CA2083507A1 - Nicotinylalanine as a therapeutic agent acting on the central nervous system - Google Patents

Abstract

Nicotinylalanine or physiologically equivalent derivatives thereof are used for the preparation of a medicament for the treatment of conditions characterised by an abnormal increase in the metabolism of tryptophan and abnormal excitatory neurotransmission.

Description

W09ltl7750 PCT/EP91/~0950 ~- 2 0 8 3 ~

NICOTINYLALANINE AS A THERAPEUTIC AGENT ACTING ON THE
CENTRAL NERVOUS SYSTEM

The present invention xelates to the use of nico-tinylalanine as a therapeutic agent in the txeatment of diseases of the central nervous system.
Nicotinylalanine, which has the following structu- ;
s ral formula:

~ ,.
having an asy~etric carbon, exists in enantiomeric (R
or S) or racemic form. The invention relates either to the use of one of the two enantiomers or to the use of the racemic mixture.
The single enantiomers of nicotinylalanine can be obtained by conventional methods of optical resolution, starting from the racemic compound. Alternatively, they can be obtained by the stereoselective synthesis herei-nafter disclosed.
Nicotinylalanine has been known for some time:
about 30 years ago, it was believed to be a metabolic product of tryptophan and an intermediate metabolite of the so-called kynurenine pathway, which is the sequence of metabolic events leading to the opening of the indole ring of tryptophan and hence the formation of NAD.
Howevex, it was soon found that this hypothesis was not sustainable experimentally and that nicotinyla-lanine, ~though not a metabolite of tryptophan, might nevertheless intervene as an inhi~itor by virtue of i~s metabolism in at least two enzyme systems: kynurenine-ase and kynurenine hydroxy.lase (Decker et al., J. Biol.
Chem., 1963; 238; 1049-1053).
These enzymes catalyse the metabolism of kynure-S nine, first to 3-hydroxykynurenine and then to 3-hy-droxyanthranilic acid, the direct precursor of quinoli-nic acid.
Much more recently it was found that 3-hydroxyky-nurenine can be toxic for neuronal cells in culture and that quinolinic acid is a toxin capable of stimulating neurones and possibly causing their death, both in in vitro preparations and in vivo (Stone and Connick; Neu-roscience, 1985; 15; 597-618).
The mechanism responsible for this neuronal death is one of competitive inhibition of kynurenine hy-droxylase and ~ynureninease. Along the metabolic pathway leading from tryptophan to NAD it is also pos-sible for kynurenic acid to be formed by a simple en-zymatic process that provides for the intervention of a transaminase.
This acid is capable of inhibi~ing the toxic sti-mulant effect of quinolinic acid by acting as antaaonist of the effects of the latter on the NMDA
receptors by a modulatins mechanism that has now been adequately clarified (Moroni et al., Eur. J. Pharmac., l989; l63; 123-126 ) .
Below is illustrated the metabolic pathway of tryptophan and the so-called kynurenine pathway.

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3-indolepyruvic acid _ tryptophan (indoleamine 2,3-dioxygenase) C-CH2-CI-!NH2--COOH
NH--CHO
N-formylquinurenine `:
(formamidase) :, r OH O ~ :
1~ --NH2 ~C--C'12--CH--COOH
_ I ~ NH2 ~,~ ~N~ ~COOH ( quinurenine ~' `NH~~:
-quinurenic acid- transaminase) quinurenine -~ , ...
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NH2 --~--N~2 ` COOH
OH OH

3-hydroxyquinurenine xanthurenic acid :~
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~,COOH~ COOH . ' .

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, WO9ltl77~0 PCT/EP91/00950 c~ ~` 6~
; -4 However, it was not forseeable from current bio-chemical knowledge that nicotinylalanine might have any therapeutic applications; despite being known for seve-ral decades, this substance has, in fact, never been the subject of pharmacological studies.
It has now been found that nicotinylalanine pre-vents the accumulation of toxic metabolites of tryp-tophan and increases the synthesis of metabolic sub-stances with a sedative action, as shown by the fol-lowing pharmacological experiments.
Rats or mice treated with lipopolysaccharides ex-tracted from bacteria have concentrations of quinolinic acid and kynurenic acid in their biological fluids and brain that are three ti~es hi~her than those in con-trols for approximately 70 hours. This is due to theinduction o~ he enz~e indoleamino-2,3-dioxygenase and the resultant increased formation of all the substances originating frcm the kynurenine pathway. When nico-tinylalanine (50-1000 mg/kg i.p.) is administered in addition to the bact~rial lipopolysaccharides, the in-crease in the concentrations of quinolinic acid is si-gnificantly reduced while that in the concentration of kynurenic acid is markedly enhanced. In other words, nicotinylalanine facilitates the formation of a metabo-lic product of try~tophan that has antagonistic pro-perties on the receptors for the stimulant amino acids and reduces the formation of a toxic stimulating sub-stance. Similar results were obtained when appropriate doses of nicotinylalanine were administered together with tryptophan.
~urthermore, in the DBA/2 mouse the simultaneous WOgl/~7750 PCT/~P91/OOg50 2(~3,~ari administration of tryptophan (200 mg/kg i.p.) and nico-tinylalanine (200 mg/kg i.p.) causes sedation and pro-tects the animals from audiogenic convulsions. This suggests that the accumulation of kynurenic acid in the ~rain (as demonstrated by us) is associated with the same behavioural effects in the animal.
In order to examine the versatility of nicotinyla-lanine in a variety of seizure models it was also te-sted against leptazol (pentylenetetxazol; metrazol) and electroshock induced convulsions.
Groups of 10 mice weighing 25-35 g were given ni-cotinylalanine and then leptazol administered (85 mg/kg subcutaneously) 1 hour later. At a dose of 370 mg/kg intraperitoneally, nicotinylalanine protected against lS lepta~ol seizures, increasing the latency to convulsion from 270 to 588 seconds. The leptazol test is widely used as a test system for drugs useful in petit mal epilepsy.
In separate ex~eriments groups of 10 mice were used to determine the threshold electric current needed to induce convulsions from ear electrodes. After treat-ment with nicotinylalanine 370 mg/kg, intraperitone-ally, ~0% of the test animals were protected against convulsions. This test is a frequent indicator of drug efficacy in grand mal epilepsy.
At the effective doses of 370 mg/kg the experimen-tal animals also showed signs of sedation but with mi-nimal ataxia or other motor disturbances.
Nicotinylalanine administration (200-350 mg/kg i.p.) to Swiss mice was also able to completely prevent the seizures induced by i.c.v. administration of kynu-.: : . . .. ; .. .. :. .. : .. i - .. ~ .. . . . . .. . .. .

~ 6 renine, whereas nicotinyl administration (150-400 mg/kg) to ethanol dependent C57 mice prevented withdrawal induced seizures, tremors and changes of body temperature (Ritzmann and Tabakoff 193, 158-170;
1976).
Finally, in hippocampal slices of the rat incuba-ted in the presence of kynurenine, nicotinylalanine (0.1-100 ~M) increases the neosynthesis of kynurenic acid. It has been also observed that nicotinylalanine displaces radioactive glycine from its binding sites in cortical membranes.
It is clear from the above that nicotinylalanine can be used to advantage in various diseases effecting the central nervous system that are characterised by an abnormal increase in the metabolism of tryptophan.
Examples of such conditions in which there is an imbalance petween toxic stimulant substances and their modulators or antagonists include:
1) All infectious diseases of both a bacterial and a viral nature (including ~IDS). Here the activation of the immune system causes an increase in the flow of tryptophan through the metabolic pathway of the kynurenines, which leads to an accumulation of auinolinic acid in the brain ~Heyes et al., Annals Neurol., 1989; ~eyes et al., 1988; 51; 1946-1948);
2) Neoplastic diseases, particularly those affecting the immune system (lymphomas);
3) Treatment with interferons and/or interleukins j~
(Brown f't al., Cancer Res., 1989; 49; 4941~4945);

4) Many convulsive diseases (Lapin et al. r Epilepsia, 1981; 22; 257-265) including variants of grand mal , 2 ~ r~ ~

and petit mal ePilePsiai 5) Huntington's chorea and other degenerative diseases such as Parkinson's disease and senile dementia (Schwarts et al., Life 'iciences, 1984; 35; 19-23);

6~ Liver diseases (Moroni et al., J. Neurochem., 1986;
46; 849-874);

7) Changes in the sleep-wake cycle;

8) The neuropsychiatric changes associated with pellagra and other vitamin deficiencies;

9) Chronic alcoholism where there is a reduction in the levels of kynurenic acid in the brain;

10) Numerous situations of psychiatric interest in which changes in the tryptophan metabolism have been fully documented (depression, schizophrenia, etc.);

11! Stroke and other forms of cerebral ischemia includin~ traumatic head injury;

12) Hypertension and essential tremor syndrome.
For its envisased therapeutic applications, nico-tinvlalanine or physiologically equivalent derivatives thereof (salts, esters, non-toxic amides) will gene-rally be administered in doses of between 10 and 100 mg/kg/day. The exact dosage, however, will depend on various factors, such as the patient's condition and ~5 the nature and severity of the disease.
The substance should be administered by mouth, rectally or parenterally, using conventional pharmaceu-tical formulations such as those described in - Remington's Pharmaceutical Sciences Handbook, Mack Pub.
Co., N.Y., USA, 17th Ed., 1985.
Nicotinylalanine can be prepared by the methods :
', - ':' . .

WO91/17750 ~I PCT/EP91/00950 `?~
~,~J~ 8 described in J. Biol. Chem- 238, 1049 (1963) and J.
Org. Chem. 28, 383 (1953)- Although the use of the sub-stance in the natural L steric configuration is prefer-red, the invention also relates to the use of the race-mic form and the D form of nicotinylalanine. The enan-tiomers can be prepared according to the following scheme and Examples.

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WO91/17750 PCT/EP91/~950 Preparation of S-nicotinylalanine a) S-(3-Benzyloxycarbonyl-5-oxo-4-oxazolidinyl)-acetyl-chloride (3) S-(3-Benzyloxycarbonyl-5-oxo-4-oxazolidinyl)-acetic acid ~J.M. Scholtz, P.A. Bartlett, Synthesis, (1989), 542-543] (2, 0.491 g, 1.76 mmol) was added to a solu-tion of thionyl chloride in toluene (5 ml, 1:1, v:v) and the resulting mixture was stirred at room tempera--ture for 4 h in a argon atmosphere. After evaporation of the solvent, the residue was dried in high vacuum to give pure 3 (0.513 g, 98%). lH-NMR (CDC13) ~: 3.52 (2H, d, CH2CoCl); 4.25 (lH, t, CH); 5.12 (2H, s, CH2Ph);
5.22 and 5.45 (2H, dd, CH2O); 7.30 (5H, s, aromatic's);
13c_NMR (CDC13) ~: 46.7, 51.6, 68.4, 78.4, 128.3, 128.6, 135.0, 152.5, 170.0, 171.6.
b) S-3~(3-Benzyloxycarbonyl-5-oxo-4-oxazolidinyl)-ace-tyl ~yridine (4) (PPh3)2PdC12 (0.051 g, 0.07 mmol) was added to a solu-tion of (3) (0.505 g, 1.70 mmol) and trimethylstan-nylpyridine [Y. Yamamoto, A. Yanagi, Chem. Pharm.
Bull., (1982), 30(5), 1731-I737] (0.595 g, 2.50 mmol) in benzene (20 ml) and the resulting mixture was refluxed for 12 h in a argon atmosphere. After cooling, the catalyst ~as removed by filtration over celite, the filtrate was concentrated in vacuo and then diluted with ethyl acetate (25 ml), washed with saturated sodium hydrogen carbonate solution (5 ml), water (5 ml) brine (5 ml) and dried over anhydrous magnesiumi sul-phate. After evapor2tion of the solvent the residue was submitted to ,lash chromatography: elution with ethyl WO91/17750 ~ PCT/EP9l/OO9So C~rJ

acetate-hexane 8:2 yielded pure 4 (0.276 g, 48%). lH-NMR (CDC13) ~: 3.40 and 3.70 (2H, dd, CH2CO); 4.40 (lH, t, CH), 5.10 (2H, s, CH2ph); 5.40 and 5.53 (2H, dd, CH o); 7.25 (SH, s, Ph); 7.20-7.40 (lH, m, Py); 7.95-8.10 (lH, m, Py); 8.55-8.7() (lH, m, Py); 8.95 (lH, s, Py); 13C_NMR (CDC13) ~: 36.9, 50.8, 67.7, 78.4, 123.5, 128.2, 128.5, 130.9, 131.8, 149.5, 152.5, 154.0, 171.8, 195.3.
c) S-Nicotinylalanine (5) A suspension of (4) (0.087 g, 0.24 mmol) in 6N
hydrochloric acid (10 ml) was refluxed for 8 h. The resulting solution was then washed with ether (5 ml) and the aqueous phase was evaporated off. The residue (0.083 g) was passed through a ion exchange resin column (Dowex lx8, AcO form): elution with 0.3N acetic acid gave pure (5) (0.046 g, 93%), mp 158-160C (d);
H-NMR (D2O) ~: 3.45 (2~, dd, J = 5 Hz and 1 Hz, CH2);
4.02 (lR, dt, J = 5 ~z and 1 Hz, CH); 7.33 (lH, dd, J ~
7.4 Hz and 4 Hz, H-5 Py); 8.19 (lH, dt, J 0 7.4 Hz and 1.5 Hz, H-4 ~y); 8.45 (lH, dd, J - 4 Hz and 1.5 Hz, H-6 Py); 8.78 (lH, a, J - 1.5 Hz, H-2 Py); 13C-NMR (D
39.0, 50.4, 124.6, 131.6, 136.9, 148.5, 153.3, 173.1, lq8.6; [~]D25 = +18.3 (c = 0.6, H2O).

Preparation of R-nicotinylalanine a) R-t3-Benzyloxycarbonyl-5-oxo-4-oxazolidinyl)-acetyl chloride (8) R (3-Benzyloxycarbonyl-5-oxo-4-oxazolidinyl)-acetic acid (7, 0.900 g, 3.22 mmol) was added to a solution of thionyl chloride in toluene (8 ml, 1:1, v.v) and the resulting mixture was stirred at room temperature for 4 -' .

WO9l/177~0 PCT/EP91/00950 2 ~ !~ 3 ~ ~ ~

h in a argon atmosphere. Aft:er evaporation of the sol-vent, the residue was dried in high vacuum to give pure (8) (0.945 g, 98%).
b) R-3-(3-Benzyloxycarbonyl-5-oxo-4-oxazolidinyl)-ace-tyl pyridine (9) (PPh3)2pdC12 tO.100 g, 0.13 mmol) was added to a solu-tion of 8 (0.900 g, 3.03 mmol) and trimethylstannylpy-ridine (1.10 g, 4.50 mmol) in benzene (30 ml~ and the resulting mixture was refluxed for 12 h in a argon at-mosphere. After cooling, the catalyst was removed by filtration over celite, the filtrate was concentrated in vacuo and then diluted with ethyl acetate (35 ml), washed with saturated sodium hydrogen carbonate solu-tion (10 ml), water (10 ml), brine (10 ml) and dried over anhydrous magnesium sulphate. After evaporation of the solvent the residue was submitted to flash chroma-tographv: elution with ethyl acetate-hexane 8:2 yielded pure (9) (0.279 g, 26%).
c) R-Nicotinylalanine (10) A suspension of 9 (0.270 g, 0~74 mmol) in 6N hydrochlo-ric acid (25 ml) was refluxed for 8 h. The resulting solution was then ~ashed with ether (8 ml) and the aqueous Dhase was evaporated off. The residue (0.250 g) was ~assed through a ion exchange resin column (Dowex lx8, AcO- form): elution with 0.3N acetic acid gave pure (10) (0.040 g, 31%), mp 153C (d); [~]D25 = -21 (c - 1, H20 ) . ~ , .

Claims (4)

1. Use of nicotinylalanine or physiologically equiva-lent derivatives thereof as therapeutic agents.

2. Pharmaceutical compositions containing as active principle nicotinylalanine or a physiologically equiva-lent derivative thereof.

3. Pharmaceutical compositions according to claim 2 wherein nicotinylalanine is in the L steric configura-tion.

4. Use of nicotinylalanine for the preparation of a medicament for the treatment of conditions characteri-sed by an abnormal increase in the metabolism of tryp-tophan and abnormal excitatory neurotransmission.