JPH0529207B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes nerve growth factor (Nerve growth factor).
It induces the production and secretion of NGF factor (hereinafter abbreviated as NGF) in the central tissue, activates the function of the innervating nerves, and promotes the repair of the innervating nerves or reinnervation by native nerves, thereby promoting Alzheimer's disease. Senile Dementia of Alzheimer
NGF including Type (hereinafter abbreviated as SDAT)
It is related to production and secretion promoters. [Background of the invention] Since NGF was discovered by R. Levi-Montalcini and S. Cohen, it has been the subject of numerous studies, and has already been studied in the differentiation and growth of peripheral nerve cells, especially sensory and sympathetic nerve cells during the embryonic period. , has been shown to be an essential factor for the survival and functional maintenance of mature sympathetic neurons (H.
Thoenen and YABarde: Physiol.Rev. 60 , 1284−
1335, 1980 and BAYanker and EMShooter:
Ann. Rev. Biochem. 51 , 845-868, 1982). However, NGF is a physiologically active substance in ultra-trace amounts, and its distribution and dynamics within tissues were unknown until recently.
It was not possible to directly prove its effect in vivo. In recent years, the active subunit of NGF (hereinafter referred to as β-NGF
Enzyme Linked Immunosorbent Assay
ELISA) has been developed and improved, and detection sensitivity and specificity have dramatically increased (S. Furukawa et al.
al: J. Neurochem. 40 , 734-744, 1983 and S.
Korshing and H. Thoenen: Proc. Natl. Acad. Sci.
USA 80 , 3513-3516, 1983). In addition, the NGF gene was cloned and structurally analyzed, and its messenger DNA was investigated using β-NGF complementary DNA (abbreviated as cDNA) as a probe.
A method for quantifying RNA (abbreviated as mRNA) has also been established (DL Shelton and LF Reichardt: Proc. Natl.
Acad.Sci.USA 81 , 7951-7955, 1984 and R.
Heumann et al.: EMBO J. 3 , 3183-3189, 1984). As a result, in the periphery, NGF is distributed to tissues innervated by sympathetic nerves.
In particular, it was confirmed that the mRNA content was high, and that there was a positive correlation between the amount and the norepinephrine content, that is, the degree of sympathetic innervation. More surprisingly, NGF also appears in the central nervous system of rats, particularly the hippocampus, neocortex, olfactory bulb, septal area of the basal forebrain, Broca's diagonal zone, and magnocellular basal ganglia.
was detected, and its mRNA content was found to be high in the hippocampus and neocortex, and low in the basal septal area, as well as in other areas of the brain where NGF is not detected (S. Korshing et al.: EMBO J. 4 , 1389-1393,
1985). This result was subsequently replicated one after another by other research groups (DL Shelton and LF
Reichardt: Proc.Natl.Acad.Sci.USA 83 ,
2714−2718, 1986 and S.R. Whittemore et al.:
Proc.Natl.Acad.Sci.USA 83 , 817−821,
1986). This result confirms the physiological and chemical experimental evidence that NGF acts as a so-called "neurotrophic factor" on cholinergic nerve bundles in the central nervous system. In other words, NGF is gene expressed, produced, and secreted not only in the periphery but also in the central region, especially the hippocampus, neocortex, and olfactory bulb.
It is taken up by the nerve terminals of cholinergic nerve bundles that project to this area, and reaches the nerve cell body in the nucleus of origin in the basal part of the cerebrum through reverse axonal transport. Here, NGF maintains the survival and function of the nerves, such as choline acetyltransferase (choline acetyltransferase).
It has become clear that it acts as an essential factor for the gene expression of acetyltransferase (abbreviated as CAT) (MESchwab et al.: Brain Res. 168 ,
473−483, 1979 and M. Seiler and MESchwab:
Brain Res. 300 , 33-39, 1984 and H. Gnahn
et al.: Dev.Brain.Res. 9 , 45-52, 1983). It has also been demonstrated that NGF receptors exist in the brain, and that NGF and receptor complexes are transported from the cerebral cortex to the basal ganglia, and from the hippocampus to Broca's diagonal zone and septal area (M. Taniuchi et al.: Proc. Natl. .
Acad.Sci.USA, 83 , 1950−1954, 1986), NGF
It was further confirmed that the molecule is closely and decisively involved in central physiological functions. On the other hand, there are already many findings indicating that the direct cause of SDAT, whose characteristic early symptoms are decreased memory ability and disorientation, is degenerative ataxia of the central cholinergic nervous system. That is, in SDAT patient brains, CAT
Activity is markedly reduced, but in the early stages,
A series of biochemical studies have shown that this is not due to degeneration and loss of neurons in the cerebral cortex and hippocampus, but rather to ataxia and degeneration of cholinergic nerve bundles, which supply exogenous acetylcholine to these regions. (EKPerry
et al.: Lancet, 1 , 189, 1977, et al.). Subsequently, pathological findings in the patient's brain (PJ
Whitehouse et al.: Ann. Neurol. 10 , 122-126.
(1981, et al.) and behavioral pharmacological analysis of rats with memory and learning disabilities due to basal forebrain destruction (C.
Flicker et al.: Pharmacol.Biochem.Behave. 18 ,
973-981, 1983, and others). Clinically, attempts have also been made to administer acetylcholinesterase inhibitors and precursor substances as a therapy to activate the cholinergic system in the brain of SDAT patients, and some cases of symptom improvement have been reported. Separately, there is a growing movement to examine the expression of the NGF gene in SDAT patients and directly analyze the correlation between imbalances in NGF production and secretion and the onset of SDAT (M. Goedert et al.: Mol. Brain
Res. 1 , 85-92, 1986). [Purpose of the Invention] Based on the above-mentioned two major research developments, the inventors have discovered
The direct cause of the learning and memory impairments manifested as reduced memorization and disorientation, which are the characteristic early symptoms of SDAT, is the progressive degeneration of cholinergic nerve bundles that project from the basal part of the brain to the cerebral cortex and hippocampus. Even if it is a malfunction of the innervated area, the essential cause is a deficiency in the production and secretion of NGF, a "neurotrophic factor" that is produced and secreted in the innervated area and guarantees nerve innervation. It is something. Therefore, for the purpose of preventing or treating the progression of SDAT, therapy that improves the utilization of acetylcholine cannot be expected to have more than a temporary effect; rather, it is necessary to ensure the production and secretion of NGF in the cerebral cortex and hippocampus.
We believe that the only effective way is to break the functional vicious cycle that has been established with the controlling nerves. However, in this case, human-type β can be obtained by cloning the gene.
-Although the path to large-scale preparation of NGF has been opened, it is expected that various pharmaceutical restrictions will be unavoidable in the direct application of proteins with molecular weights exceeding 10,000. The inventors searched for a low-molecular-weight compound that activates the gene expression function of NGF and increases its production and secretion, and by administering this peripherally during relatively mild symptoms, it can be used to treat the remaining parts of the central cerebral cortex and hippocampus. In addition to increasing NGF production ability and preventing the progression of degeneration of the innervating nerves, it also promotes nerve repair and reinnervation with surviving nerves, etc.
The screening method of the present invention was established with the goal of establishing a treatment method that relies on the plasticity of brain function.
Namely, two-step NGF production in vitro, secretion promotion test, acute toxicity test, NGF production in vivo,
Compounds were sequentially selected through secretion stimulation tests, and their effects were finally confirmed through behavioral pharmacology tests, leading to the completion of the present invention. Test methods and examples are shown below. In addition, in this specification, regarding the substituent R in general formula (A), a group of compounds in which R is two hydrogen atoms or two acyl groups is referred to as a compound (), and R is -
The group of compounds in which CO- is the compound (), R is -
A group of compounds that are CO・CO- are called compounds (), and a group of compounds where R is -C(CH ₃ ) ₂ - is called a compound (). These compounds are listed in general reagent catalogs. We purchased, purified and used, but those that were expected to be difficult to obtain were prepared as follows. That is, a Friedel-Crafts reaction condensate of catechol and the corresponding carboxylic acid chloride using anhydrous aluminum chloride as a catalyst, or a Friedel-Crafts reaction condensate of catechol and the corresponding carboxylic acid using BF ₃ as a catalyst is usually used. The compound () (R=H, one of R ₁ and R ₂ is a hydrogen atom and the other is an alkyl group) was obtained by reduction and purification using the method described in (a). The hydroxyl group of the thus obtained compound () (R, R ₁ = H, R ₂ = acetyl group) is acetylated, and the above reaction is repeated and deacetylated using this as a starting material in place of catechol, so that R ₁ also has an alkyl group. A series of compounds () were obtained. The ester-type substituted product of the hydroxyl group is the compound () (R
=H) is reacted with an acid anhydride in the presence of a base to form a compound () (R = acyl group), and similarly reacted with phosgene or a carbonate ester to form a compound ().
Similarly, compound () was easily obtained in high yield by reacting with oxalyl chloride. The ether-type substituted product of the hydroxyl group is the compound () (R
=H) was reacted with acetone in the presence of acid to obtain compound () in high yield. [Test 1] In vitro using mouse fibroblasts
NGF production and secretion promotion test LM cells (ATCC, CCL 1.2), an established mouse fibroblast cell line, proliferate in a serum-independent manner.
It has been shown that NGF is produced and secreted into the culture medium, and that relatively high concentrations of catecholamines promote this without mediating adrenergic receptors (Y. Furukawa et al.: J. Biol.
Chem. 261 , 6039-6047, 1986). This search method using cultured cells is suitable for searching a large number of compounds;
A test method was set up as a first-stage screening system according to the method of Furukawa et al. i.e. 199 medium supplemented with 0.5% peptone (Gibco
LM cells were precultured in a 24-well culture plate (manufactured by Falcon, 2.1 culture area per culture hole).
Sow approximately ^3x104 cells/culture hole in ^cm2 ) and incubate for 3 days.
Culture at 37°C until completely confluent (approximately 10 ⁶ cells/culture hole). The medium is replaced with 199 medium (0.5 ml/culture hole) supplemented with 0.5% bovine serum albumin (fifth fraction, manufactured by Armor). The test compound was contained in the culture medium at a predetermined concentration, and the NGF concentration in the culture medium after 24 hours was measured using a high-sensitivity ELISA method (S.
Furukawa et al.: J. Neurochem. 40 , 734-744.
(1983). The results were calculated as a magnification of the concentration in the culture medium of the test compound cultured in a medium not containing the test compound. The detection limit of this ELISA method is 0.25 pg/ml, and the control NGF concentration is
Usually 50-200pg/0.5ml/culture hole. Values are shown as the mean ± standard error of four runs using the same cell preparation. Example 1 Table 1 shows the results of examining the basic framework that promotes NGF production and secretion. This revealed that a 1,2-dihydroxybenzene ring, a so-called catechol ring, having a hydroxyl group at the ortho position is the basic structure for exerting activity.

【table】

[Table] Example 2 Side chain at position 4 of 1,2-dihydroxybenzene ring
Table 2 shows the results of examining the influence of _R2 . The effects of catecholamines were shown in group A, and the effects of other catechol derivatives were shown in group B. This results in 1,2
It has been found that the effect of the -dihydroxybenzene ring is enhanced by the side chain at the 4-position, but the degree of enhancement varies greatly depending on the structure of the side chain. That is, an amino group is not essential in the side chain (some in group B exhibit activity comparable to that in group A), and neither are hydroxyl groups or carboxyl groups.
It was also suggested that a saturated structure is preferable to an unsaturated structure, and that a structure with 3 carbon atoms has higher activity than 2 carbon atoms. Surprisingly, it has also been found that homocatechol (4-methylcatechol) of group B, although having one carbon number, exhibits high activity in a low concentration range, approaching that of compounds having three carbon atoms. Although this compound did not have the expected promoting activity when added at high concentrations, cell deformation and detachment of some cells from the vessel wall were observed in this concentration range, indicating that the effect was attenuated due to cytotoxicity. It is thought that it was done.

【table】

[Table] Example 3 Although the promoting effect on NGF production and secretion activity of L-M cells was initially discovered as an action specific to catecholamines, it is common to compounds having a 1,2-dihydroxybenzene ring (catechol ring). It was suggested that a sufficient enhancing effect could be observed even with a simpler alkyl side chain.
Therefore, the effect of the alkyl substituent of R ₁ or R ₂ of R=H in the 3-position or 4-position of 1,2-dihydroxybenzene (in the claimed compound ())

【table】

【table】

【table】

[Table] We examined the results in detail and obtained the results shown in Table 3. As a result, the difference in the position of the substituents of R ₁ and R ₂ is not decisive, and if either one has an unbranched alkyl group with 2 to 5 carbon atoms, the combination of R ₁ and R ₂ is more Compounds that are not bulky, preferably combinations in which one of the atoms is a hydrogen atom,
It was found to be as effective as or more effective than the effective catecholamines, but at lower concentrations. It has also become clear that the attenuation of the effect in the high concentration range, which is observed for compounds with side chains with a small number of carbon atoms, is no longer observed in the measured concentration range for compounds with a large number of carbon atoms. The compound that showed high activity is different from catecholamines and is not known to have adrenergic neurotransmitter activity, and was judged to be advantageous in terms of clinical application in the present invention, and has since been subjected to higher-level tests. I decided to do a further search based on this. Example 4 Compound found to have high activity in Example 3,
That is, cells in the acute phase in the high concentration range of catechols having a linear alkyl group at the 3rd or 4th position.

[Table] Chill