NO177100B - peptides - Google Patents

Description

The present invention relates to peptides. ~

In US-P. 4,190,646 and 4,261,886 various peptides are described which have thymic and immunological function. US patent 4,190,646 describes the "thymopoietin pentapeptide" (TP5), and substituted derivatives thereof, while US patent 4,261,886 describes peptide analogues of TP5 with greater potency than TP5. In the US patents, said peptides are produced by a synthesis technique in solid phase, usually described as "Merri-field synthesis". Furthermore, the patents also indicate that the classical technique, (ie solution synthesis technique) can be used to prepare certain peptides and derivatives, but no specific classical method or synthesis route is described.

Although Merrifield's solid phase synthesis technique is suitable for the production of small amounts of peptides in the laboratory, it would be impractical and generally uneconomical for the production of large amounts (ie more than 100 g) of peptide, and for such large amounts it is more appropriate to use a solution synthesis technique. This type of technique is usually far cheaper than a technique where a solid phase is used, which is due to a lower unit price of some of the reagents used. Certain special peptides have now been made available which can be used for the production of desired final peptides in a more appropriate and economical way compared to the known techniques.

According to the present invention, a dipeptide is thus provided which is characterized by the formula:

where R' is NH 2 and Y is SAR.

Furthermore, a protected dipeptide is provided, which is characterized by the formula:

alpha-T-X-omega-TJ-Z-OH — where X is SAR or epsllon-T"-LYS; Z is ASP or GLU; and T and T" are each selected from the group consisting of: a) wherein R 1 is benzyl, benzyl wherein the phenyl ring is substituted with from 1 to 3 groups selected from lower alkyl and t-butyl; and B)

where R 2 is lower alkyl of 2-4 carbon atoms;

and U is benzyl.

The invention also provides a protected tetrapeptide characterized by the formula:

where R<*> is OU or NH 2 ; U is benzyl; R 1 is NH 2 ; x is epsilon-T"-LYS and Y is VAL or X and Y are both SAR; Z is ASP; and T and T" are each a group selected from the group consisting of those defined in a)-b) above.

Furthermore, the invention provides a tetrapeptide characterized by the formula:

where R' is OU or NH 2 ; TJ is benzyl; X is epsilon-T"-LYS and Y is VAL or X and Y are both SAR; Z is ASP; and T" is a group selected from the group consisting of those defined in

a)-b) above.

Also provided is a protected pentapeptide which is

characterized by the formula:

where R' is OH or NH 2 ; U is E or benzyl; X is epsilon-T"-LYS and Y is VAL or X and Y are both SAR; Z is ASP; and T and T" are each H or a group selected from the group consisting of those defined in a)-b) above .

The invention also provides a protected tripeptide characterized by the formula:

where Z is ASP; Y is VAL; R' is OU; U is benzyl where the phenyl ring is substituted with a lower alkyl; and T is selected from the group consisting of those defined in a)-b) above.

Finally, the invention provides a dipeptide characterized by the formula: where X is SAR or epsilon-T"-LYS; T and T" are each selected from the group consisting of those defined in a)-b) above; T' is selected from the group consisting of

and other symbols used have the same meanings

as stated above.

Present peptides can be used for the production of special pentapeptides that have the formula:

where X is LYS and Y is VAL, or X and Y are both SAR, Z is ASP or GLU, and R is OH or NH2, and this method includes: A) forming a fragment I, which consists of H-Y-TYK -R', where R' is OU or NB2; Y is SAR or VAL; and U is benzyl or benzyl where the phenyl group is substituted with from 1 to 3 groups selected from halogen, nitro, C1-C3 lower alkyl, and C1-C3 lower alkoxy, by i) protecting the alpha-amino group of the Y-amino acid by allowing it to react with a reagent which will introduce the protecting group T, where the group T is selected from a) wherein R 1 is phenyl; tolyl; xylyl; adamantyl; allyl; beta-cyanoethyl; fluorenylmethyl; benzyl; benzyl wherein the phenyl ring is substituted with from 1 to 3 groups selected from halogen, nitro, lower alkyl and lower alkoxy, nitropropylmethyl; diphenylmethyl; cyclohexyl; cyclopentyl; vinyl; t-butyl; t-amyl; dimethyldifluoromethylmethyl; or dimethylbiphenylmethyl; b) where R 2 is lower alkyl of 2-4 carbon atoms or lower alkyl of 1-4 carbon atoms substituted with from 1 to 5 halogen groups; c) V is S or O, and R 3 and R 3 are each benzyl or lower alkyl; d)

where R5 and Rf, individually each are

lower alkyl or R5 and R5 together are

-CH2-C-CH2- where R7 and Rg are each

R7 R8

hydrogen or lower alkyl,

e)

wherein R 8 is hydrogen or nitro; and f)

5

where R^o is hydrogenmethyl, halogen or nitro,

provided T is monodentate when X is SAR;

ii) activating the protected Y formed in step i) with respect to nucleophilic attack at the carboxy group of an amine, to form a carboxy-activated protected Y amino acid;

iii) turnover of said carboxy-activated protected Y

with TYR-R' where R' has the meaning given above;

iv) removal of the protected group T, whereby fragment

In forming,

forms fragment II, which consists of alpha-T-(epsilon-T")X-omega-Y-Z-OH, where Y, U and Z have the meanings given above, and T" has the same meaning as T given above, by

i) production of omega-U-Z-OH, where U is a protecting group on the omega-carboxy group of the Z-amino acid, and has the above meaning;

ii) protection of the alpha-amino group (and if X is LYS,

epsilon amino group) in the X-amino acid by allowing it to react with reagent(s) which will introduce the protecting group(s) and T (and T"), where T and T" have the above meanings;

iii) activating the protected X formed in step ii) with respect to nucleophilic attack at the carboxy group of an amine to form a carboxy-activated protected X-amino acid;

iv) reaction of said acarboxy-activated-protected X-amino acid with omega-U-Z-OH, whereby fragment II is formed,

C) fragments I and II react to form a fragment III consisting of T-(epsilon-T")X-omega-U-Z-Y-TYR-R', where X, "U, Z, Y, R', T and T" has the meanings given above; D) removes the T-protecting group from fragment III to form fragment HIA, which consists of H-(epsilon-T")X-omega-U-Z-Y-TYR-R', where the symbols have the above indicate meaning;' E) produces alpha-T-omega-T'-ARG, where T is a protecting group on the alpha-amino group of L-arginine, and T<*> is a protecting group on the guanidino group of L-arginine, where T' is a group selected from the group consisting of

0

II

R^-OC- where Ri has the meaning given above, nitro, hydrochloride, p-methoxybenzylsulfonyl and tosyl, F) reacts fragment 11 IA with alpha-T-omega-T'-ARG to form alpha-T-omega-T <*->ARG-(epsilon-T'-)X-omega-U-Z-Y-TYR-R', where the symbols have the meanings indicated above; G) removes the groups U, T, T' and T" to form the peptide

E-ARG-X-Z-Y-TYR-R; and

H) isolate and purify the resulting peptide.

If X is LYS, then of course its epsilon-amino group must also be suitably protected by means of the amino-protecting group T" during the preparation of fragments containing X and during their use for the preparation of the final peptide. Said T" group must be easily removed during conditions which do not destroy the resulting peptide, while said group must be stable during removal of the T groups.

The alpha-amino protecting group T may be the same or different for each of the amino acids, and must be stable against removal in the steps used to link the amino acid groups, while also being easily removable at the end of the linking steps under conditions which will not cleave any of the amide bonds in the peptide. For certain groups (e.g. BOC) this removal is brought about by means of a strong acid (e.g. trifluoroacetic acid), which results in the intermediate product being produced as the corresponding acid addition salt (e.g. trifluoroacetate).

The guanidino protecting group T" can be any suitable amino protecting group as described below, or a nitro group as well as acid addition salts such as the hydrochloride. Among the amino protecting groups, it is preferred to use urethane protecting groups (formula a below) and substituted sulfonic acid derivatives such as p- methoxybenzenesulfonyl and tosyl. The hydrochloride salt is the most preferred. This guanidine protecting group is here designated the "omega" group to indicate that it is at the end of the chain. The exact position of many guanidine protecting groups on the chain is not definitely known.

The carboxy-protecting group U should be easily removed under conditions that do not destroy the resulting peptide, while the group must be stable during removal of the T groups.

The R' group is either NH2 (for product peptides where R is NH2 ) or OU (for product peptides where R is OH).

The above-mentioned amino-protecting group f) as bidentate, can only be used for the alpha-amino groups on L-arginine or L-valine or the alpha-amino or epsilon-amino groups on L-lysln, but not for the alpha-amino group 1. sarcosine. The amino-protecting group for the sarcosine alpha-amino group must be monodentate because of the methyl substituent on this amino group. The remaining amino protecting groups can be used for all amino acids.

As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine, but chlorine and bromine are preferred. The terms lower alkyl and lower alkoxy respectively include saturated aliphatic hydrocarbons with from one to six carbon atoms such as methyl, ethyl, isopropyl, t-butyl, n-hexyl and the like, as well as the corresponding alkoxides such as methoxy, ethoxy, isopropoxy, t-butoxy, n -hexoxy and the like. Methyl is the preferred lower alkyl group and methoxy is the preferred lower alkoxy group.

The reagents used to introduce these protecting groups (usually the corresponding acid chlorides, although other derivatives can also be used) will in some cases be referred to here as "protecting group reagents". Other suitable protective groups are, for example, described in "Protective Groups in Organic Chemistry", J.F.W McOmie, ed. Plenum Press, N.Y., 1973.

It is preferred that each T and T" are the same and may be benzyloxyoxycarbonyl (CBZ) or trifluoroacetyl (TFA). It is preferred that T- is the hydrochloride salt.

A variety of reagents can be used to prepare the carboxy-activated protecting amino acid groups described above.

One type of carboxy-activated, protected amino acid group is a reactive ester. Examples of reagents that can be used to prepare suitable activated esters are phenol, phenol where the phenyl ring is substituted with from one to five groups selected from the group consisting of halogen (eg chlorine or fluorine), nitro, cyano and methoxy; thiophenyl; N-hydroxyphthalimide; N-hydroxysuccinimide; N-Hydroxyglutaramide; N-Hydroxybenzamide; 1-hydroxybenzotriazole and the like. Other suitable agents are, for example, described in "Protective Groups in Organic Chemistry"; J. F. W. McOmie, ed., as referenced above. In the following specific examples, N-hydroxysuccinimide or 1-hydroxybenzotriazole will usually be used.

Other activation methods such as the blinded or symmetric anhydride method, the acid chloride method and the azide method are well known and are described, for example, in Bodanszky et al, "Peptide Synthesis", 2nd edition, 1976, pages 85-128. These other methods can also be used.

Of expediency groups, the following compounds will be used to denote the different amino acids:

Production and use of the present peptides is shown schematically in the following Figure 1:

An example of the preparation of H-ARG-SAR-ASP-SAR-TYR-NH2 is shown schematically in the following figure 2:

An example of the production of H-ARG-LYS-ASP-VAL-TYR-OH is shown schematically in the following Figure 3:

In the above figures, the protecting groups are represented by U, T, T<*> and T" as indicated above, while the carboxy activation of the amino acid group is indicated by the letters

"OA".

With reference to Figure 2, fragment I can usually be prepared in the following way. To protect the amino group in sarcosine, a water-soluble, basic addition salt of sarcosine in water is prepared. Conveniently, this basic addition salt can be prepared by dissolving sarcosine in a slight molar excess of sodium hydroxide. To this solution is then simultaneously added a slight excess of a reagent to introduce the protecting group T (eg the corresponding acid chloride such as benzyloxycarbonyl chloride) and a solution of a base (eg sodium hydroxide) to react with the acid (e.g. HC1) which is formed during the reaction. The reagent with the protecting group must be in solution and is preferably the acid chloride. After the reaction is complete, the excess of the reagent that introduced the protecting group was removed (eg by extraction with diethyl ether or another organic solvent immiscible with water), followed by an isolation of the protecting sarcosine from unreacted sarcosine, something that happens during treatment with acid (e.g. hydrochloric acid). The acid treatment converts the basic addition salt of unprotected sarcosine, and this is soluble in water. However, the acid treatment will convert the protected sarcosine base addition salt only to protected sarcosine, as it is now not possible to prepare an acid addition salt due to the amino protecting group. This protected sarcosine is soluble in water, and it can be easily separated from the salt of the unprotected sarcosine, for example by extraction with an immiscible organic solvent as described above. The term "immiscible organic solvent" refers to all commonly known organic solvents which cannot be mixed with water, e.g. diethyl ether, ethyl acetate, benzene, toluene, xylene and the like. The preferred protected sarcosine, N-benzyloxycarbonyl-sarcosine is a known compound. A method of its preparation is shown by R.S. Tipton and B.A. Pawson, J. Org. Chem., 26, 4698 (1961), and the compound is commercially available from Bachem, Inc., Torrance, CA.

In synthesis for the condensation of this protected sarcosine with the L-tyrosinamide molecule to produce fragment I, the amino-protected sarcosine should usually be activated in some way to promote the formation of the bond. Although it is usually preferred to carry out this activation by forming an "active ester", other activation methods can also be used, such as by means of a mixed or symmetrical anhydride, azide or acid chloride.

Any of the protected saroxins may be used, although the preferred active ester is that formed by means of hydroxysuccinimide. The active ester of the protected sarcosine is prepared by reacting equivalent amounts of the protected sarcosine and an active ester reagent in a solution of a suitable organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, pyridine and the like. To this solution is then added an equivalent amount of a coupling agent, typically dicyclohexylcarbodiimide. Although other coupling agents are also effective, dicyclohexylcarbodiimide is particularly useful because the by-product from the coupling reaction is very poorly soluble in the types of solvents used, whereby it can be easily removed by filtration and the coupled product is obtained in solution. L-tyrosinamide is commercially available (eg, from Sigma Chemical Company, St. Louis, MO) or can be prepared by known methods.

The next step in the preparation of fragment I consists in reacting a molar equivalent amount of L-tyrosinamide with a protective sarcosine-active ester in the presence of an equivalent of a salt-forming material such as an organic tertiary amine. Although any organic tertiary amine can be used, it has been found most satisfactory to use triethylamine. The solvent is a suitable organic solvent of the type described above. The unreacted amino acids are removed by treating the reaction mixture with an acid (e.g. acetic acid) or by extraction with an immiscible organic solvent as described above.

Finally, the alpha-amino protected group is removed from sarcosine, preferably with trifluoroacetic acid, thereby obtaining fragment I.

The production of fragment II usually starts with L-aspartic acid protected on its beta-carboxyl group or L-glutamic acid protected on its gamma-carboxyl group. This beta or gamma carboxy group is usually referred to as the "omega" group in accordance with common nomenclature to indicate that it sits at the end of the chain.

Examples of suitable carboxyl-protecting groups are benzyl and benzyl for which the phenyl group is substituted with from one to three, the group selected from the group consisting of halogen (e.g. chlorine or bromine), nitro, C 1 -C 3 -lower oxy (e.g. methoxy) or Ci-C3~lower alkyl (eg methyl). See above referenced book by McOmie for further description of such groups. Benzyl is the preferred group. This beta-protected L-aspartic acid or gamma-protected L-glutamic acid is commercially available from Bachem, Inc. Torrance, California, or can be prepared by known methods.

This beta-protected L-aspartic acid or gamma-protected L-glutamic acid (omega-U-Z) is then reacted with said alpha-amino-protected sarcosine which has been activated (e.g. by a conversion to an active ester) as described above, whereby one obtains fabricated fragment II. On figure 2 is

Z ASP.

Fragments I and II are linked to the protected tetrapeptide alpha-T-SAR-beta-U-ASP-SAR-TYR-NH2 (fragment III) by reacting equivalent amounts in an appropriate aprotic solvent such as dimethylformamide in the presence of a weak excess of a coupling agent such as dicyclohexylcarbodiimide. It is also preferred to carry out this reaction in the presence of a compound which causes minimal racemization in connection with the carboxyl group on the L-lysin part of fragment I, and increases the reaction rate, and one can, for example, use 1-hydroxybenzotriazole. As in connection with fragment II, the alpha-amino protecting group on the sarcosin part of fragment III is removed with trifluoroacetic acid, thereby obtaining fragment IIIA.

After a coupling reaction similar to that used to connect fragments I and II, an alpha-amino and guanidine-protected L-arginine group is finally attached to the amino end group in fragment HIA, after which all protecting groups are removed and the desired pentapeptidamide is obtained. The removal of the protected groups can, for example, be carried out by treatment with hydrogen gas in the presence of a palladium-on-carbon catalyst in a suitable reducing solvent as described above (preferably aqueous acetic acid). The hydrogen gas need not be under a pressure greater than one atmosphere, although the use of pressure is appropriate as this accelerates the rate of reduction.

Isolation is achieved by a combination of crystallization and ion exchange chromatography (preferably using ammonium acetate pH 5 as eluent), and by using thin layer chromatography to establish the identity of the compounds in each fraction. Although several examples of isolation and cleaning techniques are indicated in the following examples, it is of course understood that other methods can also be used.

Production and use of the present peptide intermediates are illustrated in the following examples.

EXAMPLE I

Preparation of fragment I: SAR-TYR-NH2

A. BOC-sarcosln-hvdroxvsuccinimide ester (BOC-SAR-OSu): BOC-sacryson (24.78 g, 0.13 mol) and N-hydroxysuccinimide (15.5 g, 0.13 mol) were dissolved in 300 mL of dry THF and cooled to -5°C. A solution of dicyclohexylcarbodiimide (26.98 g, 0.13 mol) in 100 mL of dry THF was added over 15 minutes. The reaction mixture was stirred overnight and allowed to stand at room temperature. The separated solid was filtered off, and the solvent was evaporated under reduced pressure to a white solid. This was crystallized from 250 ml of absolute ethanol at 4°C and 32 g (86%) of a white solid was obtained, melting point 121-123°C

Analysis: Calcd: C, 50.35; H, 6.34; N, 9.79

Found: C, 50.23; H, 6.44; N, 9.67

TLC: RH = 0.81, CHCl 3 /MeOH 9/1

(silica gel, G, 250 pm)

p.m.r (S, CDC13); 1.45, S, 9H, BOC; 2.83, S, 4H, -OSu; 2.93, S, 3H, N-CH3; 4.27, S, 2H, -CH2-.

M.S.: M+ 286

B. BOC-sarcosvl-L-tyrosinamide (BOC-SAR-TYR-NHo^L-tyrosinamide (2.17 g, 10 mmol) and triethylamine (1.01 g, 10 mmol) were dissolved in 25 mL of dry methanol. BOC -sarcosine-hydroxysuccinimide (2.86 g, 10 mmol) was added and the mixture was stirred overnight at room temperature.

Volatiles were removed under reduced pressure and the residue partitioned between EtOAc (50 mL) and a NaCl solution [50 mL (25 mL H 2 O + 25 mL saturated NaCl)]. The phases were separated, and the organic phase was washed twice more with the same type of NaCl solution and then dried with magnesium sulfate. The drying agent was removed by filtration and the solvent evaporated under reduced pressure. The residue was chromatographed on a 75 g, 2.5 cm wide column of Silicar CC7 using ethyl acetate as eluent. The compound was eluted at about 390 ml. The next 475 mL was collected and evaporated to 1.55 (44#) of a white solid.

Analysis: Calcd: C, 58.11; H, 7.17; N, 11.96

Found: C, 57.96; H, 6.96; N, 11.45

TLC: (Silica gel GF) Rf = 0.46 CHCl3/MeOH 9/1

C. Sarcosyl-L- tvrosinamide. Trifluoroacetate (TFA-SAR-TYR-NH2): BOC-sarcosyl-L-tyrosinamide (1.30 g, 3.7 mmol) was dissolved in 15 mL of trifluoroacetic acid at 0°C. The solution was stirred for 1 hour at 0°C and the solvent removed under reduced pressure. The resulting oil was treated with 50 mL of anhydrous ether to give 1.27 g (94%) of a white solid.

p.m.r. (5, CD30D): 2,3, S, 3H, N-CH3; 3.00, d, 2E, -CH 2 -C-; 3.7, S, 2H, -N-CH2-C=O; 6.9; q, 4H, aromatics,

EXAMPLE II

Preparation of fragment II: BOC-SAR-beta-benzyl-ASP (BOC-sarcosyl-beta-benzyl-L-aspartic acid)

Triethylamine (2.02 g, 20 mmol) and beta-benzyl-L-aspartic acid (2.23 g, 10 mmol) were stirred in 50 mL of dry THF. BOC sarcosine hydroxysuccinimide ester (2.68 g, 10 mmol) was added and the solution stirred at room temperature overnight. Solids were filtered off and the solvent removed under reduced pressure. The residue was partitioned between 100 ml of ethyl acetate and 100 ml of 2 N hydrochloric acid. The phases were separated and the organic phase was washed with 2 x 100 ml water, 1 x 100 ml saturated sodium chloride solution and then dried over magnesium sulfate. The latter was filtered off, and the solvent was removed under reduced pressure. The residue was treated with hexane. The latter was then decanted and the residue dried under reduced pressure to 2.64 g (67%) of a hygroscopic solid.

TLC: Rf = 0.73 + trace impurities at 0.48

CECl3/MeOH/EOAc 85/10/5

(Silicon dioxide gel G, 250 pm)

p.m.r. (S, CE30D): 1.45, S, 9E, BOC; 2.82, S, 3E, N-CE3; 2.95, M, 2E, -CE2-C; 3.85, S, 2E, -N-CH2-C=0; 4.85, t, 1E, -CE-; 5.08, S, 2E, -CE2; 5.46, S, 2E, -NE + CO2E; 7,3, S, 5E,

-9

[α]D17° = +13.3° (C = 1.032, MeOH)

EXAMPLE III

Preparation of fragment III: BOC-sarkosyl-beta-benzyl-L-aspartyl-sarkosyl-L-tyrosinamide (BOC-SAR-beta-Bzl-ASP-SAR-TYR-NE2)

BOC-sarcosyl-beta-benzyl-L-aspartic acid (0.52 g, 1.3 mmol) and 1-hydroxybenzotriazole monohydrate (0.18 g, 1.3 mmol) were dissolved in 10 mL of dry DMF, and the solution cooled to 0 °C. Dicyclohexylcarbodiimide (0.27 g, 1.3 mmol) in 7.5 mL of dry DMF was added, and the solution was stirred for 1 h at 0 °C. Sarkosyl-L-tyrosinamide, trifluoroacetate (0.50 g, 1.3 mmol) and diisopropylethylamine (0.17 g, 1.3 mmol) were dissolved in 5 mL of dry DMF and immediately added to the first solution. The reaction mixture was stirred overnight and allowed to stand at room temperature. The solid was filtered off, and the residue was dissolved in 25 ml of ethyl acetate. The organic phase was washed first with 2 x 25 ml 10 # citric acid, 2 x 25 ml water, 2 x 25 ml 5% NaHCO 3 , 2 x 25 ml H 2 O, 25 ml saturated sodium chloride solution and then dried over anhydrous magnesium sulfate. The drying agent was filtered off and the solvent removed under reduced pressure to give 0.6 g (73.5%) of a white solid.

TLC: Rf = 0.31

CECl 2 /MeOH 9/1

(Silica gel G, 250 pm)

p.m.r. (S, CDCl 3 ): 1.45, S, 9H, BOC; 2.95, m, 10H, 2x, N-CH3,

-CH 2 -CH (Asp), -CB 2 -CH (Tyr); 3.8, m, 4E, 2x, N-CH2-C=0; 4.6, m, 2H, -CH-CH2-, Asp, -CH-CH2- (Tyr); 5.1, m, 2H, -CH2-cp; 6.92, q, 4E, tyrosine aromatic; 7.25, S, 5H, aromatic benzyl [α]D21° - 12.4° (C = 0.1046, MeOH)

Analysis: Calculated for C31H41N50g: C, 59.32, H, 6.58, N, 11.16

Found: C, 58.73; H, 6.80; N, 10.76

EXAMPLE IV

Preparation of fragment IIIA: Sarkosyl-beta-benzyl-L-aspartyl-sarkosyl-L-tyrosinamide, trifluoroacetate (TFA-SAR-beta-Bzl-ASP-SAR-TYR-NE2)

BOC -sarcosyl-beta-benzyl-L-aspar ty 1-sarcosyl-L-tyrosinamide (0.48 g, 0.76 mmol) was dissolved in 10 mL of TFA at 0°C and stirred for 1 h. The solvent was removed under reduced pressure and the residue treated overnight with 50 ml of anhydrous ether. The suspension was filtered and the solid washed with ether and dried under vacuum to 0.4 g (82 µg) of a white solid.

TLC: Rf = 0.56

n-BUOH/HOAc/H 2 O

(Silica Gel GF)

EXAMPLE V

Preparation of pentapeptide

A. Alpha-phenylmethoxycarbonyl-L-arginyl(HC1 )-sarcosyl-beta-benzyl-L-aspartyl-sarcosyl-L-tyrosinamide

[CBZ-ARG(CE1)-SAR-beta-Bzl-ASP-SAR-TYR-NH2]

Alpha-phenylmethoxycarbonyl-L-arginine HCl (2.66 g, 7.8 mmol) and 1-hydroxybenzotriazole monohydrate (106 g, 7.8 mmol) were dissolved in 20 mL of dry dimethylformamide and cooled to 0°C. Dicyclohexylcarbodiimide (1.61 g, 7.8 mmol) was dissolved in 5 mL of ether and added to the first solution. The reaction mixture was stirred for 1 hour at 0°C. The TFA salt of sarkosyl-beta-benzyl-L-aspartyl-sarcosyl-L-tyrosinamide (5.0 g, 7.8 mmol) was dissolved in 15 mL of dry DMF with triethylamine (0.79 g, 7.8 mmol) and added the first solution. The reaction mixture was stirred overnight and allowed to stand at room temperature. The solid was filtered off, and the volatile compounds removed under reduced pressure. The residue was treated with water and this gave 3 g of a residue.

TLC: Rf = 0.60

n-BU0E/H0Ac/H2O 3/1/1

(Silica Gel GF)

B. L-arginyl-sarcosyl-L-aspratyl-sarcosyl-L-tyrosinamide

(ARG-SAR-ASP-SAR-TYR-NE2)

Alpha-pheny Ime toxycarbonyl-L-asginyl (EC1 )-sarcosyl-beta-benzyl-L-aspartyl-sarcosyl-L-tyrosinamide (1.0 g) was dissolved in 100 ml of 75 10 aqueous acetic acid and reduced by 0. 5 g of 10% Pd/C at a pressure of 3.5 kg/cm<z> for 15 hours. The catalyst was filtered off and the solution lyophilized to a product of 0.8 g. This was dissolved in 7 ml of water, filtered through a 3 p multipore filter, adjusted to pH 5 with NH 4 OH (concentrated) and chromatographed on an SP-C- 25 column (2.5 x 100 cm) with 0.20 M NH4OAX, pH 5.0, 100 ml/hr, 20 ml/tube. Tubes 71 to 78 were combined and lyophilized to 0.35 g of ARG-SAR-ASP-SAR-TYR-NHg.

TLC: Rf = 0.23

n-BuOH/HOAc/H2O 3/1/1

(Silica Gel GF)

[α]D21° = +54.9 (C = 0.091, 0.1 N HOAc)

EXAMPLE VI

Using the procedures described in Examples I-V, but using equivalent amounts of suitable starting compounds, the following was prepared: BOC-epsilon-CBz-LYS-beta-benzyl-ASP-OH

BOC-epsilon-CBz-LYS-gamma-benzyl-GLU-OH

BOC-SAR-gamma-benzyl-GLU-OH

BOC-epsilon-CBZ-LYS-beta-benzyl-ASP-VAL-TYR-OE-benzyl ester BOC-epsilon-CBZ-LYS-gamma-benzyl-GLU-VAL-TYR-OH-benzyl ester BOC-SAR-gamma-benzyl- GLU-SAR-TYR-NH2

EXAMPLE VII

Preparation of fragment IVA: H-beta-benzyl-ASP-VAL-TYR-benzyl ester, trifluoroacetate (beta-benzyl-L-aspartyl-L-valyl-L-tyrosine benzyl ester, trifluoroacetate)

BOC-beta-benzyl-L-aspartic acid and a molar equivalent amount of 1-hydroxybenzotriazole monohydrate were dissolved in dry

DMF and the solution cooled to 0°C. Then—a molar equivalent amount of dicyclohexylcarbodiimide was added, and the whole was stirred for 1 hour at 0°C. The reaction mixture was then added to a solution in DMF of a molar equivalent amount of triethylamine and L-valyl-L-tyrosine benzyl ester, trifluoroacetate (prepared using the procedures of Example I, but using an equivalent amount of L-valine in place of the sarcosine mentioned therein), and the whole was stirred overnight at room temperature. The product was isolated from the reaction mixture after the reaction was complete , and the BOC group was removed, giving the desired product.

EXAMPLE VIII

Preparation of fragment III (alternatively): BOC-epsilon-CBZ-LYS-beta-benzyl-ASP-VAL-TYR-benzyl ester' (BOC-epsilon-CBZ-L-lysyl-beta-benzyl-L-aspartyl-L-valyl -L-tyrosine benzyl ester)

BOC-epsilon-CBZ-L-lysine-hydroxysuccinimide was added to a solution of molar equivalents of triethylamine and beta-benzyl-L-aspartyl-L-valyl-L-tyrosine-benzyl ester-trifluoroacetate in dry THF, and the whole was stirred overnight. After solids were removed, the product was isolated from the solution.

EXAMPLE IX

Preparation of fragment V: tri-CBZ-ARG-epsilon-CBZ-LYS-OH

(tri-CBZ-L-arginine-epsilon-CBL-L-lysine)

To a suspension of tri-CBZ-L-arginine para-nitrophenyl ester (1.40 g, 2 mmol) in 3 mL of THF was added epsilon-CBZ-L-lysine (625 mg, 2.2 mmol). Triethylamine (400 mg, 4.4 mmol) was then added and the whole was stirred for 48 hours at room temperature. Then the solvent was removed under reduced pressure, and 20 ml of methanol was added to the residue. The methanol was removed and the solid was washed with an additional 10 ml of methanol. The combined filtrate and washing solution were evaporated under reduced pressure to an oil, and this was chromatographed on a 10 ml silica gel column using 0-5 H methanol/chloroform as eluent. The other compound that came out of the column was the desired product, as could be detected by p.m.r.; yield 75 mg.

Analysis: Calculated for C^Eso^O-j^ • 2/3 CEC13:

C, 58.41; H, 5.56; N, 9.15 Found: C, 58.88; E, 5.50; N, 9.29

EXAMPLE X

Preparation of pentapeptide (other option)

Molar equivalents of tri-CBZ-L-arginyl-epslone-CBZ-L-lysine and 1-hydroxybenzotriazole were dissolved in dry DMF after which one molar equivalent of dicyclohexylcarbodiimide was added. To the solution was added a solution of molar equivalents of beta-benzyl-L-aspartyl-L-valyl-L-tyrosine benzyl ester trifluoroacetate and triethylamine in DMF, and the entire mixture was stirred overnight. The product was isolated from the reaction mixture after removal of solid residues, after which the protecting groups were removed, yielding E-ARG-LYS-ASP-VAL-TYR-OH.

Claims (7)

1. Dipeptide, characterized by the formula: where R' is NH 2 and Y is SAR. 2. Protected dipeptide, characterized by the formula: where X is SAR or epsilon-T"-LYS; Z is ASP or GLU; and T and T" are each selected from the group consisting of: a) wherein R 1 is benzyl, benzyl wherein the phenyl ring is substituted with from 1 to 3 groups selected from lower alkyl and t-butyl; and B) where R 2 is lower alkyl of 2-4 carbon atoms; and U is benzyl. 3. Protected tetrapeptide, characterized by the formula: where R' is OU or NH 2 ; U is benzyl; R 1 2 is NH 2 ; X is epsilon-T"-LYS and Y is VAL or X and Y are both SAR; Z is ASP; and T and T" are each a group selected from the group consisting of those defined in claim 2. 4. Tetrapeptide, characterized by the formula: where R' is OU or NH 2 ; U is benzyl; X is epsilon-T"-LYS and Y is VAL or X and Y are both SAR; Z is ASP; and T" is a group selected from the group consisting of those defined in a)-b) of claim 2. 5 . Protected pentapeptide, characterized by the formula: where R' is OH or NH 2 ; U is H or benzyl; X is epsilon-T"-LYS and Y is VAL or X and Y are both SAR; Z is ASP; and T and T" are each H or a group selected from the group consisting of those defined in a)-b) i requirement 2. 6. Protected tripeptide, characterized by the formula: where Z is ASP; Y is VAL; R' is OU; U is benzyl where the phenyl ring is substituted with a lower alkyl; and T is selected from the group consisting of those defined in a)-b) in claim 2. 7 . Dipeptide, characterized by the formula: wherein X is SAR or epsilon-T"-LYS; T and T" are each selected from the group consisting of those defined in a)-b) above; T' is selected from the group consisting of and other symbols used have the same meanings as stated in claim 2.