WO2000046193A2

WO2000046193A2 - Ether derivatives having neuronal activity

Info

Publication number: WO2000046193A2
Application number: PCT/US2000/002773
Authority: WO
Inventors: Thomas Brumby; Fiona Mcdonald; Eckhard Ottow; Herbert Schneider
Original assignee: Schering AG; Vertex Pharmaceuticals Inc
Current assignee: Bayer Pharma AG; Vertex Pharmaceuticals Inc
Priority date: 1999-02-03
Filing date: 2000-02-03
Publication date: 2000-08-10
Anticipated expiration: 2001-08-03
Also published as: AU3587300A; WO2000046193A3

Abstract

The invention relates to the compounds of Formula (I), as well as the process for their production and their use as pharmaceutical agents. The invention also relates to the compounds of Formula (IA), their isomers and physiologically compatible salts.

Description

Ether Derivatives

The invention relates to ether derivatives, the process for their production and their use in pharmaceutical agents.

It has been reported that compounds with an affinity for the FK506 binding protein (FKBP) that inhibit that protein's rotamase activity also possess nerve growth stimulatory activity. [Lyons et al., PNAS. 91, pp. 3191-3195 (1994)]. Many of these such compounds also have immunosuppressive activity.

FK506 (Tacrolimus), an immunosuppressive drug, has been demonstrated to act synergistically with NGF in stimulating neurite outgrowth in PC 12 cells as well as sensory ganglia [Lyons et al. (1994)]. This compound has also been shown to be neuroprotective in focal cerebral ischemia [J. Sharkey and S. P. Butcher, Nature, 371, pp. 336-339 (1994)] and to increase the rate of axonal regeneration in injured sciatic nerve [B. Gold et al, J. Neurosci.. 15, pp. 7509-16 (1995)].

The use of immunosuppressive compounds, however, has obvious drawbacks. In addition to compromising immune function, prolonged treatment with these compounds can cause nephrotoxicity [Kopp et al., J. Am. Soc. Nephrol., 1, p. 162

(1991)], neurological deficits [P. C. DeGroen et al., N. Rng. J. Med._r 317, p. 861 (1987)] and vascular hypertension [Kahan et al., N. Eng. J. Med., 321, p. 1725 (1989)].

More recently, sub-classes of FKBP binding compounds which inhibit rotamase activity, but which purportedly lack immunosuppressive activity have been disclosed for use in stimulating nerve growth [see United States patents 5,614,547; 5,696,135; WO 96/40633; WO 96/40140; WO 97/16190; J. P. Steiner et al., Proc. Natl. Acad. Sci. USA.

94, pp. 2019-23 (1997); and G.S. Hamilton et al., Bioorg. Med. Chern. Lett.. 7, pp. 1785-

90 (1997)]. While these compounds supposedly avoid certain unwanted side effects of immunosuppressive FKBP binding compounds, they still bind to FKBP and inhibit its rotamase activity. This latter property may still lead to undesirable side effects due to other roles FKBP may play in mammals.

Surprisingly, it is now known that binding to FKBP is not necessary for neuronal activity. Co-pending United States patent application Serial Nos. 08/748,447,

08/748,448 and 08/749,114, each of which is incorporated by reference in its entirety, each describe the use of non-FKBP binding, non-immunosuppressive compounds for stimulating nerve growth and preventing neurodegeneration. Due to their lack of affinity for FKBP, these compounds advantageously avoid any potential interference with FKBP- associated biochemical pathways. These compounds do, however, inhibit multi-drug resistance ("MDR") through inhibition of the p-glycoprotein and MRP. While it appears that the dosages of those compounds necessary to stimulate nerve growth and prevent neurodegeneration are lower than those that effect MDR, it would still be desirable to obtain compounds which are specific for neuronal activity, without other significant mechanisms of action.

It is known that piperidine and pyrrolidine derivatives have immunosuppressive and non-immunosuppressive properties. For example, WO 96/40633 describes that N- glyoxyl-prolylester compounds, which have an affinity to FKBP receptors, have a neurotrophic action and stimulate neuronal regeneration as inhibitors of the FKBP-

rotamase. The stimulation of neurite growth in nerve cells with piperidine derivatives is described in WO 96/41609, which is incorporated by reference herein in its entirety. The clinical use of the previously known piperidine and pyrrolidine derivatives for stimulation of neurite growth does not increase the chances of success, since the compounds are unstable in plasma and do not pass through the blood-brain barriers in sufficient amounts. WO 99/10340 also discloses compounds having neuronal activity.

Its entire disclosure is also incorporated by reference herein.

Applicants have identified several subclasses of compounds that have potent neuronal activity. The term "neuronal activity," as used herein, includes stimulation of damaged neurons, promotion of neuronal regeneration, prevention of neurodegeneration and treatment of a neurological disorder. The compounds of this invention have activity in both peripheral nerves and the central nervous system.

Applicants have discovered diverse genera of compound with neuronal activity which do not bind to FKBP, and which do not have multi-drug resistance reversal activity. Without being bound by theory, applicants believe that the compounds disclosed in this application exert their neuronal activity by increasing cytoplasmic Ca^{2 '} concentrations. This is likely achieved by interaction, either direct or indirect, with a calcium release channel, such as the ryanodine receptor or the inositol 1 ,4,5-trisphosρhate receptor, in the endoplasmic reticulum of the nerve cell.

Thus, according to one embodiment, the invention provides a method of stimulating nerve growth or preventing neurodegeneration by contacting nerve cells with a compound that: a. increases cytoplasmic Ca^2~ concentration or binds to the ryanodine receptor; b. does not bind to FKBP; and c. does not possess MDR reversal activity. According to a related embodiment, the present invention provides a compounds that: a. has neuronal activity; b. increases cytoplasmic Ca^2* concentration or bind to the ryanodine receptor; c. does not bind to FKBP; and d. does not possess MDR reversal activity.

The term "increases cytoplasmic Ca²" concentration," as used herein means a detectable increase in channel current recorded in the single channel recording assay described below in the presence of such a compound as compared to an appropriate control. Alternatively, the term "increases cytoplasmic Ca²" concentration," as used herein means a detectable shift in the fluorescence spectrum in the cell assay described herein.

The term "binds to the ryanodine receptor," as used herein, means that the compound specifically competes with ryanodine for binding to microsomes in the assay described below.

The term "does not bind FKBP," as used herein means that the compound demonstrates a Ki of 10 μM or greater in at least one of the rotamase inhibitory assays described below. The term "does not possess MDR reversal activity," as used herein means that at a concentration of 2 5 μM, the compound has an MDR ratio of less than 7 0. and preferably less than 3 0 in at least one of the MDR assays described below

Single-channel recording experiments are useful to determine if the compounds of this invention cause the requisite increase in cytoplasmic Ca² concentration These expeπments are conducted as descπbed in E Kaftan et al , Circulation Research. 78, pp

990-997 (1996), the disclosure of which is herein incorporated by reference Single channel recordings are conducted under voltage clamp conditions with a pair of Ag/AgCl electrodes contacting the solutions via CsCl junctions Vesicles are added to the cts chamber and fused with planar lipid bilayers composed of phosphatidylethanolamine/phosphatidylcholme (3 1, 30 mg/ml in decane, Avanti Polar Lipids) The trans chamber contains 250 mM HEPES and 53 mM B(OH)₂, pH 7 35, the cis chamber contains 250 mM HEPES-Tπs pH 7 35 Compounds dissolved in methanol are added to the cis chamber Channel currents are amplified using a bilayer clamp amplifier (BC-525A, Warner Instruments) and recorded on VHS tape (Dagen Corp )

Data are filtered to an eight-pole Bessel filter (Frequency Devices) to 500 Hz, digitized at 2 kHz, transferred to a personal computer, and analyzed with pClamp version 6 0 (Axon Instruments) Single channel recordings are done at least 3 times for each compound condition Ryanodine binding may be measured by incubating microsomal protein with ³H- ryanodine in buffer containing 36 mM Tπs pH 7 2 and 50 mM KCl in the absence or presence of test compounds Controls for maximum binding were done in the presence of 0 72 mM ATP and 36 μM CaCl₂ Nonspecific binding was measured in the presence of 25 μM unlabeled ryanodine Binding reactions were incubated for 2 hours at room temperature, and then centrifuged for 15 minutes at 30,000 x g. The pellets were solubilized and the radioactivity was measured by scintillation counting.

Alternatively, the flux of cytoplasmic Ca² into the cell can be followed fluorescently. For example, neuronal cells can be incubated with NGF and a calcium binding fluorescent dye, such as Fura-2, in a calcium-containing buffer. Cells are imaged continuously both before and after the addition of a test compound of this invention. The difference in fluorescent intensity before and after the addition of compounds is then plotted as a ratio of fluorescence units at 340 n and 380 nm.

Testing a compound of this invention to confirm that it binds to FKBP12 with a Ki of 10 μM or higher may be achieved using several assays known in the art. In particular, those compounds may be assayed for their ability (or lack thereof) to inhibit rotamase. Examples of assays that measure inhibition of FKBP 12 rotamase activity are those in which the isomerization of an artificial substrate — N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide ~ is followed spectrophotometrically [M. W. Harding et al., Nature, 341, pp. 758-60 (1989); by J. J. Siekierka et al., Nature, 341, pp. 755-57 (1989); and S. T.

Park et al., J. Biol. Chem.. 267, pp. 3316-24 (1992)]. The assay includes the cis form of the substrate, FKBP 12, the compound to be tested and chymotrypsin. Chymotrypsin is able to cleave p-nitroanilide from the trans form of the substrate, but not the cis form. Release of p-nitronilide is measured. Other FKBP binding assays include a competitive LH20 binding assay using labeled FK-506 as a reporting ligand. These have been described by M. W. Harding et al, Nature, 341, pp. 758-60 (1989) and by J. J. Siekierka et al, Nature, 341, pp. 755-57 (1989). To determine whether a compound according to this invention has the requisite

MDR ratio below 7.0, any of the assays described in United States patents 5,543,423,

5,717,092, 5,726,184 or 5,744,485, the disclosures of which are herein incorporated by reference in their entireties, may be utilized. In particular, cell lines which are known to be resistant to particular drug are employed. These cell lines include, but are not limited to, the L1210, P388D, HL60 and

MCF7 cell lines. Alternatively, resistant cell lines may be developed. The cell line is exposed to the drug to which it is resistant, or to the test compound; cell viability is then measured and compared to the viability of cells which are exposed to the drug in the presence of the test compound ("MDR ratio").

The compounds of Formula I are metabolically stable and pass through the blood- brain barriers and stimulate the neurite growth by itself or in combination with neuronal growth factors. Since the new compounds also do not show any significant side effects, they are suitable for treatment of the various neuropathological diseases, which are affected by neuronal regeneration and growth, such as, e.g., peripheral nervous disturbances, which are caused by physical injuries or diseases such as diabetes; physical injuries of the central nervous system (e.g., of the brain or spinal cord); strokes; neurological disorders by neurodegenerations such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. The invention relates to the compounds of Formula I and their physiologically compatible salts

h

R is hydrogen, Ar, straight-chain or branched C,-C₇ alkyl, which can be substituted with Ar or E, straight-chain or branched C₂-C alkenyl, which can be substituted with Ar or E, C₃-C-, cycloalkyl, which can be substituted with Ar or E or C₅-C₇ cycloalkenyl, which can be substituted with Ar or E,

Y is -(C=O)-(C=O)-, -SO₂-, -(C=O)NH-, -(C=S)NH-, -(C=O)-(C=O)-O-, - (C=O)-(C=O)NH-, -(C=O)-O- or -SO₂-NH-,

R is straight-chain or branched C,-C₆ alkyl, which can be substituted with phenyl or halogenated phenyl,

R is straight-chain or branched C,-C₆ alkyl, straight-chain or branched C₂-C₆ alkenyl, C₃-C ₇ cycloalkyl, C -C ₅ cycloalkenyl, cyclohexylmethyl, whereby the alkyl, alkenyl, cycloalkyl and cycloalkenyl radical can be substituted by the same or a different Ar group in one to two places, or R² and R³ together with the N-atom form a 5- to 7-membered heterocycle, which can be saturated or unsaturated and which can be substituted with

C,-C₄ alkyl and OH,

X is -O-,

R⁴ is Ar, straight-chain or branched C,-C₉ alkyl, straight-chain or branched

C₂-C₉ alkenyl, C₃-C₇ cycloalkyl, C₅-C₇ cycloalkenyl, whereby the alkyl radical and the alkenyl radical can be substituted with Ar, C_y-C-. cycloalkyl and C₅-C₇ cycloalkenyl by the same or different groups in one to two places, Ar is a C₆-C₁₂ mono- or bicyclic aromatic compound, which can contain 0 to

4 N, S or O atoms and which optionally is partially hydrogenated and which can be substituted with E in one to three places, and E is halogen, hydroxy, nitro, CF₃, CN, OCF„ amino, phenyl, methylenedioxy, phenoxy, benzyloxy, C,-C₄ alkoxy or C,-C alkyl.

The compounds of Formula I can also be present as stereoisomers, geometric isomers or stable tautomers. The invention comprises all possible isomers, such as E- and Z-isomers, S- and R-enantiomers, diastereomers, racemates and mixtures thereof. The stereochemistry of the CH group, which carries substituents R³, can be R or preferably S. Conventional methods can be used to prepare the desired structures, e.g., enzymatic, (chiral) HPLC, diastereomer formation, etc.

The physiologically compatible salts can be formed with inorganic and organic acids, such as, for example, oxalic acid, lactic acid, citric acid, fumaric acid, acetic acid, maleic acid, tartaric acid, phosphoric acid, HCl, HBr, sulfuric acid, p-toluenesulfonic acid, methanesulfonic acid, i.a.

In each case, alkyl means a straight-chain or branched alkyl group, such as, e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, tert- pentyl, neopentyl, n-hexyl, sec-hexyl, heptyl, octyl, nonyl.

The alkenyl substituents contain at least one double bond, such as, for example, the following radicals: vinyl, 2-propenyl, 1-propenyl, 2-butenyl, 1-butenyl, 1 -methyl-l - propenyl, 2-methyl-2-propenyl, 3-methyl-2-propenyl, l -penten-3-yl, n-hexenyl, 1 - hepten-4-yl.

Cycloalkyl is defined in each case as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Cycloalkenyl means, e g , cyclopentenyl, cyclohexenyl and cycloheptenyl

In each case, halogen means fluorine, chlorine, bromine or iodine In each case, in the embodiment that does not contain heteroatoms, Ar is defined as 1- and 2-naphthyl, biphenyl, indenyl and preferably phenyl In the embodiment that contains heteroatoms, Ai is a monocyclic or bicychc heteioaromatic compound, which contains 5 to 6 ring members in each ring and 1 to 4 heteroatoms and which can be partially hydrogenated, Ar preferabh means a 5- or 6-πng that contains 1 to 3 heteroatoms and that can have a benzene ring fused, whereby the bonding in general is via carbon atoms For example, the following heteroaromatic compounds can be mentioned furyl, thienyl, pyπdyl, pyrrolyl, oxazolyl, thiazolyl, isothiazolyl, lmidazolyl, pyrazolyl, isoxazolyl, tπazolyl, oxadiazolyl, pyπmidinyl, pyrazinyl, indolyl, benzimidazolyl, benzothiophenyl, qumolmyl, isoqumohnyl, quinoxahnyl, 1,2,3,4-tetrahydroqumohnyl and benzothiazolyl Preferred are pyπdmyl, thienyl, furyl, thiazolyl, indolyl, quinohnyl, and isoqumohnyl Especially preferred are 2-, 3- and 4-pyπdyl, 2- and 3-thιenyl, 2- and

3-ιndolyl, and 2-, 4- and 5-thιazolyl, and especially pyπdyl

The invention also relates to the compounds of Formula I A. then isomers and physiologically compatible salts

(I A), in which R^IA is straight-chain or branched C,-C₇ alkyl, straight-chain or branched C₂-

C₇ alkenyl, C₃-C₅ cycloalkyl, C₅-C₇ cycloalkenyl or Ar' optionally substituted with halogen, hydroxy, nitro, CF„ C,-C₄ alkoxy or C,-C₄ alkyl,

R is straight-chain or branched C,-C₇ alkyl, straight-chain or branched C

C-, alkenyl, C_3.5 cycloalkyl, C₅-C_j cycloalkenyl, whereby the alkyl radical and the alkenyl radical can be substituted by the same or a different Ar' group in one to two places, r is 1 or 2, and Ar' means C₆-C₁₂ aryl or 5- or 6-membered heteroaryl with 1 -3 N, O or S atoms, whereby the aryl radical and the heteroaryl radical can be substituted with halogen, hydroxy, C,-C₄ alkoxy, nitro, CF₃ or C,-C₄ alkyl by the same or a different group in one to three places. The embodiments of the compounds of Formula I that are mentioned as preferred also are regarded as preferred embodiments of the compounds of Formula IA.

Preferred embodiments of R¹ are Ar, straight-chain or branched C,-C₇ alkyl, which can be substituted with Ar or E, and C₃-C₇ cycloalkyl, which can be substituted with Ar or E, whereby in the case of cycloalkyl, E means in particular C,-C₄ alkyl.

Especially preferred are phenyl optionally substituted with E and straight-chain or branched C,-C₇ alkyl optionally substituted with E.

Preferred embodiments of Y are -(C=O)-(C=O)-, -SO₂-, -(C=O)NH-, -(C-S)NH-, -SO₂-NH- and especially -(C=O)-(C=O)-.

A preferred embodiment of R² is methyl, ethyl and benzyl optionally substituted with halogen in one to three places in an aromatic compound. A piefeπed embodiment of R¹ is straight-chain or branched C,-C₆ alkyl, w hich can be substituted with Ar in one to two places, whereby Ar can be substituted w ith E in one to three places For example, benzyl and 4-chloιobenzyl can be mentioned

If R² and R³ together with the nitrogen atom form a heterocycle, the latter is preferably saturated and has 5 to 7 ring members, pyrrolidine and piperidine are especially pieferred

As especially preferred embodiments of R^: and R\ R² and R¹ together form a pyrrohdinyl or pipeπdinyl ring

In each case, E pieferably means halogen, hydroxy, nitro, CF₃, CN, C,-C alkoxy and C,-C alkyl

Preferred embodiments of R⁴ are straight-chain or branched C,-C₉ alkyl, which optionally is substituted with Ar in up to four places or Ar optionally substituted w ith E

R⁴ is especially preferably in the meaning of a straight-chain or branched C, - alkyl radical, which is substituted with Ar in one to two places, whereby Ar is in particular in the terminal position

R⁴ can be mentioned with the structure

in which Ai has the above-mentioned meaning, and o and p mean 0, 1, 2 or 3. and n is O or 1

The invention also relates to the use of the compounds of Formula 1 for the production of a pharmaceutical agent for the stimulation of neuronal activin The compounds of Formula I are suitable for stimulation of neurite growth in nerve cells, for stimulation of neuronal regeneration, for prevention of neurodegeneration, for treatment of neurological diseases such as neurodegeneration and for treatment of peripheral neuropathies. The prevention and treatment of neuron cell death, which is triggered by a variety of diseases or physical trauma, is therefore made possible with the compounds of Formula I. The methods of stimulating nerve grow th and preventing neurodegeneration disclosed herein employ the above compounds either alone or in combination with a neuronal growth factor. The methods are useful in treating or preventing nerve damage caused by various neurological diseases and physical traumas and also in ex vivo nerve regeneration.

Diseases that according to the invention can be treated or prevented with the compounds of Formula I are in particular trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy, muscular trauma, progressive muscular atrophy, peripheral neuropathies, peripheral myelin disorders, Alzheimer's disease, Guillain-Barre syndrome, Parkinson's disease, amyotrophic lateral sclerosis, Tourette's syndrome, multiple sclerosis, central myelin disorders, strokes, ischemia, neural degenerative diseases, trauma and Huntington's disease.

The compounds of Formulas I and IA can be administered in one formulation or in separate formulations in combination with a neurotrophic factor or by themselves. The term "neurotrophic factor" relates to compounds that stimulate the growth and the proliferation of nerve cells. Numerous neurotrophic factors are known, such as, for example, NGF, BNDF, aFGF, bFGF, PDGF, BDNF, GDNF, CNTF, NT-3, NT-4/5 and IGF-1 and its derivatives such as gIGF-1 and Des(l-3)IGF-1. NGF is especially preferred for combined use. For use of the compounds according to the invention as pharmaceutical agents, the latter are brought into the form of a pharmaceutical preparation, which in addition to the active ingredient contains vehicles, adjuvants and/or additives that are suitable for enteral or parenteral administration. The administration can be done orally or sublingually as a solid in the form of capsules or tablets or as a liquid in the form of solutions, suspensions, elixirs, aerosols or emulsions or rectally in the form of suppositories or in the form of injection solutions that optionally also can be administered subcutaneously, intramuscularly or intravenously, or topically or intrathecally. As adjuvants for the desired pharmaceutical agent formulation, the inert organic and inorganic carrier materials that are known to one skilled in the art are suitable, such as, e.g., water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, etc. In addition, preservatives, stabilizers, wetting agents, emulsifiers or salts for changing the osmotic pressure or buffers can optionally be contained. For parenteral administration, especially injection solutions or suspensions, especially aqueous solutions of active compounds in polyhydroxyethoxylated castor oil are suitable.

Topical administration is also defined as transdermal patches, ophthalmic preparations, and aerosols for inhalation. As carrier systems, surface-active adjuvants, such as salts of bile acids or animal or plant phospholipids, but also mixtures thereof as well as liposomes or components thereof can also be used.

For oral administration, especially tablets, coated tablets or capsules with talc and/or hydrocarbon vehicles or binders, such as, for example, lactose, corn or potato starch, are suitable The administration can also be done in liquid form, such as, for example, as a juice, to which a sweetener is optionally added

The dosage of the active ingredients can vary based on the method of administration, age and weight of the patient, type and severity of the disease that is to be treated and similar factors The daily dose is 0 01-100 mg/kg of body weight/day, whereby the dose can be given as a single dose that is to be administered once or divided into two or more daily doses

If combination therapy is performed, the dose can be reduced due to the synergistic action of the active ingredients The neurotrophic factor is preferably administered in a dose of 0 01 μg - 100 mg/kg/day together with the above-mentioned dose of the active ingredients

The neurotrophic action of the compounds of Formula I and their physiologically compatible salts can be determined according to methods by W E Lyons et al , Proc Natl Acad Sci USA, 91 , pages 3191-95 (1994) The production of the compounds of Formula I is characterized in that a compound of Formula II

in which R² and R have the abo\ e-mentioned meaning and P is a protective group, a) is reacted with A-R⁴, in which A means a reactive leaving group. b) R⁴ optionally is further modified by, e.g., C-C-linkage reactions, such as, e.g., a Heck reaction and/or hydrogenation, c) the amino protective group P is cleaved off, d) Y-R¹ is introduced, and optionally the isomers are separated, and the salts are formed.

A compound of Formula II can also be reacted, however, for example with a ketone, ketal or enol ether to a compound of Formula III

in which R², R³ and P have the above-mentioned meaning, and R and R' are selected so that the desired radical R⁴ forms in the above-mentioned meaning, and then b) the double bond optionally hydrogenates, c) amino protective group P is cleaved off, d) Y-R¹ is introduced and optionally the isomers are separated and the salts are formed.

As protective group P, all known amino protective groups are suitable, such as alkoxycarbonyl groups, such as BOC, trimethylsilylalkoxycarbonyl groups, such as

TeOC, i.a.

As active group A, for example, tosylate, mesylate. halogen or triflate is suitable. The cleavage of the amino protective group and the subsequent introduction of

Y-R¹ is carried out according to known methods, which are described in, for example,

US 5,721,256 and WO 98/291 17 and WO 98/13355.

The Heck reaction is carried out according to methods that are known to one skilled in the art, as described in, e.g., Tetrahedron Letters Vol. 34, 8329 (1993).

The production of enol ether can be carried out, for example, according to the process that is described in Methoden der Organischen Chemie [Methods of Organic Chemistry] (Houben-Weyl), Thieme Stuttgart 1965, Volume VI/3 (oxygen compounds) 90 ffi The optically active compounds of Formula I can be obtained with optically active starting materials or by separation according to commonly used methods, such as, for example, crystallization, chromatography or salt formation in the enantiomers or E/Z- isomers in the intermediate or final stages.

If the production of the starting compounds is not described, the latter are known or can be produced analogously to the processes that are known or according to the processes that are described here. The compounds of Formula IA are also obtained according to processes that are known or analogously to the described processes.

Below, the production of some precursors, intermediate products and products is described by way of example. The presently disclosed compounds advantageously possess neuronal activity, without interfering with other pathways known to be affected by structurally similar compounds.

The nerve growth activity of the compounds of this invention may be initially assayed using several cell culture assays known in the art. For example, the compounds of this invention may be tested in neurite outgrowth assay using pheochromocytoma

PC 12 cells as described by Lyons et al., ENAS, 91, pp. 3191-3195 (1994). A similar assay may be carried out in SH-SY5Y human neuroblastoma cells. Alternatively, the chick dorsal root ganglia assay described in United States patent 5,614,547 or in G. S. Hamilton et al., Bioorg. Med. Chem. Lett.. (1997) and references cited therein, may be utilized.

The compounds of this invention may also be assayed for nerve growth activity in vivo using a mouse model of Parkinson's disease [J. P. Steiner et al., Proc. Natl. Acad. Sci. USA. 94, pp. 2019-23 (1997), United States patent 5,721,256] or following surgical sciatic nerve crush in rats.

These include, but are not limited to, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, muscle injury, progressive muscular atrophy, progressive bulbar inherited muscular trophy, heraiated, ruptured or prolapsed invertebrae disk syndrome's, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies, such as those caused by lead, dapsone, ticks, or porphyria, other peripheral myelin disorders, Alzheimer's disease, Gullain-Barre syndrome, Parkinson's disease and other Parkinsonian disorders, ALS, multiple sclerosis, other central myelin disorders, stroke and ischemia associated with stroke, neural paropathy, other neural degenerative diseases, motor neuron diseases, sciatic crush, neuropathy associated with diabetes, spinal cord injuries, facial nerve crush and other trauma, chemotherapy- and other medication-induced neuropathies and Huntington's disease.

In another aspect, the method is used to stimulate nerve growth ex vivo. For this aspect, the compounds or compositions described above can be applied directly to the nerve cells in culture. This aspect of the invention is useful for ex vivo nerve regeneration.

According to an alternate embodiment, the method of stimulating neurite outgrowth or preventing neurodegeneration comprises the additional step of treating a patient or ex vivo nerve cells in culture with a neurotrophic factor, such as those contained in the compositions of this invention described above.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

The entire disclosure of all applications, patents and publications, cited above, and of corresponding German application No. 199 05 254.9, filed February 3, 1999, is hereby incorporated by reference.

Example 1 a) (2S)- 1 , 1 -Dimethylethyl-2-(2-propen- 1 -yloxymethyl Vpyrrolidine- 1 -carboxylate 6.04 g (30 mmol) of Boc-prolinol is dissolved in tetrahydrofuran, and 1.40 g (45 mmol) of sodium hydride is added. After one hour of stirring at room temperature, 2.55 ml (30 mmol) of allyl bromide is added drop by drop. Then, the reaction mixture is refluxed for 30 minutes, cooled and carefully hydrolyzed with water. The water/THF mixture is extracted several times with dichloromethane, the combined organic phases are washed with salt solution, dried on magnesium and concentrated by evaporation.

6.40 g of orange oil is obtained, which after column chromatography (hexane - hexane/ethyl acetate 4:1) yields 5.44 g (75%) of the title compound as a colorless oil.

¹³C-NMR (75 MHz, CDC1₃); δ = 22.9 T, (23.8 T) C-4); 28.5 D (1 C-3); 28.8 T,

(28.0 T) (C-3); 46.4 T, (46.8 T) (C-5); 56.5 D (C-2); 71.0 T, (70.6 T) (2C-1): 72.0 T

(2C-2); 79.2 S (1C-2); 1 16.6 T (2C-4); 134.9 D (2C-3); 154.5 S (lC-l) ppm.

b) (2S)-1 , 1 -Dimethylethyl-2-G-(3-pyridyl)propoxymethyl)-pyrrolidine- 1 - carboxylate

5.40 g (22 mmol) of the compound that is obtained according to method a) is dissolved in 15 ml of DMF and 0.30 ml of triethylamine. 3.47 g (22 mmol) of 3-bromo- pyridine, 1.85 g (22 mmol) of sodium bicarbonate, 184 mg (0.66 mmol) of triphenylphosphine and 57 mg (0-10 mmol) of palladium(II) acetate are added, and the mixture is kept at 120°C for 4 hours. The black mixture is filtered, concentrated by evaporation, and the residue is chromatographed (hexane - hexane/ethyl acetate 4: 1 -

1 :1).

4.50 g (64%) of a clear, yellow oil, which is directly hydrogenated, is obtained. Hydrogenation:

0.35 g of Pd/C (10%o) is added to the solution of yellow oil in 100 ml of ethanol, and it is hydrogenated at normal pressure and room temperature. The residue is filtered off, the filtrate is concentrated, and 4.40 g (97%) of product is obtained. ¹ C-NMR (75 MHz, CDC1₃); δ = 22.9 T (23.8 T) (C-3); 25.7 S (C-4); 28.5 Q

(1C-3); 29.5 T (2C-3); 31.0 T (2C-4); 46.4 T, (46.9 T) (C-5); 56.4 D (C-2); 69.8 T (2C-

2); 71.8 T, (71.1 T) (2C-1); 79.1 S (1C-2); 123.3 D (Py-5); 135.9 D (Py-4); 137.2 S (Py-

3); 147.3 D (Py-6); 150.0 D (Py-2); 154.6 S (lC-1) ppm.

c) Methyl-2-((2S)-2-(3-(3-pyridyl)propoxymethyl)-pyrrolidin-l -yl)glyoxy1ate 4.39 g (14 mmol) of the compound that is obtained according to method b) is mixed with 21.3 ml of trifluoroacetic acid in 125 ml of dichloromethane while being cooled with ice. It is stirred for 2 more hours at room temperature, mixed with 2N NaOH and extracted with dichloromethane. The combined extracts are washed with salt solution, dried on MgSO₄ and concentrated by evaporation. 2.79 g (92%>) of yellow oil, which is directly further processed, is obtained.

Acylation:

The oil is dissolved in 30 ml of dichloromethane, cooled to 0°C and mixed with 3.50 ml (16 mmol) of triethylamine. A solution of 1.70 ml (18 mmol) of methyloxalyl chloride in 20 ml of dichloromethane is added drop by drop. The mixture is stirred at 0°C for 1.5 hours, then poured into water and extracted with dichloromethane. After working-up and after concentration by evaporation, 3.54 g (91%>) of product is obtained. ^,3C-NMR (75 MHz, CDC1₃); δ = 24.3 T, (21.7 T) (C-3); 27.3 T, (28.3 T) (C-4); 29.4 T, (29.2 T) (2C-3); 30.9 T, (30.5 T) (2C-4); 48.1 T (46.1 T) (C-5); 52.6 Q, (52.5 Q) (1C-3);

57.3 D, (57.2 D) (C-2); 69.9 T, (69.7 T) (2C-2); 70.2 T, (72.4 T) (2C-1); 123.3 D, (123.3 D) (Py-4); 135.9 D, (135.8 D) (Py-3);

137.1 S, (136.8 S) (Py-2); 147.3 D, (147.4 D) (Py-5); 149.9 D, (149.9 D) (Py-1); 158.7 S, (159.2 S) (1C-2); 162.6 S (lC-1) ppm (by-product in ()).

d) 3,3-Dimethy1-1 -t(2SV2-G-(3-pyridyl)propoxymethyl)-pyrrolidin-l -yl)-pentane-

1 ,2-dione

3.54 g (11 mmol) of the compound that is obtained according to method c) is dissolved in 50 ml of THF and cooled to -78°C. 15 ml (15 mmol) of a solution of 1,1- dimethylpropylmagnesium chloride (IM in ether) is added drop by drop and stirred for

2 more hours at this temperature. The reaction mixture is brought to room temperature and allowed to stand overnight. It is quenched with saturated NH₄C1 solution and extracted with ethyl acetate. The extracts are washed, dried on MgSO₄ and chromatographed (hexane - hexane/ethyl acetate 4:1). 1.96 6 (49%>) product is obtained. ^,3C-NMR (75 MHz, CDC1₃); δ = 8.9 D, (8.8 D) (1C-5); 23.5 Q, (23.4 Q) (1C-6); 23.5

Q, (23.8 Q) (1C-7); 24.5 T, (21.5 T) (C-3); 27.4 T, (28.6 T) (C-4); 29.5 T, (29.4 T) (2C- 3); 31.0 T, (30.8 T) (2C-4); 32.4 T, (32.8 T) (1C-4); 46.5 S (1C-3); 47.4 T (45.2 T) (C-5);

56.4 D, (56.9 D) (C-2); 70.0 T (2C-2); 70.0 T, (71.8 T) (2C-1); 123.3 D (Py-5); 135.9 D (Py-4); 137.0 S, (137.1 S) (Py-3); 147.3 D, (147.4 D) (Py-6); 149.9 D, (149.9 D) (Py-2); 165.6 S (lC-1); 207.6 S, (207.4 S) (1C-2) ppm (by-product in ()). Example 2 a) Tosylation

10.0 g (50 mmol) of Boc-prolinol is dissolved in 50 ml of dichloromethane and mixed with 30 ml of pyridine. After cooling to 0°C, a solution of 11.4 g (60 mmol) of p-toluenesulfonyl chloride in 60 ml of dichloromethane is added. The solution is allowed to stand overnight at room temperature. The mixture is added to water and extracted with dichloromethane. The organic extracts are dried with MgSO₄, concentrated by evaporation, and the residue is chromatographed (hexane -→ hexane/ethyl acetate 4:1). 14.1 g (79%) of tosylate is obtained.

b) Etherification

1.41 (10 mmol) of 3-(3-pyridyl)-propanol is dissolved in 30 ml of DMF and mixed with 454 mg of NaH (80%) in paraffin, 15 mmol) and stirred for 1 hour at room temperature. A solution of 3.56 g (10 mmol) of the tosylate in 10 ml of DMF and a catalytic amount of potassium iodide are added, and the reaction mixture is stirred at 120°C for 4 hours. It is poured into IN NaOH, extracted with dichloromethane, the organic phases are dried with MgSO₄ and concentrated by evaporation. After the residue is chromatographed (hexane/ethyl acetate 9:1 - 1 : 1), 673 mg (33%) of 5-(l,l- dimethylethoxy-carbonylamino)pentan-2-one and 681 mg (22%) of the title compound are obtained.

Continuation of Example 2

(2S)-2-((3-(3-Pyridyl)-propoxymethyI)-l-(3,4,5-trimethoxyphenylgIyoxyloyl)-

pyrrolidine 1. 1.60 g (5.00 mmol) of (2S)-l-(l ,l -dimethylethoxycarbonyl)-2-((3-(3- pyridyl)-propyloxymethyl)-pyrrolidιne obtained according to b) is dissolved in 20 ml of CH₂C1₂, and 7.7 ml of trifluoroacetic acid is added.

After 22 hours at ambient temperature, the mixture is concentrated by evaporation in a vacuum, dissolved again in CH₂C1 and washed with 2N

NaOH. The organic phase is dried on Na₂SO₄ and concentrated by evaporation in a vacuum. 1.02 g (83%) of crude product is obtained.

2. 6.00 g (25 mmol) of 3,4,5-trimefhoxyphenylglyoxylic acid is dissolved in 70 ml of toluene, and 2.3 ml (25 mmol) of 1 ,1-dichloromethylether is added. The mixture is heated at 60°C for 17 hours, cooled to room temperature and concentrated by evaporation in a vacuum.

3. The amine that is obtained in 1. is dissolved in 20 ml of CH₂C1₂, and 0.96 ml (6.9 mmol) of triethylamine is added. This solution is added to the acid chloride that is obtained in 2., and the resulting mixture is stirred at ambient temperature for 4 days. The reaction is then diluted with CH₂C1₂ and washed with 2N NaOH and brine and dried on Na₂SO₄. After the solvent is evaporated, 3.5 g of material is obtained, which is subjected to chromatography (ethyl acetate → ethyl acetate/methanol 7:3) to yield 1.42 g (64%) of the title compound as an oil.

[a]_D = -80° (0.6, CHC1₃), ^,3C-NMR (75 MHz, CDC1₃): δ = 24.4T (22.2T) (C 3),

27.5T (28.6T) (C 4), 29.4T (29.1T) (2C3), 30.9T (30.2T) (2C4), 47.5T (45.8T) (C 4), 56.3Q (1C8), 56.7D (56.7D) (C 2), 61.0Q (61.0Q) (1C9), 70.1T (69.9T() 2C2), 70.2T (72.6T) (2C1)), 107.2D (107.4D) (1C4), 123.3D (2C8), 127.9S (128.8S) (1C7), 135.9D (135.7D) (2C7), 137.1S (136.7S) (2C6), 144.1S (143.6S) (1C3), 147.3D (147.3D) (2C9).

149.9D (149.7D) (2C5), 153.3S (153.1S) (1C6), 165.2S (165.1S) (1C1), 190.2S (189.1S) ppm (1C2).

A 1.2: 1 mixture of rotomers is observed, shifts of the minor rotomer in () if observed.

Example 3

(2S)-l -(l ,l-Dimethylethoxycarbonyl)-2-((3-phenyI-l-propenyloxymethyl)- pyrrolidine

(2S)-l -(l ,l -Dimethylethoxycarbonyl)-2-((3-phenyl-2-propenyloxymethyl)- pyrrolidine

3.00 g (14.9 mmol) of Boc-prolinol is dissolved in 15 ml of THF, and 1.08 g

(22.4 mmol) of NaH (50% in paraffin) is added. The mixture is stirred for 1 hour at ambient temperature, and then a solution of 2.94 g (14.9 mmol) of cinnamyl bromide in

5 ml of THF is added. After the reaction is refluxed for 1 hour, it is allowed to cool and hydrolyzed by careful addition of water. The THF/water mixture is extracted with

CH₂C1₂ and the combined organic layers washed with brine, dried on Na_:SO₄, and the solvent is removed in a vacuum. Chromatography of the residue yields 1.21 g (26%) of the vinyl ether and 1.35 g (29%>) of the allyl ether.

Vinyl ether: ¹³C-NMR (75 MHz, CDC1₃): δ = 23.9T (23.0T) (C 4), 28.0T (28.5T) (C 3), 28.5Q (1C3), 30.2T (2C4), 47.0T (46.5T) (C 5), 56.5D (C 2); 72.9T

(72.2T) (2C1 ), 79.3S (79.7S) (1C2), 105.6D (104.9D) (2C3), 125.7D (2C8), 128.3D

(2C6), 128.3D (2C7), 141.7S (141.8S) (2C5), 146.1D (145.6D) (2C2), 154.4S (154.6S)

ppm (lCl). Allyl ether: ^,3C-NMR (75 MHz, CDC1₃): δ - 22.9T (23.8T) (C 4), 28.5T (1C3),

28.8S (28. IT) (C 3), 46.4T (46.9T) (C 5), 56.5D (C 2), 71. IT (70.8T) (2C1 ), 71.8T (2C2), 79.2S (1C2), 126.2D (2C4), 126.5D (2C6), 127.6D (2C8), 128.5D (2C7), 132.2D (2C3), 136.7S (2C5), 154.6S ppm (1C1) A mixture of rotomers is observed, shifts of the minor rotomer in () if observed.

(2S)-1-(1,1 -Dimethylethoxycarbonyl)-2-(3-phenyI-propyloxymethyl)-pyrrolidine

1.21 g of the vinyl ether that is obtained above is dissolved in 30 ml of ethanol, and 130 mg of Pd/C (10%) is added. The mixture is hydrogenated for 20 minutes at ambient temperature under normal hydrogen pressure. Filtration and evaporation of ethanol yield 1.10 g (91%) of the title compound, which is used without further purification or characterization.

(2S)-2-((3-Phenyl-propoxymethyl)-l-(3,4,5-trimethoxyphenylglyoxyloyl)-pyrrolidine

1. 855 mg (2.77 mmol) of (2S)-l -(l,l-dimethylethoxycarbonyl)-2-(3- phenyl-propyloxymethyl)-pyrrolidine is dissolved in 20 ml of CH₂C1₂, and 4.3 ml of trifluoroacetic acid is added. After 16 hours at ambient temperature, the mixture is concentrated by evaporation in a vacuum, dissolved again in CH₂C1₂ and washed with IM NaOH. The organic phase is dried on MgSO₄ and concentrated by evaporation in a vacuum. 592 mg (98%o) of crude product is obtained. 2. 2.67 g (11.1 mmol) of 3,4,5-trimethoxyphenylglyoxylic acid is dissolved in 30 ml of toluene, and 1.0 ml ( 11.1 mmol) of 1 , 1 -dichloroethyl ether is added. The mixture was heated at 60°C for 16 hours, cooled to room temperature, and concentrated by evaporation in a vacuum.

3. The amine that is obtained in 1. is dissolved in 10 ml of CH₂C1₂, 0.56 ml

(4.1 mmol) of triethylamine, and a suspension of the acid chloride that is obtained in 2. in 10 1 of CH₂C1₂ is added. The mixture is stirred at ambient temperature for 5 days. The reaction is then diluted with CH₂C1₂ and washed with 2N NaOH and brine and dried on Na₂SO₄. After the solvent is evaporated, 438 mg of material is obtained, which is subjected to chromatography (hexane → hexane/ethyl acetate 7:3) to yield 204 mg (17%) of the title compound as an oil.

[a]_D = -78° (0.5 CHC1₃), ¹³C-NMR (75 MHz, CDC1₃); δ = 24.4T (22.2T) (C 3),

27.5T (28.6T) (C 4), 31.3T (30.5T) (2C3), 32.3T (32.0T) (2C4), 47.6T (45.9T) (C 5),

56.3Q (1C7), 56.8D (C 2), 61.0Q (61.0Q) (1C8), 70.3T (70.2T) (2C2), 70.6T (72.5T)

(2C1), 107.2D (107.5D) (1C4), 125.8D (2C8), 127.9S (128.8S) (1C6), 128.3D (128.3D) (2C6), 128.3D (128.4D) (2C7), 141.5S (141.8S) (2C5), 144.0S (143.6S) (1C3), 153.4S

(153. IS) (1C5), 165.2S (1C1), 190.3S (189.2S) ppm (1C2).

A 1.1 :1 mixture of rotomers is observed, shifts of the minor rotomer in () if observed.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

1. Compounds of Formula I and their physiologically compatible salts

in which

R¹ is hydrogen, Ar, straight-chain or branched C,-C- alkyl, which can be substituted with Ar or E, straight-chain or branched C₂-C₇ alkenyl, which can be substituted with Ar or E, C₃-C₇ cycloalkyl, which can be substituted with Ar or E or C₅-C₇ cycloalkenyl, which can be substituted with Ar or E, Y is -(C=O)-(C=O)-, -SO₂-, -(C=O)NH-, -(C=S)NH-, -(C=O)-(C=O)-O-, - (C=O)-(C=O)NH-, -(C=O)-O- or -SO₂-NH-,

R² is straight-chain or branched C,-C₆ alkyl, which can be substituted with phenyl or halogenated phenyl, R³ is straight-chain or branched C,-C₆ alkyl, straight-chain or branched C₂- C₆ alkenyl, C₃-C₇ cycloalkyl, C,-C- cycloalkenyl, cyclohexylmethyl, whereby the alkyl, alkenyl, cycloalkyl and cycloalkenyl radical can be substituted with Ar by the same or a different component in one to two places, or R² and R^"' together with the N-atom form a 5- to 7-membered heterocycle, which can be saturated or unsaturated and which can be substituted with C,-C₄ alkyl and OH, X is -O-, R⁴ is Ar, straight-chain or branched C,-C₉ alkyl, straight-chain or branched -C, alkenyl, C₃-C₇ cycloalkyl, C₅-C₇ cycloalkenyl, whereby the alkyl radical and the alkenyl radical can be substituted with Ar, C₃-C₇ cycloalkyl and C₅-C₇ cycloalkenyl by the same or a different component in one to two places, and Ar is a C₆-C_l2 mono- or bicyclic aromatic compound, which can contain 0 to

4 N, S or O atoms and which optionally is partially hydrogenated and which can be substituted with E in one to three places, and E is halogen, hydroxy, nitro, CF₃, CN, OCF₃, amino, phenyl, methylenedioxy, phenoxy, benzyloxy, C,-C₄ alkoxy or C,-C₄ alkyl.

2. Compounds of Formula I according to claim 1 , in which R⁴ means a straight- chain or branched alkyl radical, which is substituted with Ar in one to two places.

3. Compounds of Formula I according to claim 1, in which R' means C,-C alkyl or Ar.

4. Compounds of Formula IA, their isomers and physiologically compatible salts

0 A), in which

R^IA is straight-chain or branched C,-C₇ alkyl, straight-chain or branched C₂- C₇ alkenyl, C₃-C₅ cycloalkyl, C₅-C₇ cycloalkenyl or Ar' optionally substituted with halogen, hydroxy, nitro, CF₃, C,-C₄ alkoxy or C,-C₄ alkyl,

R^4A is straight-chain or branched C,-C₇ alkyl, straight-chain or branched C₂- C₇ alkenyl, C_3.5 cycloalkyl, - cycloalkenyl, whereby the alkyl radical and the alkenyl radical can be substituted with Ar' by the same or a different component in one to two places, r is 1 or 2, and

Ar' means C₆-C,₂ aryl or 5- or 6-membered heteroaryl with 1-3 N, O or S atoms, whereby the aryl radical and the heteroaryl radical can be substituted with halogen, hydroxy, C,-C₄ alkoxy, nitro, CF₃ or C,-C₄ alkyl by the same or a different component in one to three places. 5. 3,3-Dimethyl-l-((2S)-2-(3-(3-pyridyl)propoxymethyl)-pyrrolidin-l-yl)- pentane-l,2-dione

(2S)-2-((3-(3-pyridyl)-propoxymethyl)-l-(3,4,5-trimethoxyphenylglyoxyloyl)- pyrrolidine

(2S)-2-((3-phenyl-propoxymethyl)- l -(3,4,

5-trimethoxyphenylglyoxyloyl)- pyrrolidine according to claim 1.

6. Pharmaceutical agent that contains a compound according to claim 1 and one or more pharmaceutically common vehicles and adjuvants.

7. Pharmaceutical agent according to claim 6 in combination with a neurotrophic factor. 8 Pharmaceutical agent according to claim 7, in which the neurotrophic factor is IGF-1 , gIGF-1 , Des(l-3)IGF-1 , aFGF, bFGF, PDGF, BDNF, CNTF, GDNF. TN-3, NT-4/5 or preferably NGF

9 Use of a compound according to claim 1 for the production of a pharmaceutical agent

10 Use according to claim 9 for treatment and prevention of neurodegeneration, for stimulation of neuronal regeneration, for treatment of neurological diseases and for stimulation of neurite growth

1 1 Use according to claim 10 for the treatment of Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntmgton's disease, ischemia, stroke, multiple sclerosis, peripheral neuropathy, neuralgia, muscular atrophy and Guillain-Barre syndrome

12 Production of the compounds of Formula I, characteπzed in that a compound of Formula II

in which P is a protective group, and R² and R³ have the above-mentioned meaning, a) is reacted with A-R⁴, in which A is a reactive leaving group, b) R⁴ optionally is further modified by C-C-linkage reactions, and/or hydrogenation, c) amino protective group P is cleaved off, d) Y-R¹ is introduced, and optionally the isomers are separated, and the salts are formed.