Journul of illcdicinal Chemistry, 1 N O , Vol. 13, -Yo. S 347
4-~~~C.\llBOX.~~~l~O-8-.~l~~Irl.Ih'C-V,ISOPIZLSSIN
PH.IKbI.\COLOGIC.~L
Vasopressin compd
PO'TEh'CILS'
OF
Tau~13I AXALOGS O F ARGININE-VASOPRESSIN .\Nu LYSINE-VASOPRESSIN
Antidiuretic (A) (rat)
Pressor (U) (rat)
Vasodepressor (fowl)
Oxytocic (rat)
Ratio
A :B
-20 1 8-Argiriirie-vasopressinb -400 -400 -60 l-Deamino-8-arginine-vasopressinc 1300 i 200 370 i 30 150 i 4 27 f 4 3.5 4-Decarboxamido-8-arginine-vasopreasiii -25 ao -760 -38 -5 l-Deamino-4-decarboxamido-8-arginine1020 i 67d 10.7 =k 1 . 3 3 . 3 f 0.4 vasopressin 9.9 i0.7 93 240 f 13 7.3 i0.2 8-Ly sine-vasopreasine 50 f 4 1 266 i 18 126 i 2 301 i 11 1-Deamino-8-lysine-vasopressin' 61 f 2 12 f 0 . 5 2.4 1 3 . 1 =k 0 . 7 1.54 i 0.10 707 i 107 4-Decarboxamido-8-lysine-vasopressine 10.2 i 0 . 6 70 l-Deamino-4-decarboxamido-8-lysine729 i 26 3.5 i0.2 vasopressine 12.6 i 0.6 1 . 5 1 i 0 . 0 5 200 Expressed in unit's per milligram as mean potencies f standard error. * R. A. Boissonnas, S. Guttmann, B. Berde, a,nd H. Konzett, Experientiu, 17, 377 (1961). It. L. Huguenin, E. St#iirmer,R. A. Boissonnas, and B. Berde, ibicl., 21, 68 (1963). d The antidiuretic action is considerably more prolonged than that of the USP posterior pituitary reference standard. e See 1). Gillessen and Y. du VigIt. D. Kimbrough, Jr., W. L). Cash, L. -4. Branda, W. Y. Chan, and V. du Vigneaud, J . Biol. Chem., 238, 1411 (1963). neaud.3
'
replacement of the carboxamide group of glutamine by H leads to an analog exhibiting the various activities characteristic of the hormone, although the profile of the activities is changed. The most striking change in going from arginine-vasopressin to the 4-decarboxamido analog is the twofold increase in antidiuretic activity accompanied by a tenfold decrease in pressor activity, leading to an antidiuretic pressor ratio of 20. The corresponding ratio for 4-decarboxamido-8-lysine-vasopressin is '70. The parent hormones possess a ratio of approximatelg 1. The carboxamido group of glutamine obviously plays an important role in the degree of pressor activity, whereas in the instances mentioned its absence enhances the antidiuretic activity. The replacement of the amino group 011 the halfcystine residue at position 1 of 4-decarboxamido-8arginine-vasopressin with H results in a further substantial increase in antidiuretic potency and a further decrease, approximately 3.5-fold, in the pressor potency, so that the ratio of antidiuretic to pressor activity has now become 95 for 1-deamino-4-decarboxamido-arginine-vasopressin in comparison to 20 for 4-decarboxamido-8-arginine-vasopressin. It mill be noted in contrast that a similar modification of 4-decarboxamido8-lysine-vasopressin did not lead to an increase in antidiuretic potency. However, there was a threefold decrease in pressor potency, so that the antidiuretic:pressor ratio of l-deamino-4-decarboxamido-8-lysine-vasopressin is 200. With their very high antidiuretic and their very low pressor and oxytocic potencies, both 1-deamino-4-decarboxamido-arginine-vasopressin and l-deamino-4decarboxamido-lysine-vasopressin offer attractive possibilities for clinical application as antidiuretic agents.
I
s, S
1
3
2
1
0
II
6
0
1 c=o
I NH
5
CHZ-CH-NH-CI
I
C=O CH,-N
I
I
I
0
CONH,
\ CH-CC-NH-CH ' II /
CH,--CH,
0
8
I
II
9
-C-NH-CH,-CONH,
CH,
I
CH2CHzNH-C-NH,
ll
NH Figure 1.-Structure of X-arginine-vasopressin. Numbers indicate the position of the individual amino acid residues.
portions of EteO. Aft,er being dried in vucuo over NaOH and CaC12 overnight, the hydrobromide was dissolved in 120 ml of dry RleOH and passed through a column of Iiexyii IiGl (OH) (Fisher). The column was washed with 350 ml of bleOH and, after removal of the solvent from the comhiiied eluate and waahing, the residue was dried for several hours in vacuo and then dissolved in 11 ml of IIMF. To this solutioii 2.2 g of p-nitrophenyl S-benzvloxycarbonyl-ol-amiriobutyratez was added at 5". The reaction mixture partially solidified after it was allowed to stand for 2 days a t room temperature. EtOAc, 50 ml, and EtOH, 10 ml, were added and the resulting gel was kept for 1 hr at, 2" before it was filtered off and then washed with a total of 150 ml of EtOAc and 120 ml of EtOH. After being dried over PtOj in oacuo the product weighed 4.4 g; m p 196-199" (sintering a t 189"), [ a I z 3 D -36.6" ( C 1, DRIF). Anal. (C&aNiiO&) C, H, N. N-Benzyloxycarbonylphenylalanyl- a-aminobutyrylasparaginylS-benzylcysteinylpropyl-SG-tosylarginylglycinamide.-The proExperimental Section15 tected hexapeptide described iii the preceding section (4.0 g) was S-Benzyloxycarbonyl-~-aminobutyrylasparaginyl-~-benzyl- dissolved in 10 ml of glacial AcOH and treated with 10 ml of HBr in glacial AcOH (407,, w/w) by the procedures described cysteinylprolyl SG tosylarginylg1ycinamide.-S - Benzyloxycarin the preceding section. The hydrobromide was isolated, dried, bonylabparagiiiyl-S- benzylcysteinylprolyl-LYG-tosylarginylglyciiidissolved in 80 ml of dry MeOK, and passed through Rexyn RG1 amide4 (3.6 g ) was dissolved in 20 ml of glacial AcOH, and 20 ml (OH), and the column was washed with 150 ml of RleOH. of a solution of HBr in glacial AcOH (4092, w/w) wm added. The residue obtained after evaporation of the RIeOH from the The resulting precipitate dissolved during the next 15 min. eluate and washing wad dried in vucuo, dissolved in 8 ml of DMF, After 1.5 hr a t room temperature the solution was poured into and t'hen treated with p-nitrophenyl ill-benzyloxycarbonyl400 ml of dry EtzO. The precipitated hydrobromide of the free phenylala1iinate~~,~7 (1.7 g ) at 0'. After 3 days at room temperapentapeptide was washed by decantation with three 150-ml
- -
(15) All melting points are corrected capillary melting points. Where analyses are indicated only by symbols of t h e elements, analytical results obtained for t h e elements ivere within &0.4% of t h e theoretical values.
(16) 11. Goodman, and I(. C. Stueben, J . Amer. Chem. Soc., 81, 3980 (195Y). (17) 11.Bodanscky, and V. du Vigneaud, ibid., 81, 6072 (1959).
A >ample was hydrolyzed iri 6 'Y HC1 a t 110' for 22 hr and theri tem on a Beckmati-Spitico amino acid analyzed in the ;iO-30° analyzer. The follow4rig molar ratios of amino acids arid NH3 were obtained with the value of glycine taken as 1.0: aspartic acid 1.0, proline 1.0, glycine 1.0, a-aminobutyric acid 1.0, tyrosine 0.9, phenylalaiiirie 1.0, arginine 0.9, and NH3 2.0. In addition, cystine (0.4) and the mixed disulfide of cysteine and P-mercaptopropionic acid (0.6) were present. Thebe two sulfur compounds account for the half-cystine residue i n this analog.21
Acknowledgments.-The aut'hors \\.ish to t'hank t'he following people for their assist'ance: Mr. Joseph Albert for the elemental microanalyses, A h . Roger Sebbane for the amino acid analyses, and JIrs. AIaxiIle Goldberg, L\Irs. Frances Richman, arid Miss lfargitt'a lvahrenburg for the biological assays u d e r t'he direction of Dr. W. Y. Chan.
Synthesis of Analogs of the C-Terminal Octapeptide of Cholecystokinin-Pancreozymin. Structure-Activity Relationship JOSIPl'LUS?EC, JOHX T. S H E E H A X , EMILY E'. SABO, S I N A OCT4VIA.U ICOCY, A N D AIIGUEL A. O N D E T T I Syutbb Inatilulc for X d i c a l Rtsearch,
.\-tu:
\.T71LLIAI\IS,
B r u n s w x k , -1cw Jcrscu
0800.;
Rtcciatd Decrmbcr 5 , 1069 A iiiiniber (if arialogs of the C-termiiial octapeptide of cholecystokiniii-paii~reo~~~~iiii (CCK-PZ) were syiithesized, iisirig both stepwise atid fragmeiit condensation approaches. Their gallbladder-contractile activities were conipared with those of the C-terminal octapeptide of CCK-PZ in vir0 and in vitro.
the gallbladder-contracting activity of CCK-PZ in vivo and in viti.o.'O The C-terminal pentapeptide of I is structurally identical with both the C-terminal pentapeptide of gastrin," a hormone that controls secretion of gastric acid, and with the C-terminal pentapeptide of caerulein,12 a decapeptide isolated from the skin of an amphibian, Hgla caerulea. The remarkable biological properties of I1 prompted us to study the importance of various structural features of the molecule, namely: (a) S-termiiial amino group, (b) aspartic acid residues in positions 1 arid 7, (c) methionine residues in positions 3 and 6, and (d) position of the 0-sulfate- tyrosine residue. A selected number of analogs arid derivatives of 11, which are listed in Table I, were synthesized and evaluated biologLys-(Sla~,Gly~,Pro~,Yer~)-Arg-Val-(Ile~,~letl,Ser~ )-Lvs-Asriically. ( Asx~,G1~1,Hisl,Le~~,Pro~,Ser2)-Arg-Ile-(Asp~,Serl~-Arg-AspChemistry.-Two synthetical approaches were used: SOsH (1) stepwise, starting from the C-terminal amino acid I (C;l~,,hletz,Trp~,Tyr~)-Bap-Phe-~Hsresidue (analogs 6 and 7), and ( 2 ) fragment condensaI tion (1, 2, 3, 4, 5 , 8). I n the stepuise approach, two types of active esters, ~,4,j-trichloropheny11~ The seyuerice of the amino acids of the C-terniirial ester arid p-nitrophenyl ester,I4 were used for the octapeptide of I is as follows :ti coupling steps. In the fragment condensation, the SOjH S-terminal dipeptide azide was coupled with the CI Asp-Tyr-hIet-Gly-Trp-?\Iet-Asp-Phe-?:H2 terminal hexapeptide. Both fragmerits were synthe1 2 8 4 5 6 7 8 sized stepwise. The introduction of the sulfate ester I1 group was carried out with concd H2S04in the preparaThis octapept'ide I1 possesses the biological activities tion of 6 and 7, and with pyridine-SOj ~ o m p l e x ' ~in~ ' ~ of the full hormonal moleculeYand showed S-10 times that of the other analogs. Sulfation with HzSOd was
Ivy and Oldberg' reported in 1928 the discovery of a new gallbladder-contracting principle of the small irit,estine, arid named it cholecystokinin (CCB). 9 1 most 15 years later, Harper and Raper2discovered in the mucosa of the upper intestine a substance that' caused secretion of enzymes in pancreatic juice. This substance, which they also considered to be a specific hormone, was named pancreozymin (PZ). Finally, in 1964 Jorpes and M u t t 3 isolated from the same intestiue a polypeptide that exhibited the properties of bobh cholecvstokinin arid pancreozymin. They have referred to it, at8least temporarily, as cholecystokininpancreozymin (CCIC-PZ). This hormone, for which a partial structure (I) has been p r ~ p o s e d , ~is- ~a polypeptide of 33 amino acid residues :7
(1) .'i C. . Iv? and PJ. Oidberx, d m e r . I ' h p i o l . , 86, 5Y9 (19281. ('21 .I. A . Harper and AI. 5 . Raper. J . E'hy.,aol. (London),102, 115 ( l Y - 4 3 ) . ( 3 ) .J. E. Jorpes. V. M u t t . and K. Toczko. A c t a Chem. Scard., 18, 2408 (1963). (4) V. Aliitt and .J. E. Jorpes. B i o c h e m . B i o p h y a . Res. C o m m u n . , 26, 392 (1967). (5) V. h l u t r and .J. E. .Torpen, >,hi-. .I. Bioclicn.. 6, l,56 (1968). (61 V. l l i i t t a n d .J. 15. .Jorp?a, I J r m . I u t . 1 . n i o t t I'liysiol. Sri.. 6, 1 9 3 (1963). (71 Tire ahbreviativns recommended i)y tire ICP.hC-IUI3 Commission on 13ioclicmical Somenclature (.I. B i d . Chem., 241, 2491 (1966); 555 i l U 6 i ) ) a r e used in this paper. ( 8 ) .J. E. Jorpes, G d r o e r i t e m l o y y . 66, 157 (1Y68). (9) 31. .\, Ondetti. ,J. PluSEec. E. F'.ziai>o,J. T. Slieelittn, and S . \\'illiamb, .J. ..lmrr.. C ' i i c m . Sor... 92, 195 ( l Y i 0 ) .
(10) 11. Kuliin, S. L. Engel, .I. A l . Urungis, A i . Uselakalns, E. 0. Origas, A I . H. IVaugli, a n d E. l-iacas, J . Phorm. Sci.. 68, 955 (1969). (11) J. Beacham, P. H. Bentley, R. A . Gregory, G. \V. Kenner. J. K. MacLeod, and R . C. Sheppard, N a t u r e , 209, 585 (1966). (12) .i. Anastasi, V. Ersparmer, and R. Endean, Ezperientia. 28, 699 (1967). (13) J. Pless and R . A . Roissonnas. Helr. Chim.A d a . 46, 1609 (1963). (14) 11. Rodanszky, S n t u r e , 176, 685 (1955). (15) H. C. Reits, R. E. Ferrel, H. Fraenkel-Conrat, and H. S. Olcott, .I. A m e r . C k e m . Sor.. 68, 1024 (1916): I
