of the last amino acid. The amino group is then neutralized and megacetylated with ten times the excess of N-acetylimidazole in dichloromethane or twice in a 1: 1 mixture of acetic anhydride and pyridine (reaction time: 1 hour).

For deprotection and cleavage of the peptide chain from the resin, the resin bearing the protected peptide chain is treated at 0 ° C for 45 minutes with 5 mixtures of 82% anhydrous liquid hydrofluoric acid, 10% anisole and 8% 1,2-ethane. It is composed of dithiol (10 ml of this mixture are used for 1 g of resin). After completion of the reaction, the volatiles were distilled off under reduced pressure, the free peptide was washed three times with 30 mL of diethyl ether or ethyl acetate, three times with 30 mL of 30% aqueous acetic acid, and then three times with 30 mL of distilled water. The combined aqueous extracts were freeze-dried to give the crude peptide as a white powder. The peptides thus obtained were purified by column chromatography on a cation-exchange carboxymethylcellulose, eluting with a gradual gradient of 250 ml of 0.01 M ammonium acetate (pH 4.5), followed by 250 ml of 0. 01 M ammonium acetate solution (pH 6.8), then 250 ml of 0.1 M ammonium acetate solution (pH 6.8), and finally 250 ml of 0.2 M ammonium acetate solution (pH 6.8). = 6.8). The main product (followed at 280 nm) is the last portion of a 0.01 M solution of 5M ammonium acetate (pH 6.8) and the first half of a 0.1 M ammonium acetate solution (pH 6.8). between the two. The appropriate fractions were freeze-dried to give the purified peptide as a white powder.

D The cyclic analogs of α-melanotropin of the present invention are summarized in the following tables:

Structure of α-melanotropin and its cyclic analogs

SERIAL Primer Sequence 16th 1 2 3 4 5 6 7 8 9 10 11 12 13 Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 17th Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NHA 18th Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-NH2 19th Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-NH2 20th Ac-Nle-Asp-His-D-Phe-Arg-Trp-Orn-NH2 21st Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dab-NH 2 22nd Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dpr-NH2

-10HU 200480 Β

SERIAL Analog formula 16th alpha-MSH 17th Ac- [Nle ⁴ , Glu ⁵ , D-Phe ⁷ , Lys ¹⁰ , Gly ^II ] -alpha-MSH 4 - 13 NH ₂ 18th Ac- [Nle ⁴ , Glu ⁵ , D-Phe ⁷ , Lys ^lo ] -alpha-MSH ₄ - 10NH ₂ 19th Ac- [Nle ^{4, 5-Asp,} D-Phe ^7, L} s ^lo] -alpha-MSH4-ioNH ₂ 20th Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Om ¹⁰ ] -alpha-MSH 4 -oNH ₂ 21st Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Dab ^lo ] -alpha-MSH 4 -oNH ₂ 22nd Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Drp ^lo ] -alpha-MSH 4-loNH ₂

The preparation of the cyclic analogue fragments of α-melanotropin according to the present invention is further illustrated by the following examples, without limiting the scope of the invention.

Example 11 |

Ac- [NLe ⁴ , Ghi ⁵ , D-Phe ⁷ Eys ^{1 (l.} Gly ^tl -MSH 4 - 13 NH 2

The protected, resin-bound form of the title compound was prepared in 1.0 g

The N -Boc-Val-p-MBHA resin, i.e., a p-methylbenzhydrylamine resin bearing a tert-butoxycarbonyl protected on the α-amino group of 0.7 mmol / g, It contains a t-butoxycarbonyl-protected valine on the α-amino group, and is linked stepwise to the following amino acid protected on the α-amino group by a tert-butoxycarbonyl group:

tert-butoxycarbonyl35 proline protected by t-butoxycarbonyl-tert-butoxycarbonyl-a-amino group,

- glycine protected by the a-amino group,

- on the α-amino group

and lysine protected by a 2,4-dichlorobenzyloxycarbonyl group on the epsilon-amino group,

- tryptophan protected on the α-amino group by a tertiary butoxycarbonyl group and a formyl group on the nitrogen of the indole ring system,

- arginine protected on the α-amino group by the tert-butoxycarbonyl group and on the guanidino group by the p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group,

- histidine protected on the α-amino group by a tert-butoxycarbonyl group and a p-toluenesulfonyl group on the nitrogen atom of the imidazole ring,

- gamma-benzyl ester of glutamic acid protected on the α-amino group by tert-butoxycarbonyl, and

- norleucine protected on the α-amino group by a tertiary butoxycarbonyl group.

After attachment of the last amino acid, the t-butoxycarbonyl group attached to the α-amino group is cleaved and the amino group is neutralized. followed by a 10-fold excess of N-acetylimidazole in dichloromethane (reaction time: 6-8 hours) or also in dichloromethane. The mixture was megacetylated twice with a 1: 1 mixture of acetic anhydride and pyridine (reaction time: 1-2 hours). In this way, Article 65

Ac-Nle-Glu (gamma-Bzl) -His (N ^im -Tos) -D-Phe-Arg (NS-Tos) -Trp (N ⁱ -For) -Lys (N ^e -2.4-ClZ) - Gly-Pro-Val-p-MBHA resin bearing the protected peptide is obtained. A portion of this product (1.0 g, 0.6 mmol) was treated with 10 mL of anhydrous hydrofluoric acid at 0 ° C for 45 minutes in the presence of 1 mL of anisole and 0.8 mL of 1,2-ethanedithiol. The hydrofluoric acid, anisole and 1,2-ethanedithiol were distilled off under reduced pressure, and the dried product was washed three times with 30 ml of diethyl ether and extracted three times with 30 ml of 30% acetic acid each. The aqueous extract of the peptide was freeze-dried to give 325 mg of a crude white powder.

Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH ₂ is obtained.

A portion of the crude Ac- [Nle, D-Phe ⁷ , Lys ¹⁰ , Gly ¹¹ ] -α-MSH 4-13NH 2 analogue was purified in a 150 mg portion. For this purpose, this sample of the crude peptide was dissolved in 2-4 ml of 0.01 M ammonium acetate pH 4.5 and chromatographed on a 2.0 x 25.0 cm column filled with carboxymethylcellulose. , with gradient gradient elution. As eluent, first 250 ml of 0.01 M (pH 4.5), then 250 ml of 0.01 M (pH 6.8) and finally 250 ml of 0.2 M ammonium acetate (pH 6.8) solution. The main product is monitored by measuring at 280 nm, the product coming off the column with the first half of 0.1 M ammonium acetate buffer (pH 6.8). The appropriate reactions were freeze-dried to give 104 mg of a white powder. Chromatography on carboxymethylcellulose showed that the pure Ac- [NLe ⁴ , D-Phe ⁷ , Lys ¹⁰ , Gly ¹¹ ] -a-MSH 4-13NH 2 peptide was further purified by high performance liquid chromatography using a 25 cm x 25 A semi-preparative column packed with Vydac 218TP15-16 CisRP reverse phase silica gel (0.1 mm) was used, eluting with 0.1% trifluoroacetic acid buffer and acetonitrile. 100 mg peptide purified by high performance liquid chromatography 74 mg pure,

Ac- [Nle ⁴ , D-Phe ⁷ , Lys ¹⁰ °, Gly ¹¹ ] -a-MSH 4 -I 3NH 2 are obtained.

A 40 mg sample of pure Ac- [Nle ⁴ , D-Phe ⁷ , Lys ¹⁰ .Gly ^II ] -α-MSH 4 -13 was dissolved in 1 ml 5%. aqueous hydrochloric acid, and the solution was chromatographed on a 1.0 x 15.0 cm column packed with diethylaminoethylcellulose hydrochloride, eluting with 100 mL of 5% aqueous hydrochloric acid, and

-11GB 200480 Β at 280 nm. Fractions containing the peptide were combined and freeze dried to give 35 mg of Ac- [Nle ⁴ , DPhe ⁷ , Lys ¹⁰ , Gly ^II ] -α-MSH 4 - 13 NH 2 .HCl.

This peptide salt was dissolved in 3 ml of anhydrous and secondary amine-free dimethylformamide (distilled from ninhydrin under reduced pressure), anhydrous dipotassium hydrogen phosphate was added to the solution, and the reaction mixture was cooled to 0 ° C in a salt bath. azide. The reaction mixture is then stirred at 0 'C and the vessel containing the mixture is transferred to a 12' C cold room. Here, the reaction mixture is stirred overnight at 12 ° C, and the progress of the reaction is monitored by high performance liquid chromatography. For this purpose, a 25.0 cm x 4.6 mm Vydac column was used, eluting with 0.1% trifluoroacetic acid and acetonitrile.

The ring closure is also monitored by the ninhydrin probe. That's how it got

Ac- [Nle ⁴ , Glu ⁵ , D-Phe ⁷ , Lys' ° Gly ⁿ ] -a-MSH 4-13 NH 2 is purified after the reaction is quenched with 10% aqueous acetic acid. The mixture was desalted on a P4-polyacrylamide column (80.0 cm x 1.0 cm), eluted with 30% acetic acid, and purified by semi-preparative HPLC. In this way, 16 mg

Ac-[Nle ⁴ , Glu ⁵ , D-Phe ⁷ , Lys ¹⁰ , Gly ¹ 'and-MSfU-isNHi cyclic peptide are obtained.

[α] D 259 = -57.8 ° (c = 0.045, 10% acetic acid),

Rf = 0.79 [1-butanol / acetic acid / pyridine / water (15: 3: 10: 12 tf)].

Example 72 _

Ac- [Nle ⁴ .GhiÁD ^{Phe-7, l0} Lys] -alpha-MSH -i _{0 4} NH2

The title compound was 2.0 g

N ^a -Boc-Lys- (N ^e -2,4-Cl 2 Z) -p-MBHA, i.e., t-butoxycarbonyl on the α-amino group and 2,4- on the epsilon-amino group. lysine bearing a dichlorobenzyloxycarbonyl protected p-methylbenzhydrylamine resin, 1.0 mmol / g, with a t-butoxycarbonyl group on the a-amino group and 2.4 on the epsilon-amino group. containing lysine protected by a dichlorobenzyloxycarbonyl group.

The title compound is prepared in the form of a protected and resin bound peptide by stepwise addition of the following amino acids protected by a-amino tert-butoxycarbonyl group in the following order:

- on the α-amino group with a tert-butoxycarbonyl group. and tryptophan protected with a formyl group on the nitrogen of the indole ring system,

- on the α-amino group with a tert-butoxycarbonyl group. and arginine protected on the guanidino group with a p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group, and

- histidine protected on the α-amino group by tert-butoxycarbonyl and on the nitrogen atom of the imidazole ring by p-toluenesulfonyl.

So it got,

Ac-His (N ^im- Tos) -D-Phe-Ara (NS-Tos) -Trp (N'-For) -Lys (N ^e -2.4-Cl ₂ Z) -p-MBHA protected peptide chain carrier resin is divided into two parts. To a portion of the resin bearing the protected pentapeptide (1.4 g, 0.5 mmol) is coupled, first, a t-butoxycarbonyl-gamma-benzyl ester protected on the α-amino group and then on the α-amino group. norleucine protected by a tert-butoxycarbonyl group. This affords the protected, resin-bound form of the title peptide. After attachment of the last amino acid, the t-butoxycarbonyl protecting group for the α-amino group is cleaved, the amino group is neutralized and then all. megacetylated as described in Example 1b. In this way it is

Ac-Nle-Glu (gamma-Bzl) -His (N ^im -Tos) -D-Phe-Arg (N 2 -Tos) -Trp (N'-For) -Lys (N ^e -2.4-Cl 2 Z) - A resin bearing a protected peptide chain of formula p-MBHA is obtained. A 1.0 g portion of this protected peptide resin is deprotected with liquid hydrofluoric acid, and the peptide chain is cleaved from the resin and the product obtained. .

This gave 356 mg of crude product as a white powder

Ac- [Nle ⁴ , D-Phe ⁷ , Ly ¹ °] -a-MSHa-10NH 2 is obtained. A 100.0 mg portion of this crude peptide was purified by the methods described in Example 11 to give 65 mg pure by high performance liquid chromatography.

Ac- [Nle ⁴ , D-Phe ⁷ .Lys ^to ] -a-MSH 4 -10NH 2 is obtained.

A 40 mg portion of this pure peptide was ring-sealed as described in Example 11 to give 13 mg pure by HPLC.

Ac- [Nle ⁴ , Glu ⁵ , D-Phe ⁷ , Lys 10] -a-MSH 4 -oNH 2 are obtained.

[ot] 598 "= -13.3 '(c = 0.07, 10% acetic acid),

Rp = 0.89 [butanol / acetic acid / water (4: 1: 5 v / v, upper phase)].

Example 13 _

Ac-1Nle ⁴ Asp ⁵ d) -Phe ⁷ d.ys ^{, o} ] -a-MSH 4 -] oNH 2

The title compound was prepared in protected and resin-bound form by 1.4 g (0.5 mmol)

Boc-His (N ' ^m -Tos) -D-Phe-Arg (NS-Tos) -Trp (N'-For) -Lys- (N ^e -2.4-Cl ₂ Z) -p-MBHA Stepwise, first the t-butoxycarbonyl-protected aspartic acid P-benzyl ester on the α-amino group is coupled, and then the norleucine protected on the α-amino group by tert-butoxycarbonyl. Both couplings are performed using the same procedure described above for solid phase peptide synthesis in general. After attachment of the last amino acid, the resulting product is cleaved to yield the α-amino protecting tert-butoxycarbonyl group, the amino group

The reaction mixture was neutralized and the resulting compound was all. megacetylated as described in Example 1b. In this way it is

Ac-Nle-Asp (P-Bzl) -His (N ^im -Tos) -D-Phe-Arg (Ng-Tos) -Trp (N'-For) -Lys (N ^e -2.4-CbZ) - A resin bearing a protected peptide chain of formula p-MBHA is obtained. This peptide chain-bearing resin was dried under reduced pressure, then a 1.0 g sample was subjected to a cleavage reaction and the product was processed as described in Example 11. This gave 370 mg crude

Ac- [Nle @ ^4, Asp ^'i, D-Phe ^7, Lys ^lo] -a-MSH4-ioNH2 formula product. A portion of the crude heptapeptide weighs 110 mg. The product was purified by the methods described in Example 1 to afford 82 mg of the title compound as an open powder version of the title peptide as a white powder.

A 40.0 mg portion of Ac- [Nle ⁴ , Asp +, D-Phe ⁷ , Lys ^lo ] -α-MSH 4 -oNH 2 is subjected to a ring closure reaction and the product is purified by high performance liquid chromatography. In this way, 12 mg is pure

Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Lys ^lo ] -a-MSH 4 -oNH 2 are obtained.

[a] 598 ²² = -54.2 ° (c = 0.07, 10% acetic acid);

Rf = 0.73 (1-butanol / acetic acid / pyridine / water (15: 3: 10: 12 tf)) ·

Example 14 -Ac- [Nle ⁴ Asp ⁵ , D-Phe ⁷ , Orn ¹ °] -a-MSH 4-loNH 2

To 1.0 g of p-methylbenzhydrylamine resin (containing 0.7 mmol of amino groups per gram) is coupled to the α-amino group by tert-butoxycarbonyl and the gamma- omitin protected at the amino group with a benzyloxycarbonyl group. After 1 hour, the coupling reaction is stopped, the resin is washed, neutralized, and the free amino groups of the resin are megacetylated in dichloromethane with twice the excess of a 1: 1 mixture of acetic anhydride and pyridine, for 1 hour. The following amino acids are then added to the compound thus obtained in the following order, stepwise:

- tryptophan protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the indole ring system,

- arginine protected on the α-amino group by the tertiary butoxycarbonyl group and on the guanidino group by the p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group,

- histidine protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the imidazole ring by a p-toluenesulfonyl group,

- p-benzyl ester of aspartic acid protected on the α-amino group by tert-butoxycarbonyl, and

- norleucine protected on the α-amino group by a tertiary butoxycarbonyl group.

Each coupling for solid phase peptide synthesis is accomplished using the coupling procedure generally described. After attachment of the last amino acid, the t-butoxycarbonyl protecting group of the α-amino group is cleaved, the N-terminal amino group is neutralized and megacetylated. In this way it is

Ac-Nle-Asp (P-Bzl) -His (N ^im -Tos) -D-Phe-Ara (NS-Tos) -Trp- (N ⁱ -For) -Om (NS ^amma -Z) -p-MBHA a protected peptide chain resin. This product was dried under reduced pressure and a 1.0 g portion was cleaved to give the product. Example 4a. In this way, 332 mg of crude

Ac- [Nle ⁴ , Asp \ D-Phe ⁷ , Om ¹⁰ ] -a-MSH 4-10 NH 2 is obtained.

A 105 mg portion of the crude heptapeptide was purified as described in Example 11 to give 78 mg of the open chain peptide as a white powder. The clean

A 40.0 mg portion of Ac- [Nle ⁴ , Asp5, -D-Phe ⁷ , Om ¹⁰ ] -α-MSH 4 -yl-NH 2 is alluded. The ring closure reaction described in Example 1A. After proper work-up, this gives 15 mg pure

Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Om ¹⁰ ] -a-MSH 4 -oNH 2 are obtained.

[α] 589 ²² = -37, Γ (c = 0.07, 10% acetic acid),

Rf = 0.71 [1-butanol / acetic acid / pyridine / water (15: 3: 10: 12 tf)].

Example 15 -Ac- (Nle ⁴ Asp ⁵ J) -Phe ⁷ J) ab ^{, o} ] -a-MSH 4 -toNH 2

1.0 g (0.7 mmol / g) of p-methylbenzhydrylamine resin is coupled to the t-butoxycarbonyl group on the α-amino group and to the gamma-amino group using a procedure generally described for solid phase peptide synthesis. 2,4-diaminobutyric acid protected with benzyloxycarbonyl. After a one hour coupling reaction, the reaction is stopped, the resin is washed, neutralized, and the unreacted amino groups of the resin are megacetylated with a single excess of a 1: 1 mixture of acetic anhydride and pyridine in dichloromethane for 1 hour. The following amino acids are then added to the resin stepwise in the following order:

- tryptophan protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the indole ring system,

- arginine protected on the α-amino group by the tert-butoxycarbonyl group and on the guanidino group by the p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group,

- histidine protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the imidazole ring by a p-toluenesulfonyl group,

- p-benzyl ester of aspartic acid protected on the α-amino group by tert-butoxycarbonyl, and

- norleucine protected on the α-amino group by a tertiary butoxycarbonyl group.

After attachment of the last amino acid, the t-butoxycarbonyl protecting group for the α-amino group is cleaved, the N-terminal amino group is neutralized and megacetylated as described in Example 11. In this way it is

Ac-Nle-Asp (β-Bzl) -His (N ^im -Tos) -D-Phe-Arg (NS-Tos) -Trp- (N'-For) -Dab (N8 ^amma -Z) -p- Resin bearing the protected peptide chain of formula MBHA is obtained. This product was dried under reduced pressure and a 1.0 g portion was subjected to a cleavage reaction.

-13HU 200480 Β

The reaction mixture was worked up by 318.2 mg crude

Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Dab ¹⁰ ] -α-MSH 4 -10NH 2 are obtained. A sample of 100.0 mg of this crude heptapeptide was purified to give 78.2 mg of the open-chain peptide as a white powder. The clean

A weighing 45.0 mg portion of Ac- [Nle ^{4, 5-Asp,} D-Phe ⁷ .Dab ^lo] -a-MSH4-tuna formula ₂ peptide is cyclized as described above and the resulting product was also purified in the manner described above so 13.2 mg of pure_

Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Dab ¹⁰ ] -α-MSH 4 -10NH 2 are obtained.

[α] 589 ²² = -54.5 ° (c = 0.01, 10% acetic acid),

R f = 0.80 [1-butanol / acetic acid / pyridine / water (15: 3: 10: 12 tf)].

Example 16 __,

Ac-1Nle ⁴ Asp ⁵ .D-Phe ⁷ J) pr ¹⁰ ] -a.-MSH 4 -10HH 2

1.0 g (0.7 mmol / g) of p-methylbenzhydrylamine resin is coupled to the tert-butoxycarbonyl group on the α-amino group by solid-phase peptide synthesis following the procedure generally described above. 2,3-diaminopropionic acid protected with a benzyloxycarbonyl group on an amino group. After 1 hour, the coupling reaction is stopped, the resin is washed, neutralized, and the unreacted amino groups of the resin are megacetylated with twice the excess of a 1: 1 mixture of acetic anhydride and pyridine in dichloromethane for 1 hour.

The following amino acids are then added to the resin stepwise in the following order:

- tryptophan protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the indole ring system,

- arginine protected on the α-amino group by the tert-butoxycarbonyl group and on the guanidino group by the p-toluenesulfonyl group,

- D-phenylalanine protected on the α-amino group by a tert-butoxycarbonyl group,

- histidine protected on the α-amino group by the tertiary butoxycarbonyl group and on the nitrogen atom of the imidazole ring by a p-toluenesulfonyl group,

- an aspartic acid benzyl ester protected on the α-amino group with a tert-butoxycarbonyl group, and

- norleucine protected on the α-amino group by a tertiary butoxycarbonyl group.

After attachment of the last amino acid, the α-amino protecting tert-butoxycarbonyl group is cleaved, the N-terminal amino group is neutralized and megacetylated as described in Example 11. In this way it is

Ac-Nle-Asp (-Bzl) -His (N ' ^m -Tos) -D-Phe-Arg (NS-Tos) -Trp (N'-For) -Dpr (3-Z) -p-MBHA, yielding a resin bearing a protected peptide chain.

This product was dried under reduced pressure. then a 1.0 g portion was subjected to a cleavage reaction and worked up as described in Example 1. In this way 310.8 m raw,

Ac- [Nle ⁴ , Asp \ D-Phe ⁷ , Dpr ¹⁰ ] -α-MSH 4 -I (> NH 2). A sample of crude heptapeptide (115.0 mg) was purified as described in Example 14 to give a white, powdery powder. 82.5 mg of the open-chain peptide were obtained in the form of pure material

A 38.3 g portion of the Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Dpr 10 ^O ] -α-MSH 4 -oNH ₂ ring was ring-purified and purified as described above to give 11.3 g. mg pure

Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ Dpr ¹⁰ ] -a-MSH + -10NH 2 are obtained.

[ot] 589 ²² = -33.3 "(c = 0.045, 10% acetic acid),

Rp = 0.78 (1-butanol / acetic acid / pyridine / water (15: 3: 10: 12)).

The biological activity of α-melanotropin and its open-chain and cyclic analogs is determined by measuring the ability of these compounds to stimulate the proliferation of melanosomes (granules containing melanin) in in vitro experiments on frog skin and lizard skin. This in vitro assay gives clear dose response curves in the concentration range of 2.5 x 10 ¹¹ moles to 4 x 10 ¹⁰ moles and can be used to detect very small concentrations of about 10 ¹¹ moles of α-melanotropin. The assay is based on the measurement of the centrifugal distribution of melanosomes in the processes of the melanophoric (melanin-containing) cells of the skin (resulting in a darker skin color). Prepare each assay solution for the assay by serial dilution of a 10 ⁴ M stock solution. Frogs (Rans pipiens) used for measurements are from Kons Scientific, Germantown, WI, and lizards (Anolis carolinensis) are from Snake Farm, La Piacé, LA. The biological activity of cyclic analogs of α-melanotropin is given in the table below:

analog number Biological effect Residual effect Frog F.A.Q Frog F.A.Q 16th 1.0 1.0 p (-) P (-) 17th 1.0 6.0 P (+) P (+) 18th 0.5 9.0 p (+) P (+) 19th 0.5 90.0 p (+) p (+) 20th 1.0 20.0 p (-) p (-) 21st 1.0 5.0 p (-) Ρ (-) 22nd 0.01 5.0 P (-) p (-)

Previous U.S. Patent Nos. 4,457,864 and 4,485,039, which are incorporated herein by reference in their entirety, have indicated the importance of having a fixed conformation at the site of action of α-melanotropin or adrenocorticotropic hormone. When the methionine (position 4) and glycine (position 10) at the 4 and 10 positions of the α-melanotropin peptide chain are replaced by cysteine as the amino acid by pseudoisostere substitution, the resulting fixed conformation ring structure analogue multiplies the activity of melanocytes resulting in the proliferation of melanin particles. The much greater effect of ring analogs over their open-chain relatives is obviously dependent on the biological measurement system used. In particular, we wanted to produce analogs that show strong activity when measured on lizard skin.

-14EN 200480 Β the results of this measurement are directly proportional to the effects on mammals.

The main characteristics of cyclic analogs of the formula [Cys ⁴ , Cys ¹⁰ ] -a-MSH are: they show only a weak effect on the biological value of lizard skin, whereas they have a strong effect on the frog skin; furthermore, if the number of members of the optimized sized 23-membered ring is reduced (22-membered ring) or increased (24-membered ring), this change results in a 500-fold smaller effect than the compound containing the optimally sized ring. Creating a fixed conformation in α-melanotropin analogs by constructing an amide bond between the 5 and 10 positions of the peptide chain yields a class of cyclic analogs, the production of which is an object of the present invention. The compound 19 containing the 23-membered ring showed the strongest biological effect on the skin of the lizard, and this effect did not change much in the study on the skin of the frog. The effect of the cyclic compound is about 10-fold greater than that of its open-chain analog. The activity of the 18-membered ring compound is not substantially different from that of its open-chain analog, but a significant change is that the cyclic analogue exhibits a sustained action. The tripeptide at the C-terminus, the Gly-Pro-Val moiety, does not interfere with the activity of these compounds as illustrated by Compound 17. The essential property of these cyclic lactam backbone peptides is that the cyclic compounds have a longer duration of action than the open-chain analogs. The compounds of the present invention outperform α-melanotropin in one or more of the following characteristics:

- in vitro or in vivo efficacy measured on frog and / or lizard,

the duration of the in vivo effect, and / or

- resistance to the degradation effect of enzymes in blood serum.

The compounds of the present invention may be administered transdermally (transdermally), and the compounds may be formulated into pharmaceutical forms of the desired mode of administration using methods and methods known to those skilled in the art. For example, the compounds of the present invention and various common, known ingredients may formulate formulations such as creams, ointments, gels, lotions, tablets or spray formulations, depending on the form of the desired active ingredient. patient. These compositions may be formulated using known thickeners, plasticizers, surfactants, dyes, fragrances, preservatives, fillers and emulsifiers, all of which are known in the art and commonly used in the preparation of transdermal compositions. Generally, these non-bioactive ingredients make up the greater part of the total formulation. Preferably, such compositions are prepared. so that they can be applied slowly and over a period of time.

Due to their remarkable properties, the compounds of the present invention can be used in place of the α-melanotropin and analogs of the formula [Nle ⁴ ] -α-MSH in the diagnostic, therapeutic and basic research systems currently in use. In the field of diagnostic methods, it will be appreciated that the compounds of the present invention, and in particular those substituted with radioactive iodine or coupled with gamma-emitting agents, are particularly useful for locating and / or discriminating melanoma cells on the basis of that these compounds bind to the melanotropin receptors of such cells. Furthermore, the property of the compounds of the present invention to be stable to serum makes them particularly suitable for delivery of the active compounds to selectively defined sites, such as tissues known to contain many melanotropin receptors. It is hoped that the relatively potent and sustained action of the compounds of the present invention in the phenomena of skin and coat discoloration will be further enhanced by other biological effects previously reported with the natural melanocyte-stimulating hormone and its synthetic analogues.

Thus, although the process of the present invention has been described and illustrated with respect to preferred embodiments thereof, it is to be understood that the invention may be modified and modified to some extent, and therefore, it is not intended to limit the scope thereof. modifications and modifications which will allow the process of the present invention to be used and applied in a variety of other circumstances. Accordingly, such changes and modifications are wholly within the scope of the teachings equivalent to the present disclosure, and thus within the scope of the present disclosure and the following claims.

Thus, the foregoing describes the process of the present invention, and its implementation and application, in a complete, understandable, concise and accurate form, so that any person skilled in the art or to whom it belongs can carry out and apply the present process.

Example 17

Al 1-16. The following peptides were prepared as described in Examples 1 to 8:

Ac- [Nle ^4. Asp ⁵ , D-Phe ⁷ ] α-MSH ⁴ -10-NH 2, [α] 546 D ²³ = -44.8 ° (c = O, 25, 10% acetic acid), R f = 0.01. (1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)).

Ac- [Nle ⁴ , D-Phe ⁷ , Om ¹⁰ ] α-MSH 4 -O-NH ₂ , [α] 546 ²³ = 12 ° (c = 0.04, 10% acetic acid), R f = 0.37 (1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)).

Ac- [Nle ⁴ , Asp ⁵ , D-Phe ⁷ , Om ¹⁰ ] α-MSH 4 -10-NH 2, [α] 546 ²³ = 20.0 ° (c = 0.03, 10% acetic acid), R f = 0.34. [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Ac- [Nle ⁴ , D-Phe ⁷ , Dab ¹⁰ ] α-MSH 4 -10-NH 2, [α] 2546 ²³ = 30.0 ° (c = 0.05, 10% acetic acid). R f = 0.44 [1-butanol / acetic acid / pyridine / water (5: 5: 1: 4 v / v)].

Ac- [Nle ⁴ Asp ⁵ , D-Phe ^7. Lys ¹ °, Gly ¹¹ ] O 1 -MSH 4-13 -NH 2, α] 589 ²² = 53.3 '(c = 0.04, 10% acetic acid), R 0.76 (1-butanol / acetic acid / pyridine / water (15: 3: 10: 12)).

-151

HU 200480 Β

PATENT CLAIMS

Claims (19)

A process according to formula (I) For the preparation of R ¹ -Nle-R ² -His-R ₃ -Arg-Trp-R 4 -R 5 (I) α-melanotropin derivatives and 5-10 ring analogues thereof, wherein R 1 is Ac-Ser-Tyr-Ser or Ac, R2 is Glu or Asp, R.3 Phe or D-Phe, R1 is -Gly-Pro-Val-NH2 or -NH2, and R4 is Lys or ha R 5 is -NHo and R 1Ac, R 4 is Dab, Dpr, Om or Gly, characterized in that protecting groups and / or at least one peptide of formula (I) having known protecting groups and / or bound to a known resin are present. the resin is cleaved and, if desired, the resulting open chain compound is cyclized, The process according to claim 1, is α-melanotropin Ac-Ser-Tyr-Ser-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NHI, characterized by starting with peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analog of Ac-Nle-Glu-His-D-Phe-Ara-Trp-Lys-Gly-Pro-Val-NH2, starting from peptides containing the appropriate amino acids. The process according to claim 1, is α-melanotropin Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH₂: characterized in that it is derived from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2, starting from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analogue of Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-NH2, starting from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-NH2, starting from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin Ac-Nle-Glu-His-D-Phe-Arg-Trp-Orn-NH2. characterized by starting with peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Orn-NHz, starting from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin Ac-Nle-Glu-His-D-Phe-Arg-Trp-Dab-NHi. characterized by starting with peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dab-NH2, starting from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of the open-chain analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dpr-NH2, starting from peptides containing appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of an open-chain analogue of Ac-Nle-Glu-His-Phe-Arg-Trp-Lys-NH2, starting from peptides containing appropriate amino acids. 14. The process of claim 1, is α-melanotropin For the preparation of an open-chain analog of Ac-Nle-Asp-His-Phe-Arg-Trp-Lys-NH2, starting from peptides containing appropriate amino acids. The process of claim 1, is α-melanotropin For the preparation of the cyclic analog of Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-GLy-Pro-Val-NHo, starting from peptides containing the appropriate amino acids. The process according to claim 1, is α-melanotropin For the preparation of the cyclic analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NHi, starting from peptides containing appropriate amino acids. 17. The process according to claim 1, is α-melanotropin For the preparation of the cyclic analogue of Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-NH 2, starting from peptides containing appropriate amino acids. 18. The process according to claim 1, is α-melanotropin For the preparation of the cyclic analog of Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-NH2, starting from peptides containing the appropriate amino acids. 19. The process of claim 1, is α-melanotropin Ac-Nle-Asp-His-D-Phe-Arg-Trp-Orn-NH2