WO1997044341A1

WO1997044341A1 - Cyclic polyamides of glutamic acid and aspartic acid with anti-gastrin activity, a method for their preparation and their pharmaceutical use

Info

Publication number: WO1997044341A1
Application number: PCT/EP1996/002121
Authority: WO
Inventors: Francesco Makovec; Walter Peris; Lucio Claudio Rovati; Luigi Angelo Rovati
Original assignee: Rotta Research Laboratorium S.P.A.
Priority date: 1996-05-17
Filing date: 1996-05-17
Publication date: 1997-11-27

Abstract

Compounds of general formula (I) in which: R1 is a simple phenyl group or a phenyl group mono- or di-substituted with chlorine; R2 is H or CH3; R3 is a heterocyclic spiro ring group; s is 1 or 2; R4 and R5 are selected independently from H, phenyl and linear or branched alkyl or alkoxyalkyl groups containing from 1 to 6 carbon atoms or together form a cyclic, bicyclic or spiro bicyclic ring group containing from 5 to 11 carbon atoms overall; X is S (sulphur) or O (oxygen); R6 and R7 are independently H or CH3; z is 1 or 2; u is 0 (zero) or 1; t is a whole number from 0 to 2.

Description

CYCLIC POLYAMIDES OF GLUTAMIC ACID AND ASPARTIC

ACID WITH ANTI-GASTRIN ACTIVITY, A METHOD

FOR THEIR PREPARATION AND THEIR

PHARMACEUTICAL USE

The subject of the present invention is new derivitives of glutamic acid and aspartic acid which may be represented by the general formula (I) shown below

in which:

Ri is a simple phenyl group or a phenyl group mono- or di- substituted with chlorine;

R₂ is H or CH₃;

R₃ is a heterocyclic spiro ring group represented by:

in which Y is selected independently from CH₂ and 0 (oxygen) and n is 0 (zero) or 1; s is 1 or 2;

R₄ and R5 are selected independently from H, phenyl and linear or branched alkyl and alkoxyalkyl groups containing from 1 to 6 carbon atoms, or together form a cyclic, bicyclic or bicyclic spiro group containing from 5 to 11 carbon atoms overall; X is S (sulphur) or 0 (oxygen) ; R₆ and R₇ are independently H or CH₃; z is 1 or 2; u is 0 (zero) or 1; t is a whole number from 0 to 2; the stereochemistry of the chiral centre indicated * in the general formula (I) is the D (dextro) configuration; the stereochemistry of the chiral centre indicated ** in (I) may be D (dextro), racemic (DL) or L (laevo) ; the stereochemistry of the carbon atom indicated *** in (I), which may be asymetric depending on the substituents bonded thereto, may be D (dextro) , (DL) or L (laevo) .

Preferably Ri is a phenyl group substituted with chlorine in the 3 and 5 positions, R₃ is the group 8- azaspiro [4.5]decan-8-yl, s is 2, R₄ and R₅ together form a ring with 6 carbon atoms, mono- or di- substituted with a methyl group, or R₄ is pentyl while R₅ is H, X is S (sulphur) , R₆ and R₇ are both H, z is 1, u and t are both 0 (zero) and the stereochemistry of the chiral centre indicated (**) in (I) is D (dextro) .

The compounds of the present invention have been shown to be powerful antagonists to the gastrin receptors at the peripheral level, that is in the gastrointestinal system, and to the cholecystokinin (CCK) receptors in the central nervous system (CCK-B-antagonists) . It may thus be thought that they may be usable to advantage in the treatment of various illnesses in man linked to imbalances in the physiological levels of gastrin and CCK or other bioactive polypeptides correlated therewith, both in the gastrointestinal tract and in the central nervous system (CNS) or in other organs or systems in which these bioactive peptides play a physiological or pathological role. Thus, for example, one may forsee an advantageous use of these compounds in the treatment, at the gastrointestinal level, of illnesses linked to disturbances in motility and mucous trophism such as, for example, gastritis, peptic ulcers, colitis and certain forms of gastrointestinal tumors supported by gastrin or polypeptide hormones correlated therewith and, at the level of the CNS, for treating mental disorders such as anxiety, panic attacks, psychosis, such as, for example schizophrenia, anorexia etc. Another use could be in the treatment and prevention of some pathological conditions of the eye such as, for example, myosis induced during surgical treatment for cataracts or chronic eye inflammation.

Pharmaceutical forms of the compounds which are the subject of the invention may be prepared by conventional techniques as, for example, tablets, capsules, suspensions, solutions and suppositories, and may be administered orally, parenterally, rectally, to the eyes or transdermally or in other forms suitable for achieving the therapeutic effect.

The active ingredient is administered to the patient typically in doses of from 0.01 to lOmg/kg body weight per dose. For parenteral and occular administration it is preferable to use a water soluble salt of the compounds in question such as the sodium salt or another non-toxic and pharmaceutically acceptable salt. The inactive ingredients used may be those substances commonly used in pharmaceutical preparations as eccipients, binders, aromatising compounds, dispersants, colouring agents, humectants etc. The process for the preparation of the derivatives of glutamic acid and aspartic acid according to the invention consists of the amidation of an acid derivative of formula (II) :

in which Ri, R₂, R₃ and s have the meanings indicated above, with suitable amino acids of formula (III) :

in which R₄, Rs? X/ Re/ R7, u, z, t have the meanings indicated above, to give the corresponding derivatives of formula (I), according to the general reaction below:

The amidation process is preferably carried out by the mixed anhydride method in an inert solvent, at a

temperature of between -15°C and +15°C or by other appropriate conventional methods.

The starting acid derivatives of formula (II) are prepared as described in [Makovec et al, J.Med.Chem. 3_5 (1992), 28-38] while the amino acids of formula (III) are available commercially or are described in the literature or are prepared by conventional methods described below. The following examples are given in order better to illustrate the invention.

Example 1 Preparation of (D) -3-carboxy-8-methyl-l-thia-4- azaspiro [4.5] decane [Compound (h) - Table 2] .

73g (0.61 moles) of L-cisteine were dissolved in 850mL of hot water. The solution was brought to ambient temperature and a solution of 68g (0.61 moles) of 4- methyl-cyclohexanone in 350mL of 95% ethanol were added dropwise. After 12 hours at ambient temperature the white solid formed was filtered and washed successively with water and petroleum ether, giving 83g of product with a yield of 64% (Cι₀H₁₇NO₂S) .

M.p 110-112°C; TLC (butanol/AcOH/water 5:2:2) Rf 0.64 Rotatory power [α]_D = -103°(c=1.5 in AcOH)

Example 2

Preparation of (D,D) -1-[3-carboxy-8-methyl-l-thia-4- azaspiro[4.5]decan-4-yl]-l-oxo-5- (8-azaspiro[4.5]decan-8- yl) -4- (3, 5-dichlorobenzoylamino) -5-oxopentanoic acid [Acid of Compound 9 - Table 1] .

37.3g (0.084 moles) of 5- (8-azaspiro [4.5]decan-8-yl) -4- (3, 5-dichlorobenzoylamino) -5-oxopentanoic (D) acid (CR 2194) and 12.4mL of triethylamine (0.088 moles) were dissolved at ambient temperature in 700mL of tetrahydrofuran (THF) and the solution was cooled to -5°C. This temperature was maintained while 8.4mL (0.088 moles) of ethyl chloroformate were added. At the end of the addition, the mixture was left to react for 15 minutes, still at a low temperature, and then 20g (0.093 moles) of (D)-3-carboxy-8-methyl-l-thia-4- azaspiro [4.5]decane dissolved in 300mL of THF were added slowly, the temperature still being kept at -5°C. At the end of this addition, the reaction mass was made to react at ambient temperature for about a further 12 hours. The precipitate was then removed by filtration and the solvent was evaporated under vacuum. The residue, dissolved in ethyl acetate, was washed appropriately with HCI and then with NH₄OH to remove the compound (h) and the CR 2194 which had not reacted. 36g of product was recovered after evaporation of the solvent, giving a yield of 67% (C₃ιH4iCl₂N₃0₅S) used as such in the following example.

Example 3

Preparation of: (D,D) -1- [3-carboxy-8-methyl-l-thia-4- azaspiro [4.5]decan-4-yl]-l-oxo-5- (8-azaspiro [4.5]decan-8- yl) -4- (3, 5-dichlorobenzoylamino) -5-oxopentanoic sodium salt [Compound 9 - Table 1] .

25g of the acid of compound 9 (0.039 moles) and 4.6g of sodium carbonate (0.041 moles) were mixed in IL of 95% ethanol. The suspension was refluxed for 6 hours. After this period of time, the solid in suspension was filtered off and the solvent was evaporated under vacuum. The residue was made friable with acetonitrile and filtered, giving 21g of sodium salt with a yield of 81% (C₃ιH₄oCl₂N₃Na0₅S) . Mp 229-231°C; TLC (iAmOH/acetone/water 5:2:l)Rf 0.61

Rotatory power [α]_D = -62.2°(c=2 in MeOH)

All the compounds of formula (I) were synthesised by an analogous procedure. Table 1 below gives several derivatives of formula (I) obtained in this manner together with some physico-chemical properties which identify them. It should be noted that several of these compounds, including mixtures of diastereoisomers depending on the substituents in R₄ and R₅, may give rise to splitting in thin-layer chromotography, as shown for example for the compounds 2, 5, 6, 13, 14, and 15.

Table 2 gives some physico-chemical data for the amino acid intermediates of formula (III) not previously known and used in the preparation of derivatives described in Table 1 and some data from literature regarding the known intermediates.

TABLE 1 : COMPOUNDS OF FORMULA:

TABLE 2: COMPOUNDS OF FORMULA:

Compound Rz R. Config Formula Melting TLC Rotatory CAS-Registry (") Point (°C) (RO¹ Power² Number a isopropyl H D C₇H₁₃N0₂S - - - 14347-75-2 b 2,2-dimenthyl-propyl H D C₉H₁₇N0₂S 159-160 0.71 -108.6 - c butyl H D C₈H₁₅N0₂S - - - 90205-28-0 d pentyl H D C₉H₁₇N0₂S - - - 69588-05-2 e cyclohexyl H D C,oH₁₇N0₂S - - - 1010-28-2 f phenyl H D C,oH_πN0₂S - - - 42607-21-6 g cyclohexyl D C₉H₁₅N0₂S - - - 56888-62-1 h 4-methyl-cyclohexan- 1 -yl D C₁₀H₁₇NO₂S 1 10-1 12 0.64 -103.0 - i 4,4-dimethyl-clycohexan- 1 -yl D C„H₁₉N0₂S 191-194 0.70 -98.7 -

1 spiro[5.5]undecan-3 -yl D C₁₄H₂₃N0₂S 195-197 0.68 -90.7 - m spiro[5.5]undecan-3 -yl L C₁₄H₂₃N0₂S 192-193 0.67 +88.7 -

(1): eluent.n-BuOH/AcOHΛvater 5:2:2 (v/v/v) (2): AcOH (c = 1.5)

Description of the pharmacological activity

1) Anti-cholecystokynin activity (anti-CCK-B) in vitro

The capacity of some of the compounds which are the subject of the invention to interact with the central CCK-B receptor was evaluated with the use of [3-H] [N- methyl-N-leucine]CCK-8. The binding was shown to be selective for the CCK-B receptors, having an affinity about 4000 times higher for the receptors of the cortex (CCK-B) than those of the pancreas (CCK-A) in guinea pigs [Knapp et.al; J.Pharmacol, and Exp. Therap 255 (3) (1990), 1278-1286] . The cerebral cortices of male albino guinea pigs were therefore used, the method given above being followed with slight modifications [Makovec et al.; Bioorganic & Med. Chem. Letters 3 (5) (1993) , 861-866] so as to give a membrane content corresponding to about 300mcg of protein/mL. The results obtained are given in Table 3 which shows the IC₅₀ values, that is, the concentration (in μmoles/litre) of antagonist capable of displacing 50% of the [3-H] [N-methyl-N-leucine]CCK-8 from the receptor. Table 3

Inhibition of the binding of (*H) - [N-Methyl-N-leucine] CCK-8 to the cortical membrane in guinea pigs

Compounds ICsoxlO^"^ Compounds IC₅₀xl0 ^®M

1 38.5 11 21.6 2 18.6 12 1500 3 13.4 13 36.8 4 13.7 14 15.8 5 13.5 15 56.7 6 32.5 16 38.5 7 35.5 CR 2194 240 8 23.0 Pentagastrin 0.3

9 6.0

10 3.9

From the data given in Table 3 it is seen that many of the compounds of the invention, such as, for example, the compounds 9 and 10, are powerful inhibitors of the binding of [N-methyl-N-leucine] -CCK-8 to the receptors of the- cortical membrane in guinea pigs. Some of them are in fact 50 times more powerful than their precursor, that is, the gastrin antagonist CR 2194, while they are only 10 to 15 times less active than the specific antagonist pentagastrin.

The displacement activity is strongly conditioned by the stereochemistry of the carbon atom indicated (**) in the general formula (I) . In fact, for example, the compound

11 is about 70 times more active than its L-diastereoisomer, the compound 12.

2) Anti-gastrin activity (peripheral) in rabbit gastric mucosa cells in vitro

The parietal cells of the gastric mucosa are responsible for the secretion of HCI. They present specific membrane receptors which may be activated by gastrin and which have been defined as gastrin or type-B cholecystokynin

(CCK-B) receptors.

Since it has been observed that the activation of the CCK-B receptors by gastrin results in a raising of the levels of cytosolic calcium ions, a technique has been used for measuring the increase in intracellular calcium induced by gastrin in the presence and absence of the compounds which are the subject of the invention as an index of the anti-gastrin activity of the compounds themselves. Suspensions (0.8 x 10⁶/ml) of rabbit gastric mucosa cells were prepared by conventional techniques with the use of collagenase and pronase as digestive enzymes; the estimation of the [Ca²⁺h values, basal or achieved after stimulation of the cell system, was carried out in accordance with Grynkiewicz et al [J.Biol.Chem 260 (1985), 3440]. In the control samples the cells were stimulated with 5 x 10^"8 gastrin while in the samples in which the effect of the compounds in question was evaluated, the cells were incubated therewith before stimulation with gastrin. The results are expressed as percentage increases in [Ca²⁺]ι with respect to the control value. The anti-gastrin activity of the compounds was expressed as the IC₅o value, that is, the concentration (in μmoles/litre) at which the response to the stimulus induced by the gastrin was reduced by 50%. The results thus obtained for several compounds of the invention are given in Table 4 which also gives an index formed from the ratio between the peripheral anti-gastrin activity just described and the displacement activity derived from the study of binding to the cortical receptors of guinea pigs described above. Table 4

Inhibition of the increase in cytosolic calcium induced by gastrin in rabbit gastric mucosa cells.

Compounds IC5oXlO^_8M Ratio IC₅oBinding cortex*

IC₅₀ (Gastric mucos)

I 20.6 1.9 2 4.0 4.7

3 2.3 5.8

4 2.7 5.1

5 1.1 12.3

6 3.2 10.1 7 2.3 15.4

8 1.7 13.5

9 0.3 20.0

10 0.8 4.9

II 1.7 12.7 12 30.0 50.0

13 3.0 12.3

14 5.3 3.0

15 4.2 13.5

16 5.7 6.7 CR 2194 38.0 6.3

(*) values taken from Table 3 From the results given in Table 4 it is seen that many of the compounds of the invention are powerful inhibitors of the increase in cytosolic calcium induced by gastrin in rabbit gastric mucosa cells.

The peripheral anti-gastrin activity essentially accords well with the anti-gastrin activity obtained centrally from the binding studies illustrated previously in Table 3. In fact, the compounds 9 and 10 are also, in this case, the most powerful of the compounds described, exhibiting IC₅₀ values of nanomolar order of magnitude. Generally those compounds of the series in question which have D-chirality at the chiral centre indicated (**) in formula (I) display anti-gastrin activity in this model at concentrations 2-20 times less than those obtained centrally. Again in this case, the most powerful compounds are about 50-100 times more active than their precursor, CR 2194.

3) Anti-cholecystokynin activity (anti-CCK-A)

In order to test the hypothesis that the compounds in question are specific CCK-B antagonists, all the compounds illustrated in Tables 3 and 4 were also tested for possible CCK-A antagonist activity. The experimental model used was the guinea pig gall bladder stimulated in vitro by CCK-8 according to the method described by Makovec et al. [ (Arzneim. Forsch / Drug. Res. .3_5 (⁷)' 1048-1051 (1985)] .

None of the compounds tested displayed any anti-CCK-A activity more powerful than 10 x IO^"6 M.

If these activities are compared with the anti-CCK-B activity illustrated previously in Table 4, it may be concluded that the compounds in question are specific antagonists for the CCK-B receptor, the more powerful compounds, such as the compounds 9 and 10, exhibiting an affinity at least 1000 times higher for the gastrin receptor (CCK-B) than for the cholecystokynin receptor (CCK-A) .

4) Anxiolytic activity

Among the possible therapeutic activities of the compounds in question on the central nervous system, linked to imbalances in the physiological neural levels of gastrin or other peptides correlated therewith, their potential anxiolytic activity appears particularly interesting.

It has in fact recently been postulated that the central CCK-B receptor plays an important role in anxiety. This accords with studies also carried out in man which have 1& shown that the central CCK-B mechanisms have an important function in the mediation of panic attacks [Bradwejn, J.et al; J.Psychopharmacology 6 (1992), 345] . In order to confirm this hypothesis, the anxiolytic activity of several of the most powerful CCK-B antagonists of the invention were evaluated by the method of the "elevated plus-maze" in rats, carried out in accordance with Pellow et al. [J. of Neurosc. Meth. 1_4(1985), 149-167] . A labyrinth was used in which the length of the cross arm was about 45cm placed at a height from the ground of 70cm. On this experimental model, a compound having anxiolytic activity produces a percentage increase in the stay time in the open arms and a percentage increase in the number of entries into the open arms.

The results obtained are given in Table 5 below where the activities obtained with different doses of the compound 9 administered intravenously are given in comparison with a group of animals treated with physiological saline in the same way.

TABLE 5

ANXIOLYTIC ACTIVITY IN RATS IN THE "PLUS MAZE" TEST

Compounds Dose mg/kg No. Animals Entries to open arm/ % Effect Vs Time in open arm/ % Effect Vs

IP total entries (%) controls total time ^(a/- controls

Controls 12 20.7 10.4

Compound 9 0.03 12 32.2(*) 55.5 17.7 71.2

0.3 12 31.9(*) 54.2 23.8(*) 130.0 3.0 12 34.2(*) 65.4 22.6(*) 118.0

(*): Duncan test: p<0.05 vs control group

From an examination of Table 5 it is seen that the compound 9 displays a powerful anxiolytic effect.

It is in fact seen that the compound increases the percentage entries into the open arm over the total number of entries to a significant extent at all the doses used. This percentage increase is about 50-60% compared with the control group for all the doses used.

The compound 9 also increases the stay time in the open arms at all doses; this increase is significant for the doses 0.3 and 3mg/kg. For this parameter the compound displays a bell profile typical of compounds which act on the central nervous system.

Claims

22 CLAIMS

1. Compounds which may be represented by the general formula (I) are indicated

in which Ri is a simple phenyl group or a phenyl group mono- or di- substituted with chlorine; R₂ is H or CH₃; R₃ is a heterocyclic spiro ring group represented by:

R₄ and R₅ are selected independently from H, phenyl and linear or branched alkyl and alkoxyalkyl groups containing from 1 to 6 carbon atoms, or together form a cyclic, bicyclic or bicyclic spiro ring group containing from 5 to 11 carbon atoms overall; X is S (sulphur) or 0 (oxygen) ; R₆ and R₇ are independently H or CH₃; z is 1 or 2; u is 0 (zero) or 1; t is a whole number from 0 to 2 the stereochemistry of the chiral centre indicated * in the general formula (I) is the D (dextro) configuration; the stereochemistry of the chiral centre indicated ** in (I) may be D (dextro), racemic (DL) or L (laevo); the stereochemistry of the carbon atom indicated *** in (I), which may be asymetric depending on the substituents bonded thereto, may be D (dextro) , (DL) or L (laevo) .

Preferably Ri is a phenyl group substituted with chlorine in the 3 and 5 positions, R₃ is the group 8- azaspiro[4,5]decan-8-yl, s is 2, R₄ and R₅ together form a ring with 6 carbon atoms, mono- or di- substituted with a methyl group, or R₄ is pentyl while R₅ is H, X is S (sulphur) , R₆ and R₇ are both H, z is 1, u and t are both 0 (zero) and the stereochemistry of the chiral centre indicated (**) in (I) is D (dextro) .

2. Compounds according to Claim 1 of general formula (I) in which Ri is the group 3, 5-dichlorophenyl, R₂ is H or CH₃, R₃ is the group 8-azaspiro[4, 5]decan-8-yl, s is 2, R₄ and Rs together form a simple cyclohexyl group or a cyclohexyl group mono- or di- substituted with a methyl group, R₆ and R₇ are both H, z is 1, u and t are both 0 (zero) , X is S (sulphur) and the stereochemistry of the chiral centre indicated (**) in (I) is D (dextro) .

3. Compounds according to Claim 1 of general formula (I) in which Ri is the group 3, 5-dichlorophenyl, R₂ is H or CH_3/ R₃ is the group 8-azaspiro [4, 5]decan-8-yl, s is 2, R₄ is pentyl, R5 is H, R₆ and R₇ are both H, z is 1, u and t are both 0 (zero), X is S (sulphur) and the stereochemistry of the chiral centre indicated (**) in (I) is D (dextro) .

4. A pharmaceutical preparation including at least one of the compounds of Claim 1 or a pharmaceutically acceptable salt thereof as its active ingredient.

5. A pharmaceutical preparation according to Claim 4 for therapeutic use related to its anti-ulcer activity.

6. A pharmaceutical preparation according to Claim 4 for use in the treatment of tumors supported by gastrin and other bioactive polypeptides correlated therewith.

7. A pharmaceutical preparation according to Claim 4 for use in the treatment of gastrointestinal disorders such as non-ulcerous dispepsia and irritable colon.

8. A pharmaceutical preparation according to Claim 4 for the treatment of pathological conditions of the central nervous system linked to imbalances in the physiological neural levels of gastrin or other bioactive polypeptides correlated therewith, such as, for example, dysphoria, panic attacks, psychosis, anorexia etc. or other causes correlated with the mechanism by which the compounds of Claim 1 act.

9. A pharmaceutical preparation according to Claim 4 further including pharmaceutically acceptable inactive ingredients selected from the group consisting of vehicles, binders, aromatising agents, dispersing agents, preservatives, humectants and mixtures thereof, or ingredients which facilitate transdermal absorption.

10. A pharmaceutical preparation according to Claim 6 for use in the treatment and prevention of pathological eye conditions such as myosis induced by surgical treatment for cataracts or chronic eye inflammation, or for the prevention of pathological conditions of other sensory apparatus connected to the mechanism by which the compounds according to Claim 1 act.

11. A process for the preparation of a derivitive of general formula (I) in which R_w R₂, R₃, R₄, R₅, B-βr R?, X, s, z, u, t have the meanings given in Claim 1 and in which the substituents on the chiral centre indicated (*) are in the D configuration and the substituent on the chiral centres indicated (**) and (***) are in the D, DL or L configurations, which consists of the amidation of the acid derivatives of formula (II) :

in which Rx, R₂, R₃, and s have the meanings indicated above, with suitable amino acids of formula (III) :

in which R₄, R₅, R₆, R₇, u, z, t have the meanings indicated above, in a molar ratio of 1 to 3, at a

temperature of between -15°C and +20°C by the mixed anhydride method or by other conventional equivalent methods of synthesis, in the recovery of the compounds (I) from the reaction mass as such or as pharmaceutically acceptable salts, and their purification by conventional methods.