CA1182111A

CA1182111A - D-phenylalanyl-l-propyl-l-arginine aldehyde sulfate and process for the preparation thereof

Info

Publication number: CA1182111A
Application number: CA000393981A
Authority: CA
Inventors: Sandor Bajusz; Erzsebet Szell; Eva Barabas; Daniel Bagdy
Original assignee: Richter Gedeon Vegyeszeti Gyar RT
Current assignee: Richter Gedeon Vegyeszeti Gyar Nyrt
Priority date: 1981-01-13
Filing date: 1982-01-12
Publication date: 1985-02-05
Also published as: ES508637A0; JPH0251920B2; AU544492B2; AU7944682A; FI74024C; MX156383A; PT74268B; JPS57181046A; AT383352B; NO158021B; NO158021C; DE3200812C2; NL8200105A; SE453509B; FI74024B; GB2091270A; GB2091270B; PL133451B1; FR2497799B1; PT74268A

Abstract

A B S T R A C T O F T H E D I S C L O S U R E

The invention relates to D-phenylalanyl-L--prolyl-L-arginine aldehyde sulfate, highly stable in aqueous solution, and to a process for preparing it from D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate or NG-carboxy derivative con-taining an acid-sensitive protecting group at its amino terminal,wherein the acid sensitive amino terminal protecting group or optionally the NG-carboxy group is removed with 1 to 12 N sulfuric acid, applied in 1 to 12 equivalent amounts end the resulting free tripeptide aldehyde sulfate is isolated, The D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate of the invention possesses valuable anti-coagulant activity.

Description

- 2 _ The invention relate~ to D-phenylalanyl-L-_prolyl_L-arginine aldehyde sulfa~e, highly stable in aqueous ~olution, and to a process for the pre paration thereof.

IS ia known that heparin, pvlyanions of rele_ ted ~tructurs ~heparinoids/, and coumarin deriva-tives ara being mostly applied in anticoagulant therapy at present. 1~ ia a common feature of these agents that they fail to induce direct inh~bltion oF
~he proteolytio reaction, triggering blood clotting.
Haparin as a catalyst accelera~es the ~nhibitory action of one of the plasma inhibitor~, antlthrom_ bin III, on the enzymee of the coagulation prsce~s, primarily that of thrombin, while coumarin deriva_ ~ivas are inhiblting the biosynthesis of pro~eins containing ~ ca rboxy-glutamlc acid /Glaj moleties, Th~re ara fo~r pro~eins of this ~ype which are in-volved in the blood coagulation process, one of thsm ~elng prothrombin. Blood coagulation factors having no or le~s than the normal number of Gla residùe3, are inac~ive, and are not participating in the ~oagulation process. It should be noted,how-ever, that ~hi~ inhibition cov~ra the synthesis of the entire range oF Gla contalning proteina, ia e, one of the natural inhibitors of ~he coagulation~proce~, A2L~05-67 protein C /or fac~or XIVJ, i8 al80 ~ynthe~:Lzed in ~nactive ~orm in the pre~ence Qf coumarin deriva-tive~, which ie rather di~advantageou~. It is al~o a characterletie feature that heparln i~ admini6t-ered prim~rily in i. v. infusion, as it i~ practi-cal:Ly $nactive at oral applica~ion, whil0 coumari derivatives can only be given orally. ConsequentlyJ
the ef feot of heparln may bo regle~ered rapidly, wlthin a ehor~ period of tlme" whlle ~hat of coumarln derivatives - be:ir)g eynthe3i~ inhibitor~ _ only a f ~e r 24 ~ o 36 hou re, Furthermore it i~ known that there are tripeptide aldehydes whlch also exhibit 0nti coagulant activity; howav~r~ con~rary to the abova agants they enter in~o dlrect reac~lon with thrombln, inhibit~ng ~ts proteolytic reaction~ even in the abeence of antithrombin III. D-phenylalanyl-L_prolyl.
_I_arginine aldehyde aoetate, described in Hungarlan Patent 169J 870 a~ well ae D_phenylalanyl-L-prolyl--N~_carboxy-L-arginine aldehyde, described ln Belgian Patent ~80, 8~4 are bo~h potent thrombin inhibltors, It wae observed tha~ ~he an~i~hrombin potancy of ths 2bove syn~hetic arginine_pept~de aldehyde salt~ - eepecially that of compounds having a free terminal amino group - i. e~ D phenylalanyl-L~prolyl-` 4 -L-arginine aldehyde acetate and hydrochloride - is varying and rapidly decreasing upon standing in aqueous solution, making therapeutical application impossible. Though the NG-carboxy derivative of the :Eree tripeptide aldehydes, i.e. D-phenylalanyl-L-prolyl-NG-carboxy-L-arginine aldehyde is retaining its activity in aqueous buffer solution for 20 to 24 hours, aEter several days~
however, there is already a significant reduction in potency and after several months there is a loss of original activity even in solid form.
The invention relates to a new salt of D-phenylalanyl-L-prolyl-L-arginine aldehyde which in contrary to hitherto known products is stable also in aqueous solution, and to a process for the preparation thereof.
According to one aspect of the present invention there is provided a process for preparing D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate from D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate or from D-phenylalanyl-L-prolyl-NG-carboxy-L-arginine aldehyde containing an acid-cleavable protecting group at the amino-terminal thereof, which comprises removing the acid-cleavable amino terminal protecting group and, when present, the NG-carboxy group, with sulfuric acid, and isolating the resulting free tripeptide aldehyde sulfate.
According to another aspect of the present invention there is provided D-Phenylalanyl-l.-prolyl-L-arginine aldehyde sulEate whenever prepared by the above process or by an obvious chemical equivalent thereoE.
It was found that the stability of diverse saLts of D-phenylalanyl-L-prolyl-L-arginine aldehyde was varying in aqueous solution, i.e. isotonic salt solution, to a significant degree. In the course of our tests the peptides were dissolved in concentrations of 10 mg/ml, stored at 5C~ and the ensuing change in antithrombin activity registered for 180 days. The potency was assayed in a system containing the following components:

....

~ 5 ~

0,2 ml o~ 0,5 percent bovine fibr~nogen in a 009 percent solu~ion of sodium ohlorlde, Ool ml o~ tris/hydroxyme~hyl/-amino-methane hydrochlorlde - hydrechloric ~c~d buffer /pH 7.2/ contain~ng th~ peptide 501ut ion 0~1 ml o~ US Standard Human Thrombin ~NIH, Be~hesda~
Maryland, USA/, lO Uni~/ml 501utlon.
The thrombin time of tha paptide-free eystam i~
15 5, mea6ured in the "Schnither-Gross Coagulo-meter".

The ac~ivi~y o~ the tripep~ide aldehyde eolu~
tion was arbitrarily set up 38 100~ if ~he reaction mix~ure induced a FiveFold relative thrombin time a~
a final oonoentration of 3,5 x lO 7 M tin the oaee of the tripep~ide aldehyde ~ulfate at D.175 ~g~ml~.

The test data are summarized in Table I, It is app~rent that whil~ the activity of the corr~sponding hydrochloride in isotonic ealt solution is starting to deorease after 5 days~ and that of ~he acetate, citrate~ .tartra~e and ~osyla~e already after several day~ /~imilarly to ~he N~-carboxy deriva~ive of the fre~ tripeptide aldehyde having rela~ed properties/, the D, phenylalanyl-L_prolyll-argininc aldehyde sul-fa~e i~ retaining lts anti~hrombin activi~y for 90 -- 6 ~

day3, In prolonged s~ability t rials D ph0nylalanyl_ -L-prolyl-L~arg~nine ~31dehyde sulfate proved to be table even ~or 180 daye :in aqueou~ medium, and in solid form it simil~rly fa~led to loose aotivlty for 6 months.

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In order to slmulate physiological condition~
the antithrombin ac~ivity o~ ~he ~ripepLide aldehyde ~al~s as w911 a~ that of th~ carbamic acid deriva-tive8 wa~ tes~ed al~o on human pla3ma in the following sy9t~m:
O .2 ml of human cit rate plaeme, 0~1 ml~ tris~hydroxyme~hyl~amlno-methane hydro-chloride _ hydrochloric acid buffsr ~olu tion /pH 7~2/ containing the peptide 501ut ionO and 0.1 ml~ US S~andard Human Thrombin /NIH, Bethesda Maryland, USA/, 10 Uni~s/ml solu~on.
The thrombin tlme of the peptide_ Free ~y6tem is 15 e, mca ured in the "Schnlther-Groe~ Coagulo-me~er", ~ .

- L0 _ Table 2 Antithrombin a~ti~ity of trip~ptiide aLdeh~de deri~ati~es in ~uman pLas~a ~mount of peptide (/~/~L+/ required ReLati~e to increase throm- acti~ity Peptide aLdehyde bin -time t~ofoLd immecliately fol-lowing dissoLu-tion of the pep-tide .. ... . . . ...
10 D-Phe-Pro-Arg-H.I12S04 0.020 L00 D-Phe-Pro-A~g(COOH)-H 0.042 L~
D-Phc-Pro-Arg-H~2 CH3COOH 0.06~ - o.L40 3~-lL~
D-Phe-Pro-Arg-II.2 HCL o.o60 - O.L30 33-1 Heparina O~L0~ L9 1~ aVaLue mcasured with commerciaL heparin (L32.2 U/mg, U~ S~ Pha XVII).

.~mount of the peptide in the reaction mixtureO

The data of Table 2 cLearLy demonstrate that the amount of peptLde .reclllired to aoh,ieve a t~ofoLd thrombin time inc.rease compared to the controL :is ~aryin~ aocording to the ba.tch used in the case of the acetate and the hydrochl.oride salts 9 and is a manifoLd - in the case of the carbamic acid deri~ati~e twofold 2~ _ of the amount required from the tripeptide aLdehyd~
sulfate.
'~

Tha in ViVQ trial oF the tripaptide aldehyde derivatives i~ ~ummar~zed in Table 3, D~Phenyl-alanyl-L~prolyl-L~arglnin0 aldahyde sulfate ha~ a signiflc~nt antithrombin potency in vivo, At lntra-venous ~nd ~ubcutaneous applica~ion its eff~cacy i~
in the ranga of ~hat of heparin, generally applied in ~herapy, howev0r, it has ma~or advantages com-pared ~o it, Whlla haparirl, given orally, i~ in-active, therapeutic effect may be ach~eved with oral dsses of 25 mg/kg of the ~ripeptlde aldehyde ~ulfate /but only with 50 mg/kg doses of the carbamic acid derivative/.

- 12 _ Table 3 In vivo tr~als Dose required for therapeueic effecta mg/kg/hour mg/kg Peptid~ aldehyde ~,v, infusion s~ c. p~ o.
rabblt dog rubbit doy rabbit dog D~Phe_Pro-Arg_H. ~ 100 100 o2 GH3COOH
I;)_Phe_Pro_Arg_H . ~ 100 .2 HCl D Pha~Pro-Arg/COO~/-H _ 3.0 10.0 6.0 50 50 D-Phe-Pro-Arg-H. 1.0 005 6.0 6,.0 25 25 ~I H2SOq, Heparln O

a The therapeutic effect is oharacterized by the dose required to prolong thrombin tlm0 in the whole b~ood 1,5 to 2.5fold ~ ies, A~ S~1978/
in ~ ~Melmon, K. L~ and Morrelli~ Fo F, Eds.~ ~nd Ed. pp~ 303 to 306, Macmillan Publ. Co~ Ino~ New York; and Verera~teO M.and Verwilghen, R./19BO/ in ~
Treatment Prlnci~lee and Practice of Clinical 2nd Ed,, Avery G, S, Ed. /1980/ pp~ 88g ~o 952, Edinburgh and London~.

.. . .. . .... . .

_ 13 ~

The toxiclty data of D~phonylalanyl-L-prolyl-~L~arglnine aldehyde sulfate are also more favour-able than those of either the ace~ate or the carba-mic acid deriva~ive. The acu~e toxicity da~a are summarized in Table 4; ~his amounted ~n ~he ca~e of the tripeptide aldehyde sulfate at oral admini3tra-tion to 2 g/kg~

Table 4 Acute toxicity data in mice LD50 mg/kg Peptide aldehyde i.v. i,p, 60C. p~Oo bolus _--D-Phe Pro-Arg-H ,2CH3G00~1 9 38 960 D_Phe_Pro Arg/COOH/ Ha ~ - 1200 D~Phe-Pro-Arg-lt~H2S04 45230 1800>2000 Hepa rin no literature dara available .
.. ~.. . ___ __~
a Due to poor solubility the toxicity data obt&ined at applications other than p. o, are rather uncertain, b ~t in~ravenous infusion the LD50 amounted to 58 mg/kg in the rabblt~

Coneiderlng the low ~oxicity and high potency of D-phenylalanyl-L-prolyl~L-arginine aldehyde ~ulfa~e, .. .. . . . ... .. . . .. . ... .

the therapeu~ic index, including both, and belng the most character.tstlc ind~ca~or of the therapeutical value of a drug, is more favourable than ~S9 ef the other tr~paptide aldeh yd9 derivatives, On the b~sis of intravensus infu~ion trials in dogs the doee of the human intravenous infus~on w~
~stabli~hed a~l~2mg~kg/hour.

It wa~ found tha~ D phenylalanyl~L-prolyl-L~
arginine aldshyde sul~a~e may be prepared by a ~ethod known per se, by ~ubmi~tlng benzyloxycarbonyl-_~-phenylalanyl-L~prolyl_NG.benzyloxycarbonyl~L_ -arg1nine aldehyde /~_D_Phe_Pro_Arg/Z/_H/ to hydro-genoly~i~ .in the prcsence o~ equivalen~ amoun~s of sulfuric acidO Fur~hermere it may be simply prepare~
in sufficient purity from its acid sensitive deri-vativ~ c~ntaining triphenylmethyl or t-alkyloxycar-bonyl pro~ective ~roup~ i~ c. from t bu~yloxycarbo-nyl D_phenylalanyl-L-prolyl-L-arginine aldehyde he ml-~ulfate or t-butyloxycarbonyl~D-phenylalanyl-L~pro-lyl_NG-carbcxy-L-arginine aldehyde with 1 ~o 12 N
sulfuric acid, At the same time tho following method~, known per 8e, failed to furnish satisfactory result~:

.. . . .. ..

a~ AcldoLysis of the t-b-utyLoxycarhonyL
group with suLfuric acid dissoL~ed i.n acetic acid CB0yel~an et aL,: in Pepticles, L970 (Ed,: H. Nes~adba), p. L38., North HoLLand, Amsterdam~ L~73].

b. Dj.rect con~ex~ion of the D-Phe-Pro-Arg(COOH)--H carbamic acid derivative of Belgian Patent 880,844 with suLfuric acid into the un-LO protected tripeptide aldehyde qulfate.

c. Trans~ormation o~ dî~er~e other unprotected tripaptide aldehyde saLts, i. e. of D-phenyl-aLanyL L-prolyl L-arginine aldehyde acetate o~ Hungarian Patent L6J,8~o into its 3uLfate either with an ion-exohanga r~sin or ~ith suL~uric aclcl~

The products obtained with.either o~ the a-c methocls p.ro~ed to be of poor homogeneLty and/or stabLLlty in aqueous solutLon.
Ba~ed on the above the in~ention relates to D-phenyLalanyl-L-prolyL-L arginine aLdehyde suLfate and to a proces~ for preparing it fro~ D-phenyLaLanyL-L--proLyL-L~arginine aldeh~de hemisulfate or D-phanyl-aLanyl-L-prolyl~N -carboxy-L-arginine aldeh~de, oontain-ing an acid ~ensiti~

_ 16 ~

protecting group at its amino $erminal, i.n uhich the ~cid sensitlve amlne terminal protecting group or optionally ~he NG~carboxy group :~9 removed with 1 to 12 N ~ulfurlo acid, applied ln 1 to 12 equivalent amounte, ~nd the resulting free ~ripep~ide aldehyde ~ulfa~e isolated~

Accordlng ~o a preferred process of the in-v~nt~on the L-arg;Lnine lactam, protec~ed at i~e gua-nîdino group with a benzyloxycarbonyl group, ls condensed with t-bu~yloxycarbonyl-D~phsnylalanyl-L--proline, ~h~ rssul~ing blocked tr~peptide lactam reduced, and the benzyloxycarbonyl group at ~ha guanidino group of the protected tripeptide aldehyde obta Lned~ submitted to hydroganelysie in e~h~nol or tetrahydrofuran containing 30 to 40 percent of wa~er, in the presance of an e4uivalen~ amoun~ of sulfur Lc acid. The resulting tripeptide aldehyde hemisulfate, still protected at i~s amino terminalO is dissolved ln 8 to 12 equlvalents, prefQrably 10 equiv~lents of 4 to 6 N, prefarably 5 N`sulfuric acid, and heated for 20 to 40 mLnutes, preferably 30 m:Lnutes ~o 40 to 60C, preferably to 50CO ~he solution is sub-Requen~ly neutrallzed with calcium carbonate, ~iltered, and preferably freeze-dried.

_ 17 --The product prepa red in ~hi~ way rnay eventu-ally contain 4 to 6 percen~ of calclum sul~ate, which howaver doe~ not affeet either its biological acti~
vity or its therapeutic applica~ion.

The invention i~ further illustrated by but not limited to the follew~ng Examples~

The RF valu2~ in the Examples are de~ermined by silica gel thin~layer chromatography /Kieselgel G, Reanal, Budape~t/ in ~h~ ~ollow~ng systemso 1. Ethyl ace~ate-pyridine-acetic acld-wa$er _ 480:2~
Ethyl acetate_pyridine_acetic acid~water -60:20:6:11

- 3, Ethyl acetate_pyridine~acetic ac~d-water 30:~0:6:11.

. ,. _ ... . ........... .... ..

Example 1 __ D_Phenylalanyl_L_prolyl_L_arginlne aldehyde ~ulfate ~ OButyloxycarbonyl_D_phenylalanyl_L-prolyl-L-_arginine aldehyde hemi ulfa~c /2.74 g, 5 mmoles/
is di3~01ved in water /5 ml/, 10 N ~ulfuric acid /5 ml~ added at constant stirring, and the mixture heated to 50C, The ~olution is s~irred for 15 minutes at 50C, then dllu~sd wi~h ice wa~er /25 ml/, and its pH adiu~ted to 6.5 with calcium carbonate /about 2,25 g~ at icc cool~ng. The precipit3ted calcium sulfate ls filtered, and wa~hed twice wi~h water J5 ml/. The filtrate ls extracted ~wice with N butanal /10 ~1/, conc~ntrated to abou~
30 n~ ered, if necessary, and f ree;ze dried, Yield 2,25 9 /79 percent/ o~ the title product, con_ ~aining 4~8 percen~ of calcium sulfate, RF - .,35 t o O ,40 ~o~20 w _117+1 Jc a 1~ water/.
Anfllyei3 calculated for C20H300;~N6.H2S04, 2 CaS04 /565.85/;
Calculatcd: C 42.45, H 6,77, N 14.85, S04 20~37 Ca 1,41, H20 9~55 percent.
Found: C: 42~2, H 6~9, N 14,85, S04 19~8, Ca 1~3, H20 9,75 perc~ntO

~ 19 ~ , The starting material~ are synthe~:5zed accor-ding to the following procedure:

~: t-Bu~yloxycarbonyl~D-phenylalanyl_L pro~
lyl~N ~ben~yloxyca rbonyl ~. L_a rg in:lne lactam t~Butyloxycarbonyl-NG-benzyloxycarbonyl_L_ ~arginlne lactam /8.6 9~ 22 mmoles, Belgian Patent 880~ 844~ is suspended in ethyl acetate /2û ml/, and at 5C and constant stirring a solu~ion of 4M hydro-chloric acid in ethyl acetate ~40 ml/ added to it.
The reac~ion mix~ure is ~tirred For 30 minutes under ice cooling t diluted with cool ethyl aceta~ /100 ml/~
the precipitate formed filtered, washed with ethyl acetate, and drled at reduced pressure in an ex~iccator over potassium hydroxid~, The re~ulting NG_benzyloxyc~rbonyl-L-arginine l~ctam hydrochloride is di~solved in dimethylfsrmamide ~20 ml/, and at ~10C trlethylamin~ /6~2 ml, 44 mmol~s/ added ~o it, The suspeneion Formed is added to the following mixed ~nhydrids, t~Butyloxycarbonyl-D-phenylalanyl-L~proline ~U9 Ludascher and R. Schwyzer: Helv Chim. Acta 55, 2052 ~ 2/J /7,25 90 20 mmoles/ and N-methyl-mor-phollne /2.22 ml, 20 mmole~/ are di~Rolved in di-methylformamide /20 ml/. The solu~ion is cooled to ~ 20 --L5 C, chLorofo~mic acid isobutyl. ester (2.64 mL, 20 mmoLes is added to it at stirxing and then after 5 min~tes the abo~e 301ution in dimethylfor~amide is added. The stirring is continued fvr L houx at -1~ C, and ~or 1 hour at 0 ~. 3 then the xeaction mi~ture is diluted with 'benzene (30 mL), the pxeoipitated ~alts are fiLtsred and washed twice with benzena (10 ml).
The solution of ben~ene-dimethyLfo~lamide is diluted with water (50 mL) and the phases are separated, The aqueou~ Layer i9 e~tracted twice with benzene (10 ml) 7 then the com'bined benzene e~tracts are washed three times with a solution of 10 percent sodium car'bonate (30 ml), water (30 ml), three times with 0.5 N suLfuric aoid (30 ml), twice with water (30 ml), and the so'lution e~aporated at reduced pressure following dryin~ o~er an.hydxous ~odium ~uLfate. The e~aporation residue is homogenized with petxoleum ether, fiLtered, waqhed with petroleum ether and air-dried. Yield: 9,65 g (76 pexcent) of the title product.
RF = 0.81 to o.89.

.~

21 ~

~_ t-Butyloxycarbonyl_D_phenylalanyl_L_prolyl_ -NG-benzyloxycarbonyl-L-argirline aldehyde The tripeptide lactam /9,52 9, 15 m~olos, Step 1 / i3 di~solved in te~rahydrofuran /45 ml/, and at -20C and vigorous stirring lithium aluminium hyd-ride /11~25 mmolee/ added to lt in tetrahydrofuran /about 28 ml of a 0.4 M solution/~ The prooeeding of the reductian iB cont relled by ~hin_layer chromatog-raphy ~ 1 ~ 0,71 ~o 0.77 /lactam/ and Rl ~ 0.31 to 0.3~ /aldehyds/~. If necessary, a further portion of the hydride solution i5 addcd, When the reactlon i5 concluded the te~rahydrofuran solution is cautiouely acidified with 0,5 N sulflric acid to pH 3, then dl-luted in such a way that no precipltation occurs jabout 100 ml/. The aqueous tetrahydrofuran solu~ion i8 extracted three time5 with methylene chloride /75 ml/, and the combined methylene chloride extracts washed three time~ with a solutlon of 10 percent ~odium carbonate /10 ml/, than twice with water ~10 ml/. The methylena chloride ~olution ~s ~ub-sequently dried ovor flnhydrous sodlum sulfate and evapora~ed at reduced pressure~ The evaporation rg5idue i8 di~solved in benzene /50 ml/ and the solution repeatedly evaporated at reduced pr~sure.
Then the dissolving and ev~porating i~ repe~ted onc~ more~ The evaporat~on resldue is worked up with .. ..

; - ~2 ~

ether, filtered, washed with diethyl ethar and air-_driedO Yield: 6,9 9 /72 peroent/ of ~he tltle preduct, RF = 0-3 to 094~

~_; t-Butyloxycarbonyl_D-phenylalanyl-L prolyl-;L.arginine aldehyde hemisul~ate Protsoted tr~peptide aldehyde /6.4 g, 10 mmoles, Step 2 / is dissolved in a mixture of wa~er /50 ml~, tetrahydrofuran /50 ml/ and 1 N sulfurlc acid /10 ml/ and eu~mitted to hydrogenolysis in the presence of a 10 percen~ palladium charcoal catalyst /1 9/. The proceeding of the reaction is controll0d by thln-layer chromatography /R~ a 0,95 ~o loO
~ripeptide aldehyde protected at it8 guanidino group/
and R2 3 0.45 to 0.54 /unprotec~ed tripeptide aldehyde/~O
After the reaction is concludsd the catalys~ i8 filtaredr washed wi~h a 50 p~rcent aqUeQus tatrahydro-f~ran solution /30 ml/, and the combined filtrates concentratsd at reduced pre~sure to about 60 ml, The re~idue is ex~rac~ed four t~me~ wlth n-butanol, The n-.butanol layers are combined and evaporatad flt ro-duced pressure to drynes~. The evaporation re3idue is worked up with a mlxture of dlethyl ether _ diiso-propyl ather /1:1/, filtered, washed with the above ; 23 -mix~ure~ then dried at reduced prsssure in an ex~iccator. Yield: 4.4 g j80 percent/ of the ~itle compound.
RZ = 0~45 to 0.54.
L~ 203 _65+1 / c ~ 1, wa~er/.
Analysi~ calculated for C~5H3805N6.0,5 H2S04 /5~1.64/:
Calcula~ed: C ~,43, H 7,13t N 15.23, S04 8.71 percent.
Found: C 54~5 , H 7.3 ? N 15.2 , S04 807 p~rcent.

Example 2 __ D-Phenylal~nyl-L~prolyl~L-a rginine aldehyde sulfate t-Butylsxycarbonyl-D-phenylalanyl_L-prolyl--NG~c~rboxy-L-arginine aldehyde /2,~5 9, 5 mmol~/ i3 diesolvad in wa~er /5 ml/ 3 10 N sulfurîc acid /5 ml/
added to it and heated to 50C, The solu~ion i9 s~irred for 15 m~nutes at 50C, then diluted with ice water /25 ml~, and its pH ad~usted to 6.5 with solid calcium hydroxide /about 1.6 grams/ under ~ce cooling~ The precipit~ed calcium sul~ate i~ fil~ered and washed twice with water /5 ml/. The ~iltrate is axtracted twlce with n butanol /10 ml/, concentrated at reduced pressure to about 30 ml~ ~iltered, if neceR~ary, and ~hen ~reeze-dried. Yield: 2,3 9 /81 percent/ of the title compound, contalning 4.9 per-cent of calclum sulfate.

-- 2~ --R3 = 0~35 to 0.40~, 20 ~ ~117~1 /c ~ 1, Ylater/O

Th~ startin~ material i~ prepared according ~o the following procedure:

t_Butyloxycarbonyl_D-phenylalanyl_L_prolyl_ _NG_benzyloxycarbonyl~L-arginine al~ehyde /6.4 g, 10 mmoles, Example 1,, Step 2 ~ is dissolved ln 75 percent aqueou~ ethanol /100 ml/, and submitted to hydrogenolysis in the presence of a 10 percent palla-dium charcoal catalyst ~1 g/0 The progre~s of the reaction is con~rolled by thin layer chro~atography ~R~ = 0~90 to 0.95 ~protect0d tripeptide aldehyde~
and 0045 to 0055 JNG_carboxy derivative/~, At the end of the reaction the catalyst is filtered~ washed wi~h water /30 ml/, ~nd the filtrate concentrated to 30_~0 ml 'at reduced pre~sure, The residue i~ diluted with wa~er /100 ml/~ ext racted twice with methylene chloride /20 ~1/, and freeze dried. Yiald: 5,1 g /85 percent/ of the title product, ~F a 0~45 to 0.55 Amino..acid analysis: Phe ~ 0~96 Pro 3 1 /reference amino acid/~ M. w. accordin3 to thc amino acid ana-ly~is: S70.

~ 25 -Example 3 ___ Preparation of a pharmaceutlcal composition The 2-ampoule preparation suitable for 6 or 12 hour intravenous infusion is prapared accordlng to thc followin~:

D~Phenylalanyl L_prolyl._L-arginine alçlehyde sulfate /420-840 mg/ and human albumin /40-80 mg/
~re ~ubmitted to joint freeze-drying~ The contents of the freeze dried ampoule are dissolv~d prior to use ~n ~erlle, germ~fr~ iso~onio ~alt solution 200 ~1).

.. . .... .

Claims

The EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate from D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate or from D-phenylalanyl-L-prolyl-NG-carboxy-L-arginine aldehyde containing an acid-cleavable protecting group at the amino-terminal thereof, which comprises removing the acid-cleavable amino terminal protecting group and, when present, the NG-carboxy group, with sulfuric acid, and isolating the resulting free tripeptide aldehyde sulfate.

2. A process according to claim 1 wherein 8 to 12 equivalents of 4 to 6N sulfuric acid are used.

3. A process according to claim 2 wherein 10 equivalents of 5N
sulfuric acid are used.

4. A process according to claim 1, wherein the acid-cleavable protecting group in the starting material is a t-alkoxycarbonyl group.

5. A process according to claim 2 wherein the acid-cleavable protect-ing group in the starting material is a t-alkoxycarbonyl group.

6. A process according to claim 3 wherein the acid-cleavable protect-group in the starting material is a t-alkoxycarbonyl group.

7. A process according to claim 1 wherein the acid-cleavable protect-ing group in the starting material is a t-butoxycarbonyl group.

8. A process according to claim 2 wherein the acid-cleavable protect-ing group in the starting material is a t-butoxycarbonyl group.

9. A process according to claim 3 wherein the acid-cleavable protect-ing group in the starting material is a t-butoxycarbonyl group.

10. The compound D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate whenever prepared by a process according to claim 1, 2 or 3, or by an obvious chemical equivalent thereof.

11. The compound D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate whenever prepared by a process according to claim 4, 5 or 6, or by an obvious chemical equivalent thereof.

12. The compound D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate whenever prepared by a process according to claim 7, 8 or 9, or by an obvious chemical equivalent thereof.

13. A process for preparing D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate which comprises reacting t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate with 10 equivalents of 5N sulfuric acid and isolating the D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate so formed.

14. A process for preparing D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate which comprises reacting t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-carboxy-L-arginine aldehyde with 10N sulphuric acid and isolating the D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate so formed.

15. A process according to claim 13 wherein the t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate is obtained by hydro-genolysis of t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde in the presence of a palladium charcoal catalyst.

16. A process according to claim 15 wherein the t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde is obtained by reducing t-Butyloxycarbonyl-D-phenylalanyl-L-proly-NG-benzyloxycarbonyl-L-arginine lactam with lithium aluminium hydride.

17. A process according to claim 16 wherein the t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine lactam is obtained by reacting t-Butyloxycarbonyl-NG-benzyloxycarbonyl-L-arginine lactam with hydrogen chloride and reacting the hydrochloride salt so obtained with t-Butyloxycarbonyl-D-phenylalanyl-L-proline.

18. A process according to claim 14 wherein the t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate is obtained by hydro-genolysis of t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde in the presence of a palladium charcoal catalyst.

19. A process according to claim 18 wherein the t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde is obtained by reducing t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine lactam with lithium aluminium hydride.

20. A process according to claim 19 wherein the t-Butyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine lactam is obtained by reacting t-Butyloxycarbonyl-NG-benzyloxycarbonyl-L-arginine lactam with hydrogen chloride and reacting the hydrochloride salt so obtained with t-Butyloxycarbonyl-D-phenylalanyl-L-proline.

21. The compound D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate whenever prepared by a process according to claim 13, 14 or 15, or by an obvious chemical equivalent thereof.

22. The compound D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate whenever prepared by a process according to claim 16, 17 or 18, or by an obvious chemical equivalent thereof.

23. The compound D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate whenever prepared by a process according to claim 19 or 20, or by an obvious chemical equivalent thereof.