Pyridazino [4,5-b]-quinoline 5-oxide derivatives, their preparation and their use as glycine antagonists

The present invention relates to novel chemical compounds which are 5 pyridophthalazinediones, pharmaceutical compositions containing them and their use against neurological disorders related to excitotoxicity and disorders of glutamatergic neurotransmission.

Background of the Invention and Prior Art

Glutamate is probably the main mediator of stimuli in the central nervous system, but it is also involved in many pathological and excitotoxic processes. In this regard, finding glutamate antagonists for therapeutic use is critical (see review by Dansyz W., Parsons CG,

Bresik I., Quack G. (1995): Drug News & Perspectives 8, p. 261-277). Glutamate activates three major types of ionotropic receptors, namely, α-amino-3-hydroxy-5-methyl-4-isoxazolpropanoic acid (AMPA), kainate, and N-methylD-aspartate (NMDA), and several metabotropic receptor types. Antagonists at NMDA receptors potentially have a wide range of therapeutic applications. Functional

Inhibition of NMDA receptors can be accomplished by acting on a variety of recognition domains, such as the primary transporter region, the strychnine insensitive glycine region (glycine-B), the polyamine region, and the phenylcyclidine region located inside the cation channel.

Receptor desensitization may be a physiological process that implements endogenous control mechanism to prevent long-term neurotoxic activation of glutamate receptors but allowing their short-term physiological activation. In the case of NMDA receptors, the co-agonist glycine is an endogenous ligand that inhibits such desensitization through activation of the glycine-B domain. Interestingly, ischemia increases not only extracellular glutamate levels but also glycine, and although the latter effect is less pronounced, it actually persists for longer. Thus, some full glycine-B antagonists under such conditions could restore normal synaptic transmission by increasing NMDA receptor desensitization to physiological levels. Indeed, the idea of ​​introducing into the central nervous system of laboratory animals suggests that glycine-B antagonists could offer a better therapeutic niche than agents acting at other sites on the NMDA receptor complex. Unfortunately, the poor pharmacokinetic properties of most glycine-B antagonists until recently have not confirmed this prediction when administered to the body. However, several glycine-B antagonists have been reported to have good therapeutic indices in hyperalgesic models and as anxiolytics when administered.

Description of the Invention

We have invented a series of tricyclic pyridophthalazindiones. Class I compounds have similar structures to Zeneca's patented glycine-B antagonists (ICI, EP

0516297 A1,1992.12.02). Class II compounds are N-oxides of those compounds and are neither described nor suggested by Zeneca. Class II conjugates are also potent glycine-B antagonists in vitro and show much better systemic availability and / or penetration through the blood-encephalic barrier in vivo than class I compounds. In addition, the salts of these compounds obtained, for example, by the addition of choline and tetramethylammonium (4-NH₃), has even better bioavailability.

The novel compounds of the present invention have apparent utility in the treatment of the following disorders:

1. Acute excitotoxicity such as ischemia in stroke, trauma, hypoxia, hypoglycemia and encephalopathy.

2. Chronic neurodegenerative diseases such as Alzheimer's disease, vascular dementia, Parkinson's disease, Huntington's disease, multiple sclerosis, lateral amyotrophic sclerosis, AIDS-neurodegeneration, cerebellar atrophy, Ture's syndrome, motor neuron disease, mitochondrial dysfunction, K-dysfunction.

3. Other disorders associated with long-term plastic central nervous system disorders, such as chronic pain, drug tolerance, drug dependence and exercise (eg opioids, cocaine, benzodiazepines and alcohol) and late dyskinesia.

4. Epilepsy (advanced and partial seizures), schizophrenia, anxiety, depression, muscle spasms and ringing in the ears.

The object of the invention

It is an object of the present invention to provide novel and more effective compounds, pyridophthalazinediones, pharmaceutical compositions thereof and methods of treating neurological disorders related to excitotoxicity and disorders of glutamatergic neurotransmission. It is a further object of the invention to provide novel compounds, compositions and methods that satisfy the above theoretical requirements. Additional goals will be clear below, and further goals will be clear to those skilled in the art.

Summary of the Invention

Thus, the invention includes, among others, individually or in combination, the following aspects:

A compound selected from pyridylphthalazinediones of formula

<img file="LT4591B_D0001.tif" />

wherein R 1 and R 2 are selected from the group consisting of hydrogen, halo and methoxy, or wherein R 1 and R 2 together form a methylenedioxy group, and pharmaceutically acceptable salts thereof;

a compound wherein the salt is selected from choline and 4-tetramethylammonium salts thereof;

a compound selected from the group consisting of

4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazinone [4,5-b] quinoline-5-oxide,

8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

7.8-Dichloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

7-bromo-8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide, and

7-chloro-8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide, and pharmaceutically acceptable salts of any of the foregoing; and such a compound selected from the group consisting of

Choline salts of 4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] -quinoline 5-oxide,

8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] quinoline 5-oxide choline salt,

8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide choline salt,

8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide choline salt,

7.8-Choline salts of dichloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

Choline salts of 7-bromo-8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline, and

Choline salts of 7-chloro-8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline;

further comprising a pharmaceutical composition comprising, as active ingredient, an effective glycine-B antagonistic amount of such compound;

such a pharmaceutical composition containing, as active ingredient, an effective glycine-B antagonist amount of such compound in the form of a choline salt thereof;

such a pharmaceutical composition containing as active ingredient an effective glycine-B antagonistic amount of a compound selected from the group consisting of

4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

7,8-Dichloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

7-bromo-8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide, and

7-chloro-8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide, or a pharmaceutically acceptable salt thereof;

such a pharmaceutical composition containing as active ingredient an effective glycine-B antagonistic amount of a compound selected from the group consisting of

Choline salts of 4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] -quinoline,

Choline salts of 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] -quinoline,

8-Bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide choline salt, ·: 8-fluoro-4-hydroxy-1-oxo-1,2 choline salts of -dihydropyridazine- [4,5-b] -quinoline 5-oxide,

7.8-Choline salts of 7.8-dichloro-4-hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] quinoline 5-oxide,

Choline salts of 7-bromo-8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline, and

Choline salts of 7-chloro-8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline;

further comprising applying an effective amount of such a compound, a glycine-B antagonist or pharmaceutical composition, to treat neurological disorders associated with excitotoxicity and disorders of glutamatergic transmission in animals;

such an application wherein the compound is in the form of its choline salt;

such an application wherein the compound is selected from the group consisting of

4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

7.8-Dichloro-4-hydroxy-1-oxo-1,2-dihydropyridazino [4,5-b] quinoline oxide,

7-bromo-8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide, and

7-chloro-8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide, or a pharmaceutically acceptable salt thereof;

further comprising the use of an effective amount of a glycine-B antagonist or pharmaceutical composition for treating neurological disorders associated with excitotoxicity and glutamatergic transmission disorders in animals, wherein the compound is selected from the group consisting of:

Choline salts of 4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] -quinoline 5-oxide,

8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] quinoline 5-oxide choline salt,

8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide choline salt,

8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide choline salt,

Choline salts of 7,8-dichloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline 5-oxide,

Choline salts of 7-bromo-8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline, and

Choline salts of 7-chloro-8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinoline.

Detailed Description of the Invention

The following discussion, examples and pharmacological section are intended to illustrate but not limit the present invention.

Methodologies and results

Basic structure of class I and II tricyclic pyridophthalazindiones:

<img file="LT4591B_D0002.tif" />

<img file="LT4591B_D0003.tif" />

R1 / R2 = H and / or halogen;

R1 / R2 = H and / or O-CH₃;

R1 / R2 = H and / or methylenedioxy;

Chemistry

General procedure for the preparation of quinoline-2,3-dimethyldicarboxylate 1-oxides (3).

A solution of 2-nitrobenzaldehyde 1 (25 mM) and sodium (27 mM) in dry methanol (40 mL) was cooled in an ice bath over 30 min. treated with dimethyl (diethoxyphosphinyl) succinate 2 (30 mM, prepared according to the procedure described by S. Linke et al., Lieb. Ann. Chem. 1980 (4), 542) in dry methanol (10 ml). A dark solution formed for 1.5 hours. After stirring at 0-5 ° C, the solvent was evaporated in vacuo and the residue partitioned between ethyl acetate and water. The ethyl acetate was dried over sodium sulfate and then evaporated in vacuo. The residue is recrystallized from isopropanol to give quinoline-2.39 dimethyldicarboxylate 1-oxide in the form of an off-white (or light yellow) powder.

Physical properties of compounds 3 and ¹Data for H-NMR spectra are shown in Tables 1 and 2.

a. 5-Bromo-4-chloro-2-nitrobenzaldehyde (1f).

To a mixture of sulfuric acid (40 mL) and sodium nitrate (2.66 g, 31.3 mM) was added 3-bromo-4-chlorobenzaldehyde (6.25 g, 28.5 mM) at 0-5 ° C. The resulting mixture 7 hours. stirred at room temperature, then diluted with ice water (300 mL). The precipitate is filtered off, washed with water and dried to give a powder. This material is recrystallized from isopropanol / water (2: 1) to give the title 2-nitrobenzaldehyde 1f (3.6 g, 51.5%) as a light yellow powder; I. temp. 81-82 ° C.

Analysis data C7H₃BrCINO₃: Calculated (%): C 31.79 H 1.14 N 5.30 Found (%): C 31.55 H 0.98 N 5.09 ¹1 H-NMR (CDCl 3)₃), δ: 8.22 (s, 1H), 8.23 ​​(s, 1H), 10.39 (s, 1H).

b. 4-Bromo-5-chloro-2-nitrobenzaldehyde (1g).

According to procedure (a), but starting from 4-bromo-3-chlorobenzaldehyde (2.97 g, 13.5 mM), affords the title compound 1g (1.9 g, 53.0%) as a light yellow powder; I. temp. 95-98 ° C.

Analysis data Č₇H₃BrCINO₃:

Calculated (%): C 31.79 H 1.14 N 5.30 Found (%): C 31.60 H 1.01 N 5.11 ¹1 H-NMR (CDCl 3), δ: 8.02 (s, 1H), 8.43 (s, 1H), 10.39 (s, 1H).

General procedure for the preparation of quinoline-2,3-dimethyldicarboxylates (7). A solution of N-oxide 3 (10 mM) and phosphorus trichloride (30 mM) in dry chloroform (100 mL) was refluxed for 7 hours. The solvent was removed in vacuo and the residue partitioned between ethyl acetate and water.

The organic layer was dried over sodium sulfate and then evaporated in vacuo. The residue is recrystallized from isopropanol to give the title quinoline-2,3-dimethyldicarboxylate 7 as an off-white (or light yellow) powder.

Physical Properties of Compounds 7 and ¹Data for the H-NMR spectra are presented in the 3 and 4 l entels.

General procedure for the preparation of 4-hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] quinolines 5-oxides (5).

hydrazine hydrate (15 mM) was added to a stirred solution of quinoline-2,3-dimethyldicarboxylate 1-oxide 3 (5 mM) in boiling ethanol (25 ml) under argon and the mixture was refluxed for 3 hours. dark precipitate forms over time. After cooling to room temperature, the reaction mixture is filtered and the separated solid is washed with ethanol and ether, dried to give the hydrazine salt 4. This material was stirred in acetic acid at 70-110 ° C for 3 h, cooled to room temperature, diluted with water (45 mL) and filtered. The separated solid is washed with ethanol, dried to give a dark yellow solid. This material is recrystallized several times from dimethylformamide to give the title pyridazine- [4,5-b] -quinoline 525 oxide as 5 orange powders.

Physical properties of compounds 5 and ¹H-NMR data are shown in Tables 5 and 6.

General procedure for the preparation of 1,4-dioxo-1,2,3,4-tetrahydropyridazine- [4,5-b] quinolines (9).

To a stirred solution (or suspension) of quinoline-2,3-dimethyldicarboxylate 7 (5mM) in boiling ethanol (25mL) was added hydrazine hydrate (30mM) and the mixture was refluxed for 8 hours until a precipitate formed. After cooling to room temperature, the reaction mixture is filtered and the separated solid is washed with ethanol, ether and dried; obtains hydrazine salt 8. This substance for 3 hours. stirred in acetic acid (15 mL) at 70-100 ° C, and after cooling to room temperature, the mixture was diluted with water (45 mL) and filtered. The separated solid is washed with ethanol and ether, dried to give the title pyridazine- [4,5-b] quinoline 9 as a yellow powder.

The physical properties and 'H-NMR data of compounds 9 are shown in Tables 7 and 8.

Total choline salts of 6-oxides of 4-hydroxy-1-oxo-1,2-dihydropyridazino- [4,5-b] quinolines and 1,4-dioxo-1,2,3,4-tetrahydropyridazine [4, Methods for the preparation of 5-b] quinoline choline salts (10).

| to a stirred suspension of pyridazine- [4,5-b] -quinoline 9 or N-oxide 5 (10 mM) in methanol (50 mL) was added choline hydroxide (10.5 mM, 45% w / w in methanol). The resulting solution is concentrated in a rotary evaporator and the solid residue is recrystallized from ethanol; obtains the title choline salt in the form of 10 or 6 orange (or red) hygroscopic powders.

Physical properties of compounds 6 and 10 and ¹H-NMR data are given at 9.10 and

11.12 in the tables, respectively.

<img file="LT4591B_D0004.tif" />

table. Compounds 3¹H-NMR (CDCl3) spectral data.

<td>δ (md), J (Hz)</td><td>To 3.98 (s, 3H), 4.11, (s, 3H), 7.66-8.05 (m, 3H), 8.43 (s, 1H), 8.75 (dd, Ji = 8.5, J₂ = 2.0, 1H)</td><td>3.98 (s, 3H), 4.11 (s, 3H), 7.71 (dd, Ji = 8.5, J₂ = 2.5, 1H), 7.91 (d, J = 8.5, 1H), 8.38 (s, 1H), 8.74 (d, J = 2.5, 1H)</td><td>3.91 (s, 3H), 4.07 (s, 3H), 7.13 (dd, Ji = 9.5, J₂ = 2.0.1 H), 7.44 (d, J = 2.0, 1H), 8.22 (s, 1H), 8.58 (d, J = 9.5, 1H)</td><td>z T— 0 in II &lt;N 0 o * II ~ O d co N CO Z ω T— co co z 04 e [ 04 N. N t co 't K Z co 'T Z co Ά- co CJ) co</td><td>3.97 (s, 3H), 4.10 (s, 3H), 8.08 (s, 1H), 8.28 (s, 1H), 8.83 (s, 1H)</td><td>3.97 (s, 3H), 4.09 (s, 3H), 8.26 (s, 2H), 8.82 (s, 1H)</td><td>3.97 (s, 3H), 4.09 (s, 3H), 8.06 (s, 1H), 8.27 (s, 1H), (02 9s, 1H)</td>

<td>The compound</td><td>3a</td><td>3b</td><td>3c</td><td>3d i</td><td>3e</td><td>H— ω</td><td>CD CO</td>

CO ro • g 'ω o

I = r φ

on

L.

ra

TO

Φ

Ę

I co

CM

I c

O

C j =

O • Φ

Φ · ♦ - »c

Φ

<td>Yield (%)</td><td></td><td></td><td> 61,5</td><td> 49,0</td><td> 72,0</td><td> 49,0</td><td> 41,0</td><td> 60,0</td><td> 62,5</td>

<td>Lyd. temp. (° C)</td><td></td><td></td><td> 175-176</td><td> 126-127</td><td> 168-170</td><td> 194-196</td><td> 183-186</td><td> 171-173</td><td> 206-208</td>

<td rowspan="6">Element Analysis</td><td rowspan="3">% Found</td><td>z</td><td> 5,31</td><td>CO Γ'-</td><td> 4,00</td><td> 4,95</td><td> 4,14</td><td> 3,65</td><td> 3,75</td>

<td>Z</td><td> 4,11</td><td> 3,32</td><td> 2,75</td><td> 3,38</td><td> 2,62</td><td> 2,13</td><td> 2,25</td>

<td>o</td><td> 59,84</td><td> 52,80</td><td> 45,57</td><td> 55,19</td><td> 47,18</td><td> 41,39</td><td> 41,68</td>

<td rowspan="3">Estimated (%)</td><td>z</td><td> 5,36</td><td> 4,74</td><td>i— T "</td><td> 5,01</td><td> 4,24</td><td> 3,74</td><td> 3,74</td>

<td>T</td><td> 4,24</td><td>co</td><td> 2,96</td><td> 3,60</td><td> 2,75</td><td> 2,42</td><td>CM 't OJ</td>

<td>o</td><td> 59,77</td><td> 52,81</td><td> 45,89</td><td> 55,86</td><td> 47,30</td><td> 41,69</td><td> 41,69</td>

<td>Formula (molar mass)</td><td></td><td></td><td>ΙΛ O Z CM T o CM r- O</td><td>to o ? R O) T co ' d</td><td>ΙΛ O ? CM "S z £ 2 CO T " O</td><td>m o gg o σ&gt; Z CM CO 6th</td><td>in o Z cz O ° σ&gt; CO Z £ 2 o T " o</td><td>in o z o &lt;2 - Etc. co r &lt; X £ 2 co d</td><td>m o z _ o &lt;2 ώ £ ££ 2 CO d</td>

<td>CM cc</td><td></td><td></td><td>z</td><td>o</td><td>u. m</td><td>LL</td><td>o</td><td>u. CO</td><td>d</td>

<td>oc</td><td></td><td></td><td>z</td><td>z</td><td>z</td><td>Z</td><td>o</td><td>d</td><td>k. co</td>

<td>The compound</td><td></td><td></td><td>(0 CO</td><td>Yeah CO</td><td>o co</td><td>Ό CO</td><td>Φ co</td><td>'-H- co</td><td>D) CO</td>

table. Quinoline-2,3-dimethyldicarboxylates 7.

<td>Yield (%)</td><td></td><td></td><td>o co " co</td><td> 90,0</td><td> 81,5</td><td> 85,0</td><td> 96,0</td><td> 95,0</td><td> 67,0</td>

<td>Lyd. temp. (° C)</td><td></td><td></td><td>90I - V0I.</td><td> 152-154</td><td> 155-157</td><td> 119-121</td><td> 113-115</td><td> 128-130</td><td> 142-144</td>

<td rowspan="6">Element Analysis</td><td rowspan="3">% Found</td><td>z</td><td> 5,63</td><td> 5,00</td><td> 4,26</td><td> 5,26</td><td>V "</td><td> 3,88</td><td>3.87 i</td>

<td>T</td><td> 4,52</td><td> 3,59</td><td> 3,05</td><td> 3,79</td><td> 2,85</td><td> 2,48</td><td> 2,51</td>

<td>o</td><td> 63,48</td><td> 55,74</td><td> 48,09</td><td> 59,23</td><td> 49,56</td><td> 43,60</td><td> 43,47</td>

<td rowspan="3">Estimated (%)</td><td>z</td><td> 5,71</td><td>τ- Ο in</td><td> 4,32</td><td> 5,32</td><td> 4,46</td><td>Τ- Ο) co "</td><td> 3,91</td>

<td>X</td><td> 4,52</td><td> 3,60</td><td> 3,11</td><td> 3,83</td><td> 2,89</td><td> 2,53</td><td>2.53 i</td>

<td>o</td><td> 63,67</td><td> 55,83</td><td> 48,17</td><td> 59,32</td><td> 49,71</td><td> 43,55</td><td> 43,55</td>

<td>Formula (molar mass)</td><td></td><td></td><td>o z Z in X o T " o</td><td>M · O - FT oj oj CO d</td><td>o Z cr ώ rf χ CO CO., LTD d</td><td>o ZF? u- while CO _r to X CM d</td><td>o z zc CM O σ&gt; t "I CO co ' d</td><td>o O co m co " O) xt X £ 2. CO r— O</td><td>o O co m co "0) 3 = £ 2 d</td>

<td>CM cc</td><td></td><td></td><td>X</td><td>d</td><td>k- Q0</td><td>Ll_</td><td>d</td><td>k. 00</td><td>d</td>

<td>cc</td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>d</td><td>d</td><td>ώ</td>

<td>The compound</td><td></td><td></td><td>CO</td><td>n r-</td><td>o r-</td><td>TJ b-</td><td>φ b-</td><td>b-</td><td>O) b-</td>

table. Compounds 7¹1 H-NMR (CDCl 3)₃) spectral data.

<td>δ (md), J (Hz)</td><td>3.98 (s, 3H), 4.06 (s, 3H), 7.58-8.00 (m, 3H), 8.21 (dd, Ji = 9.5, J₂ = 2.0, 1H), 8.77 (m, 2H), 8.77 (s, 3H)</td><td>3.97 (s, 3H), 4.06 (s, 3H), 7.76 (dd, Ji = 9.5, J₂ = 2.0, 1H), 7.90 (d, J = 2.0, 1H), 8.67 (s, 1H)</td><td>3.97 (s, 3H), 4.07 (s, 3H), 7.90 (dd, Ji = 9.5, J₂ = 2.0, 1H), 8.09 (m, 2H), 8.66 (s, 1H)</td><td>3.98 (s, 3H), 4.07 (s, 3H), 7.49-7.72 (m, 2H), 8.20 (dd, Ji = 10, J₂ = 5.0, 1H), 8.69 (s, 1H)</td><td>3.97 (s, 3H), 4.04 (s, 3H), 8.02 (s, 1H), 8.31 (s, 1H), 8.64 (s, 1H)</td><td>X T^-co co co " X ui co co co X T " td OJ OO X co (Λ CO O_ X co co Oops cd</td><td>3.96 (s, 3H), 4.04 (s, 3H), 8.03 (s, 1H), 8.53 (s, 1H), 8.62 (s, 1H)</td>

<td>The compound</td><td>CO r-</td><td>7b</td><td>7c</td><td>O r-</td><td>7e</td><td>M— r-</td><td>cn</td>

table. 5-Oxides of 4-Hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] quinolines 5.

<td>Yield (%)</td><td></td><td></td><td> 44,5</td><td> 88,0</td><td> 78,0</td><td> 37,0</td><td> 16,0</td><td> 15,0</td><td> 17,0</td>

<td>ΧεΡι -J Φ 4- »</td><td></td><td></td><td> &gt;300</td><td> &gt;300</td><td> &gt;300</td><td>297-298 i</td><td> &gt;300</td><td> &gt;300</td><td> &gt;300</td>

<td rowspan="6">Element Analysis i</td><td rowspan="3">% Found</td><td>z</td><td> 18,22</td><td> 15,40</td><td> 13,49</td><td> 16,90</td><td> 14,34</td><td> 11,94</td><td> 12,00</td>

<td>X</td><td> 2,93</td><td> 2,29</td><td> 1,91</td><td> 2,35</td><td>This one</td><td>1.33 _I</td><td> 1,31</td>

<td>o</td><td> 57,56</td><td> 49,34</td><td> 42,57</td><td> 53,44</td><td> 44,17</td><td> 37,93</td><td> 38,17</td>

<td rowspan="3">Estimated (%)</td><td>z</td><td> 18,33</td><td> 15,94</td><td> 13,63</td><td> 16,99</td><td> 14,10</td><td> 12,27</td><td> 12,27</td>

<td>X</td><td> 3,08</td><td> 2,29</td><td>96'l-</td><td> 2,44</td><td> 1,69</td><td>r- T ~</td><td>h- T—</td>

<td>o</td><td> 57,65</td><td> 50,11</td><td> 42,88</td><td> 55,44</td><td> 44,32</td><td> 38,57</td><td>38.57 _I</td>

<td>Formula (molar mass)</td><td></td><td></td><td>CO o _ CM σ&gt; X CM 6</td><td>CO o CO - Z to o ω &lt;OX d</td><td>CO o CO K Z Tώ g co O X £ 2- T " o</td><td>CO o £ CM LL l · - &lt;D X CM d</td><td>CO o CO ZP OJ FS ^mv. x v d</td><td>CO o CO r! cm co * o CO X d</td><td>CO o CO δ⁵? r cm CO Mm CO X ▼ - o</td>

<td>CM cc</td><td></td><td></td><td>X</td><td>d</td><td>d</td><td>Ll_</td><td>d</td><td>u. Ooo</td><td>d</td>

<td>cc</td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>d</td><td>d</td><td>ώ</td>

<td>The compound</td><td></td><td></td><td>CO in</td><td>Yeah to</td><td>o LO</td><td>TO LO</td><td>φ m</td><td>u5</td><td>cn io</td>

<td>Mrz 2 /</td><td></td><td></td><td> 499</td><td> 502</td><td> 514</td><td>516 I</td><td> 518</td><td> 551</td><td> 568</td>

table. Compounds 5¹1 H-NMR (DMSO-d₆) spectral data

<td rowspan="2">δ (md), J (Hz)</td><td>NH, OH (changing)</td><td>10.65 (pi s, 1H), 12.00 (pi s, 1H)</td><td>12.05 (pi s, 1H), 14.60 (pi s, 1H)</td><td>11.00 (pi s, 1H), 12.00 (pi s) 1H)</td><td>10.92 (pi s, 1H), 12.00 (pi s, 1H)</td><td>11.25 (pi s, 1H), 12.05 (pi s, 1H) _</td><td>12.06 (pi s, 1H), 14.28 (pi s, 1H) ___</td><td>12.13 (pi s, 1H), 14.32 (pi s, JH) _</td>

<td>aromatic protons (and OCH3)</td><td>7.88-8.28 (m, 2H), 8.46-8.79 (m, 2H), 9.07 (s, 1H)</td><td>8.07 (dd, Ji = 9.0, J₂ = 2.5, 1H), 8.59 (d, J = 9.0, 1H), 8.69 (d, J = 2.5, 1H), 9.11 (s, 1H)</td><td>T T " 0 oh II —3 tS CM CO co ' I Τ- Ο oh II “3 to O CO co ' I τ- ό oh II ~ o 0 ' σ&gt; II -5 T -d 2 S O CM O co 'oi</td><td>Z ui oh CJ “3 ui O) II “Oh TO * TO co co co ' ui 2 oi CM ..s CO uii co _θ¹¹ ui -5 * n - CM in “3 oi ui 11 oi s o '7. 0 -O 2- 2 r- lo 0 m co 'co *</td><td>8.83 (s, 1H), 8.90 (s, 1H), 9.06 (s, 1H)</td><td>8.80 (s, 1H), 9.00 (s, 1H), 9.01 (s, 1H)</td><td>8.90 (s, 1H), 9.01 (s, 1H), 9.04 (s, 1H)</td>

<td>The compound</td><td></td><td>5a</td><td>5b</td><td>5c</td><td>5d</td><td>5e</td><td>H— in</td><td>O) m</td>

table. 1,4-Dioxo-1,2,3,4-tetrahydropyridazine- [4,5-b] quinolines 9.

<td>Yield (%)</td><td></td><td></td><td> 86,0</td><td> 88,5</td><td> 82,0</td><td> 84,0</td><td> 82,5</td><td> 69,5</td><td> 88,0</td>

<td>Lyd. temp. (° C)</td><td></td><td></td><td> &gt;300</td><td> &gt;300</td><td> &gt;300</td><td> &gt;300</td><td>o o co Λ</td><td> &gt;300</td><td> &gt;300</td>

<td rowspan="6">Element Analysis</td><td rowspan="3">% Found</td><td>z</td><td> 19,16</td><td> 16,68</td><td> 14,09</td><td> 17,99</td><td>14.87 I</td><td> 12,88</td><td> 12,98</td>

<td>I</td><td> 3,45</td><td> 2,28</td><td> 2,11</td><td>&gt; - M cm</td><td> 1,70</td><td>CM 't v "</td><td> 1,40</td>

<td>o</td><td> 61,43</td><td> 32,89</td><td> 44,74</td><td> 56,73</td><td> 46,44</td><td>40.16 I</td><td> 40,23</td>

<td rowspan="3">Estimated (%)</td><td>z</td><td>19.71 I</td><td> 16,97</td><td> 14,39</td><td>co co</td><td> 14,90</td><td> 12,87</td><td> 12,87</td>

<td>z:</td><td> 3,31</td><td>cm</td><td>r- o cm</td><td> 2,60</td><td>CT&gt; r-</td><td> 1,54</td><td> 1,54</td>

<td>o</td><td> 61,97</td><td> 53,35</td><td> 45,23</td><td> 57,14</td><td> 46,84</td><td>CO o</td><td> 40,46</td>

<td>Formula (molar mass)</td><td></td><td></td><td>CM O CM rc. CO r - SS d</td><td>CM O CO z— 2 O o £ X SS d</td><td>CM O CO -. Z T- ω co O) X SS d</td><td>CM O Z &lt;S LL T &lt;P CO x SS d</td><td>CM xS r- O</td><td>CM o co r: co CQ CM in CO X t- o</td><td>CM O co §S? u! co CO £ 1Λ CO X d</td>

<td>CM OC</td><td></td><td></td><td>X</td><td>o</td><td>u- co</td><td>LL</td><td>δ</td><td>co</td><td>o</td>

<td>cc</td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>o</td><td>o</td><td>u. co</td>

<td>The compound</td><td></td><td></td><td>oh O)</td><td>n σ&gt;</td><td>o CD</td><td>U cn</td><td>ω σ&gt;</td><td>&gt; + - σ&gt;</td><td>CF) O)</td>

<td>Mrz 2 of</td><td></td><td></td><td> 585</td><td> 501</td><td> 503</td><td> 519</td><td> 515</td><td> 539</td><td> 538</td>

table. Compounds 9¹1 H NMR (DMSO-d₆) spectral data.

<td rowspan="2">δ (md), J (Hz)</td><td>ω c CO ίο o u ω .o X 2</td><td>11.60 (bs, 2H)</td><td>11.60 (ss, 2H)</td><td>11.55 (ss, 2H)</td><td>11.90 (bs, 2H)</td><td>11.60 (ss, 2H)</td><td>11.65 (bs, 2H)</td><td>11.70 (s, 2H)</td>

<td>aromatic protons</td><td>7.76-8.16 (m, 2H), 8.22-8.47 (m, 2H), 9.30 (s, 1H)</td><td>X T- in CM II -&gt; TO CM in co X 0 O) II -3 to co CM co X T— LO * CM II OJ “3 0 O) II -7 X n 'T. S 12 · C \ l CO O_ CM co σ&gt;</td><td>8.16 (m, 2H), 8.60 (s, 1H), 9.25 (s, 1H)</td><td>m cm II 04 —3 LO * σ&gt; II G? X TJ Ό 00 here X CM T. θ⁵' LO CM 3 = n T. CO LO LO Q II 0 c? II X X. oT LO O) in n 0 II. - T3 TJ. 2 ^ωS &amp; Η_ωco X r- t-</td><td>8.47 (s, 1H), 8.67 (s, 1H), 9.22 (s, 1H)</td><td>8.52 (s, 1H), 8.91 (s, 1H), 9.28 (s, 1H)</td><td>8.68 (s, 1H), 8.71 (s, 1H), 9.26 (s, 1H)</td>

<td>The compound</td><td></td><td>9a</td><td>9b</td><td>9c</td><td>9d</td><td>ω O)</td><td>M— O</td><td>cn cn</td>

table. Choline salts of 4-hydroxy-1-oxo-1,2-dihydropyridazine- [4,5-b] quinolines 5-oxides 6.

<td>Yield (%)</td><td></td><td></td><td> 52,5</td><td> 87,5</td><td> 71,5</td><td> 27,0</td><td></td><td></td><td></td>

<td>no E _ Φ oo n</td><td></td><td></td><td> 179-180</td><td> 185-188</td><td> 191-193</td><td> 201-203</td><td></td><td></td><td></td>

<td rowspan="6">Element Analysis</td><td rowspan="3">% Found</td><td>Z</td><td> 15,86</td><td> 14,24</td><td> 12,86</td><td> 14,95</td><td></td><td></td><td></td>

<td>X</td><td> 6,32</td><td> 5,47</td><td> 4,93</td><td> 5,73</td><td></td><td></td><td></td>

<td>O</td><td> 54,76</td><td> 49,31</td><td> 44,85</td><td> 51,74</td><td></td><td></td><td></td>

<td rowspan="3">• o § ^r15th to co CL &lt;</td><td>Z</td><td> 15,99</td><td> 14,55</td><td> 13,04</td><td>14.92 I</td><td></td><td></td><td></td>

<td>X</td><td> 6,32</td><td> 5,50</td><td> 4,92</td><td> 5,63</td><td></td><td></td><td></td>

<td>o</td><td> 54,84</td><td> 49,93</td><td> 44,75</td><td> 51,19</td><td></td><td></td><td></td>

<td>Formula (molar mass)</td><td></td><td></td><td>o _ z 'T 88 5 «d</td><td>o Z co O co 'O&gt; co X ss co d</td><td>o m -r- OY— X co d</td><td>o Z rt ų =, co 'x £ 2 CO Τ ' O</td><td></td><td></td><td></td>

<td>04 oc</td><td></td><td></td><td>X</td><td>d</td><td>m</td><td> 11.</td><td>O</td><td>U- CQ</td><td>o</td>

<td>cc</td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>d</td><td>d</td><td>m</td>

<td>The compound</td><td></td><td></td><td>co co</td><td>Yeah co</td><td>o co</td><td>Ό CO</td><td>φ CO</td><td>H— CO</td><td>05 CO</td>

<td>E _ 2 CM</td><td></td><td></td><td> 577</td><td> 576</td><td> 570</td><td>T ~ l · - in</td><td> 574</td><td></td><td></td>

= 1.0 (a, b, c) Table 0.5 (d). Choline salts 6¹1 H-NMR (CD₃OD) spectral data.

<td rowspan="2">δ (md), J (Hz)</td><td>aromatic protons</td><td>7.69-8.00 (m, 2H), 8.18 (d, J = 8.0, 1H), 8.59 (s, 1H), 8.76 (d, J = 8.5, 1H)</td><td>7.88 (dd, Ji = 9.0, J₂ = 2.5, 1H), 8.27 (d, J = 2.5, 1H), 8.52 (s, 1H), 8.76 (d, J = 9.0, 1H)</td><td>7.99 (dd, Ji = 9.5, J₂ = 2.0, 1H), 8.41 (d, J = 2.0, 1H). 8.53 (s, 1H), 8.64 (d, J = 9.5, 1H)</td><td>7.64-7.98 (m, 2H), 8.62 (s, 1H), 8.87 (dd, Ji = 10.0, J₂ = 5.0, 1H) ____</td><td></td><td></td><td></td>

<td>choline protons</td><td>3.20 (s, 9H), 3.47 (m, 2H), 3.98 (m, 2H)</td><td>3.22 (s, 9H), 3.50 (m, 2H), 4.01 (m, 2H)</td><td>3.20 (s, 9H), 3.48 (m, 2H), 3.99 (m, 2H)</td><td>3.22 (s, 9H), 3.51 (m, 2H), 4.02 (m, 2H)</td><td></td><td></td><td></td>

<td>The compound</td><td></td><td>Cj CO</td><td>6b</td><td>6c</td><td>T3 CO</td><td>6e</td><td>M— CO</td><td>D) CO</td>

table. Choline salts of 1,4-dioxo-1,2,3,4-tetrahydropyridazine- [4,5-b] quinolines 10.

<td>05 φ θ ′&gt; cn</td><td></td><td></td><td> 82,0</td><td>o co</td><td> 61,0</td><td> 95,0</td><td> 83,0</td><td> 92,0</td><td> 95,0</td>

<td>Lyd. temp. (° C)</td><td></td><td></td><td> 102-110</td><td> 189-191</td><td> 234-236</td><td> 229-230</td><td> 205-208</td><td> 207-209</td><td> 201-203</td>

<td rowspan="6">Element Analysis</td><td rowspan="3">% Found</td><td>Z</td><td> 14,25</td><td> 15,61</td><td> 12,73</td><td> 15,19</td><td> 13,60</td><td>12.44 i</td><td> 12,37</td>

<td>z</td><td> 7,39</td><td> 5,55</td><td> 5,14</td><td>6.23 i</td><td> 5,03</td><td> 4,60</td><td> 4,57</td>

<td>o</td><td> 54,23</td><td> 54,10</td><td> 44,07</td><td> 52,08</td><td> 47,24</td><td> 42,78</td><td> 42,66</td>

<td rowspan="3">• a&gt; 05 θ ' 4— » o * .2 O&gt; o * · g X ω? D. &lt;</td><td>z</td><td> 14,06</td><td> 15,76</td><td> 12,72</td><td> 5,74</td><td> 13,89</td><td> 12,51</td><td> 12,51</td>

<td>z</td><td> 7,59</td><td> 5,53</td><td> 5,49</td><td>15.20 _I</td><td> 4,99</td><td>in • 't</td><td> 4,5</td>

<td>o</td><td> 54,26</td><td> 54,08</td><td> 43,64</td><td> 52,16</td><td> 47,65</td><td> 42,92</td><td> 42,92</td>

<td>Formula (molar mass)</td><td></td><td></td><td>CO o - τ CO Z co ° to 're; to CO d</td><td>CO o z CO Oh o ° 2 io Z £ 2 CO V " O</td><td>CO o CO in ® G) z £ 2 CO d</td><td>CO o you know? CM z ^ co d</td><td>CO o rr co co o</td><td>CO o Tf z o t. CO CM ® MZ CO d</td><td>CO o Tf Z _ o £ CO CM to ^ J. z ~ to r- O</td>

<td>CM cc</td><td></td><td></td><td>z ··</td><td>d</td><td>u— co</td><td>LL</td><td>d</td><td>κ- ω</td><td>d</td>

<td>cc</td><td></td><td></td><td>z</td><td>z</td><td>z</td><td>Z</td><td>d</td><td>d</td><td>K- CQ</td>

<td>The compound</td><td></td><td></td><td>I 10a</td><td>10b</td><td>o o</td><td>10d Ί _</td><td>o o</td><td>H— O</td><td>O) O T "</td>

<td>L! S GJ</td><td></td><td></td><td> 604</td><td> 596</td><td> 586</td><td>572 I</td><td> 574</td><td> 598</td><td>Z6S</td>

0.25 (d) 1.0 (d, e) Table 2.5 (c). Choline salts 10¹1 H-NMR (CD₃OD) spectral data.

<td rowspan="2">δ (md), J (Hz)</td><td>aromatic protons</td><td>7.64-8.57 (m, 4H), 9.17 (s, 1H)</td><td>7.89 (dd, Ji = 9.0, J₂ 2.5, 1H), 8.23 ​​(d, J = 2.5, 1H), 8.34 (d, J =) 9.0, 1H), 9.13 (s, 1H)</td><td>7.99 (dd, Ji = 9.0, J₂ = 2.0, 1H), 8.26 (d, J = 9.0, 1H), 8.41 (d, J = 2.0.1H), 9.09 (s, 1H)</td><td>7.64-7.81 (m, 2H), 8.39 (dd, J1 = 9.5, J)₂ = 5.0, 1H), 9.12 (s, 1H)</td><td>8.46 (s, 1H), 8.55 (s, 1H), 9.14 (s, 1H)</td><td>8.53 (s, 1H), 8.64 (s, 1H), 9.13 (s, 1H)</td><td>8.43 (s, 1H), 8.73 (s, 1H), 9.13 (s, 1H).</td>

<td>choline protons</td><td>3.21 (s, 9H), 3.51 (m, 2H), 4.01 (m, 2H)</td><td>3.25 (s, 9H), 3.52 (m, 2H), 4.02 (m, 2H)</td><td>3.22 (s, 9H), 3.47 (m, 2H), 3.97 (m, 2H)</td><td>3.20 (s, 9H), 3.49 (m, 2H), 3.98 (m, 2H)</td><td>3.22 (s, 9H), 3.50 (m, 2H), 4.02 (m, 2H)</td><td>3.22 (s, 9H), 3.50 (m, 2H), 4.00 (m, 2H)</td><td>3.22 (s, 9H), 3.50 (m, 2H), 4.02 (m, 2H)</td>

<td>The compound</td><td></td><td>10a</td><td>10b</td><td>10c</td><td>10d</td><td>10e</td><td>M— O t—</td><td>ra o t—</td>

PHARMACOLOGY

In vitro

Receptor binding studies

Membrane preparations and protein determination.

Tissue preparations were prepared according to the method of Foster and Vong (Foster AC, Wong EHF (1987): Brit. J. Pharmacol. 91, pp. 403-409). Male Sprague-Dawley rats (200-250 g) were decapitated and their brains removed immediately. The dissected cerebral cortex was homogenized in 20 volumes of ice-chilled 0.32 M sucrose in a glass-Teflon homogenizer. The homogenate was centrifuged for 10 min. with 1000 x g. The pellet was resuspended in 20 volumes of distilled water and centrifuged for 20 min. with 8000 x g. The supernatant and clot light layer were then centrifuged three times (20 min, 48000 xg) in 5 mM Tris-HCl, pH 7.4. All centrifugations were performed at 4 ° C. After resuspension in 5 volumes of 5 mM Tris-HCl, pH 7.4, the membrane suspension was rapidly cooled to -80 ° C and left until the day of assays. On the day of the assays, membranes were thawed and washed four times by resuspension in 5 mM Tris-HCl, pH 7.4, and centrifugation for 20 min. with 48,000 x g. The final sediment was suspended in assay buffer.

The protein content of the final membrane preparation was determined by the Louri method with some modifications (Lovvry OH, Rosenbrough NJ, Farr AL, Randeli RJ (1951): J. Biol. Chem. 193, pp. 265-275; Hartfree EF (1972): Analytical Biochemistry 48, pp. 422-427). Three identical samples of the protein were diluted to 50 ml with 1 ml of distilled water and treated with 0.9 ml of a solution of 2 g of potassium sodium tartrate and 100 g of Na.₂CO₃ 500 mL of 1N NaOH and 500 mL of water. Comparative and standard (with bovine serum albumin) solutions were prepared in the same way. Test tubes 10 min. leave in a water bath at 50 ° C, then cool to room temperature. Add 100 μΙ of a solution of 2 g potassium sodium tartrate and 1 g CuSO₄ x 5H₂And 90 ml water and 10 ml 1N

NaOH. The samples are kept at room temperature for at least 10 minutes, after which 3 ml of Folin-Ciocalteu reagent (1 ml of reagent diluted in 15 ml of water) is added rapidly with stirring. The tubes are heated again for 10 minutes. At 50 ° C, then cooled to room temperature. Measure the absorbance in 1 cm cells at 650 nm. The final protein concentration used in our assays was between 100 and 250 pg / ml.

Incubation in both binding assays was terminated using Milliporre filters. The samples, each in triplicate, are washed three times with 2.5 ml of ice-cooled assay buffer on a glass filter purchased from Schleicher &amp; Schuell under constant vacuum. After separation and washing, the filters are immersed in scintillation fluid (5 ml; Ultima Gold) and the radioactivity remaining on the filters is measured using a standard liquid scintillation counter (Hewlett Packard, Liquid Scintillation Analyzer). "Total Binding" was the absolute amount of radioligand bound in the absence of any additive whereas "nonspecific binding" was determined at high concentrations of the competitive reagent.

[³H] 5,7-DCKA binding assay

Experiments were performed according to a modified methodology of previous researchers (Canton T., Doble A., Miquet J.M., Jimonet P., Blanchard J.C. (1992): J. Pharm. Pharmacol. 44, pp. 812-816). Membranes were suspended and incubated in 10 mM Tris-HCl buffer, pH 7.4. Incubation time - 45 min. At 4 ° C. Non-specific [³H] 5,7-DCKA binding was determined by the addition of 0.1 mM unlabeled glycine. The suspension solution consisted of 10 mM Tris-HCl and 10 mM magnesium sulfate, pH 7.4. Filter as soon as possible. Replacement experiments were performed with constant [³H] 5,7-DCKA at 10 nM concentration. Test compounds are diluted with water or DMSO and added in at least 5 different concentrations.

[³H] glycine binding assay [³H] glycine binding assays were performed according to the procedure described by Kessler et al. (Kessler M., Terramani T., Lynch G., Baudry M. (1989): J. Neurochem. 52, pp. 1319-1328). Rat cortical membranes were prepared as described above and the final sediment suspended in 50 mM Tris-acetate, pH 7.4. At least 5 different concentrations of test compounds were incubated with 20 nM [³H] glycine for 30 min. At 4 ° C in the presence of 100 μΜ of strychnine. All compounds were dissolved in water or DMSO, respectively. Nonspecific binding was determined by adding 100 μΜ of glycine to the incubation mixture. The incubation was terminated by diluting the samples with 2 ml of stop solution (50 mM Tris-HCl containing 10 mM magnesium sulfate, pH 7.4, cooled to 2 ° C) followed by washing with 2.5 ml buffer. Filtered as soon as possible.

Results

Of all compounds tested [³In the H] -DCKA study, eight had an IC50 of 1 μΜ. Strength of the six selected compounds [³The H] -glycine assay seems to be larger at first glance, but this is not reflected in the large differences in K &lt;j values ​​(not shown). For particularly interesting pairs of compounds, class II compounds have higher affinity than class I compounds, [³In the H] -glycine study.

Communication capture

Upper dorsal rootstocks were isolated from rat embryos (E20 to E21) and placed in chilled ice-buffered Hank's salt-free calcium and magnesium (Gibco) solution. Cells are mechanically dissociated in 0.05% DNAse / 0.3% ovomucoid solution (Sigma) after incubation for 15 minutes in 0.66% trypsin / 0.1% DNAse solution (Sigma). Dissociated cells are centrifuged at 18 g for 10 minutes, resuspended in a minimum volume of basic medium (Gibco) and plated on poly-L-lysine (Sigma) coated Petri dishes (Falcon) at 200,000 cells / cm² density. The cells are fed with NaHCO₃/ HEPES in basic medium supplemented with 5% fetal calf serum and 5% horse

Table 13a

<td>Mrz2 /</td><td>The compound</td><td> [³H] -DCKA IC₅while μΜ</td><td> [³H j glycine IC50 μΜ</td>

<td> 499</td><td>II</td><td> 16,0</td><td></td>

<td> 501</td><td>8-CI-1</td><td> 0,120</td><td> 0,080</td>

<td> 502</td><td>8-CI-11</td><td> 0,020</td><td> 0,013</td>

<td> 503</td><td>8-Br-1</td><td> 0,250</td><td> 0,013</td>

<td></td><td>8-Br-II</td><td> 0,010</td><td> 0,004</td>

<td></td><td>8-Fl</td><td> 1,100</td><td> 0,015</td>

<td></td><td>8-F-II</td><td> 0,300</td><td> 0,017</td>

<td></td><td>7,8-diCl-1</td><td> 0,530</td><td></td>

<td></td><td>7,8-diCl-II</td><td> 0,650</td><td></td>

Table 13b

<td>Mrz2 /</td><td>The compound</td><td> [⁵H] -DCKA IC50 μΜ</td>

<td> 572</td><td>8-Fl (Chol)</td><td> 1,14</td>

<td> 571</td><td>8-F-II (Chol)</td><td> 0,32</td>

<td> 569</td><td>8-CI-1 (Chol)</td><td> 0,97</td>

<td> 576</td><td>8-CI-II (Chol)</td><td> 0,45</td>

serum (Gibco), and incubated at 37 ° C with 5% CO₂ and 95% humidity in the atmosphere. Neuroglial mitosis is inhibited with cytosine-p-Darabinofuranoside (20 μΜ, Sigma) after approximately 7 days in vitro, followed by complete media switching. The medium is then changed twice a week. The roller coaster culture is chosen for these experiments because it produces stable responses, which is a prerequisite for potential-dependent and kinetic experiments.

In addition, relatively small neurons (body 0 15–20 μιτι) are ideally suited to mitigate buffer diffusion related problems.

Binding fixation studies in these neurons were performed using a polished glass electrode in a whole lattice model at room temperature (20-22 ° C) using an EPC-7 amplifier (List). Compounds are tested by switching channels in a conventional high-throughput system with a conventional outflow (change time -10-20 ms). The solution inside the cell was as follows (mM): CsCI (120) TEACI (20), EGTA (10), MgCl₂ (1), CaCl₂ (0.2), glucose (10), ATP (2), cAMP (0.25); The pH is brought to 7.3 with CsOH or HCl. The solutions on the outside of the grid were as follows (mM): NaCl (140), KCI (3), CaCl₂ (0,2), glucose (10), HEPES (10), sucrose (4,5), tetrodoxin (ΊΓΊΓΧ 3 * 1 θ ′)⁴). In most experiments, the solutions contained 1 μΜ of glycine. Experiments for determination of glycine dependence on pyridophthalazindiones were performed with steadily increasing glycine concentrations (1-10 μΜ).

Results

Five pairs of tricyclic pyridophthalazinediones have ICs₅and for intrinsic currents due to NMDA (200 μΜ) in the region of small μΜ, and class II compounds were generally 23 times stronger than class I compounds (see Table 14a). The strongest of these were Mrz 2/502 and Mrz 2/514. This effect occurs at the glycine-B binding site, which is shown by the parallel shift in the concentration-response curves with increasing glycine concentration. Thus, the compound of Mrz 2/502 K_b evaluated by Cheng-Prusov dependence had similar values ​​for glycine at 1, 3, and 10 μΜ (80, 124, and 118 nM, respectively. In addition, the influence of Mrz 2/501 and 2/502 was independent of potential. 3- to 10-fold stronger at steady state currents than at peak currents Choline derivatives were similar in potency to the free acids in vitro (Table 14b).

In contrast, three of these potent glycine-B antagonists were only very weak antagonists in terms of internal currents due to AMPA (100 μΜ). Mrz 2/502, 2/514 and

The IC50 values ​​of 2/516 for AMPA-induced currents were 25, 73, and 18 μΜ, respectively, but were completely inactive for plateau currents, all ICs₅₀ &gt; 100 μΜ (Table 14a). This mode of action, although very mild, is typical of competitive AMPA receptor antagonists, which preferentially block the peak non-sensitized state, the affinity state of the receptor (see Parsons CG, Gruner R., Rozental J. (1994): Neuropharmacology 33, p. 589 -604).

Table 14a

<td>Mrz2 /</td><td>The compound</td><td>Peak NMDA IC50 μΜ</td><td>Plato NMDA IC50 μΜ</td><td>Peak AMPA IC50 μΜ</td><td>Plato AMPA IC50 μΜ</td>

<td> 585</td><td>I</td><td> 65,9</td><td> 19,1</td><td></td><td></td>

<td> 499</td><td>II</td><td> 51,2</td><td> 13,8</td><td></td><td></td>

<td> 501</td><td>8-CI-1</td><td> 2,3</td><td> 0,7</td><td></td><td></td>

<td> 502</td><td>8-CI-11</td><td> 0,8</td><td> 0,3</td><td> 25,0</td><td> 150,0</td>

<td> 503</td><td>8-Br-1</td><td> 1,7</td><td> 0,6</td><td></td><td> 307,0</td>

<td> 514</td><td>8-Br-II</td><td> 0,5</td><td> 0,2</td><td> 72,7</td><td></td>

<td> 519</td><td>8-Fl</td><td> 18,0</td><td> 5,8</td><td></td><td></td>

<td> 516</td><td>8-F-II</td><td> 6,3</td><td> 1,6</td><td> 17,6</td><td> &gt;100</td>

<td> 515</td><td>7,8-diCl-1</td><td> 3,7</td><td> 0,9</td><td></td><td></td>

<td> 518</td><td>7,8-diCl-II</td><td> 3,8</td><td> 0,8</td><td></td><td></td>

<td> 539</td><td>7-CI, 8-Br-1</td><td> 5,3</td><td> 0,7</td><td></td><td></td>

<td> 551</td><td>7-CI, 8-Br-II</td><td> 2,4</td><td> 0,6</td><td></td><td></td>

<td> 538</td><td>7-Br, 8-CI-1</td><td> 93,9</td><td> 2,5</td><td></td><td></td>

<td> 568</td><td>7-Br, 8-CI-11</td><td> 10,0</td><td> 1,5</td><td></td><td></td>

<td> 554</td><td>8-O-CH 3 -I</td><td> 170</td><td> 36,2</td><td></td><td></td>

Table 14b

<td>Mrz 2 of</td><td>The compound</td><td>Long NMDA IC50 μΜ</td><td>Plato NMDA IC50 μΜ</td>

<td> 569</td><td>8-CI-1 (Chol)</td><td> 2,0</td><td> 0,5</td>

<td> 576</td><td>8-CI-II (Chol)</td><td> 1,1</td><td> 0,5</td>

<td> 586</td><td>8-Br-l (Chol)</td><td> 2,2</td><td> 0,6</td>

<td> 570</td><td>8-Br-II (Chol)</td><td> 0,6</td><td> 0,1</td>

<td> 572</td><td>8-Fl (Chol)</td><td> 12,4</td><td> 3,5</td>

<td> 571</td><td>8-F-II (Chol)</td><td> 4,9</td><td> 1,0</td>

<td> 578</td><td>8-O-CH3-II (Chol)</td><td> 101</td><td> 7,7</td>

<td> 575</td><td>7-O-CH3-I (Chol)</td><td> 94,0</td><td> 14,5</td>

Excitotoxicity in vitro

Isolation of cortical neurons proceeded in a similar manner to that described in connection fixation experiments, except that rat embryos from 17 to 19 days of gestation were used. The neurons were spread in a 24-well plate (Greiner) coated with poly-L-lysine at a density of 300,000 cells per well. Cells were cultured in modified Dulbeck's medium (DMEM, GIBGO) supplemented with heat-inactivated fetal calf serum (GIBCO). Store the culture at 37 ° C and 5% CO₂ in the atmosphere. The medium is changed for the first time after one week, and then every three days, half of the medium is replaced with fresh medium. A 17-day culture was used for the experiments.

Contact with EEA was performed in serum-free MEM-N2 medium (Bottenstein JE,

Shato GH (1979): Proc. Natl. Acad. Sci. USA 76, p. 514-517) containing 0.5 mM NMDA / 1 μΜ glycine and the drug being studied. Cells were incubated for 15 min prior to addition of NMDA. Incubate with drug and 1 μΜ glycine. After 24 or. cytotoxic effects are investigated morphologically using phase contrast microscope and quantified biochemically by LDH emanation.

LDH activity in the supernatant after 24 h. determined by the method of Wrublevsky and La Due (Vrobrobski F., La Due JS (1955): Soc. Exp. Biol. Med. 90, p. 210). Briefly, 0.1 ml of supernatant was added to 0.9 ml (pH 7.5) sodium phosphate buffer (22.7 mM) and NADH (0.8 mg / 10 ml) at room temperature. The conversion of pyruvate to lactate is recorded after 10 minutes by measuring the optical density at 340 nm on a Kontron spectrophotometer.

Results

We do not have complete response curves for concentration at this time. However, the low μΜ concentrations of Mrz 2/501 and Mrz 2/502 act as effective neuroprotective agents in vitro, and in this respect Mrz 2/502 appears to be more potent (see Table 15).

table

<td>Mrz 2 /</td><td>The compound</td><td>In vitro cytotoxicity, IC50 μΜ</td>

<td> 501</td><td>8-CI-1</td><td> &lt;5</td>

<td> 502</td><td>8-CI-11</td><td> &lt;&lt;5</td>

<td> 503</td><td>8-Br-1.</td><td> &gt;20</td>

In vivo

Antispasmodic activity

The aim is to evaluate the antagonistic properties of the investigated agents with respect to NMDA receptors by assessing antispasmodic effects. In addition, the transporting role of organic acids in the removal of test substances from the brain has also been evaluated with the use of an inhibitor, probenicide.

Methodologies

Male white Swiss mice (19-21 g), placed in cages 10-15 each, were used for the NMDA lethal test (Leander JD, Lawson RR, Ornstein PL, Zimmerman DM (1988): Brain Res. 448, p. 115). Pentylenetetrazole (PTZ) -induced convulsions were used in white Swiss male mice (25-34 g) housed in 40 cages (58x38x20 cm) and NMR female mice (18-28 g) were used in maximum electroshock and motor impairment studies at 5 cages. All animals were kept ad libitum with water and food for 12 h. light-dark cycle (light on at 6 am) at controlled temperature (20 ± 0,5 ° C). All experiments were performed between 10 hours. morning is 5 or so. afternoon. Test compounds are administered intraperitoneally for 15 min. before convulsions are induced, unless otherwise stated (see below). Mrz 2/502 was dissolved in brine with the addition of NaOH. Most other agents were dissolved in the following solvent: 0.606 g Tris, 5.0 g glucose, 0.5 g Tween 80, 95 ml water. The choline and tetramethylammonium salts were dissolved in distilled water.

In a study of NMDA-induced convulsions in mice, dose-response NMDA was first performed to determine ED₉₇ dose, which was then used to test for antagonistic properties. With the introduction of ED₉₇ At the dose of NMDA, the animals were placed in a small cage (20x28x14 cm) and observed for 20 minutes. Death after clonic convulsions and tonic seizures was a pharmacological outcome.

Pentylenetetrazole was administered intraperitoneally at a dose of 90 mg / kg. Thereafter, the presence of total tone convulsions is assessed for 30 minutes as this parameter is more sensitive to NMDA receptor antagonists than clonal convulsions. The pharmacological outcome is calculated when the hind limbs are involuntarily stretched by stretching.

MES (100Hz, shock duration 0.5 s, shock intensity 50 mA, pulse duration 0.9 ms, Ugo Basile) applied via corneal electrodes. The presence of tonic convulsions (tonal extension of the hind limbs with a minimum body angle of 90 °) is evaluated. In additional experiments, 30 min.

probenicide (200 mg / kg) is administered to experimental mice prior to the administration of test compounds to evaluate the role of organic acid transport in elimination (duration of action). The goal was to obtain ED for all parameters₅₀ values ​​using Litchfield Wc (Wilcoxon F.

(1949): J. Pharmacol. Exp. Ther. 96, p.99) for a quantitative assessment of dose response.

Results

Of the compounds tested, only four compounds, all of class II, were effective

Introduced intraperitoneally in the MES test (Mrz 2/499, Mrz 2/502, Mrz 2/516 and Mrz 2/514, see Table 16a). The corresponding class I compounds were inactive. The four compounds appear to have very short lifetimes in vivo. The PTZ assay emerged as a more sensitive model for the activity of glycine-B antagonists administered intraperitoneally, and in fact, the same class II compounds were active at doses 2-4 times lower when class I compounds remained inactive (Table 16a).

The choline salts (structure II) of the same N -oxide derivatives exhibited clear anticonvulsant activity in all three models, whereas their non-N-oxide derivatives were either inactive or weak (Table 16b). In addition, choline salts appear to have a longer duration of action. Probenicide injections significantly prolonged the anticonvulsant duration of action of all agents tested. For example, the half-lives of 2/514 and 2/570 in the absence of probenicide were approximately 40 and 80 minutes, respectively. Following the introduction of probenicide, the half-lives increased to 180 and 210 minutes, respectively. Thus, the transport of organic acids in the vascular plexus from the brain seems to play an important role due to the short duration of action of the compounds tested. The dose of probenicide used (200 mg / kg) has no independent effect on MES-induced convulsions in the pers.

Table 16a

<td>Mrz 2 of</td><td>Material</td><td>MES ip (ID50 mg / kg)</td><td>NMDAi.p. (ID50 mg / kg)</td><td>PTZ ip (ID50 mg / kg)</td>

<td> 585</td><td>I</td><td> &gt;100,0</td><td> 58,9</td><td> 59,0</td>

<td> 499</td><td>II</td><td> 87,0</td><td></td><td> 18,6</td>

<td> 501</td><td>8-CI-1</td><td> &gt;100,0</td><td> &gt;100,0</td><td> &gt;40,0</td>

<td> 502</td><td>8-CI-11</td><td> 47,6</td><td> 26,0</td><td> 8,3</td>

<td> 503</td><td>8-Br-1</td><td> &gt;100,0</td><td> &gt;100,0</td><td> &gt;100,0</td>

<td> 514</td><td>8-Br-II</td><td> 20,2</td><td> 99,0</td><td> 12,8</td>

<td> 519</td><td>8-Fl</td><td> &gt;60,0</td><td> &gt;100,0</td><td> &gt;100,0</td>

<td> 516</td><td>8-F-II</td><td> 16,6</td><td> 40,0</td><td> 7,9</td>

<td> 515</td><td>7,8-diCl-1</td><td> &gt;100,0</td><td> 98,0</td><td> &gt;100,0</td>

<td> 518</td><td>7,8-diCl-II</td><td> &gt;60,0</td><td> &gt;100,0</td><td></td>

<td> 539</td><td>7-CI, 8-Br-1</td><td> &gt;60,0</td><td> &gt;100,0</td><td> &gt;100,0</td>

<td> 538</td><td>7-Br, 8-CI-1</td><td> &gt;60,0</td><td> 106,0</td><td> &gt;100,0</td>

<td> 554</td><td>8-OCH3-I</td><td> &gt;100,0</td><td></td><td></td>

Table 16b

<td>Mrz 2 /</td><td>Material</td><td>MES ip {ID₅₀ mg / kg)</td>

<td> 577</td><td>II (Chol)</td><td> 23,7</td>

<td> 569</td><td>8-CI-1 (Chol)</td><td> &gt;50</td>

<td> 576</td><td>8-CI-II (Chol)</td><td> 7,7</td>

<td> 586 .</td><td>8-Br-l (Chol)</td><td> &gt;50</td>

<td> 570</td><td>8-Br-II (Chol)</td><td> 12,8</td>

<td> 572</td><td>8-Fl (Chol)</td><td> &gt;100</td>

<td> 571</td><td>8-F-II (Chol)</td><td> 15,5</td>

<td> 574</td><td>7,8-diCl-1 (Chol)</td><td> &gt;100</td>

<td> 578</td><td>7,8-diCl-II (Chol)</td><td> &gt;100,0</td>

<td> 575</td><td>7-OCH3-I (Chol)</td><td> &gt;100,0</td>

Microelectrophoretic administration of EAA agonists to spinal cord neurons in vivo

The ability of these glycine-B antagonists to act as NMDA receptor antagonists was investigated in vivo by intravenous administration and by measuring the response of single neurons in the rat spinal cord to the microelectrophoretic administration of AMPA and NMDA. Class II compounds Mrz 2/502 and Mrz 2/516 have been shown to be potent NMDA receptor antagonists in vivo with ID₅₀1.2 and 1.8 mg / kg, respectively, while the congeners of Class I were completely inactive up to 16 mg / kg intravenously. Three to four times higher doses also antagonized the AMPA response, although this apparent lack of selectivity contrasted with in vitro studies (Table 17a).

Table 17a

<td>Mrz 2 of</td><td>Material</td><td>Microelectrophoresis NMDA (ID₅₀ mg / kg iv)</td><td>Microelectrophoresis AMPA (ID₅₀ mg / kg iv)</td>

<td> 501</td><td>8-CI-1</td><td> &gt;16,0</td><td> &gt;16,0</td>

<td> 502</td><td>8-CI-11</td><td> 1,2</td><td> 4,9</td>

<td> 519</td><td>8-Fl</td><td> &gt;16,0</td><td> &gt;16,0</td>

<td> 516</td><td>8-F-II</td><td> 1,8.</td><td> 3,6</td>

In this model, the choline salts were approximately as potent as the free acids when administered intravenously but were slightly more selective for NMDA than AMPA (Table 17b). In addition, non-N-oxide derivatives (class I compounds) were inactive.

Table 17b

<td>Mrz 2 of</td><td>Material</td><td>Microelectrophoresis NMDA (ID₅₀ mg / kg iv)</td><td>Microelectrophoresis AMPA (ID₅o mg / kg iv)</td>

<td> 577</td><td>II (Chol)</td><td> 34,0</td><td> &gt;32,0</td>

<td> 569</td><td>8-CI-1 (Chol)</td><td> &gt;16,0</td><td> &gt;16,0</td>

<td> 576</td><td>8-CI-II (Chol)</td><td> 2,8</td><td> &gt;16,0</td>

<td> 586</td><td>8-Br-l (Chol)</td><td> &gt;16,0</td><td> &gt;16,0</td>

<td> 570</td><td>8-Br-II (Chol)</td><td> 4,5</td><td> &gt;16,0</td>

<td> 572</td><td>8-Fl (Chol)</td><td> &gt;16,0</td><td> &gt;16,0</td>

<td> 571</td><td>8-F-II (Chol)</td><td> 4,7</td><td> 9,2</td>

Discussion

The four class II compounds Mrz 2/499, 2/501, 2/514 and 2/516 are glycine-B antagonists in vitro and exhibit much better systemic and / or CNS availability in vivo than the corresponding class I compounds (Mrz 2/585). , 2/501, 2/503 and

2/519). CNS entry is a major problem for nearly all glycine-B antagonists found to date, but this new class of compounds bypasses such a major barrier and, accordingly, is therapeutically relevant glycine-B antagonists.

Access salts

The addition salts of compounds 5, 6, 7, 8, and 10 with quaternary amines (e.g., 4-tetramethylammonium, 4-tetraethylammonium), quaternary amino alcohols (e.g., choline), or quaternary amino acids (e.g., Ν) are prepared according to the procedures described above. , Ν, Νtrimethylserine salts). Choline and 4-tetramethylammonium salts significantly improve bioavailability and are preferred.

Pharmaceutical compositions

The compounds of the present invention may be incorporated into pharmaceutical compositions consisting of a pharmaceutically acceptable carrier or diluent and the active compound of the present invention. Such compositions may be administered orally or parenterally to an animal, especially a human. For example, solid preparations or pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, powders or granules. In such solid pharmaceutical compositions, the active ingredient or prodrug thereof is mixed with at least one pharmaceutically acceptable diluent or carrier, such as cane sugar, lactose, starch, talc or synthetic or natural resin, a binder such as gelatin, a lubricant such as sodium stearate and / or sodium stearate. or a disintegrating agent such as sodium bicarbonate. In order to achieve a sustained-release effect, a substance such as a hydrocolloid or other polymer may be incorporated into the pharmaceutical composition. Additional materials such as lubricants or buffers, as is conventional in the art, may be added. If desired, tablets, pills, or granules may be enteric coated. Liquids for oral administration may be in the form of liposomes, emulsions, solutions or suspensions containing commonly used inert diluents such as water. In addition, such liquid pharmaceutical compositions may contain wetting, emulsifying, dispersing or surfactant agents, as well as sweetening, flavoring or aromatic substances.

Suitable formulations for parenteral administration include, but are not limited to, sterile aqueous or non-aqueous solutions, suspensions, liposomes, or emulsions. In this form of pharmaceutical composition presentation, a number of additional substances known in the art may be used as a pharmaceutically acceptable diluent or carrier.

Depending on the desired mode of administration and the duration of treatment, the exact dosage of the active compound in the formulations of the present invention may vary, particularly depending upon the attending physician or veterinarian.

The active agent of the present invention may, when administered, generally be combined with other pharmaceutically active agents.

In the compositions of the present invention, the proportion of active agent or agents can vary within wide limits, it is only necessary that the active ingredient or prodrug of the present invention is present in an effective amount, that is, in an appropriate effective dosage form. Usually several dosage forms, as well as several individual active compounds, are administered simultaneously or even in the same pharmaceutical composition or dosage form.

As stated above, the compounds of the present invention are suitable, in particular in the form of pharmaceutical compositions or formulations thereof, for oral or parenteral administration, and the precise individual dosage as well as daily doses are determined on a case-by-case basis according to well known medical and / or veterinary principles. or as directed by your veterinarian.

In addition to oral and parenteral administration, rectal and / or intravenous administration may be employed; doses are usually significantly lower when parenteral administration is used, although oral administration is preferred. Suitable amounts of about 1-3 grams per day in multiple or divided doses. Wider ranges of about 0.5 to 10 grams per day may also be used, depending on the circumstances of the individual case. However, 500 mg of the active compound has been found to be particularly suitable for use in tablets, the individual dose may vary from about 200 to 1000 mg, and the amount of 500 mg proposed for use in tablets may be administered orally, for example 1-3 times a day. Needless to say, more than one tablet may be administered per dose if the above suggested daily oral intake of 1-3 g per day is required.

As noted above, the compound of the present invention or a prodrug thereof may be administered to an animal, including a human, by any of several routes, for example, orally in the form of capsules or tablets, parenterally in the form of sterile solutions or suspensions, or intravenously. in the form of sterile solutions. Other conventional routes of administration are subcutaneous, subcutaneous, mucosal, intramuscular, and intraperitoneal, and the particular route of administration will, as usual, be selected by the attending physician or veterinarian.

It is to be understood that the present invention is not limited to the specific compounds, compositions, methods or methods described herein, as many modifications and modifications thereof will be readily apparent to those skilled in the art with respect to the present invention. .