Large-Scale Synthesis of a Cyclic Hexapeptide Analogue of

Jun 3, 1986 - Carl F. Homnick, Willie L. Whitter, Paul Curley, Ruth F. Nutt, and Daniel F. ... (1) Veber, D. F.; Saperstein, R.; Nutt, R. F.; Freiding...
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J. Org. Chem. 1987,52, 764-769

Large-Scale Synthesis of a Cyclic Hexapeptide Analogue of Somatostatin Stephen F. Brady,* Roger M. Freidinger, William J. Paleveda, Christiane D. Colton, Carl F. Homnick, Willie L. Whitter, Paul Curley, Ruth F. Nutt, and Daniel F. Veber Merck Sharp h Dohme Research Laboratories, West Point, Pennsylvania 19486 Received June 3, 1986

A recent report from our laboratories describes the preparation and biological properties of the highly potent cyclic hexapeptide somatostatin analogue 1. Herein we report the details of the large-scale synthesis of 1 developed to provide supplies for clinical study. The synthesis was accomplished entirely via solution chemistry, by stepwise elongation of the amino acid chain, starting from tyrosine methyl ester. Use of the mixed anhydride procedure allowed coupling without the need for protection of the tyrosine hydroxyl. Several intermediates were purified by crystallization, including the protected hexapeptide 10. Cyclization was achieved in high yield employing an improved technique using diphenylphosphoryl azide, with sodium bicarbonate as base. Only a single chromatographic procedure was required, at the stage of the penultimate cyclic peptide 12. Using this synthetic route we have been able to prepare >99% pure final product 1 in batches of 40-50 g. Yields based on each of the constituent residues ranged from 17% (Boc-alanine)to 52% (Boc-Dtryptophan).The process serves as an example of the kinds of considerations important for rapid and efficient large-scale synthesis of peptides.

The highly potent cyclic hexapeptide analogue of somatostatin, cyclo-(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe)(1) was recently disclosed.' This compound is undergoing clinical evaluation as an agent for improved control of glucose levels in insulin-dependent diabetic patients. Multihundred gram quantities of peptide 1 have been needed for assessment of its safety and efficacy. Herein we report the details of the synthesis developed to supply the required amounts of this material.2 Initial quantities of analogue 1 for biological evaluation were prepared by a procedure representative of our general approach to small cyclic somatostatin analogues: wherein protected linear precursor is assembled on solid support and cyclization steps are carried out in solution, after cleavage from the resin by hydrazinolysis. While this route, as summarized in Figure 1, proved well suited for synthesizing numbers of analogues, and up to about 1 g of cyclic peptide 1 in particular, it was not feasible for larger scale work either in terms of laboratory manipulations or economics. We also realized that problems associated with the synthesis of cyclic peptides of this type, which do not contain cystine, are quite distinctive from those confronted in synthesizing somatostatin itself.3a Key limiting factors of our earlier approach include (1) the extensive reliance on chromatographic procedures of limited capacity, particularly gel filtration, to obtain final product of the requisite purity; (2) special difficulties associated with the removal of byproducts formed in liquid HF;4 (3) the need for extensive utilization of protecting groups and excesses of costly reagents during couplings on solid phase; and (4) the unique problems relating to the preparation and handling of acyl azide^.^ As a result of these considera(1) Veber, D. F.;Saperstein, R.; Nutt, R. F.; Freidinger, R. M.; Brady, S. F.; Curley, P.; Perlow, D. S.; Paleveda, W. J.; Zacchei, A. G.; Tocco, D. J.; Hoff, D. R.; Vandlen, R. L.; Gerich, J. E.; Hall, L.; Mandorino, L.; Cordes, E. H.; Anderson, P. S.; Hirschmann, R. Life Sci. 1984,34, 1371. (2) A preliminary account of this work has already been presented Brady, S. F.; Freidinger, R. M.; Paleveda, W. J.; Colton, C. D.; Homnick, C. F.; Whitter, W. L.; Curley, P.; Nutt, R. F.; Veber, D. F.; Abstracts of Papers, 188th National American Chemical Society Meeting, Philadelphia, Pennsylvania (August 26-31, 1984); ORGN 214. See also: Nutt, R. F. Eur. Pat. Appl. EP 70.021 (Jan 19, 1983), Chem. Abstr. 1983, 99, 22929F. (3)Nutt, R. F.; Veber, D. F.; Curley, P. E.; Saperstein,R.; Hirschmann, R. I d . J.Pept. Protein Res. 1983,21,66 and references therein. (a) Cf. Diaz, J.; Guegan, R.; Beaumont, M.; Benoit, J.; Clement, J.; Fauchard, C.; Galtier, D.; Millan, J.; Muneaux, C.; Muneaux, Y.; Vedel, M.; Schwyzer, R. Bioorg. Chem. 1979,8,429. See also: Rivier, J.; Kaiser, R.; Galyean, R. Biopolymers 1978,17, 1927. (4) Tam, J. P.; Heath, W. F.; Merrifield, R. B. J.Am. Chem. SOC.1983, 105, 6442. (5) Meienhofer, J. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; 1979; Vol. 1,Chapter 4, p 197.

0022-3263/87/1952-0764$01.50/0

tions, an alternative approach based entirely on solution chemistry was developed. A number of structural elements in our target 1 had to be reexamined in the design of this new route. First of all, the reactivity of the side chains of tyrosine and lysine, which typically require protection during a multistage synthesis, had to be addressed. Secondly, lability of the indole in D-tryptophan to acidic and oxidative conditions requires, if not protection, limiting its exposure to such conditions. Thirdly, N-methylalanine, a secondary amino acid, may be subject to increased steric hindrance to coupling. Finally, of six possible linear precursors, the one expected to undergo cyclization most efficiently ought to be selected. In our initial work we had already determined that the linear sequence depicted in Figure 1 cyclizes quite well, and, in addition, we had prepared a large number of cyclic somatostatin analogues from linear hexapeptide precursors having N-terminal D-tryptophan. This sequence offered the additional advantage of introduction of the sensitive tryptophan residue as late as possible. Thus, structure 11 (Figure 4) became the intermediary target of our new solution synthesis. The overall route is outlined in Figures 2-4.

Noteworthy features of the process include (1) the use of mixed anhydride conditions to give efficient coupling with low racemization and easily removed byproducts without the need to protect the tyrosine hydroxyl; (2) an improved mild cyclization technique using diphenylphosphoryl azide (DPPA) for carboxyl activation, with sodium bicarbonate as base;6 and (3) removal of the lysine side-chain protection (Cbz) quantitatively by transfer hydrogenolysis' and direct isolation of final product without resort to column chromatography. At a number of points in the synthesis we were able to purify intermediates by crystallization, and only a single chromatographic procedure was required, at the stage of penultimate cyclic peptide 12. Using this synthetic route we have been able to prepare upwards of 150 g of >99% pure final product 1. The overall yield was 23% from tyrosine methyl ester hydrochloride (11 steps) and ranged from a low of 17% based on Boc-Ala-OH (12 steps), to a high of 52% based on Boc-D-Trp-OH (7 steps), the most expensive of the six starting amino acid derivatives. (6) Brady, S.F.; Paleveda, W. J.; Arison, B. H.; Freidinger, R. M.; Nutt, R. F.; Veber, D. F. In Peptides: Structure and Function, Proceedings of the Eighth American Peptide Symposium;Hruby, V. J., Rich, D. H., Eds.; Pierce Chemical Company: Illinois, 1983, p 127. (7) Brieger, G.; Nestrick, T. J. Chem. Reu. 1974, 74, 567.

0 1987 American Chemical Society

J. Org. Chem., Vol. 52, No. 5, 1987

Cyclic Hexapeptide Analogue of Somatostatin Z-CICbz (TFA)

Cbz

Bzl I

H-2-TrpLJs-Val-Phe-N-Me- Ala-Tyr-Oa

1

I *(HCI) H-Lys-OMe 93%

1.

H2NNH2/DMF

I 2. i-AmONO/DMF-HCI,

765

-20°C

1

+C

(6)

Boc-E-TrpOH (M.A.) bz

*Boc-g-TrpLis-OMe 12

3. Et(kPr)2N, high dilution

THF-H20

C bz 2-CICbz I

I

Boc-g-TrpLys-OH

Bzl I

Cyclo-((D-TrpLys-Val-Phe-N-Me- Ala-Tyr) 8

10 I1

9

I.

HF/IO% anisole,

1.

I

7

6

1

(M.A.)

2. H-Val-Phe-N-Me-Ala-Tyr-OMe,

5

3,

OOC

C bz 2. Sephadex gel filtration, 2 E HOAc

Boc-g-TrpL)!s-Val-Phe-N-Me-Ala-Tyr-OMe 88%

(2 steps)

Cydo-(N-Me-Ala-Tyr-E-Trp-Lys-Val-Phe) (HOAc) 6

8

7

9

10

I1

Figure 1. Synthesis of cyclic hexapeptide somatostatin analogues by combined solid phase/solution appr~ach.~

1

Cbz

1.

(mixed anhydride)

Ala-Tyr-OMe

NaHC03, O°C

(2)

HCI/EtOAc

2. Boc-Phe-OH

(M.A.)

(3) 96%

1.

1

Cbz I

Cydo-(N-Me-Ala-Tyr-E-Trp-Lys-Val-Phe)

*Boc-Phe-N-Me-Ala-Tyr-OMe

I

DPPA/DMF, 0.008l peptide

2. Silica gel column

1.

L/

(10)

Figure 3. Synthesis of linear hexapeptide.

Boc-N-Me- Ala-OH

59%

79%

I

I (HCI) H-~-TrpLys-Val-PhoN-Me-Ala-Tyr-OH(II)

(HCI) H-Tyr-OMe

*Boc-N-Me-

NaOH (pH 12) THF-H20

I *Boc-E-TrpL ys-Val-Phe- N-Me- Ala-Tyr-OH

1

87%

C bz

I

50% TFA/CH2C12

1.

4.4% HCOOH/MeOH

10% Pd/C, 2 hr. 2. 50% HOAc Evaporate/lyophilize

Cydo-(N-Me-Ala-Tyr-E-TrpLys-Val-Phe) HOAc

1

(12)

2. Roc-Val-OH (M.A.)

*Boc-Val-Phe-N-Me-Ala-Tyr-OMe 95%

(9)

(4)

50% TFA/CH2CIZ

(TFA) H-Val-Phe-N-Me-Ala-Tyr-OMe

(5)

Figure 2. Synthesis of the C-terminal linear tetrapeptide frag-

ment.

Synthesis of Linear Precursor. Since tyrosine was situated at the carboxy terminus of the target sequence, we were able to have an immediate test of the reactivity of the tyrosine hydroxyl during coupling. As anticipated, with the use of stoichiometric quantities of reagents, the mixed anhydride reaction was highly selective for a-amino over hydroxyl, and tetrapeptide 4 was readily assembled in stepwise fashion (Figure 2). In practice, both the re-

(1)

Figure 4. Cyclization and purification.

quirement for only minimal excesses of reagents during coupling and the overall simplicity of operation (convenient batch scaleup, extractive workups) proved to be important advantages. Intermediates 2, 3, and 4 were purified by crystallization, which greatly facilitated execution of this sequence on a large scale, and all crystalline products were obtained in purity >98%, as determined by HPLC. The somewhat lower yield in the second coupling step (BocPhe-OH onto N-methylalanyl dipeptide (cf. 3) was not entirely unexpected, assuming some steric hindrance to acylation (see above),s and unreacted dipeptide was determined to be present to the extent of about 10%. Sig(8) Tung, R. D.; Rich, D. H. J. Am. Chem. SOC.1985,107,4342 and references therein.

766 J. Org. Chem., Vol. 52, No. 5, 1987

nificantly, a t no point in the sequence was O-acylation at tyrosine detected. Both the HCl and TFA methods were explored for each Boc removal, and the superior approach was chosen in each instance. Thus, in the case of tripeptide 3, TFA was preferred because the resulting TFA salt was found to dissolve more readily in the next coupling reaction solution, whereas it was HCl treatment of dipeptide 2 that afforded a purer (crystalline) product than did the TFA procedure. Finally, we determined that when Boc removal from peptide 4 was conducted with HC1 a more polar impurity (see Experimental Section) not seen when TFA was used, was evident. We did not pursue isolation of this material, but the possibility of racemization at the N methylalanyl residue is not unreasonable on the basis of literature reports of racemization of N-methyl amino acid residues during exposure to strongly acidic condition^.^ Continuation of the stepwise incorporation sequence by reaction of TFA salt 5 with Boc-e-Cbz-lysineto give pentapeptide product was explored, but due to its failure to crystallize, this product would have required column chromatography for purification. Hence, the alternative (2 + 4) approach to hexapeptide 9 via dipeptide 8 was evaluated with excellent results, as shown in Figure 3. The crystallinity of dipeptide ester 7 allowed its isolation in high yield and purity, and saponification provided pure acid 8 in quantitative yield. Mixed anhydride coupling of fragments 5 and 8 proceeded, as expected under the carefully controlled activation/coupling conditions,1° without discernible racemization at lysine. Our efforts to crystallize hexapeptide methyl ester 9 were not successful, but the acid 10 could be crystallized in a straightforward fashion to provide the fully assembled linear precursor three steps from the end in better than 98% purity. Removal of Boc from peptide 10 to give HC1 salt 11 (Figure 4) was accomplished under conditions shown to limit loss of Cbz to