CA1335403C - Process and intermediates for lh-rh peptides - Google Patents

Abstract

Disclosed herein is an improved process, and inter-mediates therefor, for preparing gonadorelin and certain analogs. The key intermediate is the protected nonapeptide Pyr(W1)-His(W2)-Trp(W3)-Ser(W4)-Tyr(W5)-X1-Leu-Arg(W6)-Pro-OH wherein W1 to w6 inclusive are protective groups and X is Gly, D-2-(naphthylalanyl) or D-Trp(W7) wherein W7 is a protective group.

Description

~ -1- 1335~3 PROCESS AND INTERMEDIATES FOR LH-RH PEPTIDES

Field of the Inventlon This invention relates to an efficient process for pre-paring gonadorelin and certain analogs thereof, and to intermediates for use in the process.

Background of the Invention Gonadorelin, a naturally occuring peptide produced in the hypothalamus, causes the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. The peptide has the following structure:

Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 The isolation and structure elucidation of the peptide was reported by A.V. Schally et al., Biochem. Biophys. Res.
Commun., 43, 292 and 1334 (1971). Since that report, much attention has been directed to the applications of gonadorelin and its analogs in the fields of human and veterinary medicine. Gonadorelin is used as a diagnostic tool to evaluate the pituitary component of the hypotha-lamus-pituitary-gonad axis. Several of its more important analogs, for example leuprorelin, tryptorelin and nafarelin, are indicated for the treatment of prostate cancer. Other applications are possible in the treatment of endocrino-logical disorders such as amenorrhea, hypogonadism, inferti-lity and the like. In the veterinary domain, the main interest in these peptides lies in inducing ovulation and estrus synchronization in livestock.

133~03 As a consequence of the immense interest in these peptides, there is a need for an efficient process to pro-duce these peptides on a commercial scale.

5A number of processes for producing gonadorelin and its important analogs have been reported; for example, G.R.
Flouret, U.S. patent 3,796,697, March 12, 1974; H.V. Immer _ al., U.S. patent 3,835,108, September 10, 1974; S.
Sakakibara et al., U.S. patent 3,856,769, December 24, 1974;
10D.F. Veber and S.F. Brady, U.S. patent, 3,888,836, June 10, 1975; W. Konig et al., U.S. patent 4,275,001, June 23, 1981;
and R. Uhmann and K. Radscheit, U.S. patent 4,691,008, September 1, 1987. In some of these reports, it is claimed that the process is suitable for use on the large scale, see 15 for example, Immer et al and Uhmann and Radscheit. An analysis of the previous routes, however, reveals that there is room for improvement. A commercially scaled process most desirably would avoid the use of the azide method of coupling (which liberates obnoxious hydrazoic acid), use 20 coupling methods which minimize racemization, limit the number of liquid phase coupling steps so that large amounts of solvent are not required, and still produce the desired peptide efficiently with a high degree of purity.

25The present process has the features of being simple, rapid and avoids the use of obnoxious chemicals. It efficiently and economically produces the peptide on a commercial scale and with a purity of greater than 98%. By the particular choice of reaction conditions and inter-30mediate fragments, the process yields the desired peptide free of significant racemization and troublesome by-products.

~ 3~
- ` 1335~03 Summary of thls In~entlon The process of this invention comprises the preparation of the peptide of formula l Pyr-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-NH2 (l) wherein X is Gly, D-(2-naphthylalanyl) or D-Trp, whlch comprises:
a) forming a protected nonapeptide-resin of formu-la 2 pyr(wl )_His(p~2)-Trp(w3)-ser(w4)-Tyr(w5) Arg(W6)-Pro-S-P (2) wherein W1 is a protective group for Pyr, preferably benzyloxycarbonyl~ W2 is a protective group for the imidazolyl group of Hi~, preferably tosyl or 2,4-dinitro-phenylsulfenyl, W3 is a protective group for the indole nitrogen of Trp, preferably formyl, W4 is a protective group for the hydroxyl of Ser, preferably benzyl, W5 is a protective group for the hydroxyl of Tyr, preferably benzyl or 2,5-dichlorobenzyl, H6 is a protective group for the guanidino group of Arg, preferably tosyl or mesitylene-2-sulfonyl, X1 is Gly, D-(2-naphthylalanyl) or D-Trp(W7) wherein W7 is a protective group for the -indole nitrogen of Trp, preferably formyl, S is a photosensitive spacer and P is a benzhydrylamine resin or 4-~ethylbenzhydrylamine resin; the formation of the nonapeptide-resin being effected by successively couplin~, in the order of the sequence of the nonapeptidyl portion thereof, the amino acid residues to a proline-resin of formula H-Pro-S-P wherein S and P are as defined hereinabove;

B
.

.. .... . ..

~4~ 1335~03 b) cleaving the protected nonapeptlde-resin by photolysis to obtain the corresponding protected nonapeptlde of formula 3 P (Wl) His(W2)-Trp(W3)-Ser(W4)-Tyr(W5)-X -Leu-Arg(W6)-Pro-OH (3) wherein W1 to w6 inclusive and Xl are as defined here-inabove; and c) thereafter coupling the protected nonapeptide o~ ~ormula 3 with glyclnamide to obtain the corresponding protected decapeptide of formula 4 Pyr(W1)-His(W2)-Trp(W3)-Ser(W4)-Tyr(W5)-Xl-Leu-Arg(W6)-Pro-Gly-NH2 (4) wherein Wl to w6 inclusive and Xl are as defined here-inabove, and deprotecting the protected decapeptide of formula 4 to obtain the corresponding peptide of formula l wherein X is Gly, D-(2-naphthylalanyl) or D-Trp.
~.
Another aspect of this invention relates to the key intermediate of formula 3 wherein W1 to w6 inclusive and Xl are as defined hereinabove.

A further aspect of the nvention relates to inter-mediates linked by a photosensitive spacer to a benzhydryl-amine, resin or a 4-methylbenzhydrylamine resin. These intermediates are represented by formula 2 wherein W1 to w6 inclusive, S and P are as defined hereinabove.

B ` :
... .... ~

Detail~ of the Inventlon 1335403 The term 'residue' with reference to an amino acid means a radical derived from the corresponding -amino acid by eliminating the hydroxyl of the carboxyl group and one hydrogen of the -amino group.

In general, the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, see Biochemistry, 11, 1726-1732 (1972). For instance, Pyr, His, Trp, Ser, Tyr, Gly, Leu, Arg and Pro represent the 'residues' of L-pyroglutamic acid, L-histidine, L-tryptophan, L-serine, L-tyrosine, glycine, L-leucine, L-arginine and L-proline, respectively. D-Trp represents the residue of D-tryptophan. "D-(2-naphthyl-alanyl)" also is known as "3-(2-naphthyl)-D-alanyl".

The term 'photosensitive spacer' or 'photolabile spacer', designated by the symbol 'S', as used herein means a divalent organic linking unit which, when incorporated into the peptide-resin system, links the first amino acid building block to the resin by orthogonal covalent bonds, the unit or spacer being further characterized in that the bond between the spacer and the first amino acid residue can be cleaved by photolysis to afford the peptide (or the first amino acid residue) with a C-terminal carboxyl. For examples of such spacers, see D.H. Rich and S.K. Gurwara, Canadian patent 1,108,348, September 1, 1981; J.P. Tam et al, J. Amer. Chem. Soc., 102, 6117 (1980); F.S. Tjoeng and G.A. Heavner, J. Org. Chem, 48, 355 (1983); and J. Gauthier, Canadian patent application, SN 547,394, filed September 21, 1987. When utilized herein, the spacer is first attached to the resin to give the solid support of formula Q-S-P wherein Q is bromo, chloro or iodo, and S and P are as defined herein. Preferred spacers are represented by the formulae -6- 1 335~03 -CH(CH3)C0 - ~ -OCH2C0- and -CH(CH3)C0 ~ CH2C0-.

Turning to the process of this invention, one feature of the formation of the protected nonapeptide of formula 2 is the protection of labile side chain groups of the various amino acid residues with suitable protective groups to pre-vent a chemical reaction from occurring at that site until after the completion of coupling steps to produce the pro-tected peptide of formula 4. Another common feature is the protection of ~-amino group of an amino acid while the free carboxyl group of that reactant is coupled with the free ~-amino group of the second reactant; the ~-amino protec-tive group being one that can be selectively removed to allow the subsequent coupling step to take place at the amino group from which the protecting group is removed.
The starting material for the process, a proline-resin of formula H-Pro-S-P wherein S and P are as defined herein, can be prepared as follows: An ~-amino protected proline, preferably N~-(t-butyloxycarbonyl)-proline is coupled to a solid support of formula Q-S-P wherein Q is bromo, chloro or iodo and S and P are as defined herein in the presence of potassium fluoride or cesium chloride to give the corres-ponding solid support having an ~-amino protected proline linked thereto. Therafter, the ~-amino protective group of the latter resin derivative is removed to give the desired proline-resin starting material with a free amino group. In the instance where the ~-amino protective group is t-butyl-1335~03 oxycarbonyl, 30% to 50% (v/v) trifluoroacetic acid inmethylene chloride containing 3% to 5% (v/v) ethanedithiol is an effective deprotecting agent.

The practice of the process involves as a first-stage the formation of the protected nonapeptide-resin of formula 2 from the proline-resin of formula H-Pro-S-P by stepwise coupling in the desired order the appropriate -amino pro-tected amino acids to the growing peptide-resin using a modified form of solid phase synthesis. (For a recent review of solid phase synthesis, see J.M. Stewart and J.D. Young, 'Solid Phase Peptide Synthesis', 2nd ed., Pierce Chemical Company, Rockford, Illinois, U.S.A., 1984.) More explicit-ly, coupling of the first six residues of arginine, leucine and subsequent amino acid residues up to and including the tryptophan residue is effected using benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), described by B. Castro et al., Tetrahedron Letters, I4, 1219 (1975). Subsequent couplings of the histidine and pyroglu-tamic acid residues are achieved by using dicyclohexyl-carbodiimide (optionally adding l-hydroxybenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine or N-hydroxy-succinimide) as the coupling agent, or by employing the 'mixed anhydride' activated form of the ~-amino protected acids. Each ~-amino protected amino acid is introduced into the reaction system in a relatively low excess (two molar equivalents) and complete coupling to the growing peptide-resin was achieved with 45 minutes; an exception, however, was the coupling of the arginine residue which required double coupling. The success of the coupling reaction at each stage is monitored by the ninhydrin reaction as described by E. Kaiser et al., Anal. Biochem., 34, 595 1335~03 (1970). Removal of the ~-amino protective group completes the coupling cycle. In the instance, where the ~-amino protective group is a t-butyloxycarbonyl, trifluoroacetic acid in methylene chloride is used to effect deprotection.
The efficiency (yields) of the deprotection step in the latter instance can be improved by using ethanedithiol or a similar scavenger for t-butylcarbocations, especially when the t-butyloxycarbonyl i5 being removed from the growing peptide-resin in which the terminal or last- added amino acid residue is the arginine or leucine residue, cf. D.
Le-Nguyen et al., J. Chem. Soc. Perkin Trans., 1, 1915 (1987).

The next step following the formation of the protected nonapeptide-resin of formula 2 is the cleavage of the pro-tected nonapeptide therefrom. The cleavage is achieved by photolysis. The photolysis is accomplished by dissolving or suspending the protected nonapeptide-resin in a photo-lytically stable liquid media; for example, dioxane, dimethylformamide, methanol, ethanol or N-methylpyrrolidine;
purging the solution or suspension of the nonapeptide-resin with argon or nitrogen to remove any dissolved oxygen; and then irradiating the suspension or solution with photo-lytically effective ultraviolet light. In practice, irradi-ation at a wavelength of 350 nm has been found to be very effective. In this manner, the protected nonapeptide of formula 3, a key intermediate of the present process, is obtained.

~9~ 1 3 3 5 40 3 Thereafter, the key intermediate of formula 3 is trans-formed to the peptides of formula 1 by coupling and depro-tection reactions. Thus, the protected nonapeptide of formula 3 is coupled with glycinamide. For this particular coupling the choice of coupling agent is not critical.
Coupling is readily achieved by activating the terminal carboxyl of the peptide of formula 3 with coupling agents commonly used in peptide synthesis. Examples of such coupling agents are dicyclohexylcarbodiimide toptionally with the additives noted above), methyl chloroformate, 1-hydroxybenzotriazole, N-hydroxysuccinimide and, preferably BOP. In this manner, the corresponding protected deca-peptide of formula 4 is obtained. Subsequent deprotection of the latter compound by standard methods to remove its remaining protective groups, for example by treatment with hydrogen fluoride, affords the corresponding peptide of formula 1 in which X is Gly, D-(2-naphthylalanyl) or D-Trp.

The following examples illustrate further this invention. Abbreviations used in the examples include BOC:
t-butyloxycarbonyl; BOP: benzotriazol-l-yloxytris-(dimethylamino)phosphonium hexafluorophosphate; TFA: tri-fluoroacetic acid; CH2C12: methylene chloride; DIEA:
diisopropylethylamine; DMF: dimethylformamide; EtOH:
ethanol; DCC: N,N'-dicyclohexylcarbodiimide; HOBt: 1-hydroxybenzotriazole; and MeOH: methanol. Solution percent-ages are calculated on a volume/volume basis unless stated otherwise. The following terms are trademarks: Pyrex and Rayonet.

1335~03 Example 1 Preparation of (4-(2-chloropropionyl)phenoxy)acetamido-methyl-copoly(styrene-1% divinylbenzene) A suspension of benzhydrylamine resin (301.4 g, amine content = 0.43 mM/g) in 5% DIPEA in CH2C12 (2.5L) was stirred at room temperature (20-22 C) for 20 min. The resin was collected on a filter, washed well with CH2cl2 and air-dried.
A solution of (4-(2-chloropropionyl)phenoxy)acetic acid (59.3 g) and HOBt (37.5 g) in DMF (lL) was stirred at 0 C
for 15 min. DCC (50.6 g) was added to the solution and stirring of the solution at 0 C was continued for lh.
The above resin was added to the solution. The mixture was stirred at room temperature for 18h. Thereafter, the solid material in the mixture was collected on a filter, washed with DMF, MeOH, CH2C12 and EtOH, and air-dried to give the desired solid support (322 g, substitution = 0.59 mM/g);
negative Kaiser test (E. Kaiser et al., Anal. Biochem., 34, 595 (1970).

Example 2 Preparation of the protected nonapeptide: Pyr(Z)-His(Tos)-Trp(CHO)-Ser(Bzl)-Tyr(2,6-diclBzl)-Gly-Leu-Arg(Tos)-Pro-OH

N~-Boc-Pro-OH (23.30 g, 108 mmol) and potassium fluoride (14.10 g, 243 mmol) were added to a stirred suspen-sion of the solid support of example 1 (50.00 g, 27.0 mmol) in DMF (1 L) at 70 C. The reaction mixture was stirred at 70 C for 24 hours. The mixture is then cooled and filtered. The collected solid i5 washed successively with DMF, DMF-H20 (1:1), H20, DMF-H20 (1:1), dioxane, ethanol, CH2C12 and ethanol, and dried under vacuum.
The dried amino acid support (54.10 g) showed a proline content of O. 47 mmol/g, as determined by deprotection of an aliquot followed by picric acid titration; see the method of B.F. Gisin, Anal. Chim. Acta, 58, 248 (1972).
The latter proline-solid support (5.00 g, 2.35 mmol) was used to form the amino acid sequence of the desired non-apeptide by a modification of the solid phase technique of R.B. Merrifield, J. Amer. Chem. Soc., 85, 2149 (1963). The following protocol was used: a) Boc-deprotection: 30% TFA
in CH2C12 (2 times, firstly for 1 min and then for 30 min) was used: the scavenger ethanedithiol (5% by volume of the TFA-CH2C12 solution) was used for Pro, Arg, and Leu.
b) Wash: Isopropanol (2 times, each for 0.5 min) followed by CH2C12 (2 times, each for 0.5 min) was used. In the case of Pro, Arg and Leu, the scavenger ethanedithiol (3% by volume) was added to the isopropanol and CH2C12 washes.
c) Coupling: All the amino acid couplings except for His and Pyr were done in situ by the activated ester method using the amino acid (2.0 equivalents), BOP reagent (2.0 equivalents), DIEA (12 equivalents) in CH2C12; coupling time was effected in less than 45 min as determined by the Kaiser test; only Arg was double coupled. When His or Pyr were to be coupled,a neutralization cycle was incorporated into the system prior to the coupling. Accordingly, neutralization was effected with 5% DIPEA in CH2C12 (2 times, rirstly for 1 min and then for 3 min), follow-ed by a washing with CH2C12 (3 times for 0.5 min); thereafter, the coupling of the His or Pyr (4-equ~valents) was achieved in 4h, the His or Pyr being preactivated with DCC (2 equiva-lents, 30 min). d) Wash: The post-coupling wash was effected with CH2C12 (2 times for 0.5 min), then iso-propanol (2 times for 0.5 min) and finally, CH2Cl2 (2 times for 0.5 min).

The Boc group gave Na protection for all amino acids.
Side chain protection was as follows: tosyl for Arg and His, 2,6-dichlorobenzyl for Tyr, formyl for Trp and benzyl-oxycarbonyl for Pyr.

1~ On completion of the peptide sequence, the protected nonapeptide-resin was collected on a filter, washed with EtOH and dried under vacuum over phosphorus pentoxide and then sodium hydroxide. The dried nonapeptide-resin weighed 8.35g, (95~ yield, b~sed on the proline-solid support).
Cleavage of the completed protected nonapeptide from the solid support was done by photolysis in the following manner: A stirred suspension Or the protected resin-peptide (8.57 g) in DMF: EtOH (1:4,700 mL), contained in a PYREX*
vessel, was purged with argon for one hour at 0 C.
Utilizirg a RAYONET*RPR-208 photochemical chamber (Southern New England Ultraviolet Co., Hamden, Connecticut, U.S.A.), the suspension was irradiated at 350 nm for 20 h. The reaction mixture was riltered. The filtrate was concen-trated to dryness under vacuum. The residual oil wastriturated with diethyl ether. The resulting solid was collected, washed with cold diethyl ether and dried to give 2.00 g (100% yield) cr the corresponding protected nona-peptide.

* Trade mark . -- .

~ -13- 133S403 Example 3 Preparation of Gonadorelin The protected nonapeptide of example 2 (1.87 g, 1.03 mmol) was dissolved in DMF (10 mL). BOP (579 mg, 1.30 mmol) was dissolved in DMF (3 mL). The two solutions were combined. Glycinamide trifluoroacetate (248 mg, 1.31 mmol) dissolved in DMF (6 mL) was added to the combined solution.
The mixture was stirred under nitrogen for 5 min at room temperature, and then N-methylmorpholine (0.43 mL, 3.93 mmol) was added to the mixture. The reaction mixture was ~tirred at room temperature under nitrogen for 20 h and then poured dropwise into stirred ethyl acetate (300 mL). Stir-ing is continued for 30 min. The solid in the resultant suspension is collected, washed with ethyl acetate and dried under vacuum to give the protected decapeptide Pyr(Z)-His-(Tos)-Trp(CHO)-Ser(Bzl)-Tyr(2,6-diClBzl)-Gly-Leu-Arg(Tos)-Pro-Gly-NH2 (1.36 g, 70% yield) The protected decapeptide (1.00 g) was mixed with anisole (1 mL) and ethanedithiol (1.5 mL) in a reaction flask. The mixture was cooled to -78 C. Anhydrous hydrogen fluoride (15 mL) was condensed in the reaction flask. The mixture in the reaction flask was stir-red at 0 C for lh. The HF is removed by distillation under reduced pressure in about 45 min. The residue was triturated with diethyl ether. The solid was collected, washed with diethyl ether, and dried under vacuum to give the crude product (1.26 g). The crude product was extracted with 15~ aqueous acetic acid. The extract was filtered to remove undissolved material and the filtrate was lyophilized to yield a white flocculent solid (0.64 g, 100% yield). HPLC
analysis showed this crude product to be 85% pure.

Product Purification - Preparative HPLC WâS performed on a Waters model ~00 15at 23C nm using a Vydac C18 column (2.2 x 50 cm) 15-20 y.
The gradient of acetonitrile in 0.1% aqueous TFA was 10-15d in 20 minutes followed by 15-20~ in 40 min, with a flow-rate of 20 ml/min. Pure fractions were pooled and lyophili~ed after removal of acetonitrile under reduced pressure. A
sample of crude product (340 mg) was purified to afford the trifluoroacetate salt (111 mg, 33~ yield) in tne pure form.
Conversion of this material (103 m~) through a Michel Miller (C18 VYDAC* 20-30~) column using 50~ aqueous acetic acid afforded the corresponding acetic acid addition salt (92 mg, yield 90~). Analytical HPLC analysis showed this product to be 99.7% pure.

* Trade mark ,. ..
: . . ~
.. .

Claims (6)

1. A process for preparing the peptide of formula 1 Pyr-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-NH2 (1) wherein X is Gly, D-(2-naphthylalanyl) or D-Trp, which comprises:

a) cleaving a protected nonapeptide-resin of formula 2 Pyr(W1)-His(W2)-Trp(W3)-Ser(W4)-Tyr(W5)-X1-Leu-Arg(W6)-Pro-S-P (2) wherein W1 is a protective group for Pyr, w2 is a protective group for the imidazolyl group of His, W3 is a protective group for the indole nitrogen of Trp, W4 is a protective group for the hydroxyl of Ser, W5 is a protective group for the hydroxyl of Tyr, w6 is a protective group for the guanidino group of Arg, X1 is Gly, D-(2-naphthylalanyl) or D-Trp(W7) wherein W7 is a protective group for the indole nitrogen of Trp, S is a photosensitive spacer of the formula or and P is a benzhydrylamine resin or 4-methylbenzhydrylamine resin, by photolysis to obtain the corresponding protected nonapeptide of formula 3 Pyr(W1)-His(W2)-Trp(W3)-Ser(W4)-Tyr(W5)-X1-Leu-Arg(W6)-Pro-OH (3) wherein W1 to W6 inclusive and X1 are as defined in this claim; and b) thereafter coupling the protected nonapeptide of formula 3 with glycinamide by activating the terminal carboxyl of the protected nonapeptide of formula 3 with a coupling agent to obtain the corresponding protected decapeptide of formula 4 Pyr(W1)-His(W2)-Trp(W3)-Ser(W4)-Tyr(W5)-X1-Leu-Arg(W6)-Pro-Gly-OH (4) wherein W1 to W6 and X1 inclusive are as defined in this claim, and deprotecting the protected decapeptide of formula 4 to obtain the corresponding peptide of formula 1 wherein X
is Gly, D-(2-naphthylalanyl) or D-Trp.

2. A process as claimed in claim 1 wherein the protected nonapeptide resin of formula 2 is prepared by successively coupling with a coupling agent, in the order of the sequence of the nonapeptidyl portion of the protected nonapeptide resin, the required amino acid residues to a proline-resin of formula H-Pro-S-P wherein S and P are as defined in claim 1.

3. A process of claim 1 wherein the coupling of the protected nonapeptide of formula 3 with glycinamide is effected with BOP as the coupling agent.

4. A process of claim 2 wherein the successive coupling to the proline-resin of the first six residues of Arg, Leu and subsequent amino acid residues up to and including the Trp residue is effected with BOP as the coupling agent.

5. A protected nonapeptide of formula 3 as defined in claim 1.

6. A protected nonapeptide-resin of formula 2 as defined in claim 1.