DE1935876A1 - Process for the production of previously unknown polypeptide derivatives - Google Patents

Description

Basel Case 100-2953

-— ■ - "■" patent attorneys

Dr. W. Schalk, Dipl.-Ing. P. Wirth

Di 'l.-l: ". G. Dannenberg

Dr. V. Sr :. . · ■ 'J-Kov / arzik

Dr. Γ. -, .. iiijU / Dr. D. Gudel

6 Frankfun.'M., Gr. Eschenheimer sir 39

Process for the production of previously unknown polypeptide derivatives

The present invention relates to previously unknown polypeptide derivatives of the general formula (3-Thio-2-X-propionylJ-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L- · hemicystinyl-L-valyl-L-leucyl-L-seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl- L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-LY-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, where X has the meaning -H or -NH »and Y stands for the L-norleucyl- or L-methionyl radical (see formula sheet 1), their therapeutically effective acid addition salts and heavy metal complexes and processes for their preparation.

If X has the meaning -NH ₃ , the amino acid residue (3-thio-2-X-propionyl) is in the L-form.

The previously unknown polypeptide derivatives of the above general formula can be used for the synthesis of compounds this type of well-known methods produced

9 09 884/1791

BATH ORIGINAL

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. where the amino acids are in the order specified in the above formula individually or after prior formation smaller peptide units are linked together.

In the last stage of the condensation, methods are to be used in which racemization does not occur or only slightly can be maintained, preferably by using the azides or the activated Enter, with activation being preferred is made with N-hydroxysuccinimide.

The conversion of a protected amino group into a free one Group on the L-hemicystine molecule to obtain one on the terminal L-hemicystine molecule unprotected compound, takes place by treatment with hydrolyzing or reducing Means.

In the construction of the previously unknown polypeptides or polypeptide derivatives , the tert-butyloxy group has proven useful for blocking the 7-carboxyl group, for example in the partial sequence A described below , but other protective groups, such as the methoxy, the ethoxy , the tert-amyloxy, the amide or the benzyloxy group can be used.

PUr blocking the imidazole group of the histidine residue In the partial sequence C described below, the triphenylmethyl group has proven itself, but other suitable protective groups, such as the carbo-tert.-butoxy-, the carbo-

909884/1791

ORIGINAL

tert-ainyüoxy, the carbobenzoxy or the benzyl group be used.

A tert-alkoxycarbonyl group, preferably the tert-butoxycarbonyl (carbobutoxy) group, can be used to block the co-amino group of the lysine residue in the partial sequence C described below. ^T earth.

The nitro group was used to block the guanidino group of the arginine residue in the partial sequence E described below, but other suitable protective groups such as the tosyl group, the p-nitrocarbobenzoxy group or the 2- (isopropyloxycarbonyl) -), k, 5 * 6-tetrachlorobenzoyl group can also be used be used. The protective effect of protonation of the guanidino group can also be used in the synthesis.

The benzyl radicals used to synthesize the partial sequence F1F2 or F1F5 to protect the SH groups are usually used cleaved at the end of the synthesis by treatment with sodium in liquid ammonia. It has now been found that the Cleavage of the benzyl protective groups and the production the S-S bond before the last stage leads to particularly good yields of the end product.

The starting products for the production of the new polypeptide derivatives can, provided they have not previously been

909884/1791

BATH

- '4 -' 100> -2953.

were known by the methods known for peptide chemistry are obtained, the amino acids individually or according to, prior formation of smaller peptide units are linked together.

The new polypeptide derivatives can also be in the form of their Obtain or use salts. The salts are those with organic acids, such as acetic acid, propionic acid, glycolic acid, Lactic acid, pyruvic acid, malonic acid, berry

^ stinic acid. Maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, salicylic acid, 2-phenoxy or 2-acetoxybenzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, Hydroxyethanesulfonic acid, benzene or toluenesulfonic acid, Naphthalenesulfonic acid, sulfanilic acid and polymeric acids such as tannic acid, alginic acid, polygalacturonic acid, Polyphloretin phosphate or carboxymethyl cellulose and salts with inorganic acids such as hydrohalic acid, e.g. Hydrochloric acid or hydrobromic acid, nitric acid, thio-gyan

"acid, sulfuric acid and phosphoric acid in question. As a heavy metal complex, for example, that of zinc" comes into question.

The new compounds represent an important therapeutic principle. They lower / the "calcium plasma level, in particular the increased calcium plasma level and act as antagonists of the parathyroid hormone a ^ positive calcium balance, im Bone. -

909884/179 1

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They are therefore indicated in all states, in multiple marriages a decrease in plasma calcium levels is desired, e.g. Hypercalcemia of various origins, deficiency of the endogenous Thyroid gland due to a breakdown of thyroid tissue, • Hyperfunction of the parathyroid glands.

The new compounds are indicated for all bone infections that are based on increased breakdown or for which increased calcium fixation in the bone is desired _# e.g. osteoporosis of various origins (e.g. post-climacteric, post-traumatic, caused by corticosteroid therapy or inactivity, etc.), Fractures, osteomalacia, rickets and especially for combination therapy with calcium or phosphate *

Biological testing of the new compounds revealed one Efficacy of approx. 100 to ^ O MRC units / mg peptide. The required daily dose (administered IM) in MRC units is 20 mU to 10 U, preferably 100 mU / kg animal weight.

In humans, the daily dose (administered IM) is 1 to 500 U in MRC units. Preferably, a daily dose of 5 U IM is used. administered

A particular advantage of the previously unknown polypeptides derivatives of the above general formula, in which X

- 6 - 100-2953

has the meaning -Ή or -NHp and Y for the norleucine residue is their resistance to oxidation.

The new compounds can be used as remedies, for example in the form
pharmaceutical preparations, find use. These contain the compounds mentioned in a mixture with one
organic or inorganic carrier material suitable for parenteral administration. The new compounds can also be administered in the form of a depot preparation.

The new compounds and their salts can also be used as
Find intermediate product for the production of pharmaceutical preparations use.

8AD

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The following abbreviations are used!

Z = Carbobenzoxy (benzyloxycarbonyl) BzI Benzyl BOC = 'tert ", - butyl oxycarbonyl Door - Trityl = triphenylmethyl OTB = tert-butyloxy OFP = p-nitrophenyl ester OCP 2,4,5-trichlorophenoxy OMe = Methoxy OEt = Ethoxy NO ₂ Nitro Ser - = L-seryl Äsn γτ .L-Äs ^ araginyl Leu β L-Ih-I acyl Thr 1 - "^ - reonyl VaI L ^ Yfelyl AIa L-alanyl Tyr «L-tyrosyl Trp = L-tryptophanyl Arg = L-arginyl Phe = L-phenylalanyl GIu “L-glutamyl His L-histidyl Per L-prolyl GIy Glycyl Lys L-lysyl 9 0 9 8 8 4/1791

BATH

- 8 - 10Q-295>

Met = L-methionyl

Cys =. L-cysteinyl

Never = L-norleucyl

MCP - β-mercaptopropionyl

The following examples illustrate the execution of the procedure explain, but in no way limit the scope of the invention all temperatures are given in degrees Celsius.

9Oa88A / 179T

S-W

9 0 988A / 1791

. , _r—— ι

BBC- jSar CIy Met Ply ( -OH H- j php CIy fro QIu TIT fro \ -K THl Π ' TU

,, Trt we _

Ίή— {ur ti «Tyr Try« rgj -HHUHa H- ) do Ua * «« * «« WlTp iWH «Trt- ^ Ml« Ly »Hiap HWHz ll- ^ 3er CIy Hut CIy

μ cly Kct "r P (IC Cly Per CIu ! thr Per | ₃ he cly Mead "» ... in" Cly Per SIu Thr, Pro I

Tr »- | s« r «la Tjr Tm» r «

^{> to} ">«

jI "" Ly "Ptl"

Scr CIy Wet CIy ■ 'Pnc CIy Pro CIu Thr' Pfttj -Kll ;, '.

" J

«Ar km Ua 8« rf * WWl _t W ^ TK y Cy »VI Uu | ^ "L" Trt- js.r Al. Tyr Trp «m>« n Uu ""»■""" 1 -WMH ₁ 1l- ] lll «ty» fh.

TtIIWnMMWW TIMlamyrn « ti -fdHl » j

8 «r GIy Wet CIy

aOC- ] cir «a» r> M LM ltr

Mlam

00. ■ j ——. 1 . _ _;

QJJ. BSC- jCy »aar>« n Lea »a r Tl ir C y» V »I Leu J-XWIlJ H-jr, « r Ala Tyr T

^J TelltemH-n » Ti. Η

' Tellnenuent ΛΙΈΡ »

Phe CIy Pro CIu Tn

r Proj -HII

aar AU Tyr TrP Ar » Ann Leu "at "at pho Ills Lya ma Scr Cly Mead Cly Ph * My Per CIu Thr Per , r Ala Tyr Tri> Aan Lukewarm Aan "at rna Ilia Lya Ptia Scr Cly Mat Cly Fhe Cly Per CIu Thr Per

BOC- Jc ₁

3 «r« Ml tau aar

Thr Cj · V> 1 Iw

Jar Al "Tyr Trp" r "

»· Η Liu« · η »· η rn«

Iy «« Η »

, Ph ", ply Pro g'.u Thr

he CIy Pro ClU .Ttl.r Pix. | -Sl! a,

Icy «Sor, A« n Lou aar Thr C y «V« X Lau Ser Ala Tyr Trp Arg A »n Leu Aon Aun fhe

- - - τ --r-—

111. l; y « fix

Scr .CIy. , K? T Cly

'cocn

to ο co Po co. £ ■ · »

co Z- ) scr ciy mc

100-3993,

Tr * i 4 .1 »r Al» Tyr Trp ΛΓΚ f -HHHH,

Q-ICHM! * ¹ TTt- ) | li; : .y3 TM f HHiW, H- | Srr My ill · cTr

Scr Λί »Tyr Trp Arn - Ann Leu Asn ASH ■ tM> f W; W2 Trt- Jlll3 _t l.ys I'h?

Sgr CIy »:? CIy

BIl

B0ie- ) cya a «r Am L« u TeH »ciiugnt Pg

BOC- fcya "

H- j T «r cy»: v «l! .Imf OH. . Tatlaw <iu * ng PI . ,

, ■ ■ ■ ■ Kii ■ '■ ■ ■

" ^hl ~ ^ ^{tfw ν} ^^ ^ίΒ " f t ** Trtf

3 ιί- »l« Tyr Trp Arn

Λ <η L »u A» n; ) sn ι ·! ι »

H- I 111 »iV l'Ue

Scr üiy It'lf

UIa Ly.1 PfI *

CjIy ^ TM <■ GIy

3gr Agn Lou 3er

Thr Cy «VAl 1><H WHH, H ] 3« r Al »Tyr Trp Arn ABn L = U Ann Agn

111 «t-yn, Phe ^f

Oy IJl- CIr

Tffl larnuiinz OJ

BOC-fcya Jar Λ «η Lau Jar 7Hr Cy» V »l Leu 3τ Ala Tyr? Rp Ar *

ltla l.ya 'fhff

Scr C] y HIe-. ' Gly

Cy · V »l Leu pn- ^r .: Y Pro; o; ü "THr ¹ - pro

H Sir Gi / ~); * "Sly ... l:> ? T7 .'- "j (!.; Jr frp '. | ->

TKr CIy ϊ'γλ /-.''j Tnr per H r

pnr CIy prty; i! u 'pftr rro f ? ;; <

, '.:' »■. * ■ 'Ciy'rr» CIu Tn'r ".rf.o H « _T

ϊ.

9 884/1791

, ■ "■ . '^;:''''* , "' ■ OTB "■

H »« τ oiy «« oirH W Η · ) rn »ci> rna cm rhi rro | - iM-

Trt, 'lKIC

CD

'''''','!'' ■, "'''.■' i & j S * r CIy m« CIy mc Ely l> ro a Thr fro | -KH _t

■ ■ '' , '; ■>'.' ■ -Tr * »TOC . '■ ';',, ' OTIl ■ '

»» F «1» TTT tr »» r «H WB ^ (t j Ma Uu « I »> m fly» H «i» a Irt- flU »Ija Di» (- MCT ^ m aar aiy m «; Ply . yhe CIr> ro tlu Tar fro h KH _?

C '. ■ ■ ^: ''■; ■■. _:

a «r οι> nh el »me c» fr »e» u .τ »fro

\ KSf —Γ ton twi HPt WW ^ I H '^ Hw ^ M *** tSTW f trV fT *** Tyr Try Arn A «a tau α« λ * gn fh * l UMMU K HHU Ly rtw 3er Cly Mlo Oly fh · CIy fro din Thf

'^ Ff f jf ywf »» *) f_ ^ B ■''■■''! ■ ■'.'ι"■'■',"' ^ ft ^ Bi? ^ U *? Ng AFlI ^ .''■'., ■ '■■■.' / '',,

i * Ala Ty "Trp * rg Mn Xtu *" a Ain ftf ■ HI "ty a rhc 3τ Ply HI · Oiy- yt." Cly pro 'ciu Thp

,. IMCr-MT U » UM» »γ τ» Β »Wl Uwj— B.» fWf> il iW trt »r» - ■■■ »M Mu« «» Λ »Β fM ^m * '" * ^fh * "» he Oy Ml «CIy flw Ply Γτό ~ ύΐ.ι leads Γπ

. ''-.'' T «il» «« «Mrm» ι. η . «. '"' Τ« ι 1 »« «i« n »» niePM . ■, ■, '' ■: ■, '■. ■■■■■', ■ ',

Ann Uu Bap ¹ door cy »Vi tar AL »' _t lar Al » Tyr: ' Arp Ar «Al fh « Uta COC pn « '. "■' '-S« r CIy NIa CIy Ph « CIy fro OTB Thr Per [ IMf E «r . <r «* nc» «Hi« nit Aniepnpl Ut CIu «Ml You 0 «r door eye 'Vi Tyr Ml « Th « is «r CIy IUa oiy Ph « CIy Per Thr Tr »Arc. Αι ty » Olu Pro \ inST fr Xl Uu in .Leu Am ■■■ »» ή Il Uu in Lau Aaa Aan

* - 1 * - 100-2955

Example 1; L-HemlGystinyl-L ^ seryl-L-.asparaginyl-L-leucyl-L-seryl-L.-threonyl-L-hemioystinyl-L-valyl-L-leuoyl-L-seryl-L-alanyl-L-tyrosyl- L-tryptophanyl-L-arginyl-L-asparaganyl-L-leucyl-L-asparaginyl-L-aspa- raginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-L -methionyl-glycyl- »L-phenylalanyl-glycyl-L-prolyl-L.-glutamyl.-L- threonyl-L-prollnamld · decaacetate » decahydrate (H-Cys-Ser-Asn-Leu-Ser-Thr-Cys- Val-Leu-Ser-Alaiyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NHg ♦ Decaacetate decahydrate) (formula sheet 2)

Nan dissolves 5.3 g of BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser *. Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys (BOC) -Phe-Ser-Oly-Met-Oly-Phe-Gly-Pro-Glu-Thr-Pro-NHp · hexaacetate · Decahydrate (sequence ABCDEF1F2, Example 21) under a nitrogen atmosphere in 100 ml of trifluoroacetic acid, left to stand for 15 minutes at 20 ° and evaporated to dryness. One solves in 300 ml of 0.2 M acetic acid, treated with 20 ml of araberlite-IRA-JUO (acetate), lyophilized, washed with diethyl ether and dries over potassium hydroxide la high vacuum

Cye-Sei'-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-Trp-Ai ^ g-Asn-Leu-Asn-Asn-Phe-His-Lys-Phe-Ser-Gly -Met-Gly-Phe-Gly-Pro-Glu-Thr- ^ ro-NHg · 10 CH ₃ CO ₂ H- 10 HgO, m.p. 220 ° (decomp.), -52 ° ^! in 1 N acetic acid ·.

909884/1791

- 15 -. 100-2955

1335876

Amino acid composition after acid hydrolysis (6 N, 16 hours); ^ ₁ , Arg ^, Asp ^ Cys / 2 ^,

VaI 0.9 (Trp 1.0 by spectrophotometry).

Example 2; L-hemieystinyl-Ls; ery l · ^

^! b

L-asparagiiavl-L-leüoyl ^ L-asparagi ^

-L "lysyl · ^ L · ^ phew

-L · -pr

amide »_. O efcascetat; “Decahydrate

■ Ti

Pro-Glu-Thr-Pro-HHg · Öötaac # feät · Dec (Formula sheet 5)

One dissolves 5.3 g of BOC-Cys-Ser-Asn-Leu-Ser-ihr-.Cys.-Yal-Ijeu-Set »- AIa-OYr-TrP-ArS-ASn-LeU-ASn-ASn-PhC-HiS-LyS

Nle-Gly-Phe-Gly-PrQ-Glu (OTB) - »Φhr ~ Pro-l · IH _^ · hexaacetate ·

Decahydrate (sequence AB1CDEP1P2, example 22) under a nitrogen atmosphere in 100 ml of trifluoroacetic acid, leaves 15 Stand for minutes at 20 ° and evaporate to dryness. Man dissolves in 500 ml of 0.2 N acetic acid »treated with 20 ml of amber

lit-IRA-4lO (acetate), lyophilized, washed with diethyl ' 909884/1791

- 16 - ν 100-2955 -

ether. and dries, over Kal-_iut «hydr * © xM. \ In a high vacuum * You get, JI ^ ys-rSeF-Asia- ^

* 8 CH, C0 _o K * IG H ₀ Q, Snip, ·. 220 °,

20 ° "

(Zers *) _f , IYes] ^ .. _; -.- τ- | 3 ^ ° _; in 1. N acetic acid * Aminic acid to Gamraen settlement. after acid hydrolysis (6 ll _f 1 & hrs.} r AIa,., Arg, _Λ ,

Never, .. _Λ , Phe, «. * -, Pro _<; Serw.ß, fc _, type, _, 1 * 0. .5.0; 2>1;> e · 1.9 1 * 0

(Trp 1.0 by spectrophorometry).

Example ^ ^ ß ^ MercaptQprQpiQnyl ^ L ^ seryl · -L · -asparaRln; yl- ^

I _! -prolyl-L-gl ^ tamyl-L · -threQnyl ■■ L-pr0linanlid »penta- . acetate »octahydrate

PPΌ-GXu ^^

^ Octah ^ rat) ^ ori ^ lplatt%): λ «

Dissolve 1 * 05 S Nonapeptide-Hydrazi4 (Teilseqw ^ nz FlF ^), .in a mixture of 50 ml of dimethylformamide and 1.2 ral 2 H dioxane / HCl at -20 °. Q.116 ml of tert-butyl nitrite are added and 10 is stirred Minutes at -20 °, gives 1.4 ml of triethylamine and 5.1 g of tri-

909884 / 17S1

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eosapeptide acetate (partial sequence ABCDE) and 5 ml of water and stirred for 16 hours at 0 °. It is evaporated to dryness, the residue is washed with diethyl ether, chloroform and acetone, dissolved in trifluoroacetic acid, left to stand at 25 ° for 1 hour, evaporated and washed with ethyl acetate. In this way , the crude Dotriäeofttapeptide is obtained, which is dissolved in 100 ml of 0.3 N acetic acid, treated with 20 ml of Amberlite IRA-410 (acetate) and then 50 ml of 0.6 N ammonium hydroxide is added. To set the pH to 6.5 and the resultant solution coated on a Carboxymethylcellulosesäule (10 χ 100 cm), the Amraoniumacetatpuffer solution was equilibrated with a 0.15 K. The elution is carried out with an increasing concentration and pH gradient (0.15 N; to 0.4 Hf, pH 6.5 to pH 7.0) of an ammonium acetate buffer. The combined fractions, which contain the pure peptide, are freeze-dried three times, the residue is washed with ethanol and then with diethyl ether and washed over. Potassium hydroxide dried in a high vacuum.

MCP-Ser-Asn-Leu-Ser-Thr'-Cys-Val-Leu-Ser-Ala-Tyr-Trp-Arg-Äsn-Leu-Asn-Asn-Phe-His-Lys-Phe-Ser-Gly is obtained -Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH ₂ · 5 CH ₃ CO ₂ H · 8 HgO, m.p. 2 ^ 0 ° (dec.) Ca] ^ = -6l ^p in 1 N Acetic acid. Amino acid composition after acid hydrolysis (6 N, 16 hours): ^v

LeUo Q, LyS, »«% ₀ , Phe .. _Q ,

ο (Trp 1 _Ä O dürca spectrophotometry). 'r

909834/1711

BATH ORIGINAL

- 18 - ipo-2953

1935S76

Example 4 β-Meroaptopropionyl-L-seryl-L-asnaraginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-lencyl-L-seryl-L-alanyl-L-tyrosyl -L-tryptophanyl-L-arplnyl-L-asparaginyl-L-leucyl-L-asparag.inyl-r _J -asparar; inyl-L-phenylalanyl-L-hi5tidyl-L-lysyl-τ.-phen5 'lalanyl- ^ L- ' seryl-Klycyl-L-norleucyl-fJrlycyl-L-phenylalany l- glycyl- L-prolyl-L-glutamyl-L-threonyl-L-prolinainid * Hexaacetät · ■ Dodecahydrate (MCP-Ser-Asn-Leu -Ser-Thr-Cys-Val-Leu-Ser-Äla-Tyr-Trp-Arg-Asn-Leu-Asn-Äsn-Phe-His-Lys-Phe-Ser-Gly-Nle-Gly-Phe-Gly ^ Pro -Glu-Thr-Pro-NHg

6 acetate / dodecahydrate) (formula sheet 5)

1.05 g of nonapeptide hydrazide ( Teilsequerss PlPj) are dissolved in a mixture of 50 ml of dimethylformamide and 1.2 ml of 2N dioxane / HCl at -20 °. One gives 0.116 ml of tert . -Butyl nitrite stirs for 10 minutes at -20 °, adds 1.4 ml of triethylamine and>, 1 g of tricosapeptide acetate (partial sequence ABICDE) and 5-1 of water and stirs for 16 hours at 0 °. It is evaporated to dryness, the residue is washed with diethyl ether, chloroform and acetone, dissolved in trifluoroacetic acid, left to stand at 25 ° for 1 hour, evaporated and washed with ethyl acetate. The crude dotrlacontapeptide is thus obtained, which is dissolved in 100 ml of 0.3 N acetic acid, treated with 20 ml of Amberlite IRA-410 (acetate) and then 50 ml of 0.6 N ammonium hydroxide is added. The pH is adjusted to 6.5 and the resulting solution is layered on © ine carboxymethyl cellulose column (10 100 cm), which is connected to a

909884/1791, _

100-295}

0.15 N ammonium acetate buffer solution was equilibrated. The "'elution will ^{-with a;} icing ferizentratiöns- are pH-degrees ■" (Θ, 1'5 N to ''- 0, k N>"pff6; 5 ~ ■ atuf * pH ^: f, 0j ei rie sf "" Amino nium -

..V -'.- '.: .-.,. . ■■ ■■■■ = '' .- · - · ■ - ■: ■■■ ^{■■■■■::} - ^r - ^{l.. ';} - · - ^; '· ' ■■ - ■ '.V-' = ■■: ■■ «·.-O ₁ ; .-. ■ acetate buffer, s carried out. The purified fractions, which contain the "pure peptide", are freeze-dried four times, the residue with ethanol and diethyl ether

washed and dried over Kaliuinhydno; cid in a high vacuum.

MCP-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-

Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys-Phe-Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NHg · 6 CH-CO ₂ H · 12 -H ^ O, snip. 220 ° (dec.)> Ca] _D = -57 ° in 1 -N acetic acid. Amino acid composition after acid hydrolysis (6 N, 16 hours):

2.9 '

MB

* 'tTrp "1.0 * by spectrophotometry).

■ Λ- "■

90 9 884/179 1i V.

8AD

, ■ - 20 - 100.-2953 '

The starting materials used in the examples 1 to ^ are manufactured as follows:

Example 5 ;

Partial sequences ?, Λ: L-Pl: ef.y 1 -n. Il hr> yl - ply ay 1 -L-iprc Iy I - y- b.cri «-b uty 1 -

(ll-Phe-

GIy-PrO-GIu (OT

^{Dissolve 1} kg of Z-Thr-IIIH-iilip in 2 1 1 N hydrochloric acid at -5 ° C. and add 0.55 1 1 N sodium nitrite. After 5 minutes, potassium carbonate is added to pH 9, and the azide formed extracted with ethyl acetate and a solution of 80 g of H-PrO-IiH ₂ · hydrochloride in 100 ml of water, 500 ml of dimethylformamide and 77 nil of triethylamine added. The ethyl acetate is evaporated at 20 ° in vacuo and left to stand over power at 25 ° The solution is evaporated in vacuo, the residue dissolved in ethyl acetate, ashed with water, dilute hydrochloric acid and an aqueous potassium carbonate solution and dried over sodium sulfate. Evaporated in vacuo, dissolved in warm ethyl acetate and cooled. Z-Thr-Pro is obtained -NH ₂ J m.p.

20th
148 ° i [a] _D = -72 ° in 95 % acetic acid. 90 g are then dissolved

Z-Thr-Pro-NHg in 2 l of dioxane and 2βθ ml of 1N hydrochloric acid and hydrogenated at 20 ^p and normal pressure in the presence of a palladium catalyst. It is filtered, the solution is evaporated in vacuo, the residue is washed with ethyl acetate and H-Thr-Pro- • HCl / mp. 216 °, [a] _D ° = -64 ° in 95 % acetic acid is obtained. This

909884/1791 ~

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dissolved in 500 ml of dimethylformamide, 50 ml of water and> 2 ml of triethylamine and added 11.8 g of Z-GIu (OTB) -OCP and 800 ml of tetrahydrofuran. It is left to stand over power at 20 °, evaporated in vacuo and the residue is crystallized with ethyl ether. Z-GIu (OTB) -Thr ~ Pro-NH ₀ , Srnp is obtained. 65 °

2 '

20 ''

(Dec.), [^ J ₀ = -18 ° in dimethylformamide.

80 β Z-GIu (OTB) -Thr-Pro-NBp is dissolved in 1.5 l of dioxane and 200 ml of water and hydrogenated at 20 ° and normal pressure in the presence of a palladium catalyst. The solution is filtered and evaporated in .Vakuum and the residue with Diä _; ethyl ether added, whereby H-GIu (OTB) -Thr-Pro-tfH is obtained, m.p. 65 ⁰ (dec.).,. [aj * = -28 ° in dimethylformamide. This is dissolved in 700 ml of dimethylformamide at 0 °, 200 ^; ml of acetonitrile, 68 g of Z-Phe-Gly-Pro-OH, - 18 g of N-hydroxysuocinimide and 52 g of dicyclohexylcarbodiimide added, left to stand overnight at 20 °, filtered, evaporated in vacuo and ethyl acetate added. The solution is then washed with water, dilute hydrochloric acid and aqueous potassium carbonate solution, dried over sodium sulfate, evaporated in vacuo and the residue crystallized from ethyl acetate / ethyl ether. One obtains Z-Phe-Gly-Pro-Glu (OTB) -Thr-Pro-NH ₂ of melting point 120 ° (decomp.), [A.Jjjj ⁰ '~ - -66 ° in-dimethyl-' .- " formamide, which is dissolved in 1500 ml of dioxane and 300 ml of water. 50 g of palladium halves (10 $ 6) are added and the mixture is hydrogenated at room temperature and normal pressure until hydrogen uptake is complete. It is filtered, the filtrate is evaporated and crystallized

909811/1791

- 22 u 100-2953

sized the residue from dioxane. The H-Phe-Cily-PrO-GIu (OTB) -TlIr-PrO-OTI ₂ , Snip is obtained. 153 °, [α] ^ = -79 ° in dimethylformamide.

Example 6:

Partial sequence B: H-tert-butyloxycarbonyl-L-seryl-glycyl-L · - ^v

methionyl-glycine (BOC-Ser-Gly-Met-Gly-OH)

- a) L-methionyl-giyGln-ethylester-hydrochloride '(H-Met-Gly-OEt · HCl)

65 g of BOC-Met-üII are dissolved in 000 ml of ChlOroforni, cooled to -10 °, ^ O ml of K-methylmorpholine and 35> .6 g of isobutyl chlorineate are added. For 10 minutes a solution of 30 g of glycine ethyl ester in 200 ml of chloroform is slowly added and the mixture is left to react at 20 ° for one hour. It is extracted with 0.5 N ammonium hydroxide, then with 0.2 N sulfuric acid and washed neutral with water, dried over sodium sulfate and concentrated. After recrystallization from petroleum ether, BOC-Met-Gly-OEti melting point ^ 9 °, [a3 _D = -19 ° in ethanol. It dissolves in 750 ml of 4N HCl / ethanol, lets stand for 1 hour at 25 °, evaporates, washes the residue with diethyl ether and dries to constant weight. The H-Met ~ GIy-OEt 'HCl is obtained in the form of an oil. '

b). ter ■ t .- ^ ButyiQXycarbonyl- · L- ^ seryi ^ -p; l · yein · _! 'ethyl ester (BOC-Ser-Gly-OEt)

Dissolve 12.5 g of tert-BiityXoxyearbonyl-serine in 100 ral

9098Ö4 / 11T01

^ BATH

- 25 - 100-2955

Chloroform and 6.1 g of N-methylmorpholine are added; then 8.2 g of isobutyl chloroformate are added dropwise. After 10 minutes, a solution of 6.6 g of glycine ethyl ester in 50 ml of chloroform is added and the mixture is stirred at room temperature for 1 hour. The reaction mixture is washed with dilute ammonia and then with hydrochloric acid solution, dried over sodium sulfate and the organic phase is evaporated. BOC-Ser-Gly-OEt is obtained as OeI; [a] ^ = -5 ° in dimethylformamide.

^c ) N "ter t. --Buty 1 ox · :: ·. -: ■ 'bcny 1 -L-sery 1 -r: 1 yciη - hy d razid

19.4 g of BOC-Ser-Gly-OEt are dissolved in 270 ml of ethyl alcohol, add 48.6 ml of hydrazine hydrate and leave for 2 days Stand room temperature. The solution is then evaporated and the residue is crystallized from a methyl alcohol / Ethyl ether-Geinisch (1: 5). BOC-Ser-Gly-NHNHg is obtained,

on

M.p. 157 °, [α] IT = -5 ° in dimethylformamide.

d) N-tert-butylorycarboryl-L-seryl-glycyl-L-methionyl-

glycine ethyl ester (BOC-Ser-Gly-Met-Gly-OEt) 25 ml of a 4 N hydrogen chloride solution in ethyl ether are added to 200 ml of dimethylformamide and 11 g of BOC-Ser-Gly-MNHg are dissolved therein at -10 °. Then 5.4 ml of tert-butyl nitrite are added dropwise at -10 °, first 18 nil triethylamine and then a solution of 12 g of H-Met-Gly-

909884/1791

BAD ORlGlNAU

- 2h '- ■ 100-2955

OEt · KGl in 100 ml of dimethylformamide and 6.2 ml of trietbyl -'.- autln. The mixture is "stirred at room temperature for 3 hours," after -12 hours filtered and evaporated. The residue is dissolved in chloroform, washed successively with dilute ammonia and hydrochloric acid solution, dried over sodium sulfate and evaporated. EÖG-Ser-Gly-Met-Gly-OEt, lalt = -14 ° in DiiHethylformaKiid is obtained.

(BOC-Ser-Gly-Met-Gly-OH)

f4an dissolves 24 g of BOC-Ser-Gly-Met-Gly-OEt in 250 ml of dioxane, adds 75 ^ral 1- ^K sodium hydroxide solution, stirs for 1 hour at 25 °, treated with 150 ml of Dowex-50 (H ⁺ -Por-m ), filtered, the Pilträt evaporated and the residue crystallized from ethyl acetate / diethyl ether. BOC-Ser-Gly-Met-Gly-OH, Srop. 87 ⁰ (dec.)., ■ Ca] ^ ⁰ = -17 ⁰ in 'dimethylforineneid.

Example 7:

Partial sequence BIl Benzyloxycarbonyl-.L-seryl ~ glycyl- »L-norleucyl« -

glycine (Z-Ser-Gly-Kle-Gly-OH)

a) Benzyloxyearbonyl-L-norleucyl-glycine (Z-Mle-Gly-OH) Dissolve 51.6 g of H-GIy-OH in 420 ml of 1N sodium hydroxide solution, add 84 g ml of tetrahydrofuran, then add 1> 0 g Z-NIe-OKP, shake until the solution becomes clear, leaves a

Maethe stand at 25 °. Then in a vacuum to 500 RiI

909884/1791

ß ORIGINAL

- 100-2953

1935076

centered and the resulting aqueous solution adjusted to pH 9 with 1 If NaOH, extracted twice with ether, then acidified with 1N hydrochloric acid and extracted with ethyl acetate. The ethyl acetate solution is washed with water and with 30 % sodium chloride solution, dried over sodium sulfate ■, ■ evaporated in vacuo. The residue is triturated with ether, left to crystallize for 2 hours at -20 ^ and thus Z-NIe-

on

GIy-OH, m.p. 158 ⁰ , [a] ^ = -4 ° in dimethylformamide.

b) Benzylοχγοα ^ οητί-κίγογί-ΐ— nQrleücyl-glycine (Z-Gly-Nle-

aiy-OH)

Dissolve 100 g of Z-Nle-Gly-OH In 1000 ml of 4N HBr / glacial acetic acid, can be 50 minutes at 25 °, triturated with ether, filtered off the crystals and wash gufe with ether. HBr · H-Nle-Gly-OH, Eaj £ = + 2 ^ 7 ° in acetic acid (95 %) is obtained. Dissolve 84 g HBr 'H-Nle-Gly-OH in 24o ml H _g 0, mixed with 152 ml of 4N NaOH, 8OQ ml of tetrahydrofuran and 91 g of Z-Gly-ONP.' It is shaken for 1 hour at 25 °, then left to stand for 2 days at 25 °. It is concentrated in vacuo to 500 ml, adjusted to pH 9 with 1N sodium hydroxide solution and extracted with ether. The aqueous phase is acidified with 1N hydrochloric acid, then twice with ethyl acetate extractor. It is washed with mass, a 30% sodium chloride solution, dried over sodium sulfate, evaporated in vacuo, the residue is triturated with ether., Z-Gly-Nle- Gly-OH » Smp * 155 ^G ^ Jcs} _D =» 5 * 6 ° in Di-

909 884/179 1

: - 2β - 100-2953.

methylformamide. . . , ^ _h

c) Benzyl o-äy carbonyl-L-sery 1-plycy IL-norleucy-1-glycine Gly-Nle-Gly-OH)

Dissolve 89.5 g of Z-Gly-Wle-Gly-OH in 900 ml of 4N HBr / _Elsessig: can 50 minutes at 25 ° are provided, evaporated in vacuo, v / ER 'rubs with Äetheri filtered, washed well with ether, dries - in the Valmum, dissolves approx. 650 ml of dimethylformamide in 280 ml of water,

™ gives 66.5 ml of triethylamine. to and mixed with the azide that ■ is obtained as follows:

9 ^ 0 ml of 1N hydrochloric acid are cooled to -5 °, 96 g of Z-Ser-NH-NHp are added and "" - is then added dropwise at -5 ° 560 ml of 1 .N sodium nitrite, and stirring is continued. 6 minutes at -5 °, covered with approx. 600 ml ( ethyl acetate at 0 °, adjusts to pH 8.5 with the addition of solid sodium carbonate, separates the ethyl acetate phase; extracted again, the solution dried over sodium sulfate and filtered »

I The reaction solution is evaporated in a vacuum, the

Residue 60Ö ml of water, acidified, extracted with ethyl acetate, washed with water "with 30 $ brine, cooled and filtered the crystals. One obtains Z-Ser-Gly-Nle-Gly-OH to mp.-21O ^d ■ ' (with ' decomp. ), Ία) ^ ° = -8 ° in Dime thy If ormaimid «. «

909e> 4/1-f 9 1. . ■ "-

ßAO ORIGINAL

- 27 - 100-2955

Partial sequence C: N, N., -> * Dltrityl-L-histldyl-tert. -butyloxy-

cafbonyl-L-lysyl ^ L-phenylalanine hydrazide (Trt) -HiS (Trt) -LyS (BOC) -PlIe-IiH-NH ₂

a) Z-Lys (BQC) -Phe-QMe

94.8 g of H-Phe-OMe: "* HCl are dissolved in 1 liter of ether and approx. Dissolve 50 ml of ice water and add enough while stirring and cooling Sodium carbonate added until all the water has set. It is filtered and the filtrate is steamed to Constant weight, a colorless oil obtained will.

144 g of Z-Lys (BOC) -OH are dissolved in 300 ml of acetonitrile and 150 ral of dimethylformamide and the H-Phe-OMe obtained above is added. It is then cooled to -20 ° and a * solution of 90/8 g of dieyclohexylcarboid in 100 .-, I acetonitrile added. The whole thing left to Währe with occasional Ifmschütteln M are k hours in the ice chest. The

Any precipitate is filtered off and the piltrate is evaporated. The evaporation residue is dissolved in 1 liter of ethyl acetate dissolved and then in the cold with 1 M soda solution, water, 1 K sulfuric acid, water and saturated saline solution washed. The acetic acid: ethyl ester phase, dried over sodium sulfate, is completely evaporated. After recrystallization Z-Lys (BOC) -Phe-OMej is obtained from ethyl acetate / petroleum ether Snip. 102-105 °, eat ^ ° = -7 °, eat methanol >> -12 ° in Diniethylforiaamid, 909884/1791

^v i, ... ^ o ^ m BAD ORIGINAL

-28 -. '100-2953

b) H-LysCB ^ @) j> rPhte-OMe · HCl

29 g of Z-LySfBö ©) -Pfoe-OMe · werä'eri Oi 500 mi ^v of ^methanol; · ■■ '■ <· "- ^' and nacfcZugabe ~; Vön ·, 5 <j5 ^: ml ^f 1 * If- HGl / Methänol ¹ in'A'nwesenhö ^ t · * ■" ■■ - ^j of palladium carbon hydride: - Na '"oh filtering and evaporation ·"■;' - "" the residue is kneaded in high vacuum ¹ '. · '■'"_■"'-' ■ <'''ν * -, ν * - ^c ~ .jvi- ·

0) Z ~ His-Lys (BOC) -Phe-QMe

¹ ^ 5 g of H-Lys (BOC) -Phe-OMe · HCl are dissolved in a mixture of 300 ml of acetonitrile and 50 ml of dimethylformamide, then a solution of 40 g of Z-His-N ^. in 150 ml of Essigester and 20 ml of triethylamine and allowed to "4 hours at Ö ^ö 's6enen the entirety After filtration, the filtrate is evaporated and the residue is taken up in ethyl acetate and then three times / water-saturated brine (1: 1).. washed. " The acetic acid dried over sodium sulphate

- · '-;".J'.Τι? -: Η. ··. ·' ·? '-? Vv: i" wiN j- \: ⁱ ' ^: - ^; SP ' ⁱ -; - -: "- v -"\,> ^s Λ \!>. ;; &' ■? ^Λ 1 * ^ - '4 ^; "λ-"^;'-® ^ ί ^ - ^ Λί -; ^ * ^ ethyl ester phase is completely evaporated, with Z-His-

Lys (BOC) -Phe-OMe (light beige, amorphous foam) is obtained.

[α] ^ ^υ = -11 ° in dimethylformamide.

d) H-Hls-l ^ s

9β g ZiHls ^ l ^ s (

solS; -Denatowerden ΙΟ gj

120 ⁵¹ ItIlΐ 1 ίR> ■ Acid acid mixed and ^r irti the »Εσέύη§" igög.% -

The whole thing would be veirier'i ^ stMtälg'ei ^^

rature and normal pressure subject and ahseft! l; i ^: 6-§se ^; nd ^*:; il ⁱ r ^ ^ "

Catalyst filtered off. The filtrate is again with 5 g

' ^{: i} * 9090 84/1 7 ^C

- 29 - 100-2955

10% palladium on Aktivkohlein water and subjected to hydrogenation at room temperature and normal _pressure: NaOH 5 1/2 hours is completed. Approx. 80 % of the theoretical amount of hydrogen is consumed here. The catalyst is filtered off and the piltrate is completely evaporated, H-His-Lys (BOC) -Phe-OMe * HCl being obtained in the form of an amorphous foam. '

e) Trt-His (Trt) -Lys (30C) -Phe-0Me

144 g of H-HiS-Lys (EOC) ~ Phe-OMe · HCl are dissolved in 5000 ml of pyridine dissolved, at about + 5 ° 122 ml of triethylamine are added and then a solution of 207 6 trityl chloride in 1000 ml of pyridine was added dropwise over the course of 15 minutes. After the The pH is checked from time to time and if necessary adjusted to pH 9 with triethylamine. The whole thing is after evaporated about 24 hours, the residue in chloroform and water and adjusted to pH 4 with the addition of a little 1N hydrochloric acid. The chloroform phase is separated off, wash them twice with water, dry over sodium sulfate and evaporates completely. The residue is dissolved in a little ether dissolved, precipitated with petroleum ether and then filtered off, where Trt4Iis (Trt) -Lys (B0C) -Phe-ÖMe is obtained, -S6 ° in dimethylformamide.

134 g of Trt-His (Trt) -LyS (BOC) ~ Phe-OMe are in 1000 ml

90988 4/1791

8ADORtGINAt

- 30 - 100-2955

Dissolved methanol and mixed with βθ ml of hydrazine hydrate. It is then left to stand for 2 days at room temperature. It is evaporated, the residue is dissolved in a mixture of ethyl acetate / n-butanol (91) and washed twice with water. The organic phase is dried over sodium sulfate and evaporated. The residue is then washed with ether and filtered off. It consists of Trt-His (Trt) -Lys (B0C) -Phe-NH-ΐϊH ₂ of melting point 125 ° with decomposition, [a] _D = + 10 ° in dimethylformamide.

Example 9;

Partial sequence Dr ! - (L-Asparaglnyl-L-leucyl-L-asparaglnylrL · - »

asparaKiny 1 -!, - phenylalanine) -2-benzyloxycarbonyl-hydrazld · Trlfluoraeetat (H-Asn-Leü-Asn-Asn-Phe-NH-1W-Z · CP-COOH) ^;

a) l- (N ~ tgrt. -Butyloxycarbonyl-L-phenylalantn) ^ -berayloxy-

carbonyl hydrazld (BOC-Phe-m-NH-Z)

72 g of BOC-Phe-OH and 28 g of N-methylmorpholine are dissolved in 500 ml of methylene chloride and 26 ml of methyl chloroformate are added dropwise at -5 °. After 10 minutes, 44 g of Z-NHNH ₂ in 100 ml of methylene chloride are added and the mixture is stirred for a further 4 hours at room temperature. After washing with dilute phosphorous acid, it is dried and evaporated. To

Crystallization from petroleum ether is obtained. BOC-Phe-NH-NH-Z

20th
from m.p. 117 °, ία] _β = -5 ° in Dimethylformiaraid.

909884/1791

- 31 - 100-2953

b) l- (N-tert. -Butyloxycarbonyl-L-aspara ^ inylrL-phepyjLalani.p) - ^ 2-benzyloxycarbonyl-hydrazide (BQC-Asn-Phe, rNHrNH-Z ₁ ) .., " _£ , .., , ..., Dissolve 4l gBOC-Phe-NHp-Z in 4-00 ml of rifluoroacetic acid and leave to stand for one hour at 20 °. When. Yerdampfen, the. _{; <r} trifluoroacetic acid. one obtains the ^ HP.he-NHNH-Z als.-kr, ^!? ^^! - ^, ^. ,, lines trifluoroacetate of m.p. 191 °, Ea] ₁ . = + 26.4 ° in _n dimethylformamide ,, this. Substance ^ W ^ r ^^^ together ^^ t ^^ g _, _. ₄ BOC-Asn-ONP and 30 g of IJ-MethyJLmorphoMi., Dissolved in 20Q ml. DAroethyJL- _. ^ Formamide. After 16 hours of standing at 20 °, the solvent is evaporated and the residue is successively washed with ethyl acetate and dilute phosphorous acid ^ y ^

on

receives BOC-Asn-Phe-NH-NH-Z with a melting point of 210 °, ία] ρ = -18 °

c) l- (N-tert, -Butylo ^ c.arbQ.nyl-Lras.parass.inyXrAr.asparaginyl- * L-pheny'lalanine) -2.-Jpenay 1 oxycarbonyl.r-hydrazide ,, (BOC- Asn-Asn-Phe-NH-NH-Z)

One solves 26 * g''Bbü-Äsn-Fhe-NH-NH ^ Z '* inΊ200 ■ mi ~ TrJLf lüoressig-

acid and let stand at 20 ° for 1 hour. After evaporation

->, -: -. - .-, - λ - ,,.,. .r -----, - -. ·> - "* ■. ■ - '■ - ; v— ^iA -v * ~ ;;" O ~ - ■ ·; "* J. * - · ■ · ■". ·' ·: --Λ- of the solvent, 17 g of BOC-Äsn-ONP and 15 g of N-methylmorpholine are added to 100 ml of dimethylformamide. After 16 hours at 20 ° v / ird the solvent is evaporated and the residue washed successively with ethyl acetate and dilute phosphorous acid. BOC-Asn-Asn-Phe-NH-NH-Z, melting point 240 °, [a] ^ is obtained = -28 ° in dimethylformamide.

90986t / ii 9r; Γ

BAD OBlGlNAU

52 ■ - 100-2955

asparaginyl-L-pheny! alanin) "^ (BOG-Leu-Äsn-Asn-Phe-MH-NH-Z)

22 g of BOC-Asn-Asn-Phe-NH-WH-Z are dissolved in 150 ml of trifluoro * acetic acid and left to stand for 1 hour at 20 °. After evaporation of the solvent, the residue is mixed with 12 g of BOG-Leu-ÖNP and 10 $ ■ --- N-Methylffiorpholln -in 100 ml of dimethylforraataid, left to stand for 16 hours at 20®, the Dilaethylforinaraid is evaporated and the residue is successively with ethyl acetate and diluted Washed with phosphorous acid, Man

receives BOG-Leu-Äsn-Asn-Phe-HH-MZ from m.p. 210 °, [aj | ² '«

-5% ® in ~~

Han dissolves 57 g BGG-Leu-Asn-Asn-Phe »NH-ftH» Z in 300 ml TrifluoresslgsMure and leaves for one hour at 20 °. After the solvent has evaporated, the residue is crystallized in ether. The tetrapeptide fcriflitöraeetat is dissolved in 400 snl of dimethylformamide and the solution is mixed with & J g BOG-Asn-ÖNP and 25 g N-methylmorpholine. After standing for 6 hours at 20 °, the solvent is diluted and the residue is washed in succession with Sssigester and dilute phosphorous acid. One receives BOC-Äsn »Leu * A © n-Asn« Phe »NH ~ HH-Z. From the Srap. 250 ° (decomp.), TaJ ^ ⁰ - ■ -3 ** ia Dimethyl fonaamld. 909864/1791 '

original

100 = 2953

f) irCL-Asgayaglnyl · ^

Dissolve% 5s5 iBQC'-AsjirL'ewrAsiv-.Asn ^ Phe ^ NH ^ NH-Z.iii ^ OO ' ml - .. Trifluoroacetic acid and ISs; ® * stand ^{at 2G & for one hour.} _: . After evaporation, the EüokstsaM is crippled with the help of ether. "Man-.er-hSlt H ^ Äänrtea-Aän ^ Äsri-Phia-KH ^ NH-Z '. · : ° 2r ± ~ fluorooetat * m.p. 2h2 * _s EaI ^ ⁰ ^ -22 * in Dimethy 1 forisamid. ■

^ Trity 1-L-sery 1 ^ Lral'Sttiy 1 -L * tyr0syX-L-1rypto ^

Load 35 g of H * Arg (H0 ^} - 01s ^; ■> - MCI, · 1% n_-M ^to lethyini © rpholin In 3QO ml - Whore t hy Iförmamiö, on; ■

and leaves 16 - senses! en ^ bäl -SO *. ". laels? eröääWf © ii- its -

fIHt-. nife esthete ::. ^ * ^ * Bate ^

case?-

BATH ORIGINAL

100-2953

N ^ tert ^ Butylq ^ carbonyl-L'tyrosyX-L ^ tryptophanyl-

(guaniflo) H-nifcro-agginine mefchylesfeer (BOG-Tyr ~ Trp-Arg (NOg) - ■

. n n triggers-'1S g "- BOC-Trp-ArgCNOg) -OMe in 80 ml 5K Lischer hydrochloric acid solution and leaves for 1 hour at room temperature .---stand. ·" -After evaporator ^e: n.des ! Solvent and. Treatment with ether remain, IjJTg H «1 * pp-Ar ^ {N0g) -" 0Me as hydrochloride backi Smp »120 ^ * IaI _n -. * ~ '-Ψ -lh dimethylformamide?: - This dipotide is Eusammsn with 1%; g B0G * T2fr- ^: \ OGP and 8 g Nrflethylmorpholin'.ln; Ö0O SiI Bim © tfeyiformamid;. 'Dissolved. After 16 hours of standing, feel 2ö ^& -will be the solvent

evaporated and the -btickstand recorded in R & älgester. To Wash with. -Diluted; it? "-, phpsphorigei? '- acid. And concentration of the Solvent can be done with Hilf®; von.Äether "BÖC-Tyr-Trp- ■ ■ Arg (NOg.) - OMe "isolating- Smp> '1 ^ 0 *» .. laid ^ -ii® in ■ formamide. ... '-. -. ...; "■ · .- ■■" ■ "

i ^ (BOC-Ala ~ Tyr-Trp-

15.5 g of BOC-Tyr-Trp-Arg (Kö _g ) -OPfe are dissolved in IJO ml of 5 K methanolic hydrochloric acid solution; on, can 1 hour at 25 ^ ^e are evaporated to dryness · © * in the Riickstaiid washed with dimethyl ether, filtered off, dissolved in 100 ml of dimethylformamide and treated with 8 g of BOC ö ~ Aia "öCPiiBd 3 * 0 ral TriSthyl- 'amin. After ΐβ hours at 25 ° there are 500 ml of vinegar aster

added, washes with dilute sulfuric acid and potassium bicarbonate

909884 / Ί791

ORIGINAL

- 55 - '■ 100-2953

■ ^: ".. ·. 193587B .-

solution and evaporate to dryness. After washing the residue with chloroform, BOO-Ala-Tyr-Trp-Arg (NO ₂ ) -OMe Vom Sffip is obtained. 120 ° (decomp.) »[A3 _D » -12 ° in dimethylformamide.

phanyl- (guanido) N ^ nitro-L ^ arginine methyl ester (BOC-Ser-AIa- $ yr-frp-Arg (NOg) -OMe)

Mail load 12.3 g of BOC-Ala-Tyr-Trp-AERG {NQG) -OMe in 100 ml of 5 N methanolic hydrochloric acid, can IStüMa "at 25 °, are evaporated suf dryness © in., The residue is washed with diethyl Ether, dissolve it in 100 ml dimethylformamide * mixed with - ■ "■ JA ml frllifchylarnifc and 8 * 5 g BOG" Ser "fe * 4Mt 16! Hours b & l 0 ° jgtehehj. Give IOD ml acetic ester acid w. Then add dilute sulfuric acid

dried over sodium sulfate and "läfiipft Φ & έ solvent -ab · Haoh Wasoheri the residue with chloroform to dissolve it in Essigester and precipitated by addition of DiäthyiSther" obtained 13 _a 5 g BOC * Be * Ala-type ICRP Are (SOG) - OMe # Bmp,

^ - »15

• «) JPrity 1 -L-sery 1 -1, -alany 1 ^ L ^ tyrösyl φ- tryptophany 1» L-

arginin ·,, hydrazld XTrt ^ Sei ^ Äla ^ iyr ^ rp-Arg-NHfitg) · .-

dissolves IJiί g from * i © ffl obigeö ^ @ sit ^ i | itiilester lsi 300 «ril 60 ssl ^ asger ώα i ^ 'S t PallMium charcoal and hyäriert to φΜ JnSe - ^ er -Ifasserstqffaufo .man / filtered« ^! iissapffc - ^ ibj. llsi; € ^ n Meksfcänä. in the .; . Bisethylformasild on, oampft wifederuisi & b _e um öie traces

100-2955

to eliminate acetic acid. Then you solve the Residue in 250 ml of 5 Ii methanolic hydrochloric acid, leaves 1 Stand at 20 ° for hours, evaporate, dissolve in methanol, evaporate and dissolves in 250 ml of pyridine. Then give 7 rnl Add triethylamine, cool to 0 °, give 9.5 g Triphenylehlor ™ methane and let stand at 0 ° for 16 hours. It is evaporated to dryness, dissolved in ethyl acetate and washed with a 5 #igeri NaCl solution. It is dried, evaporated, the residue is washed with a mixture of diethyl ether-petroleum ether (2: 1) and Dissolves in 20 ml of methanol, mixed with 26 ml of hydrazine hydrate and lets l6 hours steep at 40 °. You steam off, wash the residue with water until neutral and dried over sulfuric acid in a high vacuum. One obtains Trt-Ser-Ala-

20th

Tyr-Trp-Arg-NHNH ₂ , m.p. 153 ° / [α] _β = -6 ° in dimethylformamide.

S03884 / 1791

BADORiGJNAL

- 37 - 100-295: 5

Partial sequence Fl: L-threonyl-S-bengyl-L-cyst- in yl-L-valyl -L--

leucine methyl ester hydrobro.T.id (H-Thr-

Cys (Bzl) -Val-Leu-ÖMe HBr)

a) H-CyS (BzI) -VaI-LeU-OMe * HBr

21.0 g of Z-Cys (BzI) -OCP and 12.3 g of H-Val-Leu-OMe · HCl are dissolved in 120 ml of dimethylformamide. Then 5.9 ml of triethylamine are added, left to stand for 16 hours at 25 °, ethyl acetate is added, washed with dilute hydrochloric acid, dried over sodium sulfate, evaporated to dryness and the residue crystallized from ethyl acetate / diethyl ether. This gives Z-Cys (Bzl) -Val-Leu-OMe, mp. L6o °, [α] * = -28 ° in dimethylformamide, the one in 210 ml of a solution of iiigen ^J l0. Dissolves hydrogen bromide in glacial acetic acid. The mixture is left to stand at 25 ° for 1 hour, evaporated to dryness and the residue is recrystallized from isopropanol / diethyl ether. You get

on

H-CyS (BzI) -VaI-LeU-OMe · HBr, m.p. 168 °, Ea] * = + 14 ° in Dimethylformamide.

b) H-Thr-Cys (Bzl) -Val-Leu-OMe. HBr

Dissolve 20 g Z-ThT-NHNH ₂ in 350 ml of dimethylformamide, cooled to -20 °, is 100 ml to a solution of 2 N hydrochloric acid in dioxane and then a further 10 ml of tert-butyl nitrite. After 10 minutes at -20 °, 45 ml of triethylamine and 25/5 g of H-Cys (Bzl) -Val-Leu-OMe · HBr are added and the resulting mixture is shaken at 0 ° for 16 hours. It is evaporated to dryness, the residue is dissolved in a mixture of vinegar

90988 ^ / 1791

- 38 - 100-2953

1935Ö76

acid ethyester / water, washes the organic phase with it dilute hydrochloric acid, dried over sodium sulfate, evaporated and the residue recrystallized from ethyl acetate.

One obtains Z-Thr-Cys (Bzl) -Val-Leu-OMe, m.p. 208 °, Ea] J ^ '= _ -27 ° in dimethylformamide.

Dissolve 20 g of the tetrapeptide obtained above in 200 ml of a mixture of trifluoroacetic acid / ethyl acetate (l; l) on, conducts a stream of gaseous form for 1 hour at 0 ° Bronzstoffs, then evaporates and the residue crystallizes from methanol / diethyl ether. H-Thr-Cys (BzI) -Val-Leu-0Me.1.3 HBr, Snip is obtained. 202 ° / ' [α J-f- = -10 ° in dimethylformamide.

Example 12:

Partial sequence F2: tert-butyloxy carbonyl-S-benzyl-L-cysteinyl-

L-seryl-L-asparaginyl-L-leücyl-L-serine-hydra- . zid (BOC-Cys (BzI) -Ser-Asn-Leu-Ser-MH-i ^).

^a ) H-Asn-Leu-Ser-OMe . HCl

43 g of H-Leu-Ser-OMe · HCl and 53 g of BOC-Asn-ONP are dissolved in 400 ml of dimethylformamide, 22 ml of triethylamine are added, the mixture is left to stand for 16 hours at 25 °, evaporated to dryness and the residue is crystallized out Methanol around. 51.6 g of BÖC-Asn-Leu-Ser-OMe, melting point 190 °, [a] ^ ° = -24 ° are obtained in diraethylformaramide, which is dissolved in 500 ml of a 4N solution of hydrochloric acid in methanol. It is allowed to stand for 1 hour 25 ° hot, evaporated to dryness, the residue is dissolved in methanol and

909884/1791

- 59 -. 100-2955

falls with diethyl ether. H-Asn-Leu-Ser-OMe * HCl is obtained, Mp. 180 ° -,. [A] p - -23 ° in dimethylformamide,

b) H-Ser-Asn-Ieu-Ser-OMe · HCl

39.5 g of BOC - S er-1, Ή KH _{2 are dissolved} in 500 ml of dimethylformamide, the mixture is cooled to -20 °, 200 ml of a 2N solution of hydrochloric acid in dioxane and then 20 ml of tert-butyl nitrite are added 10 minutes at -20 ° v.'erden 40 ml of triethylamine and 38.0 g of H-Asn-Leu-Ser-OMe were added, then stirred for 1.6 hours at 0 °, evaporated to dryness and the residue was recrystallized from chloroform / diethyl ether. This gives BOC-Ser-Asn-Leu-Ser-OMe, m.p. 135 °, ta] * = -22 ° in dimethylformamide, which is dissolved in 420 ml of a 4N hydrochloric acid solution

solution dissolves in methanol. It is left to stand at 25 ° for 1 hour, evaporates to dryness and crystallizes from methanol / ethyl acetate around. One obtains H-Ser-Asn-Leu-Ser-'OMe · HGl, M.p. 155 ° (decomp.), Ca] ^ = "15 ° in dimethylformamide.

c) EOC-CyS (BzI) -Ser-Asn-Leu-Ser-NHMP Iq

Dissolve 18/5 U H-Ser-Asn-Leu-Ser-OMe · HCl and 18.0 g BOC-Cys (Bzl) -ONP in 100 ml dimethylformamide, add 10 ml v / water, 3/5 ml acetic acid and 5.6 ml of triethylamine, left to stand for 16 hours at 25 °, evaporated to dryness and recrystallized from methanol. 25.1 g of BOC-Cys (Bzl) seron are obtained
Asn-Leu-Ser-OMe, m.p. 182 °, [a3 _D = -IJ ⁰ in dimethylformamide / which is put in 200 ml of dimethylformamide with gentle warming

9098 84/17 9 1 ■ / '. r ;. _r & 6AD OBIGlNAL

- ΐκ> - 100-2955

solves. 200 ml of methanol and 20 ml of hydrazine hydrate are added, - lets stand for 16 hours at 30 °, falls with diethyl ether, washes the precipitate with diethyl ether / kethanol (1: 1) and dries the BOC-CysCBzl ^ Ser-Asn-Leu-Ser-NHMIg,

on

M.p. 224 °, [alp = -13 ° in dimethylformamide. Example Γ5:

? 21 S-Benzyl-ß-mercaptoproDlonyl-L-se ryl-L-

asparaginyl-L-leucyl-li-serine-hydrazid (MCP (BzI) -Ser-Asn-Leu-Ser-NHrNHg)

a) H-Asn-Leu-Ser-OMe · HCl

Kj> g of H-Leu-Ser-OMe · HCl and 53 g of BOC-Asn-ONP are dissolved in 400 ml of dimethylformamide, 22 ml of triethylamine are added, the mixture is left to stand at 25 ° for 16 hours, evaporated to dryness and the residue is crystallized from methanol. This gives 51.6 g of BOC-Asn-Leu-Ser-CMe, mp. 190 ^0, Εα] ^ ^υ = -24 ° in dimethylformamide, the one in 500 ml of a k N solution of hydrochloric acid in methanol dissolves. The mixture is left to stand at 25 ° for 1 hour, evaporated to dryness, the residue is dissolved in methanol and precipitated with diethyl ether. H-Asn-Leu-Ser-OMe * HCl, melting point 180 °, [a] ^ ^w = -25 ° in dimethylformamide is obtained.

b) K-Ser-Asn-Leu-Ser-OMe ♦ HCl

39.5 g of BOC-Ser-IiHNH _{2 are dissolved} in 500 ml of dimethylformamide,

9 0 9884/1791

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cools to -20 °, gives 200 nil of a 2 N solution of hydrochloric acid In dioxane and then 20 ml of tert-butyl nitrite to * After 10 minutes at -20 °, 40 ml of triethylamine and 38.0 g of H-Asn-Leu-Ser-OMe are added, followed by 16 hours stirred at 0 °, evaporated to dryness and the residue recrystallized from chloroform / diethyl ether. You get BOC-Ser-Asn-Leu-Ser-OMe, m.p. 135 °, [a] ^ = -22 ° in dimethyl formamide, which is dissolved in 420 ml of a 4N hydrochloric acid solution dissolves in methanol. The mixture is left to stand at 25 ° for 1 hour, evaporated to dryness and crystallized from methanol / acetic acid ethyl ester. H-Ser-Asn-Leu-Ser-OMe 'HCl is obtained, M.p. 155 ° (decomp.), [A] ^ = -15 ° in dimethylformamide.

c) MCP (BzI) -Ser-Asn-Leu-Ser-NHrlft ^

Dissolve 18.5 S H-Ser-Asn-Leu-Ser-OMe · HCl and 13.5 S MCP (BzI) -ONP in 100 ml dimethylformamide, add 10 ml v / water, 3 * 5 ml acetic acid and 5% * 6 ml of triethylarain to
left to stand for 16 hours at 25 °, evaporated to dryness and recrystallized from ethanol. 21.3 g are obtained

20 MCP (BzI) -Ser-Asn-Leu-Ser-OMe, m.p. 193 °, CaL = -18 ° in dimethylformamide, which is dissolved in 200 ml of dimethylformamide with gentle warming. 200 ml of methanol and 20 ml of hydrazine hydrate are added, the mixture is left to stand for 16 hours at 30 °, it is precipitated with diethyl ether, the precipitate is washed with diethyl ether / methanol (1: 1) and the MCP (BzI) -Ser-Asn-Leu thus obtained is dried -Ser-NH-NH ₂ , m.p. 251 °, £ a] ^ ° = -16 ° in dimethylformamide. 909884/1791

BATH

100-2953

Example l4;

Partial sequence ^ ABG ^ L-histidyl- £ "~ tert> -butyloxycarbony-lL-ly syl-

L-phenylalanyl-L-seryl-gly c yl-L-methion yl ^ | ylyQyl-L-phenylalanyl-glycyl-Ii-prolyl-L »^ gl · uta ^ myl-L-threonyl-L-prollnaraid · triaoetate (H. -His-Lys (BOC) -Phe-Ser-Gly-Met-Gly-Phe ~ Gly- Pro-Glu-Thr-Pro-ilH ₂ · 3 CH ₃ CO ₂ H)

. a) L-Seryl-glycyl-L-methionyl-glycyl-L-phenylalanyl-glycyl - L-prolyl-L-glutamyl-L-threonyl-L-prolinamide * Hyd rochloric!

(H-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-i; H ₂ 'HCl) Dissolve 6.7 g of BOC-Ser-Gly-Met-Gly-OH (partial sequence B. ), 12 g H-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ (partial sequence A) and 2.1 s N-hydroxysuccinimide in 50 ml dimethylformamide and 20 ml acetonitrile, cools to 0 °, add 4.0 g of dicyelohexylcarbod * iimide and leave to stand for 16 hours at 0 °. The solvent is evaporated off, the residue is washed with water, diethyl ether and ethyl acetate and recrystallized from chloroform. BOG-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ »mp. 121 ° (decomp.), [A] jj = -4 ° in dimethylformamide , which is dissolved in 250 ml of a solution of 8N hydrogen chloride in dioxane. The mixture is stirred for 2 hours at 25 °, evaporated to dryness and the residue is processed in diethyl ether. H-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-

Thr-Pro-NH ₂ • HCl [m.p. 130 ⁰ (dec.), Ea] * = -48 ° in dimethylformamide]. .

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b) L-Hlstidyl- ~ £ -tert ^ -butyloxycarbonyl ^ L-lysyl-L-phenylalanyl L-seryl-glycyl-L-rnethlonyl-glycyl-L-phenylalanyl-glycyl-

L-prolyl-L-glutamyl-L-threoriyl-L-prolinamide triacetate (H-His-LysiBOCO-Phe-Serr-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NHg * 3 CH ^ COOH)

9.9 g of Trt-His {Trt) -Lys (BOC) -Phe-NHNHg (partial sequence C) are dissolved in 100 ml of dimethylformamide, cooled to -20 °, 15 ml of dioxane / HCl 2 N and then 1.16 ml are added tert-butyl nitrite, stir for 10 minutes at -20 °, add 28 ml of triethylamine and 10.0 g of H-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NHg.HCl, stir for 4 hours at 0 °, filtered and evaporated to dryness. The residue is dissolved in a mixture of Essigester / methanol (8: 2), washed with dilute ammonia and then with water to neutrality, dried over sodium sulfate, concentrated to 100 ml and precipitated by addition of diethyl ether. The precipitate is again dissolved in dimethylformamide and precipitated by adding diethyl ether. This gives Trt HisCTrtO-LysiBOCj-Phe-Ser-Gly-Met-Gly-Phe-Gly

2O

Pro-Glu-Thr-Pfo-NHg [m.p. 150 ° (decomp.), [A] _D = -48 ° in dimethylformamide J, which is dissolved in 500 ml of acetic acid / water (8: 2). The mixture is left to stand for 3 hours at 40 °, evaporated, the residue is washed in diethyl ether and dried in a high vacuum over KOH chips. H ^ HiS-LyS (BOC) -Phe-Ser-Gly-Met-Gly-Phe-Gly is obtained -Pro-Glu-Thr-Pro-NHg. 3 CH ₃ COOH, m.p. 201 ° (dec.), [Aljj ⁰ . = -53 ° in acetic acid *

909884/179 1

■■: ξ ~ ι Uf- "

BATH ORIGINAL

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Example 15:

Partial sequence ABlC: L-HIstidyl- £ -tert.-butyloxycarbonyl-Ll ysyl-

L-phenylalanyl-L-seryl-glycyl-L-norleucyl- · glyoyl-L-phenylalanyl-glycyl-L-prolyl-terb. - butoxy-L-glutamyl-L-threonyl-L-prolinaniid · Triacetate (H-His-Lys (BOC) -Phe-Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro -NH ₂ 3 CH COOH)

a) L-Seryl-glycyl-L-norleucyl-glycyl ^ -L-phenylalanyl-glycyl-L-prolyl-tert.-butyloxy-L-glutamyl-L-threonyl-Lp rolinamid- (H-Ser-Gly-Nle- Gly-Phe-Gly-Pro-Glu (OTB) -Thr-Pro-NHp) Dissolve 12 g of H-Phe-Gly-Pro-Glu (OTB) -Thr-Pro-NH _g (partial sequence A) in 100 ml of dimethylformamide and 20 ml of acetonitrile at 0 ° and adds 2.1 g of N-hydroxysuocinimide, 8.0 g of Z-Ser-Gly-Nle-Gly-OH (partial sequence B1) and 4.0 g of dicyclohexylcarbodiimide, leaves overnight at 0 ° stand, filtered, evaporated in vacuo and crystallized from chloroform. Z-Ser-Gly-Nle-Gly-Phe-Gly-JPro-Glu (OTB) -Thr-Fro-NHp, melting point 125-138 ⁰ , [α3ρ = -44 ° in dimethylformamide is obtained. It is dissolved in 400 ml of dioxane and 400 ml of H ₃ O and hydrogenated at normal pressure in the presence of a palladium catalyst until the hydrogen stops. It is filtered, evaporated in vacuo, dissolved in methanol, mixed with ether and obtained H ~ Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ , melting point I30 ⁰ ( Dec.), [A] _D = -52 ° in dimethylformamide.

909884/1791

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b) L-histidyl-6ert-butyloxycarbonyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-norleucyl-glycyl-L-phenylalanyl-glycyl-L-prolyl-tert-butyloxy-L-glutamyl -L-threonyl-L-prolinamide »IPriacetate (H-His-Lys (BOC) -Phe-Ser-Gly-Nle-Gly-Phe- 'GIy-Pro-Glu (OTB) -Thr-Pro-NH ₂ · 3 CH-COgH)

9.9 g of N ^a -Trt-N ⁱⁿ -Trt-His-Lys (B0G) -Phe-NHNH _{2 are dissolved} in -100 ml of dimethylformamide, cooled to -20 °, ^ O ml of dioxane / HCl 2 N are added and then Add 1.16 ml of tert-butyl nitrite, stir for 10 minutes at -20 °, add 28 ml of triethylamine and 9.7 g of H-Ser-01y-Nle-01y-Phe-Gly-Pro-Glu (0TB) -Thr- Pro-NH ₂ · HCl, stirred for 4 hours at 0 °, filtered and evaporated to dryness. The residue is dissolved in a mixture of ethyl acetate / methanol (8: 2), washed with dilute ammonia and then with water until neutral, dried over sodium sulfate, concentrated to 100 ml and precipitated by adding diethyl ether. The precipitate is again dissolved in dimethylformamide and precipitated by adding _% diethyl ether. Trt-His (Trt) -Lys (BOC) -Phe-Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu (OTB) -

on

Thr-Pro-NHg (m.p. 135 ° decomp.), Ε «3 ^ = -31 ° in dimethylformamide, which is dissolved in 500 ml of acetic acid / water (8s2). It is left to stand at 40 ° for J hours, evaporated and washed Residue in diethyl ether and dried in a high vacuum over KOH chips. H-His-Lys (BOC) -Phe-Ser-Gly-Nle-Gly-Phe-

Gly-Pro-Glu (OTB) -Thr-Pro-NHg '3 CH ₅ COOH (m.p. 142 ° dec.) # 20

^v s -45 ° in dimethylformamide _β \

90 9 8 84 / -17.91

ORKSlNAt _n

- 46 - 100-2955

? Sii§29 ^u 2i} 5-51l Trityl -L-seryl-L-alanyl-L-tyrosyl-L-trypto-

phanyl-L-arginyl-L-asparafdny1-L-IeUCyI-L-asparaginyl-L-asparaginyl-L-phenylalanine hydrazide (Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe- NHNH ₂ · 2 CH ₃ COOH)

a) ! - (N-Trityl-L-seryl-L-alanyl-L-tyrosyi-L-tryptoDhanyl-L-arginyl-L-asparaginyl-L-le'ucyl-L-asparaginyl-L-asparaginyl-L-phenylalanine ) -2-benzyloxycarbonyl-hydrazide »diacetate (Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-NHNH-Z · diacetate)

The mixture is cooled to -20 J20 ml Dimethylforraaraid ⁰ C, 100 ml of 2 N hydrogen chloride in dioxane is added and dissolved therein 72 g of Trt-Ser-Ala-Tyr-Trp-Arg-NHNHg · acetate (partial sequence E). Then 12 ml of tert-butyl nitrate are added, the mixture is stirred for 10 minutes at -20 ° and 35 ml of triethylamine are added dropwise. The mixture thus obtained is combined with a solution formed from 8.8 g of H-Asn-Leu-Asn-As * h-.Phe-NHNH-Z · trifluoroacetate (partial sequence D) and 17 ml of triethylamine in 150 ml of dimethylformamide. After Ιβ hours at 0 ^θ , the mixture is evaporated. The residue is dissolved in ethyl acetate and washed with dilute acetic acid and soda solution. After evaporation of the solvent, the Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Äsn-

_. 2 OH ^ OOOH / _r O ₀
Asn-Phe-tüMH-2 ^ vom SmpTTö8 ^e , Ca] ^ = -1ψ ix »Dimethylformamide»

b) Trityl-L ~ @ eryl ~ L ° alanyl "Igtyrgsyl" L-tjt ^^^^ g ^ ,! 3 ^

9098 84/1191

- 47 - 100-2955

• phenylalanine hydragid diacetate (Trt-Ser-Ala-Tyr-Trp-Arg-

Asn-Leu-Asn-Asn-Phe-NHNHg.2 CH, C00H)

90 g of Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-NHm-Z are dissolved in 500 ml of dimethylformamide and hydrogenated with 10 g of Pd / C (10 #ig) until the end of the Hydrogen absorption After filtering off the catalyst, the solvent is evaporated and the residue is crystallized from a mixture of ether and ethanol containing 1% acetic acid. Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-MHNHg · 2 CH-COOH. Of melting point 215 °, ta] ^ = -53 ° in dimethylformamide is obtained.

Example 17;

Partial sequence tert. "Butyloxyc_arbpnyl ~ L-'heniicy stlnyl ~ L-5eryl"

----- L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L- ·

hemicystinyl-L-va.lyl-L-leuciii-hydrazide

-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-NHNHg) Dissolve 18.4 g of BOC-CyS (BzI) -Ser-Asn-Leu-Ser-NHNHg (partial sequence F2) in 150 ml of dimethylformamide, cools to -20 °, are ml of a 2N solution of hydrochloric acid in Dloxan and 15 ml

tert-butyl nitrite too. After 10 minutes at -20 °, 28 nil triethylamine and 24.8 g of H-Thr-Cys (Bzl) -Val-Leu-OMe ^s Ip3 HBr (partial sequence Fl) are added and the mixture is then _{stirred at 25® β for 16 hours} filtered, the solution evaporated and the residue washed through with hot methanol. You get

9 0 988Ä / t791

"'SAD ORlQlNAU

- 48 - 100-2955

BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-Thr-Cys (Bzl> -Val-Leu-OMe, m.p.

PCi

242-244 °, [aj ^ = -25 ° in dimethylformamide, which is obtained at 60 °

dissolves in 100 ml of dimethylformamide. 10 ml of hydrazine hydrate are added and let stand for 2 hours at 60 °. The resulting nonapeptide hydrazide crystallizes out. One washes with diethyl ether, V / water and methanol and dry in a high vacuum

about phosphorus pentoxide. One receives nonapeptide hydrazide from

20th
Mp. 260 ° (decomp.), [A] _D = -33 ° in dimethylformamide / water

(3: 1). The product obtained is suspended in 5000 ml of dried material Ammonia, while stirring and boiling the ammonia, adds sodium metal until it turns deep blue. For the purpose of discoloration ammonium chloride is added and then a stream of air up to passed through the solution to the negative nitroprussiate reaction. It is evaporated to dryness and the residue is washed with water and acetone through. After drying, BOC-Cys-Ser-Asn-Leu-

20th

Ser-Thr-Cys-Val-LeU-NHNH ₂ , m.p. 298 ° (dec.), Ca] ^ = -20 ° in dimethylformamide / V / water (j5: 1).

Example l8;

Partial sequence f1F3: 0 ~ mercaptopropionyl «-L-seryl-L ~ asparaginyl · -

L-leucyl-L-seryl-L-threonyl-L-hemicystinyl "

L-valyl-L-leucine hydrazide (MCP-Ser-Asn-Leu

Ser-Thr-Cys-Val-Leu-NHNHg).

16.2 g of MCP (BzI) -Ser-Asn-Leu-Ser-NH-NH ₂ (partial sequence

FJ) in ISO ffil dimethylforroamide, cools to -20 °, gives 40 ml

909884/1791

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to a 2 N solution of hydrochloric acid in dioxane and 14 ml of tert-butyl nitrite. After 10 minutes at -20 °, 28 ml of triethylamine and 24.8 g of H-Thr-Cys (Bzl) -Val-Leu-OMe are added. 1.5 HBr (partial sequence P1) and stirred for 16 hours at 25 °. Then it is filtered * the solution is evaporated and the residue is washed through with hot methanol. This gives 24.1 g of MCP (BzI) -Ser-Asn-Leu-Ser-Thr-Cys (Bz!) - Val-Leu-OMe, melting point 244 °, [a] * ^u = -22 ° in dimethylformamide, which is dissolved in 100 ml of dimethylformamide at 60 °, 10 ml of hydrazine hydrate are added and the mixture is left to stand at 60 ° for 2 hours. The resulting nonapeptide hydrazide crystallizes out. It is washed with diethyl ether, water and methanol and dried in a high vacuum over phosphorus pentoxide. You get

On

15.1 g of nonapeptide hydrazide, m.p. 2β5 °, [α] £ ^υ = -55 ° in Dime thy Iformamid / Was s er (5il). The product obtained is suspended in 5000 ml of dried ammonia, sodium metal is added until the ammonia is stirred and boiled until it becomes deep blue. Ammonium chloride is used for discoloration. added and then a stream of air passed through the solution until the nitroprussiate reaction was negative. It is evaporated to dryness and the residue is washed through with water and acetone. After drying, MCP-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-NHNHg, melting point 278 °, Ca] :: = -19 ° in dimethylformamide / water (3: X) is obtained.

909884 / T791

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Example 19;

Partial sequence ABCDE; L-Seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-

L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-E-tert. -butyloxycarbonyl-L-lysyl-L-phenyl a lany1-Ls ery 1-glyoyl-L-roethiony1-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-klutamyl-L-threonyl-L-prollnamide * hexaacetate (H. -Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys (BOC) -Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro- NHg 6 CH ₃ CO ₂ H)

15.2 g of Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-NHNHp · diacetate (partial sequence DE) are dissolved in 200 ml of dimethylformamide, cooled to -20 °, and 15 ml of dioxane are added / HCl 2 N and then 1.16 ml of tert-butyl nitrite and stir for 10 minutes at -20 °. 14 ml of triethylamine and a solution of 18.9 ^{% of the trideeapeptiöaeetats obtained in} ^ Teilsequenz ABC in dimethylformamide are added, the resulting mixture is stirred for 16 hours at 0 °, filtered and evaporated to dryness. The residue is washed with diethyl ether and chloroform, dissolved in dioxane / water (8i2), the solution obtained is treated with 100 ml of Amberlite IRA-410 (acetate form), evaporated and the residue is recrystallized from isopropanol / water. The Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-LysiBOCj-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro is obtained -NHg acetate, m.p. 128 ° (decomp.),

309884/1791

100-2953

20 ι

yy. = -40 ° in dimethylformamide, which is dissolved in 500 ml of 80% acetic acid under a nitrogen atmosphere * It is left to stand at 45 ° for 1 hour, concentrated to 100 ml and precipitated by adding 1000 ml of diethyl ether. It is filtered, the residue is dissolved in dimethylformamide and precipitated with diethyl ether. After drying over KOH chips, the H-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-Kis-I.ys (BOC) -Phe-Ser-Gly-Met-Gly- Phe-Gly-Pro-Glu-Thr-Pro-NH _a · 6 CH COgH, m.p.

(Decomp.),

20th

in acetic acid.

Example 20t

Partial seqüera; ABlCDEf

L-Seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl'-L-asparaginyl-L-leucyl-L-aspa ra-

-giB3rl- ° L "asparaginyl- L-phenylalanyl - L-histidyl ■ _m £ - ^ tertc-butyloxyearbonyl-Ll ysyl-L-phen;, '·! -

phe nylalan, Tl-p: l? ₁ rcyl-L-prolyl-'tert. -butyloxy-L'-Klutamyl-L-threonyl-L-prolinamide * Hexaeaetat (E-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-

Asn-Äsn-Pke-His-Lys (BOC) -Phe-Ser-Gly-Nle-

One l ^ st 15 _S 8 g
NHNHg 'Dlaöetat (Teilseqnesig DE) in 200 ml of dimethylformamide, cools to -20®, gives 15 nl - BioxaivföCl- 2 N and then Ic 16

tePt. «Bütyltltti * £ t sa and i'öhrfe 10 meows at -20 °.

90988A / 1791

BATH ORIGINAL

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14 ml of triethylamine and a solution of 18.6 g of des Tridecapeptide acetate obtained in partial sequence ABIC in dimethylformamide the mixture obtained is stirred for 16 hours at 0 °, filtered and evaporates to dryness. The residue is washed with diethyl ether and chloroform, dissolved in dioxane / V / water (8: 2), treats the resulting solution with 100 ml of Amberlit-IRA-410 (Acetate form), evaporated and the residue crystallized from isopropane oil / V / water. The Trt-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-LysiBOCj-Phe-Ser-Gly-Nle-

W Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ ; Acetate, Sinp. 155 °

20th
(Decomp.), [AL · = -45 ° in dimethylformamide, which is under

Dissolve a nitrogen atmosphere in 500 ml of 80% acetic acid. The mixture is left to stand at 45 ° for 1 hour, concentrated to 100 ml and precipitated by adding 1000 ml of diethyl ether. It is filtered, the residue is dissolved in dimethylformamide and precipitated with diethyl ether. After drying over KOH chips, the H-Ser- 'Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-LysiBOC) -Phe-Ser-Gly-Nle-Gly-Phe-Gly is obtained -Pro-Glu (OTB) -Thr-Pro-NK ₂ * 6 CH., COpH, m.p. 150 ° (dec.), [Α3 _β = -45 ° in acetic acid.

Example 21;

Sequence tert-butyloxycarbonyl-L-hemicystinyl-L-seryl-L " ABCDEF1F2 £ asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L -alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-'L-leucyl-L ^ asparaginyl-L-asparaginyl-L-phenylalanyl · - L-hlstiayl ^ tert-butyloxycarbonyl ^ L-lysyl- L-phenylalanyl ~ L-seryl- ^ lyc.yl-L- »methlonyl-jB: lyGyl" L ~ phenyl-

909884/1791

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alanyl-glycyl ^ L-prolyl-L-glutamyl'-L-threonyl- . L-prolinamide * hexaaoetate * decahydrate

. (BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys ^ Val-Leu-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys (BOC) -Phe- Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NHg 6 acetate decahydrate)

1.05 S nonapeptide hydrazide (partial sequence P1F2) is dissolved in a mixture of 50 ml dimethylformamide and 1.2 ml 2 N dioxane / HCl at -20 °. 0.16 ml of tert-butyl nitrite are added, the mixture is stirred for 10 minutes at -20 °, 1.4 ml of triethylamine and 5 * 1 S tricosapeptide hexacetate (partial sequence ABCDE) and 5 ml of water are added and the mixture is stirred at Q for 16 hours ° _o It is evaporated to dryness, the residue is washed with diethyl ether, chloroform and acetone. ■., -_.

The crude Dotriacontapeptide is thus obtained, which is dissolved in 100 ml of 0.5 N acetic acid, treated with 20 ml of Amberlite IRA-410 (acetate) and then 50 ml of 0.6 N ammonium hydroxide is added. The pH is adjusted to 6.5 and layers the resulting solution on a carboxymethyl cellulose column (10 100 Qm) ₉ which has been equilibrated with a 0.15 U ammonium acetate buffer solution. The elution is carried out with an increasing concentration and pH gradient (0 _# 15 N to Q ₉ h N; pH 6.5 to pH 7 * 0) of an ammonium acetate buffer. The combined fractions, the pure

9QS884 / 17S1

. '■ -. ■■. . BATH ORIGINAL

-'54'- ■ 100-2955

Containing peptide are freeze-dried three times, the Residue with ethanol and then with diethyl ether washed and dried over potassium hydroxide in a high vacuum.

You get BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn ~ Phe-His <-Lys (BOC) - Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-MHg 6 CH ₃ CO ₂ H 10 HgO,

pn

M.p. 220 ° (decomp.), Ia] Z. = -58 ° in acetic acid 1 N. Amino acid composition after acid hydrolysis (6 IJ, 16 hours) s

, 9 ' ^{Cys / 2} 1.6'

2.9 ' ^Lys i, i' ^Met i, o ' ^phe 3, o' ^Pro 2, i 'VaI 0.9 (Trp 1.0 by spectrophotometry).

Example 22? "

Sequence t ert.Butyloxycarbony l -L ^ -heπlicystinyl-L · »seryl-L ^ -

_k AB1CDEP1P2J asparaginyl-L-leucyl-L ^ seryl-L-thi'eonyl-L-hemi-

Ψ - -

cystin.γl-L ^^ · valyl -L »le ^ o yl- · L · --seΓyl-» L · -alanyl ^ -L-

tyFOsyl-L · -tryptophanyl ^ -L ■ -arginyl · ^ 'L · -aspara ^ irlyl-L-

L-histidyl- £ - tert-butoxycarbo nyl-L-lysyl-L-

phenylalanyl-L-sery l-fi lycyl-L-rtorleucy l- ^ lycyl- L-pheήylalanyl »-Klycyl» -I, -prol · y · l ^ -tert .- ^ bu to>: yI / "gl · ^ fca ^

· Hexaaceta t «X> e cahy <lrafc

80988471-78-t-

ßAD ORIGINAL

- 55 - 100-2955

(BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr ^ Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys (BOC) -Phe- Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ hexaacetate decahydrate)

A. Azide method

1.05 g of nonapeptide hydrazide (partial sequence P1F2) are dissolved in a mixture of 50 ml of dimethylformamide and 1.2 ml of dioxane / HCl 2 N at -20 °. 0.116 ml of tert-butyl nitrite are added, the mixture is stirred for 10 minutes at -20 °, 1.4 ml of triethylamine and 5.1 g of tricosapeptide hexa'acetate (partial sequence ABCDE) and 5 ml of water are added and the mixture is stirred for 16 hours 0 °. It is evaporated to dryness and the residue is washed with diethyl ether, chloroform and acetone. The crude, protected dotriacontapeptide is thus obtained, which is dissolved in 100 ml of 0.5 N acetic acid, treated with 20 ml of Amberlite IRA-40 (acetate) and treated with 50 ml of 0.6 N ammonium hydroxide. To set the pH to 6.5 and the resulting solution coated on a carboxymethyl cellulose column (10 χ 100 cm) which is equilibrated with a 0.15 N ammonium acetate solution. The elution is carried out with an increasing concentration and pH gradient (0.15 N to 0.4 N; pH 6.5 to pH 7.0) of an ammonium acetate buffer. The combined fractions, which contain the pure peptide, are freeze-dried three times, the residue is washed with ethanol and then with diethyl ether and dried over potassium hydroxide in a high vacuum.

909884/1791 -

100-2953

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys (BOC) -Phe is obtained -Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ · 6 CH-COgH · 10 HgO, m.p. 220 ° (dec.), [A] * = - 49 ° in 1 N acetic acid. Aural acid composition after acid hydrolysis (6 N, 16 hours)

^Ala l, l ' ^kr * l.fi · ^ASP 3.9' ^{CyS / 2} l, 6> ^Glu i, 2 ' ^Gly 5.0' ^HiS l, l ' ^Leu 2, ₉ ' ^LyS l, l '" ^Nle i, 0 ' ^Phe 5,0' ^Pro 2, r ^Ser 5,8 ' ^Thr l, 9'

^Tyr l 0 * ^Val 0 9 ^ ^{Trp li0 by} spectrophotometry).

B. Activated Ester Method

1.0 g of BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH is dissolved in 10 ml of dimethylformamide, add 1.5 g of N-hydroxysuccinimide and 0.52 g of dicyclohexylcarbodiimide, stir for 6 hours at 25 °, filtered and evaporated the piltrate to dryness. The residue is washed with ethyl acetate and diethyl ether and dries. 1.2 g of BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OSu are obtained, Mp. 242 ° that / one in 10 ml of dimethylformamide

►dissolves, gives 3.1 g tricosapeptide hexa'acetate (partial

1 ■ *

sequence AESCdE), 0.4 ml of triethylamine and 1.2 g of N-hydroxysuccinimide and stirred for 16 hours at 25 °. It is evaporated to dryness and the residue is washed with diethyl ether, chloroform and acetone. How to get the crude protected dotriacontapeptide, which is dissolved in 100 ml of 0.3 N acetic acid, treated with 20 ml of Amberlit-IRA-410 (acetate) and gives 50 ml Add 0.6 N ammonium hydroxide. The pH is adjusted to 6.5 and the resulting solution is layered on a carboxymethyl

909884/179 1-

.- / Γ '; BADORfGlNAL

- 57 - - 100-2953

cellulose column (10 χ 100 cm) which has been equilibrated with a 0.15 N ammonium acetate buffer solution. The elution is carried out with an increasing concentration and pH gradient (0.15 N to 0.4 N; pH 6.5 to pH 7.0) of an ammonium acetate buffer. The combined fractions containing the pure peptide are freeze-dried three times, the residue washed with ethanol and then with diethyl ether and dried over potassium hydroxide in a high vacuum. BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His-Lys (BOC) -Phe is obtained -Ser-Gly-Nle-Gly-Phe-Gly-Pro-Glu (0TB) -Thr-Pro-NH ₂ · 6 CH CO ₃ · 10 HpO, m.p. 220 ° (dec.), [Α] £ = - 49 ° in 1 N acetic acid. _{Amino acid composition} after acid hydrolysis (6 N, 16 hours): Ala lfl, ArS ₁ ^ ₀ , As ₁ ^ ₉ , CyB / 2 _{1> 6} ,
₁ , Nle _{1> 0} ,

Tyr, _n , ^Val n η (^ P ¹ ^ ⁰ by spectrophotometry).

The BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val ~ Leu-OH used as the starting material is produced as follows:

a) L-threonyl-S-benzyl-L-cysteinyl-.L-valyl-L-leucine (H-Thr-

Cys (BzI) -VaI-Leu-OH)

572 g of H-Thr-Cys (BzI) -VaI-Leu-OMe · HBr (partial sequence PI, Example 11) in 1800 ml of methanol, add 900 ml of 2N sodium hydroxide solution, leave to stand at 25 ° for 1 hour, give 240 ml Add glacial acetic acid and leave to stand for 2 hours at 0 °. Then fil-

909884/1791

BAD ORlGtNAU

- 58 - 100-2953

if the precipitated crystalline mass is triturated, it is washed first with 1N acetic acid, then with water and dried at 50 ° in a high vacuum. The H-Thr-Cys (Bzl) -Val-Leu-OH, melting point 219 °, [α] ^ ^υ = -55 ° in 1 H ammonia is obtained.

b) tert-Butyloxycarbonyl-L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucine (BOC-Cys-Ser-Asn -Leu-Ser-Thr-Cys-Val-Leu-OH) Dissolve 18.4 g of BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-NHNHg (partial sequence F2, example 12) in 150 ml of dimethylformamide, cool to -20 °, 40 ml of a 2N solution of hydrochloric acid in dioxane and 15 ml of tert-butyl nitrite are added. After 10 minutes at -20 °, 28 ml of triethylamine and 16.2 g of H-Thr-Cys (Bzl) -Val-Leu-OH are added and the mixture is stirred for 16 hours at 25 °. It is then filtered, the solution is evaporated and the residue is washed through with 1N acetic acid. Mail receives BOC-Cys (BzI) -Ser-Asn-Leu-Ser-Thr-Cys (Bzl) -Val-Leu-OH, m.p. 217 °, ία] ρ = -27 ° in dimethylformamide.

The product obtained is dissolved in 500C ral dried ammonia, while stirring and boiling the ammonia, sodium metal is added until it turns deep blue; ammonium chloride is added for the purpose of decolorization. It is evaporated to dryness and the residue is washed through with 1N acetic acid and acetone. After drying, BOC-Cys- _! Ser-Asn »Leu» Ser-Thr-Cys-Val-Leu-OH, m.p. 248 ° (decomp.), Fa] ^ ⁰ = -41 ° In dimethyl-

'9Q988471791

- 59 - 100-2955

formamide / water (7: 5).

The ionapeptide obtained is dissolved in 5000 ml of 0.01 N ammonia up, add 1N hydrogen peroxide with stirring until the nitroprussiatreaction is negative, then another 200 ml glacial acetic acid,

filtered and lyophilized. This gives BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH, ^{m.p. 258 0} (decomp.), [A] ^ ° = -Ϊ8 ⁰ in
Diethylformamide / water (7: 5).

909884/1791

BATH ORIGINAL

Claims (1)

Patent claims: /IL-.J Process for the preparation of previously unknown polypeptide derivatives of the general formula (5-thio-2-X-propionyl) -L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl -L-valyl-L-leucyl-L-seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L -histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-LY-glycyl-L-phenylalanyl- W glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, where X is -H or -NH _? and Y stands for the L-norleucyl or L-methionyl radical, its therapeutically effective acid addition salts and heavy metal complexes, characterized in that the polypeptide derivatives of the above formula are prepared according to methods known per se from peptide chemistry and then optionally in a manner known per se in Their therapeutically effective acid addition salts are converted with organic or inorganic acids or into their heavy metal complexes. 2. Process for the preparation of a polypeptide derivative of the formula L-hemieystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L -alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L- 909884/1791 ORIGINAL - 61 -. 100-2955 lysyl-L-phenylalanyl-L-seryl-glycyl-L-methionyl-glycyl-L-phenylalanyl-glyeyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, its acid addition salts and heavy metal complexes, characterized in that the peptide derivative of the above Formula according to methods known per se from peptide chemistry and then optionally on per se known way in its therapeutically effective acid addition salts with organic or inorganic acids or heavy metals all complex transferred. 5. Process for the preparation of a polypeptide derivative of Formula ß-Mercaptopropionyl-L-seryl-L-asparaglnyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L-alanyl-L-tyrosyl-L -tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-norleucyl-glycyl -L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, its acid addition salts and heavy metal complexes, characterized in that the peptide derivative of the above Formula represents according to methods known per se from peptide chemistry and subsequently, if appropriate, to methods known per se Way into its therapeutically effective acid addition salts with organic or inorganic acids or into its own Heavy metal complexes transferred. 909884/1791 - 62 - 100-2955 k. Process for the preparation of a polypeptide derivative of the formula ß-mercaptopropionyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L-alanyl -L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl -L-methionyl-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, its acid addition salts and heavy metal complexes, characterized in that one .das peptide derivative of the above formula from peptide chemistry known methods and then optionally converted into its therapeutically effective acid addition salts with organic or inorganic acids or into its heavy metal complexes using methods known per se . 5. Process for the preparation of a polypeptide derivative of the formula L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-II-hemicystinyl-L-valyl-L-leucyl-L-seryl-L -alanyl- ™ L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L- seryl-glycyl-L-norleucyl-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, its acid addition salts and heavy metal complexes, characterized in that the peptide derivative of the above formula is derived from Peptide chemistry are methods known per se. 909884/1791 - 65 ~ .100-2955 and then, if necessary, to known ones Way into its therapeutically effective acid addition salts with organic or inorganic acids or into its own Heavy metal complexes transferred. 6. Process for the preparation of the polypeptide derivative of the formula L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hernicystinyl-L-valyl-L-leucyl-L-seryl-L -alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phonylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl -glycyl-Lrmethionyl-glycyl-L-phenylalanyi-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, characterized in that tert-butyloxycarbonyl-L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl- ' I-seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histldyl-£ -tert-butyloxycarbonyl -L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-methionyl-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide splits off the protecting groups. 7. Process for the preparation of a polypeptide derivative of the formula ß-Mercaptopropionyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L-al "anyl-L ~ fcyrosyl * -L-tryptophanyl-L-arginyl-L »-asparasinyl-L- 909884/1791 BATH ORIGINAL - 64 - 100-2953 leucyl-L-asparaginyl-L-asparaginyl-L-phGnylaianyl-L-histJclyl-L-lysyX-L-phenylalanyl-L-seryl-glycyl-L-norleucyl-glycyl- 't-phenylalanyl-glycyl-L-prolyl-L -glutamyl-L-threonyl-L-prolinamide / characterized in that, according to methods known per se, from S-ßenzyl-A-raGreaptopropionyl-L-seryl-L-asfiarasinyl. L-leucyl-Ii-seryi-L-threonyl-S-benayl-L-cysteinyl-L-valyl-L-leuein-hydrazide /M-lercaptopropipnyl--L-seryl-.L-aspa:raginyl~ L-leucyl- L-seryl-L "threonyl-L-hemicystinyl-L-valyl-L-leucine hydrazide and after conversion into the corresponding azide with L-seryl-L-alanyl-L-tyrocyl-Ij-tryptophcinyl-L-arginyl -L ~ asparaginyl-L-leücyl -L- acparaginyl-L-aspareiginyl-L- phenylalanyl-L »histidyl-L-lyeyi-L-phenylalanyl-L-seryl-glyeyl-L-norleucyl-glycyl" L-pheri3 ^r lalanyl ~ glycyl-L-prolyl-I / -gl \ itamyl ~ L-tlireonyl-L-proliniunide condensed. l] _t method for producing a polypeptide derivative of the form! P-Merccptopropionyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L ~ threonyJ -L-jieijiicyfitinyl-Lv-alyl-L-leucyl ^ L-rSeryJ -L-alanyl-L ~ tyroßyl "L-tryptoiilianyl-L-arginyl-L» a "iiparaginyl" L-leiicy3; -L-asparaginyl-L- asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-methionyl-glycyl-L-phenylalanyl-glyoyl-.L «prolyl-L-glutamyl-L» threonyl-L --proline » amidj characterized in that from -S-bensyl-ß-mercaptoprop! onyl «L-snr.v.l T ariiaraginyl-L-leucyl-L-neryl-L-threonyl- ' -ij-benzyl'-L-cystelnv] -L-valyl-L-leucan hydrazide according to per se 9 0 9884/1791 ■ · ■ *: * - :. λΝΟ c-, ti '- BATH - 65 - 100-29 ^ 3 known methods ß-mercaptopropionyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucine hydrazide and after conversion into the corresponding azide with L-seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L -histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-methionyl-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide condensed. 9. Process for the preparation of the polypeptide derivative of the formula L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L -alanyl- L-tyrosyl-L-tryptophanyl-L-arsinyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl- L-norleucyl-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, characterized in that tert-butyloxycarbonyl-L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-Ii -seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L- ^ asparaginyl-L-phenylalanyl-L-histidyl- £ -tert-butyloxycarbonyl -L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-norleucyl-glycyl-L-phenylalanylglycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide splits off the protecting groups. 909884/1791 BAD ORIGiNAU - 66 " 100-2955 10. Polypeptide derivatives of the general formula (3-Thlo «2-X-propionyl) -L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl- L-seryl-L-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L- phenylaianyl-L-seryl-glycyl-LY-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide, where X has the meaning -H or -NH ₂ and Y is the L -Norleucyl- or L-methionyl radical, -their therapeutically effective acid addition salts and heavy metal complexes. 11. L-hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L-alanyl-L-tyrosyl- L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycy-lL-methionyl- glycyl-L- , phenylalanyl-glycyl-L-prolyl-L-glutemyl-L-threonyl-L-prolinamide. 12.L-Hemicystinyl-L-seryl-L-asparaginyl-L-leucyl-L-seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl-L-alanyl-L-tyrosyl- L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl- ! .- asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-I, -lysyl-L-phenylalanyl-L-seryl-glycyl-L-norleucyl-glycyl- r.-phenylalanyl-glycyl-L-prolyl-L-glutamyl-L-threonyl-L-prolinamide. 90988Λ / 1791 - 67 - 100-2955 1>. p-mercaptopropionyl-L-seryl-L-asparaginyl-L-leucyl " ^ -seryl-L-threonyl-L-hemicystinyl-L-valyl-L-leucyl-L-seryl- Ii-alanyl-L-tyrosyl-L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparaginyl-L-phenylalanyl-L-histidyl-L-lysyl-L-phenylalanyl-L- seryl-glycyl-L-methionyl- β-glycyl-L-phenylalanyl-glycyl-L-prolyl-L-glutainyl-L-threonylprolinamide. lh, p-mercaptopropionyl-L-seryl-L-asparaginyl-L-leucyl «L-seryl-L-threonyl-L-hernicystinyl-L-valyl-L-leucyl-L-SGryl ™ L-alanyl-L-tyrosyl- L-tryptophanyl-L-arginyl-L-asparaginyl-L-leucyl-L-asparaginyl-L-asparasinyl-L-phenylalaiiyl-L-histidyl-L-lysyl-L-phenylalanyl-L-seryl-glycyl-L-norleucyllycyl- L-phenylalanyl-glycyl-L-prolyl-L-glufcainyl-L-threonyl- L-prolinamide. 15. Medicinal product containing a compound according to claims 10 to 14, Patent attorney '' / ι ΊΊ ^ '- <- Ί \ £ - ^ Js (Dr., γ. Scümied-Kowar ^ lk) "- ^ - 7 / 30988A / 179 BATH ORIGINAL