CA2807162C

CA2807162C - Solid phase peptide synthesis via side chain attachment

Info

Publication number: CA2807162C
Application number: CA2807162A
Authority: CA
Inventors: Kleomenis K. Barlos
Original assignee: Chemical and Biopharmaceutical Laboratories of Patras SA
Current assignee: Chemical and Biopharmaceutical Laboratories of Patras SA
Priority date: 2013-02-21
Filing date: 2013-02-21
Publication date: 2023-03-21
Anticipated expiration: 2033-02-21
Also published as: CA2807162A1; CA3186364A1

Abstract

The present application discloses peptides and peptaibols of high purity may be obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.

Description

SOLID PHASE PEPTIDE SYNTHESIS
VIA SIDE CHAIN ATTACHMENT
SUMMARY:
[0001] Peptides and peptaibols of high purity were obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.
Definitions and Abbreviations:

[0002] "Hya" or "hydroxyl amino acid(s)" means amino acids that contain a hydroxyl (-OH) group.

[0003] N-terminus or amino terminus is the first amino acid in a peptide chain.

[0004] C-terminus or carboxy terminus is the last amino acid in the peptide chain as shown below.
amino terminal carboxy terminal or N-terminal or C-terminal amino acid amino acid NFI2-CHR-CO-Aa1-AA2 ...................... AAI-NH-CHR-CO2H
peptide

[0005] "P" or "solid support" or "resin" means an insoluble material containing a functional group(s) suitable to react and link with an amino acid or peptide. The solid support or resins are well known in the art.

[0006] "Alkyl" such as Ci.loalkyl or C1_6alkyl, means a branched or unbranched fully saturated acyclic aliphatic hydrocarbon group (i.e. composed of carbon and hydrogen containing no double or triple bonds). In some embodiments, alkyls may be substituted or unsubstituted.
Alkyls may include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like, and in some embodiment, each of which may be optionally substituted. Non-exclusive alkyl substituents may include C1.3a1koxy, halo (F, Cl, Br or I), nitro, amino, -SH and -OH,

[0007] "Attachment" means the linking of an amino acid or a peptide or peptide derivative to an insoluble support.

[0008] "Hse" means homoscrine; "Hnv" means hydroxylnorvaline.

[0009] "SPPS" or "solid phase peptide synthesis" means the synthesis of a peptide with the use of a resin as described herein.

[0010] "pNA" means 4-nitro anilide.

[0011] "DME" means dimethoxy ethane.

[0012] "Acid sensitive resin" means an insoluble material or resin containing a functional group(s) suitable to react and link with an amino acid or peptide, which may be cleaved from the peptide by acidic treatment.

[0013] "Acid sensitive protecting group" means a protecting group which may be cleaved from the amino acid or peptide or peptide derivative by acidic treatment or under acidic condition.

[0014] "Peptaibol" means a peptide which contain at its C-terminal position an amino alcohol instead of an amino acid or an amino acid amide.

[0015] "Step-by-step" means the method of peptide synthesis where any of the amino acids contained in the peptide chain is introduced individually and sequentially.
The method may or may not involve an intermediate purification step.

[0016] "Protected peptide" means the peptide with all functional groups blocked or protected by protecting groups.

[0017] "Partially protected peptide" means the peptide which contains at least one functional group blocked or protected by a protecting group.

[0018] Solid phase peptide synthesis is traditionally performed by the attachment of the C-terminal amino acid through its ct-carboxyl function on a suitable solid support and elongating the peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. Several hundred thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals.

[0019] In contrary to the attachment of the C-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side chain on suitable resins and their application in SPPS is described very briefly, in particular in less than 30 publication and patents. Most of these publications describe the attachment of the amino acids through a side chain carboxyl function of Asp and Glu. To our knowledge, the reports of the side chain attachment of amino acids through a side chain hydroxyl function and application in peptide synthesis are limited:
The side chain attachment of Fmoc-Hya-pNA (Formula Al) [A. Bernhardt, M.
Drewello and M. Schutkowski, The solid-phase synthesis of side-chain-phosphwylated peptide-nitroanilides J. Peptide Res. 50, 1997. 143-152] and their use for the synthesis of short nitroanilide substrates, the synthesis of Fmoc-Hya-Oallyl esters (Formula A2 [L. Rizzi, K.
Cendic, N. Vaiana, S. Romeo, Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation, Tetrahedron Letters 52 (2011) 2808-2811]) on 2-chlorotrityl resin with the aid of microwaves for application in the preparation of cyclic peptides and the synthesis of Fmoc-Tyr-O-methyl ester (Formula A3 [C. Cabrele, M. Langer and A.
G. Beck-Sickinger, Amino Acid Side Chain Attachment Approach and Its Application to the Synthesis of Tyrosine-Containing Cyclic Peptides, I Org. Chem. 1999, 64, 4353-4361D, attached on resins of the benzyl-type by the Mitsunobu redox-alkylation of the Tyr-phenoxy function and their application for the synthesis of short cyclic peptides. To our knowledge, the side chain attachment of Hse and Hyp have never been disclosed. In addition, the application of side chain attached Hya on acid sensitive resins for the solid phase synthesis of protected peptides, protected peptide fragments and of protected peptide amides and peptaibols have not been reported.
P

AlA2 A3 CI a lel I X
Fmoc-HN
Fmoo-HN o Fmoc-HN '20-12 Solid Phase Peptide Synthesis

[0020] In one embodiment, there is provided an improved synthesis of peptide acids, peptide amides, and peptaibols of pharmaceutical interest.
xe H
\d- -1=1 -I- 0 V I V Y
PO-1 ly a-A

[0021] Formula I Formula II

[0022] In one aspect of the present application, the peptides were produced very efficiently in high yield and purity by attaching a hydroxy amino acid through its amino acid side chain, or a small peptide which contain in its sequence a hydroxy amino acid on a resin of the trityl or benzhydryl-type, resulting in amino acid-resin conjugates or peptide resin conjugates of Formula I-TV, wherein P is a solid-phase support selected from the supports used in solid phase peptide synthesis, Pr' is H or an amino protecting group selected from Fmoc, Doe, Trt, Dde and Alloc, wherein Pr2 is an acid sensitive hydroxyl protecting group selected from Trt, Clt, Mmt, Mtt, Dpm and tBu, wherein Hya is a hydroxy amino acid selected from D- or L-Ser, Thu, Tyr, Hse, Hyp, Hnv etc ., and A is OH, an acid sensitive allcoxy group selected from OTrt, OCR, OMmt, 0Mtt, ODpm and OtBu, NH2, NHR1, NR1R2 wherein RI and R2 are independently an alkyl group a protected or semi protected peptide containing 1-10 amino acids in its sequence.

[0023] In another embodiment, we disclose that peptaibols, such as octreotide, were obtained by solid phase synthesis using the resin-bound amino alcohols of the Formula III-VI selected from amino alcohols which are derived from the naturally occurring hydroxy amino acids, wherein P, X, V, Z and Pr' are as defined above, wherein R3, R4 are alkyl, aryl or aralkyl groups, and Pr2 is an acid sensitive protecting group of the trityl, benzhydryl or benzyl type.
X
e Z

Formula III Formula IV
X7f.. Z Z
Ic>s' V V

Pr' Prf Formula V Formula VI

[0024] In addition we disclose for the first time that peptides prepared with the application of resins of the Formula I-IV, where the peptides are attached through the side chain hydroxyl function of a hydroxyamino acid on resins of the trityl type, may be cleaved from the resin by mild acidic treatment and wherein the side chain protecting groups of the tBu and benzyl-type remain intact. In one aspect, the cleavage from the resin occurs by the treatment with 1-3 %
acid solutions, such as TFA, diluted HCl solutions, optionally adding scavengers, in a solvent.
In another aspect, the cleavage may be performed in a solvent such as DCM or acetone. Such partially protected peptides have been found to be useful in the synthesis of longer peptides by fragment condensation in solution or on solid phase. The present method expands the versatility of the application of the resins described herein, and also results in significantly improving the purity of the resulting pharmaceutical peptides, and at the same time, substantially reducing the cost of their synthesis.

[0025] Several peptides of pharmaceutical interest were produced as representative of the new process described herein, either by the step-by-step procedure or by fragment condensation in solution and on solid phase; or a combination thereof. The examples below are representative and do not limit their application in any way to other peptides.
Lanreotide:

[0026] In one embodiment, Lanreotide was produced by solid phase synthesis using resin-bound Thr-amide as shown below:
P
= Br-Resin Fmoc-Thr(Resin)-NH2 DIP EAFrnoc-Thr-NH2 + H i \ OCH3 I
Br Sequential deprotection and coupliing steps, 7 cycles Boc-D-2-Nal-Cys(Trt)-Tyr(CIU-D-Trp-Lys(SAtt)-Val-Cysfirg-Thr(Resin)-NH2 darlmectu:itoann, clesaymagielefrozrionresIn I I
H-D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 Lanreotide Human insulin B-chain:

[0027] Optionally the human insulin B chain was synthesized by SPPS. In one aspect, the synthesis begins from the resin-bound Thr-t-butyl ester as described in the example, using the 4-methoxy benzhydryl resin. Optionally the synthesis may also be performed on solid phase by condensing the 1-8 partially protected Boc-Phe-Val-Asn(Trt)-Gln(Trt)-His(Trt)-Leu-Cys(Trt)-Gly-OH fragment with the resin-bound 9-30 fragment; or after the selective cleavage of the partially protected 9-30 fragment from the resin with condensation in solution of the 1-8 and 9-30 fragments.
P
= Br-Resin Fmoc-ThilResin)-0tBu DIPEAFmoc-Thr-DtBu + H 0C113 Br Sequential deprotection and coupliing steps, 22 cycles H-Phel-Val-Asn(Trt)-Gln(Trt)-His(Trt)-Leu-Cys(Trt)-Gly-Ser(tBu)-His(Trt)-Leu-Val-Glu(18u)-Ala-Leu-Tyr(tBu)-Leu-Val-Cys(7rO-Gly-Glu(t8u)-Arg(Pbf)-Gly=Phe=Phe-Tyr(tBu)-Thr(t8u)-Pro-Lys(Boc)-Thr(Resin)3'-OtBu ideprotection and cleavage from the resin H-Phel-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu=Tyr-Leu-Val-Cys-Gly-GluArg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thrm-OH human Insulin B-Chain Salmon Calcitonin:

[0028] Optionally salmon calcitonin may be produced starting the synthesis from resin bound Fmoc-Thr-Pro-NH2. The peptide chain is then elongated using Fmoc-amino acids.
P
10) -Br-Resin Fmoc-Thr(Resin)-Pro-NH, ...--_ PEA Fmoc-ThrPro-NH2 * li 0CH3 Sequential deprotection and Gout:ding steps, 22 cycles Sr al H-Lys(Boc)-Leu-Ser(tau)-GlnlTrO-Gliqtaugeu-les(Trt)-Lys(Boc)-Leu-GlniTrt)-Thr(1130-Tyr(tBu)-Pro-ArESPbf)-Thr(tau)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Tbr(Resh)-Pso-NH, as Boe-Cys-Ser(tBuyAsn(Trt)-Les-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH 1 = 1 I
Boe-Cys-Ser(lBu)-Asn(TrIgeu-Ser(tBuyThr(lBu)-Cys-Val-leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-HisfIrt)-Lyaleuan(Trt)-rhr(lau)-Tyr(tSu)-Pro-Arg(PleyThr(lBu)-Asn(Trt)-Thqtlitu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-Nli, H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-HIS-Lys-Leu-GINTenTyr-Pro-Arg-Thr-esn-Thr-Gly-Ser-Gly-Thr-PraNH2 Sal110311 Caldtonln

[0029] Optionally the resin-bound salmon calcitonin is produced by fragment condensation on the resin as shown above, for example, or in solution as shown below using 2-4 fragments.
P
oil= Br-Resin Fmoc-Thr(Resin)-Pro-NH2 CI PEA Finoc-Thr-Pro-N1-12. H A 0a-13 Sequential deprotection and coupIllnq steps, 22 cycles Br II
H-Lyseitocgeu-Ser(IBW-GliqTn)-GluitBui-Letr-lieffrq-Lys(BocKeu-StreTre-Thr(tBs)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(T1)-ThrIBu)-Gly-Seran)-Gly-Thr(ReSh)-Pro-NI-6 sa 1 2% TFArrES
II
H-Lys(Bory-reu=Ser(tfiu)-Gln(Trt}r3rultBui-Leu-His(Tre-Lys(Boc)-Leu-GeXT*
Thr(tBu)-Tyr(teu)-Pro-Arg(P10)-Thr(tSu)-Asn(Td)-Thr(tBu).01y-Ser(Trt)-Oty-Thr-Pro-N1 .
I I
Boc-Cys SerBBLO-Rsn(Tri)-Leu-Ser(lBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
no II I
Soc-Cys-SeetBu)-Asn(TrI)-Leu-Ser(eu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-leu-Ser(tBu)-GlreTrq-Gki(tBu)-Leu+lis(Tet)-Lys(Boc)-Leu-GIn(Tr)-Thr(tBu)-Tyr(tBu)-Pro-Are(Pbt)-Thr(tElu)-sasqTrO-ThreBui-Gly-Sargiti-Gly-Thr-Pro-HH2 I

H-Cys-Ser-Asn-Leu-Ser-Thr-CysNal-Leu-Gly-Lys=Leu-Ser-Gin-Glu-Leu-His-Lys-Leu-GlaIhr-Tyr-Pro-Arp-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Salmon Ceicitonin Octreotide:

[0030] In another embodiment, octreotide was efficiently synthesized by the attachment of Fmoc-threoninol-OTrt to the 4-methoxybenzhydryl resin through the side chain of threoninol as shown below, followed by the octreotide chain assembly using Fmoc-amino acids and finally cleaving octreotide from the resin with subsequent or simultaneous Cys-oxidation.
Fmoc-threoninol-OTrt is much easier to be produced than the Fmoc.-Thr(tBu)-ol which may be attached onto the resin through the hydroxymethyl group of threoninol on a suitable resin.
This is because H-Thr(OtBu)-ol, used as the starting material for the production of Fmoc-Thr(tBu)-ol, is much more difficult to be produced than Fmoc-threoninol-OTrt used in the attachment of threoninol through its side chain onto the resin.
och, 40 .Resn-lie OH H it P 0-Restn Br CH3 Fmoc.HN, OHCIS + Trt-ci 1)-1¨'EA. Fox-NH-CI-CH' Fnioc-tIN-_,314 0-Trt PEA .C-7rt 0-Resin H-D-PIN-Cys(MmtyPhe-0-Trp-Lys(PANt=PIte=Thr(Trt)-CyNMmt) -tiN
--.0-Trt TFAIDCWTIPS
OH
H-D-Ph=-Cyo-Pe-D-Trptys-Phe-Thr-Cyt-HN-CH.
\-OH
I Welton OH
I
H-O-Ithe CI-PINt-D=Trp-lye=Plte=Thr-4s 41*Octreettde OH
Exenatide:

[0031] In another example, Fmoc-Ser-NH2 was attached through its side chain on trityl resin and used for the synthesis of exenatide. The synthesis may be performed by the step-by-step manner or by fragment condensation in solution after cleavage a partially protected exenatide fragment from the resin by mild acidic treatment or on solid phase, as described below.
According to this method, most impurities typically formed during the synthesis of many Pro and Gly residues containing peptides are completely avoided and peptides of high purity are obtained. The method also allows the complete avoidance of impurities originating from the cleavage of peptides from peptide amide linkers using other methods known in the art, which significantly reduce the yields and purity of the peptide.

H 0-Resin Fmoc-HN
+ CI s- Fmoc-Ser(Resin)-NH2 P ---"' -- DIPEA Emoc-HN¨

NH2 NH, 0 = Resin-CI 0 1 step-by-step 38 cycles ti-His(Trt)-Gly-Glu(lBu)-Gly-Thr(tBuyPhe-Thr(tBuySet(tilluyAsp(tBu)-X-Lys(Boc)-Gin(Trt)-Met-Glu(tRu)-Glu(tBuy Glu(tBuI-Ala-Val-Arg(Pbfgeu-Phe-Ile-Clu(tBui-Trp(Soc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBui-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 X = Leu-Ser(Su) or Leu4Ser 1 DeprotectIon H-His-01y-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Oln-Met-Gloalu-Glis-ade-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N112 Exenatide

[0032] in one aspect, exenatide may be produced by cleavage of the partially protected peptide 12-39 from the resin and condensing it in solution as shown below with the partially protected 1-11 fragment. Alternatively, the condensation to obtain protected exenatide may be performed with the fragments 1-13 and 14-39.
OH 0-Resin DIPEA
-restr = o-Sr(sinyNH2 Finoc-HN + CI P ---..... Frnoc-HN FmoeRe -e,tr.
NH2 NH, 0 = Resin-CI 0 1step-by-step H-Gln(TrtyMet-Glu(tBu)-Glu(tBu)-Elki(tBu)-PJa-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(tau)-Ttp(Boc)-Lau-Lys(Boc)-Asn(Trl)-Gly-Gly-Pro-SentBu)-SerdBu)-Gly-Ale-Pro-Pro-Prc-Ser(Resin)-NH, 1 8 cic41 istra4uTirChtu?-1;1(TywsTocu).)7H-1"ffie*
condens4lon X= Leu-Ser(lBu) or Leu-arSer Boc-His(Trt)-Gly-GludBuyGly-Thr(tRu)-Phe-ThrletO-SergBui-AspOBLO-X-Lys(Boc)-GIMTrt)-Met-GlintBtO-Clu(tBu)-Glu(113u)-Ala-Val-Arg(Pb0-Leu=Phe-11e-Glu(tBu)-Trp(Soci-Leu-Lye(B0c)-Atm(Trt)-Gly-Gly-Pro-Sar(ta)-Ser(iBuj=Gly-Ala-Pro-Pro-Pro-Ser(Rasin)-NH2 1 *protection, cleavage from the resin H-His-Gly-Glu-Oly-ThsPhe-Thr-Ser-Asp-Leu-Ser-Lys-GIn-Met-Giu-Glu-Giu-Ala-VakArg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N142 &mode Pramlintide:

[0033] The method is also highly effective in the production of amylin peptides. In one aspect, . the side chain attachment may be performed using one of the C-terminal Ser, Thr or Tyr residues of amylin or its derivatives such as pramlintide. The synthesis may be performed in the step-by-step manner or by fragment condensation in solution or on solid phase. By incorporating pseudopro lines (W, see Mutter et al, Peptide Res. (1995 8, 145) into the growing peptide chain, the synthesis is accelerated and the purity of the peptide obtained is improved.
Fmoc-Tyr(Resin)-NH2 step-by-step Boc-Lys(Boc)-Cys(Tn)-Asn(Trt)-Thr(tBu)-Y-Cys(Trt)-Y-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phs-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trn-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(t8u)-Tyr(Resin)-NH2 Resin =2-ohlorotrityl resin 1%-TFA/DCM/12 Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(t8u)-Y-Cys-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-AsraTrt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thqtriu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Tr1)-Thr(tBu)-Tyr-NI-12 deprotection H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gin-Arg-Leu-Ala-Asn-Phe-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-pramlintide X = Ser(tBu)-Ser(teu) or Ser(tBu)-4,Ser Y Ala-Thr(tBu) or Ala-t1)Thr = pseudoprohn

[0034] Alternatively, the synthesis of pramlintide may be performed in liquid phase with equal success concerning the purity and the yield of the obtained pramlintide. In one embodiment, the protected peptide which is bound on the resin through the side chain of Fmoc-Tyr-NH2 may be quantitatively cleaved from the resin with the side chain protecting groups of the tBu-type remaining intact, using mild acidic treatment at various positions of the peptide chain. In one example, as shown below the partially protected 1-10 fragment prepared on the 2-ehlorotrityl resin in the step by step manner was condensed successfully with the partially protected 11-37 fragment amide.

[0035] Pramlintide:
Emoc-Tyr(Resin)-NH2 Map-by-step H-Arg(P10)-Leu-Als-Asn(Tr)-Phe-Leu-Vol-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(tB*Asn(Trt)-Vel.Gly-Ser(tBu)Asn(Trt)-Thr(tBe)-Tyr(Resin)-N112 Resin 2-chlorotrityl resin I ek-TFA/DCWTES
H-Arg(PIsf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(TrI)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro=Thr(tBu)-Asn(Tn)-Val-Gly-Ser(tEtu)-Asn(Trt)-Thr(tBu)-Tyr.NH2 X Ser(tBu)-Ser(tau) or Ser(ti3u)-4ner Boc-Lys(Soc)-Cys-Asn(Trt)-Thr(tEtu)-Y-Cys-Y=Gln-OH
Y Aia-Thr(tnu) or Ala-WThr condensation deprotesilon pramlintide Tetracosactide (ACTH 1-24):

[0036] In another example. ACTH 1-24 was effectively prepared starting from resin-bound Fmoc-Tyr-Pro-OtBu by the step by step procedure or by condensing the 1-10 partially protected fragment in solution with the 11-24 fragment or with the resin-bound fragment, as shown below.
Frnoc-Tyr-OH -1. H-Pro-Ot8u coupling Fmoc-Tyr-Pro-OtBu = CI-Resin IResin-CUDIPEA
CI CI P
Fmoo-Tyr(Resin)-Pro-OtBu istep-by-step H-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-ProNal-Lys(8oc)-Va/-Tyr(Resin)-Pro-018u Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Giu(tBu)-His(Th)-Phe-Arg(Pbf)-Trp(Boc)-Gly-OH
condensation step-by-step then deprotection and cleavage from resin Boo-Ser(tBu)-Tyr(t8u)-Ser(tBu)-Met-Glu(tBu)-1-1s(88-Phe-tha Arg(Pbf)-Trp(Eloc)-Gly-OH-Lys(Boc)-Pro- Val-Oly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pb1)-Pro-Val-Lys(Boc)-Val-Tyr(Resin)-Pro-OtBu 1 deprotaction, cleavage from the resin H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Oly-Lys-Lys-AnkArg-Pro-Val-Lys-Val-Tyr-Pro-OH
Tetracceactide (ACTH 1-24) Bivalirudin:

[0037] In another example, bivalirudin was produced in high yield and high purity starting from resin-bound Fmoc-Tyr-Leu-OtBu, extending the peptide chain in the step-by-step manner with Fmoc-amino acids and finally deprotecting and cleaving the peptide from the resin as shown below.

[0038] Alternatively, bivalirudin was obtained by the condensation of protected fragments on the resin or by cleaving a partially protected peptide which contain 4-15 amino acid residues from the resin and condensing it in solution with a bivalirudin fragment which contain 5-16 amino acids. The bivalirudin synthesis by fragment condensation on the resin of the 1-10 partially protected bivalirudin fragment with the resin-bound 11-20 partially protected bivalirudin fragment is described below.

_________________________ FrnooTyr-Leu-OtBu Frnoc-Tyr-OH H-Leu-013u e 44)1" is CI-Resin Resil-CVDIPEA CI P
CI is Frnoc-Tyr(Ream)-Leu-Oteu step-by-step H-Asp(teu)-Phe-Giu(tau)-Glu(tElu)-11e-Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-Otau i800-D-Phe-Pro-Aro(Pb1)-Fro-condensation stap-by-step then deprotection and cleavage from the resin Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Oly-Gly-ikan(TrI)-Guly;t0eurhe-GtB4-2.1u(tBu)-11e-OtOu deprotection, cleavorle from the resin H-D-Phe-Pro-Arg-Pro-Gly-Gly-Wy-Cly-Asn-Oly-Artp-Plie-Glu-Giu-lle-Pro-Giu-Glu-Tyr-Leu-OH
SivallrudIn EXAMPLES
Example 1:

[0039] Preparation of Fmoe-T1u(4-methoxybenzhydry1 polystyry1)-0tBu 21 Br-Resin Fmoc-Thr-OtBu + H OCH3 DIPEA= Fmoo-Thr(Resin)-Oteu Br

[0040] 30 mmol Fmoc-Thr-OtBu prepared from H-Thr-OtBu by its reaction with Fmoc-OSu following conventional methods were reacted with 20 g (30 mmol) of 4-methoxybenzhydryl polystyrene resin (product of CBL-Patras) and 60 mmol DIPEA in 250 ml THF for 10 h at RT.
To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA (85:10:5), 6X
DMF, 4X
IPA, 3X DEE and dried in vacuum to constant weight. 29 g of resin-bound Fmoc-Thr-OtBu were obtained with a loading of 0.95 mmol/g resin.
Example 2

[0041] Fmoc-Thr(4-methoxybenzhydryl polystyry1)-0-Clt = Br-Resin Trt=Thr-OMe + H
Br DIPEA OCH3 Trt-Thr(Resin)-0Me 1N-LiOH
1% TFA. H-Thr(Resin)-0Me H-Thr(ResIn)-OH
Fmoc-OSu Clt-CI - Fmoe-Thr(Resin)-OH Fmoe-Thr(Resin)-0-Clt DIPEA

[0042] 30 mmol Trt-Thr-OMe prepared from II-Thr-OMe by its reaction with Trt-CliMe3SiCI
and DIPEA following conventional methods were reacted with 20 g (30 mmol) of 4-methoxy 4'-polystyryl benzhydryl bromide resin (product of CBL-Patras) and 60 mmol DIPEA in 250 ml THF for 10 h at RT. To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA
(85:10:5), 3X DCM, 3X 1% TFA in DCM, 4X THF, 3X 1N-LiOH in THF/Water/Methanol (70:15:15), 3X THF/Water (75:25) 4X DMF and then reacted for 2 hat RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, 3X DCM and then reacted for 3h at RT
with 50 mmol Trt-CI and 50 mmol DIPEA, washed 4X DMF, 6X DEE and dried in vacuum to constant weight. 32.3 g of resin-bound Fmoc-Thr-OtBu were obtained with a loading of 0.78 mmoVg resin.
Example 3

[0043] Fmoc-Throl(4-methoxy benzhydryl polystyry1)-0-C1t

[0044] A) Starting from Fmoc-threoninol Oa%
..HesIn=Br OH
cu_ci DIPEA Fr CH3 Br Fmo mi_ctRC6.43 r.-HN npa-HN

[0045] 50 mmol commercial Fmoc-threoninol (CBL-Patras) in 350 ml DCM were reacted with 55 mmol monomeric at-C1 and 55 mmol DIPEA for 4 h at RT. The obtained mixture was extracted as usual with water and the DCM phase was dried over anhydrous sodium sulphate and filtered. To the resulting solution 30 g of 4-methoxy, 4-polystyryl benzhydryl bromide (CBL-Patras) were added and 50 mmol DIPEA and the resulting mixture was stirred for 4 h at RT. The resin was filtered and washed 6XDMF, 4X1PA and 4X DEE and dried in vacuum to constant weight. 38.4 g of resin-bound Fmoc-threoninol were obtained with a loading of 0.82 mmolig.

[0046] B) Starting from Trt-Thr(Resin)-0Me me 0-resin L1BH4 meT OH _ -o-resin 1% TFA MeTo-resin Trt¨Nir -Me 112N ___ OH
0 Trt¨HN __ ¨
Me 0-resin Fmoc-OSu Cli-CI
Fmoc¨N ___________________ OH DIPEA Fmoc 0-Ctt

[0047] 30 mmol Trt-Thr-OMe prepared from H-Thr-OMe by its reaction with Trt-C1iMe3SiC1 and DIPEA following conventional methods were reacted with 20 g (30 mmol) of 4-methoxy 4'-polystyryl benzhydryl bromide resin (product of CBL-Patras) and 60 mmol DIPEA in 250 in! THF for 10 h at RT. To the mixture were then added 60 mmol methanol and the mixture was shaken for additional 4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA
(85:10:5), 5X THF, and then reacted with 30 mmol LiBH4 in THF. The resin was then filtered and washed 6X THF, 4X DCM, 6X 1% TFA in DCM, 3X with DMF/DIPEA (97:3) and then reacted for 2 h at RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, DCM and then reacted for 3h at RT with 50 mmol at-CI and 50 mmol DIPEA, washed DMF, 6X IPA and 6X DEE and dried in vacuum to constant weight. 34.7 g of resin-bound Fmoc-Throl-O-Clt were obtained with a loading of 0.74 mmol/g resin.
Example 4

[0048] Fmoc-Ser(trityl resin)-NH2

[0049] 50 mmols of Fmoc-Ser-NH2, prepared according to standard procedures known in the art, were dissolved in 0.5 liter of DCM. To the suspension 30 g of Trityl chloride resin (36 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 6 h at RT.
Then and then 25 ml methanol and 30 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X with DC1WMe0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 41.1 g of Fmoc-Ser-b1142-containing resin were obtained with a loading of 0.71 mmol/g.
Example 5

[0050] Frnoc-Tyr(2-chlorotrityl resin)-N112

[0051] Following the above procedure, 50 mmol Fmoc-Tyr-NH2 and 30 g 2-CTC
chloride resin gave 43.7 g resin with a loading of 0.81 g Tyr/g resin.
Example 6

[0052] Frnoc-Hyp(4-methyl benzhydryl resin)-NH2

[0053] Following the above procedure 50 mmol Fmoc-Hyp-NH2 and 30 g 4-methyl benzhydryl bromide resin gave 39.8 g resin with a loading of 0.49 g Hypig resin.

Example 7

[0054] Fmoc-Thr(4-methoxybenzhydryl resin)-Pro-NH2

[0055] 50 mmols of Fmoc-Thr-Pro-NH2 prepared from coupling of Fmoc-Thr(tBu)-OH
with H-Pro-NH2 according to standard procedures known in the art, were dissolved in 0.5 liter of DME. To the resulting solution 30 g of 4-methoxy benzhydryl bromide resin (45 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 6 h at RT. Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X with DME/Me0H/DIPEA (90:5:5), 5X DMF, 4X
IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Thr-Pro-NH2 containing resin with a loading of 0.77 mmol/g was obtained.
Example 8

[0056] Fmoc-Tyr(2-ehlorotrityl resin)-Pro-OtBu

[0057] 50 mmols of Fmoc-Tyr-Pro-OtBu were prepared according to standard procedures known in the art, were dissolved in 0.5 liter of DCM. To the resulting solution 30 g of 2-chlorotrityl chloride resin (48 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 12 hat RT. Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2h at RT. The resin was then filtered and washed 3X
with DCM/Me0H/DIPEA (90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Tyr-Pro-OtBu containing resin with a loading of 0.64 mmol/g was obtained.
Example 9

[0058] Fmoc-Tyr(2-chlorotrityl resin)-Leu-OtBu

[0059] 50 mmols of Finoc-Tyr-Leu-OtBu, prepared according to standard procedures known in the art, were dissolved in 0.5 liter of THF. To the resulting solution 30 g of 2-CTC chloride resin (48 mmol) were added and 65 mmol DIPEA and the mixture was stirred for 12 h at 60 C.
Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred for additional 2 hat RT. The resin was then filtered and washed 3X with DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Tyr-Leu-OtBu-containing resin with a loading of 0.64 mmol/g was obtained.
Example 10

[0060] Solid-phase synthesis of peptides and protected peptide segments.
General procedure.

[0061] Al. Preparation of loaded 2-chlorotrityl resins, general procedure

[0062] 2-Chlorotrityl chloride resin (CTC-C1) (100 g; loading 1.6 mmol/g) of CBL-Patras, is placed in a 2 L peptide synthesis reactor and is swollen with 700 mL
dichloromethane (DCM):dimethylformamide (DMF) 1:1 for 30 mm at 25 C. The resin is filtered and a solution of 100 mmol Fmoc-amino acid and 300 mmol diisopropylethylamine (DIEA) in 500 mL DCM
is added. The mixture is stirred under nitrogen for 2 hours at 25 C. Then, the remaining active sites of the 2-CTC resin are neutralised by adding 10 mL of methanol (Me0H) and reacting for 1 hour. The resin is filtered and washed twice with 400 mL DMF.
The resin is filtered and treated twice with 500 mL 25% by volume of piperidine in DMF for 30 mm. The resin is then washed four times with 500 mL DMF. The resin is deswelled with 3 washes with 500 mL of isopropanol (IPA). The resin is dried to constant weight. On the resin was bound the 70-95% of the mmol of the used amino acid.

[0063] B. Solid-phase synthesis, a general protocol

[0064] The solid-phase synthesis was performed at 24 C with 1.0 g amino acid or peptide esterified to the resin of the trityl or benzhydryi type or attached through its side chain as described in Part A or in the examples Example 1. The following protocol was used in the synthesis.

[0065] Bl. Swelling of the resin

[0066] The resin was placed in a 15 ml reactor and treated twice with 7 mL
NMP, followed by filtration.

[0067] B2. Activation of the amino acid

[0068] The amino acid (3.0 equiv.) and 1-hydroxybenzotriazole (4.0 equiv.) was weighted and dissolved in a reactor with 2.5 their volume in NMP and cooled to 0 C. DIC was then added (3.0 equiv.) and the mixture was stirred for 15 mm.

[0069] 83. Coupling

[0070] The solution which was prepared in B2 was then added to the 81 reactor.
The reactor was washed once with one volume of DCM and was added to the reactor which was stirred for 1-3 h at 25 30 C, In a sample the Kaiser Test was performed to determine the completion of the reaction. If the coupling reaction was not completed after 3 h (positive Kaiser Test), the reaction mixture was filtered and recoupled with a fresh solution of activated amino acid.
After completion of the coupling the reaction mixture was filtered and washed 4 times with NMP (5 volumes per wash).

[0071] 84. Removal of the Fmoc-group

[0072] The resulting resin in 83 was filtered and then treated for 30 min with 5 mL of a solution which contained 25% by volume of piperidine. The resin is then washed three times with 5 mL NMP.

[0073] B5. Elongation of the peptide chain

[0074] After the incorporation of each amino acid the steps BI-B5 were repeated until the completion of the peptide chain.

[0075] For the introduction of each individual amino acid the following Fmoc-amino acids were used: Fmoc-Ala-OH, Fmoc-Arg(PM)-0H, Fmoc-Asn-OH, Fmoc-Asn(Trft-OH, Fmoc-D-Cys(Trt)-01-1, Fmoc-Cys(Teft-OH, Fmoc-Gln-OH, Fmoc-Gln(Trft-OH, Fmoc-G1u(tBu)-011, Fmoc-Gly-OH, Fmoc-His(Trft-OH, Fmoc-Hyp(tBu)-0H, Fmoc-He-OH, Fmoc-Leu-OH, Fmoc-Mct-OH, Fmoc-D-Phe-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-0H, Fmoc-Ser(Trft-OH, Fmoc-Thr(tBu)-0H, Fmoc-Ser(Trft-OH, Fmoc-D-Trp-014, Fmoc-Tip-OH, Fmoc-D-Trp(Boc)-0H, Fmoc-Trp(Boc)-0H, Fmoc-Tyr(tBu)-0H, Fmoc-Tyr(Clft-OH, Fmoc-Val-OH, Boc-D-Cys(Trft-OH, Boc-His(Trft-OH, Boc-Lys(Boc)-0H, Boc-D-2-Nal-OH, Boc-D-Phe-OH, Boc-Ser(tBu)-011.

[0076] C. General method for the acidic cleavage from the CTC- resin of peptides and of protected peptide segments, which contain Fmoc- or Boc-groups on their N-terminus.

[0077] The resin-bound peptide or peptide segment which was produced as described above in B1-B5 was washed 4 times with 5 mL NMP, 3 times with 5 ml IPA and finally 5 times with 7 ml DCM to remove completely any residual NMP or other basic components. The resin was then cooled to 0 C, filtered from DCM and was treated twice with a solution of 10 mL 1-2%
TFA/DCM at 5 C. The mixture is then stirred 20 min at 0 C and filtered. The resin is then washed three times with 10 mL DCM. Pyridine is then added to the filtrates (1.3 equiv, relative to TFA) to neutralize the TFA. The cleavage solution in DCM is then mixed with an equal volume of water. The resulting mixture is distilled at reduced pressure to remove DCM
(350 tort at 28 C). The peptide or peptide segment precipitated after the removal of DCM.
The resulting peptide is washed then with water and dried at 30-35 C under 15 Ton vacuum.
Example 11

[0078] Synthesis of resin-bound protected peptides by the condensation of an N-terminal protected fragment with a resin-bound C-terminal protected fragment.
General procedure

[0079] To a solution of 0.15 mmol/m1 of an N-terminal protected peptide fragment in DMSO/DCM (95:5) are added 0.2 mmol HOBt and the resulting solution is cooled to 5 C.
Then 0.14 nvnol DIC were added and the mixture is stirred for 20 min at 15 C
and added then to 0.1 mmol of a resin-bound C-terminal fragment and stirred for additional 6 h at RT. The completion of the condensation reaction is checked by the Kaiser test In the cases where the Kaiser test remained blue a second condensation was performed in order to drive the condensation into completion.
Example 12

[0080] Synthesis of partially protected peptides by the condensation of an N-terminal protected fragment with a C-terminal protected fragment in solution.
General procedure

[0081] To a solution of 0.15 mmol/ml of an N-terminal protected fragment in DCM are added 0.2 mmol HOBt and the resulting solution is cooled to 5 C. Then 0.15 mmol EDAC were added and the mixture is stirred for 20 mm at 15 C and added then to 0.15 mmol of a C-terminal protected fragment and stirred for additional 2-5 h at RT. The completion of the condensation reaction is checked by HPLC. In the cases where an incomplete condensation was observed an additional portion of 0.015 mmol EDAC was added and the reaction was left to proceed for an additional hour at RT.
Example 13

[0082] Deprotection and simultaneous cleavage from the resin of peptides.
General method

[0083] 1.00 g of the protected resin-bound peptide, produced as described above is treated with 20 mL TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resin is then washed 3X with the cleavage solution and the combined filtrates are then concentrated in vacuum and crude peptide is precipitated by the addition of ether, washed several times with ether and dried in vacuum until constant weight over KOH.
Example 14

[0084] Peptide Deprotection General method

[0085] 1.00 g of the protected peptide, produced as described above was treated with 20 mL
TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resulting solution is concentrated in vacuum and then the deprotected peptide was precipitated by the addition of diisopropylether and washed three times with 10 mL diisopropylether. The resulting solid was dried in vacuum (25 C, 15 Torr) until constant weight under KOH.
Example 15

[0086] Purification of crude peptides. Isolation of peptides.

General procedure

[0087] The solution of the peptides obtained as described above was concentrated in vacuum and ice water and ether were added. After separation of the organic layer the remaining water solution of the peptide was extracted for additional two times with ether and the resulting solution was sparged with nitrogen or helium, filtered and directly loaded on a semipreparative column 10x25 cm, LichrospherTM 100, RP-18, 12 micron (Merck);
Phase A = 1%-TFA in acetonitrile, phase B = 1%-TFA in water; or Kromasil. HPLC
fractions containing the purified peptide were concentrated in vacuum to remove as much as possible the contained acetonitrile and lyophilized using a standard lyophilisation program.
prepare the listed compounds.

[0088] Examples 16 to 23, as noted below, were performed using the above procedures to prepare the listed compounds.

[0089] Example 16: Lanreotide

[0090] Example 17: Insulin B-chain

[0091] Example 18: Salmon calcitonin

[0092] Example 19: Octreotide

[0093] Example 20: Exenatide

[0094] Example 21: Pramlintide

[0095] Example 22: Tetracosactide (ACTH 1-24)

[0096] Example 23: Bivalirudin

Claims

What is claimed is:

1. A resin conjugate which is selected from the formulae III-VI:
wherein:
Pr' is H or an amino protecting group, which is orthogonal to the resin and to Pr2;
Pe is H or a hydroxyl protecting group which is orthogonal to the resin;
R3 and R4 are each independently H or C1_10 alkyl;
X, Y, Z and V are each independently on the ortho, meta or para positions and selected from H, Cl, F, C1_10 alkyl and C1_10 alkoxy; and P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.

2. A method for the preparation of the resin conjugates of the formulae III
to VI as defined in claim 1 comprising the following steps:
preparing a hydroxyl containing amino alcohol that is unprotected on at least one of the side chains of the amino alcohol, or selectively deprotecting a protected hydroxyl amino alcohol at the side chain of the protected hydroxyl amino alcohol; and then attaching it to a suitable resin by its reaction with a resin halide, wherein the resin is selected from trityl type resins, and benzhydryl type resins of formula:

where Hal is a halogen, and X, Y, V, Z and P are as defined in claim 1, to form a resin conjugate of formulae III to VI; and adding an alcohol or thioalcohol to block any unreacted resin halide.

3. Use of the resin conjugates of claim 1 as functionalized resins in the solid phase peptide synthesis of biologically active free or partially protected peptides, cyclic peptides and peptaibols.

4. A partially protected peptide of the formula:
wherein:
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt and Mmt;
D- designates the chirality of the amino acid that follows as a D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm and StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt or tBu; and Resin is an acid labile resin of the following formulae:
where X, Y, Z and V are each independently on the ortho, meta or para positions selected from H, Cl, F, C1_10 alkyl and C1_10 alkoxy; and Date Recue/Date Received 2022-04-21 P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.

5. A partially protected peptide of the formula:
wherein:
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt and Mmt;
D- designates the chirality of the amino acid that follows as a D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm and StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt or tBu; and Resin is an acid labile resin of the following formulae:
where X, Y, Z and V are each independently on the ortho, meta or para positions selected from H, Cl, F, Ci_io alkyl and Ci_io alkoxy; and P is an insoluble solid support or an insoluble linker-resin conjugate suitable for the solid phase synthesis of peptides.

6. The peptide of claim 5, wherein the peptide is a resin-bound, partially protected octreotide.

7. A process for preparing octreotide, the process comprising:
treating the peptide resin conjugate of claim 6 with a mild acid; and oxidizing the obtained peptide solution using a suitable oxidizing agent selected from Date Recue/Date Received 2022-04-21 air, hydrogen peroxide, DMSO and iodine.

8. The process of claim 7, wherein the mild acid comprises a solution of trifluoracetic acid.

9. A process for preparing octreotide comprising:
treating the peptide resin conjugate of claim 6 with a mild acid, wherein the mild acid comprises a solution of trifluoracetic acid containing iodine;
deprotecting, purifying and lyophilizing and obtaining octreotide, wherein the octreotide has a purity of >99%.

Date Recue/Date Received 2022-04-21