Hoppe-Seyler's Z. Physiol. Chem.Bd. 359, S. 917-922, August 1978

Pentagastrin Analogs Containing -Aminooxy Acids, III*

Biological Studies and Structure-Activity Relationships

Istvan SCHÖN3, Lajos KISFALUDY*, Jozsef NÄFRÄDib, Läszlo VARGAb and Vince VARROb

a Chemical Works of Gedeon Richter Ltd., Budapestb First Department of Medicine, University Medical School, Szeged, Hungary

(Received 28 October 1977/17 April 1978)

Summary: The stimulation of gastric acid flow bythirty-six new pentagastrin analogs administeredintravenously, intrajejunally and intrarectally wasdetermined in rats. Some of the analogs are severaltimes more active than the pentagastrin (Peptavlon,I.C.I.) used as control. Unlike the control, someanalogs are absorbed in active form from thejejunum. The TV-terminal substitution witha-ami-

nooxy acids seems to increase the gastric secretoryresponse, probably by improving the enzymaticresistance of the molecule to various enzymes.None of the analogs tested significantly inhibitthe gastric acid output in rats induced by penta-gastrin. Structure-activity relationships are discus-sed.

Pentagastrin-Analoga mit Oi-Aminooxysäuren, III: Biologische Untersuchungen und Beziehungenzwischen Struktur und Aktivität

36 neue Pentagastrin-Analoga wurden nach in-travenöser, intrajejunaler oder rektaler Verab-reichung an Ratten auf ihre Wirkung geprüft, denMagensäurefluß zu stimulieren. Einige dieser Ver-bindungen waren mehrfach wirksamer als Penta-gastrin (Pentavlon der Fa. I.C.I.), das zur Kon-trolle verwendet wurde. Im Gegensatz zur Ver-gleichssubstanz wurden einige der Analoga in ak-tiver Form vom Jejunum resorbiert. TV-terminale

Substitution mit -Aminooxysäuren scheint dieMagensaft-Sekretion dadurch zu stimulieren, daßdie Resistenz des Moleküls gegen den Abbaudurch verschiedene Enzyme erhöht wird. Keinesder geprüften Analoga hemmt deutlich die durchPentagastrin verursachte Magensaft-Sekretion. DieBeziehungen zwischen Struktur und Wirkung wer-den diskutiert.

Key words: Gastric acid Stimulation, jejunal absorption, aminooxy acids, enzymatic resistance, structure-activityrelationships.

Abbreviations:Adc = 2-aminodecanoic acid; Phg = C-phenylglycine; Chg = C-cyclohexylglycine; Phg(4Cl) = 4-chlorophenylglycine;H2NOAc-OH = aminooxyacetic acid; r^NOPr-OH = 2-aminooxypropionic acid; 0-H2NOPr-OH = 3-aminooxyprop-ionic acid; H2NOPr(Ph)-OH = 2-aminooxy-3-phenylpropionic acid; Boc = tert.-butyloxycarbonyl.

* The results presented here are part of a thesis for a C.Sci. degree at the Hungarian Academy of Sciences byI. Schön.

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918 I. Schon, I., Kisfaludy, J. Nafradi, L. Varga and V. Varro Bd. 359(1978)

To date numerous analogs and derivatives of theactive centre of "little" gastrins, consisting of17 amino acids, have been synthesized 1 1~71.Several conclusions have been drawn on structure-activity relationships. According to recent studiesof Lin et alJ5l, there is, surprisingly, no part ofthe active centre of gastrin molecule that is in-dispensable for intrinsic biological activity. Nei-ther the C-terminal carboxamide group nor the0-carboxyl function of the aspartyl residue is es-sential for biological activity, as previously sup-posed, since the appropriate benzyl esters are alsoactive in very high doses.In Part l^ and II'7! of this series we have re-ported the synthesis of new analogs containingα-aminooxy acids. TV-Terminal aminooxy acidswere a common feature of the new analogs be-cause of their resistance to some proteolytic en-zymes in vitrol8'9'. We examined further how theincorporation of L- and D-phenylglycine, L- andD-cyclohexylglycine, L-4-chlorophenylglycine,D-2-aminooxy-3-phenylpropionic acid in place ofL-phenylalanine and the extension by aminooxyacetic acid at the C-terminus affect the biologicalactivity of the compounds containing TV-terminalaminooxy acids. It is well known that the me-thionyl residue in the active centre (H-Trp-Met--Asp-Phe-NH2) may be replaced by leucyl andnorleucyl residues without loss of biologicalactivity!1»2»10'1 ! I. As an extension of this ex-perience, norvalyl, L- and D-2-aminodecanoylanalogs have been synthesized for biologicalstudies.

Experimental

The experiments were performed in male Wistar ratsweighing 220 to 360 g. Gastric perfusion was performedaccording to the method described by Ghosh andSchildl 1 2 1 and later by Lail ! 3 1 as modified Pissidis andClark· 14'. Technical details were described in an earlierreport* 1 5 1; now we only wish to emphasize that afterthe basal secretion became constant, a reference dose of0.20Mg/100 g body weight of Peptavlon (I.C.I.) wasinjected into the penile vein in order to check the se-cretory reactivity of the animal. This injection wasrepeated again after an hour. When the secretion hadreturned to the basal level, a dose of 0.20 or 2.0 Mg ofour pcntagastrin analog to be tested per 100 g bodyweight was injected into the penile vein. In other ex-periments, a dose of 10 or 20 Mg of the same compoundper 100 g body weight was ad ministe red into the jejunum

and 20 or 50 Mg/100 g body weight was inserted rectally.Finally, the intravenous reference dose of pentagastrinwas injected to check the reactivity of the animals at theend of the experiment. An increase of about 40 to 60%of the basal acid output was considered as a definitelypositive response.Each of the analogs was .examined in groups of 4 to 12animals in each type of administration. The standarddeviation of the method is less than 20%l1 6LAnalogs with no or little secretory activity were examinedas potential inhibitory (antigastric) compounds. In thiscase, after checking the secretory reactivity of theanimal in the same manner as described above, theanalog was given together with an equal amount ofpentagastrin and the inhibition (if any) was registered.An acid output 40 to 60% per cent lower than thatproduced by pentagastrin was considered a positive in-hibitory effect.

Results and Discussion

The gastric acid secretory activity of compoundsI to XXXIII administered intravenously, intra-jejunally and intrarectally are shown in the Table,(see page 920) compared to a reference dose(0.2 Mg/100 g body weight) of commercial pen-tagastrin (Peptavlon, I.C.I.). The Figure shows the

25.0

20.0

15-°

S5.0

Γ| = Control pentagastrin (Peptavlon, I.C.I.)(V

iv

| |no

0.1 0.5 2.5Dose l \ig/100 g b.w.] ->

Figure. Dose-response relationships of analogs 1, IV andV administered intravenously as compared to Peptavlon.Gastric acid outputs represent average values from 4-14animals.

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Bd. 359 (1978) Pentagastrin Analogs Containing α-Aminooxy Acids, III 919

dose-response relationships of the three mostpotent analogs (I, IV and V).The compounds possessing weak or no secretoryactivity (VII, IX, XIII, XIV, XVI, XVII, XXII,XXVI and XXVIII) were tested in intravenouslyadministered doses of 0.2 and 2.0 pg/100 g bodyweight for potential inhibition of gastric acidsecretion induced by intravenously administeredpentagastrin in a dose of 0.2 Mg/100 g bodyweight. No inhibitory effects large enough forpractical use were observed.On the basis of these results the following con-clusions may be drawn with regard to structure-activity relationships:

1) Incorporation of a tert.-butyloxycarbonyl-aaminooxy acid at the jV-terminus increases thebiological activity as compared to pentagastrin.On the basis of the molar activity ratio of penta-gastrin to gastrin heptadecapeptide amidel17!, theactivity of the most potent analogs (I, IV and V)significantly approaches that of the native hor-mone. The lack of the tert.-butyloxycarbonylgroup decreases the activity. This behavior mayderive from easier and faster metabolism of theanalog II.

2) The methionyl residue can be replaced by aleucyl residue without changing of activity (IVand V), in agreement with data reported in theyt [1,2,10,11 j The shortening or elongation ofthe side chain in this position seems to decreasethe activity (VII and VIII) in accordance withMorley's observation^. The inversion of theconfiguration led to a total loss of activity (IX).

3) The originally proposed^8»9' enhanced en-zymatic resistance of the analogs containingα-aminooxy acids at theAf-terminus (I, IV and V)seems to be confirmed by the significant stimula-tion of gastric acid production in rats after ad-ministration of the compounds, in to the jejunum.These are the first pentagastrin analogs able tostimulate gastric acid production when adminis-tered intrajejunally. Analogs II to V, which weretested by intrarectal administration, seem to beabsorbed adequately and to stimulate gastricsecretion as well as pentagastrin itself^18'.

4) The replacement of the phenylalanyl residueby phenylglycine'in the pentagastrin analog con-taining an TV-terminal a-tert.-butyloxycarbonyl-

aminooxy propionyl residue (XII) significantlyincreases the biological activity as compared tothat of the control pentagastrin. This observationcontrasts with Morley's experience, that an in-active analog resulted from the switch of thephenyl ring to α-carbon atoml1!.The behavior of analog X seems to be contradic-tory. Its activity is about one-tenth that of thesame dose of control administered intravenously,but when administered jejunally, it is markedlyactive in a dose of 20 μ%/100 g body weight,whereas pentagastrin is totally inactive up to adose of 300 Mg/100 g body weight in rats'18'.Jejunal absorption of this compound in an activestate may be due to the N-terminal aminooxyacyl moiety, which is resistant to aminopeptidasein vitro'8»91.The configuration of phenylglycyl residue mark-edly affects the biological activity. Analogs con-taining a D-phenylglycyl residue (XI, XIII, XVand XVII) are marginally active, like the D-Pheanalog'11.The simultaneous incorporation of leucine ornorleucine and D-phenylglycine in place ofmethionine and phenylalanine, respectively, leedsto the poorly active or inactive analogs XIV toXVIII, with the exception of XIV, which is ac-tive in a dose of 2.UMg/100 g body weight. Theseresults are inconsistent with known structure-ac-tivity relationships!1»21, although Higaki et alJ2 'stated that inactive analogs resulted from morethan two substitutions in the active centre.The shortening or the significant elongation ofthe side chain at the methionyl position ofanalogs containing a phenylglycyl residue (XIXto XXI) totally destroys the activity in a dose of0.2 Mg/100 g body weight. Like pentagastrin,analogs X and XIII to XVII are absorbed in highdoses from the rectum and evoke gastric acidsecretion.

5) Analogs containing D- or L-cyclohexylglycineor L-4-chlorophenylglycine instead of L-phenyl-alanine (XXII, XXIII, XXVI and XXVII) arepractically inactive, whereas an additional sub-stitution of leucine for methionine (XXIV, XXVand XXVIII) restores significant secretory ac-tivity. Thus these analogs having two substitutionsappear to be exceptions·21.

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920 I. Sch n, L. Kisfaludy, J. Nafradi, L. Varga and V. Varro Bd. 359 (1978)

Table. Secretory activity in Ghosh-Lai rats of pentagastrin analogs I to XXXVI administered enterally and parenterallyas compared to Peptavlon (I.C.I.) administered intravenously in a dose of 0.2 Mg/100 g body weight.Gastric acid outputs represent average values from 4-12 animals.

Number

IIIIIIIVVVIVIIVIIIIXXXIXIIXIIIXIVXVXVIXVIIXVIIIXIXXXXXIXXIIXXIIIXXIVXXVXXVIXXVIIXXVIIIXXIXXXXXXXIXXXIIXXXIIIXXXIVXXXVXXXVI

Analog

Boc-NHOAc-Trp-Met-Asp-Phe-NH2

H2 Ν Ο Ac-Trp-Me t-A sp-Phe-NH 2

Boc-L-NHOPr-Trp-Met-Asp-Phe-NH2

Boc-NHOAc-Trp-Leu-Asp-Phe-NH2

Boc-D-NHOPr-Trp-Leu-Asp-Phc-NH2

Boc-D-NHOPr-Trp-Nle-Asp-Phe-NH2

Boc-D-NHOPr-Trp-Nva-Asp-Phe-NH2

Boc-NHOAc-Trp-L-Adc-Asp-Phe-NH2

Boc-NHOAc-Trp-D-Adc-Asp-Phe-NH2

Boc-NHOAc-Trp-Met-Asp-L-Phg-NH2

Boc-NHOAc-Trp-Met-Asp-D-Phg-NH2

Boc-D-NHOPr-Trp-Met-Asp-L-Phg-NH2

Boc-D-NHOPr-Trp-Met-Asp-D-Phg-NH2

Boc-NHOAc-Trp-Leu-Asp-L-Phg-NH2

Boc-NHOAc-Trp-Leu-Asp-D-Phg-NH2

Boc-D-NHOPr-Trp-Leu-Asp-L-Phg-NH2

Boc-D-NHOPr-Trp-Leu-Asp-D-Phg-NH2

Boc-D-NHOPr-Trp-Nle-Asp-D-Phg-NH2

Boc-D-NHOPr-Trp-Nva-Asp-D-Phg-NH2

Boc-NHOAc-Trp-L-Adc-Asp-D-Phg-NH2

Boc-NHOAc-Trp-D-Adc-Asp-D-Phg-NH2

Boc-D-NHOPr-Trp-Mct-Asp-L-Chg-NH2

Boc-D-NHOPr-Trp-Met-Asp-D-Chg-NH2

Boc-D-NHOPr-Trp-Leu-Asp-L<:hg-NH2

Boc-D-NHOPr-Trp-Leu-Asp-D-Chg-NH2

Boc-NHOAc-Trp-Met-Asp-L-Phg(4Cl)-NH2

Boc-D-NHOPr-Trp-Met-Asp-L-Phg(4Cl)-NH2

Boc-D-NHOPr-Trp-Leu-Asp-L-Phg(4Cl)-NH2

Boc-NHOAc-Trp-Met-Asp-Phe-NHOAc-OHBoc-D-NHOPr-Trp-Met-Asp-Phe-NHOAc-OHBoc-L-NHOPr-Trp-Met-Asp-Phe-NHOAc-OHBoc-^NHOPr-Trp-Met-Asp-Phe-NHOAc-OHBoc-NHOAc-Trp-Met-Asp-D-NHOPr(Ph)-NH2

H2NOAc-Trp-Met-Asp-D-NHOPr(Ph)-NH2

Boc-D-NHOPr-Trp-Met-Asp-D-NHOPr(Ph)-NH2

D-H2NOPr-Trp-Met-Asp-D-NHOPr(Ph)-NH2

Gastric acid output ΙμΓηοΙ/h]Intravenous

Controlbeforeanalog

6.27.8

12.25.64.4

10.68.18.29.3

10.69.24.49.2

12.37.32.35.07.07.85.67.77.2

11.88.78.57.2

10.93.66.66.87.06.6

Analog(0.2 Mg/100g)

10.57.68.97.45.34.62.34.70.70.92.18.82.60.90.11.12.71.900000.95.32.2002.71.43.14.12.0

Controlafteranalog

8.48.9

15.06.23.26.57.85.13.09.25.75.44.85.06.38.66.08.17.45.77.54.97.92.16.87.57.61.74.62.47.65.8

Controlbeforeanalog

3.41.89.45.66.7

13.4

17.25.87.01.7

13.53.46.64.68.38.9

11.06.3

10.8

9.7

11.15.88.95.4

Analog(2.0 Mg/100g)

8.57.68.1

12.912.214.1

20.53.02.5

015.50.7

00

2.10.93.70.5

4.0

0

2.23.22.31.1

Controlafteranalog

2.41.47.73.93.5

13.1

17.85.24.21.67.96.24.62.46.36.56.59.0

9.1

9.7

7.56.48.6

11.6

inactive in doses of 0.2 and 2.0 Mg/100 g body weight.

a 10 Mg/100 g body weight. b 50 Mg/100 g body weight.

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Bd. 359 (1978) Pentagastrin Analogs Containing α-Aminooxy Acids, III 921

6) Analogs XXXIII to XXXVI, containing 2-D-aminooxy-3-phenylpropionyl residues at theC-terminus are inactive, both as agonists and as

Gastric acid output [μΓηοΙ/η]Intrajejunal

Controlbeforeanalog

2.05.7

10.27.75.2

9.4

10.83.83.1

10.29.77.15.64.1

9.8

23.39.74.9

17.5

Analog(20 Mg/100g)

l.la

2.9a

4.80.31.7

0

3.700.30

-1.0-0.2

2.00

0

0.800.2

-0.4

Controlafteranalog

2.46.66.32.04.6

5.0

2.02.56.1

10.37.53.35.13.0

5.7

9.28.03.9

10.6

IntrarectalControlbeforeanalog

8.614.67.23.6

13.15.53.4

11.014.14.53.14.6

11.15.5

11.916.1

Analog(20 Mg/100g)

18.7b

4.23.61.7

7.400.42.13.12.51.41.8

2.15.22.2

-1.1

Controlafteranalog

5.710.8

3.43.3

26.210.14.88.2

21.23.93.24.2

7.54.5

12.914.6

antagonists. The incorporation of one oxygenatom into the backbone of the peptide chain nearthe C-terminus destroys not only the secretoryactivity but the binding affinity to receptor, aswell. These findings may be paralleled to the dis-appearance of the sweet taste for the dipeptideH-Asp-NHOPr(Ph)-OMe, conversely characteristicfor H-Asp-Phe-OMe (J.S. Morley, personal com-munication). Both observations constitute a fur- ·ther example for the importance of backbone-modification-induced conformational changes ofthe peptides.7) The gastric acid secretory activity of analogsXXIX to XXXII extended by an additional amino-oxy acetic acid at the C-terminus is lower by 35to 75% than the control pentagastrin. In contrastto several substituted derivatives of the C-terminalamidel1!, this modification does not destroy, butonly decreases the biological activity.

As pentagastrin has a trophic effect on gastricmucosa, the most potent, even jejunally activeanalogs may have therapeutic value in the treat-ment of peptic ulcers and in subacidity.

Literature

1 Morley, J.S. (1968) Proc. R. Soc. London, Ser. B.170,97-111.

2 Higaki, K., Danno, T. & Miyoshi, M. (1974)PharmacometricsS, 147-155.

3 von D ngen, Α., Konz, W. & Hummelt, H. (1976)LiebigsAnn. Chem. 860-875.

4 Lin, T.-M., Southard, G. L. & Spray, G. F. (1976)Gastroenterology 70, 733-736.

5 Lin, T.-M., Spray, G. F. & Southard, G. L. (1977)Gastroenterology 72, 566-568.

6 Kisfaludy, L., Sch n, L, Nafradi, J., Varga, L. &Varro, V. (1978) Hoppe-Seyler's Z. Physiol Chem.359, 887-895.

7 Schon, L, Kisfaludy, L., Nafradi, J., Varga, L. &Varro, V. (1978) Hoppe-Seyler's Z. Physiol. Chem.359, 897-916, preceding paper.

8 Kisfaludy, L., Low, M. & Devenyi, T. (\9T\)ActaBiophys. Biochim. Acad. Sei. Hung. 6, 393-403.

9 Kisfaludy, L., Low, M., Dancsi, L., Patthy, A.,Nyeki, O. & Sark zi, M. (1973) in Peptides 1972(Hanson, H. & Jakubke, H. D., eds.) pp. 409-415,North Holland Publ., Amsterdam.

10 W nsch, E., Jaeger, E., Deffner, M. & Scharf, R.(1972) Hoppe-Seyler's Z. Physiol. Chem. 353,1716-1720.

11 Jaeger, E., Thamm, Ρ.,-Schmidt, I., Knof, S.,Moroder, L. & W nsch, E. (1978) Hoppe-Seyler's Z.Physiol. Chem. 359, 155-164.

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922 I. Schön, L. Kisfaludy, J. Nafradi, L. Varga and V. Varro Bd. 359(1978)

12 Ghosh, M. H. & Schild, H. O. (1958) Br. Pharmac.Chemother. 13,54-61.

13 Lai,K.S. (1964) Gut 5, 327-341.14 Pissidis, G. A. & Clark, CG. (1967) Gut 8, 196-197.15 Varro, V. & Nafradi, J. (1970) Scand. J. Gasfroenterol.

5,273-275.

16 Varro, V. & Nafradi, J. (1970) Kiserl. Orvostud. 22,526-530.

17 Morlcy, J. S., Tracy, H. J. & Gregory, R. A. (1965)Nature (London) 207, 1356-1359.

18 Varro, V. & Nafradi, J. ( \ 9 l 4 ) A c f a Hepato-Gastroenterol. 21,441 -446.

Dr. lstvanScho'n,and Dr. Lajos Kisfaludy, Chemical Works of Gedeon Richter Ltd., Research DepartmentH-1475 Budapest 10, P.O.B. 27.Dr. Jozsef Nafradi, Dr. Laszlo Varga and Dr. Vince Varro, First Department of Medicine, Univ. Med. SchoolH-6701 Szeged.

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