AU479210B2 - New peptides and processes for their manufacture - Google Patents

Description

67, 1 33/74 The invention relates to new. Ilypocalcaemically active peptides of thne formnuala I

11 2 3 4 5 6 7~ 8 9 1011 1213 14 1-Cys-Gly-Asn-Leu-Se r-Th-ir-Cyzs- X -Leu-Gly-7.rr-TYr-Thr-Gln

16 17 18 19 20 21 22 23 24~ 25 26 27 28 AsD-Ph e-Asn-Lys-Ph-e-Hi's-Thnr-The-=-Pro-GO-7 h-,:-r-Al'Ia-Il e-Gly- 29 30 31 32 Val-Gly-Ala-Pro-NIH 2 9

i-h'erein X represents the L-methionine, L-valine, L-norvalinc,

L-leucine, b-isoleucine, L-rleucine or L-a--a'inobutyric

acid radical, above all the T -:.ethicnine or L-valine radical, 26 ceby -aprgn and wh~lerein L-alanine is re-place yLaprgn 2 and/or L-isolecn is re-clad by L-threonine, and wherein

optionally one or more of the aminoacids in positions 11, 12, 16, 19, 22 and 24 is or are replaced by another cominoacid, namely L~henn 1by L-lysine, I-tyrosine 12by L-leucine, L-phenylalanine 16by L-leucire, L-phenylalanine 19by L- 22 4 leucine, L-phenylalanine L-tyrosine and L~glutamnine 2

by L-arginine, their derivatives and acid addition salts and

complexes of these compounds, and processes for their

manufacture.

As derivatives there should above all be mentioned Na~acyl derivatives and dsfn -peptides. Acyl groups for the acylation of the Na~amino group

are the radicals of carbox'yLic acids such as aliphatic, aro- matic, araliphatic, heterocyclic and heterocyclyl-aliphatic carboxylic acids, especially of lower monobasic or dibasic alkanoic or alkenoic acids, above all lower alkanoic acids with 1-4 carbon atoms such as formic acid, acetic acid,

2 4 7 9.2 i 0

13 !j 174 propicrfc acid and but 7rc4 aLLids and also acrylic acid and succ-in--c acid, of alicyclic Ca__-_-.ylic acids such as cyclo- alkylcarboxylic acids, such as umsubst-ituted and substituted benzofc acid or phthalic acid~, of uznsubstituted and aryl- substitUted aryl-loaer alkyl- or al'kenyl--carboxylic acids such as phenylacetic acid, of _,nsubstituted or substituted monocbaSic or dibasic 57-membered to &-membered heterocyclic acids wznnitrogen, sul-ohur and/or oxygen as hetero-atoms, such as pyrnid-inecarbox-ylic acids and thiophenec arboxylic acids, or of heterocyclyl-l-.-:er alkanoic acids, such as ryriylaeti acid and iridazoclylace tic acid, wherein the sub- Jents of the rings are, f- or exp-nnle, halogen atcoms, nitro groups, lower alkyl or loaer allkoxy groups or lower carbal-Koxy groups. Further acyl radicals to be mentioned are above all acyl radicals of ~amnoacids, especially of a-am-_inzacids, such as, for exanple, glycyl; L-leucyl and L-pyrcgl1utanyl, and also acyl radicals which are derived from carbonic acid or thiocarbcnic acid or their esters or amides, for example lowaer alikoxycarbonyl groups, such as ethoxycarbonyl and tert.-butcxycarbonyl, and also benzyloxy- carbon-l -which is unsubstituted or substituted as indicated above, carba-"oyl and thiocarba-oyl and '-substituted carba- moyl ar.: thiJocarbamoyl, for example N-low,.er alkylcarbamoyl, N-pheny.lcarbhamoyl and Ni-phenrr'- -th-iocarbanioyl. As acid additi or salt~s there should in rarticular be mentioned salts of therapDeutically usable acids such as hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid and siu-hh*onic acids, such as lower alkanesuiphonic acids, benzenesulD',onic acid or toluernesulnphonic acid.

3 47 Q.

67 1 33/74 By complexes there are to be understood the com-

pounds, of as yet unclarified structure, v.hich are produced on addition of certain inorganic or organic substances to

long-chain peptides and impart a prolonged action thereto.

Such substances have been described, for example, for ACTH

and other adrenocorticotropically active peptides. As

examples there should be mentioned inorganic compounds,

which are derived from metals such as calcium, magnesium, aluminium, cobalt and especially zinc, above all sparingly

soluble salts such as phosphates, pyrophosphates and poly-

phosphates, as well as hydroxides of these metals, and also

alkali metal polyphosphates, such as, for example, "Calgon "Calgon 322", "Calgon 188" or "Polyron B 12".

Organic compounds which prolong the action are, for example, non-antigenic gelatines, for example polyhydroxygelatine, polyvinylpyrrolidone and carboxymethylcellulose, and also

sulphonic acid esters or phosphoric acid esters of alginic acid, dextran, polyphenols and polyalcohols, above all poly- phoretine phosphate and phytic acid, and also polymers and copolymers of basic or, above all, acid aminoacids, for example protamine or polyglutamic acid. The new compounds display a hypocalcaemic action. They lower the content of calcium and phosphate of the plasma

of the blood of mamals, as has been demonstrated by experi- ments on rats. They can be used in hypocalcaemias, osteo- porosis or osteitis deformans. The excretion of electro- lytes in the urine is the sane as in the case of human calcitonin, in contrast to sa-lzn calcitonin, which produces

-4- 479.2 1 0

67,133 74 powerful renal effects.

The compounds are obtained according to methods

which are in themselves known, by condensing the amrinoacids

required for their synthesis, so as to form COH- bonds, in

any desired time secuence, with functional groups not par-

ticipating in the reaction being protected transiently and

the cysteine radicals being oxidised to the cystine radical

at any desired point in time. Thus they are obtained, above

all, if the protective group or groups are eliminated from 1. compounds of the-formula I

l1 2 3 4 5 6 71 8 9 10 11 12 13 14 H-Cys-Gly-Asn-Leu-Ser-Thr-Cys- X -Leu-Gly-Thr-Tyr-Thr-Gln- 16 17 18 19 20 21 22 23 24 25 26 27 28 Asp-Fhe-Asn-Lys-Phe-Hi s-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly- 29 30 21 32 Val-Gly-Ala-Pro-NH2 26 wherein X has the indicated meaning and L-alanine is 27 replaced by L-asparagine and/or L-isoleucine is replaced

by L-threonine and wherein optionally one or more of the

amincacids in positions 11, 12, 16, 19, 22 and 24 are

replaced by the abovementioned aminoacids, or from their

derivatives, in which compounds at least one amino group or

one carboxyl group is protected by a protective group which

can be eliminated, or

2. comoounds of the formula II

5 47 9.2

6 7i 3 3/74 1 2 3 4 5 6 7 8 9 10 11 12 13 H-Cys-Gly-Asn-Leu-Ser-Thr-Cys- X -Leu-Gly-Thr-Tyr-Thr- 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-G1n-Thr-Ala-Ile-

28 29 30 31 32 Gly-Val-Gly-Ala-Pro-NH 2 II, 26 wherein X has the indicated meaning and L-alanine is 27 replaced by L-asparagine and/or L-isoleucine is replaced

by L-threonine and wherein optionally one or more of the

aminoacids in positions 11, 12, 16, 19, 22 and 24 is or are replaced by the abovementioned aminoacids, or their deriva- tives, %.-herein the mercapto groups are free or are protected by he trityl roup cty an acylamidomethyl protective group, are oxidised to disulphides and, if desired, the resulting

peptide-amides are converted into their acid addition salts or complexes. When manufacturing the starting materials for the 1st variant of the process according to the invention, and also manufacturing all intermediate products required for the 2 process variants, protective groups which can be used are in particular the protective groups which are known from the synthesis of long-chain peptides and which can be split off easily, for example by hydrolysis, reduction, aminolysis or hydrazinolysis. Examples of amino protective groups which should be mentioned are aryl-lower alkyl groups, such as diphenyl- methyl or triphenylmethyl groups, which are optionally sub- stituted, for example by halogen, nitro, lower alkyl or lower alkoxy, for example benzhydryl, trityl and di-para-

6 4 7 9.2 1 0

67,.1- 3.3 /74 methoxybenzhydryl, or acyl groups, such as formyl, tri- fluorcazcetyl, phthaloyl, D-tcluenesulphonyl, be nzenesul- phonyl, benzene sulphenyl and o-ni +ro hen'l sulphenyl, or

above all groups derived fro= carbonic acid or thiocarbonic

acid, such as carbobenzoxy groups which are optionally sub-.

stituted in the aromatic radical by halogen atoms, nitro groups or lower alkyl, leralkcxy or lower carbalkoxy

groups, for example carbobenzoxy, r.-bromocarbobienzoxy or

p-chlorocarbobenzoxy, p-nitrooarbobenzoxy and p-methoxy- caroobenzoxy, coloured benzyloxycarbonyl groups such as

p-pher..l azo-benzyloxycarbo-nvl' and p-(p'-nethoxyr-henylazo)-

benzyloxycarbonyl, tolyloxycarbonyl, 2-phenyl-isopropyloxy-. carbonyl, 2-tolylisopropoxycarbon-yl and above all 2-(para- biphen-ylyl-)-2-propoxycarbonyl,1 and also aliphatic oxy- carbonyl groups such as, for exaMple, allyloxycarbonyl, cyclopentyloxycarbonyl, tert. -amyloxycarbonyl, adamantyloxy-

carbonyl, isobornylox-ycarbonyl, 2,2, 2-trichloroethoxy- carbonyl, 2-iodoethoxycarbonyl and above all tert.-butoxy- carbonyl, as well as, for example, carbamoyl, thiocarbarnoyl, N-phenylcarbamoyl and N-pherylthi ocarbarnoyl. The amino groups can also be protected by forming

enamines, obtained by reaction of the amino group with 1,3- diketcnes, for example benzoylac etone, acetylacetone or dimedone. Carboxyl groups are protected, for example, by amide or hydrazide formation or by esterification. The amide and hydrazide groups can optionally be substituted, the az:ide group, for example, by the 3 4-dimethloxybenzyl or

7 4 7 9.2 1 0

.67,133/74 bi s- thoxyhe-i-l) -me thy! group arnd the hydrazide group

by, for exam:ple, the carbobernzoxy group, the trichioro-

ethoxycarbonyl group, the trifluoroacetyl group, the trityl

group, the te-rt. -butoxyc arbonyl group. or the 2-(p-bipheflylyl)- isopropoxyIcarbonYl group. Suitable compounds for esteri-

fication are, for example, low..er opt.Lonall' substituted

alkanols such as methanol, ethanol, evanomethyl alcohol,

2,2, 2-trichloroetha.-nol, 2-Jiodoethaol, benzoylmethyl alcohol or especially tert.-butanol, and also aralkanols such as aryl-lower alkanols, for example benZyl or benzhydryl

alcohols oTtionally substituted by loweer alkyl or low-.er

alkoxy groups or halogen atoms, such as p-nitrobenzyl

alcohol, p-methoxybenzyl alcohol or 2,4,6-trimethylbel~

alcohol, phenols and thiophenols which are optionally sub- stituted by electron-attracting substituents, such as phenol, thiophenol, thiocresol, p-nitrothiophenol, 2,4,5- and 2,4,6- trichlorophenol, pentachlorophelol, p-nitrophenol, 2,4- dinitrophenol, p-cyanophenol or p-methanesulpholylphelol, and also, for example, N-hydroxy-succiflimide, IN-hydroxy- phthalimide, N-hydrox3ypiperidile and 8-hydroxyquinolile. The hydroxyl'groups of the serine, threonine and tyrosine radicals can be protected by, for example, esterifi- cation or etherification. Examples of suitable acyl radi-

cals for the esterification are lower alkanoyl radicals ouch as acetyl, aroyl radicals such as benzoyl and above all radicals-derived from carbonic acid such as benzyloxycarbonyl or ethioxycarbonyl. Groups suitable for the etherification are, for example, benzyl, tetrahy.-ropyranyl or tert.-butyl

8 79.2~41 0

67, 1 3 3 /74 radicals. The 2,2 tri f'1-uoro-l--ter-t.-but oxycarbonylaino-

or -l-b-ernzyloxy.carbonyl!P--inoathy1 grus described in

Ber. 100 (1 967), 3838 7,S~49 (Iveygand) are also suitable

for protection of the hydIroxyl groups. However, the

hydroxyl groups do not necessarily have to be protected.

The ercato groups of the cysteirne radicals are protected by, for example, acylation or alkylation. Exannles of' suitable radicals for +.he acylation are the

acetyl or benzoyl radical- the ethnylcarba-moyl radical or

the ontionally substituted carbcben-zoxV radical. Examples

of suitable radicals for tzne alkylaticn are -the tert.-butyl

or benzylthio-.ethyl radical or optionally substituted aryl-

meznyl groups such as benz-Yl, p-nitrobenzyl, diphenylmethyl,

dirrnethoxybenzhydryl or trityl, as w..ell as phenylcyclohexyl-

thienyl(2)-cyclohexyl and others, comoare Ber. 101 (1968),

681, and also, for example, an acylani~domethyl radical,

compare Tetrahedron Letters No. 26 (1968), page 3,057,

cr DOS 2,060,969. Acylamidomethyl is, for example, a

group of the formula -CH -UHJ-CO-R, %.he,-rein C0-R denotes the

acyl radical of a carbox")li acid such as of an aliphatic, aromatic, araliphatic or heterocyclic carboxylic acid or of a carbonic acid derivative, such as a carbonic acid ester or carbamic acid radical. Above all, R is an optionally substituted low,.er alkyl radical, for example a methyl, ethyl,

propyl, isopropyl, n-butyl or tert.-butyl radical, w-.hich can, for example, contain chlorine, trifluor'omethyl or the nitro group as substituents. Further, R is, for example, an optionally Fubstituted cycloalkyl radical with 3-8,

9 4 7 9;.2 1 0

67.1 3 n7 preferably 5-6, ring atoms, such as the cyclopentyl or cyclohexyl radical, or an optic-nally substituted aromatic or araliphatic radical, wherein the aromatic ring is prefer- ably the benzene ring, above all optionally substituted phenyl or benzyl, for exp-le phenyl or benzyl which are unsubstituted or substituted in the phenyl radical by lower

alkyl, lo-;er alkoxy, halogen or nitro, or a preferably monocyclic hoterocyclyl radical which is optionally substi- tuted as mentioned, for e.xm-ple a thienyl or furyl radical. In particular, acylamido-.ethyl represents acetylai.idomethyl.

It is not essential for the imino group of the histidine to

be protected but it can be advantageous to protect it, for example by benzyl, trityl, carbobenzoxy, adamantyloxy-

carbonyl or the abovementicned Weygand's groups.

These protective groups can be split off in a known manner. Thus, the carbobenzoxy group can be split off by hydrcgenolysis, the N-trityl group with mineral acids such

as hydrogen halide acids, for example hydrogen fluoride or

preferably hydrogen chloride, or with an organic acid such

as formic acid, acetic acid, chloroacetic acid or trifluoro-

acetic acid in aqueous or absolute trifluoroethanol as the

solvent, or with aqueous acetic acid, the tert.-butoxycarbo-

nyl group with trifluoroacetic acid or hydrochloric acid,

and the 2-(p-biphenylyl)-isopropoxycarbonyl group with

aqueous acetic acid or, for example, with a mixture of

glacial acetic acid, formic acid (82.8% strength) and

water The methyl ester can be converted into the

hydrazide by means of hydrazine hydrate. The methyl ester

10 479,210

6 7,1 3 3/74 or ethyl ester group is hydrolysed with dilute sodium

hydroxide solution. The tert.-butyl ester is split with, for example, trifluoroacetic acid, as is the tert.-butyl

ether.

Preferably, in the 1st variant of the process

according to the invention, the tert.-butyl ester group is

used to protect the carboxyl group of the side chain and

optionally the terminal carboxyl group, the tert.-butoxy-

carbonyl group is used to protect the amino group of.the

side chain, the tert.-butyl ether group is used to protect

the hydroxyl groups of the serine, threonine and tyrosine

radicals, if these are protected at all, and, if desired,

the 2,2,2-trifluoro-1-tert-butoxycarbonylaminoethyl group is

used to protect the imino group of the histidine. All

these protective groups can, if desired, be split off in

one step by acid hydrolysis, for example by means of tri-

fluoroacetic acid, hydrochloric acid or hydrogen fluoride.

In the 2nd process variant of the process according

to the invention, the open-chain peptide used as the starting

material can preferably again be prepared with the protected

groups mentioned for variant 1).

The mercapto groups are preferably protected by

benzyl, trityl or acylamidomethyl. The S-trityl groups can

be split from the protected peptide selectively (whilst

retaining the groups which can be split off with trifluoro-

acetic acid) in organic solution by means of mercuric

acetate and hydrogen sulphide. The S-benzyl groups can be

split selectively from the protected peptide by means of

11 479.210

67.1 3 3 4 sodium in liquid ammonia. The S-acylamidomethyl groups

can be split from the protected peptide by stirring the pep-

tide, preferably at room temperature, in an aqueous solution

of a water-soluble hea ymal st, such as an acetate or

nitrate, of mercury, silver, cadmiumA tin, and the 1'D or an organo-metal salt of a.heavy metal. Not only water

but also other solvents such as methanol, dimethylformamide

or mixtures of these solvents can be used. In all cases,

the protected peptide is obtained vith free mercapto groups.

This can be oxidised to the protected disulphide, for

example with iodine in glacial acetic acid with diiodoethane

in organic solvents or with atmospheric oxygen in liquid

ammonia. It is particularly advantageous to protect the

mercapto groupsby trityl or acylamidomethyl groups and to

remove these from the protected peptide, with simultaneous

formation of the disulphide bridge, by means of iodine,

for example in methanol or 'acetic acid. The

formation of the disulphide ring can be effected at the

stage of a partial sequence containing the two cysteine

radicals, for example the decapeptide 1-10, or at the stage

of the dotriacontapeptide-amide. When manufacturing the N-acyl derivatives, the

acyl group can be used as the amino protective group.

The peptides obtained can subsequently be converted

into their acid addition salts a-/ccormplexes in a manner which

is in itself known.

The formation of acid addition salts is effected in

a knovwn manner.

12 4 7 9.2

67,1 3 3/74 The formation of complexes is also effected according to known methods or methods equivalent thereto.

Complexes with inorganic compounds such as sparingly

soluble metal compounds, for example aluminium or zinc com-

pounds, are preferably manufactured in an analogous manner

to that knovwn for ACTH, for example by reaction with a sol-

uble salt of the metal in question, for example zinc chloride or zinc sulphate, and precipitation with an alkali metal

phosphate and/or alkali metal hydroxide. Complexes with organic compounds such as polyhydroxygelatine, carboxy-

methylcellulose, polyvinylpyrrolidone, polyphloretine phos- phate polyglutamic acid and th litk are obtained by mixing these substances with the peptide in aqueous solution.

Insoluble compounds with alkali metal polyphosphates can

also be manufactured in the same manner.

The invention also relates to those embodiments of

the process in which an intermediate product obtaiable at any

stage of the process is used as the starting material and the

missing steps are carried out or the process is discontinued

at any stage and/or a starting material is formed in situ and/

or is used in the form. of a salt.

The peptides used as starting materials are obtained

by linking the aminoacids, using if required or desired protective groups which can easily be split off, in the

sequence mentioned, individually or after prior formation of

smaller peptide units, the disulphide bridge being formed,

if appropriate, at a suitable stage of the synthesis.

Appropriately, the methods of linking suitable for the

13 4 7 9,2 1 0

671 3 3n4 manufacture of long-chain peptides whilst taking the disul-

phide bridge into account, such as are known from the litera-

ture, are used.

The linking of the aminoacid and/or peptide units is effected, for example, by reacting an aminoacid or a

peptide having a protected a-amino group and activated

terminal carboxyl group with an aminoacid or a peptide

having a free a-amino group and free or protected, for

example esterified or amidised terminal carboxyl group, or

reacting an aminoacid or a peptide having an activated a-

amino group and protected terminal carboxyl group with an

aminoacid or a peptide having a free terminal carboxyl

group and a protected a-amino group. The carboxyl group can be activated, for example, by conversion into an acid

azide, acid anhydride, acid imidazolide or acid isoxazolide

or an activated ester, such as a cyanomethyl ester,

carboxymethyl ester, p-nitrophenyl thioester, p-nitrophenyl

ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl ester,

N-hydroxysuccinimide ester, N-hydroxyphthalimide ester,

8-hycdroxyquinoline ester or N-hydroxypiperidine ester or

by reaction by means of a carbodiimide (optionally with the

addition of N-hydroxysuccinimide or a 1-hydroxybenzotriazole

which is unsubstituted or substituted by, for example,

sey) or N,N'-carbonyl-diimidazole and halogen, methyl or e

the amino group, for example, by reaction with a phosphite-

amide. The most customary methods to be mentioned are the

carbodiimide method, the azide method, the method of the

activated esters and the anhydride method, as well as the

14 47 9.2

67.1 3 3 n4 Merrifield method and the method of the N-carboxyanhydrides

or N-thiocarboxyanhydrides.

When manufacturing the starting materials it is advantageous to start from a sequence which comprises the

first 10 N-terminal aminoacids and to condense the entire

remaining sequence with this N-terminal structure, prefer- ably according to the Weygand-W.unsch method.

It is however also possible to link the said N- terminal sequence with, for example, the fragment up to the

28th aminoacid (glycine) having a free terminal carboxyl

group and to condense the octacosapeptide with the tetra-

peptide of the aminoacids 29 32, for example by the 1I Weygand-Wunsch method.

The manufacture of the peptide of the formula I in which X represents L-methionine is explained below as an

example of a preferred embodiment.

The N-terminal decapeptide (1-10) can be manu- factured, for example, according to Helv. 53, 556 (1970).

The sequence to be linked to the N-terminal

sequence 1-10 and comprising aminoacid 11 to 32 is syn-

thesised, for example, from sequences 11-16, 17-20, 21-23,

24-28 and 29-32.

Sequence 24-28 can be manufactured, for example,

by linking, preferably in each case by the activated ester

method, the 27th aminoacid (Thr) to the 28th aminoacid (Gly),

coupling this 2-unit sequence to the 26th aminoacid (Asn),

and bonding the resulting 3-unit sequence to the 25th amino-

acid (Thr) and finally to the 24th aminoacid (Gln). The

15 47 9.2

67,133n4 hydroxyl groups of the threonine radicals can, in doing so, be protected by, for example, the tert.-butyl ether group. The sequence 24-28 with a protected amino group of the glutamine radical and a free carboxyl group of the glycine radical is linked to the sequence 29-32 H-Val-Gly-Ala-Pro-NH2 [Helv. 53, 2135 (1970)], preferably by the carbodiimide-N-hydroxysuccinimide method. After splitting off the amino protective group of the glutamine radical of the sequence 24-32, the sequence 21-23 Z-qr(tBu)-Phe-Pro-OH [Helv. r3, 2135 (1970)] is coupled to the sequence 24-32, preferably by the carbo- diimide method in the presence of N-hydroxysuccinimide. The bonding of the sequence 17-20 Z-Asn-Lys(Boc)- Phe-His-NH-NH2 [Helv. 53, 2135 (1970)] to the sequence 21-32 which has been freed from the amino protective group Z is- effected, for example, by means of the azide method. After splitting off the amino protective group Z of the asparagine radical of the sequence 17-32, the requisite fragment 11-32 is completed by coupling the sequence 11-16 Z-Thr(tBu)-Tyr(tBu)-Thr-(tBu)-Gln-Asp(OtBu)-Phe-NHNH2 [Helv. 53, 2135 (1970)] to the sequence 17-32, preferably by means of the azide method. The linking of the sequence 11-32, just described, to the sequence 1-10 Boc-Cys-Gly-Asn-Leu-Ser(tBu)-Thr (tBu)-Cys-Iet-Leu-Gly-0H [Helv. 53, 556 (1970)] is preferably effected by the carbodiimide-N-hydrcxysuccinimide method. The removal of protective groups still present is preferably effected in trifluoroacetic acid or concentrated

16 4 7 9,2 1 0

67,1 33 n4 hydrochloric acid and leads to Asn26-Thr27-calcitonin-H. Depending on the procedure used, the new compounds are obtained in the form of bases or of their salts. The

bases can be obtained from the salts in a manner which is in itself knowm. From the bases, salts can be obtained by reaction with acids which are suitable for forming thera- peutically usable salts, for example salts with inorganic acids such as hydrogen halide acids, for example hydro- chloric acid or hydrobromic acid, perchloric acid, nitric acid or thiocyanic acid, sulphuric acids or phosphoric acids, or with organic acids, such as formic acid, acetic acid, propionic acid, glycollic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, hydroxymaleic acid, dihydroxymaleic acid, benzoic acid, phenylacetic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, anthranilic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, methanesul- phonic acid, ethanesulphonic acid, hydroxyethanesulphonic acid, benzenesulphonic acid, p-toluenesulphonic acid, naphthalenesulphonic acid or sulphanilic acid. The peptides obtained in accordance with the pro- cess can be used in the form of pharmaceutical preparations. These contain the peptides, mixed with a pharmaceutical, organic or inorganic, excipient suitable, for example, for intravenous, intramuscular, subcutaneous or intranasal administration. The excipient can concist of materials

17 479,2

67,1 3 3 4 which do not react with the polypetides, such as, for example, gelatine, agar-agar, tragacanth, cellulose, for

exar-.le "Avicel" (micro-crystalline cellulose) and cellulose

derivatives, such as carboxy-ethylcellulose or cellulose

ethers such as methylcellulose or ethylcellulose, polyalkyl-

ene glycols, such as propylene glycols, water, monohydric or

polyhydric alcohols such as ethanol, isopropanol, glycerol

and hexitol, vegetable oils and other fatty acid esters such

as groundnut oil, cottonseed oil, almond oil, olive oil,

castor oil, ethyl oleate, isorropyl myristate, isopropyl

palmitate, "Cetiol V" (oleic acid esters of liquid fatty

alcohols), "-iglyol" or "'Labrafac" (mrrixre of triglycerides of

fatty acids with 8-12 carbon atoms), "Labrafil M 2735" or

"Labrafac WL 1219" (mixtures of glycerol anid polyoxy-

ethylene fatty acid esters), "Arlacel" (sorbitane fatty acid

esters), "Tween" (polyoxyethylene sorbitane monooleate),

silicone oils such as dimethylsilicone oil or other known

medicinal excipients. The pharmaceutical preparations can

be, for example, in the form of a lyophilisate or in a

liquid form as solutions, suspensions, emulsions or sprays,

compare, for example, German Offenlegungsschrift 2,212,315.

They are optionally sterilised and/or contain auxiliaries,

such as preservatives, stabilisers, wetting agents or emulsifiers. They can also contain yet other therapeu-

tically valuable materials. The invention is described in the examples which follow.

The follow.ing systems are used in the thin layer

18 479.210

.67,133/74 chromatograph!y:

System 43C: Tert. amyl alcohol-isopropanol-water (51:21:28)

System 45 Sec. butanol-3% strength aqueous ammonia

(70:30)

System 52 n-Butanol-glacial acetic acid-water (75:7.5:21)

System 52A: n-Butanol-glacial acetic acid-water (67:10:23)

System 70 Ethyl acetate-pyridine-water (40:20:40), upper

.phase

System 89 Ethyl acetate-acetone-wvater (72:24:4)

System 96 Sec.-butanol-glacial acetic acid-water

(67:10:23)

System 10i0: Ethyl acetate-pyridine-glacial acetic acid-

water (62:21:6:11)

System 10 1A: n-Butanol-pyridine-glacial acetic acid-water (42:24:4:30) acid- System 102A: Ethyl acetate-methyl ethyl ketone-formic water (50:30:10:10)

System 10 7 Ethyl acetate-pyridine-water (48:24:27) (44:24:2:20) System 11 2E: n-Butanol-pyridine-formic acid-water

System 12 1: Isopropanol-25% strength aqueous ammonia-water

(70:10:20)

System 12 1A: Isopropanol-25% strength aqueous ammonia-water (85:51z0) Rf data for thin layer chromatography:

DS: On SL 254 ready-prepared silica gel plates of

Messrs. Antec,Birsfelden. DC: On ready-prepared cellulose plates of Messrs.

Merck, Darmstadt.

19 di0J

6 7,13 3 nd DA: On Alox Dlates (45, g of A12 3 o ess ang i.7uttenz, 3.5 g Of gypsuMn, thickness 0.3 mm). The follow,.ing abbreviations are used in the

examples:

Boc tert butoxycarbonyl

Z carbobenzox'jf

OtBu tert.-butyl ester

O1,p p-nitrophenyl ester O'r',e methyl ester

OSu hydroxysucc'nimide ester

tBu tert.-butyl ether

BmnD 6-.nercaptcrpropiolyl DlMF dime thylfornamide. ExamDle 1 H-Cy s-Gly-Asn--Leu-Ser-Thr-CY-i4C t-Leu-Gly--Thr-Tyr-

Tihr-Gln-Asp-Phe-Asn-Lys-Phe-Hiis-Thr-Phe-Pro-Gln-

Thlr-Asn-Thr-Gly-Val-Gly-Ala-Pro-N, H 2 (hydrochloride) 26_ 27_actn M (Asn -Thr -actnnN

mg of Bo-y-l-s-e-e~tu-lrtu-IS lKet-Leu-Gly-Thr (tBu) -Tyr t~u)-Gln-Asp(C)-tBu)-Phe-

Asn'-Lys(Bc-h-i rr',u-hePoGnTr-B)Ar-h (t-kBu)-Gly-Val-Gly-Ala-Pro-'-K2 are dissolved in 1.2 ml of 0 highest purity 12 N hydrochloric acid at 0 C (duration approx. 1-2 minutes) and th.-e solution is briefly flushed

with 1 and left to stand for 10 minutes at 0 C. It is

then cooled with solid carbon dioxide, subjected to a high vacuum and concentrated to a syrup whilst slowl..Iy raising the temnerature. The syrup is dissolved in 2 ml. of water and

20 4 79,2 1 0

6 7,1 3 3/74 lyophilised, and the r--sidue is agai lyophilised from 2 ml

of water and finally equilibrated vit the humidity in the

atmosphlere. The product thus obtained (hydrochloride of 27 As2 6 hi calcitonin M) is an am.orrhous, water- soluble

powder. DC: Rf(4 5) 0.45; Rf(l0lA) 0.53; Rf(112E)=

0.39. in thin layer electrlophoresis (on a cellulose plate) at pH 1.9 and 16 volt/cm the compound migrates approx. 3.7 cm tow..ards the cathode in 11 hours. poected dotriacontaeptide-amide is prepared, Th for example, as follows: a) Z-Thr (tP~u )-Gly-OI-e 22.3 g of Z-Thr(t--u)-OSu and 8.29 g of HC1 H-Gly-

W~e are dissolved or suspended in 100 ml of dimethylformamide, 7.49 i-l of 11-methylmorpholine are added and the mixture is stirred for 1 day at 22CGand then concentrated to dryness in a high vacuum. The residue is taken up in ethyl acetate and the solution is washed with citric acid strength),

M~C03 (55' strength) and water, dried over sodium sulphate and concentrated to dryness. The oily residue crystal-

lises on being left to stand overnight at 000C; -melting

point 4800C. The dipeptide is chromatographically a single substance and is processed further direct. DS: Rf( 4 5) =0.77; Rf(chloroform-meth-Iaol

0.70 b) F-7,hr(-tBu)-Glv-0M-'e .HUl

g of Z-Thr(tBu)-Gly-0~e are dissolved in 200)ml of methanol and after addition of 13.2 ml of 1 N HUl and

21 479.2

6711 3 3/74 g of palladium on charcoal Pd) the compound is

hydrogenated, with absorption of C02 until saturated.

The catalyst is then filtered off ard the filtrate is

concentrated to dryness, whereupon a white foam is obtained.

DS: Rf(45) =0.64; Rf(chlorofform-methanol 9:1)

0.41; Rf(52) 0.42..

c) Z-Asn--Thr( t3u)-Gly-1.e

18.9 g of Z-Asn-ONp and 12.5 g of H--Thr(tBu)-Gly-

KCl are dissolved in 95 mql of~ 4.6 ml1 of N-mnethyl- morpholine are added and the mixture is left to stand for hours at room temperature. The solvent is removed in a high vacuum, the residue is powde-red by trituration with ethyl acetate, filtered off and crystallised from methanol- chlorofor-m-petroleum ether and froM ethanol-water; melting point 165 OC. DS: Rf(45) =0.68; Rf(chloroform-methanol 9:1)- 0.38, Rf(52) 0.70. d) H-Asn-TIhr (tBu )-Gly-OMe g of Z-.Asn-Thr(tBu)-Cly-OMe are hydrogenated in 210 ml of methanol in the presence of palladium on char- coal (10% of palladium). .The product is an amorphous 0 foam, melting point approx. 60 C.

DS:. Rf(45) =0.42; Rf(592) =0.27

e) Z-Thr(tBu)-Asn-Thr(t~u)-Gl-14ie

A solution of 7.51 g of Z-Thr(tBu)-OSu and 5.55 g

of H-Asn-Thr(tBu)-Gly-0loe in 50 ml of DI-IF is left overnight at 22 0 C and is th-en concentrated to dryness in a high vacuum. The residue is dissolved in ethyl acetate/n-butanol

22 4 7 9.2 1 0

67, 13 3 n4 the solution' is waShed with citric acid (%strength),

KHCO3 strengthL) and w.ater and evaporated to dryness, and

the solid residue is crystallised from methanol-ether; 0 melting point 145-146 C.

DS: Rf(45) 0.73; Rf(52) 0.75-

g of Z--Thr(tBu)-Asn-Thr(tBu)-Gly-0.1e in 20 ml

of methanol are hydrogenated in the usual manner until' saturation is reached, an azornohous (foamy) product being obtained. DS: Rf(45) 0 .41; Rf(52) 0.34

g) Z-Gln-Tir( ti ks-htu)Gy0e A soluticni of 5.33 g of H-.Thr(tBu)-Asfl-Thr(tBu)-

Gly-OMe and 4.95 Z of Z-Gln--07..p in 83 ml of DI.T is left to 0 stand overnight at 22 C, the pentapeptide being formed starting to crystallise out even after a short time. Finally, 500 ml of, vater are added, the mixture is filtered at 00 C and the residue is dried and purified by suspending it in acetone-acetcoitrile, 1:1. The crystalline product 0 has a melting point of 220 C. DS: R..(l00) 0.65; Rf(chloroform-mathanol 8;2)=

0.35; Rf(52) 0.58 h) Z-Gln-Thir(tllu).Asn-Thr(t3u)-Gly-OH 6.42 g of pentapeptide methyl ester (from are

dissolved in 58 r-1 of trifluoroethanol with warming, 25 ml of 1 N sodium hydroxide solution are added at room tempera- 0 After ture and -the mixture is stirred for 6 hours at 22 C. addition of 25 ml of 1 N 1iCl the saponified pentapeptide

23 3 7 9.2z i -4 0

6 7 3 3 i4 begins to crystallise out. The mixture is concentrated to

ml and the product is filtered off and washed with water

until free of chloride.

DS: Rf(52) 0.46; Rf(100) 0.29.

i) Z-G1n-Thr tBu)-Asn-Thr(tBu) a!l-Val-Gly-Ala-Pro-NH 2

500 mg of Z-Gln-Thr(tBu)-Asn-Thr(tBu)-Gly-OH, 335 mg

of H-Val-Gly-Ala-Pro-NH 2 (Helv. 53, 2135 (1970)) and 105 mg

of N-hydroxy-succinimide are dissolved in 4 ml of DMF with

warning. After addition of 202 mg of dicyclohexyl-carbo-

diimide, the mixture is stirred for 4 hours at 40 C after

which the reaction product is precipitated as a jelly by

introduction into a mixture of 100 ml of ether and 100 ml

of petroleum ether, and is filtered off. The crude product

is purified by twice dissolving i.t in methanol-water (85:15)

and precipitating it with ether, melting point 198-199 C.

DS: Rf(96) 0.41; Rf(107) 0.50, Rf (chloroform-

methanol 7:3) 0.35

j) ;-Gln-Thr( tBu)-Asn-Thr(tBu)-Gly-Val-Gly-Ala-Pro-NH 2

3.1 g of the abovementioned Z-derivative are hydro- genated in 120 ml of 80% strength trifluoroethanol using

310 mg of palladium on charcoal (10% of Pd), with absorption

of C02, until saturation is reached. The filtrate is con-

centrated to approx. 5 ml and after addition of 40 ml of

water the mixture is left to stand overnight at 0 C, where-

upon the nonapeptide crystallises out in the pure form,

melting point approx. 240°C (decomposition).

DS: Rf(96 0.20; Rf(107) 0.32; Rf(70) 0.18.

24 47 9,2 1 0

6 7,13 :3/14 1 k) Z-Tri( tm.u) he-Pro-Gln-Th r( tEu) Asn- Thr(-t u) -Gly-Va -Gly- Al a-Pro-ITH2 517 mg of Z-Thr(tZu)-Phe-ro-OH (Kelt. 53, 2135 enqd 74 mg (1970)), 410 mg of the abovementioned nonapeptide 4 ml of 1-hydroxy-succifimide are dissolved or suspended in of trifluoroethflol-chlorofo'm with gentle warmi ng, 220 C and 177 mg of dicyclohexyl-carbodiimide are added at the mixture is stirred for 20 hours. Tne reaction mixture repre- is then concentrated to dryness and the residue is Th-e crude cipitated once from chloroform-petroleufl ether. product thus obtained is purified in a Craig distribution in the solvent system methano&-b.afer-chloroform-carbon

-tetrachloride, 10:10:3:5:4 (buffer: 28.6 ml of glacial

acetic, acid 19.25 mg of ammonium acetate 960 ml of w-.ater) a time. through 420 stages, using phase volumes of 5 ml at No. Concentrating the contents of distribution elements 135-184 (rmax K= 0.61) to daryness and subliming away 160; the P~roniumL- acetate in a high vacum at gives the point pure dodecapeptide as en amorphous powrder of melting approx. 19500C (decomposition). DS: Rf(9 6 =0.44; Rf(70) 0.51; Rf(52A) 0.38

1) H-E 3)PePr l-hrtu-snT -tBu)-Gly-Val-Gly- Ala-Pro-NH2

504 mg of the abovementiofled.Z-dodecapeptide are are hydro- -dissolved in 30 ml of methanol with warming and mg of genated in the usual manner after addition of 100 palladium on charcoal. The product obtained on concentra- powder. tina the filtr-ate to dryn-ess is an amcrphnous, white

25 4 7 9,2 1o

671 33 n4 DS: Rf(43C) 0.25; Rf(70) 0.35; Rf(52A) 0.20. m) Z-Asn-Lys(Boc)Phe-His-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn- Thr(tBu)-Gly-Val-Gly-Ala-Pro-NH 2

378 pl of 3.2 N HC1 in dioxane and 90 ill of tert.- butyl nitrite are added to a solution of 479 mg of Z-Asn- Lys(Boc)-Phe-His-N1TH2 (Helv. 53, 2135 (1970)) in 4.5 ml of DMF at -20 0 C. After stirring for 15 minutes at 410 mg of H-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-Thr(tBu)-Gly- Val-Gly-Ala-Pro-NH 2 dissolved in 6 ml of DMF, are added, followed by 260 1 of ethyl-diisopropylamine. The mixture

is left to stand overnight at OC and the crude product is then precipitated by dropwise addition to 100 ml of ether. It is pifed ,cysibjectig it O a Craig istributn in the system methanol-buffer-chloroform-carbon tetrachloride, 10:5:10:5

[buffer as for k] through 200 stages, using phase volumes of 3 ml at a time. The pure hexadecapeptide is obtained as an amorphous powder from distribution elements No. 18-37 (rmaxx 27; K 0.16) by concentrating to dryness and sub- 0 liming away the buffer in a high vacuum at 40 C. DS: Rf(43C) 0.40; Rf(52) 0.20; RP(70) 0.58.

n) H-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Phe-Pro-Gln-'hr(tBu)-

Asn-Thr(tBu)-Gly-Val-Gly-Ala-Pro-NH 2 acetate

270 mg of the abovementioned Z-hexadecapeptide in

27 ml of 80% strength acetic acid are hydrogenated for

2 hours with 50 mg of Pd on charcoal, the catalyst is fil-

tered off and the filtrate is concentrated to approx. 3 ml

and lyophilised.

DS: Rf(52) 0.07; Rf(96) 0.28; Rf(107) 0.65.

26 47 9,2

67, 13 74 o) Z-Thr(t-'u)-Tyr(-* u)-Thr(tBu)-Gln-.sp(OtP-)-Phe-Asn-Lys

(Boc)-Phe-His-m7;,-r(tBu)-Phe-Pro-Gi -Th-(tBu)-Asn-T-hr

(tBU) -31y-Val- Gir-Ala-Pro-NH 2

117 P1 of 3.24 N HCl in dioxane and 21 pl of tert.- butyl nitrite are added to a solution of 165 mg of Z-Thr

(tu)-Tyr(tu)-TrtBu)-Gn-Asp(tBu)-Phe-NIEI (Helv. 53,

2135 (1970)) in 2 ml of iF -at -200 C. The mixture is

stirred for 15 minutes at -100 C, a solution, precooled to 00C,

of 198 m of E-Asn-Ls(Boc)-Phe-His-Thr(tBu)-Phe-Pro-Gln-

Thbr(tBu)-Asn-~Thr(tSBu)-Gly-Val-Gly-A a-Pro-i-YI2 (acetate)' in

4 ml of D.IF and 80 ul of ethyl-diisopropyla-mine are added

and the whole is left to stand overnight at 00C. The crude

product is precipitated out by drop;:ise addition of .the

reaction solution to a mixture of 40 ml of ether and 20 ml

of petroleum ether and is subsequently subjected to a Craig

distribution in the system methanol-buffer-chloroform-

carbon tetrachloride 20:5:10:10 [buffer as in through

300 stages, with phase volumes of 3 ml at a time. The

chromatographically pure docosapeptide is obtained as an

amorphous pow~der from distribution elements No. 98-117

rma 107;. K 0.55) by concentrating to dryness and subliming away the buffer in a high vacuum at 400C. DS: Rf(43C) 0.39; Rf(2) 0.30; Rf(96 0.48. p) H-Thr(tu)-T-yr(tBu)-Thr(tBu)-Gln-Asp(OtBu)-Phe-Asn-Lys (Boc)-Ph-e-His-Thr(tBu)-Phe-Pro-Gln-'nr(tIu)-Asn-Thr(t3u)-

Gly-Val-gly-Ala-Pro-NH 2

260 =g of Z-docosapeptide in 26 ml of 80% strength acetic acid are hydrogenated with 50 mg of Pd on charcoal for

27 479,2

67,133/74 filtrate is hours, the catalyst is filtered off and the To remove concentrated to approx. 5 ml and.lyonhilised. from the acetic acid, the residue is twice reprecipitated methancl-sodii= bicarbonate solution. DS: Rf(43C) 0.31; Rf(LCO) 0.29. I I -1 I

q) Boc-Cys-'Sy-sn-Leu-Ser tI3u)--inni tiu) -Cys-Met-Leu-Gly- Thrkfr(t-Dua)-Tyr (tBu)-Thr(tBu)-Gln-fAsP(0tcu)PheAsnLys -Asn-Tnr (Boc -Ph-e-HKi s-Thr tBu) -Phe-Pro-Gln-Thr tBu) -G y.-'Va'l-G1 y-Ala-Pro-NH2

described 170 mg of the hydrogenated d30sapeptide

and 101 mg of Boc-Cys-G!--.-.sn-Leu-Ser (tBu)-Thr under p)

(tu)Cs-K- Le-H lv-OH (Helv. 53, 556 (1970)) are dissolved

in 1.2 ml of Di-F whilst warming, 15.8 mg of N-hydroxy- are succinimide and 21.3 mg of dicycloOxYl-carbodiimide for 31 added and the mixture is stirred under nitrogen 0 hours at 45 G. The crude product is then precipitated by 20 ml of dropw:ise addition of the reaction mixture to it to a peroxide-free ether and is purified by subjecting

Craig distribution in the system methanol-buffer-chlloroform- through 450 carbon tetrachloride 11:3:6:7 [buffer as in The pure, stages with phase volumes of 3 ml at a time. isolated from protected dotriacontapeptide derivative is

elements No. 190-210 (rmax 200; K 0.8) as an anorphous the buffer. pow.der on concentration, and sutlimation of 0.53; Rf(lCO)= 0.35; Rf(52A) 0.29. DS: Rf(70) ExMo-le 2

B m-Gly-Asnr-LeutiSe- ThrCys-Met-Leu-Gly-il rTyrThr-Gln-Asp- l--Asn Thr-cy-vaN y- Phe-Asn--Lys-Phe- ii 5s-Thnr-Phe-Pro-'GUiZ-ii;--LA-Lr

28 47 9,2

6 7 ,1 33i4 26 27 Ala-Pro-N~H2 (hydrochloride) (Desam-ino-Asn -Thr- calcitonin M4) mg of Bmp-Gly,-Asr-Leu-Ser(t~u)-Thr(tBu)-Cy-s-Pe t- Leu-Gly-Txhrk'tBu)-Tyr( tBu)-'Tr( t3u)-Gln-Asp (OtLBu)-Phe-Asn- Ly(o)PeHsilrtu-h-roGn-hkt~)A~--i~~) Gl-a-l-AaPoVH are converted into the above peptide by acidolys is with 12 N HCl at 00C, as described in. Example 1. DC: Rf(45) 0.50; Rf(101A) =0.59; Rf(1-12E)

0.43. Thin layer electrophores-is: pH 1.9, 16 volt/cm, 1-2 hours: M-igration distance approx. 2.4 cm toiards the cathode. The protected dotriacontapeptide-amide is prepared, for example, as follows: 61. mg of B p-Gly-Asn-Leu-Ser(t.Bu)-Thr(tBu)-Cys-Met- Leu-Gly-1- (DOS 2,050,434), 113 mg of H-Thr(tBu)-Tyr(tBu)- Thr(tBu)-Gln-Asp(OtBu)-Phie-Asn-Lys(Boc )-Phe-His-.Thr(tBu)- Phe-Pro-Gln-T-hr (tBu )-Asn-T'nr (tBu) -Gly-Val-Gly-Ala-Pro-NH', 2

(Example 1D) and 10.5 mg of N-hYdroxy-succinimide are dissolved in 0.9 ml of Dj'.F. After addition of 14 mg of dicyclohexyl-carbodiimide, the reaction vessel is flushed with N2, closed and stirred for 312 hours at" 45 OC. The

crude product is precipitated by addition of 15 ml of peroxide-free ether and is purified by means of Craig dis- tributi on in the system metha:nol-buf'fer-chJiioroform-c arbon tetrachloride 11:3:6:7 [buffer as in Example 1 th.1rough

240 stages, using phase volumes of 3 ml at a tirie. The

29 4 7 9.2

67,333 '1 pure product is isolated as an amorphous powder from dis- tribution elements No. 108-129 (rma 118; K 0.97) by concentration and sublimation of the buffer. DS: Rf(52) 0.29; Rf(70) 0.65; Rf(100) 0.38. Examole 3 Bap-Gly -Asn-Leu-S e-T r-Cys-Val-Leu-Gly-rTs-Leu-T r-Gln-Asp- Phe-Asn-Lys-Phe -Hi s-Thr-Tyr-Pro-Gln-rr-Asn-Thir-Gly-Val-Gly- Ala-Pro-?-,! (hydrochloride), LLys 11-Leu 12 Desamio-V8- Tyr22_. sn2 26__'27_ 6 7hr27-calcitonin M)

ng of Bmp o-Gl-Asn-Leu-Ser(tBu)-Thr(tz u)-Cys-Val-

Leu-Gly-Ly (Boo )-Leu-Thr( tBu)-Gln-Asp (Otiu)-Phe-Asn-Lys

(Boc)-Phe-His-Thr(tau)-Tyjr(tBu)-Pro-Gln-Thr(tBu)-Asn-Thr

(tBu)-Gly-Val-Gly-Ala-Pro-NH 2 are converted into the above pep-tide by acidolysis with 12 N HCl at 0 0 C, as described in

Example 1.

DC: Rf(lOlA) 0.51; Rf(112E) 0.45. Thin layer electrophoresis: pH 1.9, 16 volt/cm, hours: Iigration distance approx. 3.7 cm towards the cathode.

The protected dotriacontapeptide-amide can be pre- pared, for example, as follows:

a) Z-Leu-Thr t-u)-Gln--Asp (OtBu)-Phe-OMe

4.15 g of H-Tbhr(tBu)-Gln-Asp(OtBu)-Phe-OIle

(Helv. 5, 2135 (1970)) are dissolved in 40 ml of D1F,

3.26 g of Z-Leu-0 p are added and the mixture is left to stand overnight at 22 0 C. The pentapeptide produced is caused to crystallise out by adding water and is purified by crystallisaticn from methanol-chloroform-petroleuma ether

30 4 7 9.2 1 0

67, 13 3n4 and from methanol-water; melting Doint 204 0 C. DS: Rf(chloroform-methanol 9:1) =0.39; Rf(89)

0.52. b) H--Leu-Th r (t~u -G'n-Asn (0t-Bu)- P e ,i~e 4.8 g of Z-Dentapeptide are hydrogenated in 100 ml of methanol in the usual manner unitil satiz'ation is reached and the solution wh.Iich has been freed from catalyst by fil- tration is concentrated to dryness, w.-hereupron an amorphous, wh~ite foam is obtained. DS: Rf(chloroform-iaethanol 0.14,; R:C(89) 0.29. c) Z-Lvs (B3oc )-Leu-Th-r (tBu -Glin-A is-(Otl~ -Phe-Olie 4gof H-Leu-Thr( tBu) -Gln-Asp -Phe-0i'oe and g of Z-Lys(Boc)-ONp are dissolved in 25 ml of D?4F and left to stand overnight at 220C. The resulting hexapep- tide is precipitated as a jelly by addition of ether and is purified by crystallisation from methanol-ethyl acetate-petroleum ether; melting point 211-212 0 C. DS: Rf(chiloroform-methanol 9:1) 0.50; Rf(89)=

0.-55. d) Z-Lys (Boc )-Leu-Tnr(tBul-Gln-Asp(tBu)-P.e-!lJH 2

g of the abovementioned methyl ester are dis- solved in 50 ml of MY.with warming, -1ml of hydrazine hydrate are added and the mixture is left to stand for hours at 2200. The product is precipitated by addition of 250 ml of water an-d the finely pulverulent precipitate is filtered off and washed w..ith water until it gives a

negative rdaction with Folin reagent. Ilelting point approx.

31 4 7 9,2 1 0

220 C (decomposition). 67 '13 3'/74 DS: R-(chloroform-methanol 9:1) 0.24; Rf(89) 0. 27.

e) Z-Thr(tBu) -Tr( tBu)-Pro-.Gln-Th.- r(tBu)'--n--Thr-

(tBu)-Gl-Val-Gly-Ala-Pro-I,:'H2

393 mig of 2-Ti1r(tB)-Tlyr(tBu)-Pro-OH (DOS 2,050,434) and 66 p1 of N-rethyl:orxpholine are dissolved in 5 EL of tetrahydrofurane, the solution is cooled to -20 0C and 82 It1 of isobutyl chloro:arbonate are acfd over the course of 1 minute. The mixture. is stirred for a furter 10 minutes 0 at -15 C, a solizion, precooled to 0 C of 300 rg of H-Cln- Thr( tBu -Asn-T r(-tBu) -Glv-Val-Gly-Ala-Prc-;H 2 [analogous to Example 1 j)l in 6 ml of trifluoroet'anol-chloroform is added, and the whole is stirred for 1 hour at OOC and left to stand overnight at 22 0C. The reaction mixture is concentratcd until a jelly-like mass forms and the

latter is reprecipitated from carbon tetrachloride-petroleun ether. The crude product is purified by subjecting it to a Craig distribution in the system methanol-buffer-chloroform-

carbon tetrachloride, 10:3:5:4 [buffer as in Exapnple 1k)] through 400 stages, using phase volumes of 3 ml at a time.

The chromatographically pure dodecapeptide is isolated as

an amorphous pow;der from distribution elements No. 118-143

(rma 130; K 0.48) by concentration to dryness and sub- limation of the buffer in a high vacuum at 400C.

DS: Rf(43C) 0.42; Rf(52A) 0.37; Rf(70) 0.65 f) H1-Thr( tBu)-Tr tBu)-Pro-Gln-Thr tBu)-Asn--Thr-( tBu)-Gly- Val-Gly-Ala-Po-f:-1i

32 4 7 9.2 1 0

67,133/ 4 1.56 g of dodecapeptide from e) are dissolved in

ml of methanol with warming, and hydrogenated in the

usual manner. On concentrating the hydrcgenation solution

to dryness, the product is obtained as an amorphous residue

of melting point 1600C. DS: Rf(430) 0.30; Rf(52A) 0.21; Rf(70) 0.56.

g) Z-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Tyr(tBu)-Pro-Gln-Thr(tBu)-

Asn-Thr Bu) -GlGly-Va l-C-y-Ala-Pro-NH2

445 mg of Z-Asn-Lys(Boc)-Phe-His-!IHNH 2 (Helv. 53,

2135 (1970)) are dissolved in 4 ml of DMF with warming and

350 pl of 3.2 N HC1 in dioxane and 83 1p of tert.-butyl 0 The mixture is nitrite are added successively at -20 C. 0 stirred for a further 15 minutes at -10 C, a solution of 400 mg of the abcvementioned hydrogenated dodecapeptide in

ml of DMF, and 192 1p of ethyl-diisopropylamine are then

added and the whole is stirred for 2 hours at 0 C. A

further 48 p1 of ethyl-diisopropylamine are then added and

the mixture is left to stand overnight at 00C. The crude

product is precipitated by dropwise addition of the reac-

tion solution to 100 ml of ether and is purified in a Craig distribution in the system methanol-buffer-chloroform-carbon

tetrachloride, 10:3:7:4 [buffer as in Example 1 through

650 stages, using phase volumes of 3 ml at a time. The

pure hexadecapeptide is isolated as an amorphous powder in the usual manner from distribution elements.No. 230-275

(rmax 255; K 0.65).

DS: Rf(43C) 0.37; Rf(52A) 0.27; Rf(70)

0.63.

33 4 7 9,2 1 0

67 133 74 h) H-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Tyr(tBu)-Pro-Gln-Thr (tBu)-Asn-Thr t3u)-Gly-Val-Gly-Ala-Pro-;H 2 acetate

250 mg of hexadecapeptide from g) are hydrogenated in the usual manner in 30 ml of 805 strength acetic acid. After filtering off the catalyst, the filtrate is concen- trated to approx. 4 ml and lyophilised. DS: Rf(43C) 0.24; Rf(52) 0.14; Rf(9 6 0.34 i) Z-Lys(Bcc)-Leu-Tir(tBu)-Gln-Asp(OtBu)-Phe-Asn-Lys(Boc)- Phe-His-Thr(tBu)-Tyr(tBu)-Pro-Gln-Thr(tBu)-Asn-Thr(tBu)-

Gly-Val-Gly-Ala-Pro-NH2

274 mg of Z-Lys(Boc)-Leu-Thr(tBu)-Gln-Asp(OtBu)-Phe-

NHNH 2 are dissolved in 2 ml of D.F with warming and 154 pl of 3.2 N HC1 in dion-ne and 37 pl of tert.-butyl nitrite are added successively at -200 C. The mixture is stirred for 0 minutes at -10 C, a solution of 275 mg of the above- mentioned hexadecapeptide-acetate in 2 ml of DMF (precooled to 00 and 85 pl of ethyl-diisopropylamine are added, and the whole is stirred for 2 hours at 0oC. After adding a further 21 pl of ethyl-diisopropylamine, the mixture is left to stand for 15 hours at o0C and thc crude product is precipitated as a pulverulent precipitate by means of ether-petroleum ether Itis pifed by subjecting it toa Cradg distribution in the system methanol-buffer-chloroform- carbon tetrachloride, 10:3:5:5 [buffer as in Example 1 k)] through 345 stages, using phase volumes of 3 ml at a time. The chromatographically pure docosepeptide is isolated in the usual manner from distribution elements No. 98-132

34 47 9,2

67 i 3 3/74 (rma 115; K DS: Rf(43C) 0.39; R (52) 0.29; Rf(95) 0.43. j) H-Ls(Boe)- r u spP A(O

GlyVai-Gly-Ala-Pro-1TH 2

173 mg of docosapeptide from i) are hydrogenated for 15 hours in 3 ml of 803' strength acetic acid, using

mg of Pd on charcoal, and the filtrate is concentrated

to epprox. 3-4 ml and lyophilised. To remove the acetic

acid, the lyophilisate is reprecipitated from trifluoro- ethziicl-sodium bicarbonate solution strength).

DS: Rf(52) 0.21; Rf(70) 0.63; Rf(l00)

0.17. t3u k) Brnp-Gly-Asn-Leu-Ser( hr(th u)-Cys-Val-Leu-Gly-Lys

(Boc)-Leu-Tir(tBu)Glnksp(0tBu)Phe AsnLys(Boc)Phe

His-ihr (tBu)-Tryr(tBu)-Pro-Gln-Thr( tBu)lAsn-Thr(t~u)-Gly" Val-Gly-Ala-Pro-NH 2

127 mg of the docosapeptide described under j), nmg of B!rD-Gly-Asn-Leu-Ser(tBu) -Thr( tBu) -Cs-Val-Leu-Gly- CH (DDS 12 mg of 11-hydroxy-succinimide and

17.5 mg of dicyclohexyl-carbodiiaide are dissolved or sus- 0 pended in 0.5 ml of DI4F and stirred for 3j hours at 45 C. The crude product is precipitated by adding 53 ml of ether and is purified by means of a Craig distribution in the system methanol-buffe,chloroform-carbon tetra-

chloride, 10:3:5:4 [buffer as in Example 1 throughi 250

stages, using phase volumes of 3 ml at a tine. The pro-

tected dotriacontapeptide, which chromatographically is a

47 92 1

67, 133/74 single product, is isolated as an amorphous powder in the usual manner from distribution elements No. 88-110 (r max 100; K 0.67).

DS: Rf(52) 0.28; Rf(70) 0.65; Rf(10)

0.27. Examnole 4 B p-i y-Asn-Leu-Ser-Tnr-C's--et-Leu-Gly-Lvs-Leu-Tlr-Gln-Asp-

Leu-Asn-Lys-Leu-Hi s-Th-ir-Tyr-Pro-Gln-Thr-Asn-Thr-Gly-Val--Gly-

Ala-Pro-NH 2 hydrochloride. (Desaino-Lys 11 -Leu 12619 '2 22 -_Tyr 2 26 -Thr 27 "itnin N

mg of B -Gly-Asn-Leu-Ser(tBu)-Thr( tBu)-.Cy's-Met- Leu-'ly-Lys(Boc)-Leu-Thr tflu)-Cln-Asp(O-tBu)-Leu-Asn-Lys(Boc)-

Leu-His-%Thr(t~u)- nyr( tBu)-Pro-Gln-hr(tBu)-Asn-Th r( tBu)-Gly-

Val-Gly-Ala-Pro-NH. are split, to give the free peptide, by- treatment with 12 N HCl at OOC, as described in Examle 1.

DC: Rf(45) 0.47; Rf(101A) 0.50; Rf(112E)

b.42.

Thin layer electrophoresis: pH 1.9, 16 volt/cm, 1-2 hours: Migration distance approx. 3.7 cm towards the cathode. The protected dotriacontapeptide-amide can be

prepared, for example, as follows:

a) Ht-Leu-Thr( tBu)-Gln-Aso(OtBu)-Leu-0Me

3.6 g of Z-Leu-hr( tBu)-Jln-sp(OtBu)-Leu--ONe (DOS

2,030,434) in 90 ml of methanol are hydrogenated using

360 mg of Pd on charcoal, with CO2 absorption, until satur-a- tion is reached, -the catalyst is filtered off and the fil- trate is ccncentrated to dryness.

36 4 7 9.2

67,13 3/74 DS: Rf(chloroforn-methanol 9:1) 0.15; R£(89) 0.32.

b) Z-Lysv (Boc)-Leu-Thr(t. u -Gn-As(0tu)-Leu-Oe 840 mg of H-Leu-Thr(tBu)-Gln-Asp(OtBu)-Leu-O-e are dissolved in 5 ml of DIM, 765 mg of Z-Lys(Boc)-O::p are added

and the mixture is left to stand overnight at 22 0 C. It is then concentrated in a high vacuum to give a syrup and the

crude product is precipitated as a jelly by addition of ml of ether. It is purified by reprecipitation from methanol-ethyl acetate-petroleum ether; the pure hexa- peptide derivative has a melting point of 218-220 0 C. DS: Rf(chloroform-methanol 9:1) 0.41; Rf(89) 0.61. c) Z-Lys(Boc)-Leu-Thr(tBu)-Gln-Asp(OtBu)-Leu-%HN.H2

720 mg of the hexapeptide methyl ester described under b) are dissolved in 40 ml of absolute methanol and

after adding 3.6 ml of hydrazine hydrate the mixture is

left to stand for 5 hours at 22°C. The resulting hexa- peptide-hydrazide is precipitated by adding 75 ml of water, filtered off, well washed with water and dried; melting point 215-2180 C.

DS: Rf(chloroform-methanol 8:2) 0.55; Rf(89) 0.29.

d) Z-Asn-Lys(Boc)-Leu-His-Thr(tBu)-Tyr(tEu)-Pro-Gln-Thr(tBu)-

Asn-Thr(tBu)-Gly-Val-Gly-Ala-Pro-NH 2

636 mg of Z-Asn-Lys(Boc)-Leu-His-NHN'TH2(DOS 2,050,434)

37 47 9,2 1 0

67,133 1 are dissolved in 10 ml of DMF with warming, the solution is cooled to -200 C and 525 pl of 3.2 N HC1 in dioxane and 119 1 of tert.-butyl nitrite are added successively. The

mixture is stirred for 15 minutes at -15°C and a solution,

precooled to OOC, of 600 mg of H-Thr(tBu)-Tyr(tBu)-Pro-Gln-

Thr(tBu)-Asn-Thr(tBu)-Gly-Val-Gly-Ala-Pro-NH 2 [Example 3 f)]

in 4.5 ml of DKF, and 230 !l of N-methylmorpholine, are then

added. The mixture is stirred for 2 hours at 0OC, a further

46 pl of 17-methylmorpholine are added and the whole is left

to stand overnight at 0 C. The crude product is precipita-

ted with 100 ml of ether and subjected to a Craig distribu-

tion in the system acetonitrile-buffer-chloroform-methanol,

1:1:1:1 [buffer as in Example 1 through 500 stages, using

phase volumes of 3 ml at a time. The pure hexadecapeptide

is obtained as an amorphous powder from distribution

elements No. 38-62 (rma x 50; K 0.11) by concentration 0 to dryness and sublimation of the buffer at 40 C in a high vacuum.

DS: Rf(43C) 0.38; Rf(52A) 0.28; Rf(70) 0.51.

e) H-Asn-Lys(Boc)-Leu-His-Thr(tBu)-Tyr(tBu)-Pro-Gln-Thr(tBu)- Asn-Th r(tBu)-Gly-Val-Gly-Ala-Pro-NH 2 acetate

380 mg of hexadecapeptide from in 45 ml of

strength acetic acid are hydrogenated using 50 mg of Pd on

charcoal until the starting product has disappeared (as

checked by thin layer chromatography). After filtering off the catalyst, the filtrate is concentrated to approx. 4 ml and lyophilised.

DS: Rf(43C) 0.29; Rf(70) 0.40.

38 479.2

67,1 3 3n4 f) Z-Lys(Eoc)-Leu-Thr(tBu)-Gln-Asp(OtBu)-Leu-Asn-Lys(-oc)- Leu-:-His-Tnr(tBu)-Tyr(tBu)-Pro-Gln-Thr(tBu)-Asn-Thr(tBu)-

Gly-Val-Gly-Ala-Pro-NH 2

230 mg of Z-Lys(Boc)-Leu-Thr(tBu)-Gln-Asp (OtBu)-

Leu-NHNH2 are dissolved in 2 ml of DIIF and 134 pi of 2.3 N

HC1 in dioxane and 32 pl of tert.-butyl nitrite are added

successively at -20°C. The solution is stirred for

minutes at -10 C, after which a solution of 306 mg of the 0

hexadecapeptide-acetate described under e) in 3 ml of DMF

and 63 pl of N-methylmorpholine are added. The mixture is

stirred for 2- hours at 0 C, a further 15 pl of N-

methylmorpholine are added, the whole is left to'stan overnight at ml of 0OC and the crude product is precipitated by adding 60

ether and 30 ml of petroleum ether. It is purified by 240 stages in subjecting it to a Craig distribution through the system methanol-buffer-chloroform-carbon tetrachloride,

10:3:5:5 [buffer as in Example 1 The pure docosa-

isolated as an amorphous powder in the usual peptide is manner from distribution elements No. 83-107 (rmax

K 0.65). DS: Rf(52) 0.27; Rf(70) 0.59; Rf(l00)

0.41.

g) H-Lys(Boc)-Leu-Thr(tBu)-Gln-Asp(OtBu)-Leu-Asn-Lys(Boc)- Leu-His-Thnr(tBu)-Tyr(tBu)-Pro-G-n-Thr(tBu)-Asn-Thr(tBu)-

Gly-Val-Gly-Ala-Pro-H 2

100 mg of docosapeptide from f) in 16 ml of mg strength acetic acid are hydrogenated-overnight with 15

of Fd on charcoal, the catalyst is filtered off and the

39 47 9.2

6 i 3 3 /74 filtrate is ccncentrated to approx. 2 ml and lyophilised.

To remove the acetic acid, t~he residue is dissolv.fed in 0.6 ml

of triandcetancl and reprecipJitated by dropw.ise additicn

to 5 m- of sodi- bicarbonate solution (55. strength).

DS: 0.18; R-(70) Rf(100) 0.20.

h) Bw7p-Gly-sn-le-ui-Ser(tu)-Thr tbu)-Cy s-Hyet-Leu-Gly-Lys

t3u )-Grn-Asp(0t'. u)-Leu-sr-Lyso3O)-Leu-

hlis-Th z3u)-'Thyr(tBu)-Pro-Gln-Th-r(t3u)-Asn-Tqr(tBu)-Gly-

Vl-Gv-la Pro-2-1 2

43'g of -C3ly-sn-Leu-Ser(tu)jtC-sy'e

Leu-Gy-C~ (DOS 2,C50,454), 80 mg of tihe abovemontioned

h-dogenated docosaeptide, 7.5 mg of N-hydroxysuccinimide

and 10 mg of dicyclchexylcarbodiimide are dissolved in 0.6 ml 0 of CEUP and the mixture is stirred for hours at 45 C in a

N2 atmosphere. The crude product is then precipitated by

addition of 15 ml of ether and is purified by Craig distri-

bution in the system me thanol-buffer- chloro form-carbon

tetrachloride, 11:3:6:7 [buffer as in Example 1 through

200 stages, using phase volumes of 3 ml at a time. The

pure substance is isolated as an amorphous po-:der in the

usual manner from distribution elements No. 90-115 (rma

100; K

DS: Rf(52) 0.27; Rf(70) 0.66; Rf(100)= 0.31.

-xamnle

H-Cs-Gly- 1sn;-Leu-Se r--CThr-Cys-iNlet-Le-Gly-Lys-Leu-Tihr-Gl-

Asr-Le u-A-Lsn-Lys-Le',:-Hi sThr-Tyr-Pro-Gln-Th-r-Asf-Thr-Gly-Val- 11 '19 16 19 2 G1- Alla-Pro-N 2 hydrochloride (Lys -Leu -Tyr 22

27 .i nin le).

40 47 9,21 0

67,133/74 mg of BOCS-Cs-Gly-Asn-Leu-Ser-tlu)-ThLrktBu)- s- Met-Leu-Gly-Lys(Boc)-Leu-Thr('t u) GlAsp(0tsu)-Leu-Asn-

Lys (Boc ttu)-T),r tu)-Pro-Gin-r(-ulu)-Asn-

Thr(tBu) -GI y-7al--ly-Ala Pro-;0_' e splttoietefe -2are lit to give the free 0 peptide by treatment with 12 N HC1 at 0 C, as described in Example 1.

DC: Rf(45) 0.43; Rf(l01A) 0.43; Rf(112E)

0.38. in layer electrophoresis: pH 1.9; 16 volt/cm,

1T'hours, migration distance approx. 4.9 cm towards the

cathode.

The prctected dotriacontapeptide-amide can be

prepared as follows: 110 mg of Boc-Cys-Gly-Asn-Leu-Ser

(tBu)-Trnt3u)-Cys-et-Leu-Gly-OH (Helv. 53, 556 (1970)), 185 mg of H-Lys(oc)-Leu-Tnr(tBu)-Gn-Asp(Ot~u)-Leu-Asn-

Lys(Boc)-Leu-is-hr(tBu)-Tyr(tBu)-Pro-Gln-r(-tu)-Asl-

Thr(tBu)-Gly-Val-Gly-Ala-Pro-NHTP 2 [Example 4 and 17.2 mg

of N-hydrox-ysuccinimide are dissolved in 1.5 ilof Di4F with

warming, 23 rg of dicyclohexylcarbodiimide are added and 0 the mixture is stirred for 3j hours at 45 C under N2 The

crude product is then precipitated by adding 30 ml of-

peroxide-free ether and is purified in a Craig distribution through 350 stages in the system methanol-buffer-chioro-

forn-carbon tetrachloride, 11:3:6:7 [buffer as in Example

1 The chromatographically pure protected dotriacoiita-

peptide is isolated from distribution elements No. 140-179

(rm 159; K 0.83) by concentration to dryness and 0 sublimaticn of the buffer in a high vacuum at 45 C.

41 479,2 10

67, 133/74 DS: Rf(52A) 0.25; -Rf(70) =0.54; Rf(100)= 0.29. zExar'&le 6: H-CN Ser- Lhr---C-ys-Meat-Leu-Gly--Thr-Tyr-Thr-Gln-

AsDPh-An-L.'-Pe--.H1 sTrPe-Fro-C-ln-Thr-Ala-Thr-Gly-

Va1-~y-~a-rc-3E~(Thr 2 7 _c:2i citonin M).

202 rz= of BCOC-Cys-Gl-Asn-Leu-Ser(t~u)-Thir(tBu)-

Cys-M.'et-Leu- cu)-Ti-l-~u )-Thrr(tBu)-Gln-Asp(0tBu)-

Phe- Asn-Lys c-he s-Th -he-Pro-Gn-Thr (tBu)-Ala- Thr~~u)G~vY~L~1yA~a-Prc-'M 2 are dissolved in 6 ml of

905 strength _,flz~oacetic acid and the solution is

degassed wihnitrogen and left to stand for 90 minutes

at 23 0C. It is then poured into 110 ml of ice-cold perox- ide-free ether and the precipoitatLe is filtered off, rinsed w~ithl- ether and d-ried in vacua at 40 C. 0 185 mg of Thr2 7 calcitonin IMare obtained as the trifluoroacetate. This is converted into the acetate by elution using a column.

of Merck ion exchanger No. !I (weakly basic, acetate form);

the yield is 155 mg of lyophilised acetate.

For purification, 60 mg of -the acetate are dis- solved in 5 all of w-.ater and a total of 1.2 ol of Merck ion exchanger 'No. 1T (weakly basic, free base form) is added whilst stirringS, whereurion the pH assumes a value of approx. 6.5 and a fine precipitate separates out. To com- plete the precipitation, the mixture is stirred for 2 hours, during which the pH~ of the solution rises to 7.1. The nrecinitate and ion exchanger are then filtered off together and tnoroughly rinsed with water, and the

42 27 9, 2 1 0

67, 13 3,r74 peptide urhich has precipitated is eluted from the ion exchanger by addition of warm- 905.' strength acetic acid. The ion exc-an is Iilte red off and rinsed with 905' strength acetic acid, and tUhe acetic acid solution is lyo- philised. ThEis gives 74 ~gof Thr 2 7 -calcitonin 1-4as the acetic acid salt,wh.'ich accordirn to thin layer charornato- graphy and electrophoresis diisplays a high degree of purity. DA: R--Lk5;2) 0.49 (calcitonin M: P1(52) 0.55); DC: C.52 (calcitonin Rf(101A)=

0.57). In thin layer electr-ophoresis (cellulose plates, pH 1.9, 2 ho-"s, 280 V) the rnigration distance is 4.5 cm towards the cathode (calcitonin M4: KI-gration distance cm). In aminoacid analysis after total hydrolysis (6 N H1, 110'C, 24 hours), the product shows the calcu- lated aminoacid ratio). The starting material can be prepared as follows: a) Z-Ala-Thr(tBu) -G-ly-0Me A rixture of 18.9 g of Z-Ala-0N'%p, 13.4 g of

H-Thr(tBu)-Gly-011e. HEd, 100 ml of DI-T and 6.2 ml of N- methylmorpholifle is stirred for 17 hours at room tempera- ture, the scolvent is removed in a high vacuum, the residue is taken =n in ethyl acetate, the solution is washed and dried as described in EvXa=nnle 1 under and evap~orated, and the residue is crystallised from ether-petroleum ether. 0 Melting point 104-106 C; 16 g.

43 4 7 9.210

671 333/74 b) H-Ala-Thr(t3u)-Gly-OKe

13.3 g of the product obtained under a) are hydrogenated in meth-nol in the presence of 1.5 g of palladium on charcoal (10% of Pd). 9.4 g of colourless

resin are obtained.

c) Z-Thr(tBu)-..la-hr(tu)-Gly-OMe

9.4 g of the product obtained under b) and 13.2 g

of Z-Thr(tBu)-OSu are dissolved in 100 ml of IVF and the

solution is left to stand for 17 hours at room temperature.

The product is then precipitated by pouring the solution

into 500 m= of ice ater, and is filtered off, dried and

crystallised frcz acetone-petroleum ether, with addition of

a little water. Melting point 193 1950C; 13.6 g.

d) H-Tnr(tBu)-Ala-Thr(tBu)-Gly-OMe

13.1 g of the product from in 750 ml of

methanol, are hydrogenated using 2.6 g of palladium on

charcoal. 9.95 g of colourless oil are obtained.

e) Z-Gln-Thr(tBu)-Ala-Thr(tBu)-Gly-OMe

This copound is prepared analogously to the

pentapeptide derivative described in Example 1 under g).

It crystallises from methanol. Melting point 198-202oC;

10.7 g. f) H-Gln-Thr(tEu)-Ala-Thr(tBu)-Gly-OMe

g of the product from in 1 litre of methanol, are hydrogenated using 2 g of palladium on charcoal. The

product (8.2 g) is i-mediately processed further.

g) Z-Thr(tu)-Phe-?ro-GLn-Thr(tBu)-Ala-Thr(tBu)-Gly-OMe

8.2 g of the product from f)-are mixed with 8.3 g

44 4792

P 67,1 3 3n 4 of Z-Thr(tsu)-Fhe- ro-OH (Helv. Chin. Acta 2135 (1970)),

100 ml of 4.0 g of t!-hydroxysuccinimide and 4.2 g of

dicyclohexylcardiiide are added and the mixture is stirred for 17 hours at room temperature. The dicyclo-

hexylurea is then filtered off, the filtrate is poured into is fil- 1.3 1 of ice water and after 2 hours the precipitate tered off, d-ied ard stirred with 120 ml of ethyl acetate.

The insoluble ow-der is filtered off, washed with ethyl

acetate and dried. 8.5 g of a product having the follow-

ing Rf values are obtained: DS: Rf (121A) 0.78;

Rf(102A) G.35; Rf(4AC) 0.71.

h) Z-Thr(t2--)-Phe--ro-Gln-Thr(tBu)-Ala-Thr(tBu)-Gly-OH

5.15 g of the product from g) are dissolved in

110 ml of 903; strength methanol with warming, the solution

is cooled to 23°C and 13.5 ml of 1.0 N aqueous sodium

hydroxide solution are added. After 10 minutes, 45 ml of

water are added, followed by a further 25 ml after a further

minutes. After 20 minutes, the solution is poured into

270 ml of ice-cold 0.05 N aqueous hydrochloric acid, and

the precipitate is filtered off, washed with ice water and

with water-acetonitrile and dried. In the DS, the

product shows the following values: Rf(45) 0.27;

Rf(100) 0.33; Rf(43C) 0.28; 4.1 g.

i) H-Thr tu)-Phe-Pro-Gln-Thr tBu)-Ala-Thnr tBu)-Gly-OH 3.65 g of the product from h) are suspended in

125 ml of and hydrcgenated in the presence of 0.5 g of

palladium cr. charcoal (10% of Pd). The product (2.62 g)

is triturated with w-.ater, filtered off and dried.

45 47 9.2 1 0

6 7, 3 3 /74 ES: Rfk'1CD) 0.12; Rf(52) 0.23. j) Z- Asn-LS (3:oc) -Ph-Hi1s-Tr( t3u) Phe-Pro-GlnThr(tBi) -Aa-.

7.2 E of Z-Asn-Lys(Bzc)-Phe-Hi-*s-NHt-NH!-! 2 (Helv. Chim. Acta 53, 2135 (1970)) are dissolved in 30 ml of DPIF whilst

warming to P:)0 C, the* solution is cooled to -30 0 C and 7.74 ml1 of 3.7 N hydrogen chloride in dioxane are added. 1.42 ml of tert.-but,l nitrite are then added at -30 C, 0 the mixture is allcw.-ed tO rea ct for 10 minutes at -10 0 C and is again 0 cooled to C, and a solution of 6.45 g of H-Thr(tBu)- -DCl-I-r(l.u Aa .Mu GyO and 7.8 ml of N- ethl-diso roylainein 70 mU of DIN' is then added drop- w-.ise. The solution i4s left to stand for 18 hours at 000C

and is then poured into 4-00 Ul of ice-cold l~z strength

acet'ic acid. The precimita-te is filtered off, washed with water and dried in vacuo over calcium chloride. The pro- duct is digested Nwifth acetonitrile, filtered off and-dried.

DS: Rf(70) 0.33; Rf(l00) 0.21. k) Z-A sn-Ly s(Boc) Pe-Hi s-ir(tBu)p-he Prolnmr (tBu) -Ala-

Thr( t'u)-G'ly-Va--Gly-Ala-.ro-.rn 2 1.llg of dicyclohexylcarbodiLimide are added to a mixture of 7.5 g of Z-Asn-Lyvs(Boc)-Pe--Iis-Thr(tBu)-Phe-Pro-

Gln-Thrr(tSu)-Ala-Thr(tBu)-Gly-OH, 0.99 g of N-hydroxy-

succinimzide, 1.84 c-of' H-a-l-l-r-T (Helv. Chim. Acta, loc .cit.) and, 70 ml of MII and thre whole is stirred for 5 hours at 45 0 C. The mixture is then poured into 1.2 litcE of ice-cold ether and the precipitate is filtered off

and dried. The crude product is stirred with twice 2C0 ml

46 4 79,2 1 0

67,13 j /14 of ace toni tri le-eu aol filtered off and dried. DS: 0.50; Rf(96) 0.46; p1T(100)=

0.29; Rf(12l) 0.83; yield 8,5 g.

Ala-Th -l-a-yAa-PoK 2 ace tate.

7.05 g of the product described un-der k) are hydrogenated in 409 ml of 80% strength acetic acid in the presence of 1 g of ralladiun on charcoal (10% of Pd). The poroduct 0) shoNw.s the follo-wina value=s in DS:

Rf (96) 0.35; PZ (121) 0.72; P1 (451) 0.27.

mn) nr(-t tBu)-Gl-Asp(O-t u)-Phe-Asn-

565 mg of' 7-Thr(tBu)-Tyr(t3u)- Thr(tBu)-Glfl-

Asp(tu-h-B- 2 (Helv. Chim. Acta, loc. cit.) are

dissolved in 4 ml of D2I' hrilst warnmixg to 8D C, the solu- 0 tion is cooled to -2 0C, 0.35 ml of 3.7 N hydrogen chloride in dioxane and 0.067 ml of tert..-butyl nitrite are added, the mixture is stirred for 10 minutes at -15OC and a solu- 0 tion, cooled to 0 C, of 600 mg of H-AIsn-Lys(Boc)-Phe-His-

Thr(tBu)-Phne-Pro-Gi' rTr(tBu)-Ala-Thr( tBu)-Gly-Val-Gly-Ala- Pro-NH2 -acetate in 5 ml of DMF a nd 0.33 r.1 of N-ethyl- diisopropylamne is introduced dropwise. The whole is

stirred for 5 minutes at -15 0C and thien allowed. to warm to 23 0 C and stirred for 6 hours. It is then poured into 100 ml nrecipitate is filtered off, washed of ice -eater and the_- with water and dried in vacuo over calciu-- chloride. It is

purified by reprez-fpcfzatiLng from met-anol-water and

47 4 7 9,2 1 0

6 7,1.33/7~4 trituratin.- the -ecioitated product, after drying, -Aith

acetone. 650 mg of product are obtained.

D3: P(4 C 0.33; Ri' (70) 0.60.

n) (tu)-T=r(tBu)-Gln-Asp (Ot-Bu)- Phe -Asn--Lys

610 v of the product obtained under m) are hydro-

genat ed 25 ml o-f 80-,0 strength acetic acid in the presence

of Dalladiun cni c.--rcoal. The product is dissolved in 50 ml

of I-buta-cI sat-urated it water and to remcve acetic acid

thne solu-zcI:n is w,,aSh-ed twvice with dilute sodium carbonate

solution, wvith half-saturated sodium chloride -hent-ice

solution and finally with water. The butanol phase is then

evauorated and 605 mg of the product are obtained as a colourless foam.

DS: Rf (100) 0.20; 'Ri' (96) 0.40.

Tihr(-tBu)-Tyr(t3u)-Thr(tBu)-Gln-Asp(tBu)Phe-Asn Lys

of diicyclohexylcarbodiimide are added to a

mixture of 605 ES cf' H-ihr(tBu)-Tyr(tBu)-Thr(tBu)>Gll-Asp (OtBu) -Phe-A*sn-L, (Boc )-Phe-Hi s-T-hr (tBu) -Phe-Pro-Gln-Thr ,7z)GlVlGyAaPo', 290 mg of the

decapeptide Eoc-Cys-Gly-Asn-Leu-Ser( tBu)-Thr(tBu)-Cys-Mlet-

Leu-Glv-0?i (Iielv. Chim. Acta 53, 556 (1970)), 46 mg of

1-Ihydroxf..,'en-zctriazole and 10 ml of D!4F, the mixture is 0 0 stirred for 9) m-inutes at 4i0C and 18 hours at 23 C, the

48 4 7 9,2 1 0

67. 131!74 dicyclohexylu2'a -w-ich has separated out is filtered off

and the filtrate .s ::oured into 100 ml of ice-cold peroxide-

free ether. The rciitate is filtered off, rinsed with

ether and dried. 'Th resulting crude product is subjected

to preliminary v iffiation by reprecipitating twice from

Di:-ether and twice lrom D.17-water. The precipitated pro-

duct is subjected tc a counter-current distribution through

300 stages (solvent system 3.66 litres of methanol, 1 litre of buffer, 2 litres cf chloroform and 2.32 litres of carbon

tetrachloride; bufer: 29 ml of glacial acetic acid and

19 g of anniun aze a-te in 1 litre of water). The pure

substance is present in fractions 123-137, distribution

index K 0.76.

DS: 0.32; Rf(52A) 0.41.

Ex&'mnle 7:

H-C 7s-G1y-A,n-Leu- S "-r--Cys-Met-L

Asp-Phe-Asn-Lys-PLZ -H!is-T r-Phe-Pro- 2 2 7 Gly-Ala-Pro-H 2 acetate (leul Thr calcitonin M)

493 mg of 3oc-Cys-Gly-Asn-Leu-Ser(tBu)-Thr(tBu)-

Cys-Iret-Le-u-Gly-- ,n u) -Leu-Thr( tBu) -Gln-Asp (OtBu) -Phe-Asn-

Ly (Boc )-Pe-His-7n.- ('Uu)-Plie-Pro-Gln-Thr(t~u)-Ala-llr(tu)

Gly-Val-G1y-Ala-Pro-,Zi 2 are introduced into 12 ml of ice-

cold 905 snrength trifluoroacetic acid and the solution is

degassed with nitroSen and warmed to room temperature.

The solution is left to stand for 90 minutes at room

temperatureand -is then again cooled to 0 C and Doured into

150 ml of ice-cold zeroxide-free ether, the mixture is

and the fine precipitate is left to st-nd for 2 hours at 00C

49 4 7 9.2

6 7,1 3 3/74 filtered cff, w.ashed with ether end dried. It is then

dissolved in 15 ml of 5% strength acetic acid and the

solution is filtered through a column of MIerck ion exchan-

ger No. II, weakly basic, acetate form, to remove the tri-

fluoroacetic acid. The column is rinsed with 3 times 10 ml of 5% strength acetic acid and the eluates are combined and

lyophilised. 432 mg of Leu12-Thr27-calcitonin 1-acetate are obtained as a colourless, light powder. DC: Rf(l01A) 0.50; DA: Rf(52) 0.45.

In thin layer electrophoresis on cellulose plates (pH 1.9, 280 V, 2 hours) the migration distance is 4.5 cm towards the cathode. The starting material can be prepared as follows: a) Z-Thr( t3u)-Leu-Thr(tBu)-Gln-Asp(OtBu)-Phe-NH-NH 2

12.7 g of H-Leu-Thr(tBu)-Gln-Asp(OtBu)-Phe-OMe are dissolved in 100 ml of DMF, 9.0 g of Z-Thr(tBu)-OSu are added and the mixture is left to.stand for 17 hours at 0 C. The product is then precipitated with water, fil- tered off, dried and crystallised from ethyl acetate, yield 14.2 g; for further purification, the product is 0 reprecipitated from methanol-water, melting point 197-199 C. For conversion into the hydrazide, 13.9 g of the Z-Thr(tBu)- Leu-Thr(tu)-Gln-Asp(OtBu)-Phe-O:e obtained are dissolved in 700 ml of methanol, 70 ml of hydrazine hydrate are added and the mixture is left to stand for 45 minutes at roon tempera-

ture. It is then concentrated in vacuo to a volume of

300 ml and the product is precipitated by adding 500 ml of

50 47 9,210

67.133n4 water. It is filtered off, washed with water until

neutral and dried. This gives 13.5 gof crude hydrazide.

The latter is p-urified by one reprecipitation from Di.F-

water, and crystallisation from methanol. 11 g of crystal-

line product, melting point 225-227oC, are obtained.

b) Z-Thr(tBu)-Leu-Thr(tBu)-Gln-Asp(OtBu)-Phe-Asn-Lys(Boc)-

Phe-His-Th'r(tBu)-Phe-Pro-Gln-Thr(tBu)-Ala-Thr(tBu)-

Gly-Val-Gly-Ala-Pro-NH 2

A suspension of 4.00 g of Z-Thnr(tBu)-Leu-Thr(tBu)-

Gln-Asp(Ot3u)-Ph e-N.H 2 and 25 ml of DMF is warmed to 700C,

whilst stirring, until all has dissolved, the solution is

cooled to -20 0 C and 2.50 ml of 3.70 N hydrogen chloride in

dioxane are added. 0.48 ml of tert.-butyl nitrite is then

added dropwise whilst stirring and cooling to -20OC, and the 0 mixture is allowed to rise to -10 C and stirred at this

temperature for a further 15 minutes. A solution, pre-

cooled to -100 C, of 4.2 g of H-Asn-Lys(Boc)-Phe-His-Thr(tBu)-

Phe-Pro-Gln-Thr(tBu)-Ala-Thr(tBu)-Gly-Val-Gly-Ala-Pro-NH 2

acetate in 50 ml of DiMF and 1.6 ml of N-ethyl-diisopropyl-

amine are then added and the mixture is allowed to rise to 0 C

and stirred for a further 60 minutes, whilst slowly adding

dropwise a further 0.8 ml of N-ethyl-diisopropylamine. 0 The mixture is allowed to react for 17 hours at 23 C,

whereupon a jelly-like precipitate forms. 500 ml of ice-

cold peroxide-free ether are now added and the precipitate

is filtered off, rinsed with ether and dried. The product

is triturated with 200 ml of ice-water, again filtered off,

dried a-d reprecipitated three times from DMF-water and

51 47 9,2

67, 1 3 3 f74 twice from Dv-ethyl acetate. 4.98 g of product are thus obtained. DS: Rf(70) 0.61; Rf(100).= 0.38. As n c) -Leu-Tir( tBul-Gin-,so(OtLu)-Phe-j_,-Lys (Boc)- Phe-His-Thir(t-u)-Phe-Pro-Gln-T-r( tBu)-Ala-Thr tBu)- Gly-Val-Gly-Ala-Pro-NH 2

g of the product obtained under b) are dissolved in 200 ml of 9050' strength acetic acid and hydrogenated in the usual iner in the cresence cf 0.5 g of palladium on charcoal (10%5 Pd). To renoe the acetic acid, the result-

ins acetic acid salt is dissolved in 200 ml of n-butanol saturated ith water and the solution is repeatedly washed with dilute sodium carbonate solution, dilute sodium chloride solution and finally water. The strong emulsions

which are formed on washing the butanol solution are separated off by centrifuging. The butanol solution is

then evaporated to dryness and 2.15 g of H-Thr(tBu)-Leu- rrnr(tBu)-Gln-Asp(0tBu)-Ph--e-Asn-Lys (Boc)-Phe-His-Thr~tBu)- Plie-Pro-Glrn-7-r C tBu)-Ala-Thr (tBu)-Gly-Val-Gly-Ala-Pro-NH 2 are obtained as a colourless resin. DS: Rf(96) 0.37; Rf(100) 0.19.

d) Boc-Cys-Gly-Afsn-Leu-Ser(tlu)-Thr(tBu) -Cys-IIet-Leu-Gly-

Phe-His-r(t~u)-Phe-Pro-Gln-Bu-Aa-r(t~u)-Gy Val-Gly-Ala-Pro-NH 2

400 mg of dicyclohexylcarbodiimide are added to a

mixture of 2.01 g of the product obtained under 1.00 g

52 47 9,2

67 133/74 o Boc-Cy s- et-Leu-ly-OH,

250 mg of !-h~rzxysuzcinimide and 30.m l of DIF, whilst

stirring, and the niiture is then stirred for 6 hours at 0 43 0 C. Thereafter, it is cooled to 0 C for 3 hours, the

dicyclohexylurea wich has separated out is filtered off

and the filtrate -s poured into 250 ml of ice-cold peroxide-

free ether. The preipitate is filtered off, rinsed with

ether and dried. The product is purified by reprecipita-

ting it three times from D!NF-water and subsequently three

times from DM-acetone. This gives 1.72 g of purified, Prte'e TU12 1 27_ 1 protected Leu -Thr-calcitonif M.

DS: Rf(52A) 0.33; Rf(100) 0.30; Rf(107)

0.58. Examnole 8:

H-Cys-Gly-Asn-Leu-SC r-Tihr-Cys-4e t-Leu-Zly-Thr-Leu-Thlr-Glf-

Asp-Phe-Asn-Lys-Ph -H-i s-Thr-Pe-Pro-Gln-Thr-A sn-Thr-Gly-

Val-Gly-kla-Pro-NTH2 (Leu12-Asn26-Thr27calcitonin M).

170 mg of Bcc-Cys-Gly-Asn-Leu-Ser(tBu)-Thr-

tLu)-Cys-!Mett-Leu-Gly,-Thr(tBu)-Leu--hr( tBu)-Gln-Asp(OtBu)-

Phe-Asn-Lys(Toc )-Phne-His-Thr(tBu)-Phie-Pro-Gln-Thr(tBu)-

Asn-hr(tu)-Gly-a-Gly-Ala-Pro-NH 2 are dissolved in 5 ml

of 90% strength trifluoroacetic acid, the solution is left 0 to stand for 90 minutes at 25 C and the peptide-trifluoro-

acetate is crecinitated with peroxide-free ether, filtered

off, rinsed with ether, dried and dissolved in l5: strength

acetic acid. It is then filtered through a colun of

Ierck ion exchanger (weakzly basic, acetate form) and eluted

ith 1- tr-ng- acetic acid, a-ld the eluate is lyophilised

53 47 9.2

67,1 3 3 n4 (116 mg). The resulting acetate is purified by dissolving

it in 5 ml of water (pH of the solution 4.2) and adding

a total of 2.8 ml of Merck ion exchanger No. II (weakly

basic, base form) in portions, whilst stirring. In the

course thereof, the pH of the solution slowly rises and

when it reaches a value of approx. 6.7 the free peptide

begins to precipitate. The mixture is stirred for a

total of 2 hours, during which a pH value of 7.1 is

reached. The precipitate and the ion exchanger are then

filtered off together and rinsed with water, and the peptide

is then dissolved out of the ion exchanger by stirring with

90D3 strength acetic acid which has been warmed to 600C.

The mixture is filtered, the residue is rinsed with

strength acetic acid and the filtrate is lyophilised. 2 26 2 98 ng of Leu 1 -Asn -Thr 7-calcitonin i are obtained as the

acetate, in high purity. DC: Rf(lO1A) 0.33; DA: Rf(52)

0.47. The process of purification according to the

invention, described here, entailing precipitation of the

peptide in the isoelectric range, forms the subject of U.S. ;TJi. 3,94 9876.

The protected peptide used as the starting

material can be prepared as follows:

a) Z-Thr(tBu)-Leu-Thr(tBu)-Gln-Asp.(OtBu)-Phe-Asn-Lys(Boc)-

Phe-His-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-Thr(tBu)-

Gly-Val-Gly-Ala-Pro-NH 2

380 mg of Z-Thr(tBu)-Leu-Thr(t.Bu)-Gln-Asp(0tBu)-

Phe-INH2 (compare Example 7) are dissolved in 5 ml of 0 DIF whilst stirring and warning to 70 C, the solution is

54 4 7 9.2' o

67.13 3 n cooled to -20 C, 0.265 ml of 3.71 N hydrogen chloride in 0

dioxane is added followed by 0.051 ml of tert.-butyl

nitrite, and the reaction is allowed to take place for

minutes at -10 C. A solution, cooled to -10°C, of H-Asn-

Lys(Eoc)-Phe-His-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-Thr(tBu)-

Gly-Val-Gly-Ala-Pro-?NH2. acetate (compare Example 1, sub. n)

in 5 ml of D.M and .0.20 ml of i-ethyldiisopropylamine is

then added and the mixture is stirred for 1 hour at 0 C.

During this time, a further 0.07 ml of N-ethyl-diisopropyl-

am.ine is added and the mixture is then allow.ed to warm to 0 room tencerature and left to stand for 1 hour at 25 C and

hours at 50C, whereupon a jelly-like precipitate forms.

Precinitation is then effected by adding 100 ml of ice water

and the precipitate is filtered off after 1 hour at O0C,

rinsed with water and dried. The powder is triturated with

ether, filtered off and again dried. The crude product from (colourless powder) is purified by one reprecipitation

DMFi-water and two from D:IF-acetone. 440 mg of product are

obtained. DS: Rf(70) 0.52; Rf(lO0) 0.31; Rf(96)

0.40. b) H-Tnr(tBu)-Leu-T7ir(tBu)-Gln-Asp(OtEu)-Phe-Asn-Lys(Boc)- Phe-His-Thr(t3u)-Phe-Pro-Gln-Tnr( tBu)-Asn-Tnr(tBu)-Gly-

Val-Gly-Ala-Pro-NH 2

260 mg of the product obtained under a) are dis-

solved in 25 ml of 80% strength acetic acid and are hydro- of genated in the usual manner in the presence of 50 mg

palladium on charcoal (10% of Pd). The product (266 mg

55 47 9.2

67.133 374 of resin) is dissolx'ed in 20 -l of ,ater-saturated n-

butanol, in order to remove acetic acid. The solution

is ;:ased re_. aaedly with sodiu= carbonate solution, dilute

chlcride solution and water, and the butanol phase

is then evacated. Yield: 199 ng.

DS: Rf(100) 0.13; Rf(96) 0.36. 1 c) BccCys-2:1-.-Asl-cn-L)eu-PSr tu)-Thr( tBu) -Cys-Net-Leu-Gly- Th~r(t~u' Thr(tBu)-CGln-Asp,(Ot~u)-Phe-Asn-Ly5 (Boc)-

V lC 1y- _L a-P ro-H 22

27 r- of dicyclohaxxryl cabod-i iide are added to a

mxt-ure of 1C2 rg of Boc-C s-Gy-sn-Leu-Ser(tu)-Thr(tkBu)- 199 mg of ::-Tnhr(t3u)-Lcu-Thr(tBu)-Gln-

As; (Otfu)-Fhei -Asrn-Lys (Boo)-Phe--i s-Thr( tBi) Phe-Pro-Gln-

Trf (tU)-Asni-i,-T (t3U) -Gly-Val-rly-Ala-Pro-NH7-1 2 16 mg of N-

hydroxysucc-inimide and 3 ml of DXTh whilst stirring, the

mixture is stirred for 6 hours at 45 C and poured into ice-

cold ether, and after 2 hours at 000 the precipitate is

filtered off. It is rinsed with ether and dried, the

powder is di solved in 5 ml of DlMFT whilst warming, the

solution is cooled to room temperature and the product is

precipitated by adding 50 ml of ethyl acetate. It is fil-

tered off, rinsed with ethyl acetate and dried, the powder

is then di s-lved in 10 ml of DF-7 ethano whilst

warmLing, the sDlution is cooled in ice and the product is

precipit-ated_ addition to ice water. After 17 hours at

000, the fine vrecipitate is filtered off, rinsed with water

and dried in :cuo over calcium chloride. 175 mg of the

-56 4 7 9.2 1 0

67,1 3 3/74 protected dotriacontapeptide mentioned in the title are

obtained.

DS: Rf(107) 0.60; Rf(52A) 0.23; Rf(100) 0.22.

Examole 9:

H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-

Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Asn-Ile-Gly-

Val-Gly-Ala-Pro-NH 2 (Asn26-calcitonin M).

115 mg of finely powdered BOC-Cys-Gly-Asn-Leu- Ser(tBu)-Thr(tBu)-Cys-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr

(tBu)-Gin-Asp(OtBu)-Phe-Asn-Lys(BOC)-Phe-His-Thr(tBu)-Phe-

Pro-Gln-Thr(tBu)-Asn-Ile-Gly-Val-Gly-Ala-Pro-l42 are

added to 2.5 ml of ice-cold 90% strength trifluoroacetic

acid whilst stirring and degassing with nitrogen. After

the peptide has dissolved, the solution is allowed to rise

to 21°C and is left to stand for 90 minutes. It is then

poured into 50 ml of ice-cold peroxide-free ether and the product is filtered off and dried. 84 mg of the tri-

fluoroacetic acid salt of Asn -calcitonin M are obtained.

To convert this into the acetate, it is dissolved in 1%

strength acetic acid and filtered through an ion exchange

column of Merck ion exchanger No. II (weakly basic,

acetate form). The eluate is lyophilised and 75 mg of

Asn -calcitonin M are obtained as the acetate. After

total hydrolysis (6 N HC1, 24 hours at 110 0 the pro-

duct shows the -expected aminoacid ratio.

In thin layer chromatography.on cellulose plates,

the product shows the following Rf values (the comparative

57 479.210

67,133/74 values for calcitonin M are shown in brackets): Rf(lO0A) 0.55 Rf(112A) 0.58 (0.62). The protected peptide used as the starting material can be prepared as follows: a) Z-Asn-I1e-Gly-OMe A mixture of 9.1 g of H-Ile-Gly-OMe.HCl, 16.3 g of Z-Asn-ONp, 50 ml of DMF and 4.1 ml of N-ethylmorpholine is left to stand for 18 hours at room temperature. Here- upon, the solution solidifies to a jelly. 300 ml of ether are then added, the product is triturated, filtered off and dried, and the insoluble powder is triturated with 200 ml of water, filtered off, dried and crystallised from ethyl acetate-hexane. 15 g of Z-Asn-Ile-Gly-OMe of melt- ing point 218-2190 C are obtained. b) H-Asn-Ile-Giy-OMe 14 g of Z-Asn-Ile-Gly-OMe are suspended in 500 ml of DMF and hydrogenated with 1 g of palladium on charcoal of Pd).Hereupon, the starting material dissolves. After completion of the hydrogenation, the catalyst is fil- tered off and the filtrate is evaporated to dryness. 9.7 g of H-Asn-lle-Gly-OMe, which is free from starting material, are obtained. c) Z-Thr(tBu)-Asn-Ile-Gly-OMe A mixture of 9.5 g of H-Asn-Ile-Gly-OMe, 80 ml of DMF and 13.6 g of Z-Thr(tBu)-OSu is left to stand for 18 hours at 250 whereupon it solidifies completely. 300 ml of ether are added and the product is triturated, filtered off and dried. It is then triturated with 200 ml of water,

58- 47 9.2

67.1 3 3 n filtered off and dried. The residue is crystallised from ethanol; 12.4 g, melting point 207-208°C. d) H-Thr(tBu)-Asn-Ile-Gly-OMe 11.0 g of Z-Thr(tBu)-Asn-Ile-Gly-OMe are dissolved in 500 ml of DE' with warming and are hydrogenated in the presence of 1 g of palladium on charcoal (10% of Pd). After the absorption of hydrogen has ceased, the catalyst is filtered off and the filtrate is evaporated to dryness. g of H-Thr(tBu)-Asn-Ile-Gly-OMe are obtained; in thin layer chromatography on silica gel plates Rf(43A) 0.12; Rf 0.28 in the system chloroform-methanol (70:30). e) Z-Gln-Thr(t3u)-Asn-Ile-Gly-OMe. g of H-Thr(tBu)-Asn-Ile-Gly-OMe are dissolved in 100 ml of DMF and 7.7 g of Z-Gln-ONp are added. The reaction mixture slowly solidifies to a solid mass. After 18 hours at room temperature, 375 ml of ether are added and the product is thoroughly triturated, filtered off, dried, triturated with 250 ml of water, filtered off and again dried. It is then precipitated from trifluoroethanol-water; 11 g are obtained; melting point 275 277°C. The.product is very sparingly soluble in the customary organic solvents. f) H-Gln-Thr(tBu)-Asn-Ile-Gly-OMe

g of Z-Gln-Thr(tBu)-Asn-Ile-Gly-OMe are sus- pended in 500 ml of DMF and hydrogenated in the presence of g of palladium on charcoal of Pd). In the course

thereof, the initially insoluble starting material dissolves. After completion of the hydrogenation, the catalyst is

59 47 92 1 0 filtered off and the6 i4,s evaporated; iAA 6.5 g of H-Gln-Tnr(tBu)-Asn-Ile-Gly-OMe are obtained as a resin. In thin layer chromatography on silica gel plates, Rf(43C) 0.20; Rf(lO1A) 0.16. g) Z-Thr(t3u)-Phe-Pro-Gln-Tnr(tBu)-Asn-Ile-Glv-OMe g of H-Gln-Thr(tBu)-Asn-Ile-Gly-OMe are dis- solved in 500 ml of DIMF and 5.8 g of Z-Thr(tBu)-Phe-Pro-OH;

2.3 g of N-hydroxysuccinimide and 3 g of dicyclohexylcarbo-

diimide are added. The reaction mixture is stirred for 16 hours at room temperature, the dicyclohexylurea is fil- tered off and the filtrate is concentrated to a volume of 200 ml and poured into 1.5 litres of ether. The mixture is then left to stand for 3 hours at OOC and the fine precipitate is filtered off (10 g of powder). The powder is triturated with 200 ml of water, filtered off and dried.

9.6 g of crude product are obtained and are next .triturated

with ethyl acetate for further purification. The product is filtered off, dried, suspended in acetone, again fil-

tered off and dried. The insoluble product (9.3 g) is sufficiently pure for further conversion. In thin layer chromatography on silica gel plates, Rf(102A) 0.66. h) Z-Thr(tBu)-Phe-Pro-Gln-The(tBu)-Asn-Ile-Gly-OH

3.1 g of Z-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-Ile- Gly-OMe are dissolved in a mixture of 155 ml of methanol,

ml of dioxane and 55 ml of water, with warming. The mixture is then cooled to room temperature, 8 ml of 1.0 N NaOH are added and the whole is left to stand for 30 min-

utes at room temperature.

60 47 9.210

It is now cooleo 0 C and 150 ml of ice water

nd 24 ml of 1.0 N HCI are added. The precipitate is fil-

ered off, rinsed with ice water and dried. 2.56 g of

owder are obtained and prove .to be a single substance ccording to thin layer chromatography; Rf(45) 0.21;

f(100) 0.19.

H-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-Ile-Glv-OH 2.37 g of Z-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn- le-Gly-OH are dissolved in 60 ml of 80% strength acetic cid and hydrogenated in the presence of 200 mg of alladium on charcoal (10% of Pd). After completion of

he hydrogenation, the catalyst is filtered off, the fil- rate is evaporated and the residue is dried. 2.28 g of

esin are obtained and prove, in thin layer chromato- raphy, to be free of non-hydrogenated starting material; f(52) 0.21; Rf(107) 0.55.

Z-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Phe-Pro-Gln-Tnr(tBu)- Asn-Ile-Gly-OH 1.41 g of Z-Asn-Lys(BOC)-Phe-His-NHHi 2 are dis- olved in 20 ml of DMF and the solution is cooled to -30 C. 5 ml of 3.2 N HC1 in dioxane are then added, followed )y 0.29 ml of tert.-butyl nitrite. The mixture is then 0 illowed to rise to -12 C and is stirred for 15 minutes at -12°C. A solution, cooled to 0°C, of 1.65 g of H-Thr ;tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-Ile-Gly-OH and 1.2 ml of a 0% strength solution of trimethylbenzylammonium hydroxide .n methanol, in 30 ml of DMF and 6 ml of water, is then

added dropwise. The mixture is stirred whilst cooling with

61 479.210

6 7,13 374 ice and additionally 1.1 ml of N-ethyldiisopropylamine are

introduced over the course of 30 minutes.

The mixture is left to stand for 5 hours at OOC, during which .time a jelly-like precipitate separates out.

The reaction mixture is then poured into 300 ml of 1%

strength acetic acid, the whole is stirred for 2 hours at

0°C, and the precipitate is filtered off. It is rinsed with water and dried, 50 ml of methanol are added and the mixture is stirred for 2 hours at room temperature. The methanol-insoluble Z-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Phe-Pro-

Gln-Thr(tBu)-Asn-Ile-Gly-OH is then filtered off.

399 mg are obtained; in thin layer chromato- graphy on silica gel plates, Rf(100) 0.18; Rf(52) 0.24; 0.33. 1) Z-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-

Asn-Ile-Gly-Val-Gly-Ala-Pro-NH 2

62 mg of dicyclohexylcarbodiimide and 103 mg of H-Val-Gly-Ala-Pro-NH2 are added to a mixture of 420 mg of Z-Asn-Lys(Boc)-Phe-His-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-

Ile-Gly-OH, 55 mg of N-hydroxysuccinimide and 7 ml of DMF, whilst stirring. The mixture is then warmed to 450C and stirred for 5 hours at this temperature. It is then cooled to 0 0 C and poured into 80 ml of ice-cold ether. The precipitate is filtered off, dried and further purified by reprecipitating it once from DMF-acetone and then from DIMF-acetonitrile; 420 mg of the hexadecapeptide mentioned in the title are obtained; in thin layer chromatography on silica gel plates, Rf(l00) 0.25; Rf(107) 0.60.

62 4 79.210

6 1 33 114 ra)

A S-le-Gly-Va 1 -Giy-m Ala-

395 mg of the nrodtct obtained under 1) are dis-

solve,; in 25 n of 60c, strength acetic acid and hydrogen-

ated in tne 7Dresence of 100 mr- of rpalladiur- on charcoal

of Pd). After comnletion of the hydrogenation, the

catalyst is filtered off and the filtrate is evaporaed to dryn-ess. 330 m--gof oroduct -hich is free from starting .a e-s l according to thin layer chromla tography on silica gel -oates are obtained; Rf(107) 0.30.

n) Z-Tr tLu)-Tyr( tBu)-Thr( tLBu) -Gin-Asp-(OtBu)-Phe-Asn-Lys (Boc)-Fhe-H-:is-Thr( tBu)-Phe-Pro-Gln-Thr' tBu)-Asn-Ile-Gly- Val-Gy-Ala-Pro-NH2

356 mg of Z-Tir(tBu)-Tyr(tBu)-Thr(t3 u)-GllAsp

(Otu)-Phe--I-I 2 are dissolved in 3 in. of D4?P by warminng,

the solution is cooled to -200 C and 0.26 ml of 3.2 N HC1 in

dioxane is added, followed by 0.045 ml of t-butyl nitrite. 0 The mixture is stirred for 15 minutes at -15 C. A solution,

cooled to 0 C, of 380 mg of the product obtained under m), 0

in a mixture of 10 ml of 2 ml of ater, 1 ml of aceto-

nitrile, 2 ml of dimethylsulphoxide and 0.2 mi of N-ethyl-.

diisopropylamine is then added and the whole is stirred for

18 hours at OOC. The product ehich has precipitated is

filtered off and is purified by twice reprecipitating it

from D-F-ethyl acetate. The powder is then stirred with

rl of methanol and the m-ethanol-insoluble product is filtered off and dried. 240 mg of the peptide mentioned

63 4 7 9.2

67, 13 1 /74 in thle title are obtained. In thin layer chromatography on slicak gel plates, the product sh-o*,s Rf(43C) 0.40; Rf(103) 0.28; Rf(96) 0.42. Th-r(tBu) -Tyr (tBu) -Thr (tBu) -Glnr_'Sp (Ot-Eu) -he-Asn- o) IT-

Lys (Boc)-Phe-His- Thr( tBu)-Phe-Pro-Gln-Tr(t-uAsnIle.

C-,--Val-G ly-Al a-Pro-NHT~

220 mg of the product obtained under n) are dis-

solv..ed in 15 iml of 805%O' strength acetic acid and hydrogenated in -t*he r-esence of 20 mog of palledium cn ch'a,-,rcoal

of Pd). Aftcr co.nolotjion of tnr~ hy-.'-ge.tion, tire cata- lyst is filtered off and the fit--c is evaiorated to dry- ness. 210 mg of residue are obtaired. To remove the acetic acid, the residue is dissolvet n1 lo -uao

sat-L-,ated N-with water and the solution is washed with dilute L'aI-i0 solution and thereafter repeatedly with %%rater. The butan-ol phase is then-evaporated to dryness, whereby 1 202 mg- o f the peptide mentioned in the title are obtained free of acetic acid. Thin layer chromatography on silica gel plates shows'Rf(96) 0.40; Rf(43C) =0.32.

p) Boc-Cys-.Gly-Asn-Leui-Ser(t~u) -Thr (tBu) 1 e- Gy

T'nr(t~u)-Tyr(tBu)-Thr(tBu)-Gln-A sp (0tBu)-Phe-Asn--Lys(Boc)-

Phe-His-Thr( tBu)-Phe-Pro-Gln-Thr(tBu)-Asn-ITle-Gly-Val-Gly-

Ala-Pro-NHT. 2

mg of dicyclohiexylcarbodiimide are added to a

mixt-ure of.97 mg of Boc-Cvs-Gly-Asn-Leu-Ser(tflu)-Th-r(tBu)- I Cys-.'Met-Leu-Gly-O11, 195 mg of the Deptide obtained under o),

16 ms of N-h droxybenzotriazole and 3 ml of D!4F and the

ihole is stirred for 5 hours at 45 C under nitrogen. The

64 64 -49,2

67. 13 3 n74 ice-cold reaction mixture is then poured into 50 ml of is filtered peroxide-free ether and the fine, precipitate off. it is purified by reprecipitating it tw~ice from 120 mg of gl*W-,w.ater and twice from D-F-ethyl acetate. are thus the protected! peptide mentioned in the title obtain~ed. in thin layer chromatography on silica gel Rf(l00) 0.17; Rf(107) 0.65. plates, Rf(52A) =0.31; I/i~J~lia~.SCALG-OM, AVICrL, CEIfIOL, ARLAC6'L and TWEV&x

65 5 7 9.2 10

67, 1 3 3n/4 THE CLAIMS DEFININO! T7=~ TNV7N'I1-N AR; AS FOLL0WSI-

1. Peptide-amides ofL the formula I.

l4 2 -3 4 5 6 7-1 8 9 10 11 12 13 14 H-Cys-G-ly-Asn-Leu-Ser-ThrCr s- X -Leu-Gly- Fr-Tr-TFhr-Gln. 16 17 18 19 20 21 22 23 214 25 26 27 28 Asp-Phne-Aisn-Lys-Phe-is-Thr-P~e-Pro-GinVir-Al,-leGly"~

29 30 31 32 Va1-Gly-A1a-Pro,.xr1I 2 *.herein X represents L-methionine, L-valine, L-norvaline, L-leucine, L-i soleucine, L-no-rleucine or L-ca--minobutyric acid radi cal ani e~hLalan2 6 is. replaced by L-asr'arag-:ine and/or L-isoleucine 2 7 is replaced by L-threoni'ie an 'horein, otionally, one or more of the arinoacids in positions 11, 12, 16, 19, 22 -and 24 is or are replaced by another a'ninoacid, na-nely L-threonine 1 1 by L-lysine, L-tyrosine 1 2 b lecnL-phenylalanine16 b

L-leucine, L-pnenylala-nine9 .22 by L-leucine, L-phenylalanine by L-tyrosine and L-*glutamine 24 by L-arginine, their deriva- tives and acid addition salts and complexes ofL these compounds. 2. Asn 2 6 -Thr 2 7-calcitonin M, its derivatives, acid addition salts and complexes. 3. Desamino-Asn -6Thr 27calcitonin I4, its acid addition salts and complexes. 4. Desamino-Val -Lys -Leu-Tyr -2Asn6Thr 7 calcitcnin its acid addition salts and complexes. Deamn-Ls-Leu ''-Tyr -Asn Th calcitcnin 1.1, its acid addition salts and comlexes. 11 1.2,16,!9_ 22_ 26_.2 7 actn 14 6. Lys -Leu '~Tyr -isn -Thr -aconn

its derilatives, acid addition Salts and com-olexes.

T ir 2 7caicitonin 1.1, its derivativcs, acid addition

4 7 92 1 0

67, 13 3'71 salts and co'molexes. 12_ 27 ieai -Tr -calcitonin M, its derivativs ai 8. Leu addition salts anid complexes.

Leu -Asn -calcitolin Iflr 12_ its derivatives, acid addition salts and complexes. Asn 26 calcitonin its derivatives, acid addition

salts and complexes. to 11. Na-Acyl derivatives of the peptides according Claims 1, 2 or 6-10. 12. Na-Lower alkano-l derivatives of the p epotides

according- to Claims 1,2 or 6-10. Claims 1.- 13. Complexes of the compounds accordin- to zinc 12 eith zinc phosphate, zinc pyrophosphate and/or hydroxide. to Claims 1 -14. Complexes of the compounds according 12 -with polyhydroxygelatifle, polyphloretifle phosphate, polyphosphates or polyglutamic acid. compounds Pharmaceutical preparations containing according to Claims 1-14. of the 16. Process for the manufacture of new peptides formula I

2 3 4 5, 6 7 8 9 10 11 12 13 14 11 X -LuGyIr~rTrGn H-y-l-s-euSr frCs 25 26 27 28 16 17 18 19 20 21 22 23 21 s'Thr-PheIPrOarnIla-Ile-Gly-

29 30 31 32 Val-GyAlaPro-''-' ,wherein X renresents L-m~ethiofine, LTisoleucirne, L-.norleucifle L-valine, L-norvaline, L-leucine,

-67- 47 9,2

67, 13 3V'4 cr L-a-aininob &tyli c acid rad cnlan cb.L-alanine2 is replaced by L-amz~ra-ine andf/cr L-isolc-ucino 2 7 4s replaced Ly-threonine- and whe-I-rein, optionally, one or more of the a:-'noacids in positions 11, 12, 16, 19, 22 and 24j is or are rep)laced by another anminoacid, namzely L-threonine 1 1 by L-lysine, L-tyrosine by L-1oucine, Lpeyaa16 b

L-1 eucine, L--henvialanine 19y L-leucine, L-rhenylalanine22 by L-tyrosine and L-glutamine 2 4 by L-arginine, their deriva-- tives and acid addition salts, and co=miexes of these comi-

pounds, o;rein

a) the Protecti-ve group or groups are split off

from compounds of the formula I

1i 2 3 4 5 6 71 8 9 10 11 12 13 14 H-_Cys-Gly-Asn-Leu-Ser--Thr-Cvs- X -Leu--Gly-Thr-CTyr-Thr-Gln- 16 17 18 19 20 21 22 23 24 25 26 27 28 Asp-Plir-A'sn-Lys-Ph-',e- i s-Th-Ph--: 'e-Pro-Gln--Thr-Ala--Ile-Gly- 29 30 31 32 Val-Gly-Ala-Pi'o-'H 2 .wherein X has the indicated meaning and w..herein L-alanine 26is replaced by L-aspara.gine and/or L-isoleucine 2 7 is replaced by L-thr.eonine and -,.herein oot icnally one or more of the aminoacids in positions 11,

12, 16, 19, 22 and 24 isor are r-er1&zed bythe above-mentioned

aninoacids, or from their derivativcs, _in -which. compounds at-

least one amino group or one carbox*yl group is protected by a protective grcup which can be split off, or

b) compounds of the formula II 1 2 3 4 5 6 7 8 9 10 11 12 13 14 H--Cys-G-y--Asn-Leu-Scr-Thr-Cys- X -Leu.-Gly-Thr-Tyr-Thr-Gln-

16 17 18 19 20 21 22 23 24 25 26 27 28 ~cp-he-An-Ly-Phc-Mi -Th -n-ro-Gln-Thr-AIla-Ile-Gly--

63 479,2

Claims (1)

1. 7,13 '/74 23 2 710 31 32 -2 26 wherein~ X has the indicarca meaning an-d wherein L-alanine is re-:,1aced by L-asparagine and/or L-isoleuc-ne 27is re- placed by L-threonine and w,.herein optionally one or mor.e of 12, 16, 19, 22 and 24 is or are the airnoacids in positions 11,

   replaced by the abovemrrenti cried aminoaci-ds, or their deriva-

   tives, wherein the mercapto groups are free or protected

   by the trityl or acylamidomethyl group, are oxidized to give

   disulphides and, if desired, the resulting peptide-amides

   are converted into their acid addition salts or complexes.

   17. Process for the manufacture of new dotriaconta-

   peptide-amides as defined in claim 1 and described in the

   examples.

   Dated this. 2 day of_/i

   CIBA-CE!CY AC

   EDWPWATERS&SONS PATENT ATTORNEYS RUSSELL STREET MELBOURNE AUSI FiALIA

   -69- 479.2