Amidic derivatives of glutamic, aspartic and 2-amino adipic acids, a process for preparing same, and anti-gastrin composition containing the derivatives

The subject of the present invention is compounds with activities antagonistic towards gastrin or other peptides related thereto, which can be represented by the general formula indicated below: ##STR2## in which r is from 1 to 3, and R.sub.1 is selected from mono-, di-substituted phenyl groups, in which the substituents are selected from chlorine, linear and branched C1-C4 alkyl groups, cyano, nitro, methoxy, and trifluoromethyl groups, the 2(beta)-naphthyl group, and heterocyclic, monocyclic and bicyclic groups containing 1 heteroatom such as nitrogen, oxygen or sulphur and selected from furyl, thiophenyl, pyrrolyl, pyridinyl, and pyridinyl-N-oxide groups unsubstituted or mono- or disubstituted with methyl and chlorine, indolyl (2- or 3-yl), thionaphthyl (2- or 3-yl), benzofuranyl (2- or 3-yl), quinolinyl (2- or 3-yl), or isoquinolinyl (3-yl) groups and in which R.sub.2 is selected independently from:
1) a heterocyclic spiro group represented by ##STR3## in which m and n are selected independently and may assume values between 1 and 3 provided that the ring formed consists of at least 5 atoms,
x and y are selected independently from (CH--R.sub.3).sub.z' (CH.dbd.CH), TCH.sub.2 and CH.sub.2 T in which T is O, S, NH--C(O) or C(O)--NH, and in which R.sub.3 is a group selected independently from H, CH.sub.3, C.sub.2 H.sub.5, OCH.sub.3, OC.sub.2 H.sub.5, CH.sub.2 OH and OH and z may assume values of from 0 to 3, provided that the ring formed consists of at least 3 atoms.
2) a bicyclic spiro amino group represented by: ##STR4## in which m, n, x, y and R.sub.3 have the meanings given in point 1 above and Q maybe selected independently from H and CH.sub.3.
3) a bicyclic amino group (condensed) represented by: ##STR5## in which m and n are selected independently and may assume values of between 1 and 3 and x, y and R.sub.3 have the meanings given in point 1 above.
4) a bicyclic amino group (condensed) represented by: ##STR6## in which m and n are selected independently and may assume values of between 1 and 3 and x, y R.sub.3 and Q have the meanings given in point 1 above.
5) an adamantyl alkylamino group represented by: ##STR7## w is between 0 and 2, L is H, a methyl or methoxyl group and Ad is an adamantyl (1- or 2-yl) group.
The stereochemistry of the compounds claimed at the chiral centre marked with an asterisk in the formula (I) may be racemic (R, S) or, preferably, rectus (R); r is preferably 2, R.sub.1 is preferably selected from the group consisting of 3-chloro-phenyl and 3,5-dichlorophenyl, and R.sub.2 is preferably Selected from the group consisting of 8-azaspiro[4.5]decan-8-yl and 1-adamantyl-2-aminoethane.
The compounds of the present invention show potent antagonistic activity towards gastrin ("little gastrin" or G-17) and towards pentagastrin, which is its biologically active terminal peptide sequence.
Gastrin is a polypeptide hormone secreted by the cells of the antral and duodenal mucosae, its secretion being induced, in particular, by a nervous mechanism by means of vagal excitation.
Once secreted, the gastrin reaches the parietal cells, which are situated mainly in the fundic mucosa, via the bloodstream and activates the receptors of the cell membranes by binding thereto. This activation starts the process of the formation of HCl and its secretion into the gastric cavity.
Hyperactivity of this system results in the hypersecretion of plastric acid, which may have consequences which are harmful to the organism but can be eliminated with the use of specific antagonists.
The compounds of the invention show a high level of activity against the secretion of acid in vivo in the various animal species tested, blocking the secretory stimulus induced by pentagastrin in a dose-dependent manner. The activity is specific, since it does not block secretions induced by carbachol or histamine.
As already stated, the compounds can therefore be used, to advantage, in the treatment and prevention of various diseases in man, for example, ulcers or gastro-duodenitis resulting from excessive gastric secretion induced by a hypergastrinaemic condition, in Zollinger-Ellison syndrome, or in the treatment of some kinds of tumours which are activated by hypergastrinaemia, in which it is advantageous to block gastrin's activity in stimulating the secretion of acid.
On the basis of the considerable activity they show in vitro as inhibitors of the binding of gastrin at the level of the CNS, the compounds of the invention might also be shown to be effective for some kinds of mental disorders attributable to an imbalance in the physiological neuron levels of gastrin or other bioactive peptides related thereto such as, for example, cholecystokinin (CCK).
The remarkable anti-CCK activity of some of the compounds of the invention, which depends in particular on suitable substitutions at R.sub.1, combined with their antigastrin activity, could also make them particularly. suitable for treating diseases in man such as biliary diskinesia, Coliris, pancreatitis or for the treatment and prevention of some eye conditions such as, for example, myosis induced in the course of the surgical treatment of cataracts or by chronic eye inflammation.
The method of preparing the compounds of the invention which have their chiral centres in the R (rectus) form is characterised by steps which may be represented as follows:
a) reacting the gamma-benzyl ester of N-carbobenzoxy-D-glutamic acid with an amine of the formula H--R.sub.2, in which R.sub.2 has the meaning given above, by the mixed anhydride method, in an inert anhydrous solvent at a temperature between -15.degree. and +15.degree. to give the compounds of formula (III), (see the diagram below)
b) debenzylating and decarbobenzoxylating the compound of formula (III), dissolved in an inert solvent, in a single step by reacting it with hydrogen at ambient temperature and atmospheric pressure in the presence of a catalytically effective quantity of a hydrogenation catalyst to produce the derivatives of formula (II) (see the diagram below),
c) reacting the derivatives of formula (II) under Schotten-Bauman conditions with a equivalent quantity of an acyl chloride of the formula R.sub.1 --COCl, in which R.sub.1 has the meaning given above, at a temperature from 0.degree. to 15.degree. C. and recovering the amides of formula (I) from the reaction mass.
The series of steps of the method according to the invention is illustrated as a whole by the following diagram: ##STR8##
The amidation step (a) is carried out at a temperature preferably between -15.degree. and -10.degree. C. for from 1 to 24 hours, preferably 6 hours, with a stoichiometric ratio between the reagents. The preferred solvent for effecting the reaction is selected from chloroform, dioxane and tetrahydrofuran.
The hydrogenation step (b) is preferably carried out in the presence of between 0.02 and 0.001 atoms of palladium per mole of compound (III), supported on carbon, in methanolic solution at ambient temperature and pressure in a stream of hydrogen for a period of from 1 to 12 hours, preferably 6 hours. The acylation step (c) is preferably carried out at a temperature of about 5.degree. C. for from 1 to 24 hours, preferably 12 hours.
The following examples are given to illustrate the invention further.

EXAMPLE 1
Preparation of the gamma-benzyl ester of (R) 4-carbobenzoxy-amino-5-(8-azaspiro[4.5]decan-8-yl)-5-oxopentanoic acid, (compound A.sub.1 in Table A).
37.1 g (0.1 moles) of the gamma-benzyl ester of N-carbobenzoxy-D-glutamic acid were dissolved in 250 ml of anhydrous tetrahydrofuran, the solution was cooled to -10.degree. C. and 10.1 g (0.1 moles) of triethylamine were added with stirring; 10.8 g (0.1 moles ) of ethyl chloroformate were then added, still at -10.degree. C. The temperature was kept at -10.degree. C. for 20 minutes and 13.9 g (0.1 moles) of 8-azaspiro[4.5]decane were then added. The mixture was stirred for a further 6 hours whilst the temperature rose to ambient temperature; the mixture was then evaporated to dryness and the residue taken up with ethyl acetate.
It was then washed with 2N HCl, sodium bicarbonate and, finally, with water and then dried over anhydrous Na.sub.2 SO.sub.4. Concentration to a small volume produced an oily residue. The crude product thus obtained was pure enough to be used directly in the subsequent step without further purification.
TLC: Rf 0.59 (benzene-ethyl acetate 7/3: v/v) Product 39.4 g. Yield 80%.
All the compounds of formula III were synthesised by the same method (see the diagram above). Table A below shows some of the compounds obtained with some of their identifying characteristics.
TABLE A__________________________________________________________________________derivatives of the formula ##STR9## MELTING TLC (Rf) POINT Benzene EthylCOMPOUNDS R2 FORMULA (.degree.C.) acetate 7:3 (V/V)]__________________________________________________________________________A1 8-Azaspiro[4.5]decan-8-yl C29H36N2O5 oil 0.59A2 3-Azaspiro[5.5]undecan-3-yl C30H38N2O5 oil 0.54A3 2-Azaspiro[4.4]nonan-2-yl C28H34N2O5 oil 0.39A4 2-Azaspiro[4.5]decan-2-yl C29H36N2O5 oil 0.41A5 3-Amino-spiro[5.5]undecano C31H40N2O5 oil 0.66A6 1.4-dioxa-8-Azaspiro[4.5]de- C27H32N2O7 oil 0.26 can-8-ylA7 Isoquinolin-2-yl-decahydro C29H36N2O5 oil 0.48A8 1-Adamantyl-2-amino-ethane C32H40N2O5 76-77 0.66__________________________________________________________________________
EXAMPLE 2
Preparation of (R) 4-amino-5-(8-azaspiro [4.5]decan-8-yl)-5-oxopentanoic acid, (compound B.sub.1 in Table B) .
49.2 g (0.1 moles) of the benzyl ester of (R) 4-carbobenzoxy-amino-5-(8-azaspiro[4.5]decan-8-yl-5-oxopentanoic acid (compound A1) were dissolved in 500 ml of methanol, to which 1 g of 10% palladiated carbon had been added, and hydrogenated with a stream of hydrogen at ambient temperature for 6 hours. The catalyst was filtered out and the methanol distilled under vacuum. The oily residue obtained (row 286.4) crystallised when taken up with acetone, and was filtered out to give a compound with a chromatographic purity greater than 95%.
TLC: Rf 0.54 [n-butanol-acetic acid-H.sub.2 O 5/2/2 (V/V)]. Melting-point: 174.degree.-175.degree. C. Rotatory power: (alpha).sup.20 D=-10.degree. (c=3% in methanol). Product 23.6 g. Yield 88%.
All the compounds of formula II were synthesised by the same method (see diagram).
Table B below shows the compounds thus obtained with some of their identifying characteristics.
TABLE B__________________________________________________________________________derivatives of the formula ##STR10## MELTING TLC (Rf) Rotary power POINT [ButOH-Acetic Acid [.alpha.]DCOMPOUNDS R2 FORMULA (.degree.C.) H2O: 5/2/2 (V/V)] (3% in MetOH)__________________________________________________________________________B1 8-Azaspiro[4.5]decan-8-yl C14H24N2O3 174-175 0.54 -10.degree.B2 3-Azaspiro[5.5]undecan-3-yl C15H26N2O3 142-143 0.64 -11.2.degree.B3 2-Azaspiro[4.4]nonan-2-yl C13H22N2O3 130-131 0.63 -8.3.degree.B4 2-Azaspiro[4.5]decan-2-yl C14H24N2O3 136-137 0.58 -9.6.degree.B5 3-Amino-spiro[5.5]undecano C16H28N2O3 142-143 0.57 -11.7.degree.B6 1.4-dioxa-8-Azaspiro[4.5]de- C12H20N2O5 149-150 0.45 -14.degree. can-8-ylB7 Isoquinolin-2-yl-decahydro C14H24N2O3 160-161 0.71 -8.6.degree.B8 1-Adamantyl-2-amino-ethane C17H28N2O3 169-170 0.82 -7.3.degree.__________________________________________________________________________
EXAMPLE 3
Preparation of 4-[3-(chlorobenzoyl)-amino]-5-(8-azaspiro 4.5]decan-8-yl)-5-oxopentanoic acid (R=rectus). (Compound C-1 in Table C).
26.8 g (0.1 moles) of (R) 4-amino-5-(8-azaspiro[4.5]decan-8-yl)-5-oxopentanoic acid (compound B1 ) were suspended in 300 ml of water and then dissolved with stirring by the addition of 10.6 g (0.1 moles) of sodium carbonate. 17.5 g (0.1 moles) of 3-chlorobenzoyl chloride were then added over a period of 1 hour at 0.degree. C. with stirring.
The mixture was left to react for 12 hours.
The mixture was then acidified to Congo red with dilute HCl and the precipitate thus formed was filtered out and crystallised with acetonitrile.
M.P.: 145.degree.-146.degree. C. TLC [methylene chloride-ethanol 1:1 (V/V)]: Rf 0.81 Product 33.4 g Formula: C.sub.21 H.sub.27 ClN.sub.2 O.sub.4 (mw 406.9) Yield 82%. Rotatory power: [alpha].sup.20 D=-15.degree. C. (c=3.0% in methanol).
All the compounds of formula I (see diagram) were synthesised by the same method.
Table C below shows the compounds thus obtained with some of their identifying characteristics, as well as the yields obtained (calculated for the method as a whole).
HPLC analysis was used to check the optical purity of the compounds obtained by the stereo-conservative synthesis method described above with the use of an ODS-C-18 column as the stationary phase, 0.025M phosphate buffer (pH 6.5)-acetonitrile as the mobile phase, and L-phenylalaninamide as the differentiating solvent.
The compounds given in Table C were found, in all cases, to have optical purities greater than 95%. The compound C1, for example, had a retention time of 11.45 minutes and an optical purity of 99.6% under the conditions described. For comparison, the compound C-32, which is an S-series (sinister) derivative and is the optical antipode of the compound C1, has a retention time of 10.8 minutes under the same experimental conditions.
TABLE C__________________________________________________________________________derivatives (D series) of the formula ##STR11## MELTINGCOMPOUNDS R1 R2 FORMULA POINT (.degree.C.)__________________________________________________________________________C1 3-chloro-phenyl 8-Azaspiro[4.5]decan-8-yl C21H27ClN2O4 149-150C2 3-chloro-phenyl 3-Azaspiro[5.5]undecan-3-yl C22H29ClN2O4 94-95C3 3-chloro-phenyl 2-Azaspiro[4.4]nonan-2-yl C20H25ClN2O4 69-70C4 3-chloro-phenyl 2-Azaspiro[4.5]decan-2-yl C21H27ClN2O4 88-89C5 3,5-dichloro- 8-Azaspiro[4.5]decan-8-yl C21H26Cl2N2O4 185-186 phenylC6 3,5-dichloro- 2-Azaspiro[4.5]decan-2-yl C21H26Cl2N2O4 112-113 phenylC7 3,5-dichloro- 2-Azaspiro[4.4]nonan-2-yl C20H24Cl2N2O4 111-113 phenylC8 3,5-dichloro- 8-Azaspiro[4.5]decan-8-yl C21H26Cl2N2O4 91-92 phenylC9 2,3-dichloro- 8-Azaspiro[4.5]decan-8-yl C21H26Cl2N2O4 105-167 phenylC10 2-Naphthyl 8-Azaspiro[4.5]decan-8-yl C25H30N2O4 145-147C11 3-methyl phenyl 8-Azaspiro[4.5]decan-8-yl C22H30N2O4 148-150C12 3-ethyl phenyl 8-Azaspiro[4.5]decan-8-yl C23H32N2O4 119-120C13 3,5-dimethyl 8-Azaspiro[4.5]decan-8-yl C23H32N2O4 183-185 phenylC14 3-nitrophenyl 8-Azaspiro[4.5]decan-8-yl C21H27N3O6 182-183C15 3-methoxy 8-Azaspiro[4.5]decan-8-yl C22H30N2O5 115-116 phenylC16 3-cyano phenyl 8-Azaspiro[4.5] decan-8-yl C22H27N3O4 154-155C17 3-trifluoromethyl 8-Azaspiro[4.5]decan-8-yl C22H27F3N2O4 122-123 phenylC18 4-isopropyl 8-Azaspiro[4.5]decan-8-yl C24H34N2O4 157-158 phenylC19 quinolinyl (3-yl) 8-Azaspiro[4.5]decan-8-yl C24H29N3O4 104-105C20 3,4-dichloro- 3-Azaspiro[5.5]undecan-3-yl C22H28Cl2N2O4 91-92 phenylC21 quinolinyl (3-yl) 3-Azaspiro[5.5]undecan-3-yl C25H31N3O4 119-120C22 3-chlorophenyl 3-amino-spiro[5.5]undecano C23H31ClN2O4 145-146C23 3,5-dichloro- 3-amino-spiro[5.5]undecano C23H30Cl2N2O4 103-105 phenylC24 furyl(2-yl) 8-Azaspiro[4.5]decan-8-yl C19H26N2O5 98-99C25 2-chloro 8-Azaspiro[4.5]decan-8-yl C20H26ClN3O4 167-168 pyridyl (3-yl)C26 indolyl (2-yl) 8-Azaspiro[4.5]decan-8-yl C23H29N3O4 194-195C27 3-chlorophenyl 1,4-dioxa-8-azospiro[4.5]deca- C19H23ClN2O6 72-73 n-8-ylC28 3,5-dichloro- 1,4-dioxa-8-azaspiro[4.5]deca- C19H22Cl2N2O6 110-111 phenyl n-8-ylC29 3-chlorophenyl isoquinolin-2- C21H27ClN2O4 84-85 decahydroC30 3-chlorophenyl 1-adamantyl-2- C24H31ClN2O4 185-186 amino ethaneC31 3,5-dichloro- 1-adamantyl-2- C24H30Cl2N2O4 179-182 phenyl amino ethaneC32 3-chlorophenyl 8-Azaspiro[4.5] C21H27ClN2O4 138-139(**) decan-8-yl__________________________________________________________________________derivatives (D series) of the formula ##STR12## TLC (Rf) methylene- chloride- CRYSTALLISATION Ethanol Rotatory power [.alpha.]DCOMPOUNDS SOLVENT 1:1 (V/V) (3% in MetOH) Yield % (*)__________________________________________________________________________C1 Acetonitrile 0.81 -15.0.degree. 60C2 Acetonitrile 0.78 -18.4.degree. 55C3 Acetonitrile- 0.66 -14.7.degree. 45 H2O(4:1)C4 Acetonitrile-Isopr. 0.73 -16.6.degree. 54 ether (5:1)C5 Acetonitrile 0.80 -11.9.degree. 61C6 Acetonitrile- 0.81 -15.0.degree. 56 Acetone (1:1)C7 Acetonitrile 0.79 -12.0.degree. 48C8 Acetonitrile 0.76 -18.4.degree. 68C9 Acetonitrile 0.87 -8.5.degree. 63C10 Acetonitrile 0.86 -29.5.degree. (**) 44C11 Acetonitrile 0.90 -13.3.degree. 53C12 Acetonitrile 0.77 -14.8.degree. 48C13 EtOH-H2O (1:1) 0.93 -11.8.degree. 47C14 Acetonitrile 0.75 -13.3.degree. 53C15 Acetonitrile 0.91 -18.3.degree. 36C16 Acetonitrile 0.73 -18.5.degree. 45C17 Acetonitrile 0.76 -15.0.degree. 35C18 Acetonitrile 0.89 -18.3.degree. 48C19 Acetonitrile-H2O 0.42 -10.0.degree. 43 (5:1)C20 Acetonitrile 0.85 -19.2.degree. 52C21 Acetonitrile 0.45 -9.6.degree. 58C22 Acetonitrile 0.84 -10.0.degree. 59C23 Acetonitrile 0.88 -12.2.degree. 57C24 Acetonitrile-isopr. 0.75 -9.3.degree. 53 ether (1:4)C25 Acetonitrile 0.69 -5.3.degree. 49C26 Acetonitrile 0.80 -8.2.degree. 23C27 Acetonitrile-isopr. 0.68 -13.4.degree. 44 ether (1:1)C28 Acetonitrile 0.67 -15.6.degree. 50C29 Acetonitrile 0.64 -19.5.degree. 56C30 Acetonitrile 0.75 +9.6.degree. (NaOH) 58C31 Acetonitrile-H2O 0.76 +11.7.degree. (NaOH) 55 (5:1)C32 Acetonitrile 0.80 +14.5.degree. 52(**)__________________________________________________________________________ (*) The yield shown is the overall yield calculated for the method as a whole, starting from the benzyl ester of Ncarbobenzoxy-D-glutamic acid (**) Rotatory power in CHCl.sub.3 (**) The compound C32 is an Sseries (sinister) derivative which is the optical antipode of the compound Cl and is shown for comparison.
The method of preparing the derivatives of the invention which have chiral centres in the racemic form (R, S) is characterised by steps which may be represented as follows:
a) reacting an internal anhydride of the formula: ##STR13## in which r and R.sub.1 have the meanings given above, prepared by conventional methods as already described in the literature [Makovec et al., Eur.J.Med.Chem. 21 (1986), 9-20] with an amine of the formula H--R.sub.2, in which R.sub.2 has the meaning given above, in a molar ratio of from 1 to 5 in a solvent such as water, ethyl acetate, dioxane, acetonitrile, tetrahydrofuran, or a mixture thereof, at a temperature between -20.degree. and 30.degree. C. and recovering the compounds of formula I from the reaction mass by fractional crystallisation or other conventional methods.
The following example is given below to illustrate the invention further.
Preparation of (R,S) (.+-.)-4-[(3-chlorobenzoyl) amino]-5-(1.4-dithia-8-azaspiro[4.5]decan-8-yl)-5-oxopentanoic acid. (Compound D.sub.1 of Table D)
26.8 g (0.1 moles) of (D, L) 3-chlorobenzoyl glutamic anhydride were loaded into a reactor and suspended in 100ml of water.
The suspension was cooled to about 5.degree. C. and 35 g (0.2 moles) of 1,4-dithia-8-azaspiro[4.5]decan-8-yl were added dropwise over a period of about 15 minutes. The mixture was left to react for 3 hours at this temperature and acidified with glacial acetic acid. It was then filtered, washed with water until neutral, and dried. The crude product thus obtained was crystallised with acetonitrile. 32.3 g were obtained.
Formula: C.sub.19 H.sub.23 ClN.sub.2 O.sub.4 S.sub.2 (mw 442.8) Yield 73%. M.P.: 169.degree.-17.degree. C. TLC (isoamyl alcohol-acetone-H.sub.2 O: 5/2/1 -V/V): Rf 0.79.
Some of the compounds thus synthesised are given by way of example in Table D below with some of their identifying characteristics as well as the yields obtained.
TABLE D__________________________________________________________________________(R, S Series) derivatives of the formula ##STR14## TLC isoamyl CRYST- alcohol MELTING ALLIS- acetone-COM- POINT ATION H2O: YIELDPOUNDS R1 R2 r FORMULA (.degree.C.) SOLVENT 5:2:1 %V/V)__________________________________________________________________________D1 3 chlorophenyl 1,4-dithia-8-Azaspiro[4.5] 2 C19H23ClN2O4S2 169-170 Acetonitrile 0.79 73 decan-8-ylD2 3-chlorophenyl 1,4-dioxa-2-hydroxymethyl 2 C20H25ClN2O7 138-140 Acetonitrile 0.51 48 8-azaspiro[4.5]decan-8-ylD3 3-chlorophenyl 1-oxa-4-thia-8-azaspiro[4.5] 2 C19H23ClN2O5S 168-169 Acetonitrile 0.63 70 decan-8-ylD4 3-chlorophenyl 8-Azaspiro[4.5]decan-8-yl 1 C20H25ClN2O4 108-109 Acetonitrile 0.60 66D5 3-chlorophenyl 8-Azaspiro[4.5]decan-8-yl 3 C22H29ClN2O4 95-98 Acetonitrile 0.81 38 isopropyl ether (1:1)D6 3-chlorophenyl 2-amino-naphthalene- 2 C22H29ClN2O4 216-217 Ethanol 0.71 57 decahydroD7 3,5-dichloro- 2-amino-naphthalene- 2 C22H28Cl2N2O4 246-247 Ethanol 0.84 53 phenyl decahydro__________________________________________________________________________
An analysis of the pharmacological activities of the compounds of the invention with regard, in particular, to their activities against acid secretion performed by an antigastrin mechanism, was then carried out by means of a series of pharmacological tests carried out both in vivo and in vitro and is documented below.
Antisecretive activity in anaesthetised rats
Antisecretive activity in rats was determined with the use of male animals each weighing about 200 g, anaesthetised with urethane. Gastric secretion was stimulated with pentagastrin. The method of K. S. Lai (Gut 5, (1964), 327-341) was used, slightly modified.
After tracheotomy, cannulae were inserted in the oesophagus and the duodenum. A tepid solution. (37.degree. C.) of 0.25 mM NaOH was perfused and passed through the stomach by means of a peristaltic pump at a constant flow rate of 1 ml/minute. After stabilisation for 20 minutes, the stimulating agent, dissolved in a physiological solution, was perfused for 120 minutes at a dose of 30 mcg/Kg/h in a volume of 0.95 ml/hour. After perfusion for 60 minutes (the basal stimulation), the product under test was administered as an intravenous bolus (I.V.) and the perfusion of the stimulant continued for a further 60 minutes. Acid secretion was recorded continuously as a function of time.
The activity of the product was evaluated as the percentage reduction in the secreted acidity after the administration of the product compared with the base acidity measured during the first 60 minutes of collection during which only the pentagastrin was present.
Different doses of the antagonist compounds tested were administered so that an ID50, that is, the dose (in mg/Kg I.V.) capable of inhibiting the effect of pentagastrin by 50%, could be calculated.
The results thus obtained are illustrated in the following table (Table 1) in which the activities of the compounds are expressed as the ID50s under the stimulus of 30 mcg/Kg/h of pentagastrin.
TABLE 1______________________________________Antagonistic activity (ID50 mg/Kg IV) towards gastric acidsecretion induced by pentagastrin (30 mcg/Kg/h) in rats______________________________________COMPOUND C1 20 COMPOUND C21 IN (>75)COMPOUND C2 33 COMPOUND C22 55COMPOUND C3 75 COMPOUND C23 40COMPOUND C4 49 COMPOUND C24 IN (>75)COMPOUND C5 15 COMPOUND C25 69COMPOUND C6 45 COMPOUND C26 IN (>75)COMPOUND C7 60 COMPOUND C27 81COMPOUND C8 41 COMPOUND C28 70COMPOUND C9 40 COMPOUND C29 35COMPOUND C10 45 COMPOUND C30 25COMPOUND C11 38 COMPOUND C31 20COMPOUND C12 38 COMPOUND C32 IN (>100)COMPOUND C13 33 COMPOUND D1 IN (>75)COMPOUND C14 68 COMPOUND D2 85COMPOUND C15 46 COMPOUND D3 67COMPOUND C16 66 COMPOUND D4 70COMPOUND C17 55 COMPOUND D5 32COMPOUND C18 28 COMPOUND D6 60COMPOUND C19 68 COMPOUND D7 48COMPOUND C20 64 Proglumide 500 Lorglumide IN (>100)______________________________________
The antigastrin activity was particularly favourable in the case of the glutamic acid (r=2) derivatives and when R.sub.1 was 3-chlorophenyl or 3,5-dichlorophenyl and when the amine group R.sub.2 was the azaspiro[4.5]-8-yl group or the 2-amino-1-adamantyl ethyl group (see compounds C1, C5, C30 and C31). It should be noted that, in this experimental model, the most active of the compounds of the invention were about 30 times more active than the reference antigastrin compound proglumide. It is also interesting to note that the CCK antagonist lorglumide is completely inactive up to a dose of 100 mg/Kg, as is the compound C-32 which is the S-series optical antipode of the compound C-1, showing that the antigastrin activity is stereospecific. The activity of these compounds against gastric secretion is linked specifically to their antigastrin activity. In fact, they have neither anticholinergic nor antihistamine (anti H.sub.2) activity since, in the experimental model described above, they were completely inactive when carbachol (30 mcg/Kg/h) or histamine (2.3 mg/Kg/h) were used as the stimulants. The activity seems even more remarkable if one takes account of the low toxicity of the compounds of the invention. For example, in rats, the compound C5 has an LD50 IV of about 400 mg/Kg. The average lethal dose (LD50) in this case is therefore about 25 times greater than the antigastrin dose (the ID50) for the same method of administration.
Antisecretive activity in conscious dogs
In order to confirm the antigastrin activity in rats, some of the most active compounds claimed were to be tested in a second animal species, the dog, in order to exclude species-specific phenomena.
Male beagles weighing about 10 Kg were used and carried gastric fistulae at the level of the greater curvature, connected near the antral region, steel cannulae 5 cm long and 2 cm wide being inserted for collecting the gastric juices externally. After a period of about one month to recover from the operation, and after suitable conditioning, the animals were treated with pentagastrin at a dose of 3 mcg/Kg/h. The Stimulant, dissolved in a physiological solution, was perfused into the femoral vein for 180 minutes at a rate of 1.9 ml/h. After perfusion for 60 minutes (the basal stimulation) the product under test, dissolved in a physiological solution (1 ml/Kg), was administered in a bolus into the contra-lateral vein and, at the same time, the perfusion of the stimulant continued for a further 120 minutes. The acid secretion was recorded continuously as a function of time.
The activity of the product was evaluated as the percentage reduction in the secreted acidity after the administration of the product compared with the base acidity measured during the first 60 minutes of collection during which only the pentagastrin was present, for each of the periods 0-60 and 60-120 minutes after the product had been administered in a bolus.
Different doses of the compounds (C-1 and C-5) tested were administered so that an ID50, that is the dose (in mg/Kg I.V.) capable of inhibiting the effect of the pentagastrin by 50% could be calculated.
The results obtained are given in the following table (Tab. 2) in which the % variations from the base (the mean .+-. standard deviations calculated on the values obtained from 4 animals) are given for the various doses administered under the stimulus of 3 mcg/Kg/h of pentagastrin, as well as the activities of the compounds, calculated as their ID50s, and the correlation coefficients of the regression lines calculated on the basis of the experimental data.
TABLE 2______________________________________Antagonistic activity (ID50 mg/Kg IV) towardsgastric acid secretion induced by pentagastrin(30 mcg/Kg/h) in conscious dogs carryinggastric fistulae Variation % (Mean .+-. SD) fromCOM- DOSE base (0-60 min)POUND (mg/Kg) (period 60-120 min) (period 120-180 min)______________________________________C1 5 -18.2 .+-. 0.3 -22.8 .+-. 16.7 10 -38.9 .+-. 7.9 -14.3 .+-. 8.5 20 -49.5 .+-. 4.2 -26.1 .+-. 6.4 30 -61.1 .+-. 12.5 -56.7 .+-. 19.5 40 -63.4 .+-. 11.5 -43.4 .+-. 10.3 60 -74.2 .+-. 5.6 -79.9 .+-. 1.6 ID50 mg/Kg = 19.7 ID50 mg/Kg = 31.5 (n = 6; r = 0.995) (n = 6; r= 0.83)C5 5 -36.0 .+-. 16.3 -20.5 .+-. 13.1 10 -59.5 .+-. 5.0 -31.9 .+-. 1.3 30 -67.8 .+-. 4.8 -48.1 .+-. 1.7 ID50 mg/Kg = 8.9 ID50 mg/Kg = 32.5 (n = 3; r = 0.916) (n = 3; r = 0.998)______________________________________ [r = regression correlation coefficient calculated an n values]-
The compounds C-1 and C-5 were shown also to be potent inhibitors of gastric acid secretion induced by pentagastrin in dogs. In fact, they block the acid output during the first hour after their administration in a bolus in a dose-dependent manner with ID50s of about 20 and 10 mg/Kg for the compounds C-1 and C-5, respectively.
The activity remains noteworthy even during the second hour after the intravenous administration of the bolus (during the period 120-180 minutes) with an ID50 of about 30 mg/Kg for both compounds.
IN VITRO TESTS
Tests on binding to cerebral cortex membranes of mice
The capacities of some of the compounds of the invention to inhibit the binding of pentagastrin to the gastrin receptor sites on the cerebral cortex membranes of mice was to be evaluated. Pentagastrin tritiate[beta-alanyl-3-3H(N)] was used as the ligand.
Mouse cortical tissue Was homogenised cold in 20 volumes of tris buffer (pH 7.7). After washing and centrifuging, the final pellet was resuspended in 20 volumes of binding buffer containing, inter alia, TRIS, BSA and Bacitracin. 0.4 ml of the membrane thus obtained were then incubated with a radioactive tracer and the compounds under test for 40 minutes at 37.degree. C.
After the supernatant liquid had been removed by centrifuging, the radioactivity associated with the pellet was determined with a liquid scintillator (a beta-counter). The specific binding was the difference between the binding in the absence and in the presence of 10-5M pentagastrin.
The results obtained are given in Table 3 which gives the IC50s of the compounds tested, that is, the concentration (in moles/liter) of the antagonist which can displace 50% of the pentagastrin tritiate from the receptor.
It can be seen from the data given in Table 3 that some of the compounds of the invention are potent inhibitors of the binding of pentagastrin to the receptors of the cortical membranes of mice. The most active compounds are in fact about 10 times less potent than the specific antagonist (pentagastrin) and about 100 times more potent than the CCK-antagonist lorglumide. The derivatives belonging to the R series (for example, the compound C-1) were about 50 times more active than the corresponding S enantiomers (compare the compounds C1 and C32).
In general, there is some correlation between the peripheral and central antigastrin activities of the compounds of the invention, and this leads to the assumption that the two types of receptors are quite similar. However, the two types of activity, that is the inhibition of gastric secretion stimulated by pentagastrin (see Table 1) and the inhibition of the binding of pentagastrin to the cortical membranes (see Table 3) do not always completely correspond. Thus, for example, the compound C1 which is about 1.5 times more active than the compound C2 on the peripheral gastrin (gastric secretion) receptor is only one third as active as C2 centrally. Similarly, the compound C5, which is slightly more active than the compound C-30 as a peripheral pentagastrin inhibitor is three times less active than that compound on the central receptor.
The considerable activity of some of the compounds of the invention in displacing pentagastrin from the central receptor sites may be assumed to be of possible practical interest since it suggests the possible use of the products of the invention in the treatment of cerebral disorders attributable to an imbalance in the physiological neuron levels of gastrin.
TABLE 3______________________________________Inhibition of the binding of pentagastrinto mouse cortical membranes IC50 IC50COMPOUNDS (moles/liter) COMPOUNDS (moles/liter)______________________________________Pentagastrin 2.5 .times. 10-9 C23 3.2 .times. 10-7C1 4.7 .times. 10-7 C29 8.0 .times. 10-7C2 1.4 .times. 10-7 C30 3.0 .times. 10-8C5 1.1 .times. 10-7 D1 2.6 .times. 10-6C6 3.2 .times. 10-7 D2 IN (>10-5)C8 2.0 .times. 10-7 D4 6.8 .times. 10-6C10 1.0 .times. 10-7 D5 1.5 .times. 10-6C14 8.1 .times. 10-6 Lorglumide (1) 6.1 .times. 10-6C21 5.1 .times. 10-7 C32 (1) 2.6 .times. 10-5______________________________________ Note (1): these compounds were tested for comparison
Anticholecystokinin (anti-cc, K) activity
In order to confirm the hypothesis that the molecular conformations of the compounds of the invention are suitable to provide them with antagonistic activity towards CCK as well as towards gastrin, the anticontracturant activities of some of the compounds of the invention were tested on guinea-pig gall bladders stimulated by CCK-8 in vitro.
A longitudinal strip of guinea-pig gall bladder was placed in a bath for isolated organs in the presence of Krebs at a temperature of 32.degree. C. and oxygenated continuously with an oxygen-CO.sub.2 mixture (95-5 V/V).
The isometric contractions were detected by means of a force transducer and recorded.
The gall bladder was contracted with the use of a concentration of 10 ng/ml of CCK-8; the antagonistic activities of the compounds towards the contracturant effect of the CCK was determined with the use of different concentrations, thus determining the IC50 values, that is, the concentration of the compound in mcg/ml which can antagonise the contracturant effect of CCK by 50%.
The results obtained are given in the following table which shows the compounds tested and the IC50s found, which were calculated by the regression method on a set of at least 3 tests for each compound tested.
TABLE 4______________________________________Anti-CCK activity (concentration 10 ng/ml)expressed as the IC50 IN mcg/ml on guinea piggall bladders in vitro.COMPOUND ACTIVITY IC50 mcg/ml)______________________________________C1 20C2 4.9C3 43C5 34.8C8 1.7C10 1.0C19 1.5C20 0.5C21 0.2C29 234C30 59D2 108D4 36Lorglumide 0.06______________________________________
It can be seen from an examination of the table that some of the compounds claimed, such as, for example, the compounds C-8, C-10,C-19, C-20 and C-21, antagonise the contracturant activity of CCK-8 at concentrations no greater than those shown by lorglumide which was the specific CCK antagonist selected for comparison.
Inhibitory effect on the rate of growth of tumorous colic cells induced by pentagastrin
Gastrin has a trophic effect on the digestive epithelium. The chronic administration of gastrin (or pentagastrin, the biologically active portion of the physiological hormone) to rats causes hyperplasia of the fundic and colic mucosae. It has recently been shown that gastrin stimulates the growth of a transplantable mouse colic tumour produced by chemical induction [Winsell et al. --Surgical Forum, 33, 384 (1982)].
Upon the basis of this premise, it was wished to investigate whether the gastrin-specific antagonists of the invention could antagonise the growth of experimentally-induced gastrointestinal tumours.
One of the gastrin antagonists of the invention which was amongst the most active in the tests described above, that is the compound C-5, was selected for this purpose. Male mice weighing 20-25 g were inoculated subcutaneously in the interscapular region with a suspension of 8.times.10.sup.4 tumour cells of a mouse colic adenocarcinoma.
4 groups each of 8 animals were used, that is: a control group, a group of animals which were treated with pentagastrin 250 mcg/Kg twice a day, and two groups of animals which were treated with the compound C-5 at doses of 3 and 10 mg/Kg i.p. respectively twice a day in addition to pentagastrin as described above.
After 21 days, the animals were killed and the fundic mucosa and the tumours were removed, weighed and extracted to determine the DNA. The results obtained are given in Table 5 below.
TABLE 5__________________________________________________________________________Antagonistic activity of the compound C-5 on colic tumorous growthinduced by pentagastrin__________________________________________________________________________ WEIGHT OF THE No. MUCOSA FUNDICA Student's FUNDIC DNA Student'sGROUPS ANIM. Mean (mg) .+-. SD t-test Mean (mg) .+-. SD t-test__________________________________________________________________________A): Control 8 18.4 .+-. 1.95 -- 0.237 .+-. 0.03 --B): Pentagastrine (PEG) 8 32.0 .+-. 3.50 VsA: 10.3 (***) 0.330 .+-. 0.05 VsA: 4.96 (***)C): PEG + C-5 (3 mg/Kg- 8 23.85 .+-. 3.48 VsA: 3.34 (**) 0.268 .+-. 0.03 VsA: 2.39 (*)2 .times. dle) VsB: 4.35 (***) VsB: 3.47 (**)D): PEG + C-5 (10 mg/Kg- 8 18.90 .+-. 2.54 VsA: 0.52 0.252 .+-. 0.03 VsA: 1.062 .times. dle) VsB: 9.21 (***) VsB: 4.17__________________________________________________________________________ (***) WEIGHT OF THE COLIC TUMOROUS No. TUMOR Student's DNA Student's GROUPS ANIM. Mean (mg) .+-. SD t-test Mean (mg) t-testD__________________________________________________________________________ A): Control 8 520.1 .+-. 36.2 -- 2.73 .+-. 0.35 -- B): Pentagastrine (PEG) 8 657.0 .+-. 97.7 VsA: 3.97 (**) 3.62 .+-. 0.50 VsA: 4.42 (***) C): PEG + C-5 (3 mg/Kg- 8 601.5 .+-. 91.7 VsA: 2.49 (*) 3.24 .+-. 0.42 VsA: 2.05 (*) 2 .times. dle) VsB: 1.25 VsB: 1.74 D): PEG + C-5 (10 mg/Kg- 8 554 .+-. 61.7 VsA: 1.44 2.94 .+-. 0.31 VsA: 1.36 2 .times. dle) VsB: 2.69 (*) VsB: 3.51__________________________________________________________________________ (**) (*): P < 0.05 (**): P <0.01 (***): P <0.001
The data of Table 5 show that pentagastrin induces significant hyperplasia in the fundic mucosa and a significant increase in the weight of the colic tumour and the DNA content. The compound C-5 can inhibit both the aforementioned trophic effects of pentagastrin in a significant and dose-dependent manner.

Number	Name	Date	Kind
4791215	Rovati et al.	Dec 1988
5064853	Gasc et al.	Nov 1991

Number	Date	Country
0383690	Aug 1990	EPX
2160869	Jan 1986	GBX
9106529	May 1991	WOX

Amidic derivatives of glutamic, aspartic and 2-amino adipic acids, a process for preparing same, and anti-gastrin composition containing the derivatives

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